JP2007525162A5 - - Google Patents

Description

Regulation of cell signaling initiated by telomeres

(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. While cancer progresses, including Me resistant to old of Contact and apoptosis is an important aspect of the method of the interaction of normal cells and their tissue specificity environment is controlled, from a negative regulatory control, tumor cells It is, ing to be more and more independent.

Cell aging has been suggested to be an important defense against cancer. Many facts, that means the impairment of shortening or telomere progressive telomere caused by not replicate the 3 'ends of chromosomes definitive 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. Telomeres are linked repeats of TTAGGG is given a fold at the proximal telomere duplex DNA, telomeric repeat binding factors (TRF), has a 3 'overhang of the stabilized about 150-300 bases, especially in TRF2 It ends with a single-stranded loop structure. Ectopic expression of a dominant negative form of TRF2 (TRF2 ^DN) is destroyed the telomere loop structure, 3 'to expose the protruding portion, cause DNA damage response, resulting in primary fibroblasts and fibrosarcoma ages.

Aging can be accelerated rapidly by extensive DNA damage or overexpression of certain oncogenes. Either maintain telomere length enzymatically, can bypass senescence by ectopic expression of telomerase reverse transcriptase catalytic subunit constructs (TERT), immortalizes certain human cell types thereby 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 pathway and pRb pathways. Tumor suppressor p53 is a variety of different stimuli, such as DNA damage, rolling Utsushima other deregulation of replication, oncogene transformation, and deregulation of cell growth arrest of certain chemotherapeutic agents in the resulting microtubule Or by converting to apoptosis, it plays a key role in the cellular stress response mechanism. When activated, p53 causes cell growth arrest or programmed cell suicide, while serving as an important regulatory mechanism for genome stability. In particular, p53 controls the stability of the genome 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 but maintains a wild-type p53 and pRb genes, HPV E6 and E7 proteins will interrupt the p53 pathway and pRb pathways, respectively. Ectopic expression of viral E2 protein suppresses HPV E6 and E7 gene transcription, induces a rapid and prominent senescent response to cervical carcinoma cell lines, which also an important role of p 53 and pRb in cancer cell senescence It is to confirm.

is possible to suppress only the p53 pathway and pRb pathway is not sufficient for fibroblasts to bypass replicative senescence. Indeed, human fibroblasts were introduced the combination of suppressing infected or p53 pathway and pRb pathways SV40T antigen adenovirus E 1 A + E 1 B or HPV E6 + E7, the life is prolonged, escape replicative 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 telomeric 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, hematopoietic and intestinal epithelia. Other side effects include hair loss. More effective cancer treatment safer, always particularly avoid Keru alternative treatment some of these side effects, or all by which is normal to preferentially target malignant cells to proliferating cells that continues to be needed.

(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 . Nucleic acid substrate Ru Ah Ri obtained in oligonucleotides having at least 50% nucleotide sequence identity _{(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, cellular morphology, SA-b-Gal activity, and include phosphorylation of p53 or p95 method comprising the step of measuring the cell properties, but not limited to, et al. 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 is that obtained possess exonuclease activity, fragments of Mre11, homolog, may be expressed as an analog or variant.

The present invention also relates to a method for in vitro screening for tankyrase modulators comprising the step of contacting a candidate modulator with tankyrase in vitro in the presence of a substrate for tankyrase and measuring the ribosylation of the substrate. Regulators can be identified by altering ribosylation relative to controls . Substrates, Ru Oh Ri obtained in a peptide or polypeptide may be a TRF. The ribosylation of substrate may more measured labeling of UV absorber or substrate.

  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, cellular morphology, SA-b-Gal activity, and including p53 phosphorylation or p95 phosphorylation which method comprises the step of measuring the cell properties, but not limited to, et al. Modulators can be identified by changing properties compared to controls . Candidate modulators, Ru obtained Ri tankyrase and drug der which specifically binds, as described above. Tankyrase, that obtained have a Riboshiraze activity, fragments of tankyrase, homolog, analog or may be expressed as a modified variant.

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. By varying the shape formed of the MRN complex as compared with control, may identify modulators. Candidate modulators that obtained by contacting with the presence of the inhibitor nucleic acid substrates or Mre11 Mre11, Rad50 and Nbs1. 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 regulators of DNA damage pathways, wherein cells expressing Mre11 and tankyrase in the presence of oligonucleotides and candidate regulators under conditions where the regulator is taken up by the cells. contacting the cell proliferation, cell viability, cell morphology, SA-b-Gal activity, and including p53 phosphorylation or p95 phosphorylation which method comprises the step of measuring the cell properties, but not limited to, et al. Modulators can be identified by changing properties compared to controls . Oligonucleotides that obtained have at least 50% nucleotide sequence identity with _{(TTAGGG) n (n = 1~20} ). Mre11 is that obtained possess exonuclease activity, fragments of Mre11, homolog, may be expressed as an analog or variant. Tankyrase, that obtained have a Riboshiraze activity, fragments of tankyrase, homolog, may be expressed as an analog or variant.

The cells used in the above cell-based screening method may be cancer cells. 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. Activators _{(TTAGGG) n (n = 1~20} ) and may oligonucleotides activator of Mre11 that obtained have at least 50% nucleotide sequence identity. About 1 to about 10 of the first 3 ′ nucleotide bonds can be hydrolyzed with a 3 ′ → 5 ′ nuclease.

The present invention is also, M re 11, tankyrase, related to the use of a composition comprising an inhibitor of 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. Inhibitor may be an oligonucleotide inhibitor of Mre11 that obtained have at at least 50% nucleotide sequence identity _{(TTAGG G) 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 present invention also relates to a a _{(TTAGG G) n (n =} 1~20) at least 50% nucleotide sequence identity, compositions with an oligonucleotide having a coupling between at least one non-hydrolyzable nucleotides . 'From 1 nucleotide binding by about 103 such as M RE11' first three may be hydrolyzed in → 5 'nuclease. Oligonucleotides that obtained have at least 50% nucleotide sequence identity with TTAGG G. The oligonucleotide may have the sequence GTTAGGGTTTAG. The non-hydrolyzable bond may be a phosphorothioate. The oligonucleotide may be PNA.

(Detailed description of the invention)
The present invention, 3 'Mre11 mediated hydrolysis of telomere overhang sequence is aging, it is based on the discovery that starts key signaling cascade protective response of the cells to sunburn and DNA damage such as 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 begins to hydrolyze the telomere protrusion from then 3 'end, Rad50 Causes activation of 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, M re 11, tankyrase, activators of DNA damage pathway or MRN complex formation, DNA damage response pathway regardless of the presence of DNA damage or telomere loop disruption Is expected to activate. 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 cause 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. As used in the specification and appended claims, the singular forms "a", "an" and "the", it should be noted that including a plurality of reference subjects unless clearly indicated by the context otherwise .

Throughout this application, to cite the patents or publications, incorporated disclosure of the full text of these publications to explain the state of the art to which this invention belongs more fully by reference into the present application.

(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 the dosage of a modulator means a single dose or multiple doses of the drug.

As used herein, the term “analog” when used in the sense to 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 to an oligonucleotide, refers to an oligonucleotide having one or more internucleotide linkages other than phosphodiester internucleotide linkages.

The term "antibody" as used herein, classes IgG, IgM, IgA, IgD Moshiku's IgE antibodies, or _{Fab,, F (ab ')} 2, Fd -containing Me, fragments Moshiku derivatives, and single chain antibodies, bispecific antibodies, bispecific antibodies, it means a divalent antibody and its these derivatives. Antibodies exhibit sufficient binding specificity to a desired epitope or sequence derived from the epitope, monoclonal antibodies, polyclonal antibodies, it may be a mixture of affinity purified antibodies or their these. The antibody may be a chimeric antibody. One or more known chemical substances in the art, the antibody may be derivatized by attachment with the peptide or polypeptide species. The antibody may be conjugated to a compound moiety .

As used herein, the term “apoptosis” refers to gradual 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 including DNA cleavage into nucleosome size fragments determined by centrifugal sedimentation analysis, but not limited to, it meant some form of cell death. Cell death occurs when cell membrane integrity is lost (eg, membrane blebbing) , with engulfment of intact cell fragments (apoptotic bodies) by phagocytic cells.

The term "treatment of cancer", as used herein, including chemotherapy and radiotherapy, but not limited to, et al., Refers to any known treatment in the art.

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 terms 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, 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 other peptides via a side chain functional group, a peptide or polypeptide bound to the polypeptide or compound, and include other modifications appear to Ru is understood in the art, but are not limited to, et al.

The term “fragment” as used herein, when used in the context of a peptide or polypeptide, preferably has a length of about 5 to about 300 amino acids, 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 , 3 1 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 4 1 , 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, inhibiting or eliminating the condition. To preventing a Condition, the composition of the present invention comprises administering to a mammal animal before developing the condition. Suppressing a condition includes administering the composition of the present invention to a mammal after induction of the condition but before its clinical symptoms appear. To suppress disease state, to prevent that or exacerbated reducing disease-like, the composition of the present invention involves administering to a mammal after appear clinical symptoms of the condition. To remove the medical condition involves such mammal is no longer developing the condition, the composition of the present invention after the pathology of clinical symptoms appeared administered to a mammal.

As used herein, the term “variant”, 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. It means a peptide or polypeptide, when used in the context of an oligonucleotide, insertion of nucleotides, but nucleotide sequences by deletion or conservative substitution is different, refers to an oligonucleotide that maintains at least one biological activity. For the purposes of the present invention, the "biological activity" includes but is capable of binding to a singular antibody, but is not limited thereto.

(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. Modulator may be a compound present in the artificially synthesized compound or a naturally. Modulator may be a low molecular weight compound, an oligonucleotide, a polypeptide Moshiku peptide or its these fragments, analogs, homologs, may be a variant or derivative conductor.

Oligonucleotide modulator Ru obtained Ri Ah with oligonucleotides having at least about 50% to about 100% nucleotide sequence identity with _(TTAGGG) n (n = about 1 to about 333). Oligonucleotides single-stranded, double-stranded, or can be of a form that may include not the combination, them to the not limited. 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 having 3 ′ non-hydrolyzable internucleotide linkages act as antagonists or inhibitors of Mre11. Other factors determining the level of Mre11 activity, 3 'the total concentration of binding between nonhydrolyzable nucleotide, including but base sequence and G content, but are not limited to, et al.

(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 linkage is a 3 'to its binding, if linkage is a non-hydrolyzable by Mre11 under physiological conditions, linkages, for the purpose of the present invention It is considered non- hydrolyzable. Representative examples of coupling between nonhydrolyzable nucleotide, but are phosphorothioate linkages and peptide nucleic acid binding (PNA) is not limited thereto, et al.

In certain embodiments of the invention, the oligonucleotide has hydrolyzable internucleotide linkages. Oligonucleotides can have from 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 a non-hydrolyzable binding. Oligonucleotides can have from about 1 to about 200 non-hydrolyzable internucleotide linkages. Oligonucleotides may also have between hydrolyzable linkage, oligonucleotide, it has a between about 0 to about 5 hydrolyzable linkages. Preferred oligonucleotides, herein, and references to be incorporated herein, is the T- oligo described in U.S. Patent Application No. 10 / 122,630 of co-pending, filed Apr. 12, 2002 .

(B. Regulator of tankyrase)
The present invention also relates to tankyrase activity regulators. This modulator that able to induce tankyrase activity. This modulator may also inhibit tankyrase activity. Modulator, Ru compound der Ri obtained that exist artificially synthesized compound or a naturally. Modulator may be a low molecular weight compound, polypeptide Moshiku peptide or its these fragments, analogs, homologs, variants Moshiku may be a derivative.

(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. Modulator is a compound present in artificially synthesized compound or a naturally. Modulator may be a low molecular weight compound, polypeptide Moshiku peptide or its these fragments, analogs, homologs, variants Moshiku may be a derivative.

(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. Modulator is a compound present in artificially synthesized compound or a naturally. Modulator may be a low molecular weight compound, polypeptide Moshiku peptide or its these fragments, analogs, homologs, variants Moshiku may be a derivative.

(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 a number of regulators from one or the other therapeutic agent and co.

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 may contain binders, fillers, lubricants, and disintegrating agents and wetting agents may include conventional excipients including, but not limited to, et al. The binding agent, syrup, acacia gum, gelatin, sorbitol, tragacanth gum, including but starch paste and polyvinylpyrrolidone, but not limited to, et al. Fillers include lactose, sugar, microcrystalline cellulose, corn starch, including but calcium phosphate, and sorbitol, but are not limited to, et al. Lubricants include magnesium stearate, stearic acid, talc, polyethylene glycol and silica, but are not limited to, et al. Disintegrants include, but are potato starches and sodium starch glycolate, but is not limited thereto, et al. Wetting agents include but are sodium lauryl sulfate, but are not limited to, et al. The tablets can be coated according to methods well known in the art.

The compositions of the present invention, aqueous or oily suspensions, solutions, emulsions, may be a liquid formulation, but not limited thereto, et al 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 suspending agents, emulsifying agents, including non-aqueous vehicles and preservatives may contain additives including, but not limited to, et al. Suspensions, sorbitol syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl an ethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and includes but is hydrogenated edible oils, but not limited to, et al. The emulsifiers, lecithin, but are sorbitan monooleate and acacia gum, but not limited to, et al. Non-aqueous vehicles, edible oils, almond oil, fractionated coconut oil, oily esters, including but propylene glycol and ethyl alcohol, but are not limited to, et al. Preservatives include, but are p- hydroxybenzoate or p- hydroxybenzoic acid propyl Le Contact and sorbic acid, but not limited to, et al.

The compositions of the present invention may also be formulated as suppositories, including cocoa butter or glycerides may include suppository base, but not limited to, et al. The compositions of the present invention may also be formulated for inhalation, solutions may be administered as a dry powder, including suspensions or emulsions, may be a shape that is not limited to, or dichlorodifluoromethane Moshiku may be a shape of Eazo Lumpur using propellant such as trichlorofluoromethane. The compositions of the present invention may also creams, ointments, lotions, pastes, medical plasters, including patch or film may be formulated as a transdermal preparation comprising an aqueous or non-aqueous vehicles but are not limited to, et al.

The compositions of the present invention may also be formulated for parenteral administration by including injection or continuous infusion, but not limited to, et al method. Formulations for injection may be a suspension of oil-soluble or aqueous vehicles, the solution or may be I-shaped der of emulsions, suspensions, including stabilizers and dispersants limited thereto, et al, Which may not 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 composition of the invention may be formulated as a stored preparation that can be administered by implantation or intramuscular injection. The composition may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in oil capable of acceptance), or an ion-exchange resin, or as sparingly soluble derivatives (for example, as a sparingly soluble salt) may be formulated.

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. 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, most of the film are made of non-ionic lipids, some forms effective to transport the compound across the stratum corneum is a lipid vehicle similar to liposomes.

(4. Treatment method)
(A. Mre11 activator, tankyrase activator, DNA damage pathway activator or MRN complex formation activator)
Mre11, tankyrase, modulators of the present invention that induce or increase the activity of DNA damage pathway or MRN complex formation alone or in combination with other treatments, growth arrest, of pathologies associated with impaired apoptosis or proliferation senescence Can be used for treatment. Representative examples of such conditions, the cells exceeding the normal best of such lymphocyte subsets in cases of cancer and for example keratinocytes or fibroblasts hypertrophic 瘢 scars and keloids in psoriasis or certain autoimmune disorders, benign including but hyperproliferative diseases proliferation such as but not limited to, et al. 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. Types of cancer cells the therapeutic aim is also an breast, lung, liver, prostate, pancreas, ovary, bladder, uterine, colon, brain, esophagus, even in the 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. obtain substitute despite taking the steroid therapy in the treatment of the disease.

(B. Inhibitors of Mre11, tankyrase, DNA damage pathway or MRN complex formation)
Mre11, tankyrase, modulators of the present invention to inhibit or reduce the DNA damage pathway or MR N active double coalescence formation are used alone or in combination with other treatments, growth arrest, apoptosis-related or proliferative senescence pathology Can be treated. Representative examples of such pathologies 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, the oligonucleotides of the present invention having a coupling between non-hydrolyzable nucleotides, by administering the oligonucleotide to a patient in need of such treatment, a condition associated with growth arrest or apoptosis 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 condition selected from the group consisting of side effects of cancer treatment, such as exposure, as well as chemotherapy and radiation therapy to the UV radiation.

(C. Administration)
Oral compositions of the present invention, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, administration of buccal, also may be administered by any method but are not limited to, et al combinations thereof . Parenteral administration includes intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, Kensayanai, and including the joint is not limited thereto, et al.

(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 dosage of Nozomu Tokoro is either dosed in a single dose, multiple doses administered at appropriate intervals, for example twice per day, three times, useful as four or more subdoses that could be administered to. 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, 950μg / kg, including 975μg / kg or 1 mg / kg, may be any dosage that is not limited thereto, et al.

(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. The screening methods in vitro, cell-based, may be performed in genetic and including in vivo analysis is not limited thereto, et al various formats.

The Mre11 modulators or tankyrase modulators may be identified by screening the quality ones that specifically binds to Mre11 or tankyrase as the case may be. Including immunoprecipitation and affinity chromatography using a number of standard techniques including, but not limited to, et al., The skilled artisan can identify a specific binding Substance in vitro. Also, including yeast two-hybrid and phage display using genetic screening using a number of standard techniques including, but not limited to, et al., One skilled in the art can also identify specific binding Substance. Moreover, those skilled in the art using chip (such as glass, plastic or silicon) including be attached to a solid support such as but not limited to high-throughput methods Mre11 or tankyrase also to identify specific binding Substance obtain.

Mre11 modulators or tankyrase modulators also be identified by screening in vitro for quality ones that modulate the activity of Mre11 or tankyrase as the case may be. Depending on the case, it is contacted with a modulator to the expected Mre11 or tankyrase, modulators that are expected by determine whether to alter the activity of Mre11 or tankyrase may identify modulators. By measuring the hydrolysis of a nucleic acid substrate of Mre11, it may determine the activity of M re 11. Measurement of UV absorption Hydrolysis of a nucleic acid substrate, and preferably may determine by methods including recovery of the labeled oligonucleotide gel analysis or non-precipitating nucleotide bases not limited thereto, et al. The activity of tankyrase may determine by including TRF1 measuring the phosphorylation of limiting peptide or polypeptide thereto.

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 . Centrifugation may measure the formation of MRN complex using a number of standard techniques known to those skilled in the art including coprecipitation and nondenaturing electrophoresis, but not limited to, et al.

By screening quality ones that modulate the activity of Mre11 or tankyrase in cell-based analysis can identify Mre11 modulators or tankyrase modulators. Regulators of the DNA damage pathway can be identified as well. Cells are contacted with suspected modulator, suspected modulators apoptosis, or a and determine how to change the level of phosphorylation of senescence, or p53 or p95, may identify modulators. As discussed above, the candidate modulator may I substances der that specifically binds to Mre11 or tankyrase. Adjusting the measurement of the size of the _sub-G 0 / _{G 1} peak in FACS analysis of apoptosis, TUNEL analysis, DNA ladder analysis may be measured by methods annexin analysis, or including ELISA analysis but not limited to, et al. By measuring the measurement of the associated β- galactosidase activity aging, or ³ H- thymidine after increasing inability or pRb phosphorylation impossible or mitogen stimulation of cell yields uptake impossible, it may determine the regulation of senescence. By gel shift assay by p53 promoter driven CAT or luciferase construct read, or by the induction of p53- regulated genes products such as p21, by measuring the phosphorylation of p53 at serine 15 or serine 37, determine a modulation of p53 activity Can be determined. The movement of the p95 band in Western blot analysis, or by FACS analysis to detect the S-phase arrest, it may determine the modulation of p95 activity by measuring the phosphorylation of p95 at serine 343. 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. In cells for use in the present invention preferably include mammalian cells, it is more preferable to include primate cell of human and non-human. 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 including but strain or transformed mouse cell lines is not limited thereto, et al. Colorimetrically the amount of protein phosphorylation, light emission analysis may be measured by standard techniques in the art including, fluorescent analysis and Western Blot but not limited to, et al.

Conditions by mixing such a modulator expected you added to the cells, if other regulatory compounds that interfere with apoptosis or signaling does not exist basically a condition in which cells can undergo apoptosis or signaling. Valid conditions allowing cell proliferation, a suitable medium, temperature, but are pH and oxygen conditions, but is not limited thereto, et al. Suitable media are typically growth factors and assimilable carbon, other nitrogen and phosphorus source, a solid or liquid media containing the appropriate salt, minerals, metals, and other nutrients, such as 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 with 10% fetal bovine serum .

Each cell tissue culture flasks, test tubes, microtiter dishes, and including Petri plates can be cultured in a variety of containers including, but not limited to, et al. 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, cellular expression (ie using expression systems including naked nucleic acid molecules , recombinant viruses, retroviral expression vectors and adenoviral expression), factors added to the medium, use of ion pairing agent, and include the use of detergents for cell permeabilization.

Candidate modulators, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, DNA and DNA fragments bets, a polynucleotide, including RNA and RNA fragments and the like , lipids, retinoids, steroids, glycopeptides, glycoproteins, natural to the molecule exists such proteoglycans; natural and "small molecule" organic compounds; or peptide analog or derivative molecules present in natural mimetics such as It may be a molecule that does not exist in The term "low-molecular organic compound" generally a molecular weight of about 1000 or less, preferably, refers to an organic compound having about 500 or less.

Candidate modulators, combinatorial chemistry techniques, fermentation methods, plant and including cell extraction, etc. that could be made by adjusted by means including, but not limited to, et al., Or in the resulting obtained that library (i.e. a collection of compounds) Can exist. Creating method of combinatorial library is a peripheral knowledge. For example, E.I. R. Felder, Chimia, 1994, 48, 512-541; Gallop et al., J. MoI. Med. Chem. 1994, 37, 1233-1251; A. Goughten, Trend s Genet. 1993,9,235-239; Houghton et al., Nature, 199 1, 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. N atl . 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)
Telomere projecting repeat sequence (TTAGGG; SEQ ID NO: 1) homologous oligonucleotide, sequence (11mer -1: pGTTAGGGTTAG; SEQ ID NO: 2) oligonucleotide, complementary oligonucleotide to the sequence (11-mer -2: pCTAACCCTAAC; SEQ ID nO: 3), and oligonucleotides that are not related to the telomere sequence (11mer -3: pGATCGATCGAT; SEQ ID nO: 4), the Jurkat cells, a human T cell line has been reported to perform apoptosis in response to telomere disruption culture Added to the product . Compared is the with a control cells 25-30% within 48 hours, 50% of the cells treated with SEQ ID NO: 5 of 40μM is integrated in phase S (p <0.0003, Nonpea t-test, FIG. 1A~1D reference), as compared to the control is 2-3%, 13% of these cells were apoptotic to determine a _sub-G 0 / _{G 1} content at 72 hours (p <0.0 07, Nonpea t-test, see Figure 1E~1H). Control Compared to the 3 to 5 percent, at 96 hours, 11 20 ± 3% of the mer -1-treated cells were apoptotic (p <0.0001, Nonpea t-test). To exclude preferential uptake of 11-mer -1 as an illustration of its especial effect, and treated with oligonucleotides labeled with fluorescein phosphoramidite Jurkat cells 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 is p53 increased in 24 hours after the addition 11mer -1, 11 without increasing the mer-2 or 11-mer -3, level of E2F1 transcription factor showed that 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)
Diluent Cultures of Jurkat human T cells, 11 mer-1 (SEQ ID NO: 1) or phosphorothioate 11 96 hours with either mer -1 (11-mer -1-S), cells were collected for FACS analysis Processed. Two concentrations of oligonucleotide were tested: 0.4 μM (FIGS. 2A-2C) and 40 μM (FIGS. 2D-2F). In 0.4 .mu.M, it did not affect in any oligonucleotide de also logarithmically growing cell cycle profile of the expected Jurkat cells. In 40 [mu] M, 11 mer-1 induced a wide range of apoptosis represented by the _sub-G 0 / _{G 1} peak, 11-mer -1-S had no effect.

(Example 3)
(11 Phosphorothioate Version of mer-1, interfere with the induction of S phase arrest by phosphate backbone 11mer -1)
Culture of keratinocytes lines (SSC12F, 100,000 cells / 38cm ²⁾ a 11-mer -1 (SEQ ID NO: 2) alone or in the presence of 11-mer -1-S increasing concentrations, 11-mer -1 For 48 hours. As indicated above in Example 1, 11-mer -1 induced S phase arrest represented 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 added to these cultures to increasing concentrations of the phosphorothioate 11mer-1, the number of stops cells was less (Figure 3A~3 G). Complete inhibition of this arrest 11mer -1 ratio of 11-mer -1-S of 2: observed when 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)
S91 cultures mouse melanocytes (100,000 cells / 38 cm ²⁾ and 100μM of pTpT or phosphorothioate pTpT (pTspT) (Figure 4), or 40μM of 11mer-1 or the phosphorothioate 11mer-1 (11-mer - 1-S) (Figure 5) for 6 days, cells were collected and counted and analyzed for melanin content. pTpT and 11mer-1 has been stimulated melanogenesis in these cells (FIGS. 4 and 5), pTspT and 11mer -1-S did not stimulate (FIGS. 4 and 5) . Furthermore, pTspT (Figure 4) and 11mer -1-S (Figure 5) reduced the constitutive pigmentation in these cells. This Exposure of this arrangement chronically during telomere repair / replication can provide signals for the steady low level for melanogenesis, that this signal is disturbed by pTspT and 11mer -1-S Suggests.

(Example 5)
(Phosphorothioate pTspT inhibits UV-induced melanogenesis)
Duplicate 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, Nwebburyport, Mass.) And 1 kW xenon arc solar simulator (XMN1000-21, Op t ical radiation, Azuza, CA) was irradiated with sun simulation light 5 mJ / cm ² from. Add the pTspT of 100μM to two sham-irradiated plates were treated with pTspT similarly two irradiation cultures. 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, the constitutive pigmentation of these cells was decreased in sham-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 one week in the presence of an oligonucleotide, and then stained for senescence-associated β- galactosidase activity of cells (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 “ early” reaction stimuli 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 one week, but do not induce phosphorylation in 48 hours, and the 3 ′ → 5 ′ nuclease sensitivity increases senescence-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 10 picomoles of Mre11. were treated with either siRNA or 10 pmol of control (not found homologous resistance in human sequences expressed). The culture dish was approximately 60% confluent on the day of siRNA transfection . Following the protocol supplied by the manufacturer, transfection was performed using a Lipofectamine2000 (Invitr o gen, Ca r lsbad, CA). 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, treated with diluent alone as a dual culture T- oligo or negative control. After 48 hours, cells were collected and 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 Technol ). Proteins were analyzed by Western blot using and p53 specific antibodies (Oncogene, San Diego, CA ) (FIG. 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. Analyzed autoradiography graph with a densitometer, and represents the value for the T- oligo sample as the value for the diluent treated samples (FIG. 10 and FIG. 11). Corrected by regarding the loading, the cells MRE11 levels were significantly reduced, phospho-p53 reaction is decreased for T- oligo, it is clear that there is no phospho-p95 / Nbs1 reaction.

(Example 8)
(P53 pathway and pRb pathways both inactivation is required to escape the T- oligo-induced senescence in R2F fibroblasts)
(Oligonucleotide)
Two DNA oligonucleotides were used . One homologous to the telomere protrusion:; a (T-oligo p GTTAGGGTTAG SEQ ID NO: 2) and one, complementary to Re its (PCTAACCCTAAC; SEQ ID NO: 3) der is, use as a negative control It was . These oligo was synthesized by Midland Certified Reagent Company (Midland, Texas ). The oligonucleotides were prepared as placing serial to previously (Eller, et al., [2003] telomere 3 'induction of p95 / Nbs-1 mediated S phase checkpoint by projecting specific DNA (Induction of a p95 / Nbs -1- (Mediated S-Phase Checkpoint by Telomere 3'overhanging Specific DNA), Faseb J17, 152-162).

(Thin 胞源 and culture)
R2F neonatal dermal fibroblasts, and induced ^{p53DD, cdk4 R24C} and p 53DD ^{/ cdk R24 C} shape quality transductant (generous gift from James G.Rheinwald Dr. (Harvard Medical School)), the functional p53 pathway , The pRb pathway and both pathways are each missing.

(Aging-related β-galactosidase staining)
Cells diluent only, 1 week complementary oligo T- oligo or 40 [mu] M of 40 [mu] M, were treated once without replacing the liquid. The cells were then treated with 2% formaldehyde / 0. Fixed 3-5 minutes in 2% glutaraldehyde, literature (Dimri et al., Biomarkers to identify aging human cells in-aging skin in [1995] culture and in vivo (A Biomarker that Identifies Senescent Human Cells in Culture a n d in aging Skin in vivo), Proc.N atl .Acad.Sci.USA, 92,9363-9367) overnight at 37 ° C. (CO ₂ in the atmosphere) as fresh age-related beta-Gal ( (SA-β-Gal) staining solution was incubated.

(Western blot analysis and antibodies)
Described previously (Eller, et al., [1996] DNA damage promotes camera two emissions form (DNA Damage Enhances Melanogenesis), Proc.N atl .Acad.Sci.USA, 93,1087-1092) Western As Blot analysis was performed. The following antibodies were used: DO-1 (Ab-6) anti-p53 (Oncogene Research Products, Cambridge, MA), anti-phospho p53 (ser15) (Cell Signaling Technology, Beverly, MA), anti-phospho pRb ( ser7 ) , Ser807 / 811) (Cell Signaling Technology, Beverly, MA), anti-cdk4 (Cell Signaling Technology, Beverly, MA) and anti-actin (Santa Cruz Biotechnology, CA).

(Clonogenic assay)
Human fibroblasts only diluent, treated 1 week complementary oligo T- oligo or 40 [mu] M of 40 [mu] M, were counted trypsinized. 300 cells were seeded in triplicate in culture dishes 60 mm, were incubated for 2 weeks by exchanging the medium twice a week with complete culture ground. 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)
Permanox chamber slides onto diluent HT-1080 fibrosarcoma cells cultured in, for 4 days with complementary oligo T- oligo and 40 [mu] M of 40 [mu] M, 5-bromo-2'-deoxyuridine according to the protocol provided 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, the purified genomic DNA of 1μg was digested with Hinf1 / Rsa1, 0.8% agarose gel to separate the DNA fragments by Le, then transferred onto a nylon membrane for Southern blot, specific digoxigenin telomere repeat 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, described previously (Harley et al., Telomeres shorten during aging [1990] Human fibroblasts (Telomer e s Shorten during Ag e ing of Human Fibroblast s), Nature , was calculated as has been 345,458-460).

(result)
Senescent fibroblasts characteristically large shows a flat form, show an increase in aging-related β- galactosidase (SA-β-Gal) activity. TRF2 ^DN ectopic expression of destroys telomere loop structure, induces senescence in normal human fibroblasts by activating the p53 pathway and pRb pathways. To prevent TRF2 ^DN induction aging, it is necessary to interfere with both the p53 pathway and pRb pathway in human cells.

p53 pathway and / or pRb pathway have been used to analyze signal transduction pathways you involve cell lines that have been genetically engineered to lack the 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. The p53 transcriptional target, p21 / SD I 1 protein, was below detection levels 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 (FIG. 12a) , which is a previous report that human keratinocytes are 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 treatment R2F fibroblasts exhibited increased compared with larger spread morphology and SA-β-Ga l active and diluent treated control cells (65 ± 7%, respectively, and 8 ± 1% SA -β- Gal positive cells, p <0.01: FIGS. 12b and 12c). Similarly, in p53DD R2F fibroblasts, T-by 1 week of exposure to oligo, T-oligo treatment R2F fibroblasts large spread morphology as compared to diluent treatment RiHoso cells and SA-beta- Ga l showed an increase in activity (45 ± 4% and 6 ± 2% of SA - beta-Gal-positive cells, respectively, p <0.01: Figure 12b and 12c), inactivation of p53 alone pathway, T- It shows that it is insufficient for suppression of oligo-induced aging. T-oligo induces an aging phenotype in cd k 4 ^R24C R2F fibroblasts compared to diluent-treated cells (60 ± 5% and 7 ± 3%, 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 cd k 4 ^R24C , T-oligo cannot induce the senescence phenotype compared to diluent-treated cells. (7 ± 1% and 5 ± 2%, respectively, p> 0.0 5: Figure 12b and 12c), completely T- oligo-induced senescence be weakened both the p53 pathway and pRb pathway in human fibroblasts It is necessary to suppress. Therefore, T-conditions oligo-induced senescence is the same as the conditions of replication aging after continuous subculture or senescence induced by TRF2 ^DN.

Example 9
(P53 pathway and pRb pathways both inactivation is required to escape the T- oligo induced senescence of HT-1080 cells)
TRF2 ^DN has been reported to induce an aging phenotype in human fibrosarcoma HT-1080 cells. Since the exposure to the telomere 3 'overhang DNA (T-oligo) determining whether induces senescence in these cells, HT-1080 cells (American Type C ulture Collection; Manassas , VA) diluent alone , Treated with T-oligo or complementary oligo as a control for 4 days to evaluate 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 from diluent or complementary control oligo treated cultures (80 ± 7%, respectively, 3 ± 2% and 6 ± 3%, p <0 .01; FIG. 13b). Further, as indicated by the significant reduction in BrdU incorporation, nor even control oligo treated cells with diluent, T-only oligo-treated cells did not proliferate (respectively 7 ± 2%, 90 ± 8 % and 85 ± 10%, p <0.01; FIGS. 13c and 13d).

(Example 10)
(Telomeric oligonucleotide blocks phosphorylation of pRb)
HT-1 0 80 cells have been known to have a functional pRb, p53 pathway, p16 is missing it as a result of lacking. We next examined whether T-oligo treatment activates pRb by blocking its phosphorylation in HT-1080 cells. Western blot analysis, T-response to oligo serine 780 and pRb phosphorylation on serine 79 5 and serine 807/811 was found to significantly selectively reduce (Figure 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. cdk2 is no activation of Cdk4 phosphorylates extremely to efficiently to pRb on serine 780 and serine 795. 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)
For eliminating the T- oligo to test whether reversing senescence phenotype of fibrosarcoma cells, concurrent cultures of HT-1080 cells diluent or 40μM of T- oligo or complementary control oligo 40μM Treated for 4 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).

T- To determine the long-term effects on cell proliferation of oligo treatment, HT-1080 human fibrosarcoma cells only diluent, treated 1 week with either T- oligo or 40 [mu] M complementary control oligo 40 [mu] M, then the same number of 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 complementary oligo-treated cells (90.5 ± 9.4% of the control treated with diluent), clonogenic capacity of cells pretreated with T- oligo was almost completely inhibited (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 average telomere length (MTL) Effect T- oligos against the HT-1080 cells, which corresponds to the time senescent phenotype were readily observed, analyzed the treated cells 4 days T- oligo did. Compared to diluent treatment (5.61Kb) or complementary oligo treated controls (5.51kb), T- oligo did not alter MTL (5.56kb) (Figure 16). A difference of less than 100 bp in the calculated MTL is within experimental variation and is not significant. This is because, as seen after the destruction of the telomere by TRF2 ^DN, treatment with T- oligo fibroblasts up to 1 week is consistent with the observation that not result in degradation of the telomere 3 'overhang (data Not shown). Destruction of telomere loop, 'since it is known to produce digestion of protrusions, T-oligo does not affect the MTL, or 3' rapid shortening and 3 of MTL similar without causing digestion of the projecting portion Or the fact that it initiates the same signaling indicates that the T-oligo mimics the exposure of the 3 ′ overhang, even 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. 4 hours after the addition T- oligo or diluent (D), we were given an additional dose of each inhibitor to the cells. Fibroblasts were treated with 3AB and T-oligo and then cells were collected for Western blot after 48 hours . T- oligo-induced up-regulation of phosphorylation of total p53, p21 and p53 serine 15 (indicating p53 activation) were all reduced in the presence of 3AB (Figure 17A).

Fibroblasts pretreated with IQ also, T-oligo added after 16, 20, (data not shown) similarly indicated the induction of phosphorylation of p53 on the total p53 and serine 15 in 24 hours. The effect of IQ on the interference of T-oligo-mediated induction of total p53, p53 phosphoserine 15 and p21 persisted through 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 AdGFP as AdTRF2D N or negative controls. Cells were treated with diluent, 3AB (2.5 mM) or IQ (100 μM) 2 hours prior to infection. After 3 days, c-myc-tag TRF2DN (to confirm the infection), the cells were collected for Western analysis about the 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. Was treated with S ao s-2 cell line diluent or 40μM of the specified oligonucleotide, cells were harvested after 48 hours 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 telomere-negative cells is the same as the reaction of telomere-positive malignant cells lines.

(Example 16)
(Down-regulation of PARP tankyrase protein level interferes with T-oligo reaction)
Human fibroblast pair cultures were either treated once with tankyrase siRNA, non-specific siRNA (control) or mock transfected as a second control . After two days, when the tankyrase level tankyrase siRNA treated cells decreased significantly, 11mer -1 culture; supplemented with (PGTTAGGGTTAG SEQ ID NO 2) or a complementary sequence 11-mer -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, the cells having a normal tankyrase levels, T-oligo treated cells, the increase in the amount of phosphorylated p95 was doubled, control oligo treated cells or diluent-treated cells, 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 t-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 diluent or 40 [mu] M 4,6,8,19,24 and 48 hours (11-mer -1) were collected for Western blot analysis using p53 phosphoserine 37 specific antibody. Sham and IR irradiating (10 Gy) fibroblasts, respectively were used as negative and positive controls. Faster increase in the band intensity corresponding to the p53 serine 37 in Western blots also detected in 8 hours, and in 48 hours, very prominent in compared to diluent (D) treated samples T- oligo (T) processing specimen during Met.

As indicated above, T-oligo produces phosphorylation of p53 on serine 15 . Phosphorylation of p53 at serine 15 is mediated by the ATM. Figure 20, T-oligos have shown that also occur phosphorylation of p53 on serine 37. Phosphorylation of p53 at serine 37 is mediated by either an ATM-related (ATR) kinase or DNA-PK kinases, that are mediated by ATM is not known. Therefore, demonstration of p53 serine 37 is also another marker of pathway activation, a downstream target of one or both Mre11 activation of these kinases. 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.

FIG 1A~1H a diluent (FIGS. 1A and 1E); 11-mer 40 [mu] M -1 PGTTAGGGTTAG (SEQ ID NO: 2: Figure 1B and 1F); 11-mer 40 [mu] M -2 PCTAACCCTAAC (SEQ ID NO: 3: FIGS. 1C and 11G); 11-mer 40μM -3 p GATCGATCGAT: FACS analysis of (SEQ ID NO: 4 Figure 1D and 1H) treated with propidium iodide staining Jurkat cells (immortalized T lymphocytes) Show. Analysis before 48 hours (Fig. 1A to 1D) or 72 hours Jurk a t cells were treated for 48 hours at mentioned reagents (Figure 1E~1H). FIG 2A~2F is a graph showing the results of fluorescence activated cell sorting for addition to the following cell: FIG. 2A, diluent; Figure 2B, 0.4 .mu.M of 11mer-1; Fig. 2C, 0. 4μM of 11-mer -1-S; FIG. 2D, diluent; Figure 2E, 40 [mu] M of 11-mer -1; Figure 2F, 40 [mu] M of 11-mer -1-S. 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 11 11mer -1-S-mer -1 and 1 [mu] M; Figure 3D, 11 mer-1 and 5 [mu] M 11-mer -1-S of 10 [mu] M; FIG. 3E, a 11-mer -1 and 10 [mu] M of 10 [mu] M 11-mer -1-S ; Figure 3F, 20 mu M of 11-mer -1 -S; Figure 3G, 10 [mu] M of 11-mer -1-S. Figure 4 is a bar graph showing diluent, melanin content of cells treated with pTpT or p TSPT a (pg / cell). 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 administration of pTspT. 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. Figure 8 is shown in FIG. 7, beta-galactosidase activity represented by pairs cells staining positive in, phospho b thio benzoate oligonucleotides 1, 2, 3 and 4 resulting aging in cultures of normal neonatal human fibroblasts It is a bar graph which shows the result of the test of an effect. Oligonucleotide "11-1" indicates the whole fibroblast cultures treated with SEQ ID NO: 2 synthesized in phosphodiester bond. "Dill (Dil)" indicates fibroblast cultures treated with diluent not containing oligonucleotide. 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. Figure 12 shows that both the p53 pathway and pRb pathways contribute to T- oligo-induced senescence in human fibroblasts. FIG. 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 . Figure 12 shows that both the p53 pathway and pRb pathways contribute to T- oligo-induced senescence in human fibroblasts. Figure 12b: contribution to p53 pathway and pRb pathways T- oligo derived 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. Figure 12 shows that both the p53 pathway and pRb pathways contribute to T- oligo-induced senescence in human fibroblasts. Figure 12c: quantified analysis of SA-β-Gal positive cells. Counting the cells expressing SA-β-Gal activity was presented as a percentage of total cells in culture. From 3 times each independent experiments were calculated mean and standard deviation of three representative fields. 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. Counting the cells expressing SA-β-Gal activity was expressed as a percentage of total cells in 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. The BrdU-positive cells was calculated and expressed as a percentage of total cells in 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, using 30 μg total protein and 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. Lane D, T and C, diluents, T-oligo and complementary oligo protein sample from cells treated with each. 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. Was treated as described in Figure 13a the concurrent culture. 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. Was treated as described in Figure 13a the concurrent culture. 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. Was treated as described in Figure 13a the concurrent culture. 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 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. Figure 15 b: the clonogenicity quantification. Colonies of three cultures were counted and plotted as a percentage of the diluent treated 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, includes a diluent (D), T-oligo (T) or treated with complementary oligo (C) from cells genomic DNA. 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%. Figure 9f: lane 1, diluents, 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. Figure 18b: Western blot using specific antibodies against the phospho-p95 / Nbs1. Lanes 1, 2 and 3, respectively diluent, it contains do the proteins from cells treated with 11-mer-1 or 11mer-2. Control cells were irradiated (+) with 10 Gy IR or sham irradiated (−) (3 hours) . Figure 19 shows that down-regulation of motor Nki hydrolase protein levels interfere with T- oligo reactions. The upper panel shows densitometer readings and the lower panel shows Western blots. Figure 20 shows that results in phosphorylation of p53 above T- oligo 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.

Images