US20060046250A1 - Novel use of aim 3 acting as a tumor suppressor - Google Patents
Novel use of aim 3 acting as a tumor suppressor Download PDFInfo
- Publication number
- US20060046250A1 US20060046250A1 US10/536,257 US53625705A US2006046250A1 US 20060046250 A1 US20060046250 A1 US 20060046250A1 US 53625705 A US53625705 A US 53625705A US 2006046250 A1 US2006046250 A1 US 2006046250A1
- Authority
- US
- United States
- Prior art keywords
- aim3
- cancer
- polypeptide
- atm
- atr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 title abstract description 8
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 title abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 194
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 138
- 238000000034 method Methods 0.000 claims abstract description 82
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 51
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 51
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 51
- 230000001404 mediated effect Effects 0.000 claims abstract description 37
- 230000006907 apoptotic process Effects 0.000 claims abstract description 36
- 201000010099 disease Diseases 0.000 claims abstract description 34
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 34
- 230000035755 proliferation Effects 0.000 claims abstract description 26
- 230000001105 regulatory effect Effects 0.000 claims abstract description 19
- 210000004027 cell Anatomy 0.000 claims description 248
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 claims description 103
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 claims description 103
- 230000014509 gene expression Effects 0.000 claims description 93
- 206010028980 Neoplasm Diseases 0.000 claims description 92
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 88
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 78
- 229920001184 polypeptide Polymers 0.000 claims description 76
- 230000000694 effects Effects 0.000 claims description 60
- 210000001519 tissue Anatomy 0.000 claims description 58
- 201000011510 cancer Diseases 0.000 claims description 54
- 108020004414 DNA Proteins 0.000 claims description 47
- 101000891649 Homo sapiens Transcription elongation factor A protein-like 1 Proteins 0.000 claims description 31
- 101000596402 Mus musculus Neuronal vesicle trafficking-associated protein 1 Proteins 0.000 claims description 31
- 101000800539 Mus musculus Translationally-controlled tumor protein Proteins 0.000 claims description 31
- 101000781972 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Protein wos2 Proteins 0.000 claims description 31
- 101001009610 Toxoplasma gondii Dense granule protein 5 Proteins 0.000 claims description 31
- 102100040250 Transcription elongation factor A protein-like 1 Human genes 0.000 claims description 30
- 239000000126 substance Substances 0.000 claims description 30
- 239000012634 fragment Substances 0.000 claims description 20
- 239000008194 pharmaceutical composition Substances 0.000 claims description 17
- 102100034533 Histone H2AX Human genes 0.000 claims description 16
- 101001067891 Homo sapiens Histone H2AX Proteins 0.000 claims description 16
- 230000004913 activation Effects 0.000 claims description 16
- 210000004881 tumor cell Anatomy 0.000 claims description 14
- 230000001939 inductive effect Effects 0.000 claims description 12
- 230000033616 DNA repair Effects 0.000 claims description 10
- -1 Rad17 Proteins 0.000 claims description 9
- 230000003213 activating effect Effects 0.000 claims description 9
- 230000002401 inhibitory effect Effects 0.000 claims description 9
- 101100220616 Caenorhabditis elegans chk-2 gene Proteins 0.000 claims description 8
- 206010009944 Colon cancer Diseases 0.000 claims description 8
- 239000004480 active ingredient Substances 0.000 claims description 8
- 201000007270 liver cancer Diseases 0.000 claims description 8
- 208000014018 liver neoplasm Diseases 0.000 claims description 8
- 230000027455 binding Effects 0.000 claims description 7
- 208000029742 colonic neoplasm Diseases 0.000 claims description 7
- 230000022983 regulation of cell cycle Effects 0.000 claims description 7
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 6
- 230000037041 intracellular level Effects 0.000 claims description 6
- 210000003734 kidney Anatomy 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 201000009030 Carcinoma Diseases 0.000 claims description 5
- 101000971203 Homo sapiens Bcl-2-binding component 3, isoforms 1/2 Proteins 0.000 claims description 5
- 101000971209 Homo sapiens Bcl-2-binding component 3, isoforms 3/4 Proteins 0.000 claims description 5
- 201000004681 Psoriasis Diseases 0.000 claims description 5
- 108050002772 E3 ubiquitin-protein ligase Mdm2 Proteins 0.000 claims description 4
- 102000012199 E3 ubiquitin-protein ligase Mdm2 Human genes 0.000 claims description 4
- 206010039491 Sarcoma Diseases 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 238000003745 diagnosis Methods 0.000 claims description 4
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 4
- 201000005202 lung cancer Diseases 0.000 claims description 4
- 208000020816 lung neoplasm Diseases 0.000 claims description 4
- 230000004936 stimulating effect Effects 0.000 claims description 4
- 206010046766 uterine cancer Diseases 0.000 claims description 4
- 101150050673 CHK1 gene Proteins 0.000 claims description 3
- 206010060862 Prostate cancer Diseases 0.000 claims description 3
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims description 3
- 102100027833 14-3-3 protein sigma Human genes 0.000 claims description 2
- 102100040302 39S ribosomal protein L41, mitochondrial Human genes 0.000 claims description 2
- 101150028668 APO1 gene Proteins 0.000 claims description 2
- 206010000830 Acute leukaemia Diseases 0.000 claims description 2
- 102100032305 Bcl-2 homologous antagonist/killer Human genes 0.000 claims description 2
- 206010005003 Bladder cancer Diseases 0.000 claims description 2
- 206010005949 Bone cancer Diseases 0.000 claims description 2
- 208000018084 Bone neoplasm Diseases 0.000 claims description 2
- 206010006143 Brain stem glioma Diseases 0.000 claims description 2
- 206010006187 Breast cancer Diseases 0.000 claims description 2
- 208000026310 Breast neoplasm Diseases 0.000 claims description 2
- 101100447653 Caenorhabditis elegans phg-1 gene Proteins 0.000 claims description 2
- 206010007953 Central nervous system lymphoma Diseases 0.000 claims description 2
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 2
- 108050006400 Cyclin Proteins 0.000 claims description 2
- 108010058546 Cyclin D1 Proteins 0.000 claims description 2
- 102000002431 Cyclin G Human genes 0.000 claims description 2
- 108090000404 Cyclin G1 Proteins 0.000 claims description 2
- 102100039524 DNA endonuclease RBBP8 Human genes 0.000 claims description 2
- 108050008316 DNA endonuclease RBBP8 Proteins 0.000 claims description 2
- 101000802895 Dendroaspis angusticeps Fasciculin-1 Proteins 0.000 claims description 2
- 102100033996 Double-strand break repair protein MRE11 Human genes 0.000 claims description 2
- 102100034597 E3 ubiquitin-protein ligase TRIM22 Human genes 0.000 claims description 2
- 208000001976 Endocrine Gland Neoplasms Diseases 0.000 claims description 2
- 206010014733 Endometrial cancer Diseases 0.000 claims description 2
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 2
- 102100022466 Eukaryotic translation initiation factor 4E-binding protein 1 Human genes 0.000 claims description 2
- 108050000946 Eukaryotic translation initiation factor 4E-binding protein 1 Proteins 0.000 claims description 2
- 101150089023 FASLG gene Proteins 0.000 claims description 2
- 102000013601 Fanconi Anemia Complementation Group D2 protein Human genes 0.000 claims description 2
- 108010026653 Fanconi Anemia Complementation Group D2 protein Proteins 0.000 claims description 2
- 102100024165 G1/S-specific cyclin-D1 Human genes 0.000 claims description 2
- 102000054184 GADD45 Human genes 0.000 claims description 2
- 208000017604 Hodgkin disease Diseases 0.000 claims description 2
- 208000010747 Hodgkins lymphoma Diseases 0.000 claims description 2
- 101000723509 Homo sapiens 14-3-3 protein sigma Proteins 0.000 claims description 2
- 101001104225 Homo sapiens 39S ribosomal protein L41, mitochondrial Proteins 0.000 claims description 2
- 101000591400 Homo sapiens Double-strand break repair protein MRE11 Proteins 0.000 claims description 2
- 101000848629 Homo sapiens E3 ubiquitin-protein ligase TRIM22 Proteins 0.000 claims description 2
- 101000997053 Homo sapiens Glycosyl-phosphatidylinositol-anchored molecule-like protein Proteins 0.000 claims description 2
- 101001066158 Homo sapiens Growth arrest and DNA damage-inducible protein GADD45 alpha Proteins 0.000 claims description 2
- 101000672316 Homo sapiens Netrin receptor UNC5B Proteins 0.000 claims description 2
- 101001098352 Homo sapiens OX-2 membrane glycoprotein Proteins 0.000 claims description 2
- 101000735459 Homo sapiens Protein mono-ADP-ribosyltransferase PARP9 Proteins 0.000 claims description 2
- 101000713813 Homo sapiens Quinone oxidoreductase PIG3 Proteins 0.000 claims description 2
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 claims description 2
- 101000723893 Homo sapiens Zinc finger matrin-type protein 3 Proteins 0.000 claims description 2
- 101000851815 Homo sapiens p53-regulated apoptosis-inducing protein 1 Proteins 0.000 claims description 2
- 208000005016 Intestinal Neoplasms Diseases 0.000 claims description 2
- 206010061252 Intraocular melanoma Diseases 0.000 claims description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 2
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 claims description 2
- 206010052178 Lymphocytic lymphoma Diseases 0.000 claims description 2
- 102100040289 Netrin receptor UNC5B Human genes 0.000 claims description 2
- 102100024403 Nibrin Human genes 0.000 claims description 2
- 108050003990 Nibrin Proteins 0.000 claims description 2
- 102100037589 OX-2 membrane glycoprotein Human genes 0.000 claims description 2
- 206010033128 Ovarian cancer Diseases 0.000 claims description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 2
- 102100037606 P2X purinoceptor 6 Human genes 0.000 claims description 2
- 101710190089 P2X purinoceptor 6 Proteins 0.000 claims description 2
- 108091007960 PI3Ks Proteins 0.000 claims description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 2
- 101001024685 Pandinus imperator Pandinin-2 Proteins 0.000 claims description 2
- 208000000821 Parathyroid Neoplasms Diseases 0.000 claims description 2
- 208000002471 Penile Neoplasms Diseases 0.000 claims description 2
- 206010034299 Penile cancer Diseases 0.000 claims description 2
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 claims description 2
- 208000007913 Pituitary Neoplasms Diseases 0.000 claims description 2
- 201000005746 Pituitary adenoma Diseases 0.000 claims description 2
- 206010061538 Pituitary tumour benign Diseases 0.000 claims description 2
- 102100036691 Proliferating cell nuclear antigen Human genes 0.000 claims description 2
- 102100034930 Protein mono-ADP-ribosyltransferase PARP9 Human genes 0.000 claims description 2
- 208000015634 Rectal Neoplasms Diseases 0.000 claims description 2
- 206010038389 Renal cancer Diseases 0.000 claims description 2
- 102100038013 Ribonucleoside-diphosphate reductase subunit M2 B Human genes 0.000 claims description 2
- 101710118360 Ribonucleoside-diphosphate reductase subunit M2 B Proteins 0.000 claims description 2
- 102100026715 Serine/threonine-protein kinase STK11 Human genes 0.000 claims description 2
- 101710181599 Serine/threonine-protein kinase STK11 Proteins 0.000 claims description 2
- 208000000453 Skin Neoplasms Diseases 0.000 claims description 2
- 206010041067 Small cell lung cancer Diseases 0.000 claims description 2
- 208000021712 Soft tissue sarcoma Diseases 0.000 claims description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 2
- 108010033711 Telomeric Repeat Binding Protein 1 Proteins 0.000 claims description 2
- 102100036497 Telomeric repeat-binding factor 1 Human genes 0.000 claims description 2
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 2
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 claims description 2
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 claims description 2
- 206010046431 Urethral cancer Diseases 0.000 claims description 2
- 206010046458 Urethral neoplasms Diseases 0.000 claims description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 2
- 208000002495 Uterine Neoplasms Diseases 0.000 claims description 2
- 201000005969 Uveal melanoma Diseases 0.000 claims description 2
- 102100028482 Zinc finger matrin-type protein 3 Human genes 0.000 claims description 2
- 201000005188 adrenal gland cancer Diseases 0.000 claims description 2
- 208000024447 adrenal gland neoplasm Diseases 0.000 claims description 2
- 210000000436 anus Anatomy 0.000 claims description 2
- 208000025997 central nervous system neoplasm Diseases 0.000 claims description 2
- 201000010881 cervical cancer Diseases 0.000 claims description 2
- 230000001684 chronic effect Effects 0.000 claims description 2
- 208000024207 chronic leukemia Diseases 0.000 claims description 2
- 208000030381 cutaneous melanoma Diseases 0.000 claims description 2
- 201000011523 endocrine gland cancer Diseases 0.000 claims description 2
- 201000003914 endometrial carcinoma Diseases 0.000 claims description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 2
- 201000004101 esophageal cancer Diseases 0.000 claims description 2
- 206010017758 gastric cancer Diseases 0.000 claims description 2
- 201000005787 hematologic cancer Diseases 0.000 claims description 2
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 claims description 2
- 201000010982 kidney cancer Diseases 0.000 claims description 2
- 210000003292 kidney cell Anatomy 0.000 claims description 2
- 208000029559 malignant endocrine neoplasm Diseases 0.000 claims description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 2
- 208000026037 malignant tumor of neck Diseases 0.000 claims description 2
- 208000026045 malignant tumor of parathyroid gland Diseases 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 claims description 2
- JTSLALYXYSRPGW-UHFFFAOYSA-N n-[5-(4-cyanophenyl)-1h-pyrrolo[2,3-b]pyridin-3-yl]pyridine-3-carboxamide Chemical compound C=1C=CN=CC=1C(=O)NC(C1=C2)=CNC1=NC=C2C1=CC=C(C#N)C=C1 JTSLALYXYSRPGW-UHFFFAOYSA-N 0.000 claims description 2
- 201000002575 ocular melanoma Diseases 0.000 claims description 2
- 210000003101 oviduct Anatomy 0.000 claims description 2
- 102100036520 p53-regulated apoptosis-inducing protein 1 Human genes 0.000 claims description 2
- 201000002528 pancreatic cancer Diseases 0.000 claims description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 2
- 208000021310 pituitary gland adenoma Diseases 0.000 claims description 2
- 208000016800 primary central nervous system lymphoma Diseases 0.000 claims description 2
- 108050008067 rad9 Proteins 0.000 claims description 2
- 102000000611 rad9 Human genes 0.000 claims description 2
- 206010038038 rectal cancer Diseases 0.000 claims description 2
- 201000001275 rectum cancer Diseases 0.000 claims description 2
- 201000007444 renal pelvis carcinoma Diseases 0.000 claims description 2
- 201000000849 skin cancer Diseases 0.000 claims description 2
- 201000003708 skin melanoma Diseases 0.000 claims description 2
- 208000000587 small cell lung carcinoma Diseases 0.000 claims description 2
- 208000037959 spinal tumor Diseases 0.000 claims description 2
- 201000011549 stomach cancer Diseases 0.000 claims description 2
- 201000002510 thyroid cancer Diseases 0.000 claims description 2
- 201000000360 urethra cancer Diseases 0.000 claims description 2
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 2
- 208000012991 uterine carcinoma Diseases 0.000 claims description 2
- 206010046885 vaginal cancer Diseases 0.000 claims description 2
- 208000013139 vaginal neoplasm Diseases 0.000 claims description 2
- 201000004916 vulva carcinoma Diseases 0.000 claims description 2
- 208000013013 vulvar carcinoma Diseases 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 23
- 102100027308 Apoptosis regulator BAX Human genes 0.000 claims 1
- 108050006685 Apoptosis regulator BAX Proteins 0.000 claims 1
- 102100021572 Bcl-2-binding component 3, isoforms 1/2 Human genes 0.000 claims 1
- 101100044298 Drosophila melanogaster fand gene Proteins 0.000 claims 1
- 101100042886 Drosophila melanogaster snk gene Proteins 0.000 claims 1
- 101150064015 FAS gene Proteins 0.000 claims 1
- 102100034338 Glycosyl-phosphatidylinositol-anchored molecule-like protein Human genes 0.000 claims 1
- 101100325746 Homo sapiens BAK1 gene Proteins 0.000 claims 1
- 101000655540 Homo sapiens Protransforming growth factor alpha Proteins 0.000 claims 1
- 101000693970 Homo sapiens Scavenger receptor class A member 3 Proteins 0.000 claims 1
- 101000669895 Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H) Replication factor A Proteins 0.000 claims 1
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 claims 1
- 101150011368 Plk2 gene Proteins 0.000 claims 1
- 101100335198 Pneumocystis carinii fol1 gene Proteins 0.000 claims 1
- 102100032350 Protransforming growth factor alpha Human genes 0.000 claims 1
- 102100027192 Scavenger receptor class A member 3 Human genes 0.000 claims 1
- 108010046722 Thrombospondin 1 Proteins 0.000 claims 1
- 102000007614 Thrombospondin 1 Human genes 0.000 claims 1
- 101150099624 AIM3 gene Proteins 0.000 abstract description 53
- 108700025716 Tumor Suppressor Genes Proteins 0.000 abstract description 6
- 102000044209 Tumor Suppressor Genes Human genes 0.000 abstract description 6
- 102000000872 ATM Human genes 0.000 description 125
- 101000785063 Homo sapiens Serine-protein kinase ATM Proteins 0.000 description 125
- 241000699670 Mus sp. Species 0.000 description 115
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 74
- 239000013598 vector Substances 0.000 description 51
- 229940009456 adriamycin Drugs 0.000 description 37
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 34
- 238000001262 western blot Methods 0.000 description 31
- 230000005778 DNA damage Effects 0.000 description 26
- 231100000277 DNA damage Toxicity 0.000 description 26
- 239000013604 expression vector Substances 0.000 description 26
- 238000011282 treatment Methods 0.000 description 24
- 241000699666 Mus <mouse, genus> Species 0.000 description 23
- 241000282414 Homo sapiens Species 0.000 description 22
- 230000022131 cell cycle Effects 0.000 description 22
- 230000006698 induction Effects 0.000 description 21
- 238000004458 analytical method Methods 0.000 description 19
- 230000004663 cell proliferation Effects 0.000 description 19
- 230000001965 increasing effect Effects 0.000 description 19
- 230000026731 phosphorylation Effects 0.000 description 18
- 238000006366 phosphorylation reaction Methods 0.000 description 18
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 17
- 229960001948 caffeine Drugs 0.000 description 17
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 17
- 230000019491 signal transduction Effects 0.000 description 16
- 230000001028 anti-proliverative effect Effects 0.000 description 14
- 230000006870 function Effects 0.000 description 14
- 230000003993 interaction Effects 0.000 description 14
- 210000000952 spleen Anatomy 0.000 description 14
- 230000002950 deficient Effects 0.000 description 12
- 230000001419 dependent effect Effects 0.000 description 12
- 210000000056 organ Anatomy 0.000 description 12
- 210000004988 splenocyte Anatomy 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 11
- 108700020796 Oncogene Proteins 0.000 description 11
- 239000012071 phase Substances 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 238000003757 reverse transcription PCR Methods 0.000 description 11
- 230000030570 cellular localization Effects 0.000 description 10
- 210000004185 liver Anatomy 0.000 description 10
- 210000004072 lung Anatomy 0.000 description 10
- 230000035882 stress Effects 0.000 description 10
- 239000000758 substrate Substances 0.000 description 10
- 238000013518 transcription Methods 0.000 description 10
- 230000035897 transcription Effects 0.000 description 10
- 108700028939 Amino Acyl-tRNA Synthetases Proteins 0.000 description 9
- 102000052866 Amino Acyl-tRNA Synthetases Human genes 0.000 description 9
- 230000005764 inhibitory process Effects 0.000 description 9
- 238000005259 measurement Methods 0.000 description 9
- 230000009467 reduction Effects 0.000 description 9
- 230000014616 translation Effects 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 206010025323 Lymphomas Diseases 0.000 description 8
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 230000010261 cell growth Effects 0.000 description 8
- 230000008859 change Effects 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- 108010085238 Actins Proteins 0.000 description 7
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 7
- 108060001084 Luciferase Proteins 0.000 description 7
- 150000001413 amino acids Chemical group 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 230000001766 physiological effect Effects 0.000 description 7
- 230000008439 repair process Effects 0.000 description 7
- 102000007469 Actins Human genes 0.000 description 6
- 238000010240 RT-PCR analysis Methods 0.000 description 6
- 208000009956 adenocarcinoma Diseases 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 238000000749 co-immunoprecipitation Methods 0.000 description 6
- 230000006378 damage Effects 0.000 description 6
- 210000004408 hybridoma Anatomy 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 238000001243 protein synthesis Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 108700028369 Alleles Proteins 0.000 description 5
- 208000005623 Carcinogenesis Diseases 0.000 description 5
- 230000036952 cancer formation Effects 0.000 description 5
- 231100000504 carcinogenesis Toxicity 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 239000003937 drug carrier Substances 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000003125 immunofluorescent labeling Methods 0.000 description 5
- 238000003780 insertion Methods 0.000 description 5
- 230000037431 insertion Effects 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 208000032839 leukemia Diseases 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 210000003079 salivary gland Anatomy 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 208000036762 Acute promyelocytic leukaemia Diseases 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 4
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 4
- 102100021573 Bcl-2-binding component 3, isoforms 3/4 Human genes 0.000 description 4
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 4
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 4
- 239000005089 Luciferase Substances 0.000 description 4
- 206010071541 Metastatic lymphoma Diseases 0.000 description 4
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 4
- 238000010222 PCR analysis Methods 0.000 description 4
- 208000033826 Promyelocytic Acute Leukemia Diseases 0.000 description 4
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 230000012820 cell cycle checkpoint Effects 0.000 description 4
- 230000032823 cell division Effects 0.000 description 4
- 231100001129 embryonic lethality Toxicity 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 231100000024 genotoxic Toxicity 0.000 description 4
- 230000001738 genotoxic effect Effects 0.000 description 4
- 238000010324 immunological assay Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000000411 inducer Substances 0.000 description 4
- 210000001165 lymph node Anatomy 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000004017 serum-free culture medium Substances 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229940104230 thymidine Drugs 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 239000012623 DNA damaging agent Substances 0.000 description 3
- 230000004543 DNA replication Effects 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 230000035519 G0 Phase Effects 0.000 description 3
- 230000010190 G1 phase Effects 0.000 description 3
- 108010068250 Herpes Simplex Virus Protein Vmw65 Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000002105 Southern blotting Methods 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 230000033115 angiogenesis Effects 0.000 description 3
- 230000035578 autophosphorylation Effects 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000025084 cell cycle arrest Effects 0.000 description 3
- 239000013611 chromosomal DNA Substances 0.000 description 3
- 201000010897 colon adenocarcinoma Diseases 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000013537 high throughput screening Methods 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000005865 ionizing radiation Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 230000001177 retroviral effect Effects 0.000 description 3
- 210000001625 seminal vesicle Anatomy 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 201000010653 vesiculitis Diseases 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 2
- 101100161473 Arabidopsis thaliana ABCB25 gene Proteins 0.000 description 2
- 108010034798 CDC2 Protein Kinase Proteins 0.000 description 2
- 102000009728 CDC2 Protein Kinase Human genes 0.000 description 2
- 229940126074 CDK kinase inhibitor Drugs 0.000 description 2
- 101100297347 Caenorhabditis elegans pgl-3 gene Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102100034770 Cyclin-dependent kinase inhibitor 3 Human genes 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 101000945639 Homo sapiens Cyclin-dependent kinase inhibitor 3 Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 108010003060 Methionine-tRNA ligase Proteins 0.000 description 2
- 102000000362 Methionyl-tRNA synthetases Human genes 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 230000006369 cell cycle progression Effects 0.000 description 2
- 108091092356 cellular DNA Proteins 0.000 description 2
- 230000036755 cellular response Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 2
- 239000002875 cyclin dependent kinase inhibitor Substances 0.000 description 2
- 229940043378 cyclin-dependent kinase inhibitor Drugs 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 239000002270 dispersing agent Substances 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 230000008713 feedback mechanism Effects 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000012224 gene deletion Methods 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 235000003642 hunger Nutrition 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 230000000951 immunodiffusion Effects 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 2
- 201000005249 lung adenocarcinoma Diseases 0.000 description 2
- 201000005296 lung carcinoma Diseases 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 2
- 108700025694 p53 Genes Proteins 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000010379 pull-down assay Methods 0.000 description 2
- 239000000941 radioactive substance Substances 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 230000025915 regulation of apoptotic process Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 230000028617 response to DNA damage stimulus Effects 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- 238000005204 segregation Methods 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 230000037351 starvation Effects 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 239000000375 suspending agent Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- 231100000588 tumorigenic Toxicity 0.000 description 2
- 230000000381 tumorigenic effect Effects 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 239000012827 ATM inhibitor Substances 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000000058 Anaplasia Diseases 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 108010014885 Arginine-tRNA ligase Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 108010065272 Aspartate-tRNA ligase Proteins 0.000 description 1
- 102100028820 Aspartate-tRNA ligase, cytoplasmic Human genes 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241000700663 Avipoxvirus Species 0.000 description 1
- 208000004736 B-Cell Leukemia Diseases 0.000 description 1
- 108700020463 BRCA1 Proteins 0.000 description 1
- 102000036365 BRCA1 Human genes 0.000 description 1
- 101150072950 BRCA1 gene Proteins 0.000 description 1
- 108700020462 BRCA2 Proteins 0.000 description 1
- 102000052609 BRCA2 Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 101150008921 Brca2 gene Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- 101000702579 Crotalus durissus terrificus Snaclec crotocetin Proteins 0.000 description 1
- 108010060385 Cyclin B1 Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 231100001074 DNA strand break Toxicity 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- 101100300807 Drosophila melanogaster spn-A gene Proteins 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 description 1
- 230000004707 G1/S transition Effects 0.000 description 1
- 102100032340 G2/mitotic-specific cyclin-B1 Human genes 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 102100024977 Glutamine-tRNA ligase Human genes 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 101000642971 Homo sapiens Cohesin subunit SA-1 Proteins 0.000 description 1
- 101000620365 Homo sapiens Protein TMEPAI Proteins 0.000 description 1
- 101000742054 Homo sapiens Protein phosphatase 1D Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 102000028555 IgG binding proteins Human genes 0.000 description 1
- 108091009325 IgG binding proteins Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108010092041 Lysine-tRNA Ligase Proteins 0.000 description 1
- 102000017737 Lysine-tRNA Ligase Human genes 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 102000038030 PI3Ks Human genes 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 1
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 102100036134 Probable arginine-tRNA ligase, mitochondrial Human genes 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010009341 Protein Serine-Threonine Kinases Proteins 0.000 description 1
- 102000009516 Protein Serine-Threonine Kinases Human genes 0.000 description 1
- 102100022429 Protein TMEPAI Human genes 0.000 description 1
- 102100038675 Protein phosphatase 1D Human genes 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102100029538 Structural maintenance of chromosomes protein 1A Human genes 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 102000006747 Transforming Growth Factor alpha Human genes 0.000 description 1
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 description 1
- 102000015098 Tumor Suppressor Protein p53 Human genes 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000005735 apoptotic response Effects 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000003123 bronchiole Anatomy 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000007960 cellular response to stress Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 101150113535 chek1 gene Proteins 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000008029 eradication Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 208000018685 gastrointestinal system disease Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000005861 gene abnormality Effects 0.000 description 1
- 238000012637 gene transfection Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 108010051239 glutaminyl-tRNA synthetase Proteins 0.000 description 1
- ZNNLBTZKUZBEKO-UHFFFAOYSA-N glyburide Chemical compound COC1=CC=C(Cl)C=C1C(=O)NCCC1=CC=C(S(=O)(=O)NC(=O)NC2CCCCC2)C=C1 ZNNLBTZKUZBEKO-UHFFFAOYSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 201000004514 liver lymphoma Diseases 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- KNJDBYZZKAZQNG-UHFFFAOYSA-N lucigenin Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.C12=CC=CC=C2[N+](C)=C(C=CC=C2)C2=C1C1=C(C=CC=C2)C2=[N+](C)C2=CC=CC=C12 KNJDBYZZKAZQNG-UHFFFAOYSA-N 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000021332 multicellular organism growth Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 230000006654 negative regulation of apoptotic process Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000000123 paper Substances 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 108060006184 phycobiliprotein Proteins 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000032029 positive regulation of DNA repair Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000000861 pro-apoptotic effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 230000025600 response to UV Effects 0.000 description 1
- 230000006335 response to radiation Effects 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229940043230 sarcosine Drugs 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 108010004731 structural maintenance of chromosome protein 1 Proteins 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 239000000225 tumor suppressor protein Substances 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/53—Ligases (6)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/10—Drugs for disorders of the urinary system of the bladder
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
- A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/20—Dermatological disorders
- G01N2800/205—Scaling palpular diseases, e.g. psoriasis, pytiriasis
Definitions
- the present invention relates to a novel tumor suppressor, and particularly to a novel tumor suppressor that activates ATM or ATR.
- Cells have a variety of fail-safe mechanisms, one of which is to arrest the cell division of damaged chromosomal DNA and to repair the damage, thus preventing mutations from settling.
- chromosomal DNA damaged by UV and the like is continued to undergo cell division in a condition where the damage is not repaired, the damaged chromosomal DNA will be replicated so as to accumulate mutations. This leads to an increase in the incidence of cancer cells.
- cells operate a process of repairing the damage and an intracellular feedback mechanism of arresting the cell division until the repair of DNA damage is over, followed by inhibiting the development of cancers. Such a feedback mechanism is mediated by checkpoints in each cycle of cell division.
- checkpoints The overall function of these checkpoints is to detect damaged or abnormally structured DNA and to coordinate cell-cycle progression with DNA repair (Robert T. Genes & development, 15:2177-2196, 2001).
- cell-cycle checkpoint activation slows or arrests cell-cycle progression, thereby allowing time for appropriate repair mechanisms to correct genetic lesions before they are passed on to the next generation of daughter cells.
- checkpoint proteins link DNA strand breaks to apoptotic cell death via the induction of p53 (Robert T. Genes & development, 15:2177-2196, 2001).
- ATM and ATR are highly homologous to each other and use the same substrate. However, they are different in that their activities are increased by different genotoxic stresses. ATM responds to agents, such as IR (ionizing radiation) that breaks double strands DNA, whereas ATR responds to agents (including IR) that cause bulky adducts on DNA or single strand DNA. Furthermore, ATM and ATR are activated by different methods. ATM activation requires autophosphorylation that results in the disruption of an ATM dimer (Bakkenist, C. J. et al., Nature, 421:499-506, 2003). How autophosphorylation of ATM triggered is still unknown.
- agents such as IR (ionizing radiation) that breaks double strands DNA
- ATR responds to agents (including IR) that cause bulky adducts on DNA or single strand DNA.
- ATM and ATR are activated by different methods. ATM activation requires autophosphorylation that results in the disruption of an ATM dimer (Bakkenist, C. J.
- ATR may also be autophosphorylated, but it is not evident that ATR forms either an inactive dimer or an active monomer in cells. Also, it is not yet clear that other subunits or cofactors are required for the activation of ATM/ATR. In addition, the intracellular biochemical mechanism of a signal transduction system where the DNA damage causes the activation and operation of ATM/ATR was not completely established.
- Target proteins known to be phosphorylated directly by ATM/ATR include p53, chk1, chk2, c-Abl, RPA and the like, of which p53 is phosphorylated on serine 15 by ATM/ATR. It was reported that the over-expression of p53 arrests G2 and suppresses the synthesis of two proteins, CDK1 (cyclin-dependent kinase 1) and cyclin B1, which are required for the entry of cells from G2 to M. Thus, p53 does not only the function of inhibiting the abnormal division and proliferation of cells, but also the function of arresting the cell cycle so as to repair the damaged DNA when DNA was damaged.
- p53 activates the transcription of p21, another tumor suppressor gene, thereby inhibiting the G1/S transition and causing the p53-dependent apoptosis.
- p21 which is expressed by p53 was known to be a kind of a CKI (cyclin-dependent kinase inhibitor) which functions to inhibit the division and proliferation of cells. Accordingly, efforts for developing new anticancer agents using cell-cycle regulation factors or substances of activating the factors are now continued.
- ARSs aminoacyl-tRNA synthetases
- MRS methionyl-tRNA synthetase
- QRS glutaminyl-tRNA synthetase
- RRS arginyl-tRNA synthetase
- KRS Lysyl-tRNA synthetase
- DRS DRS(aspartyl-tRNA synthetase) and so on
- p43 is known to play an important role as a cytokine in immune response and angiogenesis (Ko et al., J. Biol. Chem., 276:23028-32303, 2001b; Park et al., J. Biol. Chem., 277:45234-45248, 2002). Furthermore, p38 was found to downregulate c-myc, a protoocogene, and to be involved in lung differentiation (Kim et al., Nat. Genet., 34:330-336, 2003).
- p18 shows sequence homology to elongation factor subunits (EF-1) (Quevillon and Mirande, FEBS Lett., 395:63-67, 1996). Given this, p18 is presumed to be involved in protein synthesis. However, the biological functions of p18 are not yet clearly understood, and particularly, there is no study on the relation between p18 and cancer.
- p18 which had been known as a cofactor of an aminoacyl-tRNA synthetase (ARS) complex to “AIM3 (ARS-interacting multifunctional protein 3)”. Accordingly, p18 will hereinafter be referred to as “AIM3”.
- ARS aminoacyl-tRNA synthetase
- the present invention provides a method for activating ATM, ATR and proteins regulated by ATM or ATR, in the cell, tissue and individual, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of the following:
- the present invention provides a method for inducing the expression of p53 or its target genes in the cell, tissue or individual, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of following:
- the present invention provides a method for inhibiting the proliferation of tumor cells, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of the following:
- the present invention provides a method for stimulating apoptosis in the cell, tissue or individual, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of the following:
- the present invention provides a method for treating or preventing ATM- or ATR-mediated diseases, comprising administering to a subject in need thereof an effective amount of one selected from the group consisting of the following:
- the present invention provides a method for screening a substance having the effect of treating or preventing ATM- or ATR-mediated diseases, the method comprising the steps of:
- the present invention provides a method for identifying a subject having the risk of ATM- or ATR-mediated diseases, comprising the steps of:
- the present invention provides a kit for the diagnosis of ATM- or ATR-mediated diseases, comprising one selected from the group consisting of an AIM3 protein-encoding nucleic acid, a fragment thereof, a peptide encoded by the nucleic acid or its fragment, and an antibody to the peptide.
- compositions comprising, as an active ingredient, one selected from the group consisting of the following:
- AIM3 p18
- ARS aminoacyl-tRNA synthetase
- AIM3 is moved into nuclei and induced at a high level.
- AIM3 shows an anti-proliferation activity against cells.
- AIM3 induces apoptosis.
- AIM3 induces the expression of tumor suppressor gene p53 and its target genes.
- AIM3 interacts directly with ATM/ATR so as to activate ATM, ATR and proteins which are regulated by ATM or ATR.
- AIM3 a reduction in the expression level of AIM3 induces tumorigenesis, and it is expressed at a low level in cancer cell lines and tissues isolated from cancer patients.
- the present invention provides a method for activating one selected from the group consisting of ATM, ATR and proteins regulated by ATM or ATR using an AIM3 protein or a nucleic acid encoding the AIM3 protein.
- the term “activating” means the phosphorylation of proteins or the structural or chemical mutation of proteins.
- the activation of ATM/ATR is mediated by the biding of AIM3, which causes a variety of intracellular responses involved in ATM/ATR. Such intracellular responses include, but are not limited to, DNA repair, cell cycle regulation and apoptosis induction.
- AIM3 apoptosis-inducing signal transduction pathway caused by DNA damage.
- the ATM/ATR-regulated proteins include proteins which are directly phosphorylated by ATM/ATR, and proteins which are sequentially phosphorylated in signal transduction pathways by the phosphorylation of said proteins.
- Preferred examples include, but are not limited to, H2AX (Burma S. et al., J. Biol. Chem., 9;276(45):42462-42467, 2001), p53 (Saito S, et al., J. Biol. Chem., 12;277(15):12491-12494, 2002), chk2 (Matsuoka S, et al., Proc. Natl. Acad. Sci.
- the proteins may be H2AX, p53 or chk2.
- the present invention provides a method for inducing the expression of p53 or its target gene using the AIM3 protein or a nucleic acid encoding the AIM3 protein.
- the term “p53-target gene” refers to a gene located in downstream of p53, whose expression is induced by p53.
- the p53-target gene may be a gene involved in at least one mechanism selected from the group consisting of p53 control, cell cycle regulation, DNA repair, apoptosis, angiogenesis, cellular stress response and determination of cell fate.
- Preferred examples of this target gene include, but are not limited to, p21 (Fujioka S, et al., J. Biol. Chem., Apr.
- IGF-BP3 Mendoza-Rodriguez C A, et al., Rev. Invest. Clin., 53(3):266-273, 2001
- PAG608 Higashi Y, et al., J. Biol. Chem., 1;277(44):42224-42262, 2002
- DR5/KILLER Takimoto R, et al., Oncogene, 30;19(14):1735-1743, 2000
- GML Higashiyama M, et al., Eur. J.
- TSP-1 Harada H, et al., Cancer Lett., 28;191(1):109-119, 2003
- BAL1 Nakamura Y., Cancer Sci., 95(1):7-11, 2004
- CSR Nakamura Y., Cancer Sci., 95(1):7-11, 2004
- PIG3 Gaampieri S, et al., Oncogene, Apr. 12, 2004; Contente A, et al., Nat. Genet., 30(3):315-320, 2002
- Apaf-1 Gaampieri S, et al., Oncogene, Apr.
- the target gene may be p21 or PUMA.
- AIM3 of the present invention inhibits the proliferation of tumor cells through signal transduction pathways mediated by ATM/ATR and stimulates apoptosis caused by DNA damage. Accordingly, the present invention provides methods to inhibit the proliferation of tumor cells and to stimulate apoptosis, using the AIM3 protein and a nucleic acid encoding the AIM3 protein.
- all the methods described above comprise administering an effective amount of the AIM3 protein or the nucleic acid encoding the protein to cells or tissues.
- the term “effective amount” refers to the amount of AIM3, which shows an effect selected from the group consisting of the following: the activation of ATM/ATR in cells or tissues; the increase of phosphorylation of ATM/ATR-regulated proteins; the induction of expression of p53 or its target gene; the inhibition of proliferation of tumor cells; and the promotion of apoptosis.
- the AIM3 proteins used in the present invention include natural or recombinant AIM3 proteins, or proteins having the substantially equivalent physiological activity of the natural or recombinant AIM3 proteins.
- the amino acid sequence of the AIM3 protein is known in the art and preferably derived from mammals, including human beings.
- the AIM3 protein of the present invention preferably has an amino acid sequence shown in SEQ ID NO: 1. Proteins having the substantially equivalent physiological activity of AIM3 include natural/recombinant AIM3 proteins, their functional equivalents and their functional derivatives.
- the term “the substantially equivalent physiological activity” means the activity of: activating ATM/ATR or ATM/ATR-regulated proteins; inducing the expression of p53 or its target gene; inhibiting the proliferation of tumor cells; and/or stimulating apoptosis.
- the term “functional equivalents” refers to amino acid sequence variants with a substitution of some or all of the amino acids of a natural AIM3 protein or a deletion or addition of some of the amino acids, which have a physiological activity substantially equivalent to the natural AIM3 protein.
- the term “functional derivatives” refers to those having a physiological activity substantially equivalent to natural AIM3 protein, as proteins modified to increase or reduce the physicochemical properties of the AIM 3 protein.
- the proteins having a physiological activity substantially equivalent to the AIM3 protein have a homology of at least 70%, preferably at least 80%, and more preferably at least 90%, with the polypeptide shown in SEQ ID NO: 1.
- the AIM3 protein used in the present invention can be prepared by any genetic engineering method known in the art.
- the inventive pharmaceutical composition containing the AIM3 protein as an active ingredient can be administered to human beings and animals by oral route or by parenteral route, such as an intravenous, subcutaneous, intranasal or intraperitoneal route.
- Oral administrations include sublingual application.
- Parenteral administrations include injection techniques, such as subcutaneous injection, intramuscular injection and intravenous injection, as well as drip infusion.
- the pharmaceutical composition can be formulated into various forms with a pharmaceutically acceptable carrier by a conventional method.
- the term “pharmaceutically acceptable” carrier means a substance which is physiologically acceptable and, when administered to human beings, generally does not cause allergic reactions, such as gastrointestinal disorder and dizziness, or similar reactions thereto.
- the pharmaceutically acceptable carriers in the case of oral administration, there may be used binders, lubricants, disintegrants, excipients, solubilizers, dispersing agents, stabilizers, suspension agents, pigments and flavors, and in case of injection agent, there can be used buffers, preservatives, analgesics, solubilizers, isotonics and stabilizers, and in case of formulations for local administration may include bases, excipients, lubricants and preservatives.
- the inventive pharmaceutical composition containing the AIM3 protein may be formulated into various forms with the pharmaceutically acceptable carriers.
- the inventive composition may be formulated into the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and so on, and for injection agent, it may be formulated into unit dose ampoules or multiple dose products.
- a total effective amount of the AIM3 protein of the present invention can be administered to patients in a single dose or can be administered by a fractionated treatment protocol, in which multiple doses are administered over a more prolonged, period of time.
- the amount of the AIM3 protein or a nucleic acid encoding the AIM3 protein in the inventive pharmaceutical composition may vary depending on the severity of diseases, the protein or the nucleic acid may be generally administered several times a day at an effective dose of 1 ⁇ g-10 mg.
- a suitable dose of the AIM3 protein in the inventive pharmaceutical composition may depend on many factors, such as the age, body weight, health condition, sex, disease severity, diet and excretion of patients, as well as the route of administration and the number of treatments to be administered.
- any person skilled in the art may determine an effective dose for treating or preventing ATM/ATR-mediated diseases.
- the inventive pharmaceutical composition containing the AIM3 protein has no special limitations on its formulation, administration route and/or administration mode insofar as it shows the effects of the present invention.
- nucleic acids encoding the AIM3 protein of the present invention include DNA or RNA.
- they refer to DNA encoding AIM3 proteins derived from mammals, particularly human beings.
- the human AIM3 gene is known in the art (GenBank accession No. AB011079).
- the nucleic acid of the present invention is shown in SEQ ID NO: 2.
- the nucleic acids also include nucleic acids encoding functional equivalents to the AIM3 protein.
- the present invention can be included nucleic acids having a sequence homology of at least 80%, preferably at least 90%, and more preferably at least 95% with either a nucleic acid encoding the AIM3 protein or a nucleic acid comprising the complementary nucleotide sequence thereof.
- the nucleic acid encoding the AIM3 protein may be used for gene therapy by inserting it into an expression vector, such as a plasmid or viral vector, and then introducing the expression vector into a target cell by any method known in the art, such as infection or transduction.
- an expression vector such as a plasmid or viral vector
- a gene transfer method using a plasmid expression vector is a method of transferring a plasmid DNA directly to human cells, which is an FDA-approved method applicable to human beings (Nabel, E. G., et al., Science, 249:1285-1288, 1990). Unlike viral vectors, the plasmid DNA has an advantage of being homogeneously purified.
- Plasmid expression vectors which can be used in the present invention include mammalian expression plasmids known in the pertinent art. For example, they are not limited to, but typically include pRK5 (European Patent No. 307,247), pSV16B (PCT Publication No. 91/08291) and pVL1392 (PharMingen).
- the plasmid expression vector containing the nucleic acid according to the present invention may be introduced into target cells by any method known in the art, including, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun methods, and other known methods for introducing DNA into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).
- virus expression vectors containing the nucleic acid according to the present invention include, but are not limited to, retrovirus, adenovirus, herpes virus, avipox virus and so on.
- the retroviral vector is so constructed that non-viral proteins can be produced within the infected cells by the viral vector in which virus genes are all removed or modified.
- the main advantages of the retroviral vector for gene therapy are that it transfers a large amount of genes into replicative cells, precisely integrates the transferred genes into cellular DNA, and does not induce continuous infections after gene transfection (Miller, A. D., Nature, 357:455-460, 1992).
- the retroviral vector approved by FDA was prepared using PA317 amphotropic retrovirus packaging cells (Miller, A. D. and Buttimore, C., Molec. Cell Biol., 6:2895-2902, 1986).
- Non-retroviral vectors include adenovirus as described above (Rosenfeld et al., Cell, 68:143-155, 1992; Jaffe et al., Nature Genetics, 1:372-378, 1992; Lemarchand et al., Proc. Natl. Acad. Sci. USA, 89:6482-6486, 1992).
- the main advantages of adenovirus are that it transfers a large amount of DNA fragments (36 kb genomes) and is capable of infecting non-replicative cells at a very high titer.
- herpes virus may also be useful for human genetic therapy (Wolfe, J. H., et al., Nature Genetics, 1:379-384, 1992).
- any suitable virus vector known in the art may be used.
- a vector capable of expressing the AIM3 gene may be administered by a known method.
- the vector may be administered locally, parenterally, orally, intranasally, intravenously, intramuscularly or subcutaneously, or by other suitable routes.
- the vector may be injected directly into a target cancer or tumor cell at an effective amount for treating the tumor cell of a target tissue.
- the inventive pharmaceutical composition can be injected directly into the hollow organ affected by the cancer or tumor using a needle, a catheter or other delivery tubes.
- any effective imaging device such as X-ray, sonogram, or fiberoptic visualization system, may be used to locate the target tissue and guide the needle or catheter tube.
- inventive pharmaceutical composition comprising the nucleic acid encoding the AIM3 protein may be administered into the blood circulation system for treatment of a cancer or tumor which cannot be directly reached or anatomically isolated.
- the pharmaceutical composition comprising the nucleic acid encoding the AIM3 protein as an active ingredient may additionally comprise pharmaceutically acceptable carriers or excipients. These carriers or excipients include dispersing agents, wetting agents, suspending agents, diluents and fillers.
- the ratio of the particular pharmaceutically acceptable carrier and the expression vector contained in the inventive pharmaceutical composition can be determined by the solubility and chemical properties of the composition, and the particular administration mode.
- the therapeutic or preventive effective amount of the inventive pharmaceutical composition containing the AIM3 protein-encoding nucleic acid may be suitably selected depending on the subject to be administered, age, individual variation and disease condition.
- the present invention provides a method for treating or preventing ATM/ATR-mediated diseases using the AIM3 protein or a nucleic acid encoding the AIM3 protein.
- the present invention provides a method for treating or preventing ATM- or ATR-mediated diseases, which comprise administering to a subject requiring treatment an effective amount of one selected from the group consisting of the following: (a) an isolated polypeptide of an AIM3 protein; (b) a polypeptide having at least 70% homology with the polypeptide (a); and (c) an isolated nucleic acid encoding the polypeptide (a) or (b).
- the term “subject” means mammals, particularly animals including human beings. The subject may be a patient requiring treatment.
- ATM- or ATR-mediated diseases refers to diseases induced by the inactivation or activation reduction of ATM/ATR, i.e., diseases induced by abnormalities occurring in signal transduction pathways mediated by ATM/ATR, due to the inactivation or activation reduction of ATM/ATR.
- the signal transduction pathways mediated by ATM/ATR include signal transduction pathways mediated by ATM/ATR themselves or ATM/ATR-regulated proteins.
- the signal transduction pathways may be signal transduction pathways in DNA repair, cell cycle regulation, apoptosis, p53 regulation, angiogenesis and/or intracellular stress response.
- the ATM/ATR-mediated diseases may be caused by the over-proliferation of cells, such as cancers or psoriasis.
- the cancers include, but are not limited to, breast cancer, rectal cancer, lung cancer, small-cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine carcinoma, ovarian cancer, colorectal cancer, cancer near the anus, colon cancer, oviduct carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulva carcinoma, Hodgkin's disease, esophagus cancer, small intestinal tumor, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, uterine cancer, penis cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or urethra cancer, kidney cell carcinoma, kidney pelvis carcinoma, CNS tumor, primary CNS lymphoma, spinal tumor, brain stem glioma, and pituitary adenoma, and a combination of one or more thereof.
- the treating or preventing method according to the present invention is effective in treating or preventing cancers caused by p53 gene abnormalities.
- the dose (effective amount) and administration mode of the AIM3 protein or the nucleic acid encoding the AIM3 protein are the same as described above.
- the AIM3 protein of the present invention interacts directly with ATM/ATR so as to activate ATM/ATR and various proteins regulated by ATM/ATR.
- the AIM3 protein shows the activity of inducing the expression of p53, one of the ATM/ATR-regulated proteins, and the expression of its target genes, so as to stimulate the apoptosis of cells for DNA damage and to inhibit the proliferation of tumor cells.
- Such characteristics of AIM3 may be used to screen a substance effective for treating/preventing ATM/ATR-mediated diseases, particularly cancer.
- the present invention provides a method for screening a substance effective for treating or preventing ATM/ATR-mediated diseases, which comprise the step of: (a) culturing the AIM3 protein or a recombinant cell expressing the AIM3 protein together with a candidate substance; and (b) determining the effect of the candidate substance on an increase in the activity of AIM3 or the intracellular level thereof.
- activity of the AIM3 protein refers to the binding activity with ATM/ATR, the activity of promoting the phosphorylation of ATM/ATR or proteins regulated by ATM/ATR, and/or the activity of inducing the expression of p53 and its target genes.
- the term “increase in the intracellular level of the AIM3 protein” means the increase in the concentration of the AIM3 protein by the increase of expression of the AIM3 gene or the inhibition of the degradation of the AIM3 proteins.
- the expression of the AIM3 gene includes process for the transcription of the AIM3 gene and the translation into proteins. Accordingly, the substances screened in the present invention has the property of: promoting the binding of AIM3 to ATM/ATR; activating ATM/ATR or proteins regulated by ATM/ATR; inducing the expression of p53 and its target genes; and/or increasing the intracellular level of the AIM3 protein. These substances include not only proteins but also naturally occurring or chemically synthesized compounds or extracts.
- the activity and intracellular level of the AIM3 protein can be measured by various methods known in the art. Exemplary methods include, but are not limited to, co-immunoprecipitation, enzyme-linked immunosorbent assay, radioimmunoassay (RIA), immunohistochemical assay, Western blotting, and fluorescence activated cell sorter (FACS) analysis.
- exemplary methods include, but are not limited to, co-immunoprecipitation, enzyme-linked immunosorbent assay, radioimmunoassay (RIA), immunohistochemical assay, Western blotting, and fluorescence activated cell sorter (FACS) analysis.
- HTS high throughput screening
- the HTS is a method for screening the biological activities of a number of candidate substances simultaneously or almost simultaneously by testing the candidate substances simultaneously.
- cell lines are cultured in a 96-well microtiter plate or a 192-well microtiter plate, into which a number of candidate substances are added and then measured for the expression of AIM3 by an immunohistochemical method.
- 96 independent tests may be simultaneously performed in a single 8 cm ⁇ 12 cm plastic plate containing 96 reaction wells. The wells require an assay volume of 50-500 ⁇ l typically.
- a number of gauges, instruments, pipetters, robots, plate washers and plate readers are commercially available in order to make the 96-well format suitable for a wide range of homogeneous and heterogeneous assays.
- the expression level of the AIM3 gene or protein in biological samples can be compared with that of normal persons so as to diagnose (identify) subjects having the risk of ATM/ATR-mediated diseases.
- biological samples e.g., blood, serum, sputum, urine and/or tumor biopsies
- ATM/ATR-mediated diseases may be identified.
- the present invention provides a method for identifying a subject having the risk of ATM/ATR-mediated diseases, which comprise the steps of: (a) measuring the expression level of AIM3 in a tissue sampled from a subject; and (b) comparing the level of AIM3 in the tissue with a normal AIM3 level.
- the methods for identifying such a disease include those which are capable of detecting the expression of AIM3 at a transcriptional or translational level (such as RT-PCR, Northern blotting, Western blotting, immunological assays and so on). This method is very effective for diagnosing cancer among ATM/ATR-mediated diseases.
- the present invention provides a kit for the diagnosis of ATM/ATR-mediated diseases, which comprises one selected from the group consisting of a AIM3-encoding nucleic acid, a fragment thereof, a peptide encoded by them, and an antibody to the peptide.
- the AIM3 protein-encoding nucleic acid and a fragment thereof may be synthesized with reference to the known sequence of the AIM3 gene.
- the fragment of nucleic acid is preferably a primer capable of amplifying the AIM3 gene.
- the peptide encoded by the AIM3 protein-encoding nucleic acid or its fragment may be synthesized by any technique known in the art (Creighton, Proteins: Structures and Molecular Principles, W.H.
- the peptide can be produced by the conventional stepwise liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Williams et al., Eds., Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press, Boca Raton Fla., 1997; Atherton ⁇ Sheppard, Eds., A Practical Approach, IRL Press, Oxford, England, 1989).
- the antibody to the peptide can be produced using the AIM3 protein or its fragment as an antigen by any conventional method widely known in the immunological field.
- the antibodies include polyclonal antibodies and monoclonal antibodies.
- the polyclonal antibodies can be prepared from a variety of warm-blooded animals, such as horses, cattle, goats, sheep, dogs, fowl, turkeys, rabbits, mice or rats, by any conventional technique known in the art. Namely, the animals are immunized by intraperitoneal, intramuscular, intraocular or subcutaneous injection of an antigen.
- the immunogenicity to the antigen can be increased by the use of an adjuvant, for example Freund's complete adjuvant or incomplete adjuvant.
- an adjuvant for example Freund's complete adjuvant or incomplete adjuvant.
- a small serum sample was collected and tested for the reactivity to the target antigen. Once the animal's titer reaches a stagnant state in view of its reactivity to the antigen, a large amount of the polyclonal antibodies can be obtained by bleeding the animal at one-week intervals or by blood-letting the animal.
- the monoclonal antibodies can also be produced by a known method (Kennettm McKearn, and Bechtol(eds.), Monoclonal Antibodies, Hybridomas; A New Dimension in Biological Analyses, Plenum Press, 1980).
- the monoclonal antibodies can be produced by immunizing an animal with the AIM3 protein or its fragment as an immunogen, fusing the splenocytes of the immunized animal with myeloma cells to produce a hybridomas, screening a hybridoma that selectively recognizes the AIM3 protein, culturing the screened hybridoma, and isolating antibodies from the hybridoma culture.
- the monoclonal antibodies according to the present invention may also be prepared by injecting said hybridoma into an animal, and after a given period of time, isolating antibodies from the collected ascites of the animal.
- the antibody contained in the inventive diagnostic kit is preferably immobilized onto a solid substrate.
- the antibody can be immobilized by various techniques described in literatures ( Antibodies: A Labotory Manual, Harlow ⁇ Lane; Cold SpringHarbor, 1988).
- Suitable solid substrates include those supported by rods, synthetic glass, agarose beads, cups, flat packs, or other solid support or those having a film or coating attached to them.
- other solid substrates include cell culture plates, ELISA plates, tubes and polymeric films.
- the diagnostic kit according to the present invention may contain, in addition to an antibody selectively recognizing the AIM3 protein, reagents which are used in immunological assays.
- the immunological assays may include methods capable of measuring the binding of an antigen to the antibody of the present invention. These methods are known in the art and include, for example, immunocytochemical assays, immunohistochemical assays, radioimmunoassays, ELISA (enzyme linked immunoabsorbent assay), immunoblotting, Farr assays, precipitin reaction, turbidimetry, immunodiffusion, counter-current electrophoresis, single radical immunodiffusion and immunofluorescence.
- Reagents which are used in the immunological assays include a suitable carrier, a labeling substance capable of emitting detectable signals, a solubilizer and a washing agent. Furthermore, if the labeling substance is enzyme, a substrate capable of measuring enzymatic activity and a reaction stopping agent may be used.
- Suitable carriers include, but are not limited to, soluble carriers, for example, one of biologically acceptable buffers known in the art (e.g., PBS), insoluble carriers, for example polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, polymers, such as latex containing magnetic fine particles plated with metal, paper, glass, metal, agarose and combinations thereof.
- PBS biologically acceptable buffers known in the art
- insoluble carriers for example polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, polymers, such as latex containing magnetic fine particles plated with metal, paper, glass, metal, agarose and combinations thereof.
- Labeling substances capable of emitting detectable signals include enzymes, fluorescent substances, luminescent substances and radioactive substances.
- the enzymes include peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, glycose oxidase, maleate dihydrogenase, glucose-6-phosphodihydrogenase, invertase and so on.
- the fluorescent substances include fluorescein isothiocyanate and phycobili-protein.
- luminescent substances isolucinol and lucigenin and so on can be used.
- radioactive substances I 131 , C 14 , H 3 and so on, can be used.
- the ATM/ATR-mediated diseases which can be diagnosed with the inventive kit are the same as described above, and preferably, may be lung cancer, colon cancer, liver cancer, lymphoma and leukemia.
- AIM3 gene-deficient mice were produced by a gene trap method. Then, genomic DNA mutated by the insertion of a gene trap vector was introduced into the embryonic stem cell of the mice so as to construct a mutant library. Clones containing the mutated AIM3 gene were searched from the library and used to prepare AIM3 heterozygous mutant mice.
- the sequence of an AIM3 allele in the mutant mice was analyzed.
- the results showed that the gene trap vector was integrated between the first and second exons in the AIM3 gene (see FIG. 1 a ).
- genomic PCR and Southern blotting were performed to determine AIM3 mutation ( FIGS. 1 b and 1 c ), and the expression level of AIM3 by the mutation was determined using Western blot (see FIG. 1 d ).
- AIM3 ⁇ mice The post-natal genotype of progenies obtained by crossbreeding the AIM3 heterozygous mutant mice (hereinafter, referred to as “AIM3 ⁇ mice”) and the genotype of embryos with the passage of time were examined and the results showed that the AIM3 ⁇ mice appeared at a similar ratio to that of wild-type littermates (see Table 1). This indicates that about 50% of the AIM3 ⁇ mice were dead in the pre-natal stage. AIM3 homozygous mice (hereinafter, referred to as “AIM3 ⁇ / ⁇ mice”) would die during the early embryonic stage (see Tables 1 and 2). This suggests that AIM3 performs an important role in vivo.
- the AIM3 protein is related to a multi-tRNA synthetase complex involved in protein synthesis (Han et al., Biochem. Biophys. Res. Commun., 303:985-993, 2003), the inventors expected that a reduction of the AIM3 level would have an effect on the overall body growth of mice. However, it was interestingly shown that the growth rate of the AIM3 ⁇ mice was similar to or slightly higher than that of wild-type mice regardless of their sex (data not shown). This suggests that protein synthesis is not inhibited by a reduction of the AIM3 level.
- a rapid cell cycle is a typical indication for tumorigenesis (Evan and Vousden, Nature, 411:342-348, 2001).
- cells isolated from the AIM3-deficient mice increased faster than those of wild-type mice and showed faster cell cycle (see FIGS. 3 a and 3 b ).
- the expression of AIM3 in cell cycles was examined by Western blot analysis and flow cytometry, and the results showed that AIM3 was significantly induced during DNA synthetic phase (see FIGS. 3 c and 3 d ).
- FIGS. 3 c and 3 d To understand the functional reason for the AIM3 induction during DNA synthetic phase, the cellular localization of AIM3 in growth arrest state and proliferation condition was examined.
- AIM3 was detected mainly in cytoplasm when the cell growth was suppressed by serum starvation. However, it was detected in nucleus when the cells resumed growth (see FIG. 3 e ). This indicates that, during DNA synthesis, AIM3 is not only induced but also translocated into nuclei. Such results suggest that AIM3 can perform novel functions within the nuclei.
- Cell responses to DNA damage include cell cycle arrest, apoptosis, and direct activation of DNA repair networks (Zhou B B et al., Cancer Biol. Ther., S (4 Suppl 1):S16-22, 2003). Also, the resistance to apoptosis, one of cell responses, is a typical indication for tumorigenesis (Evan and Vousden, Nature, 411:342-348, 2001). Thus, in order to examine whether AIM3 is involved in apoptosis regulation, the response of AIM3 ⁇ mouse-derived cells was examined using adriamycin that induces DNA damage. The AIM3 ⁇ mouse-derived cells show the resistance to apoptosis (see FIG. 4 a ).
- AIM3 is induced at a high level during DNA synthetic phase and when DNA get damaged, and has anti-proliferation activity similar to other DNA repair proteins (Falck et al., Nature, 410:842-847, 2001; Lim et al., Mol. Cell, 7:683-694, 2000), it can be found that AIM3 is functionally involved in signal transduction pathways that respond to the repair of DNA damage caused by the DNA replication or stress.
- p53 a tumor suppressor gene
- p53 does functions of not only inhibiting the abnormal division and proliferation of cells but also arresting the cell cycle in the case of cellular DNA damage so as to repair the damaged DNA
- p53 is involved in cell proliferation and apoptosis to prevent DNA from being unlimitedly amplified
- the inventors examined the functional connection between AIM3 and p53. The results showed that, in cells transfected with the AIM3 gene, the levels of not only p53 but also its target gene, p21, were increased (see FIGS. 6 a and 6 b ).
- the present inventors examined whether the activity of ATM/ATR is enhanced by the association with AIM3.
- the results showed that the phosphorylation level of H2AX in the AIM3 ⁇ mouse-derived cells, which is a substrate for ATM/ATR, was significantly lower than that of H2AX in wild-type mouse cells (see FIG. 9 a ).
- the phosphorylation of H2AX was blocked by the expression of antisense-AIM3 (As-p18) (see FIG. 9 b ).
- AIM3 increased the phosphorylation of ATM and its target proteins (p53 and chk2) through various tests ( FIG. 9 c and data not shown).
- the present inventors examined the expression level of AIM3 in various cancer cell lines. The results indicated that the expression level of AIM3 was reduced in some cancer cell lines (see FIG. 10 a ). To have a clue to the possible cause for the results, the present inventors compared the DNA content for AIM3 gene using genomic PCR analysis. As a result, it was confirmed that some cancer cell lines appeared to contain less amount of DNA than other cells. This indicates that the cell lines have loss of one allele for AIM3 (see FIG. 10 b ).
- AIM3 is a tumor suppressor gene and particularly, a haploinsufficient tumor suppressor gene acting in signal transduction pathways including ATM/ATR and p53.
- FIG. 1 a is a schematic representation of a gene trap vector inserted into an AIM3 gene.
- FIG. 1 b shows the results of genomic PCR analysis to determine the insertion of a gene trap vector.
- FIG. 1 c shows the results of Southern blot analysis to determine the insertion of a gene trap vector.
- FIG. 1 d shows the results of Western blot analysis to determine the expression level of AIM3 in various organs of wild-type mice (+/+) and AIM3 heterozygous mice ( ⁇ ).
- FIG. 2 a shows the results of immunohistochemical staining of various tissues and organs isolated from AIM3 heterozygous mice.
- FIG. 2 b illustrates the results using an anti-B220 monoclonal antibody, which shows that lymphoma cells metastasized into liver and lung.
- FIG. 2 c shows the results of analysis of the incidence of tumors at different ages (months) in wild-type mice (+/+) and AIM3 heterozygous mice ( ⁇ ).
- FIG. 3 a shows the results of cell counting to measure the proliferation rate of the splenocytes and thymocytes isolated from wild-type mice (+/+) and AIM3 heterozygous ( ⁇ ).
- FIG. 3 b shows the results of analysis of the cell cycle of splenocytes isolated from wild-type mice (+/+) and AIM3 heterozygous mice ( ⁇ ).
- FIG. 3 c shows the results of Western blot analysis to determine the expression level of AIM3 in each phase of the cell cycle.
- FIG. 3 d shows the results of FACS analysis to determine the expression level of AIM3 at different cell cycle.
- DNA content(Y-axis) and the expression of AIM3 (X-axis) are analyzed by FACS and the density of cell is illustrated in contour lines.
- the “S” portion represents cells in the DNA synthetic phase on the basis of DNA content
- the “G1” portion represents cells in the G1/G0 phase.
- Right panel the expression level of AIM3 in “G1” and “S” portion respectively in the left panel is shown in histograms.
- the X-axis represents the expression level of AIM3, and the Y-axis represents cell number.
- FIG. 3 e shows the results of observation of the cellular localization of AIM3 at different proliferation conditions of cells.
- CM cell culture in complete media
- FIG. 4 a shows the results of flow cytometry to examine the apoptotic responses of splenocytes of AIM3 heterozygous mice ( ⁇ ) to adriamycin treatment(Adr), as compared to those of wild-type mice (+/+).
- FIG. 4b shows the results of flow cytometry to examine the response of wild-type mice- and AIM3 heterozygous mice ( ⁇ )-derived cells to adriamycin treatment, which caused cell growth arrest.
- FIG. 4 c shows the results of RT-PCR analysis and Western blot analysis to examine changes in the expression level of AIM3 by treatment with adriamycin (Adr), at different time.
- Bars represent the population of G1/G0 phase cells and numbers represent the percentage of G1/G0 phase cells
- FIG. 4 d shows the results of immunofluorescent staining to observe the cellular localization of AIM upon exposure to UV.
- FIG. 5 a illustrates the results of thymidine incorporation to measure the cell proliferation rate of mouse embryonic fibroblasts (MEFs) isolated from wild-type mice (+/+) and AIM3 heterozygous mice ( ⁇ ).
- MEFs mouse embryonic fibroblasts
- FIG. 5 b illustrates the results of immunofluorescent staining using an anti-Ki67 antibody (green color), to measure the cell proliferation rate of various tissues isolated from wild-type mice (+/+) and AIM3 heterozygous mice ( ⁇ ).
- FIG. 5 c illustrates the results of thymidine incorporation to measure the proliferation rate of cells transfected with an AIM3 gene.
- HCT116 cells transfected with an empty vector containing no AIM3 gene
- AIM3 HCT 116 cells transfected with an AIM3 expression vector
- FIG. 6 a shows the results of Western blot analysis to examine the effect of AIM3 on p53 expression in mouse embryonic fibroblasts (MEFs) derived from AIM3 heterozygous mice ( ⁇ ) and HCT116 cells transfected with an AIM3 expression vector.
- HCT116 cells transfected with an empty vector containing no AIM3 gene
- AIM3 HCT116 cells transfected with an AIM3 expression vector
- FIG. 6 b shows the results of RT-PCR to examine the effect of AIM3 on the p53-dependent transcription of p21.
- FIG. 6 c shows the results of luciferase assay using a vector containing a p21 promoter-fused luciferase gene, to examine the effect of AIM3 on the p53-dependent transcription of p21.
- HCT116 cells transfected with an empty vector containing no AIM3 were not treated with anything
- EV+Adr HCT116 cells transfected with an empty vector containing no AIM3 were treated with adriamycin
- AIM3 HCT116 cells transfected with an AIM3 expression vector were not treated with anything
- AIM3+Adr HCT116 cells transfected with an AIM3 expression vector were treated with adriamycin
- FIG. 6 d illustrates the effect of ATM3 on the proliferation of wild-type HCT116 cells (WT), p53 gene-null HCT116 cells (p53 ⁇ / ⁇ ) and p21 gene-null HCT116 cells (p21 ⁇ / ⁇ ).
- HCT116 cells transfected with an empty vector containing no AIM3 gene
- AIM3 HCT116 cells transfected with an AIM3 expression vector
- FIG. 6 e shows the effect of AIM3 on p53 induction caused by exposure to UV and treatment with adriamycin (Adr).
- EV HCT116 cells transfected with an empty vector containing no antisense-AIM3 (As-AIM3)
- As-AIM3 HCT116 cells transfected with a vector containing antisense-AIM3
- FIG. 7 a illustrates the effect of caffeine on the anti-proliferation activity of AIM3.
- HCT116 cells transfected with an empty vector containing no AIM3 gene
- AIM3 HCT116 cells transfected with an AIM3 expression vector
- FIG. 7 b shows the effect of caffeine on AIM3-induced apoptosis.
- FIG. 7 c shows the results of Western blot analysis to examine the effect of AIM3 on the induction of p53, after treatment with caffeine, an ATM inhibitor.
- FIG. 7 d shows the results of Western blot analysis to examine the effect of AIM3 on the induction of p53, after introducing a KD-ATM domain, a specific inhibitor of ATM activity, into cells.
- FIG. 8 a shows the results of co-immunoprecipitation to determine the interaction between AIM3 and ATM, after treatment with UV and adriamycin.
- FIG. 8 b shows the results of in vitro pull-down assay to determine the direct interaction between AIM3 and ATM.
- FIG. 8 c shows the results of co-immunoprecipitation to determine the interaction between AIM3 and ATR, after exposure to UV.
- FIG. 9 a shows the results of Western blot analysis to measure the phosphorylation level of H2AX, a substrate of ATM, in splenocytes and thymocytes isolated from wild-type mice (+/+) and AIM3 heterozygous mice ( ⁇ ).
- FIG. 9 b shows the results of Western blot analysis to examine the effect of AIM3 on the phosphorylation of H2AX, a substrate of ATM, using antisense-AIM3 (As-AIM3).
- FIG. 9 c shows the results of Western blot analysis to examine the effect of AIM3 on the phosphorylation of ATM and its target proteins (p53 and Chk2).
- p-p53 phosphorylated p53
- FIG. 10 a shows the results of RT-PCR to measure the expression level of AIM3 in different human cancer cell lines.
- FIG. 10 b shows the results of genomic PCR to examine the DNA content for AIM3 gene in different human cancer cell lines.
- FIG. 10 c shows the results of Western blot analysis to measure the expression level of AIM3 and p21 in tissues isolated from 9 leukemia patients.
- APML acute promyelocytic leukemia
- CML chronic myelocytic leukemia
- FIG. 10 d shows the results of RT-PCR to measure the expression levels of AIM3 in normal tissues and cancer tissues isolated from 9 liver cancer patients.
- the present inventors generated AIM3-deficient mice by a gene trap method (Zambrowicz, B. P. et al., Nature, 392:608-611, 1998).
- a gene trap method Zambrowicz, B. P. et al., Nature, 392:608-611, 1998.
- the embryonic stem cell library of 129/SvEvBrd mouse in which the gene trap vector was randomly introduced (OmniBank Library, Lexicon Genetics)
- the OST377244 clone including AIM3 genes mutated by the integration of the gene trap vectors was found out.
- C57BL6/albino AIM3 heterozygous mice were generated following the standard protocol of Lexicon Genetics, Inc. The heterozygous mice were interbred to generate the homozygous offspring.
- the site of a gene trap vector insertion in an AIM3 mutant allele was determined by sequencing analysis.
- the sequencing was performed by Pangenomics, a sequencing company.
- the sequencing results indicated that the gene trap vector was inserted between exon I and exon II of the AIM3 gene.
- genomic DNA was isolated from the tail of each of the mice generated in ⁇ Example 1>. Then, about 1.5-kb DNA fragment containing the exon I region of the AIM3 gene was amplified by PCR with a primer pair of p18F-1 and p18R-1 (SEQ ID NO: 3 and SEQ ID NO: 4) (see FIG. 1 a ). In addition, about 0.8-kb DNA fragment containing a part of the AIM3 gene and a part of the gene trap vector was amplified by PCR with the p18F-1 primer and an LTR primer (SEQ ID NO: 5) binding to the gene trap vector (about 5.7 kb) integrated into the genome (see FIG. 1 a ). The PCR reaction consisted of the following: denaturation of template DNA at 94° C. for 5 min; and then, 30 cycles of 1 min at 94° C., 1 min at 54° C., and 2 min at 72° C.
- genomic DNA was isolated and digested with SacI, followed by gel electrophoresis to separate the digested DNA fragments. Then, a PCR product amplified with p18F-2 and p18R-2 primers shown in SEQ ID NO: 6 and SEQ ID NO: 7, which contains the exon II region of the AIM3 gene, was labeled with a radioactive isotope (see FIG. 1 ), and the labeled probe was hybridized with the digested DNA fragments (southern, E. M., J. Mol. Biol., 98:503, 1975).
- mice were wild type (+/+) and 148 mice were heterozygous ( ⁇ ). None of surviving mice was homozygous ( ⁇ / ⁇ ). Particularly, the heterozygous mice were born at a similar ratio with the wild-type littermates, indicating that about 50% of the heterozygous mice would die during the pre-natal stage. As shown in Table 2, among total of 38 embryos isolated at 7.5-9.5 days after fertilization, only one embryo at 8.5 days containing the homozygous genotype was detected. This indicates that the AIM3 homozygous mice would be early embryonic lethal.
- proteins were isolated from various organs, such as small intestines, kidneys, heart and spleen. Then, according to the method described in Park S. G., et al. (Park S. G., et al., J. Biological Chemistry 274:16673-16676, 1999), Western blot analysis was performed using a polyclonal rabbit anti-AIM3 antibody.
- the anti-AIM3 antibody was prepared according to the method described in Kim, T. et al. aim, T. et al., J. Biol. Chem., 275:21768-21772, 2000).
- the present inventors isolated tissues and organs from the AIM3 ⁇ mice and analyzed the histological characteristics of the isolated tissues and organs.
- mice As a result, various tumors were found in the AIM3 ⁇ mice (see Table 3 and FIG. 2 a ). Interestingly, among 18 tumor-developing AIM3 ⁇ mice, 14 mice contained lymphoma which originated from the spleen or lymph node, and 5 mice had complex tumors.
- adenocarcinoma was found in the breasts of 15-month-old AIM3 ⁇ mice (B-63) and 23-month-old AIM3 ⁇ mice (B-95), adenocarcinoma in the seminal vesicles of 19-month-old AIM3 ⁇ mice (B-103), and hepatocarcinoma and sarcoma of unknown origin in 22-month-old AIM3 ⁇ mice (B-207). All of these cancers showed the typical malignant phenotypes, such as anaplasia and invasiveness.
- lymphoma was found in the lymph nodes of 22-month-old AIM3 ⁇ mice (B-232) and well-differentiated carcinoma which originated from the bronchiole epithelium was observed in 17-month-old AIM3 ⁇ mice (B-14).
- lymphomas metastasized into other organs, such as the liver, kidneys, lungs and salivary glands (see FIG. 2 b ).
- the incidence of these tumors was remarkably increased after 15 month-old (see FIG. 2 c and Table 3).
- a rapid cell cycle is a typical indicatin for tumorigenesis (Evan and Vousden, Nature, 411:342-348, 2001). Accordingly, it was addressed whether AIM3 could play a role in cell cycle control.
- the present inventors examined the cell proliferation rate of AIM3 ⁇ mouse-derived cells, compared to that of wild-type mouse cells. For this purpose, from 4-week-old wild-type mice and AIM3 ⁇ mice, the splenocytes and thymocytes were isolated, and the number of cell according to culture time was counted. As shown in FIG. 3 a, the results showed that the AIM3 ⁇ mice-derived cells proliferated faster than wild type mice cells.
- FACS analysis was performed.
- the splenocytes isolated from 4-week-old wild-type mice and AIM3 ⁇ mice were incubated overnight.
- the incubated cells were fixed with 1% PFA (paraformaldehyde) and stained with PI (propidium iodide).
- FACS analysis was conducted on 20,000 cells per sample. As shown in FIG. 3 b, the splenocytes isolated from the AIM3 ⁇ mice showed faster cell cycle than the wild-type mice cells.
- AIM3 was examined whether AIM3 is expressed depending on the cell cycle. HCT116 cells incubated in a serum-free medium for 24 hours and then incubated them again in a serum-containing medium to synchronize cell cycle. The expression level of AIM3 of the synchronized cells in different time under serum-deprivation and serum-re-fed conditions was measured by Western blot analysis. As a result, the AIM3 was remarkably induced during the DNA synthetic phase (see FIG. 3 c ).
- HCT116 cells Human colon adenocarcinoma cell line
- HCT116 cells Human colon adenocarcinoma cell line
- an anti-AIM3 monoclonal antibody Then, the cells were cultured with a FITC-conjugated anti-mouse goat IgG antibody (Pierce). And then, the cells were co-stained with PI, followed by FACS analysis.
- AIM3 was remarkably induced in the DNA synthetic phase (see FIG. 3 d ). This coincides with the result of Western blot analysis. All of these results indicate that AIM3 is induced in the DNA synthetic phase.
- DU145 cells prostate cancer cell line
- CM complete media
- SF serum-free media
- AIM3 when the cell growth was suppressed by serum starvation, AIM3 was mainly located in cytoplasm, whereas, when the cell growth was resumed, AIM3 was located in nuclei. Given thus, it could be found that, during the DNA synthetic phase of the cell cycle, AIM3 was not only induced but also translocated into nuclei. These results suggest that AIM3 could have novel functions in the nuclei.
- the splenocytes were isolated from wild-type mice and AIM3 ⁇ mice. To induce apoptosis, the isolated splenocytes were treated with 0.2 ⁇ g/ml of adriamycin (Adr, Sigma) for 2 hours. Then, the cells were cultured with FITC-conjugated annexin V (Roche) for 5 minutes. And then, the cells were washed with PBS and subjected to FACS analysis under a FL-1H detector. In this analysis, 20,000 cells per sample were used.
- Adr adriamycin
- apoptotic cells were significantly increased by treatment with adriamycin in the wild-type cells, however the AIM3 ⁇ cells showed the resistance to apoptosis induced by adriamycin. This indicates that AIM3 is required for sensitivity of cell to apoptosis induced by DNA damage. From this, it can be found that AIM3 promotes apoptosis caused by DNA damage.
- adriamycin In order to examine the importance of AIM3 in cell growth arrest caused by adriamycin, flow cytometry was performed. First, the thymocytes were isolated from wild-type mice and AIM3 ⁇ mice, and then treated with 0.2 ⁇ g/ml of adriamycin (Adr, Sigma) for 6 hours. Next, the cells were subjected to FACS analysis in the same method as in Example ⁇ 5-1>. As shown in FIG. 4 b, the growth of AIM3 ⁇ mouse cells was slightly suppressed by treatment with adriamycin, whereas that of wild-type mouse cells was arrested.
- HCT116 cells were treated with 0.2 ⁇ g/ml of adriamycin. Then, the cells were collected at different time and dissolved in Sol D solution (4 M guanidine thiocyanate, 1% laurosarcosine, 25 mM sodium citrate, and 0.1% b-mercaptoethanol). The cell extracts were incubated in acidic phenol and chloroform containing 4% isoamylalcohol, and vortexed. The mixture was centrifuged at 14,000 rpm. The upper layer was collected and added with isopropanol so as to precipitate RNA.
- Sol D solution 4 M guanidine thiocyanate, 1% laurosarcosine, 25 mM sodium citrate, and 0.1% b-mercaptoethanol.
- the cell extracts were incubated in acidic phenol and chloroform containing 4% isoamylalcohol, and vortexed. The mixture was centrifuged at 14,000 rpm. The upper
- RNA was washed with 100% ethanol, and 1 ⁇ g of RNA was dissolved in distilled water and used as a template for RT-PCR. Then, RT-PCR was performed with primers shown in SEQ ID NO: 8 and SEQ ID NO: 9. The expression level of GADPH was also measured in order to quantitatively compare that of AIM3.
- both the transcription and translation of AIM3 were induced in response to adriamycin.
- the induction of AIM3 was also observed by other DNA-damaging agents, such as UV, actinomycin D (Act.D) and cisplatin (CDPP) (data not shown).
- AIM3 was induced within 5-10 minutes after exposure to UV or adriamycin (data not shown).
- the cellular localization of AIM3 upon DNA damage was examined using U2OS cells containing large nuclei.
- the U2OS cells (osteosarcoma cell line) were treated with 254-nM wavelength UV-C (UV cross linker) at 50 J/m 2 .
- the cells were cultured in a complete medium for 30 minutes and collected.
- the same method as in Example ⁇ 4-3> was performed so as to examine the cellular localization of AIM3 by immunofluorescent staining.
- the UV-irradiated cells showed a remarkable increase in nuclear foci formed by AIM3.
- Mouse embryonic fibroblasts isolated from wild-type mice and AIM3 ⁇ mice were cultured in a medium containing 1 ⁇ Ci/ml [ 3 H] thymine. The cultured cells were washed with cold PBS and incubated in 10% TC A solution for 30 minutes so as to precipitate nucleic acids. Then, the cells were dissolved in 0.1 N NaOH, and the amount of radioactive thymidine incorporated in the precipitate was quantified by a liquid scintillation counter. The experiments were repeated three times and the data were averaged.
- MEFs isolated from the AIM3 ⁇ mice had a higher proliferation rate than the wild-type MEFs.
- Example ⁇ 6-1> a reduction in the expression of AIM3 resulted in an increase in cell proliferation.
- AIM3 results in the suppression of cell proliferation.
- the AIM3 gene (SEQ ID NO: 2) was inserted into a pcDNA3 (Invitrogen) vector so as to prepare an AIM3 expression vector. Then, the expression vector was transfected into HCT116 cells (human colon adenocarcinoma cell line). The cell proliferation rate of the transfected cells was examined in the same method as in the part a) of Example ⁇ 6-1>. As a control group, HCT116 cells transfected with pcDNA3 vector containing no AIM3 gene (empty vector; EV) were also used.
- Tumor suppressor protein p53 plays a major role in regulation of DNA damage-induced cell cycle arrest and apoptosis (Levine, Cell, 88:323-331, 1997; Vousden, Cell, 103:691-694, 2000). Thus, the functional relation between AIM3 and p53 was examined.
- the expression levels of p53 and AIM3 in mouse embryonic fibroblasts (MEFs) isolated from AIM3 ⁇ mice and wild-type mice were measured with Western blot analysis according to the same method as in Example ⁇ 5-3>. Also, the expression levels of AIM3 and p53 in the transfected HCT116 were measured with Western blot analysis after the AIM3 expression vector prepared in Example ⁇ 6-2> was transfected into HCT116 cells.
- the expression level of p53 in the MEFs of the AIM3 ⁇ mice was lower than that in the MEFs of the wild-type mice.
- the level of p53 in the HCT116 cells transfeted with the AIM3 gene was increased as compared to that in a control group cells transfected with an empty vector containing no AIM3 gene. This indicates that the ectopic expression of AIM3 elevates the expression of p53.
- Example ⁇ 7-1> The HCT116 cells transfected with the AIM3 gene (1 ⁇ g/ml) in Example ⁇ 7-1> were cultured for 24 hours. Then, RT-PCR analysis was performed in the same method as in Example ⁇ 5-3>. As a result, as shown in FIG. 6 b, the expression of p21 in the HCT116 cells transfected with the AIM3 gene was enhanced.
- luciferase assay was performed using a vector containing a p21 promoter fused to luciferase gene.
- HCT116 cells were co-transfected with a pGL-3 vector (Promega) engineered that the luciferase gene would be expressed under p21 promoter, and a recombinant AIM3 expression vector(1.2 ⁇ g/ml) containing the AIM3 gene.
- control group cells were co-transfected with the pGL-3 vector and an empty vector containing no AIM3 gene. Then, the transfected cells of each group were treated with 0.2 ⁇ g/ml of adriamycin for 2 hours. After cells were lyzed, the cell extract were incubated with substrate of luciferase for 30 minutes at room temperature. 5 ⁇ l of each sample was transferred to luminometer plate and luciferase activity was measured following the manufacturer's protocol (Promega).
- the luciferase activity regulated by the p21 promoter was highly increased by transfection with AIM3 and the luciferase activity was further increased by the additional treatment with adriamycin.
- Examples ⁇ 7-1> and ⁇ 7-2> demonstrated that the expressions of p53 and p21 depend on AIM3. Thus, it was examined whether AIM3 suppresses the proliferation of tumor cells via p53 and p21.
- the AIM3 expression vector or empty vector (2 ⁇ / ⁇ ) prepared in Example ⁇ 6-2> was transfected into each of HCT116 cells (human colon adenocarcinoma cell line), p53-null HCT116 cells and p21-null HCT116 cells. Then, the proliferation rate of each of the transfected cells was examined according to the same method as in the part a) of Example ⁇ 6-1>.
- the present inventors inhibited the expression of AIM3 by the use of antisense-AIM3 (As-AIM3) and then examined if the induction of p53 is influenced by the inhibition of the AIM3 expression.
- the N-terminal 176-bp region of the ATG-containing AIM3 gene was amplified by PCR.
- the PCR product was inserted into a pcDNA 3.1 vector in reverse orientation.
- 2 ⁇ g/ml of a vector containing antisense-AIM3 was transfected into HCT116 cells.
- the transfected cells were cultured for 24 hours.
- the cells were treated with UV and 0.2 ⁇ g/ml of adriamycin, respectively.
- Western blot analysis was performed in the same method as in Example ⁇ 2-5>. At this time, the expression level of actin was also measured in order to quantitatively compare the expression level of AIM3 and p53.
- the level of p53 was increased by treatment with UV or adriamycin, whereas the suppression of AIM3 by As-AIM3 inhibited the induction of p53.
- AIM3 is required for increasing the expression of p53.
- the transcription of PUMA an immediate early target gene of p53, was also increased by irradiation with WV, and its induction was blocked when AIM3 was suppressed by As-AIM3 (data not shown).
- AIM3 is an important upregulator of p53 that mediates the induction of p53 caused by DNA damage.
- ATM/ATR are substances directly activating p53 in response to DNA damage (Canman et al., Science, 281:1677-1679, 1998; Banin S et al., Science, 11;281 (5383):1674-7, 1998). Thus, the present inventors examined whether AIM3 acts via ATM/ATR.
- AIM3 can regulate p53 via ATM/ATR
- the present inventors first checked the anti-proliferation activity of AIM3 in the presence of caffeine known as an inhibitor of ATM/ATR.
- HCT116 cells were transfected with each of the AIM3 expression vector and the empty vector(2 ⁇ g/ml, respectively) for 24 hours. Then, the cells were added with 20 mM caffeine and cultured for 4 hours. Control group cells were added with PBS. The cell proliferation rate of the cells of each group was examined according to the same method as in the part a) of Example ⁇ 6-1>.
- FIG. 7 a the anti-proliferation activity of AIM3 was abolished by caffeine, an inhibitor of ATM/ATR. This demonstrates that AIM3 has anti-proliferation activity via ATM/ATR.
- HCT116 cells were transfected with each of the AIM3 expression vector or the empty vector(4 ⁇ g/ml, respectively) for 24 hours. Then, the cells were added with 20 mM caffeine and cultured for 12 hours. Control group cells were added with PBS. After staining the cells with PI, we checked apoptosis with measuring for the portion (%) of sub-G1 cells. As a result, as shown in FIG. 7 b, apoptosis was induced by the expression of AIM3, and this effect was relieved by treatment with caffeine.
- HCT116 cells were transfected with each of the AIM3 expression vector and the empty vector(2 ⁇ g/ml, respectively) for 24 hours. Then, the cells were added with 20 mM caffeine and cultured for 12 hours. Control group cells were added with PBS and cultured. Then, in order to examine the levels of AIM3 and p53, Western blot analysis was performed in the same method as in Example ⁇ 7-5>. At this time, the expression level of actin was also measured in order to quantitatively compare the expression levels of AIM3 and p53.
- HCT116 cells were transfected with the kinase-dead domain of ATM (KD-ATM) (Canman et al., Science, 281: 1677-1679, 1998).
- KD-ATM kinase-dead domain of ATM
- each of vectors containing the KD-ATM domain or wild-type ATM respectively was introduced into HCT116 cells with the AIM3 expression vector(2 ⁇ g/ml).
- each of these vectors was introduced into HCT116 with the empty vector containing no AIM3 gene.
- the expression levels of p53 and AIM3 in the cells of each group were examined by Western blot analysis according to the same method as in Example ⁇ 6-5>. At this time, the expression level of actin was also measured in order to quantitatively compare the expression levels of AIM3 and p53.
- AIM3 was expressed as GST fusion protein and purified according to the manufacturer's protocol (Pharmacia). Meanwhile, since it was difficult to synthesize the whole ATM due to its large size, the present inventors tested the interaction between the functional domain of ATM and AIM3. For this purpose, a fragment consisting of 612 amino acids, including the FAT domain of an ATM structure, was amplified by PCR with primers shown in SEQ ID NO: 12 and SEQ ID NO: 13. Also, a fragment (control group) consisting of 145 amino acids, including the C-terminal domain, was amplified by PCR with primers shown in SEQ ID NO: 14 and SEQ ID NO: 15.
- the amplified PCR products were subdloned into pcDNA3.1 (Invitrogen), a vector suitable for in vitro transcription and transition.
- the protein was synthesized by in vitro translation in the presence of radioactive methionine.
- 10 ⁇ l of the synthesized TNT product was incubated with the GST- or GST-AIM3 fusion protein-immobilized glutathion-sepharose beads for 5 minutes.
- the beads were washed six times with a binding buffer (PBS containing 0.2% sarcosine and 0.2% Triton X100), and dissolved in 10% SDS-PAGE. The binding of the GST-fused AIM3 to each domain was determined by autoradiography.
- the GST-fused AIM3 protein bound to the FAT domain, a functional domain, but not to the C-terminal domain of ATM. This suggests that AIM3 interacts directly with ATR.
- the FAT domain is found in not only ATM but also ATR (Abraham R, Genes Dev., 15:2177, 2001). Thus, the interaction between AIM3 and ATR was tested by co-immunoprecipitation.
- Example ⁇ 9-1> the same method as the part a) of Example ⁇ 9-1> was performed except that an anti-FLAG antibody (Sigma) was used in place of the anti-ATM antibody.
- AIM3 was co-immunoprecipitated with the flag-tagged ATR, and this interaction was further enhanced upon exposure to UV. This suggests that AIM3 also interacts with ATR as it acts on ATM.
- the present inventors examined whether the activity of ATM/ATR is enhanced by the association with AIM3.
- ATM/ATR The activity of ATM/ATR was examined using H2AX known as a substrate of ATM/ATR (Burma et al., J. Biol. Chem., 276: 42462-42467, 2001; Ward, I. M. et al., J. Biol. Chem., 276: 47759-47762, 2001; Irene M. Ward et al., J. Biol. Chem., 279(11):9677-9680, 2004).
- the present inventors treated cells with VP16, a DNA-damaging agent (Clarke et al., Nature, 362:849-852, 1993), and examined whether the phosphorylation of H2AX is inhibited by AIM3 inhibition in the presence of antisense-ATM3 (As-AIM3).
- Antisense AIM3-containing vector(2 ⁇ g/ml) prepared in Example ⁇ 7-5> was introduced into HCT116 cells. The transfected cells were cultured for 24 hours and then treated with 100 ⁇ M of VP16 (Sigma), an apoptosis-inducing agent, for 4 hours.
- the present inventors examined the phosphorylation of ATM and its target proteins, p53 and chk2, by Western blot analysis.
- Cells isolated from wild-type mice and AIM3 ⁇ mice were treated with 0.2 ⁇ g/ml of adriamycin. Then, using each of an anti-phospho-serine antibody of ATM, an anti-p53 antibody and an anti-chk2 antibody, Western blot analysis was performed (Bakkenist and Kastan, Nature, 421:499-506, 2003).
- the phosphorylation of ATM and its target proteins in the wild-type cells was enhanced by treatment with adriamycin, whereas that in the AIM3 ⁇ cells was inhibited.
- the level of expression of AIM3 was lower in HCT116, A549 and H460 cell lines. Specifically, the level of AIM3 was low in the cells containing active p53 (p53(+), i.e., HCT116, A549 and H460 cell lines), while it was normal in the cells lacking active p53(p53( ⁇ ), i.e., SW460, H23, H157 and K-562 cell lines). Also in Raji cells containing partially activated p53(Bhatia et al., FASEB J., 7:951-956, 1993), the level of AIM3 was in the middle of that of p53(+) cells and p53( ⁇ ) cells.
- the present inventors compared the DNA content for the AIM3 gene by PCR. On H157, H460, HCT116, A549 and DU145 cell lines, genomic DNA analysis was performed in the same method as in Example ⁇ 2-2>. As a control group, an actin gene was used.
- the H460 and A549 cell lines contained less amount of AIM3 DNA than that of other cell lines. This indicates that the two cell lines may have lost one allele for AIM3.
- AIM3 ⁇ mice Since solid tumors were also found in AIM3 ⁇ mice although the frequency was much lower, the present inventors also compared the expression levels of AIM3 in the cancer region with that in normal tissue isolated from liver cancer patients by RT-PCR. As a control group, the expression level of actin was also measured. From the analysis of 25 different patient samples, a cancer-specific reduction of AIM3 was observed in 12 samples. The results for exemplary 8 samples are shown in FIG. 10 d.
- AIM3 acts as a powerful tumor suppressor.
- the AIM3 protein binds to the FAT domain of ATM/ATR so as to activate ATM/ATR, thus inducing the expression of p53, tumor suppressor protein. Accordingly, the AIM3 protein or a nucleic acid encoding the protein will be useful for cancer therapy. Furthermore, it will be useful as targets for the development of anticancer drugs and as diagnostic markers of various cancers.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Wood Science & Technology (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Oncology (AREA)
- Marine Sciences & Fisheries (AREA)
- General Engineering & Computer Science (AREA)
Abstract
The present invention relates to novel uses of AIM3 acting as a tumor suppressor, and more particularly to methods for using an AIM3 protein or a nucleic acid encoding the protein to activate ATM or ATR and to treat ATM- or ATR-mediated diseases. The AIM3 protein according to the present invention interacts directly with ATM/ATR so as to activate ATM/ATR and proteins regulated by ATM/ATR. Also, the AIM3 protein upregulates tumor suppressor gene p53 and its target genes so as to not only inhibit the proliferation of cells but also to induce apoptosis.
Description
- The present invention relates to a novel tumor suppressor, and particularly to a novel tumor suppressor that activates ATM or ATR.
- Cells have a variety of fail-safe mechanisms, one of which is to arrest the cell division of damaged chromosomal DNA and to repair the damage, thus preventing mutations from settling. When chromosomal DNA damaged by UV and the like is continued to undergo cell division in a condition where the damage is not repaired, the damaged chromosomal DNA will be replicated so as to accumulate mutations. This leads to an increase in the incidence of cancer cells. Accordingly, when DNA is damaged, cells operate a process of repairing the damage and an intracellular feedback mechanism of arresting the cell division until the repair of DNA damage is over, followed by inhibiting the development of cancers. Such a feedback mechanism is mediated by checkpoints in each cycle of cell division. The overall function of these checkpoints is to detect damaged or abnormally structured DNA and to coordinate cell-cycle progression with DNA repair (Robert T. Genes & development, 15:2177-2196, 2001). Typically, cell-cycle checkpoint activation slows or arrests cell-cycle progression, thereby allowing time for appropriate repair mechanisms to correct genetic lesions before they are passed on to the next generation of daughter cells. In certain cells, such as thymocytes, checkpoint proteins link DNA strand breaks to apoptotic cell death via the induction of p53 (Robert T. Genes & development, 15:2177-2196, 2001).
- Cell-cycle checkpoints which are initiated by DNA damages are mainly regulated by ATM (ataxia-telangiectasia-mutated) and ATR (ATM- and Rad3-ralated) proteins (Shiloh, Y. Curr. Opin. Gent. Dev., 11:71-77, 2001; Abraham, R. T. Genes Dev., 15:2177-2196, 2001). Such proteins play a key role in the early signal transduction via the cell-cycle checkpoints. ATM- and ATR-deficient cells showed defects in arresting the cell cycle in response to radiation. Particularly, the ATM-deficient cells showed serious defects in G1, S and G2 checkpoints (Robert T. Genes & development, 15:2177-2196, 2001), and serious damages called “double strand breaks” occurred in the ATR-deficient cells. Furthermore, it was known that the incidence of tumor is greatly increased by the mutation of ATM/ATR.
- ATM and ATR are highly homologous to each other and use the same substrate. However, they are different in that their activities are increased by different genotoxic stresses. ATM responds to agents, such as IR (ionizing radiation) that breaks double strands DNA, whereas ATR responds to agents (including IR) that cause bulky adducts on DNA or single strand DNA. Furthermore, ATM and ATR are activated by different methods. ATM activation requires autophosphorylation that results in the disruption of an ATM dimer (Bakkenist, C. J. et al., Nature, 421:499-506, 2003). How autophosphorylation of ATM triggered is still unknown. ATR may also be autophosphorylated, but it is not evident that ATR forms either an inactive dimer or an active monomer in cells. Also, it is not yet clear that other subunits or cofactors are required for the activation of ATM/ATR. In addition, the intracellular biochemical mechanism of a signal transduction system where the DNA damage causes the activation and operation of ATM/ATR was not completely established.
- Target proteins known to be phosphorylated directly by ATM/ATR include p53, chk1, chk2, c-Abl, RPA and the like, of which p53 is phosphorylated on
serine 15 by ATM/ATR. It was reported that the over-expression of p53 arrests G2 and suppresses the synthesis of two proteins, CDK1 (cyclin-dependent kinase 1) and cyclin B1, which are required for the entry of cells from G2 to M. Thus, p53 does not only the function of inhibiting the abnormal division and proliferation of cells, but also the function of arresting the cell cycle so as to repair the damaged DNA when DNA was damaged. Recently, the mutation and loss of p53 genes are recognized as one of the most frequent genetic mutations, which is found not only in any certain cancer but in almost all types of cancer in human. Moreover, p53 activates the transcription of p21, another tumor suppressor gene, thereby inhibiting the G1/S transition and causing the p53-dependent apoptosis. p21 which is expressed by p53 was known to be a kind of a CKI (cyclin-dependent kinase inhibitor) which functions to inhibit the division and proliferation of cells. Accordingly, efforts for developing new anticancer agents using cell-cycle regulation factors or substances of activating the factors are now continued. - Meanwhile, aminoacyl-tRNA synthetases (ARSs) which are important enzymes catalyzing the first step in protein synthesis are multifunctional proteins involved in various biological functions (Ko et al., Proteomics, 2:1304-1310, 2002). Among them, various mammalian tRNA synthetases, such as MRS (methionyl-tRNA synthetase), QRS (glutaminyl-tRNA synthetase), RRS (arginyl-tRNA synthetase), KRS (Lysyl-tRNA synthetase), DRS(aspartyl-tRNA synthetase) and so on, bind to three non-enzyme cofactors, designated as p43, p38 and p18, to form a macromolecular protein complex (Han et al., Biochem. Biophys. Res. Commun., 303:985-993, 2003). Since ARSs are enzymes necessary for protein synthesis, this complex deems to be formed in order to facilitate protein synthesis.
- Among the non-enzyme cofactors binding to ARSs, p43 is known to play an important role as a cytokine in immune response and angiogenesis (Ko et al., J. Biol. Chem., 276:23028-32303, 2001b; Park et al., J. Biol. Chem., 277:45234-45248, 2002). Furthermore, p38 was found to downregulate c-myc, a protoocogene, and to be involved in lung differentiation (Kim et al., Nat. Genet., 34:330-336, 2003). The last cofactor, p18, shows sequence homology to elongation factor subunits (EF-1) (Quevillon and Mirande, FEBS Lett., 395:63-67, 1996). Given this, p18 is presumed to be involved in protein synthesis. However, the biological functions of p18 are not yet clearly understood, and particularly, there is no study on the relation between p18 and cancer.
- Therefore, it is an object of the present invention to provide novel uses of a p18 (ARS-interacting multifunctional protein 3) protein.
- The present inventors renamed p18 which had been known as a cofactor of an aminoacyl-tRNA synthetase (ARS) complex to “AIM3 (ARS-interacting multifunctional protein 3)”. Accordingly, p18 will hereinafter be referred to as “AIM3”.
- To achieve the above object, in one aspect, the present invention provides a method for activating ATM, ATR and proteins regulated by ATM or ATR, in the cell, tissue and individual, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of the following:
- (a) an isolated polypeptide of AIM3 (ARS-interacting multifunctional protein 3);
- (b) an isolated polypeptide having at least 70% homology with the polypeptide (a); and
- (c) an isolated nucleic acid encoding the polypeptide (a) or (b).
- In another aspect, the present invention provides a method for inducing the expression of p53 or its target genes in the cell, tissue or individual, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of following:
- (a) an isolated polypeptide of AIM3 protein;
- (b) an isolated polypeptide having at least 70% homology with the polypeptide (a); and
- (c) an isolated nucleic acid encoding the polypeptide (a) or (b).
- In still another aspect, the present invention provides a method for inhibiting the proliferation of tumor cells, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of the following:
- (a) an isolated polypeptide of AIM3 protein;
- (b) an isolated polypeptide having at least 70% homology with the polypeptide (a); and
- (c) an isolated nucleic acid encoding the polypeptide (a) or (b).
- In still another aspect, the present invention provides a method for stimulating apoptosis in the cell, tissue or individual, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of the following:
- (a) an isolated polypeptide of AIM3 protein;
- (b) an isolated polypeptide having at least 70% homology with the polypeptide (a); and
- (c) an isolated nucleic acid encoding the polypeptide (a) or (b).
- In still another aspect, the present invention provides a method for treating or preventing ATM- or ATR-mediated diseases, comprising administering to a subject in need thereof an effective amount of one selected from the group consisting of the following:
- (a) an isolated polypeptide of AIM3 protein;
- (b) an isolated polypeptide having at least 70% homology with the polypeptide (a); and
- (c) an isolated nucleic acid encoding the polypeptide (a) or (b).
- In still another aspect, the present invention provides a method for screening a substance having the effect of treating or preventing ATM- or ATR-mediated diseases, the method comprising the steps of:
- (a) culturing AIM3 (ARS-interacting multifunctional protein 3) or a recombinant cell expressing the protein, together with a candidate substance; and
- (b) determining the effect of the candidate substance on an increase in the activity of
AIM 3 or the intracellular level thereof. - In yet another aspect, the present invention provides a method for identifying a subject having the risk of ATM- or ATR-mediated diseases, comprising the steps of:
- (a) measuring the expression level of ATM3 protein in tissue sampled from a subject; and
- (b) comparing the level of the AIM3 protein in the tissue with a normal AIM3 protein level.
- In still another aspect, the present invention provides a kit for the diagnosis of ATM- or ATR-mediated diseases, comprising one selected from the group consisting of an AIM3 protein-encoding nucleic acid, a fragment thereof, a peptide encoded by the nucleic acid or its fragment, and an antibody to the peptide.
- In another further aspect, the present invention provides pharmaceutical compositions comprising, as an active ingredient, one selected from the group consisting of the following:
- (a) an isolated polypeptide of AIM3 protein;
- (b) an isolated polypeptide having at least 70% homology with the polypeptide (a); and
- (c) an isolated nucleic acid encoding the polypeptide (a) or (b).
- Hereinafter, the present invention will be described in detail.
- In the present invention, novel activities of AIM3 (p18) known as a cofactor of an aminoacyl-tRNA synthetase (ARS) complex were identified. The physiological activities (functions) of AIM3 identified in the present invention are as follows:
- First, in the DNA synthesis step and upon DNA damage, AIM3 is moved into nuclei and induced at a high level.
- Second, AIM3 shows an anti-proliferation activity against cells.
- Third, AIM3 induces apoptosis.
- Fourth, AIM3 induces the expression of tumor suppressor gene p53 and its target genes.
- Fifth, AIM3 interacts directly with ATM/ATR so as to activate ATM, ATR and proteins which are regulated by ATM or ATR.
- Sixth, a reduction in the expression level of AIM3 induces tumorigenesis, and it is expressed at a low level in cancer cell lines and tissues isolated from cancer patients.
- Accordingly, the present invention provides a method for activating one selected from the group consisting of ATM, ATR and proteins regulated by ATM or ATR using an AIM3 protein or a nucleic acid encoding the AIM3 protein.
- As used herein, the term “activating” means the phosphorylation of proteins or the structural or chemical mutation of proteins. The activation of ATM/ATR is mediated by the biding of AIM3, which causes a variety of intracellular responses involved in ATM/ATR. Such intracellular responses include, but are not limited to, DNA repair, cell cycle regulation and apoptosis induction. Thus, the activation of ATM/ATR by AIM3 accompanies activation of downstream proteins which are involved in DNA repair signal transduction pathways induced by DNA replication or damage, a checkpoint signal transduction pathway in each cell cycle, and/or an apoptosis-inducing signal transduction pathway caused by DNA damage. The ATM/ATR-regulated proteins include proteins which are directly phosphorylated by ATM/ATR, and proteins which are sequentially phosphorylated in signal transduction pathways by the phosphorylation of said proteins. Preferred examples include, but are not limited to, H2AX (Burma S. et al., J. Biol. Chem., 9;276(45):42462-42467, 2001), p53 (Saito S, et al., J. Biol. Chem., 12;277(15):12491-12494, 2002), chk2 (Matsuoka S, et al., Proc. Natl. Acad. Sci. U.S.A., 12;97(19):10389-10394, 2000), chk1 (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), BRCA1 (Xu B, et al., Cancer Res., 15;62(16):4588-4591, 2002; Cortez D, et al., Science, 5;286(5442):1162-1166, 1999), c-Abl (Baskaran R, et al., Nature, 29;387(6632):516-519, 1997), PHAS-1 (Chan D W et al., J. Biol. Chem., 17;275(11):7803-7810, 2000), RPA (Chan D W et al., J. Biol. Chem., 17;275(11):7803-7810, 2000), RAD9 (Chen M J et al., J. Biol. Chem., 1 1;276(19):16580-16586, 2001), MDM2 (Maya R, et al., Genes Dev., 1;15(9):1067-1077, 2001), MRE11 (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), Rad17 (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), WRN (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), PTS (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), CtIP (Li S, et al., Nature, 13;406(6792):210-215, 2000), eIF-4E binding protein 1 (Yang D Q, et al., Nat. Cell. Biol., 2(12):893-898, 2000), LKB1 (Sapkota G P, et al., Biochem J., 1;368(Pt 2):507-516, 2002), FANCD2 (Taniguchi T, et al., Cell, 17;109(4):459-472, 2002), SMC1 (Yazdi P T, et al., Genes Dev., 1;16(5):571-582, 2002), Rad17 (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), Nibrin (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), NBS (Wu K. et al., Nature, 25;405(6785):477-482, 2000), p95 (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), Pin2/TRF1 (Kishi S. et al., J. Biol. Chem., 3;276(31):29282-29291, 2001), DNA 5B (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), BRCA2 (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999) and phosphatidylinositol 3-kinase (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999). More preferably, the proteins may be H2AX, p53 or chk2.
- Furthermore, the present invention provides a method for inducing the expression of p53 or its target gene using the AIM3 protein or a nucleic acid encoding the AIM3 protein. As used herein, the term “p53-target gene” refers to a gene located in downstream of p53, whose expression is induced by p53. The p53-target gene may be a gene involved in at least one mechanism selected from the group consisting of p53 control, cell cycle regulation, DNA repair, apoptosis, angiogenesis, cellular stress response and determination of cell fate. Preferred examples of this target gene include, but are not limited to, p21 (Fujioka S, et al., J. Biol. Chem., Apr. 21, 2004; Nayak B K, et al., Oncogene, 17;21(47):7226-7229, 2002), PUMA (Gu J, et al., Oncogene, 12;23(6):1300-1307, 2004; Yu J, et al., Cell, 7(3):673-682, 2001), GADD45 (Nayak B K, et al., Oncogene, 17;21(47):7226-7229, 2002; el-Deiry W S., Semin Cancer Biol., 8(5):345-57), 14-3-3 sigma (el-Deiry W S., Semin Cancer Biol., 8(5):345-57), WIP1 (Choi J, et al., Genomics., 15;64(3):298-306, 2000), mdm-2 (Freedman and Levine, Cancer Research, 59:1-7, 1999), EGFR (Tokino and Nakamura, Crit. Rev. Onc. Hem., 33:1-6, 2000), PCNA (Tokino and Nakamura, Crit. Rev. Onc. Hem., 33:1-6, 2000), Cyclin D1 (Tokino and Nakamura, Crit. Rev. Onc. Hem., 33:1-6, 2000), Cyclin G (Tokino and Nakamura, Crit. Rev. Onc. Hem., 33:1-6, 2000), TGFα (Inoue Y, et al., Hepatology, 36(2):366-344, 2002), BAX (Gu J, et al., Oncogene, 12;23(6):1300-1307, 2004; Nayak B K, et al., Oncogene, 17;21(47):7226-7229, 2002), BAK (Gu J, et al., Oncogene, 12;23(6):1300-1307, 2004), FAS1 (Gu J, et al., Oncogene, 12;23(6):1300-1307, 2004), Fas/APO1 (el-Deiry W S., Semin Cancer Biol., 8(5):345-57), FASL (Mendoza-Rodriguez C A, et al., Rev. Invest. Clin., 53(3):266-273, 2001), IGF-BP3 (Mendoza-Rodriguez C A, et al., Rev. Invest. Clin., 53(3):266-273, 2001), PAG608 (Higashi Y, et al., J. Biol. Chem., 1;277(44):42224-42262, 2002), DR5/KILLER (Takimoto R, et al., Oncogene, 30;19(14):1735-1743, 2000), GML (Higashiyama M, et al., Eur. J. Cancer., 36(4):489-495, 2000; Nakamura Y., Cancer Sci., 95(1):7-11, 2004), p53AIP1 (Nakamura Y., Cancer Sci., 95(1):7-11, 2004), STAG1 (Nakamura Y., Cancer Sci., 95(1):7-11, 2004), p53R2 (Nakamura Y., Cancer Sci., 95(1):7-11, 2004), p53RFP (Nakamura Y., Cancer Sci., 95(1):7-11, 2004), P2XM (Nawa G, et al., Br. J. Cancer., 80(8):1185-1189, 1999), TSP-1 (Harada H, et al., Cancer Lett., 28;191(1):109-119, 2003), BAL1 (Nakamura Y., Cancer Sci., 95(1):7-11, 2004), CSR (Nakamura Y., Cancer Sci., 95(1):7-11, 2004), PIG3 (Giampieri S, et al., Oncogene, Apr. 12, 2004; Contente A, et al., Nat. Genet., 30(3):315-320, 2002), Apaf-1 (Giampieri S, et al., Oncogene, Apr. 12, 2004), p53RDL1 (Nakamura Y., Cancer Sci., 95(1):7-11, 2004), Staf50 (Obad S, et al., Oncogene, 20;23(3):4050-4059, 2004), CD200 (Rosenblum M D, et al., Blood, 1;103(7):2691-2698, 2004) and Snk/PIk2 (Bums T F, et al., Mol. Cell. Biol., 23(16):5556-5571, 2003). More preferably, the target gene may be p21 or PUMA.
- AIM3 of the present invention inhibits the proliferation of tumor cells through signal transduction pathways mediated by ATM/ATR and stimulates apoptosis caused by DNA damage. Accordingly, the present invention provides methods to inhibit the proliferation of tumor cells and to stimulate apoptosis, using the AIM3 protein and a nucleic acid encoding the AIM3 protein.
- All the methods described above comprise administering an effective amount of the AIM3 protein or the nucleic acid encoding the protein to cells or tissues. As used herein, the term “effective amount” refers to the amount of AIM3, which shows an effect selected from the group consisting of the following: the activation of ATM/ATR in cells or tissues; the increase of phosphorylation of ATM/ATR-regulated proteins; the induction of expression of p53 or its target gene; the inhibition of proliferation of tumor cells; and the promotion of apoptosis.
- The AIM3 proteins used in the present invention include natural or recombinant AIM3 proteins, or proteins having the substantially equivalent physiological activity of the natural or recombinant AIM3 proteins. The amino acid sequence of the AIM3 protein is known in the art and preferably derived from mammals, including human beings. The AIM3 protein of the present invention preferably has an amino acid sequence shown in SEQ ID NO: 1. Proteins having the substantially equivalent physiological activity of AIM3 include natural/recombinant AIM3 proteins, their functional equivalents and their functional derivatives. As used herein, the term “the substantially equivalent physiological activity” means the activity of: activating ATM/ATR or ATM/ATR-regulated proteins; inducing the expression of p53 or its target gene; inhibiting the proliferation of tumor cells; and/or stimulating apoptosis. The term “functional equivalents” refers to amino acid sequence variants with a substitution of some or all of the amino acids of a natural AIM3 protein or a deletion or addition of some of the amino acids, which have a physiological activity substantially equivalent to the natural AIM3 protein. Furthermore, the term “functional derivatives” refers to those having a physiological activity substantially equivalent to natural AIM3 protein, as proteins modified to increase or reduce the physicochemical properties of the
AIM 3 protein. The proteins having a physiological activity substantially equivalent to the AIM3 protein have a homology of at least 70%, preferably at least 80%, and more preferably at least 90%, with the polypeptide shown in SEQ ID NO: 1. The AIM3 protein used in the present invention can be prepared by any genetic engineering method known in the art. - The inventive pharmaceutical composition containing the AIM3 protein as an active ingredient can be administered to human beings and animals by oral route or by parenteral route, such as an intravenous, subcutaneous, intranasal or intraperitoneal route. Oral administrations include sublingual application. Parenteral administrations include injection techniques, such as subcutaneous injection, intramuscular injection and intravenous injection, as well as drip infusion. In addition, the pharmaceutical composition can be formulated into various forms with a pharmaceutically acceptable carrier by a conventional method. As used herein, the term “pharmaceutically acceptable” carrier means a substance which is physiologically acceptable and, when administered to human beings, generally does not cause allergic reactions, such as gastrointestinal disorder and dizziness, or similar reactions thereto.
- As the pharmaceutically acceptable carriers, in the case of oral administration, there may be used binders, lubricants, disintegrants, excipients, solubilizers, dispersing agents, stabilizers, suspension agents, pigments and flavors, and in case of injection agent, there can be used buffers, preservatives, analgesics, solubilizers, isotonics and stabilizers, and in case of formulations for local administration may include bases, excipients, lubricants and preservatives. As described above, the inventive pharmaceutical composition containing the AIM3 protein may be formulated into various forms with the pharmaceutically acceptable carriers. For example, for oral administration, the inventive composition may be formulated into the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and so on, and for injection agent, it may be formulated into unit dose ampoules or multiple dose products.
- A total effective amount of the AIM3 protein of the present invention can be administered to patients in a single dose or can be administered by a fractionated treatment protocol, in which multiple doses are administered over a more prolonged, period of time. Although the amount of the AIM3 protein or a nucleic acid encoding the AIM3 protein in the inventive pharmaceutical composition may vary depending on the severity of diseases, the protein or the nucleic acid may be generally administered several times a day at an effective dose of 1 μg-10 mg. However, a suitable dose of the AIM3 protein in the inventive pharmaceutical composition may depend on many factors, such as the age, body weight, health condition, sex, disease severity, diet and excretion of patients, as well as the route of administration and the number of treatments to be administered. In view of these factors, any person skilled in the art may determine an effective dose for treating or preventing ATM/ATR-mediated diseases. The inventive pharmaceutical composition containing the AIM3 protein has no special limitations on its formulation, administration route and/or administration mode insofar as it shows the effects of the present invention.
- Meanwhile, nucleic acids encoding the AIM3 protein of the present invention include DNA or RNA. Preferably, they refer to DNA encoding AIM3 proteins derived from mammals, particularly human beings. The human AIM3 gene is known in the art (GenBank accession No. AB011079). Preferably, the nucleic acid of the present invention is shown in SEQ ID NO: 2. The nucleic acids also include nucleic acids encoding functional equivalents to the AIM3 protein. The present invention can be included nucleic acids having a sequence homology of at least 80%, preferably at least 90%, and more preferably at least 95% with either a nucleic acid encoding the AIM3 protein or a nucleic acid comprising the complementary nucleotide sequence thereof.
- The nucleic acid encoding the AIM3 protein may be used for gene therapy by inserting it into an expression vector, such as a plasmid or viral vector, and then introducing the expression vector into a target cell by any method known in the art, such as infection or transduction.
- A gene transfer method using a plasmid expression vector is a method of transferring a plasmid DNA directly to human cells, which is an FDA-approved method applicable to human beings (Nabel, E. G., et al., Science, 249:1285-1288, 1990). Unlike viral vectors, the plasmid DNA has an advantage of being homogeneously purified. Plasmid expression vectors which can be used in the present invention include mammalian expression plasmids known in the pertinent art. For example, they are not limited to, but typically include pRK5 (European Patent No. 307,247), pSV16B (PCT Publication No. 91/08291) and pVL1392 (PharMingen).
- The plasmid expression vector containing the nucleic acid according to the present invention may be introduced into target cells by any method known in the art, including, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun methods, and other known methods for introducing DNA into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).
- In addition, virus expression vectors containing the nucleic acid according to the present invention include, but are not limited to, retrovirus, adenovirus, herpes virus, avipox virus and so on.
- The retroviral vector is so constructed that non-viral proteins can be produced within the infected cells by the viral vector in which virus genes are all removed or modified. The main advantages of the retroviral vector for gene therapy are that it transfers a large amount of genes into replicative cells, precisely integrates the transferred genes into cellular DNA, and does not induce continuous infections after gene transfection (Miller, A. D., Nature, 357:455-460, 1992). The retroviral vector approved by FDA was prepared using PA317 amphotropic retrovirus packaging cells (Miller, A. D. and Buttimore, C., Molec. Cell Biol., 6:2895-2902, 1986).
- Non-retroviral vectors include adenovirus as described above (Rosenfeld et al., Cell, 68:143-155, 1992; Jaffe et al., Nature Genetics, 1:372-378, 1992; Lemarchand et al., Proc. Natl. Acad. Sci. USA, 89:6482-6486, 1992). The main advantages of adenovirus are that it transfers a large amount of DNA fragments (36 kb genomes) and is capable of infecting non-replicative cells at a very high titer.
- Moreover, herpes virus may also be useful for human genetic therapy (Wolfe, J. H., et al., Nature Genetics, 1:379-384, 1992). In addition, any suitable virus vector known in the art may be used.
- A vector capable of expressing the AIM3 gene may be administered by a known method. For example, the vector may be administered locally, parenterally, orally, intranasally, intravenously, intramuscularly or subcutaneously, or by other suitable routes. Particularly, the vector may be injected directly into a target cancer or tumor cell at an effective amount for treating the tumor cell of a target tissue. Particularly for a cancer or tumor present in a body cavity such as in the eye, gastrointestinal tract, genitourinary tract, pulmonary and bronchial system and so on, the inventive pharmaceutical composition can be injected directly into the hollow organ affected by the cancer or tumor using a needle, a catheter or other delivery tubes. Any effective imaging device, such as X-ray, sonogram, or fiberoptic visualization system, may be used to locate the target tissue and guide the needle or catheter tube. In addition, the inventive pharmaceutical composition comprising the nucleic acid encoding the AIM3 protein may be administered into the blood circulation system for treatment of a cancer or tumor which cannot be directly reached or anatomically isolated.
- The pharmaceutical composition comprising the nucleic acid encoding the AIM3 protein as an active ingredient may additionally comprise pharmaceutically acceptable carriers or excipients. These carriers or excipients include dispersing agents, wetting agents, suspending agents, diluents and fillers. The ratio of the particular pharmaceutically acceptable carrier and the expression vector contained in the inventive pharmaceutical composition can be determined by the solubility and chemical properties of the composition, and the particular administration mode. The therapeutic or preventive effective amount of the inventive pharmaceutical composition containing the AIM3 protein-encoding nucleic acid may be suitably selected depending on the subject to be administered, age, individual variation and disease condition.
- In another aspect, the present invention provides a method for treating or preventing ATM/ATR-mediated diseases using the AIM3 protein or a nucleic acid encoding the AIM3 protein. Specifically, the present invention provides a method for treating or preventing ATM- or ATR-mediated diseases, which comprise administering to a subject requiring treatment an effective amount of one selected from the group consisting of the following: (a) an isolated polypeptide of an AIM3 protein; (b) a polypeptide having at least 70% homology with the polypeptide (a); and (c) an isolated nucleic acid encoding the polypeptide (a) or (b). As used herein, the term “subject” means mammals, particularly animals including human beings. The subject may be a patient requiring treatment. Furthermore, the term “ATM- or ATR-mediated diseases” refers to diseases induced by the inactivation or activation reduction of ATM/ATR, i.e., diseases induced by abnormalities occurring in signal transduction pathways mediated by ATM/ATR, due to the inactivation or activation reduction of ATM/ATR. The signal transduction pathways mediated by ATM/ATR include signal transduction pathways mediated by ATM/ATR themselves or ATM/ATR-regulated proteins. The signal transduction pathways may be signal transduction pathways in DNA repair, cell cycle regulation, apoptosis, p53 regulation, angiogenesis and/or intracellular stress response. The ATM/ATR-mediated diseases may be caused by the over-proliferation of cells, such as cancers or psoriasis. The cancers include, but are not limited to, breast cancer, rectal cancer, lung cancer, small-cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine carcinoma, ovarian cancer, colorectal cancer, cancer near the anus, colon cancer, oviduct carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulva carcinoma, Hodgkin's disease, esophagus cancer, small intestinal tumor, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, uterine cancer, penis cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or urethra cancer, kidney cell carcinoma, kidney pelvis carcinoma, CNS tumor, primary CNS lymphoma, spinal tumor, brain stem glioma, and pituitary adenoma, and a combination of one or more thereof. Particularly, the treating or preventing method according to the present invention is effective in treating or preventing cancers caused by p53 gene abnormalities. In this method, the dose (effective amount) and administration mode of the AIM3 protein or the nucleic acid encoding the AIM3 protein are the same as described above.
- The AIM3 protein of the present invention interacts directly with ATM/ATR so as to activate ATM/ATR and various proteins regulated by ATM/ATR. Particularly, the AIM3 protein shows the activity of inducing the expression of p53, one of the ATM/ATR-regulated proteins, and the expression of its target genes, so as to stimulate the apoptosis of cells for DNA damage and to inhibit the proliferation of tumor cells. Such characteristics of AIM3 may be used to screen a substance effective for treating/preventing ATM/ATR-mediated diseases, particularly cancer. Accordingly, the present invention provides a method for screening a substance effective for treating or preventing ATM/ATR-mediated diseases, which comprise the step of: (a) culturing the AIM3 protein or a recombinant cell expressing the AIM3 protein together with a candidate substance; and (b) determining the effect of the candidate substance on an increase in the activity of AIM3 or the intracellular level thereof. As used herein, the term “activity of the AIM3 protein” refers to the binding activity with ATM/ATR, the activity of promoting the phosphorylation of ATM/ATR or proteins regulated by ATM/ATR, and/or the activity of inducing the expression of p53 and its target genes. The term “increase in the intracellular level of the AIM3 protein” means the increase in the concentration of the AIM3 protein by the increase of expression of the AIM3 gene or the inhibition of the degradation of the AIM3 proteins.
- The expression of the AIM3 gene includes process for the transcription of the AIM3 gene and the translation into proteins. Accordingly, the substances screened in the present invention has the property of: promoting the binding of AIM3 to ATM/ATR; activating ATM/ATR or proteins regulated by ATM/ATR; inducing the expression of p53 and its target genes; and/or increasing the intracellular level of the AIM3 protein. These substances include not only proteins but also naturally occurring or chemically synthesized compounds or extracts.
- The activity and intracellular level of the AIM3 protein can be measured by various methods known in the art. Exemplary methods include, but are not limited to, co-immunoprecipitation, enzyme-linked immunosorbent assay, radioimmunoassay (RIA), immunohistochemical assay, Western blotting, and fluorescence activated cell sorter (FACS) analysis.
- In addition, for the screening method using the
AIM 3 of the invention as a target gene, high throughput screening (HTS) can be applied. The HTS is a method for screening the biological activities of a number of candidate substances simultaneously or almost simultaneously by testing the candidate substances simultaneously. In a certain embodiment, cell lines are cultured in a 96-well microtiter plate or a 192-well microtiter plate, into which a number of candidate substances are added and then measured for the expression of AIM3 by an immunohistochemical method. In this format, 96 independent tests may be simultaneously performed in a single 8 cm×12 cm plastic plate containing 96 reaction wells. The wells require an assay volume of 50-500 μl typically. In addition to the plate, a number of gauges, instruments, pipetters, robots, plate washers and plate readers are commercially available in order to make the 96-well format suitable for a wide range of homogeneous and heterogeneous assays. - Meanwhile, the expression level of the AIM3 gene or protein in biological samples (e.g., blood, serum, sputum, urine and/or tumor biopsies) collected from subjects can be compared with that of normal persons so as to diagnose (identify) subjects having the risk of ATM/ATR-mediated diseases. Specifically, using one selected from the group consisting of an AIM3 protein-encoding nucleic acid, a fragment thereof, a peptides encoded by them, and an antibody to the peptide as a primer or probe, ATM/ATR-mediated diseases may be identified. Accordingly, the present invention provides a method for identifying a subject having the risk of ATM/ATR-mediated diseases, which comprise the steps of: (a) measuring the expression level of AIM3 in a tissue sampled from a subject; and (b) comparing the level of AIM3 in the tissue with a normal AIM3 level. The methods for identifying such a disease include those which are capable of detecting the expression of AIM3 at a transcriptional or translational level (such as RT-PCR, Northern blotting, Western blotting, immunological assays and so on). This method is very effective for diagnosing cancer among ATM/ATR-mediated diseases.
- In still another aspect, the present invention provides a kit for the diagnosis of ATM/ATR-mediated diseases, which comprises one selected from the group consisting of a AIM3-encoding nucleic acid, a fragment thereof, a peptide encoded by them, and an antibody to the peptide. The AIM3 protein-encoding nucleic acid and a fragment thereof may be synthesized with reference to the known sequence of the AIM3 gene. The fragment of nucleic acid is preferably a primer capable of amplifying the AIM3 gene. The peptide encoded by the AIM3 protein-encoding nucleic acid or its fragment may be synthesized by any technique known in the art (Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman and Co., NY, 1983). The peptide can be produced by the conventional stepwise liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Williams et al., Eds., Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press, Boca Raton Fla., 1997; Atherton □ Sheppard, Eds., A Practical Approach, IRL Press, Oxford, England, 1989).
- The antibody to the peptide can be produced using the AIM3 protein or its fragment as an antigen by any conventional method widely known in the immunological field. The antibodies include polyclonal antibodies and monoclonal antibodies.
- The polyclonal antibodies can be prepared from a variety of warm-blooded animals, such as horses, cattle, goats, sheep, dogs, fowl, turkeys, rabbits, mice or rats, by any conventional technique known in the art. Namely, the animals are immunized by intraperitoneal, intramuscular, intraocular or subcutaneous injection of an antigen. The immunogenicity to the antigen can be increased by the use of an adjuvant, for example Freund's complete adjuvant or incomplete adjuvant. Following booster immunization, a small serum sample was collected and tested for the reactivity to the target antigen. Once the animal's titer reaches a stagnant state in view of its reactivity to the antigen, a large amount of the polyclonal antibodies can be obtained by bleeding the animal at one-week intervals or by blood-letting the animal.
- The monoclonal antibodies can also be produced by a known method (Kennettm McKearn, and Bechtol(eds.), Monoclonal Antibodies, Hybridomas; A New Dimension in Biological Analyses, Plenum Press, 1980). The monoclonal antibodies can be produced by immunizing an animal with the AIM3 protein or its fragment as an immunogen, fusing the splenocytes of the immunized animal with myeloma cells to produce a hybridomas, screening a hybridoma that selectively recognizes the AIM3 protein, culturing the screened hybridoma, and isolating antibodies from the hybridoma culture. Alternately, the monoclonal antibodies according to the present invention may also be prepared by injecting said hybridoma into an animal, and after a given period of time, isolating antibodies from the collected ascites of the animal.
- The antibody contained in the inventive diagnostic kit is preferably immobilized onto a solid substrate. The antibody can be immobilized by various techniques described in literatures (Antibodies: A Labotory Manual, Harlow □ Lane; Cold SpringHarbor, 1988). Suitable solid substrates include those supported by rods, synthetic glass, agarose beads, cups, flat packs, or other solid support or those having a film or coating attached to them. In addition, other solid substrates include cell culture plates, ELISA plates, tubes and polymeric films.
- The diagnostic kit according to the present invention may contain, in addition to an antibody selectively recognizing the AIM3 protein, reagents which are used in immunological assays. The immunological assays may include methods capable of measuring the binding of an antigen to the antibody of the present invention. These methods are known in the art and include, for example, immunocytochemical assays, immunohistochemical assays, radioimmunoassays, ELISA (enzyme linked immunoabsorbent assay), immunoblotting, Farr assays, precipitin reaction, turbidimetry, immunodiffusion, counter-current electrophoresis, single radical immunodiffusion and immunofluorescence.
- Reagents which are used in the immunological assays include a suitable carrier, a labeling substance capable of emitting detectable signals, a solubilizer and a washing agent. Furthermore, if the labeling substance is enzyme, a substrate capable of measuring enzymatic activity and a reaction stopping agent may be used.
- Suitable carriers include, but are not limited to, soluble carriers, for example, one of biologically acceptable buffers known in the art (e.g., PBS), insoluble carriers, for example polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, polymers, such as latex containing magnetic fine particles plated with metal, paper, glass, metal, agarose and combinations thereof.
- Labeling substances capable of emitting detectable signals include enzymes, fluorescent substances, luminescent substances and radioactive substances. The enzymes include peroxidase, alkaline phosphatase, β-D-galactosidase, glycose oxidase, maleate dihydrogenase, glucose-6-phosphodihydrogenase, invertase and so on. The fluorescent substances include fluorescein isothiocyanate and phycobili-protein. As luminescent substances, isolucinol and lucigenin and so on can be used. And, as radioactive substances, I131, C14, H3 and so on, can be used. However, the above examples are only examples and anything used in immunoassay can be used. The ATM/ATR-mediated diseases which can be diagnosed with the inventive kit are the same as described above, and preferably, may be lung cancer, colon cancer, liver cancer, lymphoma and leukemia.
- In one embodiment of the present invention, in order to identify the biological functions of AIM3, AIM3 gene-deficient mice were produced by a gene trap method. Then, genomic DNA mutated by the insertion of a gene trap vector was introduced into the embryonic stem cell of the mice so as to construct a mutant library. Clones containing the mutated AIM3 gene were searched from the library and used to prepare AIM3 heterozygous mutant mice.
- In another embodiment of the present invention, the sequence of an AIM3 allele in the mutant mice was analyzed. The results showed that the gene trap vector was integrated between the first and second exons in the AIM3 gene (see
FIG. 1 a). Furthermore, genomic PCR and Southern blotting were performed to determine AIM3 mutation (FIGS. 1 b and 1 c), and the expression level of AIM3 by the mutation was determined using Western blot (seeFIG. 1 d). - The post-natal genotype of progenies obtained by crossbreeding the AIM3 heterozygous mutant mice (hereinafter, referred to as “AIM3± mice”) and the genotype of embryos with the passage of time were examined and the results showed that the AIM3± mice appeared at a similar ratio to that of wild-type littermates (see Table 1). This indicates that about 50% of the AIM3± mice were dead in the pre-natal stage. AIM3 homozygous mice (hereinafter, referred to as “AIM3−/− mice”) would die during the early embryonic stage (see Tables 1 and 2). This suggests that AIM3 performs an important role in vivo. Particularly, considering that the genetic eradication of proteins, such as Rad51, Chk1/2 and ATR involved in the DNA-damaging response and repair system, causes early embryonic lethality (de Klein et al., Curr. Biol., 10:479-482, 2000; Lim and Hasty, Mol. Cell Biol., 14:7133-7143, 1996; Takai et al., Genes Dev., 14:1439-1447, 2000), it was believed that AIM3 would be involved in the DNA-damaging response and repair system.
- Since the AIM3 protein is related to a multi-tRNA synthetase complex involved in protein synthesis (Han et al., Biochem. Biophys. Res. Commun., 303:985-993, 2003), the inventors expected that a reduction of the AIM3 level would have an effect on the overall body growth of mice. However, it was interestingly shown that the growth rate of the AIM3± mice was similar to or slightly higher than that of wild-type mice regardless of their sex (data not shown). This suggests that protein synthesis is not inhibited by a reduction of the AIM3 level.
- In another embodiment of the present invention, we examined the histological characteristics of tissues and organs isolated from the AIM3± mice in order to identify the function of the AIM3 gene. The results showed that various tumors were found in the tissues and organs isolated from the AIM3± mice, and the incidence of the tumors was significantly increased when their age passed over 15 months (see
FIGS. 2 a to 2 c and Table 3). Particularly, in the AIM3± mice, lymphomas have developed at a high frequency (see Table 3). This is consistent with the previous report that the loss of DNA repair functions can evoke lymphomas (Bassing et al., Cell, 114:359-370, 2003; Celeste et al., Cell, 114:371-383, 2003). From the result that various tumors spontaneously formed in the AIM3-deficient mice, it is suggested that AIM3 is a powerful tumor suppressor involved in the tumorigenic pathway. - A rapid cell cycle is a typical indication for tumorigenesis (Evan and Vousden, Nature, 411:342-348, 2001). Thus, in another embodiment of the present invention, we examined whether AIM3 is involved in cell cycle control. As a result, cells isolated from the AIM3-deficient mice increased faster than those of wild-type mice and showed faster cell cycle (see
FIGS. 3 a and 3 b). Furthermore, the expression of AIM3 in cell cycles was examined by Western blot analysis and flow cytometry, and the results showed that AIM3 was significantly induced during DNA synthetic phase (seeFIGS. 3 c and 3 d). To understand the functional reason for the AIM3 induction during DNA synthetic phase, the cellular localization of AIM3 in growth arrest state and proliferation condition was examined. According to the results, AIM3 was detected mainly in cytoplasm when the cell growth was suppressed by serum starvation. However, it was detected in nucleus when the cells resumed growth (seeFIG. 3 e). This indicates that, during DNA synthesis, AIM3 is not only induced but also translocated into nuclei. Such results suggest that AIM3 can perform novel functions within the nuclei. - Cell responses to DNA damage include cell cycle arrest, apoptosis, and direct activation of DNA repair networks (Zhou B B et al., Cancer Biol. Ther., S (4 Suppl 1):S16-22, 2003). Also, the resistance to apoptosis, one of cell responses, is a typical indication for tumorigenesis (Evan and Vousden, Nature, 411:342-348, 2001). Thus, in order to examine whether AIM3 is involved in apoptosis regulation, the response of AIM3± mouse-derived cells was examined using adriamycin that induces DNA damage. The AIM3± mouse-derived cells show the resistance to apoptosis (see
FIG. 4 a). Moreover, the growth of wild-type mouse cells was completely arrested by adriamycin, whereas that of the AIM3± mouse-derived cells was slightly inhibited (seeFIG. 4 b). A change in the AIM3 level caused by DNA damage was examined and the results showed that the expression of AIM3 was induced at both transcriptional and translational levels by treatment with a DNA-damaging agent such as adriamycin (seeFIG. 4 c). In addition, the cellular localization of AIM3 caused by DNA damage was examined. The results showed that nuclear foci formed by AIM3 were remarkably increased in UV-irradiated cells (seeFIG. 4 d). All of these results suggest that AIM3 is involved in the responses to DNA damage induced by genotoxic stress, and it is translocated into the nuclei when DNA is damaged. - In still another embodiment of the present invention, it was examined whether AIM3 is involved in cell proliferation. The results showed that cells and tissues derived from the AIM3-deficient mice had a cell proliferation rate higher than those of wild-type mouse cells (see
FIGS. 5 a and 5 b). In addition, the level of proliferation of cells transfected with the AIM3 gene was lower than that of wild-type mouse cells (seeFIG. 5 c). This suggests that AIM3 shows the anti-proliferation activity against tumor cells. - According to the above results indicating that AIM3 is induced at a high level during DNA synthetic phase and when DNA get damaged, and has anti-proliferation activity similar to other DNA repair proteins (Falck et al., Nature, 410:842-847, 2001; Lim et al., Mol. Cell, 7:683-694, 2000), it can be found that AIM3 is functionally involved in signal transduction pathways that respond to the repair of DNA damage caused by the DNA replication or stress.
- Meanwhile, it is known that p53, a tumor suppressor gene, does functions of not only inhibiting the abnormal division and proliferation of cells but also arresting the cell cycle in the case of cellular DNA damage so as to repair the damaged DNA, and p53 is involved in cell proliferation and apoptosis to prevent DNA from being unlimitedly amplified (Levine, Cell, 88:323-331, 1997; Vousden, Cell, 103:691-694, 2000). Thus, the inventors examined the functional connection between AIM3 and p53. The results showed that, in cells transfected with the AIM3 gene, the levels of not only p53 but also its target gene, p21, were increased (see
FIGS. 6 a and 6 b). This increase in the gene level was further increased by treatment with adriamycin that induces apoptosis (seeFIG. 6 c). The level of proliferation of the AIM3 gene-transfected cells was lower than that of wild-type cells, and anti-proliferation activity of AIM3 was abolished in p53- or p21-deficient cancer cells (seeFIG. 6 d). The induction of p53 by UV or adriamycin was blocked when AIM3 was suppressed (seeFIG. 6 e). This indicates that AIM3 upregulates the expression of p53 induced by DNA damage and its target gene p21, thus inhibiting the proliferation of cancer cells. - It is known that mammalian ATM and ATR playing a key role in cell cycle checkpoints initiated by DNA damage are serine-threonine kinases which are involved in DNA repair processes responding to other genotoxic stresses (Yang et al., Carcinogenesis, 24: 1571-1580, 2003). Furthemore, ATM and ATR not only activate directly p53 in response to DNA damage but also regulate the cell cycle via p53 (Abraham, Genes Dev., 15:2177-2196, 2001). Thus, in order to examine whether AIM3 regulates p53 via ATM/ATR, the present inventors examined whether ATM/ATR inhibitors inhibit the activity of AIM3. The results indicated that the anti-proliferation activity of AIM3, the apoptosis induced by AIM3, and the AIM3-dependent expression of p53, were all inhibited by caffeine, inhibitor of ATM/ATR (see
FIGS. 7 a to 7 c). Moreover, the AIM3-dependent expression of p53 was also blocked by the expression of the kinase-dead domain of ATM (KD-ATM) that inhibits specifically the activity of ATM (seeFIG. 7 d). These results suggest that AIM3 acts through ATM/ATR. - In order to examine the relation between AIM3 and ATM/ATR in more detail, the present inventors analyzed the interaction between AIM3 and ATM/ATR.
- The results indicated that the interaction between the AIM3 and ATM/ATR was enhanced by stresses, such as the exposure to UV, adriamycin treatments, etc., and the interaction was done by the specific binding of AIM3 to the FAT domain of ATM/ATR (
FIGS. 8 a to 8 c). - Then, the present inventors examined whether the activity of ATM/ATR is enhanced by the association with AIM3. The results showed that the phosphorylation level of H2AX in the AIM3± mouse-derived cells, which is a substrate for ATM/ATR, was significantly lower than that of H2AX in wild-type mouse cells (see
FIG. 9 a). Furthermore, the phosphorylation of H2AX was blocked by the expression of antisense-AIM3 (As-p18) (seeFIG. 9 b). In addition, the inventors found that AIM3 increased the phosphorylation of ATM and its target proteins (p53 and chk2) through various tests (FIG. 9 c and data not shown). These results suggest that AIM3 interacts directly with ATM/ATR to activate not only ATM/ATR but also ATM/ATR-regulated proteins. - Finally, in order to examine the functional association between AIM3 and ATM/ATR-mediated diseases, the present inventors examined the expression level of AIM3 in various cancer cell lines. The results indicated that the expression level of AIM3 was reduced in some cancer cell lines (see
FIG. 10 a). To have a clue to the possible cause for the results, the present inventors compared the DNA content for AIM3 gene using genomic PCR analysis. As a result, it was confirmed that some cancer cell lines appeared to contain less amount of DNA than other cells. This indicates that the cell lines have loss of one allele for AIM3 (seeFIG. 10 b). Furthermore, we examined the expression level of AIM3 in tissues isolated from 9 leukemia patients and, as a result, found that AIM3 was expressed at a low level in the tissues of three patients. In this case, the expression of p21, a p53 target gene, was also strongly suppressed (seeFIG. 10 c). The level of AIM3 in normal tissues and cancer tissues isolated from liver cancer patients was analyzed by RT-PCR. As a result, it was confirmed that the level of AIM3 in the cancer tissues was cancer-specifically reduced (seeFIG. 10 d). These results suggest that the low level expression of AIM3 is association with various cancer cell lines and the tissues of cancer patients at high frequency. - As described above, it was first found in the present invention that AIM3 is a tumor suppressor gene and particularly, a haploinsufficient tumor suppressor gene acting in signal transduction pathways including ATM/ATR and p53.
-
FIG. 1 a is a schematic representation of a gene trap vector inserted into an AIM3 gene. -
FIG. 1 b shows the results of genomic PCR analysis to determine the insertion of a gene trap vector. - M: molecular weight marker
- +/+: wild-type mice
- ±: AIM3 heterozygous mice
-
FIG. 1 c shows the results of Southern blot analysis to determine the insertion of a gene trap vector. - +/+: wild-type mice
- ±: AIM3 heterozygous mice
-
FIG. 1 d shows the results of Western blot analysis to determine the expression level of AIM3 in various organs of wild-type mice (+/+) and AIM3 heterozygous mice (±). -
FIG. 2 a shows the results of immunohistochemical staining of various tissues and organs isolated from AIM3 heterozygous mice. -
FIG. 2 b illustrates the results using an anti-B220 monoclonal antibody, which shows that lymphoma cells metastasized into liver and lung. -
FIG. 2 c shows the results of analysis of the incidence of tumors at different ages (months) in wild-type mice (+/+) and AIM3 heterozygous mice (±). - White bar: the numbers of autopsied wild-type mice (+/+)
- Gray bar: the numbers of autopsied AIM3 heterozygous mice (±)
- Black section: the numbers of mice with tumors (tumor +)
-
FIG. 3 a shows the results of cell counting to measure the proliferation rate of the splenocytes and thymocytes isolated from wild-type mice (+/+) and AIM3 heterozygous (±). -
FIG. 3 b shows the results of analysis of the cell cycle of splenocytes isolated from wild-type mice (+/+) and AIM3 heterozygous mice (±). -
FIG. 3 c shows the results of Western blot analysis to determine the expression level of AIM3 in each phase of the cell cycle. -
FIG. 3 d shows the results of FACS analysis to determine the expression level of AIM3 at different cell cycle. - Left panel: DNA content(Y-axis) and the expression of AIM3 (X-axis) are analyzed by FACS and the density of cell is illustrated in contour lines. The “S” portion represents cells in the DNA synthetic phase on the basis of DNA content, and the “G1” portion represents cells in the G1/G0 phase.
- Right panel: the expression level of AIM3 in “G1” and “S” portion respectively in the left panel is shown in histograms. The X-axis represents the expression level of AIM3, and the Y-axis represents cell number.
-
FIG. 3 e shows the results of observation of the cellular localization of AIM3 at different proliferation conditions of cells. - SF: cell culture in serum-free media
- CM: cell culture in complete media
-
FIG. 4 a shows the results of flow cytometry to examine the apoptotic responses of splenocytes of AIM3 heterozygous mice (±) to adriamycin treatment(Adr), as compared to those of wild-type mice (+/+). - M1: annexin V-FITC positive populations
FIG. 4b shows the results of flow cytometry to examine the response of wild-type mice- and AIM3 heterozygous mice (±)-derived cells to adriamycin treatment, which caused cell growth arrest. -
FIG. 4 c shows the results of RT-PCR analysis and Western blot analysis to examine changes in the expression level of AIM3 by treatment with adriamycin (Adr), at different time. - Bars represent the population of G1/G0 phase cells and numbers represent the percentage of G1/G0 phase cells
-
FIG. 4 d shows the results of immunofluorescent staining to observe the cellular localization of AIM upon exposure to UV. -
FIG. 5 a illustrates the results of thymidine incorporation to measure the cell proliferation rate of mouse embryonic fibroblasts (MEFs) isolated from wild-type mice (+/+) and AIM3 heterozygous mice (±). -
FIG. 5 b illustrates the results of immunofluorescent staining using an anti-Ki67 antibody (green color), to measure the cell proliferation rate of various tissues isolated from wild-type mice (+/+) and AIM3 heterozygous mice (±). -
FIG. 5 c illustrates the results of thymidine incorporation to measure the proliferation rate of cells transfected with an AIM3 gene. - EV: HCT116 cells transfected with an empty vector containing no AIM3 gene
- AIM3: HCT 116 cells transfected with an AIM3 expression vector
-
FIG. 6 a shows the results of Western blot analysis to examine the effect of AIM3 on p53 expression in mouse embryonic fibroblasts (MEFs) derived from AIM3 heterozygous mice (±) and HCT116 cells transfected with an AIM3 expression vector. - +/+: wild-type mice
- ±: AIM3 heterozygous mice
- EV: HCT116 cells transfected with an empty vector containing no AIM3 gene
- AIM3: HCT116 cells transfected with an AIM3 expression vector
-
FIG. 6 b shows the results of RT-PCR to examine the effect of AIM3 on the p53-dependent transcription of p21. - −: HCT116 cells transfected with an AIM3 expression vector
- +: HCT116 cells transfected with an empty vactor containing no AIM3 gene
-
FIG. 6 c shows the results of luciferase assay using a vector containing a p21 promoter-fused luciferase gene, to examine the effect of AIM3 on the p53-dependent transcription of p21. - EV: HCT116 cells transfected with an empty vector containing no AIM3 were not treated with anything
- EV+Adr: HCT116 cells transfected with an empty vector containing no AIM3 were treated with adriamycin
- AIM3: HCT116 cells transfected with an AIM3 expression vector were not treated with anything
- AIM3+Adr: HCT116 cells transfected with an AIM3 expression vector were treated with adriamycin
-
FIG. 6 d illustrates the effect of ATM3 on the proliferation of wild-type HCT116 cells (WT), p53 gene-null HCT116 cells (p53−/−) and p21 gene-null HCT116 cells (p21−/−). - EV: HCT116 cells transfected with an empty vector containing no AIM3 gene
- AIM3: HCT116 cells transfected with an AIM3 expression vector
-
FIG. 6 e shows the effect of AIM3 on p53 induction caused by exposure to UV and treatment with adriamycin (Adr). - EV: HCT116 cells transfected with an empty vector containing no antisense-AIM3 (As-AIM3)
- As-AIM3: HCT116 cells transfected with a vector containing antisense-AIM3
-
FIG. 7 a illustrates the effect of caffeine on the anti-proliferation activity of AIM3. - EV: HCT116 cells transfected with an empty vector containing no AIM3 gene
- AIM3: HCT116 cells transfected with an AIM3 expression vector
-
FIG. 7 b shows the effect of caffeine on AIM3-induced apoptosis. -
FIG. 7 c shows the results of Western blot analysis to examine the effect of AIM3 on the induction of p53, after treatment with caffeine, an ATM inhibitor. - −: HCT116 cells transfected with an empty vector containing no AIM3 gene
- +: HCT116 cells transfected with an AIM3 expression vector
-
FIG. 7 d shows the results of Western blot analysis to examine the effect of AIM3 on the induction of p53, after introducing a KD-ATM domain, a specific inhibitor of ATM activity, into cells. - −: HCT116 cells transfected with an empty vector containing no AIM3 gene
- +: HCT116 cells transfected with an AIM3 expression vector
-
FIG. 8 a shows the results of co-immunoprecipitation to determine the interaction between AIM3 and ATM, after treatment with UV and adriamycin. -
FIG. 8 b shows the results of in vitro pull-down assay to determine the direct interaction between AIM3 and ATM. -
FIG. 8 c shows the results of co-immunoprecipitation to determine the interaction between AIM3 and ATR, after exposure to UV. -
FIG. 9 a shows the results of Western blot analysis to measure the phosphorylation level of H2AX, a substrate of ATM, in splenocytes and thymocytes isolated from wild-type mice (+/+) and AIM3 heterozygous mice (±). - p-H2AX: phosphorylated H2AX
-
FIG. 9 b shows the results of Western blot analysis to examine the effect of AIM3 on the phosphorylation of H2AX, a substrate of ATM, using antisense-AIM3 (As-AIM3). - −: no treatment with
VP 16, an apoptosis-inducer - +: treatment with
VP 16, an apoptosis-inducer -
FIG. 9 c shows the results of Western blot analysis to examine the effect of AIM3 on the phosphorylation of ATM and its target proteins (p53 and Chk2). - p-ATM: phosphorylated ATM
- p-p53: phosphorylated p53
- p-Chk2: phosphorylated Chk2
- actin: loading control
-
FIG. 10 a shows the results of RT-PCR to measure the expression level of AIM3 in different human cancer cell lines. -
FIG. 10 b shows the results of genomic PCR to examine the DNA content for AIM3 gene in different human cancer cell lines. -
FIG. 10 c shows the results of Western blot analysis to measure the expression level of AIM3 and p21 in tissues isolated from 9 leukemia patients. - APML: acute promyelocytic leukemia
- CML: chronic myelocytic leukemia
-
FIG. 10 d shows the results of RT-PCR to measure the expression levels of AIM3 in normal tissues and cancer tissues isolated from 9 liver cancer patients. - N: normal tissues at tumor-adjacent sites
- T: liver cancer tissues
- Hereinafter, the present invention will be described in detail by the following examples. It is to be understood, however, that these examples are given for illustrative purpose only and are not construed to limit the scope of the present invention.
- Generation of AIM3 Gene-deficient Mutant Mice
- The present inventors generated AIM3-deficient mice by a gene trap method (Zambrowicz, B. P. et al., Nature, 392:608-611, 1998). Among the embryonic stem cell library of 129/SvEvBrd mouse in which the gene trap vector was randomly introduced (OmniBank Library, Lexicon Genetics), the OST377244 clone including AIM3 genes mutated by the integration of the gene trap vectors was found out. Using this clone, C57BL6/albino AIM3 heterozygous mice were generated following the standard protocol of Lexicon Genetics, Inc. The heterozygous mice were interbred to generate the homozygous offspring.
- Examination of Genotypic and Phenotypic Characteristics of AIM3 Gene-deficient Mice
- <2-1> Determination of Site of Gene Trap Vector Insertion in AIM3 Allele
- The site of a gene trap vector insertion in an AIM3 mutant allele was determined by sequencing analysis. Here, the sequencing was performed by Pangenomics, a sequencing company. As shown in
FIG. 1 a, the sequencing results indicated that the gene trap vector was inserted between exon I and exon II of the AIM3 gene. - <2-2> Genomic PCR Analysis
- From the tail of each of the mice generated in <Example 1>, genomic DNA was isolated. Then, about 1.5-kb DNA fragment containing the exon I region of the AIM3 gene was amplified by PCR with a primer pair of p18F-1 and p18R-1 (SEQ ID NO: 3 and SEQ ID NO: 4) (see
FIG. 1 a). In addition, about 0.8-kb DNA fragment containing a part of the AIM3 gene and a part of the gene trap vector was amplified by PCR with the p18F-1 primer and an LTR primer (SEQ ID NO: 5) binding to the gene trap vector (about 5.7 kb) integrated into the genome (seeFIG. 1 a). The PCR reaction consisted of the following: denaturation of template DNA at 94° C. for 5 min; and then, 30 cycles of 1 min at 94° C., 1 min at 54° C., and 2 min at 72° C. - Interestingly, all of the generated mutant mice were heterozygote (AIM3± mice) producing both of 1.5 and 0.8 kb DNA fragments (see
FIG. 1 b). On the other hand, in the case of wild-type mice (AIM3+/+ mice), only the 1.5-kb band could be found. - <2-3> Southern Blot Analysis
- From the tail of each mouse, genomic DNA was isolated and digested with SacI, followed by gel electrophoresis to separate the digested DNA fragments. Then, a PCR product amplified with p18F-2 and p18R-2 primers shown in SEQ ID NO: 6 and SEQ ID NO: 7, which contains the exon II region of the AIM3 gene, was labeled with a radioactive isotope (see
FIG. 1 ), and the labeled probe was hybridized with the digested DNA fragments (southern, E. M., J. Mol. Biol., 98:503, 1975). - As shown in
FIG. 1 c, a band of about 12 kb was detected in wild-type mice but additional band of about 3 kb was detected in the heterozygous mice. - <2-4> Determination of Induction of Embryonic Lethality Caused By AIM3 Gene Deletion
- In the analysis in Examples <2-2> and <2-3>, offspring with a homozygous genotype could not be found. Thus, in order to examine whether the deficiency of the AIM3 gene induces embryonic lethality, the genotype of post-natal mice and the genotype of embryos on different time after fertilization were examined by genomic PCR according to the same method as in Example <2-2>. The results are shown in Tables 1 and 2 below.
- As shown in Table 1, among a total of 262 survival mice, 114 mice were wild type (+/+) and 148 mice were heterozygous (±). None of surviving mice was homozygous (−/−). Particularly, the heterozygous mice were born at a similar ratio with the wild-type littermates, indicating that about 50% of the heterozygous mice would die during the pre-natal stage. As shown in Table 2, among total of 38 embryos isolated at 7.5-9.5 days after fertilization, only one embryo at 8.5 days containing the homozygous genotype was detected. This indicates that the AIM3 homozygous mice would be early embryonic lethal.
TABLE 1 Post-natal segregation ratio of genotype from the offspring generated by the intercrosses between the C57BL6 AIM3 heterozygous mice Total +/+ +/− −/− Number of surving mice 262 114 148 0 % 100 43.5 56.5 0 -
TABLE 2 Embryonic segregation ratio of genotype from the offspring generated by the intercrosses between the C57BL6 AIM3 heterozygous mice Day of Gestation Total +/+ +/− −/− Resorbed 7.5 days 28 7 15 0 6 8.5 days 34 11 16 1 6 9.5 days 21 8 8 0 5 Total 83 26 39 1 17 % 31.3 47.0 1.2 20.5 - The results suggest that loss of AIM genes leads to embryonic lethality.
- <2-5> Western Blot Analysis
- According to the method described in Ziak, M, et al. (Ziak, M, et al., Biochem. Biophys. Res. Commun. 280:363-367, 2001), proteins were isolated from various organs, such as small intestines, kidneys, heart and spleen. Then, according to the method described in Park S. G., et al. (Park S. G., et al., J. Biological Chemistry 274:16673-16676, 1999), Western blot analysis was performed using a polyclonal rabbit anti-AIM3 antibody. The anti-AIM3 antibody was prepared according to the method described in Kim, T. et al. aim, T. et al., J. Biol. Chem., 275:21768-21772, 2000).
- As shown in
FIG. 1 d, although the degree of reduction varied depending on the organs, the expression level of AIM3 in the organs of the AIM3± mice was significantly lower than that in the organs of wild-type mice. - Examination of Histological Characteristics of AIM3± Mice
- In order to determine the functions of the AIM3 gene, the present inventors isolated tissues and organs from the AIM3± mice and analyzed the histological characteristics of the isolated tissues and organs.
- At first, after sacrifing mice at given time intervals, various tissues were isolated and fixed with 10% formalin. The fixed tissues were embedded in paraffin, followed by subjecting into H&E staining. In order to determine B cell metastasis, immunohistochemical staining for surface marker B220 was performed with paraffin slide. After de-paraffin using xylene, the slide was incubated in a blocking buffer (1:100, 5% BSA and 0.1
% Tween 20/PBS) containing an anti-B220 antibody (Santacruz Biotech.) for 2 hours. After the slide was washed with PBS, the tissues fixed to the slide were incubated again with an avidin-conjugated secondary antibody and DAB solution. - As a result, various tumors were found in the AIM3± mice (see Table 3 and
FIG. 2 a). Interestingly, among 18 tumor-developing AIM3± mice, 14 mice contained lymphoma which originated from the spleen or lymph node, and 5 mice had complex tumors. Specifically, adenocarcinoma was found in the breasts of 15-month-old AIM3± mice (B-63) and 23-month-old AIM3± mice (B-95), adenocarcinoma in the seminal vesicles of 19-month-old AIM3± mice (B-103), and hepatocarcinoma and sarcoma of unknown origin in 22-month-old AIM3± mice (B-207). All of these cancers showed the typical malignant phenotypes, such as anaplasia and invasiveness. Furthermore, lymphoma was found in the lymph nodes of 22-month-old AIM3± mice (B-232) and well-differentiated carcinoma which originated from the bronchiole epithelium was observed in 17-month-old AIM3± mice (B-14). - It was found that some of lymphomas metastasized into other organs, such as the liver, kidneys, lungs and salivary glands (see
FIG. 2 b). The incidence of these tumors was remarkably increased after 15 month-old (seeFIG. 2 c and Table 3).TABLE 3 Tumors found in AIM3+/− mice Mouse Age ID Tumor locus Metastasis (month) Single B-2 Liver, dysplasia −* 8 solid B-268 Liver − 23 tumor B-63 Breast − 15 (adenocarcinoma) B-233 Seminal vesicle − 21 (adenocarcinoma) Lymphoma B-191 Spleen Salivary gland, 25 kidneys, lungs B-262 Spleen Lungs 20 B-275 Mesenteric lymph Liver, spleen 17 node B-148 Spleen − 24 B-264 Spleen Salivary gland 15 B-143 Cervical lymph node Liver, lungs 25 B-226 Spleen Lungs 22 B-261 Spleen Lungs 20 B-321 Spleen Liver 1 Multiple B-103 Seminal vesicle Spleen, kidneys 19 tumor (adenocarcinoma), metastatic lymphoma B-14 Lung adenocarcinoma, Salivary gland 17 metastatic lymphoma B-95 Breast Salivary gland, 23 adenocarcinoma spleen (solid tumor), metastatic lymphoma B-232 Lung adenocarcinoma − 22 (solid tumor), lymphoma (lymph node) B-207 Metastatic sarcoma Spleen, liver, 22 (liver), hepatic lungs, salivary carconoma (liver), gland metastatic lymphoma
*negative
- As shown in the above results, all of various tumors spontaneously formed in the AIM3-deficient heterozygous mice led us to suspect that AIM3 is a strong tumor suppressor involved in general tumorigenic mechanisms.
- Identification of Relation Between Cell Cycle and AIM3
- A rapid cell cycle is a typical indicatin for tumorigenesis (Evan and Vousden, Nature, 411:342-348, 2001). Accordingly, it was addressed whether AIM3 could play a role in cell cycle control.
- <4-1> Examination of Change of Cell Cycle in AIM3± Mouse-derived Cells
- First, the present inventors examined the cell proliferation rate of AIM3± mouse-derived cells, compared to that of wild-type mouse cells. For this purpose, from 4-week-old wild-type mice and AIM3± mice, the splenocytes and thymocytes were isolated, and the number of cell according to culture time was counted. As shown in
FIG. 3 a, the results showed that the AIM3± mice-derived cells proliferated faster than wild type mice cells. - Then, in order to examine the cell cycle of the AIM3± mice-derived cells, FACS analysis was performed. The splenocytes isolated from 4-week-old wild-type mice and AIM3± mice were incubated overnight. The incubated cells were fixed with 1% PFA (paraformaldehyde) and stained with PI (propidium iodide). FACS analysis was conducted on 20,000 cells per sample. As shown in
FIG. 3 b, the splenocytes isolated from the AIM3± mice showed faster cell cycle than the wild-type mice cells. - <4-2> Examination of Change in Expression Level of AIM3 with Change in Cell Cycle
- In order to determine the functions of AIM3 during the cell cycle, it was examined whether AIM3 is expressed depending on the cell cycle. HCT116 cells incubated in a serum-free medium for 24 hours and then incubated them again in a serum-containing medium to synchronize cell cycle. The expression level of AIM3 of the synchronized cells in different time under serum-deprivation and serum-re-fed conditions was measured by Western blot analysis. As a result, the AIM3 was remarkably induced during the DNA synthetic phase (see
FIG. 3 c). - In order to confirm this fact further, the present inventors performed FACS analysis. HCT116 cells (Human colon adenocarcinoma cell line) were fixed with 1% PFA and neutralized, and were cultured with an anti-AIM3 monoclonal antibody. Then, the cells were cultured with a FITC-conjugated anti-mouse goat IgG antibody (Pierce). And then, the cells were co-stained with PI, followed by FACS analysis. As a result, AIM3 was remarkably induced in the DNA synthetic phase (see
FIG. 3 d). This coincides with the result of Western blot analysis. All of these results indicate that AIM3 is induced in the DNA synthetic phase. - <4-3> Examination of Cellular Localization of AIM3 Caused By Cell Proliferation
- In order to understand the functional reason of AIM3 induction during the DNA synthetic phase, the present inventors investigated the cellular localization of AIM3 in cell growth arrest and cell proliferation conditions. For this purpose, DU145 cells (prostate cancer cell line) were cultured in each of a 10% serum-containing RPMI-1640 medium (complete media (CM)) and a serum-free media (SF), fixed with 100% Me-OH and reacted with an anti-AIM3 monoclonal antibody. Then, they were reacted with anti-mouse goat IgG-FITC (Pierce), and stained with PI. The cellular localization of AIM3 was examined under a fluorescence microscope.
- As shown in
FIG. 3 e, when the cell growth was suppressed by serum starvation, AIM3 was mainly located in cytoplasm, whereas, when the cell growth was resumed, AIM3 was located in nuclei. Given thus, it could be found that, during the DNA synthetic phase of the cell cycle, AIM3 was not only induced but also translocated into nuclei. These results suggest that AIM3 could have novel functions in the nuclei. - Determination of Relation Between DNA Damage and AIM3
- The damage of DNA by stresses and so on generally induces apoptosis and cell cycle arrest (Zhou B B et al., Cancer Biol. Ther., S(4 Suppl 1):S16-22, 2003). Thus, the present inventors investigated the role of AIM3 in the response of cells to the stress-induced apoptosis and cell growth arrest.
- <5-1> Examination of Effect of AIM3 Gene Deletion on Apoptosis Regulation
- Using adriamycin that induces DNA damage, the response of AIM3± mouse-derived splenocytes to pro-apoptotic stress was examined.
- First, the splenocytes were isolated from wild-type mice and AIM3± mice. To induce apoptosis, the isolated splenocytes were treated with 0.2 μg/ml of adriamycin (Adr, Sigma) for 2 hours. Then, the cells were cultured with FITC-conjugated annexin V (Roche) for 5 minutes. And then, the cells were washed with PBS and subjected to FACS analysis under a FL-1H detector. In this analysis, 20,000 cells per sample were used.
- As shown in
FIG. 4 a, apoptotic cells were significantly increased by treatment with adriamycin in the wild-type cells, however the AIM3± cells showed the resistance to apoptosis induced by adriamycin. This indicates that AIM3 is required for sensitivity of cell to apoptosis induced by DNA damage. From this, it can be found that AIM3 promotes apoptosis caused by DNA damage. - <5-2> Examination of Change in Cell Growth Caused By Apoptosis-inducer
- In order to examine the importance of AIM3 in cell growth arrest caused by adriamycin, flow cytometry was performed. First, the thymocytes were isolated from wild-type mice and AIM3± mice, and then treated with 0.2 μg/ml of adriamycin (Adr, Sigma) for 6 hours. Next, the cells were subjected to FACS analysis in the same method as in Example <5-1>. As shown in
FIG. 4 b, the growth of AIM3± mouse cells was slightly suppressed by treatment with adriamycin, whereas that of wild-type mouse cells was arrested. - <5-3> Examination of Change in AIM3 Level Caused By Apoptosis-inducer
- It was examined by RT-PCR analysis and Western blot analysis whether the level of AIM3 is affected by treatment with adriamycin.
- For this purpose, HCT116 cells were treated with 0.2 μg/ml of adriamycin. Then, the cells were collected at different time and dissolved in Sol D solution (4 M guanidine thiocyanate, 1% laurosarcosine, 25 mM sodium citrate, and 0.1% b-mercaptoethanol). The cell extracts were incubated in acidic phenol and chloroform containing 4% isoamylalcohol, and vortexed. The mixture was centrifuged at 14,000 rpm. The upper layer was collected and added with isopropanol so as to precipitate RNA. The precipitated RNA was washed with 100% ethanol, and 1 μg of RNA was dissolved in distilled water and used as a template for RT-PCR. Then, RT-PCR was performed with primers shown in SEQ ID NO: 8 and SEQ ID NO: 9. The expression level of GADPH was also measured in order to quantitatively compare that of AIM3.
- Meanwhile, for Western blot analysis, cells treated with adriamycin were dissolved in RIPA containing protease cocktail. The solution was centrifuged at 14,000 rpm for 30 minutes. 20 μg of the extracted proteins were separated by SDS-PAGE. Then, according to the method described in Park S. G., et al. (Park S. G., et al., J. Biol. Chem., 274:16673-16676, 1999), Western blot analysis was performed using a polyclonal rabbit anti-AIM3 antibody. The expression level of tubulin was also measured in order to quantitatively compare that of AIM3.
- As shown in
FIG. 4 c, both the transcription and translation of AIM3 were induced in response to adriamycin. Moreover, the induction of AIM3 was also observed by other DNA-damaging agents, such as UV, actinomycin D (Act.D) and cisplatin (CDPP) (data not shown). Particularly, AIM3 was induced within 5-10 minutes after exposure to UV or adriamycin (data not shown). These results indicate that AIM3 is functionally involved in signal transduction pathways which respond to DNA repair caused by DNA replication or DNA damage. - <5-4> Cellular Localization of AIM3 Upon DNA Damage
- The cellular localization of AIM3 upon DNA damage was examined using U2OS cells containing large nuclei. The U2OS cells (osteosarcoma cell line) were treated with 254-nM wavelength UV-C (UV cross linker) at 50 J/m2. The cells were cultured in a complete medium for 30 minutes and collected. Then, the same method as in Example <4-3> was performed so as to examine the cellular localization of AIM3 by immunofluorescent staining. As a result, as shown in
FIG. 4 d, the UV-irradiated cells showed a remarkable increase in nuclear foci formed by AIM3. - All of these results indicate that AIM3 is involved in responses to DNA damage induced by genotoxic stress.
- Identification of Relation Between Cell Proliferation and AIM3
- The present inventors found in
- <6-1> Examination of Change in Cell Proliferation Caused By Deletion of AIM3 Gene
- a) Thymidine Incorporation
- Mouse embryonic fibroblasts (MEFs, E14.5d) isolated from wild-type mice and AIM3± mice were cultured in a medium containing 1 μCi/ml [3H] thymine. The cultured cells were washed with cold PBS and incubated in 10% TC A solution for 30 minutes so as to precipitate nucleic acids. Then, the cells were dissolved in 0.1 N NaOH, and the amount of radioactive thymidine incorporated in the precipitate was quantified by a liquid scintillation counter. The experiments were repeated three times and the data were averaged.
- As a result, as shown in
FIG. 5 a, MEFs isolated from the AIM3± mice had a higher proliferation rate than the wild-type MEFs. - b) In Situ Immunofluorescence Staining
- From AIM3± mice, the intestines, testes, spleens and thymuses were isolated. Then, to examine the cell proliferation rate of the isolated tissues, in situ immunofluorescence staining was performed using Ki-67, cell proliferation marker (Gerdes J. et al., J. Immunol., 133:1710-1715, 1984).
- As a result, as shown in
FIG. 5 b, the proliferation of cells in the AIM3± mouse-derived tissues was higher than that in the wild-type mouse-derived tissues. - <6-2> Examination of Change in Cell Proliferation Rate With Increase in AIM3 Expression
- The present inventors found in Example <6-1> that a reduction in the expression of AIM3 resulted in an increase in cell proliferation. Thus, it was examined whether an increase in the expression of AIM3 results in the suppression of cell proliferation.
- The AIM3 gene (SEQ ID NO: 2) was inserted into a pcDNA3 (Invitrogen) vector so as to prepare an AIM3 expression vector. Then, the expression vector was transfected into HCT116 cells (human colon adenocarcinoma cell line). The cell proliferation rate of the transfected cells was examined in the same method as in the part a) of Example <6-1>. As a control group, HCT116 cells transfected with pcDNA3 vector containing no AIM3 gene (empty vector; EV) were also used.
- As a result, as shown in
FIG. 5 c, proliferation of cells was reduced in the cells introduced with the AIM3 gene. This suggests that AIM3 shows anti-proliferation activity against tumor cells. - Identification of Function of AIM3 as Upregulator of p53
- Tumor suppressor protein p53 plays a major role in regulation of DNA damage-induced cell cycle arrest and apoptosis (Levine, Cell, 88:323-331, 1997; Vousden, Cell, 103:691-694, 2000). Thus, the functional relation between AIM3 and p53 was examined.
- <7-1> Measurement of p53 Level Caused By AIM3
- The expression levels of p53 and AIM3 in mouse embryonic fibroblasts (MEFs) isolated from AIM3± mice and wild-type mice were measured with Western blot analysis according to the same method as in Example <5-3>. Also, the expression levels of AIM3 and p53 in the transfected HCT116 were measured with Western blot analysis after the AIM3 expression vector prepared in Example <6-2> was transfected into HCT116 cells.
- As a result, as shown in
FIG. 6 a, the expression level of p53 in the MEFs of the AIM3± mice was lower than that in the MEFs of the wild-type mice. Meanwhile, the level of p53 in the HCT116 cells transfeted with the AIM3 gene was increased as compared to that in a control group cells transfected with an empty vector containing no AIM3 gene. This indicates that the ectopic expression of AIM3 elevates the expression of p53. - <7-2> Measurement of p21 Level Caused by AIM3
- In order to determine whether the increase of AIM3 would enhance the p53-dependent transcription, the AIM3-dependent transcription of p21 known as a target gene of p53 was examined.
- The HCT116 cells transfected with the AIM3 gene (1 μg/ml) in Example <7-1> were cultured for 24 hours. Then, RT-PCR analysis was performed in the same method as in Example <5-3>. As a result, as shown in
FIG. 6 b, the expression of p21 in the HCT116 cells transfected with the AIM3 gene was enhanced. - <7-3> Measurement of p21 Level Caused by AIM3 and Adriamycin
- Thereafter, in order to examine the effect of AIM3 and/or adriamycin on the transcription of p21, luciferase assay was performed using a vector containing a p21 promoter fused to luciferase gene.
- HCT116 cells were co-transfected with a pGL-3 vector (Promega) engineered that the luciferase gene would be expressed under p21 promoter, and a recombinant AIM3 expression vector(1.2 μg/ml) containing the AIM3 gene. Also, control group cells were co-transfected with the pGL-3 vector and an empty vector containing no AIM3 gene. Then, the transfected cells of each group were treated with 0.2 μg/ml of adriamycin for 2 hours. After cells were lyzed, the cell extract were incubated with substrate of luciferase for 30 minutes at room temperature. 5 μl of each sample was transferred to luminometer plate and luciferase activity was measured following the manufacturer's protocol (Promega).
- As a result, as shown in
FIG. 6 c, the luciferase activity regulated by the p21 promoter was highly increased by transfection with AIM3 and the luciferase activity was further increased by the additional treatment with adriamycin. - <7-4> Identification of Relation Between Anti-proliferation Activity of AIM3 and p53 and p21
- Examples <7-1> and <7-2> demonstrated that the expressions of p53 and p21 depend on AIM3. Thus, it was examined whether AIM3 suppresses the proliferation of tumor cells via p53 and p21.
- The AIM3 expression vector or empty vector (2 □/□) prepared in Example <6-2> was transfected into each of HCT116 cells (human colon adenocarcinoma cell line), p53-null HCT116 cells and p21-null HCT116 cells. Then, the proliferation rate of each of the transfected cells was examined according to the same method as in the part a) of Example <6-1>.
- As a result, as shown in
FIG. 6 d, the anti-proliferation activity of AIM3 was abolished by the absence of functional p53 and p21. This indicates that AIM3 suppresses the proliferation of tumor cells via p53 and p21. - <7-5> Measurement of Reduction in p53 Level Caused By Inhibition of AIM3 Expression
- The present inventors inhibited the expression of AIM3 by the use of antisense-AIM3 (As-AIM3) and then examined if the induction of p53 is influenced by the inhibition of the AIM3 expression.
- First, using primers shown in SEQ ID NO: 10 and SEQ ID NO: 11, the N-terminal 176-bp region of the ATG-containing AIM3 gene was amplified by PCR. The PCR product was inserted into a pcDNA 3.1 vector in reverse orientation. 2 μg/ml of a vector containing antisense-AIM3 was transfected into HCT116 cells. The transfected cells were cultured for 24 hours. Then, the cells were treated with UV and 0.2 μg/ml of adriamycin, respectively. Next, using an anti-AIM3 antibody or an anti-p53 antibody (Santacruz), Western blot analysis was performed in the same method as in Example <2-5>. At this time, the expression level of actin was also measured in order to quantitatively compare the expression level of AIM3 and p53.
- As a result, as shown in
FIG. 6 e, the level of p53 was increased by treatment with UV or adriamycin, whereas the suppression of AIM3 by As-AIM3 inhibited the induction of p53. This indicates that AIM3 is required for increasing the expression of p53. Moreover, the transcription of PUMA, an immediate early target gene of p53, was also increased by irradiation with WV, and its induction was blocked when AIM3 was suppressed by As-AIM3 (data not shown). - These results indicate that AIM3 is an important upregulator of p53 that mediates the induction of p53 caused by DNA damage.
- Determination of Mechanism of AIM3
- ATM/ATR are substances directly activating p53 in response to DNA damage (Canman et al., Science, 281:1677-1679, 1998; Banin S et al., Science, 11;281 (5383):1674-7, 1998). Thus, the present inventors examined whether AIM3 acts via ATM/ATR.
- <8-1> Analysis of Caffeine-induced Inhibition of Anti-proliferation Activity of AIM3
- In order to explore the possibility, that AIM3 can regulate p53 via ATM/ATR, the present inventors first checked the anti-proliferation activity of AIM3 in the presence of caffeine known as an inhibitor of ATM/ATR. HCT116 cells were transfected with each of the AIM3 expression vector and the empty vector(2 μg/ml, respectively) for 24 hours. Then, the cells were added with 20 mM caffeine and cultured for 4 hours. Control group cells were added with PBS. The cell proliferation rate of the cells of each group was examined according to the same method as in the part a) of Example <6-1>. As a result, as shown in
FIG. 7 a, the anti-proliferation activity of AIM3 was abolished by caffeine, an inhibitor of ATM/ATR. This demonstrates that AIM3 has anti-proliferation activity via ATM/ATR. - <8-2> Analysis of Caffeine-induced Inhibition of Apoptosis Induced by AIM3
- Thereafter, the present inventors checked whether AIM3-induced apoptosis is inhibited by caffeine, an inhibitor of ATM/ATR. HCT116 cells were transfected with each of the AIM3 expression vector or the empty vector(4 μg/ml, respectively) for 24 hours. Then, the cells were added with 20 mM caffeine and cultured for 12 hours. Control group cells were added with PBS. After staining the cells with PI, we checked apoptosis with measuring for the portion (%) of sub-G1 cells. As a result, as shown in
FIG. 7 b, apoptosis was induced by the expression of AIM3, and this effect was relieved by treatment with caffeine. - <8-3> Analysis of Caffeine-induced Inhibition of AIM3-dependent p53 Induction
- Thereafter, the present inventors examined whether the AIM3-induced expression of p53 is inhibited by caffeine. First, HCT116 cells were transfected with each of the AIM3 expression vector and the empty vector(2 μg/ml, respectively) for 24 hours. Then, the cells were added with 20 mM caffeine and cultured for 12 hours. Control group cells were added with PBS and cultured. Then, in order to examine the levels of AIM3 and p53, Western blot analysis was performed in the same method as in Example <7-5>. At this time, the expression level of actin was also measured in order to quantitatively compare the expression levels of AIM3 and p53.
- As a result, as shown in
FIG. 7 c, the AIM3-induced expression of p53 was suppressed by caffeine. Moreover, the expression of PUMA, a target gene of p53, was also induced by AIM3, however it was suppressed by caffeine (data not shown). These results indicate that ATM/ATR play an important role in the AIM3-dependent induction of p53. - <8-4> Analysis of KD-ATM-induced Inhibition of AIM3-dependent p53 Induction
- In order to more specifically determine that ATM/ATR play an important role in the AIM3-dependent induction of p53, HCT116 cells were transfected with the kinase-dead domain of ATM (KD-ATM) (Canman et al., Science, 281: 1677-1679, 1998). The KD-ATM suppresses specifically the activity of ATM.
- First, each of vectors containing the KD-ATM domain or wild-type ATM respectively (provided by Micheal Kastan, St. Jude Children's Hospital), was introduced into HCT116 cells with the AIM3 expression vector(2 μg/ml). Also, as a control group for the AIM3 expression, each of these vectors was introduced into HCT116 with the empty vector containing no AIM3 gene. Then, the expression levels of p53 and AIM3 in the cells of each group were examined by Western blot analysis according to the same method as in Example <6-5>. At this time, the expression level of actin was also measured in order to quantitatively compare the expression levels of AIM3 and p53.
- As a result, as shown in
FIG. 7 d, the p53 induction caused by an increase in the AIM3 expression was blocked by KD-ATM, whereas not by the wild-type ATM. These results further support that ATM is required for the AIM3-dependent induction of p53. - All of these results confirm that AIM3 has anti-proliferation activity, apoptosis-inducing activity and p53-upregulating activity, via ATM/ATR.
- Analysis of ATM/ATR Activation Caused By AIM3
- <9-1> Analysis of Interaction Between AIM3 and ATM
- a) Co-immunoprecipitation
- In order to examine the interaction between AIM3 and ATM, co-immunoprecipitation was performed. First, from HCT116 cells treated with each of UV and 0.2 μg/ml adriamycin, proteins were extracted at different times. The protein extracts were incubated with normal IgG and protein A/G-agarose for 2 hours and centrifuged to remove nonspecific IgG binding proteins. After centrifugation, the supernatant was taken, added with 2 μg of an anti-ATM antibody (Santacruz) and incubated at 4° C. for 2 hours with agitation. And then, protein A/G-agarose was added. After washing twice with cold PBS and once with PIRA, the precipitates were dissolved in an SDS-sample buffer, and separated by 6% SDS-PAGE. The proteins separated by the SDS-PAGE were transferred to a PVDF membrane, followed by reacted orderly with an anti-AIM3 single antibody and a horseradish peroxidase conjugated secondary antibody.
- As a result, as shown in
FIG. 8 a, the interaction between AIM3 and ATM was increased within 5 minutes in response to UV and adriamycin. The dissociation kinetics of AIM3 appeared to be much slower in adriamycin-treated cells possibly because adriamycin is present in the media throughout the cultivation while UV stress would affect the cells only temporarily. - b) In Vitro Pull Down Assay
- To examine the direct interaction between AIM3 and ATM, GST full-down assay was performed.
- First, AIM3 was expressed as GST fusion protein and purified according to the manufacturer's protocol (Pharmacia). Meanwhile, since it was difficult to synthesize the whole ATM due to its large size, the present inventors tested the interaction between the functional domain of ATM and AIM3. For this purpose, a fragment consisting of 612 amino acids, including the FAT domain of an ATM structure, was amplified by PCR with primers shown in SEQ ID NO: 12 and SEQ ID NO: 13. Also, a fragment (control group) consisting of 145 amino acids, including the C-terminal domain, was amplified by PCR with primers shown in SEQ ID NO: 14 and SEQ ID NO: 15. Then, the amplified PCR products were subdloned into pcDNA3.1 (Invitrogen), a vector suitable for in vitro transcription and transition. At this time, the protein was synthesized by in vitro translation in the presence of radioactive methionine. 10 μl of the synthesized TNT product was incubated with the GST- or GST-AIM3 fusion protein-immobilized glutathion-sepharose beads for 5 minutes. Then, the beads were washed six times with a binding buffer (PBS containing 0.2% sarcosine and 0.2% Triton X100), and dissolved in 10% SDS-PAGE. The binding of the GST-fused AIM3 to each domain was determined by autoradiography.
- As a result, as shown in
FIG. 8 b, the GST-fused AIM3 protein bound to the FAT domain, a functional domain, but not to the C-terminal domain of ATM. This suggests that AIM3 interacts directly with ATR. - <9-2> Analysis of Interaction Between AIM3 and ATR
- The FAT domain is found in not only ATM but also ATR (Abraham R, Genes Dev., 15:2177, 2001). Thus, the interaction between AIM3 and ATR was tested by co-immunoprecipitation.
- First, the 293 cell was transfected with an ATR vector (provided by Elledge S., Harvard University) containing flag-tagged ATR. The transfected 293 cell was treated with UV, from which proteins were extracted at different time. Next, the same method as the part a) of Example <9-1> was performed except that an anti-FLAG antibody (Sigma) was used in place of the anti-ATM antibody.
- As a result, as shown in
FIG. 8 c, AIM3 was co-immunoprecipitated with the flag-tagged ATR, and this interaction was further enhanced upon exposure to UV. This suggests that AIM3 also interacts with ATR as it acts on ATM. - Accordingly, it could be found from the above results that AIM3 interacts directly with ATR/ATM.
- <9-3> Analysis of ATM/ATR Activation by AIM3
- The present inventors examined whether the activity of ATM/ATR is enhanced by the association with AIM3.
- a) Measurement of Phosphorylation Level of H2AX in AIM3± Mouse-derived Cells
- The activity of ATM/ATR was examined using H2AX known as a substrate of ATM/ATR (Burma et al., J. Biol. Chem., 276: 42462-42467, 2001; Ward, I. M. et al., J. Biol. Chem., 276: 47759-47762, 2001; Irene M. Ward et al., J. Biol. Chem., 279(11):9677-9680, 2004).
- After isolating splenocytes and thymocytes from wild-type mice and AIM3± mice, the phosphorylation level of H2AX in the isolated cells was measured by Western blot analysis in the same method as in Example <7-5>. As a result, as shown in
FIG. 9 a, the phosphorylation of H2AX (p-H2AX) was significantly reduced in the AIM3± mice-derived cells. - b) Analysis of H2AX Phosphorylation Inhibition Caused By Antisense AIM3
- Thereafter, the present inventors treated cells with VP16, a DNA-damaging agent (Clarke et al., Nature, 362:849-852, 1993), and examined whether the phosphorylation of H2AX is inhibited by AIM3 inhibition in the presence of antisense-ATM3 (As-AIM3). Antisense AIM3-containing vector(2 μg/ml) prepared in Example <7-5> was introduced into HCT116 cells. The transfected cells were cultured for 24 hours and then treated with 100 μM of VP16 (Sigma), an apoptosis-inducing agent, for 4 hours. Next, using each of an anti-53 antibody, an anti-AIM3 antibody and an anti-p-H2AX antibody (Cell signaling), Western blot analysis was performed in the same method as in Example <7-5>. At this time, the expression level of actin was also measured in order to quantitatively compare that of each protein. As a result, as shown in
FIG. 9 b, the phosphorylation of H2AX was enhanced by treatment with VP16, but inhibited by the expression of antisense-AIM3. - c) Analysis of Effect of AIM3 on ATM Activation
- In order to analyze the effect of AIM3 on the autophosphorylation of ATM, the present inventors examined the phosphorylation of ATM and its target proteins, p53 and chk2, by Western blot analysis. Cells isolated from wild-type mice and AIM3± mice were treated with 0.2 μg/ml of adriamycin. Then, using each of an anti-phospho-serine antibody of ATM, an anti-p53 antibody and an anti-chk2 antibody, Western blot analysis was performed (Bakkenist and Kastan, Nature, 421:499-506, 2003). As a result, as shown in 9 c, the phosphorylation of ATM and its target proteins in the wild-type cells was enhanced by treatment with adriamycin, whereas that in the AIM3± cells was inhibited.
- All of these results indicate that AIM3 is required for the activation of ATM/ATR and its target proteins.
- Identification of Functional Relation Between Cancers and AIM3
- <10-1> Measurement of Expression Level of AIM3 in Human Cancer Cell Lines
- a) RT-PCR Analysis
- To identify the functional relation between human cancers and AIM3, the present inventors measured the level of AIM3 in various cancer cell lines shown in Table 4, by RT-PCR. The RT-PCR analysis was performed in the same method as in Example <5-3>.
TABLE 4 Derived p53 Cell line name from function 1 HCT116 (human colon carcinoma cell line) Colon + 2 SW480 (human colon cancer cell lines) − 3 H23 (non-small cell lung cancer cell line) Lungs − 4 H157 (non-small cell lung cancer cell line) − 5 A549 (human lung carcinoma cell line) + 6 H460 (human lung carcinoma cell line) + 7 Raji (B-cell leukemia cell line) Lympho- +/− 8 K-562 (human leukemia cell line) cytes − - As a result, as shown in
FIG. 10 a, the level of expression of AIM3 was lower in HCT116, A549 and H460 cell lines. Specifically, the level of AIM3 was low in the cells containing active p53 (p53(+), i.e., HCT116, A549 and H460 cell lines), while it was normal in the cells lacking active p53(p53(−), i.e., SW460, H23, H157 and K-562 cell lines). Also in Raji cells containing partially activated p53(Bhatia et al., FASEB J., 7:951-956, 1993), the level of AIM3 was in the middle of that of p53(+) cells and p53(−) cells. These results suggest that the expression level of AIM3 has a functional relation with p53. Also, these results further support that the aberration in either one of AIM3 or p53 may be sufficient to transform the cells, and AIM3 and p53 work in the same signal transduction pathway. - Furthermore, the expression level of AIM3 was analyzed by Western blot only in the lung cancer cell lines among the cell lines in Table 4. And the results coincided with that of the above RT-PCR analysis(data not shown).
- These results suggest that the expression level of AIM3 in some cancer cell lines is reduced.
- b) Genomic PCR Analysis
- To have a clue to the possible cause for the low expression level of AIM3 in some cancer cell lines, the present inventors compared the DNA content for the AIM3 gene by PCR. On H157, H460, HCT116, A549 and DU145 cell lines, genomic DNA analysis was performed in the same method as in Example <2-2>. As a control group, an actin gene was used.
- As a result, as shown in
FIG. 10 b, the H460 and A549 cell lines contained less amount of AIM3 DNA than that of other cell lines. This indicates that the two cell lines may have lost one allele for AIM3. - <10-2> Measurement of Expression Levels of AIM3 and p21 in Tissues Isolated From Cancer Patients
- Thereafter, the expression levels of AIM3 and p21 in the tissues isolated from cancer patients were examined. Total RNA was extracted from the leukocytes of 9 leukemia patients (five patients: acute promyelocytic leukemia (APML), and four patients: chronic myelocytic leukemia (CML)). Then, RT-PCR was performed in the same method as in Example <5-3>. In RT-PCR for p21, primers shown in SEQ ID NO: 16 and SEQ ID NO: 17 were used.
- As a result, as shown in
FIG. 10 c, the low level of AIM3 was observed in the tissues of 3 patents. In this case, the expression of p21, a target gene of p53, was also strongly suppressed. This demonstrates again that AIM3 is functionally involved in the regulation of p53. - <10-3> Comparative Measurement of Expression Levels of AIM3 in Normal Tissue and Cancer Region From Liver Cancer Patients
- Since solid tumors were also found in AIM3± mice although the frequency was much lower, the present inventors also compared the expression levels of AIM3 in the cancer region with that in normal tissue isolated from liver cancer patients by RT-PCR. As a control group, the expression level of actin was also measured. From the analysis of 25 different patient samples, a cancer-specific reduction of AIM3 was observed in 12 samples. The results for exemplary 8 samples are shown in
FIG. 10 d. - All these results in this Example suggest that a low level of expression of AIM3 is associated with various human cancer cell lines and patient tissues at high frequency. Also, these results indicate that the measurement of the expression level of AIM3 allows for the diagnosis of cancers.
- As described above, it was found in the present invention that AIM3 acts as a powerful tumor suppressor. The AIM3 protein binds to the FAT domain of ATM/ATR so as to activate ATM/ATR, thus inducing the expression of p53, tumor suppressor protein. Accordingly, the AIM3 protein or a nucleic acid encoding the protein will be useful for cancer therapy. Furthermore, it will be useful as targets for the development of anticancer drugs and as diagnostic markers of various cancers.
Claims (24)
1. A method for activating one selected from ATM, ATR and proteins regulated by ATM or ATR, in the cell, tissue or individual, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 (ARS-interacting multifunctional protein 3) having an amino acid sequence shown in SEQ ID NO: 1;
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide (a); and
(c) an isolated nucleic acid encoding the polypeptide (a) or (b).
2. The method of claim 1 , wherein the activation of ATM or ATR is mediated by the binding of AIM3.
3. The method of claim 1 , wherein the proteins regulated by ATM or ATR are selected from the group consisting of H2AX, p53, chk2, chk1, BRC AI, c-Abl, PHAS-1, RPA, RAD9, MDM2, MRE11, Rad17, WRN, PTS, CtIP, eIF-4E binding protein 1, LKB1, FANCD2, SMCl, Rad17, Nibrin, NBS, p95, Pin2/TRF1, DNA 5B, BRC A2 and phosphatidylinositol 3-kinase.
4. The method of claim 1 , wherein the activation of ATM, ATR and proteins regulated by ATM or ATR is related with DNA repair, cell cycle regulation and/or apoptosis.
5. A method for inducing the expression of p53 or its target genes in the cell, tissue or individual, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 (ARS-interacting multifunctional protein 3) having an amino acid sequence shown in SEQ ID NO: 1;
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide (a); and
(c) an isolated nucleic acid encoding the polypeptide (a) or (b).
6. The method of claim 5 , wherein the target genes of p53 are selected from the group consisting of p21, PUMA, GADD45, 14-3-3 sigma, WIPI, mdm-2, EGFR, PCNA, Cyclin D1, Cyclin G, TGFA, BAX, BAK, FAS1, Fas/APO1, FASL, IGF-BP3, PAG608, DR5/KILLER, GML, p53AIP1, p53R2, P2XM, TSP-1, BAL1, CSR, PIG3, Apaf-1, p53RDL1, Staf50, CD200 and Snk/PIk2.
7. A method for inhibiting the proliferation of tumor cells, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 (ARS-interacting multifunctional protein 3) having an amino acid sequence shown in SEQ ID NO: 1;
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide (a); and
(c) an isolated nucleic acid encoding the polypeptide (a) or (b).
8. A method for stimulating apoptosis in the cell, tissue or body, comprising administering to the cell, tissue or individual an effective amount of one selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 (ARS-interacting multifunctional protein 3) having an amino acid sequence shown in SEQ ID NO: 1;
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide (a); and
(c) an isolated nucleic acid encoding the polypeptide (a) or (b).
9. A method for treating or preventing ATM- or ATR-mediated diseases, comprising administering to a subject in need thereof an effective amount of one selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 (ARS-interacting multifunctional protein 3) having an amino acid sequence shown in SEQ ID NO: 1;
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide (a); and
(c) an isolated nucleic acid encoding the polypeptide (a) or (b).
10. The method of claim 9 , wherein the diseases are cancers or psoriasis.
11. The method of claim 10 , wherein the cancers include breast cancer, rectal cancer, lung cancer, small-cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine carcinoma, ovarian cancer, colorectal cancer, cancer neat the anus, colon cancer, oviduct carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulva carcinoma, Hodgkin's disease, esophagus cancer, small intestinal tumor, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, uterine cancer, penis cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or urethra cancer, kidney cell carcinoma, kidney pelvis carcinoma, CNS tumor, primary CNS lymphoma, spinal tumor, brain stem glioma, and pituitary adenoma, and combinations of one or more thereof.
12. A method for screening a substance having the effect of treating and/or preventing ATM- or ATR-mediated diseases, comprising the steps of:
(a) culturing AIM3 (ARS-interacting multifunctional protein 3) or a recombinant cell expressing the protein, together with a candidate substance; and
(b) determining the effect of the candidate substance on an increase in the activity of AIM3 or intracellular level thereof.
13. The method of claim 12 , wherein the ATM- or ATR-mediated diseases are cancers or psoriasis.
14. A method for identifying a subject having the risk of ATM- or ATR-mediated diseases, comprising the steps of:
(a) measuring the expression level of AIM3 (ARS-interacting multifunctional protein 3) in tissue sampled from a subject; and
(b) comparing the AIM3 level in the tissue with a normal AIM3 level.
15. The method of claim 14 , wherein the ATM- or ATR-mediated diseases are cancers or psoriasis.
16. A kit for the diagnosis of ATM- or ATR-mediated diseases, comprising one selected from a AIM3 protein-encoding nucleic acid, its fragment, a peptide encoded by the nucleic acid or its fragment, and an antibody to the peptide.
17. The method of claim 16 , wherein the ATM- or ATR-mediated diseases are cancers or psoriasis.
18. A pharmaceutical composition for activating one selected from ATM, ATR and proteins regulated by ATM or ATR, in the cell, tissue or individual, comprising, as an active ingredient, one selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 having an amino acid sequence shown in SEQ ID NO: 1;
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide (a); and
(c) an isolated nucleic acid encoding the polypeptide (a) or (b).
19. A pharmaceutical composition for inducing the expression of p53 or its target genes in the cell, tissue or individual, comprising, as an active ingredient, one selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 having an amino acid sequence shown in SEQ ID NO: 1;
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide (a); and
(c) an isolated nucleic acid encoding the polypeptide (a) or (b).
20. A pharmaceutical composition for inhibiting the proliferation of tumor cells, comprising, as an active ingredient, one selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 having an amino acid sequence shown in SEQ ID NO: 1;
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide (a); and
(c) an isolated nucleic acid encoding the polypeptide (a) or (b).
21. A pharmaceutical composition for stimulating apoptosis in the cell, tissue or individual, comprising, as an active ingredient, one selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 having an amino acid sequence shown in SEQ ID NO: 1;
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide (a); and
(c) an isolated nucleic acid encoding the polypeptide (a) or (b).
22. A pharmaceutical composition for treating or preventing ATM- or ATR-mediated diseases, comprising, as an active ingredient, one selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 having an amino acid sequence shown in SEQ ID NO: 1;
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide (a); and
(c) an isolated nucleic acid encoding the polypeptide (a) or (b).
23. An isolated polypeptide for use as an active therapeutic substance, being selected from the group consisting of the following:
(a) an isolated polypeptide of AIM3 having an amino acid sequence shown in SEQ ID NO: 1; and
(b) an isolated polypeptide having an amino acid sequence homology of at least 70% with said polypeptide.
24. An isolated nucleic acid for use as an active therapeutic substance, encoding an isolated polypeptide of AIM3 having an amino acid sequence shown in SEQ ID NO: 1 or an isolated polypeptide having a sequence homology of at least 70% with said polypeptide.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/902,641 US7902165B2 (en) | 2004-04-27 | 2007-09-24 | Use of AIM3 acting as a tumor suppressor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020040029205A KR100599454B1 (en) | 2004-04-27 | 2004-04-27 | 3 Novel use of AIM3 acting as a tumor suppressor |
PCT/KR2004/002202 WO2005102395A1 (en) | 2004-04-27 | 2004-09-01 | Novel use of aim3 acting as a tumor suppressor |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/902,641 Division US7902165B2 (en) | 2004-04-27 | 2007-09-24 | Use of AIM3 acting as a tumor suppressor |
Publications (1)
Publication Number | Publication Date |
---|---|
US20060046250A1 true US20060046250A1 (en) | 2006-03-02 |
Family
ID=35196744
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/536,257 Abandoned US20060046250A1 (en) | 2004-04-27 | 2004-09-01 | Novel use of aim 3 acting as a tumor suppressor |
US11/902,641 Expired - Fee Related US7902165B2 (en) | 2004-04-27 | 2007-09-24 | Use of AIM3 acting as a tumor suppressor |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/902,641 Expired - Fee Related US7902165B2 (en) | 2004-04-27 | 2007-09-24 | Use of AIM3 acting as a tumor suppressor |
Country Status (5)
Country | Link |
---|---|
US (2) | US20060046250A1 (en) |
EP (1) | EP1617875B1 (en) |
JP (1) | JP4772782B2 (en) |
KR (1) | KR100599454B1 (en) |
WO (1) | WO2005102395A1 (en) |
Cited By (38)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100028352A1 (en) * | 2008-06-26 | 2010-02-04 | Atyr Pharma, Inc. | COMPOSITIONS AND METHODS COMPRISING GLYCYL-tRNA SYNTHETASES HAVING NON-CANONICAL BIOLOGICAL ACTIVITIES |
US20100092434A1 (en) * | 2008-06-11 | 2010-04-15 | Atyr Pharma, Inc. | Thrombopoietic activity of tyrosyl-trna synthetase polypeptides |
US20100297149A1 (en) * | 2009-03-16 | 2010-11-25 | Atyr Pharma, Inc. | Compositions and methods comprising histidyl-trna synthetase splice variants having non-canonical biological activities |
US20100310576A1 (en) * | 2009-03-31 | 2010-12-09 | Adams Ryan A | COMPOSITIONS AND METHODS COMPRISING ASPARTYL-tRNA SYNTHETASES HAVING NON-CANONICAL BIOLOGICAL ACTIVITIES |
US20110150885A1 (en) * | 2009-12-11 | 2011-06-23 | Atyr Pharma, Inc. | Aminoacyl trna synthetases for modulating hematopoiesis |
RU2465330C1 (en) * | 2008-08-18 | 2012-10-27 | Сеул Нэшнл Юниверсити Индастри Фаундейшн | METHOD FOR INHIBITION OF MALIGNANT TUMOUR METASTASIS OR MALIGNANT CELL MIGRATION BY REDUCING CELL LEVEL OF LYSYL-tRNA-SYNTHETASE (VERSIONS), COMPOSITION AND APPLICATION OF EXPRESSION VECTOR OR KRS ANTIBODY FOR INHIBITION OF MALIGNANT TUMOUR METASTASIS OR MALIGNANT CELL MIGRATION |
US8828395B2 (en) | 2009-12-11 | 2014-09-09 | Atyr Pharma, Inc. | Antibodies that bind tyrosyl-tRNA synthetases |
US8835387B2 (en) | 2012-02-16 | 2014-09-16 | Atyr Pharma, Inc. | Histidyl-tRNA synthetases for treating autoimmune and inflammatory diseases |
US8945541B2 (en) | 2010-05-14 | 2015-02-03 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-beta-tRNA synthetases |
US8946157B2 (en) | 2010-05-03 | 2015-02-03 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of seryl-tRNA synthetases |
US8961960B2 (en) | 2010-04-27 | 2015-02-24 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of isoleucyl tRNA synthetases |
US8962560B2 (en) | 2010-06-01 | 2015-02-24 | Atyr Pharma Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Lysyl-tRNA synthetases |
US8961961B2 (en) | 2010-05-03 | 2015-02-24 | a Tyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related protein fragments of arginyl-tRNA synthetases |
US8969301B2 (en) | 2010-07-12 | 2015-03-03 | Atyr Pharma Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of aspartyl-tRNA synthetases |
US8981045B2 (en) | 2010-05-03 | 2015-03-17 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of methionyl-tRNA synthetases |
US8980253B2 (en) | 2010-04-26 | 2015-03-17 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of cysteinyl-tRNA synthetase |
US8986681B2 (en) | 2010-04-27 | 2015-03-24 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of threonyl-tRNA synthetases |
US8986680B2 (en) | 2010-04-29 | 2015-03-24 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Asparaginyl tRNA synthetases |
US8993723B2 (en) | 2010-04-28 | 2015-03-31 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of alanyl-tRNA synthetases |
US8999321B2 (en) | 2010-07-12 | 2015-04-07 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glycyl-tRNA synthetases |
US9029506B2 (en) | 2010-08-25 | 2015-05-12 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tyrosyl-tRNA synthetases |
US9034320B2 (en) | 2010-04-29 | 2015-05-19 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Valyl-tRNA synthetases |
US9034321B2 (en) | 2010-05-03 | 2015-05-19 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-alpha-tRNA synthetases |
US9034598B2 (en) | 2010-05-17 | 2015-05-19 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of leucyl-tRNA synthetases |
US9062302B2 (en) | 2010-05-04 | 2015-06-23 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of p38 multi-tRNA synthetase complex |
US9062301B2 (en) | 2010-05-04 | 2015-06-23 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glutamyl-prolyl-tRNA synthetases |
US9068177B2 (en) | 2010-04-29 | 2015-06-30 | Atyr Pharma, Inc | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glutaminyl-tRNA synthetases |
US9127268B2 (en) | 2009-12-11 | 2015-09-08 | Atyr Pharma, Inc. | Aminoacyl tRNA synthetases for modulating inflammation |
US9399770B2 (en) | 2010-10-06 | 2016-07-26 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tryptophanyl-tRNA synthetases |
US9422539B2 (en) | 2010-07-12 | 2016-08-23 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of histidyl-tRNA synthetases |
US9453214B2 (en) | 2009-02-27 | 2016-09-27 | Atyr Pharma, Inc. | Polypeptide structural motifs associated with cell signaling activity |
US9587235B2 (en) | 2013-03-15 | 2017-03-07 | Atyr Pharma, Inc. | Histidyl-tRNA synthetase-Fc conjugates |
US9688978B2 (en) | 2011-12-29 | 2017-06-27 | Atyr Pharma, Inc. | Aspartyl-tRNA synthetase-Fc conjugates |
US9714419B2 (en) | 2011-08-09 | 2017-07-25 | Atyr Pharma, Inc. | PEGylated tyrosyl-tRNA synthetase polypeptides |
US9796972B2 (en) | 2010-07-12 | 2017-10-24 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glycyl-tRNA synthetases |
US9816084B2 (en) | 2011-12-06 | 2017-11-14 | Atyr Pharma, Inc. | Aspartyl-tRNA synthetases |
US9822353B2 (en) | 2011-12-06 | 2017-11-21 | Atyr Pharma, Inc. | PEGylated aspartyl-tRNA synthetase polypeptides |
US11767520B2 (en) | 2017-04-20 | 2023-09-26 | Atyr Pharma, Inc. | Compositions and methods for treating lung inflammation |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3508505A1 (en) * | 2007-12-24 | 2019-07-10 | ID Biomedical Corporation of Quebec | Recombinant rsv antigens |
KR100895256B1 (en) | 2008-04-30 | 2009-04-29 | 재단법인서울대학교산학협력재단 | The Screening Method of Apoptotic Regulator by Controling of ATM/ATR |
US8510665B2 (en) * | 2009-03-16 | 2013-08-13 | Apple Inc. | Methods and graphical user interfaces for editing on a multifunction device with a touch screen display |
KR101123766B1 (en) * | 2009-04-17 | 2012-03-15 | 서울대학교산학협력단 | Composition for preventing or treating aging or age-related diseases comprising AIMP3 si-RNA or anti-AIMP3 antibody |
CN113874361B (en) * | 2019-03-29 | 2024-07-12 | 医药生命融合研究团 | Compound with anticancer activity and preparation method thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999046292A1 (en) * | 1998-03-12 | 1999-09-16 | Shanghai Second Medical University | A human p18 gene (cbdara04) |
CA2405525A1 (en) * | 2000-04-12 | 2001-10-25 | Principia Pharmaceutical Corporation | Albumin fusion proteins |
-
2004
- 2004-04-27 KR KR1020040029205A patent/KR100599454B1/en active IP Right Grant
- 2004-09-01 WO PCT/KR2004/002202 patent/WO2005102395A1/en not_active Application Discontinuation
- 2004-09-01 JP JP2007510600A patent/JP4772782B2/en not_active Expired - Fee Related
- 2004-09-01 US US10/536,257 patent/US20060046250A1/en not_active Abandoned
- 2004-09-01 EP EP04774463.6A patent/EP1617875B1/en not_active Expired - Lifetime
-
2007
- 2007-09-24 US US11/902,641 patent/US7902165B2/en not_active Expired - Fee Related
Cited By (92)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9499810B2 (en) | 2008-06-11 | 2016-11-22 | Atyr Pharma, Inc. | Thrombopoietic activity of tyrosyl-tRNA synthetase polypeptides |
US20100092434A1 (en) * | 2008-06-11 | 2010-04-15 | Atyr Pharma, Inc. | Thrombopoietic activity of tyrosyl-trna synthetase polypeptides |
US20100028352A1 (en) * | 2008-06-26 | 2010-02-04 | Atyr Pharma, Inc. | COMPOSITIONS AND METHODS COMPRISING GLYCYL-tRNA SYNTHETASES HAVING NON-CANONICAL BIOLOGICAL ACTIVITIES |
US9157076B2 (en) | 2008-06-26 | 2015-10-13 | Atyr Pharma, Inc. | Compositions and methods comprising glycyl-tRNA synthetases having non-canonical biological activities |
US9585946B2 (en) | 2008-06-26 | 2017-03-07 | Atyr Pharma, Inc. | Compositions and methods comprising glycyl-tRNA synthetases having non-canonical biological activities |
US8747840B2 (en) | 2008-06-26 | 2014-06-10 | Atyr Pharma, Inc. | Compositions and methods comprising glycyl-tRNA synthetases having non-canonical biological activities |
US8404471B2 (en) | 2008-06-26 | 2013-03-26 | Atyr Pharma, Inc. | Compositions and methods comprising glycyl-tRNA synthetases having non-canonical biological activities |
RU2465330C1 (en) * | 2008-08-18 | 2012-10-27 | Сеул Нэшнл Юниверсити Индастри Фаундейшн | METHOD FOR INHIBITION OF MALIGNANT TUMOUR METASTASIS OR MALIGNANT CELL MIGRATION BY REDUCING CELL LEVEL OF LYSYL-tRNA-SYNTHETASE (VERSIONS), COMPOSITION AND APPLICATION OF EXPRESSION VECTOR OR KRS ANTIBODY FOR INHIBITION OF MALIGNANT TUMOUR METASTASIS OR MALIGNANT CELL MIGRATION |
US9453214B2 (en) | 2009-02-27 | 2016-09-27 | Atyr Pharma, Inc. | Polypeptide structural motifs associated with cell signaling activity |
US8404242B2 (en) | 2009-03-16 | 2013-03-26 | Atyr Pharma, Inc. | Compositions and methods comprising histidyl-tRNA synthetase splice variants having non-canonical biological activities |
US8753638B2 (en) | 2009-03-16 | 2014-06-17 | Atyr Pharma, Inc. | Compositions and methods comprising histidyl-TRNA synthetase splice variants having non-canonical biological activities |
US10526419B2 (en) | 2009-03-16 | 2020-01-07 | Atyr Pharma, Inc. | Compositions and methods comprising histidyl-tRNA synthetase splice variants having non-canonical biological activities |
US10017582B2 (en) | 2009-03-16 | 2018-07-10 | Atyr Pharma, Inc. | Compositions and methods comprising histidyl-trna synthetase splice variants having non-canonical biological activities |
US11078299B2 (en) | 2009-03-16 | 2021-08-03 | Atyr Pharma, Inc. | Compositions and methods comprising histidyl-tRNA synthetase splice variants having non-canonical biological activities |
US9605265B2 (en) | 2009-03-16 | 2017-03-28 | Atyr Pharma, Inc. | Compositions and methods comprising histidyl-tRNA synthetase splice variants having non-canonical biological activities |
US10941214B2 (en) | 2009-03-16 | 2021-03-09 | Atyr Pharma, Inc. | Compositions and methods comprising histidyl-tRNA synthetase splice variants having non-canonical biological activities |
US20100297149A1 (en) * | 2009-03-16 | 2010-11-25 | Atyr Pharma, Inc. | Compositions and methods comprising histidyl-trna synthetase splice variants having non-canonical biological activities |
US9896680B2 (en) | 2009-03-31 | 2018-02-20 | Atyr Pharma, Inc. | Compositions and methods comprising aspartyl-tRNA synthetases having non-canonical biological activities |
US20100310576A1 (en) * | 2009-03-31 | 2010-12-09 | Adams Ryan A | COMPOSITIONS AND METHODS COMPRISING ASPARTYL-tRNA SYNTHETASES HAVING NON-CANONICAL BIOLOGICAL ACTIVITIES |
US9127268B2 (en) | 2009-12-11 | 2015-09-08 | Atyr Pharma, Inc. | Aminoacyl tRNA synthetases for modulating inflammation |
US9540628B2 (en) | 2009-12-11 | 2017-01-10 | Atyr Pharma, Inc. | Aminoacyl tRNA synthetases for modulating inflammation |
US20110150885A1 (en) * | 2009-12-11 | 2011-06-23 | Atyr Pharma, Inc. | Aminoacyl trna synthetases for modulating hematopoiesis |
US8828395B2 (en) | 2009-12-11 | 2014-09-09 | Atyr Pharma, Inc. | Antibodies that bind tyrosyl-tRNA synthetases |
US9328340B2 (en) | 2009-12-11 | 2016-05-03 | Atyr Pharma, Inc. | Amino acyl tRNA synthetases for modulating inflammation |
US9943577B2 (en) | 2009-12-11 | 2018-04-17 | Atyr Pharma, Inc. | Aminoacyl tRNA synthetases for modulating inflammation |
US8980253B2 (en) | 2010-04-26 | 2015-03-17 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of cysteinyl-tRNA synthetase |
US10717786B2 (en) | 2010-04-26 | 2020-07-21 | aTye Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Cysteinyl-tRNA synthetase |
US9540629B2 (en) | 2010-04-26 | 2017-01-10 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Cysteinyl-tRNA synthetase |
US10030077B2 (en) | 2010-04-26 | 2018-07-24 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of cysteinyl-tRNA synthetase |
US10150958B2 (en) | 2010-04-27 | 2018-12-11 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of threonyl-tRNA synthetases |
US8961960B2 (en) | 2010-04-27 | 2015-02-24 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of isoleucyl tRNA synthetases |
US9896515B2 (en) | 2010-04-27 | 2018-02-20 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of isoleucyl tRNA synthetases |
US9580706B2 (en) | 2010-04-27 | 2017-02-28 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of threonyl-tRNA synthetases |
US10563192B2 (en) | 2010-04-27 | 2020-02-18 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of threonyl-tRNA synthetases |
US8986681B2 (en) | 2010-04-27 | 2015-03-24 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of threonyl-tRNA synthetases |
US9528103B2 (en) | 2010-04-27 | 2016-12-27 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of isoleucyl tRNA synthetases |
US9320782B2 (en) | 2010-04-28 | 2016-04-26 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of alanyl tRNA synthetases |
US8993723B2 (en) | 2010-04-28 | 2015-03-31 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of alanyl-tRNA synthetases |
US9068177B2 (en) | 2010-04-29 | 2015-06-30 | Atyr Pharma, Inc | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glutaminyl-tRNA synthetases |
US9556425B2 (en) | 2010-04-29 | 2017-01-31 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Valyl-tRNA synthetases |
US8986680B2 (en) | 2010-04-29 | 2015-03-24 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Asparaginyl tRNA synthetases |
US9034320B2 (en) | 2010-04-29 | 2015-05-19 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Valyl-tRNA synthetases |
US9623093B2 (en) | 2010-04-29 | 2017-04-18 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of asparaginyl tRNA synthetases |
US10189911B2 (en) | 2010-04-29 | 2019-01-29 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Valyl-tRNA synthetases |
US8961961B2 (en) | 2010-05-03 | 2015-02-24 | a Tyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related protein fragments of arginyl-tRNA synthetases |
US10179906B2 (en) | 2010-05-03 | 2019-01-15 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-alpha-tRNA synthetases |
US9034321B2 (en) | 2010-05-03 | 2015-05-19 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-alpha-tRNA synthetases |
US8981045B2 (en) | 2010-05-03 | 2015-03-17 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of methionyl-tRNA synthetases |
US9422538B2 (en) | 2010-05-03 | 2016-08-23 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of methionyl-tRNA synthetasis |
US8946157B2 (en) | 2010-05-03 | 2015-02-03 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of seryl-tRNA synthetases |
US9340780B2 (en) | 2010-05-03 | 2016-05-17 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of seryl-tRNA synthetases |
US9593322B2 (en) | 2010-05-03 | 2017-03-14 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of arginyl-trna synthetases |
US9593323B2 (en) | 2010-05-03 | 2017-03-14 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-alpha-tRNA synthetases |
US9574187B2 (en) | 2010-05-04 | 2017-02-21 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glutamyl-prolyl-tRNA synthetases |
US9404104B2 (en) | 2010-05-04 | 2016-08-02 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of P38 multi-tRNA synthetase complex |
US10160814B2 (en) | 2010-05-04 | 2018-12-25 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glutamyl-prolyl-tRNA synthetases |
US9062302B2 (en) | 2010-05-04 | 2015-06-23 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of p38 multi-tRNA synthetase complex |
US9062301B2 (en) | 2010-05-04 | 2015-06-23 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glutamyl-prolyl-tRNA synthetases |
US9687533B2 (en) | 2010-05-14 | 2017-06-27 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-beta-tRNA synthetases |
US8945541B2 (en) | 2010-05-14 | 2015-02-03 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-beta-tRNA synthetases |
US10220080B2 (en) | 2010-05-14 | 2019-03-05 | aTyr Pharam, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-beta-tRNA synthetases |
US10179908B2 (en) | 2010-05-17 | 2019-01-15 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of leucyl-tRNA synthetases |
US9790482B2 (en) | 2010-05-17 | 2017-10-17 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of leucyl-tRNA synthetases |
US9034598B2 (en) | 2010-05-17 | 2015-05-19 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of leucyl-tRNA synthetases |
US9347053B2 (en) | 2010-05-27 | 2016-05-24 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glutaminyl-tRNA synthetases |
US8962560B2 (en) | 2010-06-01 | 2015-02-24 | Atyr Pharma Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Lysyl-tRNA synthetases |
US9322009B2 (en) | 2010-06-01 | 2016-04-26 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Lysyl-tRNA synthetases |
US9796972B2 (en) | 2010-07-12 | 2017-10-24 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glycyl-tRNA synthetases |
US10669533B2 (en) | 2010-07-12 | 2020-06-02 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Histidyl-tRNA synthetases |
US9315794B2 (en) | 2010-07-12 | 2016-04-19 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of aspartyl-tRNA synthetases |
US9637730B2 (en) | 2010-07-12 | 2017-05-02 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of histidyl-tRNA synthetases |
US8999321B2 (en) | 2010-07-12 | 2015-04-07 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glycyl-tRNA synthetases |
US8969301B2 (en) | 2010-07-12 | 2015-03-03 | Atyr Pharma Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of aspartyl-tRNA synthetases |
US9422539B2 (en) | 2010-07-12 | 2016-08-23 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of histidyl-tRNA synthetases |
US10196628B2 (en) | 2010-07-12 | 2019-02-05 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of histidyl-tRNA synthetases |
US10196629B2 (en) | 2010-07-12 | 2019-02-05 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glycyl-tRNA synthetases |
US9029506B2 (en) | 2010-08-25 | 2015-05-12 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tyrosyl-tRNA synthetases |
US9428743B2 (en) | 2010-08-25 | 2016-08-30 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tyrosyl-trna synthetases |
US9399770B2 (en) | 2010-10-06 | 2016-07-26 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tryptophanyl-tRNA synthetases |
US10563191B2 (en) | 2010-10-06 | 2020-02-18 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related protein fragments of tryptophanyl tRNA synthetases |
US9714419B2 (en) | 2011-08-09 | 2017-07-25 | Atyr Pharma, Inc. | PEGylated tyrosyl-tRNA synthetase polypeptides |
US9822353B2 (en) | 2011-12-06 | 2017-11-21 | Atyr Pharma, Inc. | PEGylated aspartyl-tRNA synthetase polypeptides |
US9816084B2 (en) | 2011-12-06 | 2017-11-14 | Atyr Pharma, Inc. | Aspartyl-tRNA synthetases |
US9688978B2 (en) | 2011-12-29 | 2017-06-27 | Atyr Pharma, Inc. | Aspartyl-tRNA synthetase-Fc conjugates |
US8835387B2 (en) | 2012-02-16 | 2014-09-16 | Atyr Pharma, Inc. | Histidyl-tRNA synthetases for treating autoimmune and inflammatory diseases |
US9273302B2 (en) | 2012-02-16 | 2016-03-01 | Atyr Pharma, Inc. | Histidyl-tRNA synthetases for treating autoimmune and inflammatory diseases |
US10093915B2 (en) | 2013-03-15 | 2018-10-09 | Atyr Pharma Inc. | Histidyl-tRNA synthetase-Fc conjugates |
US10711260B2 (en) | 2013-03-15 | 2020-07-14 | Atyr Pharma, Inc. | Histidyl-tRNA synthetase-Fc conjugates |
US10472618B2 (en) | 2013-03-15 | 2019-11-12 | Atyr Pharma, Inc. | Histidyl-tRNA synthetase-Fc conjugates |
US11072787B2 (en) | 2013-03-15 | 2021-07-27 | Atyr Pharma Inc. | Histidyl-tRNA synthetase-Fc conjugates |
US9587235B2 (en) | 2013-03-15 | 2017-03-07 | Atyr Pharma, Inc. | Histidyl-tRNA synthetase-Fc conjugates |
US11767520B2 (en) | 2017-04-20 | 2023-09-26 | Atyr Pharma, Inc. | Compositions and methods for treating lung inflammation |
Also Published As
Publication number | Publication date |
---|---|
KR100599454B1 (en) | 2006-07-12 |
EP1617875A1 (en) | 2006-01-25 |
US20080200382A1 (en) | 2008-08-21 |
JP2007534748A (en) | 2007-11-29 |
KR20050104045A (en) | 2005-11-02 |
US7902165B2 (en) | 2011-03-08 |
WO2005102395A1 (en) | 2005-11-03 |
EP1617875A4 (en) | 2007-10-10 |
EP1617875B1 (en) | 2017-08-16 |
JP4772782B2 (en) | 2011-09-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7902165B2 (en) | Use of AIM3 acting as a tumor suppressor | |
JP2004113151A (en) | Oncogene and its application | |
JP2010246550A (en) | Method and composition for diagnosis of cancer susceptibility and defective dna repair mechanism and treatment thereof | |
JP2011501741A (en) | TAZ / WWTR1 for cancer diagnosis and treatment | |
KR100553300B1 (en) | Promotion or Inhibition of Angiogenesis and Cardiovascularization | |
US20080020461A1 (en) | Kinesin-like proteins and methods of use | |
JP2005520543A (en) | Novel compositions and methods in cancer | |
US20080248009A1 (en) | Regulation of acheron expression | |
US7160681B2 (en) | Method for regulating cell growth and assays related thereto | |
US20080253632A1 (en) | Method of Detecting Precancerous Lesions | |
US6861442B1 (en) | PYK2 and inflammation | |
JP4415145B2 (en) | Screening method for drugs that modulate p53 protein activation | |
US20030054418A1 (en) | Gene and sequence variation associated with cancer | |
US20030064372A1 (en) | Gene and sequence variation associated with lipid disorder | |
US20030157481A1 (en) | Diagnosing and treating cancer cells using T-HR mutants and their targets | |
US20030198951A1 (en) | Novel methods of diagnosing colorectal cancer and/or breast cancer, compositions, and methods of screening for colorectal cancer and/or breast cancer modulators | |
AU2008201172B2 (en) | Methods and Compositions for the Diagnosis of Cancer Susceptibilites and Defective DNA Repair Mechanisms and Treatment Thereof | |
WO2005061704A1 (en) | Preventive/remedy for cancer | |
WO2001009319A1 (en) | Gene expressed specifically in human fetal heart muscle | |
CA2431313A1 (en) | Methods of diagnosing colorectal cancer and/or breast cancer, compositions, and methods of screening for colorectal cancer and/or breast cancer modulators | |
JP2002508180A (en) | Human RAD1 nucleic acids, polypeptides, assays, therapeutic methods and means | |
JP2004267118A (en) | Oncogene and application thereof | |
KR20050053628A (en) | Promotion or inhibition of angiogenesis and cardiovascularization | |
JP2003277288A (en) | Preventive/remedial agent for cancer | |
JP2004135618A (en) | Oncogene and its use |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: SEOUL NATIONAL UNIVERSITY INDUSTRY FOUNDATION, KOR Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:KIM, SUNGHOON;REEL/FRAME:017108/0417 Effective date: 20050509 |
|
AS | Assignment |
Owner name: SCHWARZ, HERBERT, SINGAPORE Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SCHWARZ, HERBERT;WITTMANN, MARGARETHE;REEL/FRAME:017716/0966;SIGNING DATES FROM 20050709 TO 20050718 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |