Impact of DNA damage repair defects on response to radium-223 and overall survival in metastatic castration-resistant prostate cancer.

PURPOSE: Radium-223 is a targeted alpha radiation therapy for metastatic castration-resistant prostate cancer. DNA damage repair (DDR) defective prostate cancers, specifically genetic aberrations leading to homologous recombination deficiency (HRD), accumulate irreparable DNA damage following genotoxic treatment. This retrospective study assessed DDR mutation status in patients treated with radium-223, investigating their association with efficacy and overall survival (OS). PATIENTS AND METHODS: Included patients were treated with radium-223 and had results from primary or metastatic tumour tissue of a comprehensive next-generation sequencing panel of DDR genes, including canonical HRD genes. Patients were grouped by presence (DDR+) or absence (DDR-) of pathogenic somatic or germline aberrations in DDR genes. We evaluated OS, time to ALP progression (TAP), time to initiation of subsequent systemic therapy (TST) and biochemical responses between DDR groups. RESULTS: Ninety-three patients were included. Twenty-eight (30%) patients had DDR mutations, most frequently in ATM (8.6%), BRCA2 (7.5%) and CDK12 (4.3%) genes. DDR+ patients showed prolonged OS (median 36.3 versus 17.0 months; HR 2.29; P = 0.01). Median TAP and TST in the DDR+ and DDR- patients was 6.9 versus5.8 months (HR = 1.48; P = 0.15), and 8.9 versus7.3 months (HR = 1.58; P = 0.08), respectively. DDR+ patients more frequently completed radium-223 therapy (79% versus 47%; P = 0.05). No difference in biochemical responses were seen. CONCLUSION: Patients harbouring DDR aberrations showed significant OS benefit, and more commonly completed radium-223 therapy. These findings need prospective confirmation and support strategies of genotoxic agents such as radium-223 in patients harbouring DDR defects.

Efficient UV repair requires disengagement of the CSB winged helix domain from the CSB ATPase domain.

The ATP-dependent chromatin remodeler CSB is implicated in a variety of different DNA repair mechanisms, including transcription-coupled nucleotide excision repair (TC-NER), base excision repair and DNA double strand break (DSB) repair. However, how CSB is regulated in these various repair processes is not well understood. Here we report that the first 30 amino acids of CSB along with two phosphorylation events on S10 and S158, previously reported to be required for CSB function in homologous recombination (HR)-mediated repair, are dispensable for repairing UV-induced DNA damage, suggesting that the regulation of CSB in these two types of repair are carried out by distinct mechanisms. In addition, we show that although the central ATPase domain of CSB is engaged in interactions with both the N- and C-terminal regions, these interactions are disrupted following UV-induced DNA damage. The UV-induced disengagement of the C-terminal region of CSB from the ATPase domain requires two conserved amino acids W1486 and L1488, which are thought to contribute to the hydrophobic core formation of the winged helix domain (WHD) at its C-terminus. Failure to undergo UV-induced dissociation of the C-terminal region of CSB from the ATPase domain is associated with impairment in its UV-induced chromatin association, its UV-induced post-translational modification as well as cell survival. Collectively, these findings suggest that UV-induced dissociation of CSB domain interactions is a necessary step in repairing UV-induced DNA damage and that the WHD of CSB plays a key role in this dissociation.

Repair gene O6 -methylguanine-DNA methyltransferase is controlled by SP1 and up-regulated by glucocorticoids, but not by temozolomide and radiation.

Therapy of malignant glioma relies on treatment with the O6 -methylating agent temozolomide (TMZ) concomitant with ionizing radiation followed by adjuvant TMZ. For the treatment of recurrences, DNA chloroethylating drugs are also used. The main killing lesion induced by these drugs is O6 -alkylguanine. Since this damage is repaired by O6 -methylguanine-DNA methyltransferase (MGMT), the repair enzyme represents a most important factor of drug resistance, limiting the therapy of malignant high-grade gliomas. Although MGMT has been shown to be transcriptionally up-regulated in rodents following genotoxic stress, it is still unclear whether human MGMT is subject to up-regulation. Here, we addressed the question whether MGMT in glioma cells is enhanced following alkylating drugs or ionizing radiation, using promoter assays. We also checked the response of glioma cell lines to dexamethasone. In a series of experiments, we found no evidence that the human MGMT promoter is significantly up-regulated following treatment with TMZ, the chloroethylating agent nimustine or radiation. It was activated, however, by dexamethasone. Using deletion constructs, we further show that the basal level of MGMT is mainly determined by the transcription factor SP1. The high amount of SP1 sites in the MGMT promoter likely prevents transcriptional up-regulation following genotoxic stress by neutralizing inducible signals. The regulation of MGMT by miRNAs plays only a minor role, as shown by DICER knockdown experiments. Since high dose dexamethasone concomitant with temozolomide is frequently used in glioblastoma therapy, induction of the MGMT gene through glucocorticoids in MGMT promoter unmethylated cases might cause further elevation of drug resistance, while radiation and alkylating drugs seem not to induce MGMT at transcriptional level.

Dynamic changes in subcellular localization of cattle XLF during cell cycle, and focus formation of cattle XLF at DNA damage sites immediately after irradiation.

Clinically, many chemotherapeutics and ionizing radiation (IR) have been applied for the treatment of various types of human and animal malignancies. These treatments kill tumor cells by causing DNA double-strand breaks (DSBs). Core factors of classical nonhomologous DNA-end joining (C-NHEJ) play a vital role in DSB repair. Thus, it is indispensable to clarify the mechanisms of C-NHEJ in order to develop next-generation chemotherapeutics for cancer. The XRCC4-like factor (XLF; also called Cernunnos or NHEJ1) is the lastly identified core NHEJ factor. The localization of core NHEJ factors might play a critical role in regulating NHEJ activity. The localization and function of XLF have not been elucidated in animal species other than mice and humans. Domestic cattle (Bos taurus) are the most common and vital domestic animals in many countries. Here, we show that the localization of cattle XLF changes dynamically during the cell cycle. Furthermore, EYFP-cattle XLF accumulates quickly at microirradiated sites and colocalizes with the DSB marker γH2AX. Moreover, nuclear localization and accumulation of cattle XLF at DSB sites are dependent on 12 amino acids (288-299) of the C-terminal region of XLF (XLF CTR). Furthermore, basic amino acids on the XLF CTR are highly conserved among domestic animals including cattle, goat and horses, suggesting that the CTR is essential for the function of XLF in domestic animals. These findings might be useful to develop the molecular-targeting therapeutic drug taking XLF as a target molecule for human and domestic animals.

How reliable is immunohistochemical staining for DNA mismatch repair proteins performed after neoadjuvant chemoradiation?

Immunohistochemistry (IHC) testing for mismatch repair proteins (MMRP) is currently being used primarily in colorectal cancer resection specimens. We aimed to compare the results of IHC staining performed on biopsy specimens obtained at endoscopy with that performed on surgical specimens after neoadjuvant therapy. Thirty-two rectal cancer subjects had paired preneoadjuvant and postneoadjuvant tissue available for IHC staining (MLH1, MSH2, MSH6, and PMS2), whereas 39 rectosigmoid cancer patients who did not receive neoadjuvant treatment served as controls. Each slide received a qualitative (absent, focal, and strong) and quantitative score (immunoreactivity [0-3] × percent positivity [0-4]). The quantitative scores of MMRP from the operative material were significantly lower in the neoadjuvant group than in the control (P < .05 for all).The scores of all MMRP from endoscopic biopsies were not significantly different between the neoadjuvant and the control groups. Disagreement between the endoscopic biopsy and the operative material was evident in 23 of 128 stains (18.5%) in the neoadjuvant group and in 12 of 156 stains (7.7%) in the control group (P = .009). In the neoadjuvant group, a disagreement pattern of "endoscopic strong operative focal" was observed in 28.1% for PMS2, 12.5% for MSH6, 12.5% for MLH1, and 6.3% for MSH2, and in the control group, this same disagreement pattern was found in 12.8% for PMS2, 7.7% for MSH6, 7.7% for MLH1, and 0% for MSH2. Based on our findings, we suggest that for rectal cancer, the endoscopic material rather than the operative material should serve as the primary material for IHC staining.

Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways.

Telomeres are at the ends of chromosomes. Previous evidence suggests that laser-induced deoxyribose nucleic acid (DNA) breaks at chromosome ends during anaphase results in delayed cytokinesis. A possible explanation for this delay is that the DNA damage response (DDR) mechanism has been activated. We describe a live imaging method to study the effects of DDR activation following focal point near-infrared femtosecond laser microirradiation either at a single chromosome end or at a chromosome arm in mitotic anaphase cells. Laser microirradiation is used in combination with dual fluorescent labeling to monitor the co-localization of double-strand break marker γH2AX along with the DDR factors in PtK2 (Potorous tridactylus) cells. Laser-induced DNA breaks in chromosome ends as well as in chromosome arms results in recruitment of the following: poly(ADP-ribose) polymerase 1, checkpoint sensors (p-Chk1, p-Chk2), DNA repair protein Ku70/Ku80, and proliferating cell nuclear antigen. However, phosphorylated p53 at serine 15 is detected only at chromosome ends and not at chromosome arms. Full activation of DDR on damaged chromosome ends may explain previously published results that showed the delay of cytokinesis.

Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks.

Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ((788)QIKLNELW(795)). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390-490 and 787-846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.

Targeting nonhomologous end-joining through epidermal growth factor receptor inhibition: rationale and strategies for radiosensitization.

DNA double-strand breaks (DSBs) are the most lethal type of DNA damage induced by ionizing radiation or chemotherapeutic drugs used to eradicate cancer cells. The ability of cancer cells to effectively repair DSBs significantly influences the outcome of therapeutic regimens. Therefore, a new and important area of clinical cancer research is the development of DNA repair inhibitors that can be used as radio- or chemosensitizers. Nonhomologous end joining (NHEJ) is the predominant pathway for the repair of radiation-induced DSBs. A series of recent reports indicates that the epidermal growth factor receptor (EGFR) or its downstream components may modulate NHEJ through direct interaction with the DNA repair enzyme, DNA-dependent protein kinase. Because EGFR is overexpressed or activated in many cancers, these findings provide a compelling rationale for combining radiotherapy with therapies that block EGFR or its downstream signaling components. In this review, we delineate how these novel connections between a cell-surface receptor (EGFR) and a predominantly nuclear event (NHEJ) provide vulnerable nodes that can be selectively targeted to improve cancer therapy.

Transcriptome analysis applied to survival of Shewanella oneidensis MR-1 exposed to ionizing radiation.

The ionizing radiation (IR) dose that yields 20% survival (D20) of Shewanella oneidensis MR-1 is lower by factors of 20 and 200 than those for Escherichia coli and Deinococcus radiodurans, respectively. Transcriptome analysis was used to identify the genes of MR-1 responding to 40 Gy (D20). We observed the induction of 170 genes and repression of 87 genes in MR-1 during a 1-h recovery period after irradiation. The genomic response of MR-1 to IR is very similar to its response to UV radiation (254 nm), which included induction of systems involved in DNA repair and prophage synthesis and the absence of differential regulation of tricarboxylic acid cycle activity, which occurs in IR-irradiated D. radiodurans. Furthermore, strong induction of genes encoding antioxidant enzymes in MR-1 was observed. DNA damage may not be the principal cause of high sensitivity to IR, considering that MR-1 carries genes encoding a complex set of DNA repair systems and 40 Gy IR induces less than one double-strand break in its genome. Instead, a combination of oxidative stress, protein damage, and prophage-mediated cell lysis during irradiation and recovery might underlie this organism's great sensitivity to IR.

Selective DNA damage responses in murine Xpa-/-, Xpc-/- and Csb-/- keratinocyte cultures.

Cellular DNA damage responses (DDRs) are induced by unrepaired DNA lesions and constitute a protective back-up system that prevents the expansion of damaged cells. These cellular signaling pathways trigger either growth arrest or cell death and are believed to be major components of an early anti-cancer barrier. Cultures of C57BL/6J keratinocytes with various defects in NER sub-pathways allowed us to follow the kinetics of DDRs in an isogenic background and in the proper (physiologically relevant) target cells, supplementing earlier studies in heterogenic human fibroblasts. In a series of well-controlled parallel experiments we have shown that, depending on the NER deficiency, murine keratinocytes elicited highly selective DDRs. After a dose of UV-B that did not affect wild-type keratinocytes, Xpa(-/-) keratinocytes (complete NER deficiency) showed a rapid depletion of DNA replicating S-phase cells, a transient increase in quiescent S-phase cells (not replicating DNA), followed by massive apoptosis. Csb(-/-) keratinocytes (TC-NER deficient) responded by a more sustained increase in QS-phase cells and appeared more resistant to UV-B induced apoptosis than Xpa(-/-). In irradiated Xpc(-/-) keratinocytes (GG-NER deficient) the loss of replicating S-phase cells was associated with a gradual build-up of both QS-phase cells and cells arrested in late-S phase, in complete absence of apoptosis. Our analysis complements and extends previous in vivo investigations and highlights both similarities and differences with earlier fibroblast studies. In vitro cultures of murine keratinocytes provide a new tool to unravel the molecular mechanisms of UV-induced cellular stress responses in great detail and in a physiologically relevant background. This will be essential to fully appreciate the implications of DDRs in tumor suppression and cancer prevention.

Comparative analysis of differentially expressed genes in Shewanella oneidensis MR-1 following exposure to UVC, UVB, and UVA radiation.

We previously reported that Shewanella oneidensis MR-1 is highly sensitive to UVC (254 nm), UVB (290 to 320 nm), and UVA (320 to 400 nm). Here we delineated the cellular response of MR-1 to UV radiation damage by analyzing the transcriptional profile during a 1-h recovering period after UVC, UVB, and UVA exposure at a dose that yields about a 20% survival rate. Although the SOS response was observed with all three treatments, the induction was more robust in response to short-wavelength UV radiation (UVB and UVC). Similarly, more prophage-related genes were induced by short-wavelength UV radiation. MR-1 showed an active detoxification mechanism in response to UVA, which included the induction of antioxidant enzymes and iron-sequestering proteins to scavenge reactive oxygen species. In addition, a great number of genes encoding multidrug and heavy metal efflux pumps were induced following UVA irradiation. Our data suggested that activation of prophages appears the major lethal factor in MR-1 following UVC or UVB irradiation, whereas oxidative damage contributes greatly to the high UVA sensitivity in MR-1.

Micronuclei in humans induced by exposure to low level of ionizing radiation: influence of polymorphisms in DNA repair genes.

Understanding the risks deriving from protracted exposure to low doses of ionizing radiation has remarkable societal importance in view of the large number of work settings in which sources of IR are encountered. To address this question, we studied the frequency of micronuclei (MN), which is an indicator of DNA damage, in a population exposed to low levels of ionizing radiation and in matched controls. In both exposed population and controls, the possible influence of single nucleotide polymorphisms in XRCC1, XRCC3 and XPD genes on the frequency of micronuclei was also evaluated. We also considered the effects of confounding factors, like smoking status, age and gender. The results indicated that MN frequency was significantly higher in the exposed workers than in the controls [8.62+/-2.80 versus 6.86+/-2.65; P=0.019]. Radiological workers with variant alleles for XRCC1 or XRCC3 polymorphisms or wild-type alleles for XPD exon 23 or 10 polymorphisms showed a significantly higher MN frequency than controls with the same genotypes. Smoking status did not affect micronuclei frequency either in exposed workers or controls, while age was associated with increased MN frequency in the exposed only. In the combined population, gender but not age exerted an influence on the yield of MN, being higher in females than in males. Even though there is a limitation in this study due to the small number of subjects, these results suggest that even exposures to low level of ionizing radiation could have genotoxic effects and that XRCC3, XRCC1 and XPD polymorphisms might contribute to the increased genetic damage in susceptible individuals occupationally exposed to chronic low levels of ionizing radiation. For a clear conclusion on the induction of DNA damage caused by protracted exposure to low doses of ionizing radiation and the possible influence of genetic polymorphism in DNA repair genes larger studies are needed.

Intracellular redistribution and modification of proteins of the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shock.

Mre11, Rad50, and Nbs1form a tight complex which is homogeneously distributed throughout the nuclei of mammalian cells. However, after irradiation, the Mre11/Rad50/Nbs1 (M/R/N) complex rapidly migrates to sites of double strand breaks (DSBs), forming foci which remain until DSB repair is complete. Mre11 and Rad50 play direct roles in DSB repair, while Nbs1 appears to be involved in damage signaling. Hyperthermia sensitizes mammalian cells to ionizing radiation. Radiosensitization by heat shock is believed to be mediated by an inhibition of DSB repair. While the mechanism of inhibition of repair by heat shock remains to be elucidated, recent reports suggest that the M/R/N complex may be a target for inhibition of DSB repair and radiosensitization by heat. We now demonstrate that when human U-1 melanoma cells are heated at 42.5 or 45.5 degrees C, Mre11, Rad50, and Nbs1 are rapidly translocated from the nucleus to the cytoplasm. Interestingly, when cells were exposed to ionizing radiation (12 Gy of X-rays) prior to heat treatment, the extent and kinetics of translocation were increased when nuclear and cytoplasmic fractions of protein were analyzed immediately after treatment. The kinetics of the translocation and subsequent relocalization back into the nucleus when cells were incubated at 37 degrees C from 30 min to 7 h following treatment were different for each protein, which suggests that the proteins redistribute independently. However, a significant fraction of the translocated proteins exist as a triple complex in the cytoplasm. Treatment with leptomycin B (LMB) inhibits the translocation of Mre11, Rad50, and Nbs1 to the cytoplasm, leading us to speculate that the relocalization of the proteins to the cytoplasm occurs via CRM1-mediated nuclear export. In addition, while Nbs1 is rapidly phosphorylated in the nuclei of irradiated cells and is critical for a normal DNA damage response, we have found that Nbs1 is rapidly phosphorylated in the cytoplasm, but not in the nucleus, of heated irradiated cells. The phosphorylation of cytoplasmic Nbs1, which cannot be inhibited by wortmannin, appears to be a unique post-translational modification in heated, irradiated cells, and coupled with our novel observations that Mre11, Rad50, and Nbs1 translocate to the cytoplasm, lend further support for a role of the M/R/N complex in thermal radiosensitization and inhibition of DSB repair.