WO2004005514A1 - Proto-oncogenese humaine 6 et proteine codee dedans - Google Patents

Proto-oncogenese humaine 6 et proteine codee dedans Download PDF

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Publication number
WO2004005514A1
WO2004005514A1 PCT/KR2002/001264 KR0201264W WO2004005514A1 WO 2004005514 A1 WO2004005514 A1 WO 2004005514A1 KR 0201264 W KR0201264 W KR 0201264W WO 2004005514 A1 WO2004005514 A1 WO 2004005514A1
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protooncogene
fragment
protein
seq
hcc
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PCT/KR2002/001264
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English (en)
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Jin Woo Kim
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Jin Woo Kim
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Priority to PCT/KR2002/001264 priority Critical patent/WO2004005514A1/fr
Priority to AU2002345414A priority patent/AU2002345414A1/en
Publication of WO2004005514A1 publication Critical patent/WO2004005514A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes

Definitions

  • the present invention relates to a novel protooncogene and protein encoded therein, which can be used in diagnosis of various cancers, preparation of transgenic animal, anti sense gene therapy and anticancer drug development.
  • DD mRNA differential display
  • Liang and Pardee is effective in elucidating the nature of tumor suppressor genes, cell cycle-related genes and transcriptional regulatory genes that control apoptosis
  • tumorigenesis is caused by various genetic changes such as the loss of chromosomal heterozygosity, activation of oncogenes and inactivation of tumor suppressor genes, e.g., p53 gene (Bishop, J. M., Cell 64: 235-248, 1991; and Hunter, T., Cell 64: 249-270, 1991). Further, it has been reported that 10 to 30% of human cancer arises from the activation of oncogene through amplification of protooncogenes.
  • protooncogenes plays an important role in the etiology of many tumors and there has existed a need to identify protooncogenes.
  • the present inventor has endeavored to unravel the mechanism involved in the tumorigenesis of cervical cancer; and, has unexpectedly found that a novel protooncogene, Human Cervical Cancer 6 (HCC-6), is specifically overexpressed in cancer cells.
  • HCC-6 Human Cervical Cancer 6
  • This protooncogene can be effectively used in diagnosis, prevention and treatment of various cancers, e.g., leukemia, cervix, lymphoma, colon, lung and skin cancers.
  • the primary object of the present invention is to provide a novel protooncogene and a fragment thereof.
  • Other objects of the present invention are to provide: a recombinant vector containing said protooncogene or a fragment thereof and a microorganism transformed therewith; a protein encoded in said protooncogene and a fragment threrof; a kit for diagnosing cancer which comprising said protein or a fragment thereof; an antisense gene having a base sequence complementary to that of said protooncogene or a fragment thereof; and a process for treating or preventing cancer by using said antisense gene.
  • a novel protooncogene having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof.
  • a recombinant vector containing said protooncogene or a fragment thereof and a microorganism transformed with said vector.
  • a protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof derived from said protooncogene or a fragment thereof.
  • Fig. 1 the results of DDRT-PCR for CA245 expressed in normal cervix tissue, primary cervical cancer tissue, metastatic lymph node tissue and CUMC-6 cervical cancer cells;
  • Fig. 2A the results of northern blot analysis for HCC-6 protooncogene expressed in cervical cancer tissues
  • Fig. 2B the results obtained with the same sample of Fig. 2A hybridized with ⁇ -actin;
  • Fig. 3A the results of northern blot analysis for HCC-6 protooncogene expressed in normal human 12-lane multiple tissues
  • Fig. 3B the results obtained with the same sample of Fig. 3A hybridized with ⁇ -actin
  • Fig. 4A the result of northern blot analysFis for HCC-6 protooncogene expressed in human cancer cell lines
  • Fig. 4B the result obtained with the same sample of Fig. 4A hybridized with ⁇ -actin;
  • Fig. 5 the sodium dodecyl sulfate (SDS)-PAGE results showing protein expression patterns before and after the IPTG induction in HCC-6 transformed E.coli cells.
  • HCC-6 protooncogene human cervical cancer 6
  • HCC-6 protooncogene consists of 899 base pairs and has the nucleotide sequence of SEQ ID NO: 1.
  • SEQ ID NO: 1 the open reading frame corresponding to base Nos. 174 to 854 (852-854: termination codon) is a full-length protein encoding region and the predicted amino acid sequence derived therefrom is shown in SEQ ID NO: 2 which consists of 226 amino acids (hereinafter "HCC-6 protein").
  • the present invention also includes, in its scope, a polynucleotide having substantially the same base sequence as the inventive protooncogene, and a fragment thereof.
  • substantially the same polynucleotide refers to a polynucleotide whose base sequence shows 80% or more, preferably 90% or more, most preferably 95% or more homology to the protooncogene of the present invention.
  • the protein expressed in vertebrate mitochondria from the protooncogene of the present invention consists of 266 amino acids and has the amino acid sequence of SEQ ID NO: 2.
  • protein synthesis is stopped at TGA termination codon (315-317 of SEQ ID NO: 1; tryptophane codon in mitochondria), which results in the production of HCC-6 protein consisting of 47 amino acids (1-47 of SEQ ID NO: 2) and having the molecular weight of about 5 kDa.
  • TGA termination codon 315-317 of SEQ ID NO: 1; tryptophane codon in mitochondria
  • the present invention includes, in its scope, a polypeptide having substantially the same amino acid sequence as the protein derived from the oncogene of the present invention and a fragment thereof.
  • substantially the same polypeptide refers to a polypeptide whose amino acid sequence shows 80% or more, preferably 90% or more, most preferably 95% or more homology to the amino acid sequence of SEQ ID NO: 2.
  • the protooncogene or the protein of the present invention can be obtained from human cancer tissues or synthesized using a conventional DNA or peptide synthesis method. Further, the gene thus prepared may be inserted to a conventional vector to obtain an expression vector, which may, in turn, be introduced into a suitable host, e.g., a microorganism such as an E. coli or yeast, or an animal cell such as a mouse or human cell. A transformed host may then be used in producing the inventive DNA or protein on a large scale. For example, E. coli DH5 ⁇ was transformed with expression vector pCEV-LAC containing the inventive HCC-6 gene to obtain an E.
  • coli transformant designated DH5 ⁇ /HCC-6/pCEV-LAC which was deposited on April 10, 2001 with the Korean Collection for Type Cultures (KCTC) (Address: Korea Research Institute of Bioscience and Biotechnology (KRIBB), #52, Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) under the accession number, KCTC 0989BP, in accordance with the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.
  • expression-control sequences e.g., promoter, terminator, self-replication sequence and secretion signal, are suitably selected and combined depending on the host cell used.
  • the overexpression of the protooncogene of the present invention occurs not in normal cervical tissues but in cervical cancer tissues. This suggests that the protooncogene of the present invention induces the uterine cervical cancer.
  • the overexpression of the protooncogene of the present invention is also observed in various cancer tissues, e.g., leukemia, uterine, colon, lymphoma, and skin cancer tissues (see Figs. 2 and 4). Therefore, the protooncogene of the present invention is believed to be a factor common to all forms of cancers and it can be advantageously used in the diagnosis of various cancers and the production of a transgenic animal as well as in an antisense gene therapy.
  • a diagnostic method that can be performed using the protooncogene of the present invention may comprise, for example, the steps of hybridizing nucleic acids separated from the body fluid of a subject with a probe containing the protooncogene of the present invention or a fragment thereof, and determining whether the subject has the protooncogene by using a conventional detection method in the art.
  • the presence of the protooncogene may be easily detected by labeling the probe with a radioisotope or an enzyme. Therefore, a cancer diagnostic kit containing the protooncogene of the present invention or a fragment thereof is also included in the scope of the present invention.
  • a transgenic animal produced by introducing the protooncogene of the present invention into a mammal, e.g., mice, is also included in the scope of the present invention.
  • the transgenic animal can be advantageously used in screening for carcinogens or anticancer agents such as chemotherapeutic drugs.
  • an antisense gene which is useful in a gene therapy.
  • an antisense gene means a polynucleotide comprising a base sequence which is fully or partially complementary to the sequence of the mRNA which is transcribed from the protooncogene having the base sequence of SEQ ID NO: 1 or a fragment thereof, said nucleotide being capable of preventing the expression of the open reading frame (ORF) of the protooncogene by way of attaching itself to the protein- binding site of mRNA.
  • ORF open reading frame
  • the present invention also includes within its scope a process for treating or preventing cancer in a subject by way of administering a therapeutically effective amount of the inventive antisense gene thereto.
  • the antisense gene of the present invention is administered to a subject in a conventional manner to prevent the expression of the protooncogene.
  • the antisense oligodeoxynucleotide (ODN) is mixed with a hydrophobicized poly-L-lysine derivative by electrostatic interaction in accordance with the method disclosed by Kim, J. S. et al. (J. Controlled Release 53: 175-182, 1998) and the resulting mixed antisense ODN is administered intravenously to a subject.
  • the present invention also includes within its scope an anti-cancer composition comprising the antisense gene of the present invention as an active ingredient, in association with pharmaceutically acceptable carriers, excipients or other additives, if necessary.
  • the pharmaceutical composition of the present invention is preferably formulated for administration by injection.
  • the amount of the antisense gene actually administered should be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age and weight of the individual patient, and the severity of the patient's symptoms.
  • the protein expressed from the inventive protooncogene may be used in producing an antibody useful as a diagnostic tool.
  • the antibody of the present invention may be prepared in the form of a monoclonal or polyclonal antibody in accordance with any of the methods well known in the art by using a protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.
  • Cancer diagnosis may be carried out using any of the methods known in the art, e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, western blot or immunoassay blot on polyacrylic gel, to assess whether the protein is expressed in the body fluid of the subject. Therefore, a cancer diagnostic kit containing the protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof is also included in the scope of the present invention.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • sandwich assay sandwich assay
  • a continuously viable cancer cell line may be established by using the protooncogene of the present invention, and such a cell line may be obtained, for example, from tumor tissues formed on the back of a nude mouse by injecting fibroblast cells transformed with the protooncogene of the present invention.
  • the cell lines thus prepared may be advantageously used in searching for anti- cancer agents.
  • Example 1 Cultivation of tumor cells and separation of total RNA
  • mRNA normal cervical tissues, untreated primary cervical cancer tissues and metastatic common iliac lymph node tissues were obtained from cervical cancer patients who underwent radical hysterectomy.
  • the human cervical cancer cell line used in the differential display method was CUMC-6 cell line described by Kim et al, (Gynecol. Oncol. 62: 230-240, 1996). Cells obtained from the above-described tissues and CUMC-6 were maintained on Waymouth's MB 752/1 medium (Gibco) supplemented with 2 mM/£of glutamine, 100 IU/m# of penicillin, 100 ⁇ glv of streptomycin, and 10% of fetal bovine serum (Gibco).
  • Step 2 Isolation of total RNA and differential display of mRNA
  • RNAs were extracted from normal cervical tissues, primary cervical cancer tissues, metastatic common iliac lymph node tissues and CUMC-6 cells obtained in Step 1 using a commercial system (RNeasy total RNA kit) provided by Qiagen (Qiagen Inc., Germany) and the removal of DNA contaminants from the RNAs was accomplished using Message clean kit (GenHunter Corp., Brookline, MA).
  • RT-PCR reverse transcription-polymerase chain reaction
  • PCR was conducted using the same anchored 3' primer of SEQ ID NO: 3 and the arbitrary 5' primer of SEQ ID NO: 4 (H-AP24 primer of RNAimage primer sets 1-4, H-AP 1-32) in the presence of 0.5 mM [ ⁇ - 35 S]- labeled dATP (1200 Ci/mmol).
  • the PCR thermal cycle was repeated 40 times, the cycle being composed of: 95 °C for 40 sec, 40 °C for 2 min and 72 °C for 40 sec, and finally the reaction was carried out at 72 ° C for 5 min.
  • PCR-amplified fragments were resolved in 6% polyacrylamide sequencing gels. Differentially expressed fragments were identified by inspection of autoradiograms.
  • the band of fragment CA245 cDNA of size 147 bp (nucleotide Nos. 705- 851 of SEQ ID NO: 1), were excised from the dried sequencing gel.
  • the cDNAs were eluted by boiling for 15 min and reamplified with the same primer pairs and PCR conditions as used in the above amplification step except that no [ ⁇ - 35 S]-labeled dATP and 20 ⁇ M dNTPs were used.
  • the reamplified CA245 PCR product obtained as above was inserted into the pGEM-T Easy vector using TA cloning system (Promega, USA) in accordance with the manufacturer's instructions.
  • the ligation reaction mixture was incubated overnight at 14 ° C .
  • E. coli JM109 (Promega, WI, USA) was incubated in 10 mi of LB broth (Bacto-trypton 10 g, Bacto-yeast extract 5 g, NaCl 5 g) until the optical density at 600 nm reached about 0.3 to 0.6.
  • the cultured mixture was kept at 0 ° C for 10 minutes and centrifuged at 4,000 rpm at 4 ° C for 10 min. The supernatant was removed and cells were harvested.
  • the harvested cell pellet was exposed to 10 mi of 0.1 M CaCl 2 at 0 ° C for 30 min to 1 hour to obtain competent cells.
  • the resultant was centrifuged at 4,000 rpm at 4 ° C for another 10 min and the collected cells were suspended in 2 mi of 0.1 M CaCl 2 at 0°C .
  • LB agar plates containing ampicillin were prepared by spreading 25 ⁇ i of X-gal (40 mg/ i stock in dimethylformamide) on top of agar with a glass spreader. 25 ⁇ i of the transformed cells thus obtained was spread thereon and the plates were incubated at a 37 ° C incubator overnight. White colonies were loaded on an LB agar plate containing ampicillin and transformed E. coli, i.e., JM109/CA245 were selected and incubated in a terrific broth (TDW 900 mi, Bacto-trypton 12 g, Bacto-yeast extract 24 g, glycerol 4 ml, 0.17 M KH 2 P0 4 , 0.72 N K 2 HP0 4 100 mi).
  • TDW 900 mi Bacto-trypton 12 g
  • Bacto-yeast extract 24 g glycerol 4 ml, 0.17 M KH 2 P0 4 , 0.72 N K 2 HP0 4 100 mi).
  • the CA245 plasmid DNA of the transformed E. coli was separated by employing WizardTM Plus Minipreps DNA Purification Kit (Promega, USA) in accordance with the manufacturer's instructions.
  • a portion of the plasmid DNA thus separated was treated with ECoRI enzyme and subjected to gel electrophoresis to confirm the insertion of CA245 gene in the plasmid.
  • the CA245 PCR product obtained in Example 2 was subjected to PCR in accordance with the conventional method and the cloned, reamplified CA245 PCR fragments were subjected to sequence analysis according to the dideoxy chain termination method using a Sequenase version 2.0 DNA sequencing kit (United states Biochemical, Cleveland, OH) in accordance with the manufacturer's instructions.
  • the base sequence of the DNA corresponds to nucleotide Nos. 705-851 in
  • SEQ ID NO: 1 SEQ ID NO: 1 and is designated "CA245".
  • the differential display reverse transcription polymerase chain reaction (DDRT-PCR) of the 147 bp cDNA fragment, i.e., CA245 obtained above was carried out using the 3' anchored primer H-T11A of SEQ ID NO: 3 and the 5' arbitrary primer H-AP24 of SEQ ID NO: 4 and resolved by electrophoresis.
  • Identification of altered gene expression by DD in the primary cervical cancer, metastatic lymph node tissue and CUMC-6 cells is shown in Fig 1.
  • the 147 bp cDNA fragment, i.e., CA245 was expressed in the cervical cancer, metastatic tissue and CUMC-6 cervical cancer cells but not in the normal tissue.
  • a bacteriophage ⁇ gtl l human lung embryonic fibroblast cDNA library (Miki, T. et al., Gene 83: 137-146, 1989) was screened by plaque hybridization with 32 P-labeled CA245 as a probe.
  • the full-length HCC-6 cDNA clone, containing a 899 bp insert in pCEV-LAC vector was obtained from the human lung embryonic fibroblast cDNA library.
  • HCC-6 clone inserted into ⁇ pCEV vector was excised out of the phage in the form of the ampicilline-resistant pCEV-LAC phagemid vector (Miki, T. et al., supra) by Not I cleavage.
  • pCEV-LAC vector containing HCC-6 gene was ligated with T4 D ⁇ A ligase and ligated clone was transformed into E. coli DH5 ⁇ .
  • the full open reading frame of the HCC-6 protooncogene of the present invention corresponds to nucleotides Nos. 174 to 854 and is predicted to encode amino acid sequence shown in SEQ ID NO: 2 which consists of 266 amino acids.
  • Example 7 Northern blot analysis of the HCC-6 gene in various cells Total RNAs were extracted from normal cervical tissues, cervical cancer tissues and cervical cancer cell lines (CaSki and CUMC-6) as in Example 1.
  • HCC-6 gene expression 20 ⁇ g denatured total RNAs from each tissue or cell lines were electrophoresed through 1% formaldehyde agarose gel and transferred to nylon membranes (Boehringer- Mannheim, Germany). The blots were hybridized with a 32 P-labeled random- primed HCC-6 full cDNA probe which was prepared using a Rediprime II random prime labeling system (Amersham, England). The northern blot analysis was repeated twice and the results were quantified by densitometry and normalized with ⁇ -actin.
  • Fig. 2A shows the results of northern blot analyses for HCC-6 gene expressed in normal cervical tissues, cervical cancer tissues and cervical cancer cell lines (CaSki and CUMC-6). As can be seen in Fig. 2A, the transcription level of HCC-6 is high in the cervical cancer tissues and cancer cell lines (CaSki (ATCC CRL 1550) and CUMC-6), but very low or undetactable in the normal cervical tissues.
  • Fig. 3A shows the results of northern blot analyses for HCC-6 gene expressed in normal human 12-lane multiple tissues; brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and leukocyte tissues (Clontech).
  • Fig. 3B shows the results obtained with the same samples hybridized with a ⁇ -actin probe to confirm mRNA integrity.
  • HCC-6 mRNA ( ⁇ 1.0 kb) is weakly present or absent in many normal tissues.
  • FIG. 4A shows the results of northern blot analyses for HCC-6 gene expressed in human cancer cell lines; HL-60, HeLa, K-562, MOLT-4, Raji, SW480, A549 and G361 (Clontech).
  • Fig. 4B shows the results obtained with the same samples hybridized with a ⁇ -actin probe to confirm mRNA integrity. As can be seen in Fig.
  • HCC-6 is transcribed at a high level in the promyelocytic leukaemia HL-60, HeLa cervical cancer cell, chronic myelogenous leukaemia K- 562, lymphoblastic leukaemia MOLT-4, Burkitt's lymphoma Raji, SW480 colon cancer cell, A549 lung cancer cell, and G361 melanoma cell.
  • Example 8 Determination of the size of the protein expressed after the transfection of E. Coli with HCC-6 protooncogene
  • HCC-6 protooncogene of SEQ ID NO: 1 was inserted into the multiple cloning site of pGEX-4T-3 vector (Amersham Pharmacia) and the resulting pGEX-4T-3/HCC-6 vector was transfected into E. coli BL21 (ATCC 47092).
  • Glutathione S transferase GST
  • the transfected E. coli was incubated using an LB broth medium in a rotary shaking incubator, diluted by 1/100, and incubated for 3 hours. 1 mM isopropyl ⁇ -D-thiogalacto-pyranoside (IPTG, Sigma) was added thereto to induce the protein synthesis.
  • Fig. 5 shows the SDS-PAGE results, which exhibit a protein expression pattern of the E. coli BL21 strain, transfected with pGEX-4T-3/HCC-6 vector. After the IPTG induction, a significant protein band was observed at about 31 kDa. This 31 kDa fused protein contained GST protein of about 26 kDa and HCC-6 protein of about 5.
  • Tins International Dc ⁇ sitai ⁇ Authority accepts the microorganism identified under 1 above, which was received bv it on April 10 2001.
  • N,iiii Korean Collection for Type Cultures S ⁇ gnature(.s) of pcrson(s) having (he power to represent the InterniiLion.il Depositary Authority of authorized ff" MKS)'

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Abstract

L'invention concerne une proto-oncogenèse humaine présentant une séquence de base de SEQ ID NO :1 ou un fragment de ladite séquence, qui est surexprimée dans divers tissus cancéreux et peut s'utiliser dans le diagnostic de différents cancers. Un gène antisens complémentaire de ladite proto-ongogenèse humaine peut être utilisé dans le traitement de cancers.
PCT/KR2002/001264 2002-07-04 2002-07-04 Proto-oncogenese humaine 6 et proteine codee dedans WO2004005514A1 (fr)

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AU2002345414A AU2002345414A1 (en) 2002-07-04 2002-07-04 Human protooncogene 6 and protein encoded therein

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6027905A (en) * 1996-08-30 2000-02-22 Matritech, Inc. Methods for the detection of cervical cancer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6027905A (en) * 1996-08-30 2000-02-22 Matritech, Inc. Methods for the detection of cervical cancer

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 2 May 2002 (2002-05-02), KIM J.W., Database accession no. (AF368271) *
DATABASE GENBANK [online] 28 December 2001 (2001-12-28), MACA-MEYER N. ET AL., Database accession no. (AF382006) *
MADRIGAL M. ET AL.: "In vitro antigene therapy targeting HPV-16 E6 and E7 in cervical carcinoma", GYNECOL. ONCOL., vol. 64, no. 1, January 1999 (1999-01-01), pages 18 - 25, XP002234549 *
NGAN H.Y. ET AL.: "Proto-oncogenes and p53 protein expression in normal cervical stratified squamous epithelium and cervical intra-epithelial neoplasia", EUR. J. CANCER, vol. 35, no. 10, October 1999 (1999-10-01), pages 1546 - 1550, XP002974542, DOI: doi:10.1016/S0959-8049(99)00166-5 *

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