Rejoining Of Breaks Research Articles

Radiosensitization by the Histone Deacetylase Inhibitor PCI-24781

PCI-24781 is a novel broad spectrum histone deacetylase inhibitor that is currently in phase I clinical trials. The ability of PCI-24781 to act as a radiation sensitizer and the mechanisms of radiosensitization were examined. Exponentially growing human SiHa cervical and WiDr colon carcinoma cells were exposed to 0.1 to 10 micromol/L PCI-24781 in vitro for 2 to 20 h before irradiation and 0 to 4 h after irradiation. Single cells and sorted populations were analyzed for histone acetylation, H2AX phosphorylation, cell cycle distribution, apoptotic fraction, and clonogenic survival. PCI-24781 treatment for 4 h increased histone H3 acetylation and produced a modest increase in gammaH2AX but negligible cell killing or radiosensitization. Treatment for 24 h resulted in up to 80% cell kill and depletion of cells in S phase. Toxicity reached maximum levels at a drug concentration of approximately 1 micromol/L, and cells in G(1) phase at the end of treatment were preferentially spared. A similar dose-modifying factor (DMF(0.1) = 1.5) was observed for SiHa cells exposed for 24 h at 0.1 to 3 micromol/L, and more radioresistant WiDr cells showed less sensitization (DMF(0.1) = 1.2). Limited radiosensitization and less killing were observed in noncycling human fibroblasts. Cell sorting experiments confirmed that depletion of S-phase cells was not a major mechanism of radiosensitization and that inner noncycling cells of SiHa spheroids could be sensitized by nontoxic doses. PCI-24781 pretreatment increased the fraction of cells with gammaH2AX foci 24 h after irradiation but did not affect the initial rate of loss of radiation-induced gammaH2AX or the rate of rejoining of DNA double-strand breaks. PCI-24781 shows promise as a radiosensitizing agent that may compromise the accuracy of repair of radiation damage.

Impact of the comet assay in radiobiology

Until the development of single cell gel electrophoresis methods in the 1980s, measurement of radiation-induced DNA strand breaks in individual cells was limited to detection of micronuclei or chromosome breaks that measured the combined effects of exposure and repair. Development of methods to measure the extent of migration of DNA from single cells permitted detection of initial radiation-induced DNA breaks present in each cell. As cells need not be radiolabeled, there were new opportunities for analysis of radiation effects on cells from virtually any tissue, provided a single cell suspension could be prepared. The comet assay (as this method was subsequently named) was able to measure, for the first time, the fraction of radiobiologically hypoxic cells in mouse and human tumors. It was used to determine that the rate of rejoining of DNA breaks was relatively homogenous within an irradiated population of cells. Because individual cells were analyzed, heavily damaged or apoptotic cells could be identified and eliminated from analysis to determine "true" DNA strand break rejoining rates. Other examples of applications of the comet assay in radiobiology research include analysis of the inter-individual differences in response to radiation, effect of hypoxia modifying agents on tumor hypoxic fraction, the role of cell cycle position during DNA break induction and rejoining, non-targeted effects on bystander cells, and effects of charged particles on DNA fragmentation patterns.

DNA double-strand break and chromosomal rejoining defects with misrejoining in Nijmegen breakage syndrome cells

NBS1-deficient cells exhibit pronounced radiosensitivity and defects in chromosome integrity after ionizing radiation (IR) exposure, yet show only a minor defect in DNA double-strand break (DSB) rejoining, leaving an as yet unresolved enigma as to the nature of the radiosensitivity of these cells. To further investigate the relationship between radiosensitivity, DSB repair, and chromosome stability, we have compared cytological and molecular assays of DSB misrejoining and repair in NBS1-defective, wild type, and NBS1-complemented cells after IR damage. Our findings suggest a subtle defect in overall DSB rejoining in NBS1-defective cells and uniquely also reveal reduced ability of NBS1-defective cells to rejoin correct ends of DSBs. In agreement with published results, one of two different NBS1-defective cell lines showed a slight defect in overall rejoining of DSBs compared to its complemented counterpart, whereas another NBS line did not show any difference from wild type cells. Significant defects in the correct rejoining of DSBs compared to their respective controls were observed for both NBS1-defective lines. The defect in DSB rejoining and the increased misrejoining detected at the molecular level were also reflected in higher levels of fragments and translocations, respectively, at the chromosomal level. This work provides both molecular and cytological evidence that NBS1-deficient cells have defects in DSB processing and reveals that these molecular events can be manifest cytologically.

Changes in poly(ADP-ribose) level modulate the kinetics of DNA strand break rejoining

ADP-ribose polymers are rapidly synthesized in cell nuclei by the poly(ADP-ribose) polymerases PARP-1 and PARP-2 in response to DNA strand interruptions, using NAD + as precursor. The level of induced poly(ADP-ribose) formation is proportional to the level of DNA damage and can be decreased by NAD + or PARP deficiency, followed by poor DNA repair and genomic instability. Here we studied the correlation between poly(ADP-ribose) level and DNA strand break repair in lymphoblastoid Raji cells. Poly(ADP-ribose) synthesis was induced by 100 μM H 2O 2 and intensified by the 1,4-dihydropyridine derivative AV-153. The level of poly(ADP-ribose) in individual cells was analyzed by quantitative in situ immunofluorescence and confirmed in whole-cell extracts by Western blotting, and DNA damage was assessed by alkaline comet assays. Cells showed a ∼100-fold increase in poly(ADP-ribose) formation during the first 5 min of recovery from H 2O 2 treatment, followed by a gradual decrease up to 15 min. This synthesis was completely inhibited by the PARP inhibitor NU1025 (100 μM) while the cells treated with AV-153, at non-genotoxic concentrations of 1 nM–10 μM, showed a concentration-dependent increase of poly(ADP-ribose) level up to 130% after the first minute of recovery. The transient increase in poly(ADP-ribose) level was strongly correlated with the speed and efficiency of DNA strand break rejoining (correlation coefficient r ≥ 0.92, p < 0.05). These results are consistent with the idea that poly(ADP-ribose) formation immediately after genome damage reflects rapid assembly and efficient functioning of repair machinery.

Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage

Adefective response to DNA damage is observed in several human autosomal recessive ataxias with oculomotor apraxia, including ataxia-telangiectasia. We report that senataxin, defective in ataxia oculomotor apraxia (AOA) type 2, is a nuclear protein involved in the DNA damage response. AOA2 cells are sensitive to H2O2, camptothecin, and mitomycin C, but not to ionizing radiation, and sensitivity was rescued with full-length SETX cDNA. AOA2 cells exhibited constitutive oxidative DNA damage and enhanced chromosomal instability in response to H2O2. Rejoining of H2O2-induced DNA double-strand breaks (DSBs) was significantly reduced in AOA2 cells compared to controls, and there was no evidence for a defect in DNA single-strand break repair. This defect in DSB repair was corrected by full-length SETX cDNA. These results provide evidence that an additional member of the autosomal recessive AOA is also characterized by a defective response to DNA damage, which may contribute to the neurodegeneration seen in this syndrome.

Critical role of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of rejoining following γ-radiation-induced damage to pMTa4 DNA in E. coli

Purpose: This study was undertaken to understand the roles of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of the process following exposure of E. coli to γ-radiation in vivo.Materials and methods: A plasmid DNA construct, pMTa4, was transformed into isogenic repair proficient (wild) and deficient (recF and recA) E. coli strains and γ-irradiated up to 30 Gy in vivo. The plasmid DNA was isolated under repair non-permissive (R−)and permissive (R+) conditions and analyzed by gel electrophoresis for the yields of single strand breaks (SSB) and double strand breaks (DSB) and their repair. The clonogenic survival of the E. coli was also recorded. The effects of γ-irradiation on recA reconstituted with cell free extract of wild strain or ultra-violet (UV)-irradiation were also monitored.Results: None of the strains used in this investigation showed effects of radiation-induced oxidative base damage. The dose dependent increase in SSB and DSB on pMTa4 in wild and recF mutants in R− condition were abolished upon repair incubation. The recA mutant exhibited a disturbed yield of SSB and DSB along with formation of γ-radiation-induced ‘ladder’. The ‘ladder’ was not observed after repair incubation, UV-irradiation or γ-irradiation in presence of cell-free extract of wild strain. The survival of recA mutants was seriously compromised.Conclusions: Wild, recF and recA strains of E. coli could repair γ-irradiation-induced oxidative damage to base or nucleotide (NT) in vivo. In absence of either RecA or RecF proteins, efficiency of rejoining of strand went down; RecA proteins seemed more critical than RecF in this. High fidelity or correct rejoining of strand breaks, on the other hand, seemed to require simultaneous presence of both RecA and RecF proteins.

Differing Effects of T4 DNA Ligase in the Modulation of the Damage Induced in Mammalian Cells by either X-Rays or Restriction Endonucleases

Background: Of the different lesions induced by X-rays, DNA double-strand breaks (DSBs) are considered the main cause of chromosomal aberrations and cell death. Restriction endonucleases (REs) induce only DNA DSBs and have frequently been used to mimic the effects of ionizing radiation in the study of DNA damage and repair. Methods: The present work makes use of clonogenic and cytogenetic assays to study the effect of T4 DNA ligase on modulating the damage induced by either X-rays or an RE (MspI) that produces breaks with cohesive ends. A CHO cell line defective in ligase III activity (EM9) and its corresponding parental line (AA8) were used. Results: Our results show that T4 DNA ligase increased cell survival and decreased chromosomal aberrations in cells treated with MspI, suggesting that most RE-induced DSBs can be repaired by a simple ligation. This enzyme was, however, unable to promote repair of the DNA damage induced by X-rays. Analysis of the ratios of exchange-type aberrations to chromatid break-type aberrations indicated that T4 ligase increased misrejoining of the DNA damage induced by X-rays. The results were similar in EM9 and AA8 cells, although the effect was greater in the cells deficient in DNA strand break rejoining. In addition, depending on whether the end strand break structure is 3′-hydroxyl and 5′-phosphoryl (REs) or more complex (X-rays), T4 DNA ligase could either promote the correct repair or, conversely, increase misrejoining. Conclusion: The present results confirm the idea that DNA DSBs induced by cohesive cutting RE are repaired by different mechanisms than those induced by X-rays causing cell lethality.

Relationship between DNA double-strand break rejoining and cell survival after exposure to ionizing radiation in human fibroblast strains with differing ATM/p53 status: Implications for evaluation of clinical radiosensitivity

To better understand the impact of defects in the DNA damage-surveillance network on the various cell-based assays used for the prediction of patient radiosensitivity. We examined noncancerous human fibroblast strains from individuals with ataxia telangiectasia (ataxia telangiectasia mutated [ATM] deficient) or Li-Fraumeni syndrome (p53 deficient) using the neutral comet, H2AX phosphorylation, and clonogenic survival assays. Using the comet assay, we found that, compared with normal fibroblasts, cells lacking either ATM or p53 function exhibited a reduced rate of double-strand break (DSB) rejoining early (< or =4 h) after exposure to 8 Gy of gamma-radiation and also exhibited high levels of unrejoined DSBs later after irradiation. ATM-deficient and p53-deficient fibroblasts also exhibited abnormally increased levels of phosphorylated H2AX (gamma-H2AX) at later intervals after irradiation. In the clonogenic assay, ATM-deficient cells exhibited marked radiosensitivity and p53-deficient cells had varying degrees of radioresistance compared with normal fibroblasts. Regardless of whether ataxia telangiectasia and Li-Fraumeni syndrome fibroblasts are DSB-repair deficient per se, it is apparent that p53 and ATM defects greatly influence the cellular phenotype as evidenced by the neutral comet and gamma-H2AX assays. Our data suggest that the gamma-H2AX levels observed at later intervals after irradiation may represent a reliable measure of the overall DSB rejoining capabilities of human fibroblasts. However, it appears that using this parameter as a predictor of radiosensitivity without knowledge of the cells' p53 status could lead to incorrect conclusions.

258 POSTER Tricyclic triazin 1,4-dioxides: a new class of hypoxia-selective cytotoxins with improved extravascular transport compared to tirapazamine

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a bioreductive agent which exhibits highly selective killing of hypoxic cells in a variety of mammalian cell lines in vitro and in murine tumors in vivo. The selective toxicity of the drug results from its one-electron reduction under hypoxic conditions to form a free radical intermediate capable of damaging DNA, through the formation of strand breaks. Using the neutral filter elution assay, SR 4233 was found to be more efficient at producing DNA double strand breaks in Chinese hamster ovary (CHO) cells than an equitoxic dose of γ-rays. Drug and radiation sequencing experiments were also performed, with both cell survival and DNA strand break rejoining used as endpoints. As a result of these studies, we now describe two additional properties of SR .4233: (a) radiosensitization of aerobic cells in culture produced by hypoxic incubation with drug either before or after irradiation, and (b) the inhibition of subsequent rejoining of radiation-induced DNA double strand breaks after hypoxic pretreatment with drug. The magnitude of the radiosensitization produced did not vary for drug treatments which, when given alone, reduced cell survival over a range from 30% to 2%. The extent of DNA repair inhibition increased with increasing severity of the SR 4233 pretreatment, but was quite small for non-lethal drug exposures.

150 Wortmannin, an ihibitor of dna double-strand break rejoining, sensitizes human cells to radiation but protects against the cytotoxic effect of cisplatin: Relevance for radiochemotherapy?

150 Wortmannin, an ihibitor of dna double-strand break rejoining, sensitizes human cells to radiation but protects against the cytotoxic effect of cisplatin: Relevance for radiochemotherapy?

Induction and quantification of γ-H2AX foci following low and high LET-irradiation

Purpose: To investigate quantitatively the induction and rejoining of DNA double strand breaks (DSB) in V79-4 and xrs-5 Chinese hamster cells and HF19 human fibroblast cells, using the phosphorylation of the histone protein H2AX (γ-H2AX) as an indicator of DSB, exposed to low doses of either low linear energy transfer (LET) 60Co γ-rays or high LET α-particles.Materials and methods: Cells were irradiated with low or high LET (20 – 2000 mGy). The γ-H2AX foci were detected using immunohistochemistry and quantified by image analysis.Results: The number of DSB determined 30 min post γ-irradiation at 37°C is 12.2 (±1.5), 13.5 (±1.6) and 19.1 (±1.7) foci/cell/Gy for V79-4, xrs-5 and HF19 cells respectively, comparable with levels detected in V79-4 cells using pulse field gel electrophoresis. 6 h post γ-irradiation, γ-H2AX foci levels in V79-4 and HF19 cells approach control levels but remain higher in DSB repair deficient xrs-5 cells. γ-H2AX foci levels remain significantly higher than controls at 6 h in α-irradiated cells.Conclusions: γ-radiation and α-radiation induced the phosphorylation of H2AX in response to DSB at low doses; the variation in the rate of dephosphorylation of induced foci are dependent both on radiation quality and cell characteristics.

Nonhomologous End Joining in Yeast

Nonhomologous end joining (NHEJ), the direct rejoining of DNA double-strand breaks, is closely associated with illegitimate recombination and chromosomal rearrangement. This has led to the concept that NHEJ is error prone. Studies with the yeast Saccharomyces cerevisiae have revealed that this model eukaryote has a classical NHEJ pathway dependent on Ku and DNA ligase IV, as well as alternative mechanisms for break rejoining. The evolutionary conservation of the Ku-dependent process includes several genes dedicated to this pathway, indicating that classical NHEJ at least is a strong contributor to fitness in the wild. Here we review how double-strand break structure, the yeast NHEJ proteins, and alternative rejoining mechanisms influence the accuracy of break repair. We also consider how the balance between NHEJ and homologous repair is regulated by cell state to promote genome preservation. The principles discussed are instructive to NHEJ in all organisms.

Evidence for the Direct Binding of Phosphorylated p53 to Sites of DNA Breaks In vivo

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.

Growth of V79 Cells as Xenograft Tumors Promotes Multicellular Resistance but does not Increase Spontaneous or Radiation-Induced Mutant Frequency

A Chinese hamster V79 xenograft model was developed to determine whether cells subjected to a hypoxic tumor microenvironment would be more likely to undergo mutation at the HPRT locus. V79-171b cells stably transfected with VEGF and EGFP were grown subcutaneously in immunodeficient NOD/ SCID mice. V79-VE tumors were characterized for host cell infiltration, doubling time, hypoxic fraction, vascular perfusion, and response to ionizing radiation. When irradiated in vitro, the mutant frequency for a given surviving fraction did not differ for cells grown in vivo or in vitro. Similar results were obtained using HCT116 human colorectal carcinoma cells grown as xenografts. However, V79-VE cells grown as xenografts were significantly more resistant to killing than monolayers. The background mutant frequency and the radiation-induced mutant frequency did not differ for tumor cells close to or distant from blood vessels. Similarly, tumor cells from well-perfused regions showed the same rate of strand break rejoining and the same rate of loss of phosphorylated histone H2AX as cells sorted from poorly perfused regions. Therefore, deleterious effects of the tumor microenvironment on DNA repair efficiency or mutation induction could not be demonstrated in these tumors. Rather, development of multicellular resistance in V79-VE tumors acted to reduce mutant frequency for a given dose of radiation.

Colcemid inhibits the rejoining of the nucleotide excision repair of UVC-induced DNA damages in Chinese hamster ovary cells

In our previous study, we found that colcemid, an inhibitor of mitotic spindle, promotes UVC-induced apoptosis in Chinese hamster ovary cells (CHO.K1). In this study, a brief treatment of colcemid on cells after but not before UV irradiation could synergistically reduce the cell viability. Although colcemid did not affect the excision of UV-induced DNA damages such as [6–4] photoproducts or cyclobutane pyrimidine dimers, colcemid accumulated the DNA breaks when it was added to cells following UV-irradiation. This colcemid effect required nucleotide excision repair (NER) since the same accumulation of DNA breaks was barely or not detected in two NER defective strains of CHO cells, UV5 or UV24. Furthermore, the colcemid effect was not due to semi-conservative DNA replication or mitosis since the colcemid-caused accumulation of DNA breaks was also seen in non-replicating cells. Moreover, colcemid inhibited rejoining of DNA breaks accumulated by hydroxyurea/cytosine arabinoside following UV irradiation. Nevertheless, colcemid did not affect the unscheduled DNA synthesis as assayed by the incorporation of bromodeoxyuridine. Taken together, our results suggest that colcemid might inhibit the step of ligation of NER pathways.

DNA Double-Strand Break Misrejoining after Exposure of Primary Human Fibroblasts to CKCharacteristic X Rays, 29 kVp X Rays and60Co γ Rays

The efficiency of ionizing photon radiation for inducing mutations, chromosome aberrations, neoplastic cell transformation, and cell killing depends on the photon energy. We investigated the induction and rejoining of DNA double-strand breaks (DSBs) as possible contributors for the varying efficiencies of different photon energies. A specialized pulsed-field gel electrophoresis assay based on Southern hybridization of single Mbp genomic restriction fragments was employed to assess DSB induction and rejoining by quantifying the restriction fragment band. Unrejoined and misrejoined DSBs were determined in dose fractionation protocols using doses per fraction of 2.2 and 4.4 Gy for CK characteristic X rays, 4 and 8 Gy for 29 kVp X rays, and 5, 10 and 20 Gy for 60Co gamma rays. DSB induction by CK characteristic X rays was about twofold higher than for 60Co gamma rays, whereas 29 kVp X rays showed only marginally elevated levels of induced DSBs compared with 60Co gamma rays (a factor of 1.15). Compared with these modest variations in DSB induction, the variations in the levels of unrejoined and misrejoined DSBs were more significant. Our results suggest that differences in the fidelity of DSB rejoining together with the different efficiencies for induction of DSBs can explain the varying biological effectiveness of different photon energies.

Pre-exposure to Low Doses: Modulation of X-Ray-Induced DNA Damage and Repair?

The adaptive response to ionizing radiation may be mediated by the induction of antioxidant defense mechanisms, accelerated repair or altered cell cycle progression after the conditioning dose. To gain new insight into the mechanism of the adaptive response, nondividing lymphocytes and fibroblasts were used to eliminate possible contributions of cell cycle effects. The effect of conditioning doses of 0.05 or 0.1 Gy followed by challenging doses up to 8 Gy (with a 4-h interval between exposures) on induction and repair of DNA damage was determined by single-cell gel electrophoresis (comet assay), premature chromosome condensation, and immunofluorescence labeling for gamma-H2AX. The conditioning dose reduced the induction of DNA strand breaks, but the kinetics of strand break rejoining was not influenced by the conditioning dose in nondividing cells of either cell type. We conclude that adaptation in nondividing cells is not mediated by enhanced strand break rejoining and that protection against the induction of DNA damage is rather small. Therefore, the adaptive response is most likely a reflection of perturbation of cell cycle progression.

DNA-PK is responsible for enhanced phosphorylation of histone H2AX under hypertonic conditions

Exposure of cells to hypertonic medium after X-irradiation results in a 3–4-fold increase in the phosphorylation of histone H2AX (γH2AX) at sites of radiation-induced DNA double-strand breaks. This increase was previously associated with salt-induced radiosensitization and inhibition of repair of DNA double-strand breaks. To examine possible mechanisms for the increase in foci size, chemical inhibitors of kinase and phosphatase activity and cell lines deficient in ATM and DNA-PK, two kinases known to phosphorylate H2AX, were examined. H2AX kinase and phosphatase activity were maintained in the presence of high salt. ATM mutant HT144 melanoma cells showed the expected 3–4-fold increase in H2AX phosphorylation in the presence of 0.5 M Na +. However, DNA-PKcs deficient M059J cells failed to respond to hypertonic treatment and M059J Fus1 cells corrected for this deficiency showed the expected increase in foci size. Although the active phosphoform of ATM, phosphoserine-1981, increased after irradiation, the level was unaffected by the addition of 0.5 M Na +. Instead, 0.5 M Na + caused a partial redistribution of serine-1981-ATM to perinuclear regions. Hypertonic medium added after irradiation was effective in inhibiting rejoining of the radiation-induced double-strand breaks even in DNA-PK deficient M059J cells. We suggest that hypertonic treatment following irradiation inhibits double-strand break rejoining that in turn maintains DNA-PK activity at the site of the break, enhancing the size of the γH2AX foci.

Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.

Deficiency in the Catalytic Subunit of DNA-Dependent Protein Kinase Causes Down-Regulation of ATM

Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKcs with small interfering RNA, Chinese hamster ovary V3 cells, lacking DNA-PKcs, had reduced levels of ATM and hSMG-1, but both were restored after transfection with PRKDC. Atm levels were also reduced in murine scid cells. Reduction of ATM in a human glioma cell line lacking DNA-PKcs was accompanied by defective signaling through downstream substrates, post-irradiation. A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences. This was accompanied by a reduction in ATM. These data were confirmed using immunocytochemical detection of the proteins. Within hours after transfection, a decline in PRKDC mRNA was seen, followed by a more gradual decline in DNA-PKcs protein beginning 1 day after transfection. No change in ATM mRNA was observed for 2 days post-transfection. Only after the DNA-PKcs reduction occurred was a reduction in ATM mRNA observed, beginning 2 days post-transfection. The amount of ATM began to decline, starting about 3 days post-treatment, then it declined to levels comparable to DNA-PKcs. Both proteins returned to normal levels at later times. These data illustrate a potentially important cross-regulation between the nonhomologous end-joining system for rejoining of DNA double-strand breaks and the ATM-dependent damage response network of pathways, both of which operate to maintain the integrity of the genome.