DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus

Double-strand breaks (DSBs) can be efficiently removed from the DNA of higher eukaryotes by nonhomologous end-joining (NHEJ). Genetic studies implicate the DNA-dependent protein kinase (DNA-PK) in NHEJ, but the exact function of this protein complex in the rejoining reaction remains to be elucidated. We compared rejoining of DNA DSBs in a human glioma cell line, M059-J, lacking the catalytic subunit of DNA-PK (DNA-PKcs), and their isogenic but DNA-PK-proficient counterpart, M059-K. In both cell lines, rejoining of DNA DSBs was biphasic, with a fast and a slow component operating with a half-life of approximately 22 min and 12 h, respectively. Deficiency in DNA-PK activity did not alter the half-times of either of these components of rejoining but increased from 17 to 72% the proportion of DNA DSB rejoining with slow kinetics. DNA DSB rejoining was nearly complete in both cell lines, and there was only a small increase in the number of unrejoined breaks in M059-J as compared with M059-K cells after 30 h of incubation. Wortmannin radiosensitized to killing M059-K cells and strongly inhibited DNA DSB rejoining. Wortmannin did not affect the radiosensitivity to killing and produced only a modest inhibition in DNA DSB rejoining in M059-J cells, suggesting that, for these end points, DNA-PK is the principal target of the drug. These observations demonstrate that DNA-PK deficiency profoundly decreases the proportion of DNA DSB rejoining with fast kinetics but has only a small effect on the fraction remaining unrejoined. We propose that in higher eukaryotes, an evolutionarily conserved, independently active, but inherently slow NHEJ pathway is stimulated 30-fold by DNA-PKcs to rapidly remove DNA DSBs from the genome. The stimulation is expected to be of local nature and the presence of DNA-PKcs in the vicinity of the DNA DSB determines whether rejoining will follow fast or slow kinetics. Structural and regulatory functions of DNA-PKcs may mediate this impressive acceleration of DNA DSB rejoining, and regions of chromatin within a certain range from this large protein may benefit from these activities. We propose the term DNA-PK surveillance domains to describe these regions.     

Nonhomologous end-joining of ionizing radiation-induced DNA double-stranded breaks in human tumor cells deficient in BRCA1 or BRCA2

Mutations in the BRCA1 or BRCA2 genes predispose to a wide spectrum of familial cancers. The functions of the proteins encoded by BRCA1 and BRCA2 remain to be elucidated, but their interaction and colocalization with hRAD51 suggest a role in homologous recombination and DNA double-strand break (DSB) repair. The role of BRCA1 and BRCA2 in the rejoining of ionizing radiation (IR)-induced DNA DSBs, which may represent a step in the overall process of repair, remains uncertain because recent reports provide conflicting results. Because elucidation of the role of these proteins in DNA DSB rejoining is important for their functional characterization, we reexamined this end point in cells with mutations in either BRCA1 or BRCA2. We show that two pancreatic carcinoma cell lines known to have either wild-type (BxPC3) or mutant forms (Capan-1) of BRCA2 rejoin IR-induced DNA DSBs to a similar extent following biphasic kinetics characterized by a fast and a slow component. Importantly, inactivation of DNA-dependent protein kinase (DNA-PK) by wortmannin generates similar shifts from the fast to the slow component of rejoining in BRCA2-proficient and BRCA2-deficient cells. This suggests that the functioning of either the fast, DNA-PK-dependent component or the slow, DNA-PK-independent component of rejoining is not affected by mutations in BRCA2. Also, a human breast cancer cell line with mutated BRCA1 shows normal rejoining of IR-induced DNA DSBs and levels of inhibition by wortmannin commensurate with the degree of DNA-PK inhibition. These observations fail to confirm a direct role for BRCA1 or BRCA2 in the rejoining of IR-induced DSBs in the genome of human tumor cells and, as a result, an involvement in nonhomologous end-joining. They are in line with similar observations with mutants deficient in genes implicated in homologous recombination and support the view that the radiosensitivity to killing of cells deficient in BRCA1 or BRCA2 derives from defects in this repair pathway.     

DNA-dependent protein kinase content and activity in lung carcinoma cell lines: correlation with intrinsic radiosensitivity

Intrinsic radiosensitivity and rejoining of radiation-induced DNA double-strand breaks (DNA-dsb) were analysed in five lung carcinoma cell lines: U-1285, U-1906, H-69, H-82 and U-1810. RS correlated with both the initial phase of DNA-dsb rejoining, at 15 min (r2 = 0.818) and the late phase, at 120 min postirradiation (r2 = 0.774), the most sensitive cell line (U-1285) showing least dsb rejoining and the most resistant (U-1810) showing most dsb rejoining of all five cell lines studied. As DNA-PK has been recognised as an important molecular component involved in DNA-dsb repair, we analysed content and activity of this kinase. We found that DNA-PK content and activity correlated with RS (r2 = 0.941 and r2 = 0.944, respectively). The lowest DNA-dependent content/activity was found in the most radiosensitive cells, U-1285 and H-69, whilst the highest content/activity was found in the most radioresistant cells U-1810. These results suggest a correlation between RS and DNA-PK content/activity in lung carcinoma cell lines.     

Adenoviral-mediated mda-7 expression suppresses DNA repair capacity and radiosensitizes non-small-cell lung cancer cells

The melanoma differentiation-associated gene-7 (mda-7) was identified by virtue of its enhanced expression in human melanoma cells induced into terminal differentiation. Enforced expression of mda-7 in human cancer cell lines of diverse origins results in the suppression of growth and induction of apoptosis. We have shown that adenoviral-mediated mda-7 (Ad-mda7) radiosensitizes non-small-cell lung cancer (NSCLC) cells by enhancing the apoptotic pathway. To identify the mechanism of this radiosensitization, we examined the level of proteins involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Western blot analysis indicated that the expression of NHEJ pathway components Ku70, XRCC4, and DNA ligase IV was downregulated in NSCLC cells--A549 with Ad-mda7 treatment. No such change was observed in normal human CCD16 fibroblasts previously shown not to be radiosensitized by Ad-mda7. The biological significance of these changes of expression of proteins critical for repair of radiation-induced DSBs was confirmed via the analysis of DSB rejoining kinetics using pulsed field gel electrophoresis and assessment of host cell reactivation capacity following Ad-mda7 treatment. Based on these results, we hypothesize that Ad-mda7 sensitizes NSCLC cells to ionizing radiation by suppressing the activity of NHEJ, a pathway essential for repair of radiation-induced DSBs.     

DNA-PK inhibition by NU7441 sensitizes breast cancer cells to ionizing radiation and doxorubicin

DNA-dependent protein kinase (DNA-PK) plays a key role in the repair of DNA double-strand breaks (DSBs) that are probably the most deleterious form of DNA damage. Inhibition of DNA-PK has been considered as an attractive approach to decrease resistance to therapeutically induced DNA DSBs. Ionizing radiation (IR) and doxorubicin, which induce DSBs, are used in the treatment of breast cancer. We determined the cellular concentration of DNA-PK and other DSB-activated kinases: ATM and ATR and the effect of DNA-PK inhibition by NU7441 on DNA repair, cell cycle, and survival after IR or doxorubicin treatment in three human breast cancer cell lines (MCF-7, MDA-MB-231, and T47D) representing different breast cancer subtypes. T47D cells had the highest expression of DNA-PKcs, ATM, and ATR and the most rapid rate of DNA DSB repair. IR caused a 10- to 16-fold increase in DNA-PK activity and two to threefold induction of ATM in all 3 cell lines. NU7441 inhibited IR-induced DNA-PK activity in all cell lines with IC₅₀s in the range 0.17–0.25 μM. NU7441 retarded the repair of DSB and significantly increased the sensitivity of all cell lines to IR (4- to 12-fold) and doxorubicin (3- to 13-fold). The greatest sensitiziation by NU7441 was observed in MDA-MB-231 cells. NU7441 affected the cell cycle distribution in all studied cell lines; increasing accumulation of cells in G2/M phase after DNA damage. Our data indicate that DNA-PK might be an effective target for chemo- and radio-potentiation in breast cancer and suggest that further development of DNA-PK inhibitors for clinical use is warranted.     

Effects of novel inhibitors of poly(ADP-ribose) polymerase-1 and the DNA-dependent protein kinase on enzyme activities and DNA repair

DNA-dependent protein kinase (DNA-PK) and poly (ADP-ribose) polymerase-1 (PARP-1) participate in nonhomologous end joining and base excision repair, respectively, and are key determinants of radio- and chemo-resistance. Both PARP-1 and DNA-PK have been identified as therapeutic targets for anticancer drug development. Here we investigate the effects of specific inhibitors on enzyme activities and DNA double-strand break (DSB) repair. The enzyme activities were investigated using purified enzymes and in permeabilized cells. Inhibition, or loss of activity, was compared using potent inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361), and cell lines proficient or deficient for DNA-PK or PARP-1. Inactive DNA-PK suppressed the activity of PARP-1 and vice versa. This was not the consequence of simple substrate competition, since DNA ends were provided in excess. The inhibitory effect of DNA-PK on PARP activity was confirmed in permeabilized cells. Both inhibitors prevented ionizing radiation-induced DSB repair, but only AG14361 prevented single-strand break repair. An increase in DSB levels caused by inhibition of PARP-1 was shown to be caused by a decrease in DSB repair, and not by the formation of additional DSBs. These data point to combined inhibition of PARP-1 and DNA-PK as a powerful strategy for tumor radiosensitization.     

Radiosensitivity of human squamous carcinoma cell lines is associated with amount of spontaneous DNA strand breaks

We asked whether the constitutive level of DNA strand breaks (SBs) in four human squamous carcinoma cell lines is associated with their radiosensitivity, measured by the clonogenic assay. Because impairment in DNA replication and the action of endogenous deoxyribonucleases are two major sources of DNA strand breaks under normal cell metabolism, we also analyzed DNA polymerase and DNA ligase activities as well as the functional status of Poly(ADP‐ribose) polymerase (PARP) and nucleolytic degradation of genomic DNA. We showed that the two relatively radioresistant cell lines, UM‐SCC‐1 and UT‐SCC‐5, had a statistically significant lower constitutive level of DNA SBs, as measured by DNA precipitation technique, compared with the two relatively radiosensitive cell lines, UM‐SCC‐14A and UT‐SCC‐9. We found that cell lines with a higher level of broken DNA tended to have a higher constitutive level of DNA polymerase α activity, measured by incorporation of [³H]dTTP in DNase I‐activated DNA. UM‐SCC‐1, UT‐SCC‐5, and UM‐SCC‐14A did not show any difference in DNA ligase activity when a nicked oligonucleotide was used as substrate. The most radiosensitive cell line, UT‐SCC‐9, had a significantly lower ligation efficiency compared to the other three cell lines. The functional status of the PARP was the same in the four cell lines. Although none of the four cell lines showed a characteristic apoptotic or necrotic degradation of genomic DNA, when tested with the “plasmid rejoining assay,” a significant degradation of the plasmid DNA in UT‐SCC‐9 was detected. We conclude that the high fraction of DNA SBs for UT‐SCC‐9, the most radiosensitive cell line, is most likely a consequence of low ligation efficiency combined with a relatively high DNA polymerase α activity and the nuclease degradation of DNA. © 2002 Wiley‐Liss, Inc.     

p53 directly enhances rejoining of DNA double-strand breaks with cohesive ends in gamma-irradiated mouse fibroblasts.

The p53 gene regulates the cell cycle response to DNA damage, which may allow time for adequate DNA repair. We asked whether p53 could directly increase the repair of defined double-strand breaks (DSBs) by nonhomologous end-joining in gamma-irradiated mouse embryonic fibroblasts with differing p53 status. By using an episomal plasmid reactivation assay, we found that presence of wild-type p53 enhanced rejoining of DSBs with short complementary ends of single-stranded DNA. p53 appeared to be directly involved in this regulation, because rejoining enhancement was dependent on the presence of nonspecific DNA binding activity as mediated by the COOH-terminal domain and was independent of transactivating function. We hypothesize that tumor cells lacking p53 and normal cells with wild-type p53 may use different pathways for repair of radiation-induced DSBs.     

DNA double-strand break rejoining in human follicular lymphoma and glioblastoma tumor cells

Follicle center cell lymphoma is among the most radioresponsive of human cancers. To assess whether this radioresponsiveness might be a result of a compromised ability of the tumor cells to accomplish the biologically-effective repair of DNA double-strand breaks (DSBs), we have measured i) the extent of the mechanical rejoining of radiation-induced DSBs in biopsy-derived follicle center cell lymphoma cells and ii) the fidelity with which nuclear protein extracts from these cells rejoin restriction enzyme-induced DSBs. Cell suspensions derived from two lymphoma biopsies, designated FCL1 and FCL2, as well as two established human glioblastoma cell lines, M059J and M059K, were exposed to 30 Gy of gamma-rays and evaluated for their ability to rejoin DSBs using a Southern transfer-pulsed-field gel electrophoresis assay. The fidelity of rejoining of restriction enzyme-induced DSBs was assessed using a cell-free plasmid reactivation assay. Both lymphoma suspensions rejoined DSBs relatively slowly and exhibited a similar phenotype to the known DSB-rejoining deficient M059J line. The level of DSB mis-rejoining in the cell-free plasmid reactivation assay was also similar in M059J and FCL2 cells and was considerably ( approximately 6-fold) higher than in M059K cells. Because of insufficient numbers of cells, we were unable to perform this assay with the FCL1 lymphoma. These limited data suggest that follicle center cell lymphoma cells may be intrinsically deficient in performing the biologically-effective rejoining of DSBs. Such a deficiency might contribute to the radioresponsiveness of this disease and may be exploitable in the development of improved treatment strategies, such as radioimmunotherapy.     

Radiosensitizer effect of wortmannin in radioresistant bladder tumoral cell lines

This work analyzes the effectiveness of wortmannin in boosting the lethality induced by different doses of X-rays, using the colorimetric assay of MTT. Bladder tumoral cell lines differing in radiosensitivity and p53 status were used. Since wortmannin is able to inhibit DNA-dependent protein kinase (DNA-PK) and rejoining of double-strand breaks (DSBs), we have analyzed the constitutive contents and expression after irradiation of the catalytic subunit of DNA-PK (DNA-PKcs) in our cell lines with the aim of explaining the differential effect of wortmannin as radiosensitizer. Considering that DNA-PK is the main protein complex involved in DNA DSB repair, the ability to remove DSBs after irradiation (with or without wortmannin) was evaluated in the different cell lines by the use of pulse-field gel electrophoresis. Our results indicate a higher radiosensitization in the radio-resistant cell line that shows both high constitutive contents of DNA-PKcs and a high rate of DNA repair by the fast component. In contrast, no radiosensitizer effect of wortmannin was observed in the radiosensitive cell line, previously characterized as defective in DSB repair by a low repair fidelity, and - as our results show - with low constitutive contents and later post-irradiation expression of DNA-PKcs. No clear effect related to p53 status of the cell line was observed. These results suggest that high constitutive contents of DNA-PKcs are indicative of radio-resistant phenotypes, and analysis of the expression of this protein could be helpful in the optimal establishment of wortmannin as radiosensitizer in bladder tumoral cell lines.     

Radiation-induced genomic rearrangements formed by nonhomologous end-joining of DNA double-strand breaks

Two major pathways for repairing DNA double-strand breaks (DSBs) have been identified in mammalian cells, nonhomologous end-joining (NHEJ) and homologous recombination (HR). Inactivation of NHEJ is known to lead to an elevated level of spontaneous and radiation-induced chromosomal rearrangements associated with an increased risk of tumorigenesis. This has raised the idea of a caretaker role for NHEJ. It is, however, not known whether NHEJ itself can also cause rearrangements. To investigate, on the DNA level, the influence of a defect in NHEJ on the formation of genomic rearrangements, we applied an assay based on Southern hybridization that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionizing radiation. After 80 Gy of X-irradiation at a high dose rate (23 Gy/min), wild-type cells repaired 50% of the induced DSBs within 24 h by incorrect rejoining. This frequency of DSB misrejoining is considerably reduced in NHEJ-deficient cells. Low-dose-rate experiments, in which the cells were exposed to 80 Gy over a period of 14 days under repair conditions, led to no detectable misrejoining in wild-type cells but revealed a misrejoining frequency of 10% in NHEJ-deficient cells. This shows that in situations of separated breaks, NHEJ deficiency leads to genomic rearrangements, in agreement with chromosomal studies. However, if multiple DSBs coincide, even wild-type cells form genomic rearrangements frequently. These repair events are absent in Ku80-, DNA-PKcs-, and DNA ligase IV-deficient cells but are present in RAD54(-/-) cells. This strongly suggests that NHEJ has, in addition to its caretaker role, also the potential to effect genomic rearrangements. We propose that it serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space.     

Non-homologous end-joining, a sticky affair

Rejoining of broken chromosomes is crucial for cell survival and prevention of malignant transformation. Most mammalian cells rely primarily on the non-homologous end-joining pathway of DNA double-strand break (DSB) repair to accomplish this task. This review focuses both on the core non-homologous end-joining machinery, which consists of DNA-dependent protein kinase and the ligase IV/XRCC4 complex, and on accessory factors that facilitate rejoining of a subset of the DSBs. We discuss how the ATM protein kinase and the Mre11/Rad50/Nbs1 complex might function in DSB repair and what role ionizing radiation-induced foci may play in this process.     

The evolution of rectal and urinary toxicity and immune response in prostate cancer patients treated with two three-dimensional conformal radiotherapy techniques

Background

The protein kinase CK2 sustains multiple pro-survival functions in cellular DNA damage response and its level is tightly regulated in normal cells but elevated in cancers. Because CK2 is thus considered as potential therapeutic target, DNA double-strand break (DSB) formation and rejoining, apoptosis induction and clonogenic survival was assessed in irradiated mammalian cells upon chemical inhibition of CK2.

Methods

MRC5 human fibroblasts and WIDR human colon carcinoma cells were incubated with highly specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB), or mock-treated, 2 hours prior to irradiation. DSB was measured by pulsed-field electrophoresis (PFGE) as well as gamma-H2AX foci formation and removal. Apoptosis induction was tested by DAPI staining and sub-G1 flow cytometry, survival was quantified by clonogenic assay.

Results

TBB treatment did not affect initial DNA fragmention (PFGE; up to 80 Gy) or foci formation (1 Gy). While DNA fragment rejoining (PFGE) was not inhibited by the drug, TBB clearly delayed gamma-H2AX foci disappearence during postirradiation incubation. No apoptosis induction could be detected for up to 38 hours for both cell lines and exposure conditions (monotherapies or combination), but TBB treatment at this moderately toxic concentration of 20 μM (about 40% survival) enhanced radiation-induced cell killing in the clonogenic assay.

Conclusions

The data imply a role of CK2 in gamma-H2AX dephosporylation, most likely through its known ability to stimulate PP2A phosphatase, rather than DSB rejoining. The slight but definite clonogenic radiosensitization by TBB does apparently not result from interference with an apoptosis suppression function of CK2 in these cells but could reflect inhibitor-induced uncoupling of DNA damage response decay from break ligation.     

Modification of non-conservative double-strand break (DSB) rejoining activity after the induction of cisplatin resistance in human tumour cells

The induction of collateral radioresistance after the development of cisplatin resistance is a well-documented phenomenon; however, the exact processes that are responsible for the cisplatin-induced radioresistance remain to be elucidated. There was no obvious difference in the level of radiation-induced DNA double strand breaks (DSBs), in DSB rejoining rates, or the level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in the cisplatin- and radiation-sensitive 2780/WT and cisplatin-resistant 2780/CP cell lines. However, there was a significantly (P < 0.01) lower level of DSB misrejoining activity within nuclear protein extracts derived from the cisplatin- and radiation-sensitive 2780/WT and OAW42/WT tumour cell lines than in similar extracts from their cisplatin- (and radiation-) resistant 2780/CP and OAW42/CP counterparts. All of the DSB misrejoining events involved deletions of between 134 and 444 bp that arose through illegitimate recombination at short repetitive sequences, such as those that arise through non-homologous repair (NHR). These data further support the notion that the radiosensitivity of DSB repair proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways. Furthermore, our data suggest that the induction of acquired cisplatin resistance is associated with a two- to threefold decrease in the activity of a non-conservative DSB rejoining mechanism that appears to be a manifestation of NHR.     

The life and death of DNA-PK

Double-strand breaks (DSBs) arise endogenously during normal cellular processes and exogenously by genotoxic agents such as ionizing radiation (IR). DSBs are one of the most severe types of DNA damage, which if left unrepaired are lethal to the cell. Several different DNA repair pathways combat DSBs, with nonhomologous end-joining (NHEJ) being one of the most important in mammalian cells. Competent NHEJ catalyses repair of DSBs by joining together and ligating two free DNA ends of little homology (microhomology) or DNA ends of no homology. The core components of mammalian NHEJ are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), Ku subunits Ku70 and Ku80, Artemis, XRCC4 and DNA ligase IV. DNA-PK is a nuclear serine/threonine protein kinase that comprises a catalytic subunit (DNA-PK(cs)), with the Ku subunits acting as the regulatory element. It has been proposed that DNA-PK is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets. The crucial role of DNA-PK in the repair of DSBs is highlighted by the hypersensitivity of DNA-PK(-/-) mice to IR and the high levels of unrepaired DSBs after genotoxic insult. Recently, DNA-PK has emerged as a suitable genetic target for molecular therapeutics such as siRNA, antisense and novel inhibitory small molecules. This review encompasses the recent literature regarding the role of DNA-PK in the protection of genomic stability and focuses on how this knowledge has aided the development of specific DNA-PK inhibitors, via both small molecule and directed molecular targeting techniques. This review promotes the inhibition of DNA-PK as a valid approach to enhance the tumor-cell-killing effects of treatments such as IR.     

Selective inactivation of DNA-dependent protein kinase with antisense oligodeoxynucleotides: consequences for the rejoining of radiation-induced DNA double-strand breaks and radiosensitivity of human cancer cell lines

The inhibition of DNA-dependent protein kinase activity with antisense-oligodeoxynucleotide (As-ODN) and its consequences for the rejoining of DNA-double-strand breaks (Dsbs) and radiation sensitivity was studied in human non-small cell lung cancer (NSCLC) cell lines. Cells were transfected with As-ODNs specific for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). In comparison, cells were treated with Wortmannin, a potent but nonspecific inhibitor of DNA-PK activity. As-ODN efficiently reduced the kinase activity with an IC50 of about 100-200 IC50 of Wortmannin was at approximately 5-10 micro M. Treatment of cells with 300 nM As-ODN increased the fraction of residual Dsb at 4 h after irradiation by a factor of 4.4, 2.6, and 1.7 in A549, H460, and H661 cells, respectively. The respective values after treatment with 20 micro M Wortmannin were 5.3, 4.3, and 2.2. Inhibition of DNA-PK activity by As-ODN and Wortmannin also decreased the surviving fraction of the NSCLC cell lines. These data show that kinase activity of DNA-PKcs can be specifically inhibited with As-ODN as effective as Wortmannin and results in marked inhibition of DNA-Dsb rejoining and radiosensitization of NSCLC cell lines.     

Niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase inhibitor,radiosensitizes human lung and breast cancer cells

The aim of this study was to assess niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast and prostate cancers were tested for radiosensitization by niraparib using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of niraparib to alter the repair of radiation-induced DNA double strand breaks (DSBs) was determined using detection of γ-H₂AX foci and RAD51 foci. Clonogenic survival analyses indicated that micromolar concentrations of niraparib radiosensitized tumor cell lines derived from lung, breast, and prostate cancers independently of their p53 status but not cell lines derived from normal tissues. Niraparib also sensitized tumor cells to H₂O₂ and converted H₂O₂-induced single strand breaks (SSBs) into DSBs during DNA replication. These results indicate that human tumor cells are significantly radiosensitized by the potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The mechanism of this effect appears to involve a conversion of sublethal SSBs into lethal DSBs during DNA replication due to the inhibition of base excision repair by the drug. Taken together, our findings strongly support the clinical evaluation of niraparib in combination with radiation.     

Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1

The DNA repair enzymes, DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase-1 (PARP-1), are key determinants of radio- and chemo-resistance. We have developed and evaluated novel specific inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361) for use in anticancer therapy. PARP-1- and DNA-PK-deficient cell lines were 4-fold more sensitive to ionizing radiation (IR) alone, and showed reduced potentially lethal damage recovery (PLDR) in G(0) cells, compared with their proficient counterparts. NU7026 (10 micro M) potentiated IR cytotoxicity [potentiation factor at 90% cell kill (PF(90)) = 1.51 +/- 0.04] in exponentially growing DNA-PK proficient but not deficient cells. Similarly, AG14361 (0.4 micro M) potentiated IR in PARP-1(+/+) (PF(90) = 1.37 +/- 0.03) but not PARP-1(-/-) cells. When NU7026 and AG14361 were used in combination, their potentiating effects were additive (e.g., PF(90) = 2.81 +/- 0.19 in PARP-1(+/+) cells). Both inhibitors alone reduced PLDR approximately 3-fold in the proficient cell lines. Furthermore, the inhibitor combination completely abolished PLDR. IR-induced DNA double strand break (DNA DSB) repair was inhibited by both NU7026 and AG14361, and use of the inhibitor combination prevented 90% of DNA DSB rejoining, even 24-h postirradiation. Thus, there was a correlation between the ability of the inhibitors to prevent IR-induced DNA DSB repair and their ability to potentiate cytotoxicity. Thus, individually, or in combination, the DNA-PK and PARP-1 inhibitors act as potent radiosensitizers and show potential as tools for anticancer therapeutic intervention.     

Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group

Rejoining of ionizing radiation (IR) induced DNA DSBs usually follows biphasic kinetics with a fast (t(50): 5-30 min) component attributed to DNA-PK-dependent non-homologous endjoining (NHEJ) and a slow (t(50): 1-20 h), as of yet uncharacterized, component. To examine whether homologous recombination (HR) contributes to DNA DSB rejoining, a systematic genetic study was undertaken using the hyper-recombinogenic DT40 chicken cell line and a series of mutants defective in HR. We show that DT40 cells rejoin IR-induced DNA DSBs with half times of 13 min and 4.5 h and contributions by the fast (78%) and the slow (22%) components similar to those of other vertebrate cells with 1000-fold lower levels of HR. We also show that deletion of RAD51B, RAD52 and RAD54 leaves unchanged the rejoining half times and the contribution of the slow component, as does also a conditional knock out mutant of RAD51. A significant reduction (to 37%) in the contribution of the fast component is observed in Ku70(-/-) DT40 cells, but the slow component, operating with a half time of 18.4 h, is still able to rejoin the majority (63%) of DSBs. A double mutant Ku70(-/-)/RAD54(-/-) shows similar half times to Ku70(-/-) cells. Thus, variations in HR by several orders of magnitude leave unchanged the kinetics of rejoining of DNA DSBs, and fail to modify the contribution of the slow component in a way compatible with a dependence on HR. We propose that, in contrast to yeast, cells of vertebrates are 'hard-wired' in the utilization of NHEJ as the main pathway for rejoining of IR-induced DNA DSBs and speculate that the contribution of homologous recombination repair (HRR) is at a stage after the initial rejoining.     

DNA-dependent protein kinase (DNA-PK), a key enzyme in the re-ligation of double-stranded DNA breaks]

Repair pathways of DNA are now better defined, and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEHJ) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEJ implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridisation in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catlytic subunit (DNA-PKCS) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immunodeficient (scid) mice are deficient in DNA-PKCS: this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylates Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival.