JP2011103889A - Modulation of telomere-initiated cell signaling transmission - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide modulation of telomere-initiated cell signaling transmission. <P>SOLUTION: The invention relates to the use of modulators of Mre 11, tankyrase, the DNA damage pathway and MRN complex formation for protecting mammals from failure of growth arrest, apoptosis or proliferative senescence. A method for screening modulators of the Mre 11 disclosed in the invention comprises the following steps: (a) a step of contacting candidate modulators with the Mre 11 in vitro in the presence of nucleic acid substrates to the Mre 11; and (b) a step of comparing with comparison by measuring hydrolysis of the substrates and identifying the modulators by changing the hydrolysis of the substrates. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

(Citation of related application)
This application is a partially pending application of International Application No. PCT / US03 / 11393 filed on April 11, 2003, the entire text of which is incorporated herein by reference.

(Background of the Invention)
(1. Technical field)
The present invention relates to the control of signal transduction pathways. More particularly, the present invention relates to the control of telomere initiation sequences, apoptosis, sunburn and DNA damage responses.

(2. Prior art)
As the population ages, the frequency of human cancer is increasing in the developed world. Despite intensive research, morbidity and mortality in recent years have not improved sufficiently at the stage of symptoms in certain types of cancer and diagnosis. Tumor cells are increasingly from negative regulatory controls, including resistance to aging and apoptosis, an important aspect of how normal cells interact with their tissue-specific environment during cancer progression Become independent.

Cell aging has been suggested to be an important defense against cancer. Many facts imply progressive telomere shortening or telomere dysfunction caused by the inability to replicate the 3 ′ end of the chromosome in aging. In germline cells and most cancer cells, immortality is associated with the maintenance of telomere length by telomerase, an enzyme complex that adds a TTAGGG repeat to the 3 ′ end of the chromosome. Telomere, a ligated repeat of TTAGGG, has a 3 ′ overhang of about 150-300 bases, stabilized with a telomeric repeat binding factor (TRF), particularly TRF2, which is hooked with proximal telomeric double-stranded DNA. It ends with a single-stranded loop structure. Ectopic expression of a dominant negative form of TRF2 (TRF2 ^DN ) disrupts the telomere loop structure and exposes the 3 'overhang, causing a DNA damage response, resulting in senescence of primary fibroblasts and fibrosarcoma.

  Aging can be accelerated rapidly by extensive DNA damage or overexpression of certain oncogenes. Enzymatic maintenance of telomere length or ectopic expression of the constructed telomerase reverse transcriptase catalytic subunit (TERT) can bypass senescence, thereby immortalizing certain human cell types However, this strongly suggests a telomere-dependent mechanism of replication aging. In addition, malignant cells generally contain mutations that express TERT and / or have cells that bypass senescence and proliferate indefinitely despite having shorter telomeres than normal senescent cells. However, certain tumor cells age in response to various anticancer drugs, suggesting that the acquisition of immortality does not necessarily mean a lack of a basic cellular response to its DNA damage.

  Senescence in human cells is highly dependent on the p53 and pRb pathways. The tumor suppressor p53 is responsible for cell growth arrest or apoptosis in a variety of different stimuli such as DNA damage, transcriptional or replication deregulation, oncogene transformation, and microtubule deregulation caused by certain chemotherapeutic agents. Transforming it plays a key role in the cellular stress response mechanism. When activated, p53 causes cell growth arrest or programmed cell suicide, while acting as an important regulatory mechanism for genome stability. In particular, p53 controls genomic stability by removing genetically damaged cells from the cell population, and one of its main functions is to prevent tumor formation.

The intact tumor suppressor pRb pathway is necessary to prevent tumorigenesis. In pRb ^{− / −} tumor cells that do not contain wild-type p53, introduction of pRb induces senescence. Cervical cancer cells often maintain wild-type p53 and pRb genes, whereas HPV E6 and E7 proteins interfere with the p53 and pRb pathways, respectively. Ectopic expression of viral E2 protein represses HPV E6 and E7 gene transcription and induces a rapid and prominent senescence response in cervical carcinoma cell lines, which also confirms an important role for p53 and pRb in cancer cell senescence To do.

  Inhibiting only the p53 and pRb pathways is not sufficient for fibroblasts to bypass replication senescence. Indeed, human fibroblasts infected with the SV40T antigen or introduced with a combination of adenovirus E1A + E1B or HPV E6 + E7 that suppress the p53 and pRb pathways prolong life and escape replication senescence.

  Double-strand breaks in DNA are very cytotoxic to mammalian cells. A highly conserved MRN complex is involved in the repair of double strand breaks in eukaryotic cells. The MRN complex attaches to the double-strand break site as soon as it is generated. The MRN complex also migrates to telomeres during the S phase of the cell cycle associated with telomere repeat binding factor (TRF).

  The MRN complex is composed of Mre11, Rad50 and NBS (p95). Mre11 as part of the Mre11 / p95 / Rad50 complex associates with telomeric 3 'overhanging DNA during the S phase of the cell cycle. Mre11 is an exonuclease that preferentially acts on the 3 'end of the DNA strand. The activity of Mre11 is believed to depend on the interaction with Rad50, a type of ATPase. Nbs1 is believed to be involved in the nuclear localization of the MRN complex as well as its association at the site of double-strand breaks.

  Typically, cancer is treated with highly toxic therapies such as chemotherapy and radiation therapy that severely damage all proliferating cells, whether normal or malignant. Side effects of such treatment include hair loss as well as severe damage to the lymphatic system, hematopoietic system and intestinal epithelium. Other side effects include hair loss. There is always a need for safer and more effective cancer treatments, especially alternative treatments that avoid some or all of these side effects by preferentially targeting malignant cells to normal but proliferating cells It continues to be.

(Summary of the Invention)
The present invention relates to an in vitro screening method for a modulator of Mre11, which comprises a step of contacting a candidate modulator with Mre11 in vitro in the presence of a nucleic acid substrate for Mre11 and measuring the hydrolysis of the substrate. Modulators can be identified by altering the hydrolysis of the substrate nucleic acid compared to the control. The nucleic acid substrate can be an oligonucleotide having at least 50% nucleotide sequence identity with (TTAGGG) _n (n = 1-20). Hydrolysis of the substrate nucleic acid can be measured by UV absorption, gel analysis of labeled oligomers, or recovery of non-precipitated nucleotide bases.

  The present invention also relates to an in vitro screening method for an agent that specifically binds to Mre11, the method comprising contacting a candidate agent with Mre11 and determining whether the candidate agent specifically binds to Mre11. Mre11 may be attached to a solid support.

  The present invention is also a cell-based screening method for a regulator of Mre11, comprising contacting a candidate regulator with a cell expressing Mre11 under conditions where the regulator is taken up by the cell, cell proliferation, cell viability, It relates to a method comprising measuring cell morphology, SA-b-Gal activity, and cellular properties including but not limited to phosphorylation of p53 or p95. Modulators can be identified by changing properties compared to controls. The candidate modulator may be an agent that specifically binds to Mre11 as identified above. Mre11 may be expressed as a fragment, homolog, analog or variant of Mre11 that may have exonuclease activity.

  The present invention also relates to an in vitro screening method for tankyrase modulators, the method comprising contacting a candidate modulator with tankyrase in vitro in the presence of a substrate for tankyrase and measuring the ribosylation of the substrate. Modulators can be identified by altering ribosylation relative to controls. The substrate can be a peptide or polypeptide that can be TRF. Substrate ribosylation may be measured by UV absorption or substrate labeling.

  The present invention also relates to an in vitro screening method for an agent that specifically binds to tankyrase, wherein the candidate agent is contacted with tankyrase to determine whether the candidate agent specifically binds to tankyrase. Tankyrase may be attached to a solid support.

  The present invention is also a cell-based screening method for a tankyrase regulator, wherein the candidate regulator and a cell expressing tankyrase are contacted under conditions where the regulator is taken up by the cell, cell proliferation, cell viability, It relates to a method comprising measuring cellular morphology, SA-b-Gal activity, and cellular properties including but not limited to p53 phosphorylation or p95 phosphorylation. Modulators can be identified by changing properties compared to controls. Candidate modulators can be agents that specifically bind to tankyrase as described above. Tankyrases may be expressed as fragments, homologues, analogs or variants of tankyrase that may have ribosylase activity.

The invention also relates to an in vitro screening method for modulators of MRN complex formation, comprising the step of contacting candidate modulators with Mre11, Rad50 and Nbs1 in vitro and measuring the formation of the MRN complex. Modulators can be identified by altering the formation of the MRN complex relative to the control. Candidate modulators can be contacted with Mre11, Rad50 and Nbs1 in the presence of nucleic acid substrates or inhibitors of Mre11. The nucleic acid may be an oligonucleotide having at least 50% nucleotide sequence identity with (TTAGGG) _n (n = 1-20). MRN complex formation can be measured by centrifugation, coprecipitation or non-denaturing electrophoresis.

The present invention is also a cell-based screening method for a regulator of a DNA damage pathway, comprising a cell that expresses Mre11 and tankyrase in the presence of an oligonucleotide and a candidate regulator under conditions in which the regulator is taken up by the cell. And measuring cell properties including but not limited to cell proliferation, cell viability, cell morphology, SA-b-Gal activity, and p53 phosphorylation or p95 phosphorylation. Modulators can be identified by changing properties compared to controls. The oligonucleotide may have at least 50% nucleotide sequence identity with (TTAGGG) _n (n = 1-20). Mre11 may be expressed as a fragment, homolog, analog or variant of Mre11 that may have exonuclease activity. Tankyrases may be expressed as fragments, homologues, analogs or variants of tankyrase that may have ribosylase activity.

  The cell used in the above cell-based screening method may be a cancer cell. Cells used in the cell-based screening methods described above can maintain telomeres by telomerase reverse transcriptase or the ALT pathway. Candidate modulators and agents described in the above in vitro and cell-based screening methods include carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, It may be a polynucleotide, lipid, retinoid, steroid, glycopeptide, glycoprotein, proteoglycan or small organic molecule.

The invention also relates to the use of a composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator. Activators can be used to treat cancer, induce apoptosis, induce cellular senescence, inhibit sunburn, promote cell differentiation or promote immunosuppression. The activator may be an oligonucleotide activator of Mre11 that may have at least 50% nucleotide sequence identity with (TTAGGG) _n (n = 1-20). About 1 to about 10 of the first 3 ′ nucleotide bonds can be hydrolyzed with a 3 ′ → 5 ′ nuclease.

The present invention also relates to the use of compositions comprising inhibitors of Mre11, tankyrase, DNA damage pathway or MRN complex formation. Inhibitors can be used to inhibit apoptosis, inhibit cell aging, promote growth, promote sunburn, inhibit cell differentiation, reduce cancer treatment side effects. The composition may be given in combination with chemotherapy or ionizing radiation. The inhibitor may be an oligonucleotide inhibitor of Mre11 that may have at least 50% nucleotide sequence identity with (TTAGGG) _n (n = 1-20). From about 0 to about 10 of the first 3 ′ nucleotide linkage can be hydrolyzed with a 3 ′ → 5 ′ nuclease.
The invention also relates to a composition comprising an oligonucleotide having at least 50% nucleotide sequence identity with (TTAGGG) _n (n = 1-20) and having at least one non-hydrolyzable internucleotide linkage. . From 1 to about 10 of the first 3 ′ nucleotide bond can be hydrolyzed with a 3 ′ → 5 ′ nuclease such as Mre11. The oligonucleotide may have at least 50% nucleotide sequence identity with TTAGGG. The oligonucleotide may have the sequence GTTAGGGTTTAG. The non-hydrolyzable bond may be a phosphorothioate. The oligonucleotide may be PNA.
The present invention further provides the following items.
(Item 1)
A method for screening for a modulator of Mre11 comprising:
(A) contacting a candidate modulator with Mre11 in vitro in the presence of a nucleic acid substrate for Mre11; and
(B) measuring the hydrolysis of the substrate, thereby identifying a modulator by altering the hydrolysis of the substrate relative to a control;
Including the method.
(Item 2)
The method according to item 1, wherein the nucleic acid substrate is an oligonucleotide having at least 50% nucleotide sequence identity with (TTAGGG) _n (n = 1 to 20).
(Item 3)
Item 2. The method according to Item 1, wherein hydrolysis of the nucleic acid substrate is measured by UV absorption or release of a radioactive label.
(Item 4)
A method for screening for a factor that specifically binds to Mre11, the method comprising:
(A) contacting the candidate factor with Mre11; and
(B) determining whether the candidate factor specifically binds to Mre11;
Including the method.
(Item 5)
Item 5. The method according to Item 4, wherein Mre11 is attached to a solid support.
(Item 6)
A method for screening for a modulator of Mre11 comprising:
(A) providing a cell expressing Mre11;
(B) contacting the candidate modulator with the cell under conditions such that the modulator is taken up by the cell; and
(C) measuring the properties of the cells selected from the group consisting of cell proliferation, cell viability, cell morphology, SA-β-Gal activity and p53 phosphorylation, p95 phosphorylation, and thereby compared to a control Identifying a modulator by altering the property;
Including a method.
(Item 7)
Item 7. The method according to Item 6, wherein the candidate modulator binds specifically to Mre11.
(Item 8)
The method according to any one of items 1 to 7, wherein the Mre11 is an Mre11 fragment, an Mre11 homolog, an Mre11 analog, or an Mre11 variant.
(Item 9)
Item 9. The method according to Item 8, wherein the Mre11 fragment, Mre11 homologue, Mre11 analog or Mre11 variant has exonuclease activity.
(Item 10)
Item 7. The method according to Item 6, wherein the cell characteristic is cell proliferation.
(Item 11)
Item 7. The method according to Item 6, wherein the cell property is cell viability.
(Item 12)
Item 7. The method according to Item 6, wherein the cell characteristic is cell morphology.
(Item 13)
Item 7. The method according to Item 6, wherein the cell characteristic is SA-β-Gal activity.
(Item 14)
Item 7. The method according to Item 6, wherein the cell characteristic is phosphorylation of p53 or phosphorylation of p95.
(Item 15)
15. The method according to any one of items 6 to 7 and 9 to 14, wherein the cell is a cancer cell.
(Item 16)
16. The method of item 15, wherein the cellular telomere is maintained by telomerase reverse transcriptase or the ALT pathway.
(Item 17)
Item 9. The method according to Item 8, wherein the cell is a cancer cell.
(Item 18)
18. The method of item 17, wherein the cellular telomere is maintained by telomerase reverse transcriptase or the ALT pathway.
(Item 19)
The candidate modulators are carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, polypeptides, proteins, nucleosides, oligonucleotides, polynucleotides, lipids, retinoids, steroids, glycopeptides, glycoproteins, proteoglycans, and organic 16. The method according to item 15, wherein the method is selected from the group consisting of small molecules.
(Item 20)
20. The method of item 19, wherein the cellular telomere is maintained by telomerase reverse transcriptase or the ALT pathway.
(Item 21)
The candidate modulators are carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, polynucleotides, lipids, retinoids, steroids, glycopeptides, glycoproteins 18. The method according to item 17, wherein the method is selected from the group consisting of: a proteoglycan, and a small organic molecule.
(Item 22)
22. The method of item 21, wherein the cellular telomere is maintained by telomerase reverse transcriptase or the ALT pathway.
(Item 23)
A method of screening for regulators of tankyrase, the method comprising:
(A) contacting the candidate modulator with the tankyrase in vitro in the presence of a substrate for tankyrase; and
(B) identifying a modulator by measuring ribosylation of the substrate, thereby altering ribosylation of the substrate relative to a control;
Including the method.
(Item 24)
24. The method of item 23, wherein the substrate is a peptide or polypeptide.
(Item 25)
25. A method according to item 24, wherein the substrate is TRF1.
(Item 26)
24. The method of item 23, wherein the ribosylation of the substrate is measured by UV absorption or labeling of the substrate.
(Item 27)
A method for screening a factor that specifically binds to tankyrase, the method comprising:
(A) contacting the candidate binding agent with a tankyrase; and
(B) determining whether the candidate factor specifically binds to tankyrase;
Including the method.
(Item 28)
28. A method according to item 27, wherein the tankyrase is attached to a solid support.
(Item 29)
A method for screening for regulators of tankyrase comprising:
(A) providing a cell expressing tankyrase;
(B) contacting the candidate modulator with the cell under conditions such that the modulator is taken up by the cell; and
(C) measuring the properties of the cells selected from the group consisting of cell proliferation, cell viability, cell morphology, SA-β-Gal activity and p53 phosphorylation, p95 phosphorylation, and thereby compared to a control Identifying a modulator by altering the property;
Including the method.
(Item 30)
30. The method of item 29, wherein the modulator binds specifically to tankyrase.
(Item 31)
31. The method according to any one of items 23 to 30, wherein the tankyrase is a tankyrase fragment, tankyrase homolog, tankyrase analog or tankyrase variant having ribosylation activity.
(Item 32)
32. The method of item 31, wherein the tankyrase fragment, tankyrase homolog, tankyrase analog or tankyrase variant has ribosylation activity.
(Item 33)
30. A method according to item 29, wherein the characteristic of the cell is cell proliferation.
(Item 34)
30. The method of item 29, wherein the cell property is cell viability.
(Item 35)
30. The method of item 29, wherein the cell property is cell morphology.
(Item 36)
30. The method of item 29, wherein the cell property is SA-β-Gal activity.
(Item 37)
30. The method of item 29, wherein the cell characteristic is phosphorylation of p53 or phosphorylation of p95.
(Item 38)
38. A method according to any of items 29-30 and 32-37, wherein the cells are cancer cells.
(Item 39)
40. The method of item 38, wherein the cellular telomere is maintained by telomerase reverse transcriptase or the ALT pathway.
(Item 40)
32. The method of item 31, wherein the cell is a cancer cell.
(Item 41)
41. The method of item 40, wherein the cellular telomere is maintained by telomerase reverse transcriptase or the ALT pathway.
(Item 42)
The candidate modulators are carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, polynucleotides, lipids, retinoids, steroids, glycopeptides, glycoproteins 40. The method of item 38, selected from the group consisting of: a proteoglycan, and a small organic molecule.
(Item 43)
44. The method of item 42, wherein the cellular telomere is maintained by telomerase reverse transcriptase or the ALT pathway.
(Item 44)
The candidate modulators are carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, polynucleotides, lipids, retinoids, steroids, glycopeptides, glycoproteins 41. The method of item 40, which is selected from the group consisting of: a proteoglycan, and a small organic molecule.
(Item 45)
45. The method of item 44, wherein the cellular telomere is maintained by telomerase reverse transcriptase or the ALT pathway.
(Item 46)
A method for screening for modulators of MRN complex formation comprising:
(A) contacting the candidate modulator with Mre11, Rad50 and Nbs1 in vitro; and
(B) measuring the formation of the MRN complex, thereby identifying the modulator by altering the formation of the MRN complex compared to the control
Including the method.
(Item 47)
49. The method of item 46, wherein the candidate modulator is contacted with Mre11, Rad50 and Nbs1 in the presence of a nucleic acid substrate or Mre11 inhibitor.
(Item 48)
48. The method of item 47, wherein the nucleic acid has at least 50% nucleotide sequence identity with (TTAGGG) _n (n = 1-20).
(Item 49)
47. A method according to item 46, wherein the formation of the MRN complex is measured by centrifugation, coprecipitation or non-denaturing electrophoresis.
(Item 50)
A method for screening for a regulator of a DNA damage pathway comprising:
(A) providing a cell expressing Mre11 and tankyrase;
(B) contacting the candidate modulator with the cell in the presence of an oligonucleotide under conditions that allow the modulator to be taken up by the cell;
(C) measuring the properties of the cells selected from the group consisting of cell proliferation, cell viability, cell morphology, SA-β-Gal activity and p53 phosphorylation, p95 phosphorylation, and thereby compared to a control Identifying a modulator by altering the property
Wherein the oligonucleotide has at least 50% nucleic acid sequence identity with (TTAGGG) _n (n = 1-20).
(Item 51)
51. A method according to item 50, wherein the Mre11 is an Mre11 fragment, an Mre11 homolog, an Mre11 analog, or an Mre11 variant.
(Item 52)
52. The method of item 51, wherein the Mre11 fragment, Mre11 homologue, Mre11 analog or Mre11 variant of the Mre11 has exonuclease activity.
(Item 53)
51. A method according to item 50, wherein the tankyrase is a tankyrase fragment, a tankyrase homolog, a tankyrase analog, or a tankyrase variant.
(Item 54)
54. The method according to Item 53, wherein the tankyrase is a tankyrase fragment, a tankyrase homolog, a tankyrase analog, or a tankyrase variant has ribosylation activity.
(Item 55)
51. A method according to item 50, wherein the characteristic of the cell is cell proliferation.
(Item 56)
51. The method of item 50, wherein the cell property is cell viability.
(Item 57)
51. The method of item 50, wherein the cell characteristic is cell morphology.
(Item 58)
51. The method of item 50, wherein the cell characteristic is SA-β-Gal activity.
(Item 59)
51. The method of item 50, wherein the cell characteristic is p53 phosphorylation or p95 phosphorylation.
(Item 60)
60. The method according to any one of items 50 to 59, wherein the cell is a cancer cell.
(Item 61)
62. The method of item 61, wherein the cellular telomere is maintained by telomerase reverse transcriptase or the ALT pathway.
(Item 62)
The candidate modulators are carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, polynucleotides, lipids, retinoids, steroids, glycopeptides, glycoproteins 51. The method of item 50, selected from the group consisting of: a proteoglycan, and a small organic molecule.
(Item 63)
A method for treating cancer comprising the steps of:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Including the method.
(Item 64)
A method for inducing apoptosis comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Including the method.
(Item 65)
A method for inducing cellular senescence comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Including the method.
(Item 66)
A method for inhibiting sunburn, the method comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Inhibition methods comprising:
(Item 67)
A method for promoting cell differentiation, the method comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Including the method.
(Item 68)
A method for promoting immunosuppression comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Including the method.
(Item 69)
The activator is an oligonucleotide activator of Mre11 having at least 50% nucleotide sequence identity with (TTAGGG) _n , wherein at least the first x 3′-nucleotide linkages are hydrolyzed by a 3 ′ → 5 ′ nuclease. 69. The method of any one of items 63-68, wherein the method is decomposable, wherein n = 1-20 and x is about 1 to about 10.
(Item 70)
A method for inhibiting apoptosis comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Inhibition methods comprising:
(Item 71)
A method for inhibiting cellular senescence comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Including the method.
(Item 72)
A method for promoting growth comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Including the method.
(Item 73)
A method for promoting sunburn, the method comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Including the method.
(Item 74)
A method for inhibiting cell differentiation comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Including the method.
(Item 75)
A method for reducing the side effects of cancer treatment comprising:
A composition comprising an Mre11 activator, a composition comprising a tankyrase activator, a composition comprising a DNA damage pathway activator, or a composition comprising an MRN complexing activator in a subject in need of such treatment The step of administering
Including the method.
(Item 76)
76. The method of item 75, wherein the composition is given in combination with chemotherapy or ionizing radiation.
(Item 77)
The inhibitor is an oligonucleotide inhibitor of Mre11 having at least 50% nucleotide sequence identity with (TTAGGG) _n, and at least the first x 3′-nucleotide linkages can be hydrolyzed by a 3 ′ → 5 ′ nuclease 77. The method of items 70-76, wherein n = 1-20 and x is from about 0 to about 10.
(Item 78)
(TTAGGG) A composition comprising an oligonucleotide having at least 50% nucleotide sequence identity with _n and at least one non-hydrolyzable internucleotide linkage, wherein at least the first x 3′-nucleotide linkages Is hydrolyzable by 3 ′ → 5 ′ nuclease, n = 1-20, and x is from about 0 to about 10.
(Item 79)
79. A composition according to item 78, wherein the 3 ′ → 5 ′ nuclease is Mre11.
(Item 80)
79. The composition of item 78, wherein the oligonucleotide has at least 50% nucleotide sequence identity with TTAGGG.
(Item 81)
81. The composition of item 80, wherein the oligonucleotide has the sequence GTTAGGGTTTAG.
(Item 82)
79. A composition according to item 78, wherein the non-hydrolyzable bond is phosphorothioate.
(Item 83)
79. A composition according to item 78, wherein the oligonucleotide is PNA.

1A-1H are diluents (FIGS. 1A and 1E); 40 μM 11mer-1 pGTTAGGGGTTAG (SEQ ID NO: 2 FIG. 1B and 1F); 40 μM 11mer-2 pCTAACCCCTAAC (SEQ ID NO: 3 FIG. 1C and 11G) Shows FACS analysis of propidium iodide stained Jurkat cells (immortalized T lymphocytes) treated with 40 μM 11mer-3 pGATCGATCGAT (SEQ ID NO: 4: FIGS. 1D and 1H). Jurkat cells were treated with the reagents mentioned for 48 hours before analysis (FIGS. 1A-1D) or 72 hours (FIGS. 1E-1H). 2A-2F are graphs showing the results of fluorescence activated cell sorting for addition to the following cells: FIG. 2A, diluent; FIG. 2B, 0.4 μM of 11mer-1; FIG. 2D, diluent; FIG. 2E, 40 μM 11mer-1; FIG. 2F, 40 μM 11mer-1-S. FIGS. 3A-3G are graphs showing the results of fluorescence activated cell sorting for addition to the following cells: FIG. 3A, diluent; FIG. 3B, 10 μM 11mer-1; FIG. 3C, 10 μM 11mer. 3D, 10 μM 11mer-1 and 5 μM 11mer-1-S; FIG. 3E, 10 μM 11mer-1 and 10 μM 11mer-1-S; FIG. 3F, 20 μM 11mer-1-S; FIG. 3G, 10 μM 11mer-1-S. FIG. 4 is a bar graph showing the melanin content (pg / cell) of cells treated with diluent, pTpT or pTspT. FIG. 5 is a bar graph showing the melanin content (pg / cell) of cells treated with diluent, 11mer-1 or 11mer-1-S. FIG. 6 shows the melanin content (pg / cell) of cells treated with mock treatment (no irradiation, no oligonucleotide), or ultraviolet (UV) treatment, or administration of pTspT without irradiation, or UV irradiation and pTspT administration It is a bar graph. FIG. 7 is a diagram of the oligonucleotide of SEQ ID NO: 2 of the nucleotide sequence synthesized with a phosphorothioate linkage. FIG. 8 shows the results of a test of the effect of phosphorothioate oligonucleotides 1, 2, 3 and 4 causing senescence in cultures of normal neonatal human fibroblasts, shown in FIG. 7 as positive for cell staining for β-galactosidase activity. It is a bar graph which shows. Oligonucleotide “11-1” represents a fibroblast culture treated with SEQ ID NO: 2, which was synthesized entirely with phosphodiester bonds. “Dil” refers to a fibroblast culture treated with a diluent that does not contain oligonucleotides. Figures 9-11 show that down-regulated Mre11 protein levels interfere with the T-oligo reaction. Figures 9-11 show that down-regulated Mre11 protein levels interfere with the T-oligo reaction. Figures 9-11 show that down-regulated Mre11 protein levels interfere with the T-oligo reaction. FIG. 12 shows that both p53 and pRb pathways contribute to T-oligo induced senescence in human fibroblasts. Figure 12a: Immunoblot analysis of p53DD and cdk4 ^R24C expression. Cells were collected for protein analysis by Western blot using 30 μg total protein and probed for total p53 and cdk4. Lanes 1, 2, 3, and 4 contain protein samples from R2F, R2F (p53DD), R2F (cdk4 ^R24C ), and R2F (p53DD / cdk4 ^R24C ) fibroblasts, respectively. Β-actin was used as a loading control. FIG. 12 shows that both p53 and pRb pathways contribute to T-oligo induced senescence in human fibroblasts. FIG. 12b: Contribution of p53 and pRb pathways to T-oligo-induced SA-β-Gal activity. R2F fibroblasts and induced transductants were treated with diluent or 40 μM T-oligo for 1 week and then analyzed for SA-β-Gal activity. FIG. 12 shows that both p53 and pRb pathways contribute to T-oligo induced senescence in human fibroblasts. FIG. 12c: Quantitative analysis of SA-β-Gal positive cells. Cells expressing SA-β-Gal activity were counted and presented as a percentage of total cells in culture. Means and standard deviations were calculated from 3 representative fields from each of 3 independent experiments. FIG. 13 shows that exposure of human fibrosarcoma HT-1080 cells to T-oligo induces senescence. FIG. 13a: Exposure to T-oligo increases SA-β-Gal activity. HT-1080 cells were treated with diluent alone or 40 μM T-oligo, or complementary control oligo for 4 days, then stained and analyzed for SA-β-Gal activity. FIG. 13 shows that exposure of human fibrosarcoma HT-1080 cells to T-oligo induces senescence. FIG. 13b: Quantitative analysis of SA-β-Gal positive cells. Cells expressing SA-β-Gal activity were counted and expressed as a percentage of total cells in the culture. Mean values and standard deviations were calculated from 3 representative fields from each of 3 independent experiments. FIG. 13 shows that exposure of human fibrosarcoma HT-1080 cells to T-oligo induces senescence. FIG. 13c: Effect of T-oligo on cell proliferation. Cells were treated for 4 days as in FIG. 12 and analyzed for DNA synthesis by BrdU incorporation. FIG. 13 shows that exposure of human fibrosarcoma HT-1080 cells to T-oligo induces senescence. FIG. 13d: Quantitative analysis of BrdU incorporation. Dark black nuclei indicate BrdU incorporated into nuclear DNA. BrdU positive cells were calculated and expressed as a percentage of total cells in the culture. Mean values and standard deviations were calculated from 3 representative fields from each of 3 independent experiments. FIG. 13 shows that exposure of human fibrosarcoma HT-1080 cells to T-oligo induces senescence. FIG. 13e: Effect of T-oligo on pRb phosphorylation. Cells were treated as in FIG. 13a and using 30 μg of total protein against pRb-ser780 ^* , ser795 ^* and ser807 / 811 ^* (pRb phosphorylated with serine 780, serine 795 and serine 807/811 respectively) Probed and collected for protein analysis by Western blot. Lanes D, T and C contain protein samples from cells treated with diluent, T-oligo and complementary oligo, respectively. β-actin was used as a loading control. FIG. 14 shows the sustained effect of T-oligo removal on the aging phenotype in human fibrosarcoma HT-1080 cells. Parallel cultures were processed as described in Figure 13a. Cells were then washed once with PBS and re-fed with complete media for 24 or 48 hours without further treatment. FIG. 14a: SA-β-Gal activity. Cells were stained for SA-β-Gal activity. FIG. 14 shows the sustained effect of T-oligo removal on the aging phenotype in human fibrosarcoma HT-1080 cells. Parallel cultures were processed as described in Figure 13a. Cells were then washed once with PBS and re-fed with complete media for 24 or 48 hours without further treatment. Figure 14b: Cell cycle arrest. BrdU incorporation was analyzed. FIG. 14 shows the sustained effect of T-oligo removal on the aging phenotype in human fibrosarcoma HT-1080 cells. Parallel cultures were processed as described in Figure 13a. Cells were then washed once with PBS and re-fed with complete media for 24 or 48 hours without further treatment. FIG. 14c: phosphorylation and activation of pRb. Immunoblot analysis was performed as described in FIG. 13e. FIG. 15 shows the effect of prolonged exposure to T-oligo on the clonogenic ability of human fibroblast sarcoma HT-1080 cells. Cells were treated with diluent, 40 μM T-oligo or complementary oligo for 1 week and analyzed. Fig. 15a: Appearance of the staining dish. FIG. 15 shows the effect of prolonged exposure to T-oligo on the clonogenic ability of human fibroblast sarcoma HT-1080 cells. Cells were treated with diluent, 40 μM T-oligo or complementary oligo for 1 week and analyzed. FIG. 15b: Quantification of clonogenic potential. Triplicate colonies were counted and plotted as a percentage of the diluent treatment control. FIG. 16 shows the effect of T-oligo on telomere mean length (MTL) in human fibrosarcoma HT-1080 cells. Cells were treated as described in Figure 13a. Lanes 1, 2 and 3 contain genomic DNA from cells treated with diluent (D), T-oligo (T) or complementary oligo (C). Lanes 4 and 5 contain high molecular weight (H) and low molecular weight (L) standard telomeric DNA. FIG. 17 shows that T-oligo and TRF ^DN initiate a DNA damage response via the same pathway. The graph shows Western blot densitometer readings with the diluent control at 100%. FIG. 9f: Lane 1, diluent, GFP; Lane 2: diluent TRF2DN; Lane 3: 3AB, GFP; Lane 4: 3AB, TRF2DN; Lane 5: IQ, GFP; Lane 6: IQ, TRF2DN. FIG. 18 shows that the effect of T-oligo is independent of telomeres. FIG. 18a: FACS graph from one representative experiment of 3 experiments, where the percentage of cells and the standard deviation at each phase of the cell cycle were calculated from 3 cultures of each condition. FIG. 18b: Western blot with an antibody specific for phospho p95 / Nbs1. Lanes 1, 2 and 3 contained proteins from cells treated with diluent, 11mer-1 or 11mer-2, respectively. Control cells were irradiated (+) with 10 Gy IR or sham irradiated (−) (3 hours). FIG. 19 shows that down-regulation of tankyrase protein levels interferes with the T-oligo reaction. The upper panel shows densitometer readings and the lower panel shows Western blots. FIG. 20 shows that T-oligo results in phosphorylation of p53 above at serine 37. After treatment for the time indicated with diluent or 40 μM, Western blot analysis was performed on normal neonatal cells using p53 phosphoserine 37 specific antibodies.

(Detailed description of the invention)
The present invention is based on the discovery that Mre11-mediated hydrolysis of the 3 ′ telomeric overhang sequence initiates a signaling cascade important for cellular protective responses against DNA damage such as senescence, sunburn and apoptosis. Without being bound by theory, we have UV radiation, DNA damage such as oxidative damage to DNA, or the production of carcinogenic adducts to DNA, or shortening of telomeres associated with aging has a TTAGGG repeat. It is believed to destabilize the telomere loop that exposes the 3 'overhang. The telomere-related protein then attaches to the overhang in a sequence-dependent manner and acts as an “anchor” for the Mre11 / p95 / Rad50 complex. Mre11 then begins to hydrolyze the telomere overhang from the 3 ′ end, causing activation of the Rad50 ATPase. Activation of Rad50 leads to activation of tankyrase by phosphorylation, certain spatial arrangement changes, or other mechanisms, and then activates other kinases such as ATM and possibly ATR. ATM then phosphorylates other DNA damage response effectors such as p95 and p53, ultimately leading to biological end points of cell cycle arrest, gene induction, apoptosis and / or senescence.

  Based on the role of Mre11 and tankyrase in the proposed signaling pathway, activators of Mre11, tankyrase, DNA damage pathway or MRN complex formation activate the DNA damage response pathway in the presence of DNA damage or telomere loop disruption It is expected to become. This is demonstrated in the examples herein that show that telomeric homolog oligonucleotides (T-oligos) are substrates for Mre11 and result in apoptosis, senescence or growth arrest without DNA damage or telomere loop disruption. ing.

  Similarly, inhibitors of Mre11, tankyrase, DNA damage pathway or MRN complex formation are expected to inhibit signaling pathways in the presence of DNA damage or telomere loop disruption. This is demonstrated in the examples herein that show that apoptosis and growth arrest are inhibited under conditions that result in DNA damage and telomere loop disruption by: (i) acting as an antagonist of Mre11 Non-hydrolyzable T-oligo, (ii) RNAi-mediated decrease in Mre11 protein level; and (iii) RNAi-mediated decrease in tankyrase protein level.

  Prior to disclosing and describing the products, compositions and methods of the present invention, the terminology used herein is for the purpose of describing particular embodiments only and is intended to be limiting. It is necessary to understand that. It should be noted that as used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. .

  Throughout this application, when patents or publications are cited, the full disclosures of these publications are incorporated herein by reference to more fully describe the state of the art to which this invention belongs.

(1. Definition)
As used herein, the term “activator” means any substance that activates or increases the activity of a protein.

  The term “administration” as used herein, when used to describe a dosage of a modulator, means a single dose or multiple doses of an agent.

  As used herein, the term “analog” when used in the sense of a peptide or polypeptide is a peptide having one or more non-standard amino acids or other structural changes from a set of normal amino acids. Or refers to a polypeptide, and when used in the sense of an oligonucleotide, means an oligonucleotide having one or more internucleotide linkages other than phosphodiester internucleotide linkages.

The term “antibody” as used herein refers to antibodies of class IgG, IgM, IgA, IgD or IgE, or fragments or derivatives thereof including Fab, F (ab ′) ₂ , Fd, and single chain antibodies, Bispecific antibodies, bispecific antibodies, bivalent antibodies and their derivatives are meant. The antibody may be a monoclonal antibody, a polyclonal antibody, an affinity purified antibody, or a mixture thereof that exhibits sufficient binding specificity for the desired epitope or a sequence derived from that epitope. The antibody may be a chimeric antibody. An antibody may be derivatized with the attachment of one or more chemical, peptide or polypeptide species known in the art. The antibody may be conjugated to a compound moiety.

  As used herein, the term “apoptosis” refers to the progressive reduction of cell volume while preserving the integrity of cytoplasmic organelles; chromatin condensation (ie, nuclear condensation) as observed with light or electron microscopy; and / or Or means a form of cell death, including but not limited to DNA cleavage into nucleosome-sized fragments as determined by centrifugal sedimentation analysis. Cell death occurs when the membrane integrity of the cell is lost (eg, membrane blebbing), with engulfment of intact cell fragments (apoptotic bodies) by phagocytic cells.

  As used herein, the term “treatment of cancer” means any treatment known in the art including, but not limited to, chemotherapy and radiation therapy.

  As used herein, the term “in combination with” when used to describe the administration of a modulator and other treatments may cause the modulator to be in conjunction with other treatments, or other treatments. It means that it can be administered after treatment or in combination.

  As used herein, the term “derivative”, when used in the context of a peptide or polypeptide, means a peptide or polypeptide that differs in other than the primary structure (amino acids and amino acid analogs); When used, it means a different oligonucleotide other than the nucleotide sequence. For example, a peptide or polypeptide derivative may differ by being glycosylated, which is a form of post-translational modification. For example, a peptide or polypeptide may exhibit a glycosylation pattern for expression in a heterologous system. If at least one biological activity is maintained, these peptides or polypeptides are derivatives according to the invention. Other derivatives include covalently modified N- or C-terminated fusion peptides or polypeptides, PEGylated peptides or polypeptides, peptides or polypeptides associated with lipid moieties, alkylated peptides or polypeptides, amino acids Peptides or polypeptides attached to other peptides, polypeptides or compounds via side chain functional groups, and other modifications as would be understood in the art, but are not limited thereto.

  The term “fragment” as used herein preferably, when used in the context of a peptide or polypeptide, is preferably about 5 to about 300 amino acids in length, more preferably about 8 to about 50 in length. Which, when used in the context of oligonucleotides, is preferably about 2 to about 250 nucleotides in length, more preferably about 2 to about 20 in length. Means any oligonucleotide fragment of one nucleotide. Representative examples of peptides or polypeptide fragments are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 in length. , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 Amino acids. Representative examples of oligonucleotide fragments are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 in length. Nucleotides.

  The term “homolog” as used herein means a peptide or polypeptide that, when used in the context of a peptide or polypeptide, shares a common evolutionary ancestry or has at least 50% identity thereto. And when used in the context of oligonucleotides, means an oligonucleotide that shares a common evolutionary ancestry or has at least 50% identity thereto.

  As used herein, the term “inhibition” when referring to the activity of a protein means to block, suppress, suppress or eliminate the activity of the enzyme.

  As used herein, the term “treatment” or “treating”, when referring to protecting a mammal from a condition, means preventing, suppressing, suppressing or eliminating the condition. Preventing a disease state includes administering the composition of the present invention to a mammal before the disease state is developed. Suppressing the condition includes administering the composition of the present invention to the mammal after induction of the condition but before its clinical manifestations. Suppressing a medical condition includes administering the composition of the present invention to a mammal after clinical symptoms of the medical condition appear so as to prevent the disease condition from being reduced or worsened. Eliminating the condition includes administering the composition of the present invention to the mammal after the clinical symptoms of the condition appear so that the mammal no longer develops the condition.

  The term “variant” as used herein, when used in the context of a peptide or polypeptide, differs in amino acid sequence due to amino acid insertions, deletions or conservative substitutions, but retains at least one biological activity. Refers to a peptide or polypeptide and, when used in the context of oligonucleotides, means an oligonucleotide that differs in nucleotide sequence by nucleotide insertion, deletion or conservative substitution, but retains at least one biological activity. For the purposes of the present invention, “biological activity” includes, but is not limited to, the ability to bind to a specific antibody.

(2. Regulatory factors)
(A. Regulatory factors of Mre11)
The present invention relates to modulators of Mre11 activity. A modulator may induce or increase Mre11 activity. A modulator may also inhibit or reduce Mre11 activity. The modulator may be an artificially synthesized compound or a naturally occurring compound. The modulator may be a low molecular weight compound, oligonucleotide, polypeptide or peptide, or a fragment, analog, homologue, variant or derivative thereof.

The oligonucleotide modulator can be an oligonucleotide having at least about 50% to about 100% nucleotide sequence identity with (TTAGGG) _n (n = about 1 to about 333). Oligonucleotides can be in forms that can include single stranded, double stranded, or combinations thereof, but are not limited thereto. Oligonucleotides are preferably from about 2 to about 2000 nucleotides, and more preferably have a single-stranded 3 ′ end of from about 2 to about 200 nucleotides. The oligonucleotide may be EST. Also specifically contemplated are oligonucleotide analogs, derivatives, fragments, homologs or variants.

  As shown in the Examples, certain oligonucleotides of the invention result in inhibition of proliferation and induction of apoptosis in the cells, while other oligonucleotides of the invention result in inhibition of growth arrest and inhibition of apoptosis. This difference in oligonucleotide activity was dependent on the number of 3 'hydrolyzable internucleotide linkages. Changing the number of 3 'hydrolyzable internucleotide linkages changed the effect of the oligonucleotide.

  Without being bound by theory, the inventors believe that the oligonucleotide is recognized by the MRN complex and becomes a substrate for the 3 'exonuclease Mre11. As a result, substrate oligonucleotides with 3 'non-hydrolyzable internucleotide linkages act as antagonists or inhibitors of Mre11. Other factors that determine the level of Mre11 activity include, but are not limited to, the total concentration of 3 'non-hydrolyzable internucleotide linkages, base sequence and G content.

  (I) when the internucleotide linkage is a phosphodiester linkage or analog thereof that is hydrolysable by Mre11 under physiological conditions, and (ii) all internucleotide linkages 3 ′ to the linkage are also hydrolyzed. If it is degradable. Regardless of the number of hydrolyzable internucleotide bonds that are 3 'to the bond, when the internucleotide bond is non-hydrolyzable by Mre11 under physiological conditions, the internucleotide bond is for purposes of the present invention. It is considered non-hydrolyzable. Representative examples of non-hydrolyzable internucleotide linkages include, but are not limited to, phosphorothioate linkages and peptide nucleic acid linkages (PNA).

  In certain embodiments of the invention, the oligonucleotide has hydrolyzable internucleotide linkages. The oligonucleotide can have about 1 to about 200 hydrolyzable internucleotide linkages. Oligonucleotides can also have non-hydrolyzable internucleotide linkages. The oligonucleotide may have about 0 to about 199 non-hydrolyzable internucleotide linkages.

  In other embodiments, the oligonucleotide has non-hydrolyzable bonds. Oligonucleotides can have from about 1 to about 200 non-hydrolyzable internucleotide linkages. Oligonucleotides may also have hydrolyzable internucleotide linkages, oligonucleotides have from about 0 to about 5 hydrolyzable internucleotide linkages. Preferred oligonucleotides are T-oligos described herein and in co-pending US patent application Ser. No. 10 / 122,630, filed Apr. 12, 2002, which is incorporated herein by reference. .

(B. Regulator of tankyrase)
The present invention also relates to tankyrase activity regulators. This regulator can induce tankyrase activity. This modulator may also inhibit tankyrase activity. The modulator can be an artificially synthesized compound or a naturally occurring compound. The modulator may be a low molecular weight compound, polypeptide or peptide, or a fragment, analog, homolog, variant or derivative thereof.

(C. Regulator of DNA damage pathway)
The invention also relates to regulators of the DNA damage pathway. Regulators can induce DNA damage pathways. This regulator may also inhibit the DNA damage pathway. A modulator is an artificially synthesized compound or a naturally occurring compound. The modulator may be a low molecular weight compound, polypeptide or peptide, or a fragment, analog, homolog, variant or derivative thereof.

(D. Regulator of MRN complex formation)
The invention also relates to modulators of MRN complex formation. Modulators can trigger the formation of MRN complexes. This modulator may also inhibit the formation of the MRN complex. A modulator is an artificially synthesized compound or a naturally occurring compound. The modulator may be a low molecular weight compound, polypeptide or peptide, or a fragment, analog, homolog, variant or derivative thereof.

(3. Composition)
The invention also relates to a composition having a modulator as described above. The composition can include an Mre11 activator. The composition may also include a tankyrase activator. The composition can also include an inhibitor of Mre11. The composition can also include an inhibitor of tankyrase. The composition may also contain more than one modulator of the present invention. The composition may also include one or more modulators along with another therapeutic agent.

  In certain embodiments of the invention, the composition comprises an oligonucleotide of the invention. The oligonucleotide may have hydrolyzable internucleotide linkages or non-hydrolyzable internucleotide linkages, or a combination thereof. In a preferred embodiment, the oligonucleotide is an activator of Mre11. In another preferred embodiment, the oligonucleotide is an inhibitor of Mre11. As described above, the activity of the oligonucleotide can be modulated to induce or inhibit Mre11 based on the total concentration of hydrolyzable internucleotide linkages.

(A. Prescription)
The composition of the present invention is in the form of a tablet or confectionery tablet formulated by a conventional method. For example, tablets and capsules for oral administration can contain conventional excipients, including but not limited to binders, fillers, lubricants, disintegrants, and wetting agents. Binders include but are not limited to syrup, gum acacia, gelatin, sorbitol, gum tragacanth, starch paste and polyvinylpyrrolidone. Fillers include but are not limited to lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate and sorbitol. Lubricants include, but are not limited to magnesium stearate, stearic acid, talc, polyethylene glycol and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycolate. Wetting agents include, but are not limited to, sodium lauryl sulfate. The tablets can be coated according to methods well known in the art.

  The compositions of the present invention may be liquid formulations including but not limited to aqueous or oily suspensions, solutions, emulsions, syrups and elixirs. The composition may also be formulated as a dry product for constitution with water or other suitable vehicle prior to use. Such liquid preparations may contain additives including but not limited to suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Suspensions include, but are not limited to, sorbitol syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible oil. Emulsifiers include, but are not limited to lecithin, sorbitan monooleate and acacia gum. Non-aqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol and ethyl alcohol. Preservatives include, but are not limited to, methyl p-hydroxybenzoate or propyl p-hydroxybenzoate and sorbic acid.

  The compositions of the present invention may be formulated as a suppository and may contain a suppository base including but not limited to cocoa butter or glycerides. The composition of the present invention may also be formulated for inhalation and may be in a form including but not limited to a solution, suspension or emulsion that may be administered as a dry powder, or dichlorodifluoromethane. Alternatively, it may be in the form of an aerosol using a propellant such as trichlorofluoromethane. The compositions of the present invention may also be formulated as transdermal preparations including aqueous or non-aqueous vehicles including but not limited to creams, ointments, lotions, pastes, medical plasters, patches or films.

  The compositions of the present invention may also be formulated for parenteral administration by methods including but not limited to injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, including but not limited to suspensions, stabilizers and dispersants. Can be included. A powder form composition may be provided for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen-free water.

  The compositions of the present invention may be formulated as storage preparations that can be administered by implantation or intramuscular injection. The composition may be formulated with a suitable polymeric or hydrophobic material (eg, as an acceptable emulsion in oil), using an ion exchange resin, or as a poorly soluble derivative (eg, as a poorly soluble salt).

  The composition of the present invention may also be formulated as a liposome preparation. Liposome preparations can include liposomes that enter the relevant cells or stratum corneum and fuse with the cell membrane to deliver the contents of the liposomes into the cells. For example, liposomes such as those described in Yarosh, US Pat. No. 5,077,211, Redziniak et al., US Pat. No. 4,621,023, or Redziniak et al., US Pat. No. 4,508,703 can be used. The compositions of the invention intended to target skin conditions can be administered before, during or after exposure of mammalian skin to agents that cause UV or oxidative damage. Niosomes can be used in other suitable formulations. Niosomes are a lipid vehicle similar to liposomes, most of which are composed of nonionic lipids, some of which are effective in transporting compounds across the stratum corneum.

(4. Treatment method)
(A. Mre11 activator, tankyrase activator, DNA damage pathway activator or MRN complex formation activator)
Mre11, tankyrase, DNA damage pathways or modulators of the invention that induce or increase the activity of MRN complex formation, alone or in combination with other treatments, for pathologies associated with growth arrest, apoptosis or growth aging failure Can be used for treatment. Representative examples of such pathologies are benign growths of cells beyond normality such as cancer and keratinocytes or fibroblast hypertrophic scars and keloids in psoriasis, or lymphocyte subsets in cases of certain autoimmune disorders. Hyperproliferative diseases such as, but not limited to. The types of cancer that are treated in these ways appear in a variety of ways, such as cervical cancer, lymphoma, osteosarcoma, melanoma, and other cancers that occur in the skin, and in various cell types and organs of the body such as leukemia. Arise. The types of cancer cells targeted by this treatment are also breast, lung, liver, prostate, pancreas, ovary, bladder, uterus, colon, brain, esophagus, stomach and thymus. Modulators can also be used to inhibit sunburn, promote cell differentiation and immunosuppression.

  In certain embodiments of the invention, an oligonucleotide of the invention having a hydrolyzable internucleotide linkage is associated with growth arrest, apoptosis or proliferative aging failure by administering the oligonucleotide to a patient in need of treatment. Used to treat medical conditions. Oligonucleotides may have non-hydrolyzable internucleotide linkages. As discussed above, the activity of the oligonucleotide can be modulated to induce growth arrest or apoptosis based on the total concentration of hydrolyzable internucleotide linkages. Oligonucleotides may be administered in combination with modulators of the invention or other treatments.

  In a preferred embodiment, the oligonucleotide is cervical cancer, lymphoma, osteosarcoma, melanoma, skin cancer, leukemia, breast cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, ovarian cancer, bladder cancer, uterine cancer, rectal cancer, brain tumor, esophagus. Used for the treatment of cancer selected from the group consisting of cancer, gastric cancer and thymic cancer.

  T-oligo can prevent the induction or induction of allergic contact hypersensitivity with the same efficacy as UV irradiation in mouse models by up-regulating TNF-α and IL10, known regulators of immunosuppression It is. Thus, local or systemic activators of Mre11 are not only lymphocyte-mediated skin diseases such as psoriasis or eczema, but also lymphocyte-mediated systemic diseases such as rheumatoid arthritis, multiple sclerosis, lupus erythematosus, and many others. Can replace steroid therapy in the treatment of other diseases.

(B. Inhibitors of Mre11, tankyrase, DNA damage pathway or MRN complex formation)
Modulators of the invention that inhibit or reduce the activity of Mre11, tankyrase, DNA damage pathway or MRN complex formation can be used alone or in combination with other treatments to treat pathologies associated with growth arrest, apoptosis or growth senescence. Can be treated. Representative examples of such medical conditions include exposure to UV radiation and side effects of cancer treatments such as chemotherapy and radiation therapy to normal tissues, or the promotion of sunburn reactions in normal skin exposed to sunlight, Not limited. Regulators can also be used to inhibit cell differentiation.

  In other embodiments, an oligonucleotide of the invention having a non-hydrolyzable internucleotide linkage may be used to administer a condition associated with growth arrest or apoptosis by administering the oligonucleotide to a patient in need of such treatment. Used to treat. The oligonucleotide may also contain hydrolyzable internucleotide linkages. As discussed above, the activity of the oligonucleotide can be modulated to inhibit growth arrest or apoptosis based on the total concentration of hydrolyzable internucleotide linkages. Oligonucleotides may be administered in combination with modulators of the present invention or other treatments.

  In certain preferred embodiments, the oligonucleotide is used to treat a medical condition selected from the group consisting of exposure to UV radiation and side effects of cancer treatments such as chemotherapy and radiation therapy.

(C. Administration)
The compositions of the present invention may be administered by any method including, but not limited to, oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal administration, and combinations thereof. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intratendon sheath, and intraosseous joint.

(D. Dosage)
The therapeutically effective amount of the composition required for treatment will vary depending on the nature of the condition being treated, the length of time required for activity, and the age and condition of the patient and will ultimately be determined by the attending physician Is done. However, dosages generally used for adult treatment typically range from 0.001 mg / kg to about 200 mg / kg per day. The dosage may be about 1 μg / kg to about 100 μg / kg per day. The desired dosage is conveniently administered as a single dose or as multiple doses administered at appropriate intervals, for example 2, 3, 4 or more sub-doses per day Can be administered. Multiple doses are desirable or necessary.

  The dosage of the modulator is about 1 μg / kg, 25 μg / kg, 50 μg / kg, 75 μg / kg, 100 μg / kg, 125 μg / kg, 150 μg / kg, 175 μg / kg, 200 μg / kg, 225 μg / kg, 250 μg / kg. kg, 275 μg / kg, 300 μg / kg, 325 μg / kg, 350 μg / kg, 375 μg / kg, 400 μg / kg, 425 μg / kg, 450 μg / kg, 475 μg / kg, 500 μg / kg, 525 μg / kg, 550 μg / kg, 575 μg / kg, 600 μg / kg, 625 μg / kg, 650 μg / kg, 675 μg / kg, 700 μg / kg, 725 μg / kg, 750 μg / kg, 775 μg / kg, 800 μg / kg, 825 μg / kg, 850 μg / kg, 875 μg / kg kg, 900 μg / kg, 925 μg / k g, any dosage including but not limited to 950 μg / kg, 975 μg / kg or 1 mg / kg.

(5. Screening method)
The present invention also relates to screening methods for identifying modulators of Mre11 activity. The present invention also relates to screening methods for identifying regulators of tankyrase activity. The invention further relates to screening methods for identifying modulators of MRN complex formation. The present invention further relates to screening methods for identifying modulators of DNA damage pathways. Screening methods can be performed in a variety of formats including but not limited to in vitro, cell-based, genetic and in vivo analysis.

  Mre11 modulators or tankyrase modulators can optionally be identified by screening for substances that specifically bind to Mre11 or tankyrase. Using many standard techniques, including but not limited to immunoprecipitation and affinity chromatography, one skilled in the art can identify specific binding agents in vitro. Also, using genetic screens using a number of standard techniques, including but not limited to yeast 2-hybrid and phage display, one skilled in the art can also identify specific binding agents. One skilled in the art can also identify specific binding agents using high-throughput methods including, but not limited to, attaching Mre11 or tankyrase to a solid support such as a chip (eg, glass, plastic or silicon). .

  Mre11 modulators or tankyrase modulators may also be identified by screening in vitro for substances that optionally modulate the activity of Mre11 or tankyrase. Optionally, a modulator can be identified by contacting Mre11 or tankyrase with an expected modulator and determining whether the predicted modulator alters the activity of Mre11 or tankyrase. By measuring the hydrolysis of the nucleic acid substrate of Mre11, the activity of Mre11 can be determined. Hydrolysis of the nucleic acid substrate can be determined by methods including, but not limited to, measurement of UV absorption, and preferably gel analysis of labeled oligos or recovery of non-precipitating nucleotide bases. Tankyrase activity can be determined by measuring the phosphorylation of a peptide or polypeptide, including but not limited to TRF1.

  Modulators of MRN complex formation can be identified in vitro by combining Mre11, Rad50 and Nbs1 and measuring the effect of candidate modulators on MRN complex formation compared to controls. The formation of MRN complexes can be measured using a number of standard techniques known to those skilled in the art including but not limited to centrifugation, coprecipitation and non-denaturing electrophoresis.

By screening for substances that modulate the activity of Mre11 or tankyrase in a cell-based assay, an Mre11 modulator or tankyrase modulator can be identified. Regulators of the DNA damage pathway can be identified as well. A cell can be contacted with a suspected regulator, and whether the suspected regulator alters the level of apoptosis, senescence, or phosphorylation of p53 or p95 can identify the regulator. As discussed above, the candidate modulator may be a substance that specifically binds to Mre11 or tankyrase. Regulation of apoptosis can be measured by methods including but not limited to measurement of the size of the sub-G ₀ / G ₁ peak in FACS analysis, TUNEL analysis, DNA ladder analysis, annexin analysis, or ELISA analysis. Modulation of senescence can be determined by measuring β-galactosidase activity associated with aging, or by measuring the inability to increase cell yield or pRb phosphorylation or ³ H-thymidine incorporation after mitogen stimulation. Determine modulation of p53 activity by measuring phosphorylation of p53 at serine 15 or serine 37 by gel shift assay, by p53 promoter driven CAT or luciferase construct readout, or by induction of p53-regulated gene products such as p21 Can do. Modulation of p95 activity can be determined by measuring phosphorylation of p95 at serine 343 by migration of the p95 band in Western blot analysis or by FACS analysis to detect S-phase arrest. In addition, Mre11 modulators or tankyrase modulators can be identified by screening for substances that modulate in vivo tumorigenesis.

  Any cell can be used in the cell-based analysis. Cells for use in the present invention preferably include mammalian cells, and more preferably include human and non-human primate cells. Representative examples of suitable cells include primary (normal) human dermal fibroblasts, epithelial keratinocytes, melanocytes, and corresponding immortalized or transformed cell lines; and primary mouse cell lines, immortalized mouse cells Strains or transformed mouse cell lines, including but not limited to. The amount of protein phosphorylation can be measured by standard techniques in the art including, but not limited to, colorimetry, luminescence analysis, fluorescence analysis and Western blot.

  The conditions under which the expected modulator is added to the cell, such as by mixing, are conditions under which the cell can undergo apoptosis or signal transduction if there is essentially no other regulatory compound that interferes with apoptosis or signal transduction. Effective conditions include, but are not limited to, appropriate media, temperature, pH and oxygen conditions that allow cell growth. Suitable media are typically solid or liquid media containing growth factors and assimilable carbon, nitrogen and phosphorus sources, as well as other nutrients such as suitable salts, minerals, metals, and vitamins, Contains an effective medium in which the cells can be cultured so that the cells can exhibit apoptosis or signal transduction. For example, in mammalian cells, the medium may include Dulbecco's modified Eagle medium containing 10% fetal bovine serum.

  Each cell may be cultured in a variety of containers including but not limited to tissue culture flasks, test tubes, microtiter dishes, and petri plates. Culturing is performed at an appropriate temperature, pH and carbon dioxide content for the cells. Such culture conditions are also within the scope of those skilled in the art.

  Methods for adding expected regulators to cells include electroporation, microinjection, cell expression (ie using expression systems including naked nucleic acid molecules, recombinant viruses, retroviral expression vectors and adenovirus expression), factors To the medium, the use of ion-pairing factors, and the use of detergents to permeabilize the cells.

  Candidate modulators include carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, DNA and DNA fragments, RNA and RNA fragments, and the like, Naturally occurring molecules such as lipids, retinoids, steroids, glycopeptides, glycoproteins, proteoglycans; or analogs or derivatives of naturally occurring molecules such as peptidomimetics; and non-naturally occurring such as “small molecule” organic compounds It may be a molecule. The term “low molecular organic compound” generally refers to an organic compound having a molecular weight of about 1000 or less, preferably about 500 or less.

  Candidate modulators may be present in a library (ie, a collection of compounds) that can be prepared or obtained by means including, but not limited to, combinatorial chemistry techniques, fermentation methods, plant and cell extraction methods, and the like. The method of creating a combinatorial library is well known. For example, E.I. R. Felder, Chimia, 1994, 48, 512-541; Gallop et al., J. MoI. Med. Chem. 1994, 37, 1233-1251; A. Goughten, Trends Genet. 1993, 9, 235-239; Houghton et al., Nature, 1991, 354, 84-86; Lam et al., Nature, 1991, 354, 82-84; Carell et al., Chem. Biol. 1995, 3, 171-183; Madden et al., Perspectives in Drug Discovery and Design, 2, 269-282; Cwirla et al., Biochemistry, 1990, 87, 6378-6382; Brenner et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 5381-5383; Gordon et al. Med. Chem. 1994, 37, 1385-1401; Lebl et al., Biopolymers, 1995, 37, 177-198; and references cited therein.

  The present invention has various aspects as shown in the following non-limiting examples.

Example 1
(Oligonucleotides can induce apoptosis)
Oligonucleotides homologous to telomeric overhanging repeats (TTAGGG; SEQ ID NO: 1), oligonucleotides of sequence (11mer-1: pGTTAGGGTTTAG; SEQ ID NO: 2), oligonucleotides complementary to this sequence (11mer-2: pCTAACCCCTAAC; SEQ ID NO: 3), and culture of Jurkat cells, a human T cell line that has been reported to undergo apoptosis in response to telomere destruction using oligonucleotides not related to the telomeric sequence (11mer-3: pGATCGATCGAT; SEQ ID NO: 4) Added to the product. Compared to 25-30% of control cells, 50% of cells treated with 40 μM SEQ ID NO: 5 accumulate in S phase within 48 hours (p <0.0003, non-pair t-test, figure 1A-1D), compared to 2-3% of controls, 13% of these cells were apoptotic as determined by sub-G ₀ / G ₁ content at 72 hours (p < 0.007, non-pair t-test, see Figures 1E-1H). At 96 hours, 20 ± 3% of 11mer-1 treated cells were apoptotic (p <0.0001, non-pair t-test) compared to 3-5% control. To eliminate preferential uptake of 11mer-1 as an explanation for its unique effect, Jurkat cells were treated with oligonucleotides labeled with fluorescein phosphoramidite at the 3 ′ end, followed by confocal microscopy and FACS analysis. It was used for. The fluorescence intensity of the cells was the same after all treatments at 4 hours and 24 hours. Western analysis showed that p53 increased 24 hours after addition of 11mer-1, but not 11mer-2 or 11mer-3, indicating that the level of E2F1 transcription factor increased simultaneously. This factor is known to cooperate with p53 in inducing apoptosis, to induce an aging phenotype in human fibroblasts depending on p53, and to control S phase checkpoints.

(Example 2)
(The phosphorothioate version of the telomeric overhanging homolog 11mer-1 does not induce apoptosis)
Jurkat human T cell cultures are treated with diluent, either 11mer-1 (SEQ ID NO: 1) or phosphorothioate 11mer-1 (11mer-1-S) for 96 hours, and the cells are collected for FACS analysis. Processed. Two concentrations of oligonucleotide were tested: 0.4 μM (FIGS. 2A-2C) and 40 μM (FIGS. 2D-2F). At 0.4 μM, none of the oligonucleotides affected the expected logarithmically proliferating cell cycle profile of Jurkat cells. At 40 μM, 11mer- ₁ induced a wide range of apoptosis as indicated by the sub-G ₀ / G ₁ peak, whereas 11mer-1-S had no effect.

(Example 3)
(The phosphorothioate version of 11mer-1 prevents the induction of S-phase arrest by the phosphate backbone 11mer-1)
A culture of a keratin cell line (SSC12F, 100,000 cells / 38 cm ² ) in the presence of 11mer-1 (SEQ ID NO: 2) alone or 11mer-1-S at increasing concentrations For 48 hours. As previously indicated in Example 1, 11mer-1 induced S-phase arrest as indicated by FACS (Becton-Dickinson FacScan). 43% of the cells were in S phase, compared to 26% of cells treated with the control diluent. However, when the concentration of phosphorothioate 11mer-1 was increased and added to these cultures, fewer cells were arrested (FIGS. 3A-3G). Complete inhibition of this arrest was seen when the ratio of 11mer-1: 11mer-1-S was 2: 1. 11mer-1-S itself did not induce S phase arrest.

Example 4
(The phosphorothioate form of telomeric oligonucleotide induces constitutive and UV-induced pigmentation and does not stimulate melanogenesis)
Cultures of S91 mouse melanocytes (100,000 cells / 38 cm ² ) were treated with 100 μM pTpT or phosphorothioate pTpT (pTspT) (FIG. 4), or 40 μM 11mer-1 or phosphorothioate 11mer-1 (11mer-1- S) (Figure 5) for 6 days, cells were collected and counted and analyzed for melanin content. pTpT and 11mer-1 stimulated melanogenesis in these cells (FIGS. 4 and 5 respectively), but pTspT and 11mer-1-S did not (FIGS. 4 and 5 respectively). . Furthermore, pTspT (FIG. 4) and 11mer-1-S (FIG. 5) reduced constitutive pigmentation in these cells. This indicates that chronic exposure of this sequence during telomere repair / replication can provide a constant low level signal for melanogenesis that is blocked by pTspT and 11mer-1-S. Suggests.

(Example 5)
(Phosphorothioate pTspT inhibits UV-induced melanogenesis)
Dual cultures of S91 cells (100,000 cells / 39 cm ² ) were sham-irradiated or measured at 285 ± 5 nm using a research radiometer (model IL1700A, International Light, Nwebport, MA), 1 kW xenon arc solar Irradiated with 5 mJ / cm ² of solar simulation light from a simulator (XMN1000-21, Optical Radiation, Azure, CA). 100 μM pTspT was added to one mock-irradiated plate and the two irradiated cultures were similarly treated with pTspT. One week later, cells were collected and counted, and the cell pellet was dissolved in 1N NaOH and analyzed for melanin content by measuring the optical density at 475 nm. UV irradiation doubled the melanin content in these cells. However, this reaction was prevented by adding pTspT (FIG. 6). Furthermore, similar to the data shown in FIGS. 4 and 5, constitutive pigmentation of these cells was reduced in mock-irradiated cultures by pTspT.

(Example 6)
(Activation requires hydrolysis of T-oligo)
An oligonucleotide based on SEQ ID NO: 2 was synthesized. Oligonucleotide 1 was synthesized completely with a phosphorothioate backbone. Oligonucleotide 2 had two phosphorothioate bonds at each end and the other bond in the center was a phosphodiester bond. Oligonucleotide 3 had two phosphorothioate linkages at the 5 ′ end (blocked at the 5 ′ end) and the remaining linkage was a phosphodiester linkage. Oligonucleotide 4 had 2 phosphorothioate linkages at the 3 ′ end (blocked 3 ′ end) and the remaining linkage was a phosphodiester linkage. See FIG.

  These oligonucleotides were added to normal neonatal fibroblast cultures. After 48 hours, cells were collected and analyzed for p53 serine 15 phosphorylation and p95 / Nbs1 phosphorylation by Western blot. Other cultures were left for 1 week in the presence of oligonucleotides and then cells were stained for senescence-related β-galactosidase activity (SA-β-Gal). β-galactosidase positive cells were counted and expressed as% of total cells (FIG. 8).

  Oligonucleotides with 3 'ends accessible to nucleases were most effective at stimulating "early" responses such as p53 and p95 / Nbs1 phosphorylation. However, oligonucleotides with 5 ′ ends that can be accessed by nucleases can also induce the senescence phenotype after 1 week, but do not induce phosphorylation in 48 hours, and 3 ′ → 5 ′ nuclease sensitivity is an aging-inducing activity. Suggests that it is preferable.

(Example 7)
(Down-regulation of Mre11 protein level interferes with T-oligo reaction)
Normal human neonatal fibroblasts were treated with either 10 pmoles of Mre11 siRNA or 10 pmoles of control (no homology was found in the expressed human sequence). The culture dish was approximately 60% confluent on the day of siRNA transfection. Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the protocol supplied by the manufacturer. The transfection mixture was allowed to act on the cells for 5 hours and then replaced with fresh medium only. The next day, the transfection protocol was repeated. The next day, double cultures were treated with T-oligo or diluent as a negative control only. Cells were collected 48 hours later, phospho p95 serine 343 specific antibody (Cell Signaling Technology, Beverly, MA), Mre11 specific antibody (GnenTex, San Antonio, TX), phosphoro p53 serine 15 specific antibody (Cell Signaling Technology) and Proteins were analyzed by Western blot using total p53 specific antibody (Oncogene, San Diego, CA) (Figure 9). Hela cell lysate was used as a positive control for Mre11. Normal fibroblasts exposed to 10 GyIR or sham-irradiated and collected 1 hour later served as positive controls for p53 and p95 / Nbs1 phosphorylation. The autoradiograph was analyzed with a densitometer, and the value for the T-oligo sample was expressed as the value for the diluent-treated sample (FIGS. 10 and 11). After correction for loading, it is clear that in cells with significantly reduced MRE11 levels, the phospho-p53 response to T-oligo is reduced and there is no phospho-p95 / Nbs1 response.

(Example 8)
(Inactivation of both p53 and pRb pathways is required to escape T-oligo-induced senescence in R2F fibroblasts)
(Oligonucleotide)
Two DNA oligonucleotides were used. One is homologous to the telomere overhang (T-oligo: pGTTAGGGTTTAG; SEQ ID NO: 2) and the other is complementary to it (pCTAACCCTAAC; SEQ ID NO: 3) and was used as a negative control. These oligos were synthesized by the Midland Certified Reagent Company (Midland, Texas). Oligonucleotides were prepared as previously described (Eller et al., [2003] Telomere 3 ′ overhang specific DNA induction of p95 / Nbs-1-mediated S-phase checkpoint (Induction of a p95 / Nbs-1-Mediated S). -Phase Checkpoint by Telomere 3'overhanging Specific DNA), Faceb J17, 152-162).

(Cell source and culture)
R2F neonatal dermal fibroblasts and induced p53DD, cdk4 ^R24C and p53DD / cdk ^R24C transductants (generous gifts from Dr. James G. Rheinwald, Harvard Medical School) have functional p53, pRb and Both paths are missing.

(Aging-related β-galactosidase staining)
Cells were treated once with diluent only, 40 μM T-oligo or 40 μM complementary oligo for 1 week without changing the solution. The cells were then fixed in 2% formaldehyde / 0.2% glutaraldehyde for 3-5 minutes and described in the literature (Dimri et al., [1995] cultures and biomarkers identifying senescent human cells in aging skin in vivo ( A Biomarker that Identities Sensational Human Cells in Culture and in Aging Skin in vivo), Proc. Natl. Acad. Sci. USA, 92, 9363-9367) at 37 ° C. in the atmosphere (CO ₂ ) Incubated with relevant β-Gal (SA-β-Gal) stain.

(Western blot analysis and antibodies)
Western blot analysis as previously described (Eller et al., [1996] DNA damage promotes melanogenesis (DNA Damage Enhancements Melanogenesis), Proc. Natl. Acad. Sci. USA, 93, 1087-1092). went. The following antibodies were used: DO-1 (Ab-6) anti-p53 (Oncogene Research Products, Cambridge, Mass.), Anti-phospho p53 (ser15) (Cell Signaling Technology, Beverly, MA), anti-phospho pRb (ser780) , Ser807 / 811) (Cell Signaling Technology, Beverly, MA), anti-cdk4 (Cell Signaling Technology, Beverly, MA) and anti-actin (Santa Cruz Biotechnology, CA).

(Clonogenic analysis)
Human fibroblasts were treated with diluent alone, 40 μM T-oligo or 40 μM complementary oligo for 1 week, and then treated with trypsin and counted. 300 cells were inoculated in triplicate in a 60 mm culture dish and incubated for 2 weeks with the medium changed twice a week in complete medium. Cells were then fixed in 100% methanol for 5 minutes. Methanol was removed and the culture dish was rinsed briefly with water. Colonies were stained for 10 minutes in 4% (w / v) methylene blue solution in PBS, washed once again with water and counted.

(BrdU incorporation analysis)
HT-1080 fibrosarcoma cells cultured on Permanox chamber slides were treated with diluent, 40 μM T-oligo and 40 μM complementary oligo for 4 days and 5-bromo-2′-deoxyuridine according to the protocol provided by the manufacturer DNA synthesis was analyzed using (BrdU) labeling and detection kit II (Roche Molecular Biochemicals, Indianapolis, IN). Briefly, cells were labeled with BrdU for 1 hour, fixed and incubated with anti-BrdU monoclonal antibody. After incubation with anti-mouse Ig alkaline phosphatase, the color reaction was detected with a light microscope.

(Telomere length)
HT-1080 fibrosarcoma cells were treated with diluent, 40 μM T-oligo or 40 μM complementary oligo for 4 days, and then genomic DNA was isolated using DNeasy Tissue Kit (Qiagen, Valencia, Calif.). The telomere length was determined using Telo TTAGGG Telomere Length Assay (Roche Molecular Biochemicals, Indianapolis, IN) according to the protocol provided by the manufacturer. Briefly, 1 μg of purified genomic DNA is digested with Hinf1 / Rsa1, the DNA fragments are separated on a 0.8% agarose gel, then transferred onto a nylon membrane for Southern blotting, and digoxigenin specific for telomeric repeats. Hybridized with (DIG) labeled probe and incubated with anti-DIG alkaline phosphatase. Terminal restriction fragments (TRF) were detected by chemiluminescence. The exposed X-ray film was scanned with a densitometer to calculate the average TRF length and was described previously (Harley et al., [1990] Telomere shortening during senescence of human fibroblasts (Telomers Shortening Aging of Human Fibroblasts). ), Nature, 345, 458-460).

(result)
Senescent fibroblasts exhibit a characteristically large and flat morphology and an increase in senescence-related β-galactosidase (SA-β-Gal) activity. Ectopic expression of TRF2 ^DN induces senescence in normal human fibroblasts by disrupting the telomeric loop structure and activating the p53 and pRb pathways. In order to prevent TRF2 ^DN- induced senescence, it is necessary to block both the p53 and pRb pathways in human cells.

Cell lines genetically engineered to lack the p53 and / or pRb pathway were used to analyze signaling pathways involved in T-oligo-induced senescence. The p53 pathway was inactivated by ectopic expression of a dominant negative mutant p53 (p53DD) that lacks the transcriptional transactivation domain of p53 and binds and inactivates endogenous wild-type p53 protein. p21 / SDI1 protein, the transcriptional target of p53, was below the level of detection in R2F fibroblasts transduced to express p53DD (data not shown). The ectopic expression of the p16-insensitive mutant cdk4 (cdk4 ^R24C ) that cannot bind p16 disrupted the pRb pathway and disrupted the control of the pRb protein. Inhibition of both pathways was performed by ectopic expression of both mutants (p53DD / cdk4 ^R24C ). The expression of p53DD and cdk4 ^R24C was confirmed by Western blot and showed overexpression of p53 and cdk4 proteins, respectively (Figure 12a), which was a previous report that human keratinocytes were transduced with these mutants Is consistent with

Cells were treated with diluent or 40 μM T-oligo for 1 week and analyzed for SA-β-Gal activity. A normal neonatal foreskin fibroblast parent line (R2F) was used as a positive control. As expected, T-oligo treated R2F fibroblasts showed greatly expanded morphology and increased SA-β-Gal activity compared to diluent-treated control cells (65 ± 7% and 8 ± respectively). 1% SA-β-Gal positive cells, p <0.01: FIGS. 12b and 12c). Similarly, with p53DD R2F fibroblasts, one week of exposure to T-oligo resulted in T-oligo-treated R2F fibroblasts having a greatly expanded morphology and SA-β-Gal activity compared to diluent-treated cells. (45 ± 4% and 6 ± 2% SA-β-Gal positive cells, respectively, p <0.01: FIGS. 12b and 12c), inactivation of the p53 pathway alone indicates T-oligo induced senescence It is shown that it is inadequate for suppression. T-oligo induces an aging phenotype in cdk4 ^R24C R2F fibroblasts compared to diluent-treated cells (60 ± 5% and 7 ± 3%, respectively, SA-β-Gal positive cells, p < 0.01: FIGS. 12b and 12c), indicating that weakening only the pRb pathway is not sufficient to suppress T-oligo-induced aging. However, when R2F fibroblasts are transduced to express both p53DD and cdk4 ^R24C , T-oligo cannot induce the senescence phenotype compared to diluent-treated cells (respectively 7 ± 1% and 5 ± 2%, p> 0.05: FIGS. 12b and 12c), because weakening both p53 and pRb pathways completely suppresses T-oligo-induced senescence in human fibroblasts It shows that it is necessary. Therefore, the conditions for T-oligo induced senescence are the same as those for continuous passage culture induced by TRF2 ^DN or replication senescence after senescence.

Example 9
(Inactivation of both p53 and pRb pathways is necessary to escape T-oligo-induced senescence in HT-1080 cells)
TRF2 ^DN has been reported to induce an aging phenotype in human fibrosarcoma HT-1080 cells. To determine whether exposure to telomere 3 'overhanging DNA (T-oligo) also induces senescence in these cells, HT-1080 cells (American Type Culture Collection; Manassas, VA) were used as diluents alone, Treatment with T-oligo or complementary oligo as a control for 4 days evaluated SA-β-Gal activity. Only the T-oligo-treated cells showed an expanded morphology and increased SA-β-Gal activity (FIG. 13a). T-oligo treated cultures contained more SA-β-Gal positive cells than diluent or complementary control oligo treated cultures (80 ± 7%, 3 ± 2% and 6 ± 3%, respectively, p <0 .01; FIG. 13b). Also, as shown by the significant reduction in BrdU incorporation, only T-oligo treated cells did not grow, neither diluent nor control oligo treated cells (7 ± 2%, 90 ± 8% and 85 ± respectively). 10%, p <0.01; FIGS. 13c and 13d).

(Example 10)
(Telomeric oligonucleotide blocks phosphorylation of pRb)
HT-1080 cells are known to have functional pRb, but the p53 pathway lacks it as a result of the loss of p16. We next examined whether T-oligo treatment activates pRb by blocking its phosphorylation in HT-1080 cells. Western blot analysis showed a significant and selective reduction of pRb phosphorylation on serine 780, serine 795 and serine 807/811 in response to T-oligo (FIG. 13e). Interestingly, in tumors lacking p16, pRb is often intact and functional. In these cells, deregulation of cdk4 results in pRb hyperphosphorylation resulting in endless cell growth and tumor formation. Activation of Cdk4 but not cdk2 phosphorylates pRb on serine 780 and serine 795 very efficiently. Thus, this finding suggests that, in the absence of p16, T-oligo inhibits cdk4 activity, possibly by induction of other INK4 family members, and p16 is an essential role in the complex network of pRb regulation And suggest that pRb cannot simply be considered an absolute downstream effector.

(Example 11)
(The effect of telomere oligonucleotides is not reversible)
To test whether removing T-oligo reversed the senescence phenotype of fibrosarcoma cells, parallel cultures of HT-1080 cells were diluted with diluent or 40 μM T-oligo or 40 μM complementary control oligo. Treated for days. The cells were then given fresh complete medium without further oligonucleotide treatment. After 1 and 2 days, T-oligo pretreated cells still showed an enlarged morphology and increased SA-β-Gal activity (FIG. 14a) and did not resume DNA synthesis (FIG. 14b). Western analysis also showed that the pRb protein was maintained in an active inhibitory state in T-oligo pretreated cells (FIG. 14c).

  To determine the long-term effect of T-oligo treatment on cell proliferation, HT-1080 human fibrosarcoma cells were treated with diluent only, either 40 μM T-oligo or 40 μM complementary control oligo for 1 week, then the same number Cells were re-plated and medium was changed twice a week for 2 weeks without further treatment and then stained with methylene blue (FIG. 15a). Compared to the complementary oligo-treated cells (90.5 ± 9.4% of the control treated with diluent), the clonogenic capacity of cells pretreated with T-oligo was almost completely suppressed (with diluent). 5.7 ± 1.9% of treated controls, p <0.01; FIG. 15b). These data indicate that T-oligo induced senescence in this malignant cell line is not reversible.

(Example 12)
(Effect of telomere oligonucleotide on average telomere length)
To determine the effect of T-oligo on mean telomere length (MTL) in HT-1080 cells, cells treated with T-oligo for 4 days, corresponding to the time at which the aging phenotype was readily observed, were analyzed. Compared to diluent treatment (5.61 kb) or complementary oligo treatment control (5.51 kb), T-oligo did not alter MTL (5.56 kb) (FIG. 16). A difference of less than 100 bp in the calculated MTL is within experimental variation and is not significant. This is consistent with the observation that treatment of fibroblasts with T-oligo for up to 1 week does not result in degradation of the telomere 3 ′ overhangs as seen after telomere destruction by TRF2 ^DN (data Not shown). Since telomere loop disruption is known to result in rapid shortening of MTL and digestion of 3 'overhangs, T-oligo does not affect MTL, or does not cause digestion of 3' overhangs. Or the fact that it initiates the same signaling indicates that the T-oligo mimics the exposure of the 3 ′ overhangs without telomere loop breakage, ie without DNA damage.

(Example 13)
(PARP activity is required for T-oligo reaction)
To investigate the role of PARP in response to T-oligo, before adding 40 μM T-oligo or an equal amount of diluent as a control, two different PARP inhibitors, 3-aminobenzamide (3AB, 2. The fibroblasts were pretreated with one of 5 mM) or 1,5-dihydroxyquinoline (IQ, 100 μM) for 2 hours. Four hours after the addition of T-oligo or diluent (D), cells were given additional doses of each inhibitor. Fibroblasts were treated with 3AB and T-oligo and then cells were collected for Western blot after 48 hours. T-oligo-induced upregulation of total p53, p21 and p53 serine 15 phosphorylation (indicating p53 activation) was all decreased in the presence of 3AB (FIG. 17A).

  Fibroblasts pretreated with IQ also showed induction of phosphorylated p53 on total p53 and serine 15, 16, 20, and 24 hours after addition of T-oligo (data not shown). The effect of IQ on the interference of T-oligo-mediated induction of total p53, p53 phosphoserine 15 and p21 persisted throughout 48 hours after addition of T-oligo. These data indicate that the p53 reaction against T-oligo requires upstream PARP activity.

(Example 14)
(PARP inhibitor blocks p53 activation and induction by TRF2 ^DN )
Neonatal fibroblasts were treated with AdTRF2DN or AdGFP as a negative control. Cells were treated with diluent, 3AB (2.5 mM) or IQ (100 μM) 2 hours prior to infection. Three days later, cells were collected for Western analysis for c-myc-tag TRF2DN (to confirm infection), p53 serine 15 phosphorylation and p21 induction. Comparison of lane 2 in FIG. 17F with lanes 4 and 6 shows that both 3AB and IQ reduced p53 phosphorylation and p21 induction in response to TRF2 ^DN .

(Example 15)
(The effect of T-oligo does not depend on telomerase)
Saos-2 cells are reportedly an osteosarcoma cell line that is telomerase negative but maintains telomeres via the ALT pathway. The Saos-2 cell line was treated with diluent or 40 μM designated oligonucleotide and cells were collected 48 hours later for FACS analysis. Only homologous nucleotides caused S phase arrest of the cells (FIG. 18a). Furthermore, telomeric overhanging oligonucleotides induced phosphorylation of p95 / Nbs1 in addition to IR (FIG. 18b). This result indicates that the effect of T-oligo in telomere negative cells is the same as the reaction in telomere positive malignant cell lines.

(Example 16)
(Down-regulation of PARP tankyrase protein level interferes with T-oligo reaction)
Human fibroblast pair cultures were either treated with tankyrase siRNA, non-specific siRNA (control) or mock transfected as a second control. Two days later, when the level of tankyrase in the tankyrase siRNA treated cells was significantly reduced, the culture was supplemented with 11mer-1 (pGTTAGGGTTTAG; SEQ ID NO: 2) or the complementary sequence 11mer-2. After an additional 24 hours, the cells were collected and processed for Western blot using an antibody specific for p95 phosphorylation at serine 343, indicating p95 modification by activated ATM kinase. The film was subjected to concentration measurement, and the diluent control for each group of cells was set to 1.0 in arbitrary units (FIG. 19). As expected, in cells with normal tankyrase levels, T-oligo treated cells doubled the amount of phosphorylated p95, whereas the increase in control oligo or diluent treated cells was Only 30-40%. However, in the tankyrase knockdown group, 11-mer-1 treated cells did not show increased p95 phosphorylation (level 1.1 versus 1.0 and 1.3 controls). These data show that the telo-oligo signal that tankerase, a telomere-associated PARP, causes ATM activation and subsequent p95 modification (phosphorylation), resulting in S-phase arrest of treated cells (Eller et al., FASEB J, 2003). It shows that it is necessary for transduction.

(Example 17)
(T-oligo produces non-ATM mediated phosphorylation of p53)
Normal neonatal fibroblasts were treated with diluent or 40 μM (11mer-1) for 4, 6, 8, 19, 24 and 48 hours and collected for Western blot analysis using p53 phosphoserine 37 specific antibodies. Mock and IR irradiated (10 Gy) fibroblasts were used as negative and positive controls, respectively. In the Western blot, an increase in band intensity corresponding to p53 serine 37 was detected as early as 8 hours, and at 48 hours it was very pronounced in the T-oligo (T) treated sample compared to the diluent (D) treated sample. there were.

  As indicated above, T-oligo produces phosphorylation of p53 on serine 15. Phosphorylation of p53 at serine 15 is mediated by ATM. FIG. 20 shows that T-oligo also phosphorylates p53 on serine 37. Phosphorylation of p53 at serine 37 is mediated by either ATM-related (ATR) kinase or DNA-PK kinase, but is not known to be mediated by ATM. Thus, the demonstration of p53 serine 37 is another marker of pathway activation, and one or both of these kinases are downstream targets of Mre11 activation. Furthermore, many of the therapeutic effects that activate the Mre11 pathway are pseudo-UV, and UV is known to activate both ATR and DNA-PK but not ATM.

Claims (1)

Invention described in this specification and drawings.

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