Correlation of carcinogen-induced unscheduled DNA synthesis and NAD reduction in fresh human lymphocytes

Incorporation of [³H]thymidine into the DNA of fresh human lymphocytes, treated with various chemical mutagens, was measured and correlated with cellular NAD levels before and after treatment. NAD levels in lymphocytes were significantly reduced following treatment with mutagenic chemicals. Reduction of cellular NAD pools was directly correlated with [³H]thymidine incorporation. As NAD levels decreased, [³H]thymidine incorporation increased. Theophylline, a known inhibitor of poly(ADP-ribose)polymerase, inhibited both the NAD reduction in cells treated with DNA damaging agents and the incorporation of [³H]thymidine into DNA. The inhibitory effect of theophylline on NAD depletion and on [³H]thymidine incorporation was dose and cell number dependent. Near normal responses to carcinogen exposure could be restored to theophylline-treated cells following the removal of theophylline. These data suggest that conversion of NAD to poly(ADP-ribose) may be necessary, or at least closely associated with, DNA repair in human lymphocytes.     

Benzo[a]pyrene diol epoxide DNA adduct formation in transformable and non-transformable human foreskin fibroblast cells in vitro

The uptake of benzo[a]pyrene (BP) by low passage (LP) and highpassage (HP) human skin fibroblast cells is followed by itstransport into the nucleus as the parent compound. When theLP and HP cells were treated with BP for 24 h and the DNA wasisolated and enzymatically digested, several DNA adducts weredetected. In both the LP and HP cells a small amount of theradiolabel was associated with the 7ß-BPDE-I—dG,7-BPDE-I—dG and BPDE-II—dG adducts. Although therewere no major qualitative differences in the adducts formedin the LP and HP cells, a higher proportion of the radiolabelwas associated with the 7ß-BPDE-I—dG adductin the LP cells. When LP or HP cells were treated with BPDE-I,the ultimate carcinogenic form of BP, similar levels of DNAmodification were observed in the two cell types and the h.p.l.c.profiles of these adducts were essentially identical. BPDE-Iinduced a carcinogenic event in the LP but not the HP cellsas measured by anchorage independent growth in soft agar andcellular invasiveness of the chick embryonic skin organ cultures.     

Metabolism of benzo[a]pyrene by cultured rat and human buccal mucosa cells

Primary cultures of epithelial and fibroblast cells derivedfrom human oral mucosa were studied for the ability to activatea tobacco smoke carcinogen, benzo[a]pyrene (BP). The cells wereexposed to benzo[a]pyrene for 18 h. The cell-free medium wasextracted with ethylacetate/acetone, and high-pressure liquidchromatography analysis of this fraction revealed that BP tetrolsand diols were the major metabolites formed by both epithelialand fibroblast cells. However, the epithelial cells had a muchhigher rate of biotransformation of BP as measured by bindingto cellular DNA. The mean binding level to human buccal mucosalDNA was among the highest observed in stratified human epithelia.The major BP - DNA adduct was formed by the reaction of the‘bay-region’ BP diolepoxide with the exocyclic 2-aminogroup in guanine. In contrast to human cells, BP phenols andBP 9, 10-diol were the major metabolites produced by primaryepithelial and fibroblast cells derived from rat buccal mucosa.The DNA binding levels of BP in the two rat cell types wereidentical, and the binding level was several-fold lower thanin the human epithelial cells. When an established rat tongueepithelial cell line (RTE 2) was treated with polycyclic aromatichydrocarbons - BP and 7,12-dimethylbenz[a]-anthracene - a slighttoxic effect was observed. Our results indicate that primarycultures of oral mucosa are able to metabolize BP into its ultimatecarcinogenic form at a rate similar to or higher than otherpotential target tissues for BP-induced carcinogenesis.     

Vaccinia-related kinase 1 (VRK1) confers resistance to DNA-damaging agents in human breast cancer by affecting DNA damage response

Vaccinia-related kinase 1 (VRK1) belongs to a group of sixteen kinases associated to a poorer prognosis in human breast carcinomas, particularly in estrogen receptor positive cases based on gene expression arrays. In this work we have studied the potential molecular mechanism by which the VRK1 protein can contribute to a poorer prognosis in this disease. For this aim it was first analyzed by immunohistochemistry the VRK1 protein level in normal breast and in one hundred and thirty six cases of human breast cancer. The effect of VRK1 to protect against DNA damage was determined by studying the effect of its knockdown on the formation of DNA repair foci assembled on 53BP1 in response to treatment with ionizing radiation or doxorubicin in two breast cancer cell lines. VRK1 protein was detected in normal breast and in breast carcinomas at high levels in ER and PR positive tumors. VRK1 protein level was significantly lower in ERBB2 positive cases. Next, to identify a mechanism that can link VRK1 to poorer prognosis, VRK1 was knocked-down in two breast cancer cell lines that were treated with ionizing radiation or doxorubicin, both inducing DNA damage. Loss of VRK1 resulted in reduced formation of DNA-damage repair foci complexes assembled on the 53BP1 scaffold protein, and this effect was independent of damaging agent or cell type. This observation is consistent with detection of high VRK1 protein levels in ER and PR positive breast cancers. We conclude that VRK1 can contribute to make these tumors more resistant to DNA damage-based therapies, such as ionizing radiation or doxorubicin, which is consistent with its association to a poor prognosis in ER positive breast cancer. VRK1 is potential target kinase for development of new specific inhibitors which can facilitate sensitization to other treatments in combination therapies; or alternatively be used as a new cancer drugs.     

Studies on the in vitro transfer of DNA binding benzo[a]pyrene metabolites from rat hepatocytes to human fibroblasts

The release and reabsorption of benzo[a]pyreme (BP) metaboliteswere studied in isolated rat hepatocytes in cubated with BP.There was a rapid uptake of BP by the hepatocytes which wasfollowed by an excretion of organic as well as water-solublemetabolltes. Upon further incubation the BP-dihydrodiols andBP-phenols were reabsorbed by the cells and finally excretedas water-soluble conjugates. The metabolism of BP by the hepatocytesaLso resulted in the formation and release of reactive intermediatesto the incubation medium. The DNA-binding intermediates releasedfrom the cells were consistent with trans-7,8-dihydroxy-7,8-dihydrobenzo-[a]pyreneand 9-hydroxybenzo 4,5-oxide(s). The BP-metabolites were foundto bind to hepatocyte and fibroblast DNA. The degree of DNAbinding was estimated as the formation of DNA strand breaks.BP metabolites formed within the hepatocytes and transferredto the fibroblasts were found to induce DNA strand breaks inthe latter cells. Addition of DNA to the medium markedly decreasedthe fraction of single stranded DNA in these cells. Under conditionswhere the excretion from hepatocytes of BP-phenols and BP dihydrodiolswere increased, the amount of DNA strand breaks in the fibroblastsincreased.     

Prevention of dacarbazine damage of human neoplastic cell DNA by aphidicolin

Treatment of human neoplastic cells with dacarbazine both inhibits DNA synthesis and induces damage in the DNA. Lysis of cells in dilute alkali and subsequent electrophoretic analysis of the isolated DNA show that the DNA of treated cells includes a high molecular weight component and a population of 2-10-kilobase single-stranded DNA fragments while untreated cells contain only high molecular weight DNA. When DNA is pulse-labeled at the beginning of the dacarbazine treatment high amounts of small DNA fragments are seen but no labeled high molecular weight DNA. Moreover the DNA fragments are not formed in cells which are treated with aphidicolin before the addition of dacarbazine. Aphidicolin is a specific inhibitor of DNA polymerase alpha, the enzyme responsible for the replicative synthesis of DNA. We conclude that dacarbazine damages DNA only in cells which are synthesizing new DNA.     

In vitro transformation of rat pleural mesothelial cells by chrysotile fibres and/or benzo[a]pyrene

Rat pleural mesothelial cells (RPMC) were treated, in vitro, in a two-stage model of carcinogenesis. RPMC obtained from passage 12th were treated once with 1 microgram/ml of benzo-[a]pyrene (BP) and from passage 13th to 40th with 0.4 microgram/cm2 of chrysotile fibres (Chr). Transformation was determined by the observation of the colonies formed when the cells were plated at low density. Colonies were classified into four classes according to the level of lack in contact inhibition between cells (0 to III). BP as well as chrysotile fibres were potent in inducing a high proportion of type III colonies which were not observed in control series. In addition, there was no synergistic effect between BP and Chr. These results would indicate that chrysotile fibres do not act as a promoter on rat pleural mesothelial cells in culture, but induce morphologically transformed colonies.     

Effect of benzo[a]pyrene-diol-epoxide-I on growth of nascent DNA in synchronized human fibroblasts

In logarithmically growing cultures of human fibroblasts, benzo[a]pyrene-diol-epoxide-I(BPDE-I) treatment inhibits both replicon initiation and DNAchain elongation. In this study we have focused on the effectof BPDE-I treatment on the growth of nascent DNA strands insynchronized human fibroblasts. Cells were collected at thebeginning of the S phase by replating confluent cultures inthe presence of aphidicolin for 24 h. At 1 h after release fromaphidicolin, cells were incubated with [³H]thymidine for 10–15min and treated with BPDE-I for 10 min. Following differentperiods of incubation with unlabeled thymidine, cells were lysedon top of alkaline sucrose gradients for analysis of the sizedistribution of nascent DNA and its average molecular weight.After a brief pulse with the radiolabded precursor, a unimodaldistribution of nascent DNA strands was observed in controlcells. Upon chase this distribution was displaced to higheraverage molecular weights at an approximate rate of 1 ?10⁶ dattons/min.Treatment with low doses of BPDE-I (<0.2µM) did notdecrease this rate. Indeed the nascent DNA in BPDE-I-treatedcells appeared to grow faster than in control cells. This higherrate of DNA strand growth may be related to the inhibition ofreplicon initiation. Treatment with 0.3µM or 0.7 fM BPDE-I,which are doses that interfere with DNA synthesis in operatingrepJteons in asynchronous cells, also inhibited the growth ofnascent DNA strands in synchronized cells by 22 and 64%, respectively.Because of the simultaneous inhibition of replicon initiation,these values are probably underestimations of the total inhibitionof DNA strand growth. The use of synchronized cells in thistype of study should aid in the elucidation of mechanisms ofreplication of carcinogen-damaged DNA.     

DNA polymerase alpha-primase complexes from carcinogen-treated Chinese hamster ovary cells

In order to investigate whether carcinogens induce alterations of the DNA polymerase alpha-primase complex we compared the physiochemical and catalytic properties and the fidelity of DNA synthesis of DNA polymerase alpha-primase complexes from carcinogen-treated and untreated Chinese hamster ovary cells. Complexes were purified by ion exchange or by immunoaffinity chromatography and both DNA-polymerizing activities and those of ancillary enzymes, such as RNA primase and exonuclease, were examined. Further characterization of the complexes included determination of the relative molecular masses, sedimentation coefficients, and diffusion coefficients, and measurements of the KmS for deoxynucleotide triphosphates and DNA templates, which were identical for the preparations from both carcinogen-treated and untreated cells. The fidelity of DNA polymerase alpha-primase complexes measured by the phi X174am3 reversion assay was also similar in carcinogen-treated and untreated cells. Thus, a carcinogen-mediated induction of a DNA polymerase alpha-primase complex with low fidelity was not observed within the detection limits of the phi X174 assay. RNA primase was found to be an ancillary enzyme activity of the DNA polymerase alpha from both carcinogen-treated and untreated cells; however, the RNA primase:DNA polymerase alpha activity ratio was significantly higher in DNA polymerase alpha-primase complexes from carcinogen-treated cells. These complexes also exhibited an at least 3 times greater velocity of synthesis with supercoiled or unprimed single-stranded DNAs as templates. Since the binding sites of DNA polymerase alpha-primase complexes for deoxynucleotide triphosphates and DNA templates were shown to be identical before and after treatment of cells with carcinogens (i.e., identical Km values for different DNA templates and Ki values for specific inhibitors), the increased synthesis catalyzed by the DNA polymerase alpha-primase complex from carcinogen-treated cells might be due to a carcinogen-induced alteration of an accessory protein of the complex.     

RNA干扰53BP1基因表达对食管癌放射敏感性的影响

背景与目的:细胞周期检测点激酶53BP1在细胞周期调控方面发挥着重要的作用,对体细胞的正常生长没有明显的调节作用,但在DNA损伤发生后被激活,引起细胞周期阻滞。本研究利用RNA干扰技术降低食管癌细胞ECA109细胞53BP1基因表达,观察其对放射线照射后细胞周期和放射敏感性的影响。方法:针对53BP1 mRNA序列,设计合成3对有效的干扰序列(siRNAl、siRNA2和siRNA3)和阴性对照序列,与载体pSIH1-H1-copGFP连接形成重组质粒,瞬时转染细胞,实时PCR(real-time PCR)和Western blot检测53BP1的表达,筛选有效干扰序列,与慢病毒包装质粒混合物共同转染293T细胞,收集病毒液,感染ECA109细胞,获得稳定转染细胞系,命名为ECA109/B,转染空载体组命名为ECA109/N。采用real-time PCR和Western blot测定53BP1在mRNA水平和蛋白水平的表达。采用MTT、流式细胞术和克隆形成实验检测RNA干扰53BP1基因对ECA109细胞放射敏感性的影响。结果:成功构建p53BP1-shRNA质粒,3对干扰序列对人53BP1基因的表达在mRNA和蛋白水平显著低于阴性对照组和空白对照组,尤以siRNA1组效果最明显,取干扰效果最好的siRNA1,利用慢病毒包装系统,共同转染293T细胞,收集病毒液,感染ECA109细胞,荧光显微镜下挑取阳性克隆,扩大培养,获得稳定转染细胞株ECA109/B,53BP1基因在mRNA和蛋白表达水平亦低于对照组;MTT结果表明53BP1低表达对细胞的增殖无明显影响,用流式细胞术分析显示5 Gy射线照射后,ECA109/B的G2/M期比例明显低于阴性对照组和空白对照组;克隆形成实验的结果显示ECA109、ECA109/N、ECA109/B细胞的D0值分别为3.06、2.90和2.07 Gy;SF2值分别为0.91、0.89和0.79;Dq值分别为1.59、1.47和1.21,可见ECA109/N、ECA109放射敏感性未见明显差异,而ECA109/B细胞的放射敏感性高于ECA109/N、ECA109。结论:RNA干扰技术可以有效地抑制食管癌细胞ECA109中53BP1基因的表达,从而增强ECA109细胞的放射敏感性。     

Excessive base excision repair of 5-hydroxymethyluracil from DNA induces apoptosis in Chinese hamster V79 cells containing mutant p53

We have demonstrated previously that the toxicity of 5-hydroxymethyl-2'-deoxyuridine (hmdUrd) to Chinese hamster fibroblasts (V79 cells) results from enzymatic removal of large numbers of hydroxymethyluracil residues from the DNA backbone [Boorstein,R. et al. (1992) Mol. Cell. Biol., 12, 5536-5540]. Here we report that a significant portion of the hmdUrd-induced cell death that is dependent on DNA base excision repair in V79 cells is apoptosis. Incubation of V79 cells with pharmacologically relevant concentrations of hmdUrd resulted in the characteristic changes of apoptosis as measured by gel electrophoresis, flow cytometry and phase contrast microscopy. However, hmdUrd did not induce apoptosis in V79mut1 cells, which are deficient in DNA base excision repair of 5-hydroxymethyluracil (hmUra). Apoptosis was not prevented by addition of 3-aminobenzamide, which inhibits synthesis of poly(ADP-ribose) from NAD, indicating that apoptosis was not the direct consequence of NAD depletion. Pulsed field gel electrophoresis indicated that hmdUrd treatment resulted in high molecular weight (2.2-4.5 Mb) DNA double-strand breaks prior to formation of internucleosomal ladders in V79 cells. Simultaneous measurement of DNA strand breaks with bromodeoxyuridine/terminal deoxynucleotidyl transferase-fluorescein isothiocyanate labeling and of cell cycle distribution indicated that cells with DNA strand breaks accumulated in late S/G(2) and that hmdUrd-treated cells underwent apoptosis after arrest in late S/G(2) phase. Our results indicate that excessive DNA base excision repair results in the generation of high molecular weight DNA double-strand breaks and eventually leads to apoptosis in V79 cells. Thus, delayed apoptosis following DNA damage can be a consequence of excessive DNA repair activity. Immunochemical analysis showed that both V79 and V79mut1 cells contained mutant p53, indicating that apoptosis induced by DNA base excision repair can be independent of p53.     

Heat shock protein 90 regulates phosphatidylinositol 3-kinase-related protein kinase family proteins together with the RUVBL1/2 and Tel2-containing co-factor complex

Heat shock protein 90 (Hsp90), a conserved molecular chaperone for a specific set of proteins critical for signal transduction including several oncogenic proteins, has been recognized as a promising target for anticancer therapy. Hsp90 inhibition also sensitizes cancer cells to DNA damage. However, the underlying mechanisms are not fully understood. Here, we provide evidence that Hsp90 is a general regulator of phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins, central regulators of stress responses including DNA damage. Inhibition of Hsp90 causes a reduction of all PIKK and suppresses PIKK-mediated signaling. In addition, Hsp90 forms complexes with RUVBL1/2 complex and Tel2 complex, both of which have been shown to interact with all PIKK and control their abundance and functions. These results suggest that Hsp90 can form multiple complexes with the RUVBL1/2 complex and Tel2 complex and function in the regulation of PIKK, providing additional rationale for the effectiveness of Hsp90 inhibition for anticancer therapy, including sensitization to DNA damage.     

ATM acts downstream of ATR in the DNA damage response signaling of bystander cells

This study identifies ataxia-telangiectasia mutated (ATM) as a further component of the complex signaling network of radiation-induced DNA damage in nontargeted bystander cells downstream of ataxia-telangiectasia and Rad3-related (ATR) and provides a rationale for molecular targeted modulation of these effects. In directly irradiated cells, ATR, ATM, and DNA-dependent protein kinase (DNA-PK) deficiency resulted in reduced cell survival as predicted by the known important role of these proteins in sensing DNA damage. A decrease in clonogenic survival was also observed in ATR/ATM/DNA-PK-proficient, nonirradiated bystander cells, but this effect was completely abrogated in ATR and ATM but not DNA-PK-deficient bystander cells. ATM activation in bystander cells was found to be dependent on ATR function. Furthermore, the induction and colocalization of ATR, 53BP1, ATM-S1981P, p21, and BRCA1 foci in nontargeted cells was shown, suggesting their involvement in bystander DNA damage signaling and providing additional potential targets for its modulation. 53BP1 bystander foci were induced in an ATR-dependent manner predominantly in S-phase cells, similar to gammaH2AX foci induction. In conclusion, these results provide a rationale for the differential modulation of targeted and nontargeted effects of radiation.     

Protein kinase CK2 is required for the recruitment of 53BP1 to sites of DNA double-strand break induced by radiomimetic drugs

The ataxia telangiectasia mutated (ATM) signaling pathway responds rapidly to DNA double-strand breaks (DSBs) and it is characterized by recruitment of sensor, mediator, transducer and repair proteins to sites of DNA damage. Data suggest that CK2 is implicated in the early cellular response to DSBs. We demonstrate that CK2 binds constitutively the adaptor protein 53BP1 through the tandem Tudor domains and that the interaction is disrupted upon induction of DNA damage. Down-regulation of CK2 results in significant reduction of (i) 53BP1 foci formation, (ii) binding to dimethylated histone H4 and (iii) ATM autophosphorylation. Our data suggest that CK2 is required for 53BP1 accumulation at sites of DSBs which is a prerequisite for efficient activation of the ATM-mediated signaling pathway.     

Foci formation of P53-binding protein 1 in thyroid tumors: activation of genomic instability during thyroid carcinogenesis

Defective DNA damage response (DDR) can result in genomic instability (GIN) and lead to the transformation into cancer. P53-binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins. Because 53BP1 molecules localize at the sites of DNA double strand breaks (DSBs) and rapidly form nuclear foci, the presence of 53BP1 foci can be considered as a cytologic marker for endogenous DSBs reflecting GIN. Although it has been proposed that GIN has a crucial role in the progression of thyroid neoplasms, the significance of GIN during thyroid tumorigenesis remains unclear, particularly in patients. We analyzed, therefore, the level of GIN, as detected with immunofluorescence of 53BP1, in 40 cases of resected thyroid tissues. This study demonstrated a number of nuclear 53BP1 foci in thyroid cancers, suggesting a constitutive activation of DDR in thyroid cancer cells. Because follicular adenoma also showed a few 53BP1 nuclear foci, GIN might be induced at a precancerous stage of thyroid tumorigenesis. Furthermore, high-grade thyroid cancers prominently exhibited an intense and heterogeneous nuclear staining of 53BP1 immunoreactivity, which was also observed in radiation-associated cancers and in mouse colonic crypts as a delayed response to a high dose ionizing radiation, suggesting increased GIN with progression of cancer. Thus, the present study demonstrated a difference in the staining pattern of 53BP1 during thyroid carcinogenesis. We propose that immunofluorescence analysis of 53BP1 expression can be a useful tool to estimate the level of GIN and, simultaneously, the malignant potency of human thyroid tumors.     

Identification of a high-molecular-weight nonfunctional protein in L1210 leukemia cells with common antigenic determinants to dihydrofolate reductase

The concentration of immunoreactive protein in the cytosol of L1210 cells measured using a specific radioimmunoassay for dihydrofolate reductase was substantially greater than the concentration of active enzyme which was measured by the binding of [3H]methotrexate. When the cytosol was subjected to gel filtration, two immunoreactive proteins were separated, a high-molecular-weight (Mr 318,000) protein which did not have catalytic activity and which did not bind [3H]methotrexate and a smaller protein (Mr approximately 20,000) which did reduce [3H]folic acid to tetrahydrofolate and did bind [3H]methotrexate. The nonfunctional high-molecular-weight protein neutralized the inhibitory effect of the antiserum on active dihydrofolate reductase. There was no spontaneous disaggregation of the big species into smaller subunits nor did 8 M urea alone, dithioerythritol alone, boiling with a mixture of 8 M urea and dithioerythritol, or RNase alter its apparent molecular weight. Trypsin, however, digested both the nonfunctional and active immunoreactive forms of the enzyme. Isoelectric focusing of the cytosol separated two nonfunctional immunoreactive isoproteins, each having the same isoelectric points as the two active isoenzymes of dihydrofolate reductase (pls of 8.0 and 8.5). Studies in rapidly replicating and stationary-phase L1210 cells showed that the concentration of the nonfunctional immunoreactive protein increased rapidly, reaching a peak on Day 2 of log growth at which time active enzyme was at a nadir, and then decreased rapidly, reaching a nadir on Day 4, at which time active enzyme was at a peak. The identical isoelectric points for the inactive and active immunoreactive proteins and the reciprocal concentration of each form in logarithmically growing cells suggest that the immunoreactive large species may be a precursor of the active enzyme.     

Lack of oestradiol-DNA binding in mouse embryo cell cultures or following rat-liver microsomal metabolism.

Oestradiol-17beta was compared to benzo[a]pyrene (BP) for its ability to bind to the DNA of mouse embryo cells in culture or to DNA added to a rat liver microsomal incubation mixture. No significant binding was found in embryo cells (at least 100-fold less than for BP). Microsomal incubation resulted in apparent oestradiol binding to a reisolated DNA-containing complex, but most of this was lost when the DNA was freed of RNA and protein.     

ATM-dependent phosphorylation of 53BP1 in response to genomic stress in oxic and hypoxic cells

The ATM kinase is activated by chromatin modification following exogenous and endogenous DSBs or cell stress, including acute anoxia. The p53 binding protein 1 (53BP1) contains multiple ATM-consensus phosphorylation sites in its N- and C-termini and may therefore be a distal read-out of ATM function. We have examined the cellular activation of these phosphorylation sites for the first time in situ following anoxic/hypoxic stress and IR-induced exogenous DSBs. We show that multiple residues of 53BP1 are phosphorylated and that these phosphoforms form discrete nuclear foci following IR or during DNA replication as exogenous or endogenous DNA double strand breaks (DSBs), respectively. Novel data pertaining to the phosphorylation of 53BP1^Ser25in situ supports its dependency on the ATM kinase; but this occurs independently of p53 function. We show that 53BP1^Ser25 is activated specifically in S-phase cells during anoxia in an ATM-dependent manner. Exogenous DSBs form discrete IR-induced foci whereas oxygen stress induced non-localized 53BP1^Ser25 activation. Our in vitro data are supported by irradiated xenograft studies in vivo whereby 53BP1^Ser25 phosphorylation does not occur in sub-regions positive for the hypoxia marker EF5. We propose a model whereby DSBs induce chromatin modification at sites of DNA damage which are tracked by the ATM substrates γ H2AX and 53BP1^Ser25 in a mechanism distinct from p53-mediated cell cycle arrest. Together this work indicates 53BP1^Ser25, and possibly other 53BP1 phosphoforms, as a bona fide DSB-biomarkers for surveying ongoing DNA-damage related signaling in oxic and hypoxic cells during clinical radiotherapy.