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Open Access article The glyoxalase system in the cytoplasm of cells provides the primary defence against glycation by methylglyoxal catalysing its metabolism to D-lactate. Methylglyoxal is the precursor of the major quantitative advanced glycation endproducts in physiological systems - arginine-derived hydroimidazolones and deoxyguanosine-derived imidazopurinones. Glyoxalase 1 of the glyoxalase system was linked to anthropometric measurements of obesity in human subjects and to body weight in strains of mice. Recent conference reports described increased weight gain on high fat diet-fed mouse with lifelong deficiency of glyoxalase 1 deficiency, compared to wild-type controls, and decreased weight gain in glyoxalase 1-overexpressing transgenic mice, suggesting a functional role of glyoxalase 1 and dicarbonyl stress in obesity. Increased methylglyoxal, dicarbonyl stress, in white adipose tissue and liver may be a mediator of obesity and insulin resistance and thereby a risk factor for development of type 2 diabetes and non-alcoholic fatty liver disease. Increased methylglyoxal formation from glyceroneogenesis on adipose tissue and liver and decreased glyoxalase 1 activity in obesity likely drives dicarbonyl stress in white adipose tissue increasing the dicarbonyl proteome and related dysfunction. The clinical significance will likely emerge from on-going clinical evaluation of inducers of glyoxalase 1 expression in overweight and obese subjects. Increased transcapillary escape rate of albumin and increased total body interstitial fluid volume in obesity likely makes levels of glycation of plasma protein unreliable indicators of glycation status in obesity as there is a shift of albumin dwell time from plasma to interstitial fluid, which decreases overall glycation for a given glycemic exposure

Repair of DNA interstrand crosslinks (ICLs) predominantly involves the Fanconi anemia (FA) pathway and homologous recombination (HR). The HR repair system eliminates DNA double strand breaks (DSBs) that emerge during ICLs removal. The current study presents a novel cell line, CL‑V8B, representing a new complementation group of Chinese hamster cell mutants hypersensitive to DNA crosslinking factors. CL‑V8B exhibits increased sensitivity to various DNA‑damaging agents, including compounds leading to DSBs formation (bleomycin and 6‑thioguanine), and is extremely sensitive to poly (ADP-ribose) polymerase inhibitor (>400‑fold), which is typical for HR‑defective cells. In addition, this cell line exhibits a reduced number of spontaneous and induced sister chromatid exchanges, which suggests likely impairment of HR in CL‑V8B cells. However, in contrast to other known HR mutants, CL‑V8B cells do not show defects in Rad51 foci induction, but only slight alterations in the focus formation kinetics. CL‑V8B is additionally characterized by a considerable chromosomal instability, as indicated by a high number of spontaneous and MMC‑induced chromosomal aberrations, and a twice as large proportion of cells with abnormal centrosomes than that in the wild type cell line. The molecular defect present in CL‑V8B does not affect the efficiency and stabilization of replication forks. However, stalling of the forks in response to replication stress is observed relatively rarely, which suggests an impairment of a signaling mechanism. Exposure of CL‑V8B to crosslinking agents results in S‑phase arrest (as in the wild type cells), but also in larger proportion of G2/M‑phase cells and apoptotic cells. CL‑V8B exhibits similarities to HR‑ and/or FA‑defective Chinese hamster mutants sensitive to DNA crosslinking agents. However, the unique phenotype of this new mutant implies that it carries a defect of a yet unidentified gene involved in the repair of ICLs.

An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In the current study we have investigated the ability of APE1 inhibitors to induce synthetic lethality in a panel of DNA double strand break (DSB) repair deficient and proficient cells; a) Chinese hamster (CH) cells: BRCA2 deficient (V-C8), ATM deficient (V-E5), wild type (V79) and BRCA2 revertant (V-C8(Rev1)). b) Human cancer cells: BRCA1 deficient (MDA-MB-436), BRCA1 proficient (MCF-7), BRCA2 deficient (CAPAN-1 and HeLa SilenciX cells), BRCA2 proficient (PANC1 and control SilenciX cells). We also tested synthetic lethality (SL) in CH ovary cells expressing a dominant–negative form of APE1 (E8 cells) using ATM inhibitors and DNA-PKcs inhibitors (DSB inhibitors). APE1 inhibitors are synthetically lethal in BRCA and ATM deficient cells. APE1 inhibition resulted in accumulation of DNA DSBs and G2/M cell cycle arrest. Synthetic lethality was also demonstrated in CH cells expressing a dominant–negative form of APE1 treated with ATM or DNA-PKcs inhibitors. We conclude that APE1 is a promising synthetic lethality target in cancer.

The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical γH2AX-staining assay. Although the sensitivity of FANCA(-/-), FANCC(-/-) and FANCA(-/-)C(-/-) cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2(-/-) cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, γH2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex.

O 6 -methylguanine (O 6 MeG) is a highly critical DNA adduct induced by methylating carcinogens and anticancer drugs such as temozolomide, streptozotocine, procarbazine and dacarbazine. Induction of cell death by O 6 MeG lesions requires mismatch repair (MMR) and cell proliferation and is thought to be dependent on the formation of DNA double-strand breaks (DSBs) or, according to an alternative hypothesis, direct signaling by the MMR complex. Given a role for DSBs in this process, either homologous recombination (HR) or non-homologous end joining (NHEJ) or both might protect against O 6 MeG. Here, we compared the response of cells mutated in HR and NHEJ proteins to temozolomide and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The data show that cells defective in HR (Xrcc2 and Brca2 mutants) are extremely sensitive to cell death by apoptosis and chromosomal aberration formation and less sensitive to sister-chromatid exchange (SCE) induction than the corresponding wild-type. Cells defective in NHEJ were not (Ku80 mutant), or only slightly more sensitive (DNA-PK cs mutant) to cell death and showed similar aberration and SCE frequencies than the corresponding wild-type. Transfection of O 6 -methylguanine-DNA methyltransferase (MGMT) in all of the mutants almost completely abrogated the genotoxic effects in both HR and NHEJ defective cells, indicating the mutant-specific hypersensitivity was due to O 6 MeG lesions. MNNG provoked H2AX phosphorylation 24–48 h after methylation both in wild-type and HR mutants, which was not found in MGMT transfected cells. The γH2AX foci formed in response to O 6 MeG declined later in wild-type but not in HR-defective cells. The data support a model where DSBs are formed in response to O 6 MeG in the post-treatment cell cycle, which are repaired by HR, but not NHEJ, in a process that leads to SCEs. Therefore, HR can be considered as a mechanism that causes tolerance of O 6 MeG adducts. The data implicate that down-regulation or inhibition of HR might be a powerful strategy in improving cancer therapy with methylating agents.

The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles. In two of these revertants, V-C8-Rev 1 and V-C8-Rev 6, the reversions lead to the wild-type Brca2 sequence. The V-C8 revertants did not gain the entire wild-type phenotype and still show a 2.5-fold increased sensitivity to mitomycin C (MMC), higher levels of spontaneous and MMC-induced chromosomal aberrations, as well as abnormal centrosomes when compared to wild-type cells. Our results suggest that Brca2 heterozygosity in hamster cells primarily gives rise to sensitivity to DNA cross-linking agents, especially chromosomal instability, a feature that might also be displayed in BRCA2 heterozygous mutation carriers.

V(D)J recombination of Ig and TCR loci is a stepwise process during which site-specific DNA double-strand breaks (DSBs) are made by RAG1/RAG2, followed by DSB repair by nonhomologous end joining. Defects in V(D)J recombination result in SCID characterized by absence of mature B and T cells. A subset of T-B-NK+ SCID patients is sensitive to ionizing radiation, and the majority of these patients have mutations in Artemis. We present a patient with a new type of radiosensitive T-B-NK+ SCID with a defect in DNA ligase IV (LIG4). To date, LIG4 mutations have only been described in a radiosensitive leukemia patient and in 4 patients with a designated LIG4 syndrome, which is associated with chromosomal instability, pancytopenia, and developmental and growth delay. The patient described here shows that a LIG4 mutation can also cause T-B-NK+ SCID without developmental defects. The LIG4-deficient SCID patient had an incomplete but severe block in precursor B cell differentiation, resulting in extremely low levels of blood B cells. The residual D(H)-J(H) junctions showed extensive nucleotide deletions, apparently caused by prolonged exonuclease activity during the delayed D(H)-J(H) ligation process. In conclusion, different LIG4 mutations can result in either a developmental defect with minor immunological abnormalities or a SCID picture with normal development.

Contains fulltext : 48605rakt.pdf (Publisher’s version ) (Closed access) Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response.

The Chinese hamster cell mutant, CL-V4B that is mutated in the Rad51 paralog gene, Rad51C (RAD51L2), has been described to exhibit increased sensitivity to DNA cross-linking agents, genomic instability, and an impaired Rad51 foci formation in response to DNA damage. To directly examine an effect of the Rad51C protein on homologous recombination (HR) in mammalian cells, we compared the frequencies and rates of spontaneous HR in CL-V4B cells and in parental wildtype V79B cells, using a recombination reporter plasmid in host cell reactivation assays. Our results demonstrate that HR is reduced but not abolished in the CL-V4B mutant. We thus, provide direct evidence for a role of mammalian Rad51C in HR processes. The reduced HR events described here help to explain the deficient phenotypes observed in Rad51C mutants and support an accessory role of Rad51C in Rad51-mediated recombination.

Functional analysis of the XRCC genes continues to make an important contribution to the understanding of mammalian DNA double-strand break repair processes and mechanisms of genetic instability leading to cancer. New data implicate XRCC genes in long-standing questions, such as how homologous recombination (HR) intermediates are resolved and how DNA replication slows in the presence of damage (intra-S checkpoint). Examining the functions of XRCC genes involved in non-homologous end joining (NHEJ), paradoxical roles in repair fidelity and telomere maintenance have been found. Thus, XRCC5-7 (DNA-PK)-dependent NHEJ commonly occurs with fidelity, perhaps by aligning ends accurately in the absence of sequence microhomologies, but NHEJ-deficient mice show reduced frequencies of mutation. NHEJ activity seems to be involved in both mitigating and mediating telomere fusions; however, defective NHEJ can lead to telomere elongation, while loss of HR activity leads to telomere shortening. The correct functioning of XRCC genes involved in both HR and NHEJ is important for genetic stability, but loss of each pathway leads to different consequences, with defects in HR additionally leading to mitotic disruption and aneuploidy. Confirmation that these responses are likely to contribute to cancer induction and/or progression, is given by studies of humans and mice with XRCC gene disruptions: those affecting NHEJ show increased lymphoid tumours, while those affecting HR lead to breast cancer and perhaps to gynaecological tumours.

Brca2 deficiency is associated with chromosomal instability and an increased risk of breast and other cancers. To examine the effect of Brca2 deficiency on mutagenesis, we measured the spontaneous mutation rate at the endogenous hprt gene in the Brca2-deficient Chinese hamster cell mutant V-C8. A 4.3-fold increase was found in the spontaneous mutation rate at this locus, indicating the importance of Brca2 in the prevention of mutagenesis. In addition, following exposure to IR, a 2.3-fold increase in mutant frequency per Gy was found for V-C8 in comparison with wild-type V79. These data suggest a potential risk from ionizing radiation for BRCA2 patients.

Purpose: Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is a potent radiosensitizer. The mechanism of dFdC-mediated radiosensitization is yet poorly understood. We recently excluded inhibition of DNA double-strand break (DSB) repair by nonhomologous end-joining (NHEJ) as a means of radiosensitization. In the current study, we addressed the possibility that dFdC might affect homologous recombination (HR)-mediated DSB repair or base excision repair (BER).Methods and Materials: DFdC-mediated radiosensitization in cell lines deficient in BER and in HR was compared with that in their BER-proficient and HR-proficient parental counterparts. Sensitization to mitomycin C (MMC) was also investigated in cell lines deficient and proficient in HR. Additionally, the effect of dFdC on Rad51 foci formation after irradiation was studied.Results: DFdC did induce radiosensitization in BER-deficient cells; however, the respective mutant cells deficient in HR did not show dFdC-mediated radiosensitization. In HR-proficient, but not in HR-deficient, cells dFdC also induced substantial enhancement of the cytotoxic effect of MMC. Finally, we found that dFdC interferes with Rad51 foci formation after irradiation.Conclusion: DFdC causes radiosensitization by specific interference with HR. (C) 2003 Elsevier Inc.

We recently described a CHO DSBR mutant belonging to the XRCC7 complementation group (XR-C2) that has the interesting phenotype of being radiosensitive, but having only a modest defect in VDJ recombination. This cell line expresses only slightly reduced levels of DNA-PKcs but has undetectable DNA-PK activity. Limited sequence analyses of DNA-PKcs transcripts from XR-C2 revealed a point mutation that results in an amino acid substitution of glutamic acid for glycine six residues from the C-terminus. To determine whether this single substitution was responsible for the phenotype in XR-C2 cells, we introduced the mutation into a DNA-PKcs expression vector. Whereas transfection of this expression vector significantly restores the VDJ recombination deficits in DNA-PKcs-deficient cells, radioresistance is not restored. Thus, expression of this mutant form of DNA-PKcs in DNA-PKcs- deficient cells substantially recapitulates the phenotype observed in XR-C2, and we conclude that this single amino acid substitution is responsible for the non-homologous end joining deficits observed in XR-C2.

Fanconi anemia (FA) is a heterogeneous autosomal recessive chromosomal instability syndrome associated with diverse developmental abnormalities, progressive bone marrow failure and a predisposition to cancer. Spontaneous chromosomal breakage and hypersensitivity to DNA cross-linking agents characterize the cellular FA phenotype. The gene affected in FA complementation group G patients was initially identified as XRCC9, for its ability to partially correct the cellular phenotype of the Chinese hamster ovary (CHO) cell mutant UV40. By targeted disruption we generated Fancg/Xrcc9 null mice. Fancg knock-out (KO) mice were born at expected Mendelian frequencies and showed normal viability. In mice, functional loss of Fancg did not result in developmental abnormalities or a pronounced incidence of malignancies. During a 1 year follow-up, blood cell parameters of Fancg KO mice remained within normal values, revealing no signs of anemia. Male and female mice deficient in Fancg showed hypogonadism and impaired fertility, consistent with the phenotype of FA patients. Mouse embryonic fibroblasts (MEFs) from the KO animals exhibited the FA characteristic cellular response in showing enhanced spontaneous chromosomal instability and a hyper-responsiveness to the clastogenic and antiproliferative effects of the cross-linking agent mitomycin C (MMC). The sensitivity to UV, X-rays and methyl methanesulfonate, reported for the CHO mutant cell line UV40, was not observed in Fancg(-/-) MEFs. Despite a lack of hematopoietic failure in the KO mice, clonogenic survival of bone marrow cells in vitro was strongly reduced in the presence of MMC. The characteristics of the Fancg(-/-) mice closely resemble those reported for Fancc and Fanca null mice, supporting a tight interdependence of the corresponding gene products in a common pathway.

Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT) are rare autosomal recessive hereditary disorders characterized by radiosensitivity, chromosomal instability, immunodeficiency and proneness to cancer. Although the clinical features of both syndromes are quite distinct, the cellular characteristics are very similar. Cells from both NBS and AT patients are hypersensitive to ionizing radiation (IR), show elevated levels of chromosomal aberrations and display radioresistant DNA synthesis (RDS). The proteins defective in NBS and AT, NBS1 and ATM, respectively, are involved in the same pathway, but their exact relationship is not yet fully understood. Stumm et al. (Am. J. Hum. Genet. 60 (1997) 1246) have reported that hybrids of AT and NBS lymphoblasts were not complemented for chromosomal aberrations. In contrast, we found that X-ray-induced cell killing as well as chromosomal aberrations were complemented in proliferating NBS-1LBI/AT5BIVA hybrids, comparable to that in NBS-1LBI cells after transfer of a single human chromosome 8 providing the NBS1 gene. RDS observed in AT5BIVA cells was reduced in these hybrids to the level of that seen in immortal NBS-1LBI cells. However, the level of DNA synthesis, following ionizing radiation, in SV40 transformed wild-type cell lines was the same as in NBS-1LBI cells. Only primary wild-type cells showed stronger inhibition of DNA synthesis. In summary, these results clearly indicate that RDS cannot be used as an endpoint in functional complementation studies with immortal NBS-1LBI cells, whereas the cytogenetic assay is suitable for complementation studies with immortal AT and NBS cells.

The molecular basis for the DNA repair dysfunction observed in mutant Chinese hamster ovary cell lines of X-ray repair cross complementing group 1 (XRCC1) is unknown and the exact role of the XRCC1 protein remains unclear. To help clarify the role of the XRCC1 gene we analyzed four mutant cell lines of this complementation group and a revertant cell line for XRCC1 protein content and for sequence alterations in the XRCC1 coding region. Immunoblot analysis of cellular extracts indicated that each of four mutant lines was lacking XRCC1 protein, whereas the repair-proficient revertant line derived from one of these mutants contained a normal level of XRCC1. Although each of these cell lines expressed XRCC1 mRNA, we found in all cases a distinct point mutation resulting in crucial alterations in the encoded XRCC1 protein sequence of 633 amino acids. Two of the mutations cause non-conservative amino acid changes, Glu102-->Lys and Cys390-->Tyr, at positions that are invariant among hamster, mouse and human XRCC1 sequences and are located in putative functional domains. A third debilitating mutation disrupts RNA splicing, generating multiple transcripts of different length that contain deletions spanning a region of >100 amino acids in the midsection of the XRCC1 coding sequence. A fourth mutation results in a termination codon that shortens the open reading frame to 220 amino acids, however, in the revertant cell line a further mutation in the same codon, Stop221-->Leu, permits translation of a full-length functional variant protein. These mutational data indicate the importance of the putative functional regions in XRCC1, such as the BRCA1 C-terminal (BRCT) domain found in common with BRCA1 and other DNA repair and cell cycle checkpoint proteins, and also regions necessary for interaction with DNA polymerase beta and DNA ligase III.

The Chinese hamster cell mutant EM-C11, which is hypersensitive to the cell killing effects of alkylating agents compared to its parental line CHO9, has been used to study the impact of base excision repair on the mutagenic effects of DNA methylation damage. This cell line has a defect in the xrcc1 gene. XRCC1 can interact with DNA polymerase-beta, thereby suppressing strand displacement, and DNA ligase III, both of which have been implicated in base excision repair. XRCC1 may, therefore, allow efficient ligation of single-strand breaks generated during base excision repair. Both EM-C11 and CHO9 cells were treated with methyl methanesulfonate (MMS), a DNA-methylating agent reacting predominantly with nitrogen atoms generating adducts which are substrates for the base excision repair pathway. EM-C11 cells are much more sensitive to the cytotoxic effects of MMS than CHO9: for EM-C11, the dose of MMS inducing 10% survival is 6-fold lower than that for CHO9. In contrast, mutation induction at the hprt locus following MMS is similar in EM-C11 and CHO9. Molecular analysis of hprt gene mutations showed that although the largest class of hprt mutations, both in EM-C11 and CHO9 cells, consisted of GC > AT transitions, most likely caused by O6-methylguanine, the size of this class was smaller in EM-C11. The fraction of deletion mutants in EM-C11, however, was twice as large as that found in CHO9 cells. These results suggest that reduced ligation efficiency of single-strand breaks generated during base excision repair, as result of a defect in XRCC1, may lead to the formation of deletions.

X-ray sensitive Chinese hamster V79 cells mutants, V-C4, V-E5 and V-G8, show an abnormal response to X-ray-induced DNA damage. Like ataxia telangiectasia (AT) cells, they display increased cell killing, chromosomal instability and a diminished inhibition of DNA synthesis following ionizing radiation. To localize the defective hamster gene (XRCC8) on the human genome, human chromosomes were introduced into the AT-like hamster mutants, by microcell mediated chromosome transfer. Although, none of the human chromosomes corrected the defect in these mutants, the defect was corrected by a single mouse chromosome, derived from the A9 microcell donor cell line. In four independent X-ray-resistant microcell hybrid clones of V-E5, the presence of the mouse chromosome was determined by fluorescent in situ hybridization, using a mouse cot-1 probe. By PCR analysis with primers specific for different mouse chromosomes and Southern blot analysis with the mouse Ldlr probe, the mouse chromosome 9, was identified in all four X-ray-resistant hybrid clones. Segregation of the mouse chromosome 9 from these hamster-mouse microcell hybrids led to the loss of the regained X-ray-resistance, confirming that mouse chromosome 9 is responsible for complementation of the defect in V-E5 cells. The assignment of the mouse homolog of the ATM gene to mouse chromosome 9, and the presence of this mouse chromosome only in the radioresistant hamster cell hybrids suggest that the hamster AT-like mutant are homologous to AT, although they are not complemented by hamster chromosome 11.

The mitomycin C (MMC)-hypersensitive Chinese hamster V79 cell mutant V-H4 has a cellular phenotype similar to Fanconi anemia (FA), and has been shown to be homologous to FA group A. To examine consequences of the defect in V-H4 cells on spontaneous mutagenesis, we studied the frequency and nature of spontaneous mutations at the hypoxanthine phosphoribosyltransferase (HPRT) locus in this mutant and the parental V79 cells. The mutation rates expressed as the number of mutations per cell per generation were 8.7X10 -7 and 3.7X10 -7 for V-H4 and V79 cells respectively. The molecular spectrum of 42 spontaneous hprt mutants of V-H4 cells was determined and compared with the previously described spectrum of spontaneous mutations at the HPRT locus of Chinese hamster V79 cells. The spectra of spontaneous mutations in the hprt gene of both cell lines are predominated by base pair substitutions and splice mutations. Among the base changes, V-H4 shows a larger frequency of transitions (13/42 ; 31%) than transversions (3/42 ; 7%), whereas in V79 transversions are observed more often than transitions (P < 0.001 ; Wilcoxon test). The frequency of splice mutations in V-H4 (17/42 ; 40%), which affects exon 4 almost exclusively, is not significantly different from V79. The fraction of deletions in V-H4 is low (6/42 ; 14%), and comparable to the level in V79. This is in contrast with the published molecular spectrum of spontaneous hprt mutants in FA (group D) cells, which consists predominantly of deletions.

The eukaryotic Rad51 protein is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination. Five paralogs of Rad51 have been identified in vertebrates, Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3, which are also implicated in recombination and genome stability. Here, we identify a mammalian cell mutant in Rad51C. We show that the Chinese hamster cell mutant, CL-V4B, has a defect in Rad51C. Sequencing of the hamster Rad51C cDNA revealed a 132 bp deletion corresponding to an alternatively spliced transcript with lack of exon 5. CL-V4B was hypersensitive to the interstrand cross-linking agents mitomycin C (MMC) and cisplatinum, the alkylating agent methyl methanesulfonate and the topoisomerase I inhibitor campthotecin and showed impaired Rad51 foci formation in response to DNA damage. The defect in Rad51C also resulted in an increase of spontaneous and MMC-induced chromosomal aberrations as well as a lack of induction of sister chromatid exchanges. However, centrosome formation was not affected. Intriguingly, a reduced level of sister chromatid cohesion was found in CL-V4B cells. These results reveal a role for Rad51C that is unique among the Rad51 paralogs.

It has been shown that the Chinese hamster cell mutant V-C8 is sensitive to different DNA damaging agents, such as mitomycin C (MMC), alkylating agents, UV light, and X-rays. We found that V-C8 is also sensitive to the following radiomimetic agents: bleomycin (approximately 2-fold, based on D10 values), H2O2 (approximately 2-fold), streptonigrin (approximately 11-fold), and etoposide (approximately 8-fold). Two independent spontaneous MMC-resistant revertants isolated from V-C8 cells show a level of cell killing by X-rays, EMS, and UV light which is similar to that of wild-type cells, suggesting that the observed pattern of cross-sensitivity of V-C8 cells to a wide spectrum of DNA damaging agents results from a single mutation. V-C8 cells also display radioresistant DNA synthesis following gamma-irradiation which, however, remained almost unchanged in the V-C8 revertants. The measurement of the level and rate of repair of DNA single- and double-strand breaks (SSBs and DSBs, respectively) by the DNA elution technique showed that the V-C8 mutant has a slower repair of DSBs induced by gamma-rays. The described unique phenotype of V-C8 cells suggested that V-C8 represents a novel type of mutant amongst X-ray-sensitive hamster cell mutants. To confirm this, complementation analysis with other X-ray-sensitive mutants was performed. V-C8 cells were fused with EM9, XR-1, xrs5, sxi-1, V-3, V-E5, irs3, and BLM2 mutant cells, representing different complementation groups. All the obtained hybrids regained X-ray resistance (or bleomycin resistance in the case of V-C8/BLM2 hybrids) similar to that of wild-type cells, indicating that V-C8 represents a new complementation group. The results presented indicate that V-C8 is defective in a gene involved in a pathway operating in the responses to different DNA damaging agents in mammalian cells.

Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG(-/-) and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ.

A Chinese hamster cell mutant (V-C8) isolated previously, which is approximately 100 fold more sensitive to mitomycin C (MMC) than its parental wild-type V79 cells (judged by D10 values), was further characterized. V-C8 cells exhibit an increased sensitivity towards other cross-linking agents, such as cis-DDP (approximately 40-fold), DEB (approximately 30-fold), and also to adriamycin (approximately 5-fold), and the monofunctional alkylating agents: MMS (approximately 5-fold) and EMS (approximately 6-fold). V-C8 cells show a higher level induction of chromosomal aberrations by cross-linking agents (MMC, cis-DDP, and DEB) and an increased level of spontaneous chromosomal aberrations in comparison to the wild-type V79 cells. To determine whether the V-C8 mutant represents a new complementation group among Chinese hamster cell mutants that also display the extreme sensitivity to MMC, V-C8 cells were fused with irs1, irs1SF, UV20, UV41, and V-H4 cells. In all cases, the derived hybrids regained the MMC sensitivity similar to wild-type cells, indicating that the V-C8 mutant belongs to a new sixth complementation group.

Several radiosensitive mutant cell lines of CHO and V79 cells have been studied to explore a possible correlation between radiation induced DNA lesions and chromosomal aberrations. In the xrs mutants which are deficient in DNA double strand break (DSB) repair, there is a correlation between the extent of the defect in repair and the frequencies of radiation induced chromosomal aberrations. In another type of radiosensitive mutant (V-C4), which has no detectable defect in DNA DSB repair, the frequencies of X-ray induced aberrations are high in comparison to wild type V79 cells. However, following treatment with restriction endonucleases or fission neutrons, the frequencies of aberrations are similar to those in V79, indicating that V-C4 cells are defective in repair of X-ray induced lesions other than DSBs. Though DSBs are the most important lesions leading to chromosomal aberrations, in repair deficient mutants, radiation induced lesions other than DSBs can lead to chromosomal aberrations.

Homologous recombination is essential for repair of DNA interstrand cross-links and double-strand breaks. The Rad51C protein is one of the five Rad51 paralogs in vertebrates implicated in homologous recombination. A previously described hamster cell mutant defective in Rad51C (CL-V4B) showed increased sensitivity to DNA damaging agents and displayed genomic instability. Here, we identified a splice donor mutation at position +5 of intron 5 of the Rad51C gene in this mutant, and generated mice harboring an analogous base pair alteration. Rad51C(splice) heterozygous animals are viable and do not display any phenotypic abnormalities, however homozygous Rad51C(splice) embryos die during early development (E8.5). Detailed analysis of two CL-V48 revertants, V4B-MR1 and V4B-MR2, that have reduced levels of full-length Rad51C transcript when compared to wild type hamster cells, showed increased sensitivity to mitomycin C (MMC) in clonogenic survival, suggesting haploinsufficiency of Rad51C. Similarly, mouse Rad51C(splice/neo) heterozygous ES cells also displayed increased MMC sensitivity. Moreover, in both hamster revertants, Rad51C haploinsufficiency gives rise to increased frequencies of spontaneous and MMC-induced chromosomal aberrations, impaired sister chromatid cohesion and reduced cloning efficiency. These results imply that adequate expression of Rad51C in mammalian cells is essential for maintaining genomic stability and sister chromatid cohesion to prevent malignant transformation. (C) 2010 Elsevier B.V. All rights reserved.

Fanconi anemia is a human inherited disease that is characterized by cellular hypersensitivity to DNA cross-linking agents. A number of potential experimental models for that disorder have been developed by selecting mutants that are hypersensitive to bifunctional mutagens. The six mutants of that class in Drosophila, all of which map to the mus308 locus, express an alteration in a mitochondrial nuclease. A recent extension of that observation to cell lines from complementation group A of Fanconi anemia has established a new cellular phenotype for that disorder. In the current study an analogous enzyme has been analyzed in eight recently isolated Chinese hamster cell lines that are hypersensitive to cross-linking agents. Among these lines. V-H4 and V-B7 are shown to exhibit an enzyme modification analogous to that observed in the mutant Drosophila and human cells. These results validate the nuclease assay as an indicator of the Fanconi defect and further establish the V-H4 cell line as a valuable cellular model for analysis of the Fanconi A defect.

A partial revertant (RH1–26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1–26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1–26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1–26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1–26 cells but different from that of V-H1 cells. Since in RH1–26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.

Radiosensitive T-B- severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-PKcs missense mutation (L3062R). The causative mutation did not affect the kinase activity or DNA end-binding capacity of DNA-PKcs itself; rather, the presence of long P-nucleotide stretches in the immunoglobulin coding joints indicated that it caused insufficient Artemis activation, something that is dependent on Artemis interaction with autophosphorylated DNA-PKcs. Moreover, overall end-joining activity was hampered, suggesting that Artemis-independent DNA-PKcs functions were also inhibited. This study demonstrates that the presence of DNA-PKcs kinase activity is not sufficient to rule out a defect in this gene during diagnosis and treatment of RS-SCID patients. Further, the data suggest that residual DNA-PKcs activity is indispensable in humans.

DNA excision repair modulates the mutagenic effect of many genotoxic agents. The recently observed strand specificity for removal of UV-induced cyclobutane dimers from actively transcribed genes in mammalian cells could influence the nature and distribution of mutations in a particular gene. To investigate this, we have analyzed UV-induced DNA repair and mutagenesis in the same gene, i.e. the hypoxanthine phosphoribosyl-transferase (hprt) gene. In 23 hprt mutants from V79 Chinese hamster cells induced by 2 J/m2 UV we found a strong strand bias for mutation induction: assuming that pre-mutagenic lesions occur at dipyrimidine sequences, 85% of the mutations could be attributed to lesions in the nontranscribed strand. Analysis of DNA repair in the hprt gene revealed that more than 90% of the cyclobutane dimers were removed from the transcribed strand within 8 hours after irradiation with 10 J/m2 UV, whereas virtually no dimer removal could be detected from the nontranscribed strand even up to 24 hr after UV. These data present the first proof that strand specific repair of DNA lesions in an expressed mammalian gene is associated with a strand specificity for mutation induction.

UV sensitive Chinese hamster mutants belonging to ERCC groups 1, 2 and 6 together with one cross-link- and one X-ray-sensitive mutant have been examined for sensitivity to 4-nitroquinoline-1-oxide (4NQO) and the ability to repair 4NQO adducts at the N2 and C8 of guanosine. Despite the fact that all of the mutants examined were hyper-sensitive to 4NQO there was little difference between the mutants V-H1, V-H4, V-C4 and UV61 and the parental cell lines as regards the ability to remove these lesions from bulk DNA. The UV5 and UV20 mutants were both defective in the ability to remove N2 guanosine adducts yet repaired the C8 guanine adduct as normal. The fact that the mutants V-H1, V-H4, V-C4 and UV61 are 4NQO sensitive but repair the above adducts suggests that either some other lesion(s) is responsible for increased toxicity in these mutants, or that some regions of the genome may not be repaired as effectively as bulk DNA in these mutants, or that the quality of the repair is less than in the parental cells. Clearly the inability to remove UV induced pyrimidine dimers and the (6-4) photoproduct associated with the UV5 and UV20 mutants correlates with the inability to repair 4NQO-N2 guanosine adducts. However, mutants capable of (6-4) photoproduct repair but not dimer repair (VH-1 and UV61) can repair this lesion. Hence it is possible that the same domains in these repair proteins are required for the recognition of (6-4) photoproduct repair and 4NQO-N2 guanosine adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

V-H4, a mitomycin C (MMC)-sensitive Chinese hamster cell mutant, is phenotypically very similar to Fanconi anemia (FA) cells. Genetic complementation analysis shows that V-H4 belongs to the same complementation group as FA group A cells. Proliferating hybrid cell lines obtained after fusion of V-H4 with normal or FA group B cells show an increased resistance to MMC. Absence of complementation was noted in V-H4 × FA group A hybrid cell lines. This was shown not to be due to the absence of a specific human chromosome. The V-H4 mutant represents the first rodent mutant that is genotypically similar to FA complementation group A cells.

It has been shown by genetic complementation analysis that a mitomycin C-sensitive mutant (V-H4) of Chinese hamster V79 cells is the first rodent equivalent of Fanconi anemia (FA) group A. The V-H4 mutant shows many typical characteristics of cells derived from FA patients. V-H4 cells exhibit increased sensitivity towards cross-linking agents as MMC (approximately 30-fold), cis-DDP (approximately 10-fold), DEB (approximately 10-fold), and PUVA (approximately 1.6-fold), but an only slightly increased sensitivity to monofunctional alkylating agents (EMS and MMS) and actinomycin D. V-H4 cells are also moderately sensitive to adriamycin (1.6-fold), and not sensitive to H2O2. The levels of chromosomal aberrations induced by MMC and cis-DDP treatment are higher (4- to 6-fold) in V-H4 cells than in the wild-type V79 cells. Genetic complementation analysis with other Chinese hamster mutants hypersensitive to MMC (irs1, irs1SF, UV20 and UV41) indicates clearly that V-H4 belongs to a different, new complementation group. This unique mutant is very stable and can serve as a vehicle to isolate the complementing FA-A gene from normal human DNA.

Faithful genome transmission requires a network of pathways coordinating DNA replication to DNA repair and recombination. Here, we used molecular combing to measure the impact of homologous recombination (HR) on the velocity of DNA replication forks. We used three hamster cell lines defective in HR either by overexpression of a RAD51 dominant-negative form, or by a defect in the RAD51 paralogue XRCC2 or the breast tumor suppressor BRCA2. Irrespectively of the type or extent of HR alteration, all three cell lines exhibited a similar reduction in the rate of replication-fork progression, associated with an increase in the density of replication forks. Importantly, this phenotype was completely reversed in complemented derivatives of Xrcc2 and Brca2 mutants. These data reveal a novel role for HR, different from the reactivation of stalled replication forks, which may play an important role in genome stability and thus in tumor protection.

The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA). To gain insight into the role of BRCA2 in the repair of DNA damage, we fused a single (BRC3, BRC4) or multiple BRC motifs to the large RPA subunit. Expression of any of these protein fusions in Brca2 mutant cells substantially improved HDR while suppressing mutagenic repair. A fusion containing a Rad52 ssDNA-binding domain also was active in HDR. Mutations that reduced ssDNA or Rad51 binding impaired the ability of the fusion proteins to function in HDR. The high level of spontaneous chromosomal aberrations in Brca2 mutant cells was largely suppressed by the BRC-RPA fusion proteins, supporting the notion that the primary role of BRCA2 in maintaining genomic integrity is in HDR, specifically to deliver Rad51 to ssDNA. The fusion proteins also restored Rad51 focus formation and cellular survival in response to DNA damaging agents. Because as little as 2% of BRCA2 fused to RPA is sufficient to suppress cellular defects found in Brca2 -mutant mammalian cells, these results provide insight into the recently discovered diversity of BRCA2 domain structures in different organisms.

Inhibition of replicative DNA synthesis by ionizing radiation is partly caused by an active, signal-mediated response termed the "S-phase checkpoint." Defects in this checkpoint were first discovered in the human inherited disorder ataxia-telangiectasia (AT). gamma-Irradiated cells from AT patients consistently display a diminished inhibition of DNA synthesis, a feature called "radioresistant DNA synthesis" (RDS). RDS has been widely used as a diagnostic marker for AT, in postnatal as well as prenatal material. The regulation and control of the S-phase checkpoint is complex and multifaceted; it is not restricted to ionizing radiation, but can occur after many genotoxic stressors. Defects in both upstream control functions, such as ATM, NBS1, and MRE11, as well as downstream modulators can provoke an RDS phenotype. Here a simple, accurate and highly reproducible experimental protocol is presented for the generation of DNA synthesis inhibition curves from cells in culture.

Mutant rodent cell lines hypersensitive to DNA-damaging agents have provided a useful tool for the characterization of DNA repair pathways and have contributed to a better understanding of the mechanisms involved in the cellular responses to mutagenic treatment. Here we present a detailed description of how to isolate mutagen-sensitive mutants from hamster "wild-type" cell lines. First, cells are treated with ethyl nitrosourea, and then the mutagenized cell populations are screened for cells with an increased sensitivity to various mutagens using a replica-plating method. Mutagen-sensitive clones are identified and then characterized by assessing their stability, degree of sensitivity to various mutagens, and by genetic complementation analysis.

Five mutant Chinese hamster cell lines deficient in DNA repair with the corresponding parental cell lines were used to determine their sensitivity to cisplatin, 5-fluorouracil and gemcitabine. The mutations in the cell lines led to defective single strand break repair (EM-C11), defective recombination mediated repair (irs1SF), defective double strand break repair (XR-V15B, a Ku-80 mutant and CR-C1, a DNA-PKcs mutant) and an AT-like mutation (VC-4). All mutant cell lines had an impaired doubling time during exponential growth and an increased sensitivity to X-irradiation. We may conclude that for cisplatin-induced cytotoxicity the homologous recombination-associated DNA repair plays an important role in the repair of the cisplatin induced lesions, confirming previous results. In 5-FU and gemcitabine induced toxicity to cells, repair processes involved with radiation-induced damage were not implicated. This is in striking contrast to the role of cisplatin in radiosensitization where inhibition of the NHEJ pathway is implicated, and to the role of gemcitabine in sensitization where specific interference with the HR pathway is implicated.

The role of DNA repair mechanisms in the cellular response to low dose rate (LDR) irradiation was studied with the aim to gain insight in the process of sublethal damage (SLD) repair. Chinese hamster cell lines mutated in either DNA single strand break (ssb) repair or DNA double strand break (dsb) repair by non homologous end joining (NHEJ) and homologous recombination (HR), or showing an AT-like phenotype, were irradiated in plateau-phase either at high dose rate (HDR, 3.3 Gy/min) or at pulsed low dose rate (p-LDR, average 1 Gy/h). Cell survival after irradiation was assessed using the clonogenic assay. A change in sensitivity when the dose rate was decreased was observed for all parental cell lines and the DNA ssb repair mutant. No difference in cell survival after p-LDR versus. HDR irradiation was observed for the two NHEJ mutants, the AT-like mutant and the HR mutant. Based on these results we conclude that single strand break repair does not play a role in the dose rate effect. The AT like protein, functional NHEJ and XRCC3 are required for the dose rate effect.

Analysis of the XRCC genes has played an important part in understanding mammalian DNA repair processes, especially those involved in double-strand break (DSB) repair. Most of these genes were identified through their ability to correct DNA damage hypersensitivity in rodent cell lines, and they represent components of several different repair pathways including base-excision repair, non-homologous end joining, and homologous recombination. We document the phenotypic effects of mutation of the XRCC genes, and the current state of our knowledge of their functions. In addition to their continuing importance in discovering mechanisms of DNA repair, analysis of the XRCC genes is making a substantial contribution to the understanding of specific human disorders, including cancer.

The Chinese hamster cell mutant V-C8 is defective in the Brca2 gene (Kraakman-van der Zwet et al., 2002, Cell Biol.; 22: 669). Here we report that V-C8 cells were 10-fold more sensitive to camptothecin, an inhibitor of topoisomerase I, than the parental V79 cells. The level of the relaxation activity of topoisomerase I in nuclear extracts was also lower (4-fold) in V-C8 than V79 cells, in spite of the fact that the level of the topoisomerase I protein was the same in these cells. The survival of V-C8 cells in the presence of camptothecin, the sensitivity of V-C8 topoisomerase I to camptothecin, and the level of the relaxation activity in V-C8 nuclear extract were almost completely restored by transfection of V-C8 cells with the murine Brca2 gene or by the transfer of human chromosome 13 providing the BRCA2 gene. These results indicate that the observed changes in the topoisomerase I activity in V-C8 are due to the defective function of the Brca2 gene.

The end-joining pathway of DNA double-strand break (DSB) repair is necessary for proper V(D)J recombination and repair of DSB caused by ionizing radiation. This DNA repair pathway can either use short stretches of (micro)homology near the DNA ends or use no homology at all (direct end-joining). We designed assays to determine the relative efficiencies of these (sub)pathways of DNA end-joining. In one version, a DNA substrate is linearized in such a way that joining on a particular microhomology creates a novel restriction enzyme recognition site. In the other one, the DSB is made by the RAG1 and RAG2 proteins. After PCR amplification of the junctions, the different end-joining modes can be discriminated by restriction enzyme digestion. We show that inactivation of the 'classic' end-joining factors (Ku80, DNA-PK(CS), ligase IV and XRCC4) results in a dramatic increase of microhomology-directed joining of the linear substrate, but very little decrease in overall joining efficiency. V(D)J recombination, on the other hand, is severely impaired, but also shows a dramatic shift towards microhomology use. Interestingly, two interstrand cross-linker-sensitive cell lines showed decreased microhomology-directed end-joining, but without an effect on V(D)J recombination. These results suggest that direct end-joining and microhomology-directed end-joining constitute genetically distinct DSB repair pathways.

The RBE of alpha-particles in different mutations of Chinese hamster cells was determined with the aim of identifying differences in the sensitivity to x-ray and alpha-particle-induced DNA damage. Two parental lines of Chinese hamster cells and four radiosensitive mutants were irradiated with different single doses of x-rays and alpha-particles and clonogenic cell survival was determined. Radiosensitivity to x-rays varied by a factor of 5 between the cell strains whereas sensitivity to alpha-particle irradiation was almost identical among all strains. The RBE is only determined by the sensitivity of the cells towards x-rays. Since cells with different defects of repair or cell cycle control have different radiosensitivities, we conclude that the effects of x-ray irradiation and the RBE are mostly determined by the activity of repair processes.

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.

The nucleotide excision repair (NER) pathway in eukaryotic cells is required for the error-free removal from DNA of lesions such as pyrimidine (6-4) pyrimidone photoproducts (PPs), cyclobutane pyrimidine dimers (CPDs), and bulky chemical adducts (1, 9, 37, 48). The molecular mechanism of NER is a complex process requiring about 25 proteins in humans. NER can be subdivided into global-genomic repair and transcription-coupled repair (TCR). These subpathways differ with respect to damage recognition and the rate at which some forms of DNA damage are repaired. For many lesions, including CPDs, the global-genomic pathway operates in all parts of the genome but acts relatively slowly. In this pathway, the XPA, RPA, and XPC-hHR23B proteins participate in damage recognition (41, 61, 62). Removal of CPDs and chemical adducts during TCR occurs more rapidly in the transcribed strand of active genes (30). Repair is initiated when an RNA polymerase II elongation complex is blocked by a lesion and requires the CSA and CSB proteins but not XPC-hHR23B (9). In both repair pathways, repair or transcription factor TFIIH is required to unwind the DNA duplex surrounding the lesion to facilitate incision by the XPG and XPF-ERCC1 single-strand or double-strand junction-specific endonucleases (10, 11, 34, 35, 38). This unwinding reaction absolutely requires the 3′ to 5′ and 5′ to 3′ helicase activities of the TFIIH subunits XPB/ERCC3 (28) and XPD/ERCC2 (42, 66), respectively. Along with its role in NER, TFIIH (reviewed in reference 15) is necessary for promoter opening during basal transcription initiation by RNA polymerase II. This nine-member protein complex has several enzymatic activities, including a cyclin-dependent kinase activity comprising subunits Cdk7, cyclin H, and Mat1. The XPB and XPD subunits of TFIIH possess single-stranded DNA-dependent helicase-associated ATPase activities. Interestingly, the helicase activity of only XPB is required for transcription. Although XPD is required for transcription, its role appears structural rather than catalytic (7, 16, 32, 51). A K48R mutation in the canonical GKS/T ATP binding motif of XPD or the Rad3 homolog abolishes repair but does not compromise transcription (43, 66). Mutations in the XPD/ERCC2 gene result in three distinct diseases (reviewed in references 3, 5, and 48): cancer-prone xeroderma pigmentosum (XP), XP in combination with Cockayne syndrome (CS; a developmental disorder), and trichothiodystrophy (TTD), which involves neurological and developmental abnormalities distinct from those of CS. Unlike the situation with most other XP genes, XPD mutations exhibit variable and complex clinical phenotypes due to the fact that XPD functions in both transcription and repair. Mutations in XPD resulting in simple XP appear to affect only the NER pathway and probably result from defective helicase activity. XPD-CS mutations show a more complex phenotype that includes defective TCR of oxidative lesions from ionizing radiation and hydrogen peroxide and increased mutations from an 8-oxo-7,8-dihydroguanine lesion in the transcribed strand in a shuttle vector (26). Mechanistically, TTD defects have been ascribed to an altered enzyme structure that reduces stability and/or interaction with other TFIIH members, thereby impairing transcription initiation (4, 7, 15). However, it is becoming apparent in the case of some XPD mutants that the clinical phenotypes require a more complex explanation than either lack of activity or lack of native XPD conformation. For example, it was reported that the R683W XPD mutant has a reduced interaction with the TFIIH p62 subunit, which normally stimulates XPD helicase activity (7). It is clear that understanding the structure-function relationships of wild-type (WT) and mutant XPD proteins will increase insight into the molecular bases of these diseases. A step in this direction was taken with the isolation of the hamster V-H1 mutant (68) and its partially UV-resistant revertants (69). The T46I substitution mutation in both alleles of V-H1 resides in helicase motif I (21), which contains an ATP binding motif found in all helicases (12). V-H1 cells are about sixfold more UV sensitive to killing than parental cells (69) and are defective in XPD helicase activity (19). In this study, we characterize three phenotypic revertants by demonstrating that these revertants have partially restored the NER activity of XPD. In each revertant, the suppressing mutation(s) was found to be R658H. Remarkably, this substitution is identical to the mutation seen in three UV-sensitive TTD patients. These results suggest that TTD mutations do in fact change the structure of XPD and that suppressor genetics is a useful tool for studying the gross structural features of the XPD helicase.

To study molecular aspects of cytotoxicity of the anticancer drug β-D-glucose-ifosfamide mustard we investigated the potential of the agent to induce apoptosis and DNA breakage. Since β-D-glucose-ifosfamide mustard generates DNA interstrand crosslinks, we used as an in vitro model system a pair of isogenic Chinese hamster V79 cells differing in their sensitivity to crosslinking agents. CL-V5B cells are dramatically more sensitive (30-fold based on D10 values) to the cytotoxic effects of β-D-glucose-ifosfamide mustard as compared to parental V79B cells. After 48 h of pulse-treatment with the agent, sensitive cells but not the resistant parental line undergo apoptosis and necrosis, with apoptosis being the predominant form of cell death (70 and 20% of apoptosis and necrosis, respectively). Apoptosis increased as a function of dose and was accompanied by induction of DNA double-strand breaks in the hypersensitive cells. Furthermore, a strong decline in the level of Bcl-2 protein and activation of caspases-3, -8 and -9 were observed. The resistant parental cells were refractory to all these parameters. Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis. From the data we hypothesize that non-repaired crosslinks induced by β-D-glucose-ifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway. British Journal of Cancer (2002) 86, 130–135. DOI: 10.1038/sj/bjc/6600027 www.bjcancer.com © 2002 The Cancer Research Campaign

To shed light on the mechanism underlying the cellular response to the radiomimetic agents calicheamicin Y(1)(1) (CAL) and neocarzinostatin (NCS), several hamster cell mutants defective in different DNA repair pathways were used. Two X-ray sensitive Chinese hamster V79 mutant cell lines, XR-V9B and V-E5 were studied for their response to the induction of cell killing, micronuclei, and G2-chromosomal aberrations relative to that of parental wild-type cells. In addition, effects of CAL and NCS on bleomycin sensitive BL-V40 cells and on UV sensitive V-H1 cells were analyzed. In general, the radiosensitive cell lines showed the highest sensitivities to CAL and NCS, but also the other mutants demonstrated differences in their responses compared to wild-type cells. With respect to cell killing, expressed as D(10)-value, enhanced sensitivities of mutants with factors up to 4.4 were recorded. For the induction of micronuclei (MN) and chromosomal aberrations (CA) all cell lines, including the parental cells, show a steep increase in the frequencies at the lowest tested doses and a leveling off at higher concentrations. Probably toxic effects at the higher exposure levels are responsible for these biphasic dose effect curves. Enhanced sensitivities of the various mutants were primarily observed at the higher exposure levels. With respect to the induction of MN increased sensitivities up to a factor of 18.1 were observed for the radiosensitive mutants, whereas for CA the mutant cell lines showed a variation from resistance (0.3) of VH-1 cells up to a 3.8-fold higher sensitivity to the radiomimetic agents. However, at the lowest tested concentrations for both MN and CA, the differences between the sensitive mutants and wild-type clearly diminished, suggesting the existence of residual and/or alternative DNA repair pathways in these mutants.

The DNA-dependent protein kinase (DNA-PK) complex plays a key role in DNA double-strand break (DSB) repair and V(D)J recombination. Using a genetic approach we have isolated cell mutants sensitive to ionizing radiation (IR) in the hope of elucidating the mechanism and components required for these pathways. We describe here, an X-ray-sensitive and DSB repair defective Chinese hamster ovary (CHO) cell line, XR-C2, which was assigned to the X-Ray Cross Complementation (XRCC) group 7. This group of mutants is defective in the XRCC7/SCID/Prkdc gene, which encodes the catalytic subunit of DNA-PK (DNA-PKcs). Despite the fact that XR-C2 cells expressed normal levels of DNA-PKcs protein, no DNA-PK catalytic activity could be observed in XR-C2, confirming the genetic analyses that these cells harbor a dysfunctional gene for DNA-PKcs. In contrast to other IR group 7 mutants, which contain undetectable or low levels of DNA-PKcs protein and which show a severe defect in V(D)J recombination, XR-C2 cells manifested only a mild defect in both coding and signal junction formation. The unique phenotype of the XR-C2 mutant suggests that a normal level of kinase activity is critical for radiation resistance but not for V(D)J recombination, whereas the overall structure of the DNA-PKcs protein appears to be of great importance for this process.

Abstract —Vascular endothelial growth factor (VEGF) is known to induce the release of nitric oxide (NO) from endothelial cells. However, the effect of NO on VEGF synthesis is not clear. Accordingly, the effect of endogenous and exogenous NO on VEGF synthesis by rat vascular smooth muscle cells (VSMCs) was investigated. Two in vitro models were used: (1) VSMCs stimulated to produce NO by treatment with interleukin (IL)-1β (10 ng/mL) and (2) VSMCs lipotransfected with pKecNOS plasmid, containing the endothelial constitutive NO synthase (ecNOS) cDNA. The synthesis of NO was inhibited by N ω -nitro- l -arginine methyl ester (L-NAME, 2 to 5 mmol/L) or diaminohydroxypyrimidine (DAHP, 2.5 to 5 mmol/L), inhibitors of NOS and GTP cyclohydrolase I, respectively. Some cells treated with L-NAME or DAHP were supplemented with l -arginine (10 mmol/L) or tetrahydrobiopterin (BH 4 ; 100 μmol/L), respectively. In addition, we studied the effect of sodium nitroprusside (SNP; 10 and 100 μmol/L) and chemically related compounds, potassium ferrocyanide and ferricyanide, on VEGF generation. IL-1β induced iNOS expression and NO generation and significantly upregulated VEGF mRNA expression and protein synthesis. L-NAME and DAHP totally inhibited NO generation and decreased the IL-1β–upregulated VEGF synthesis by 30% to 40%. Supplementation with l -arginine or BH 4 increased NO generation by L-NAME– or DAHP-treated cells, and VEGF synthesis was augmented by addition of BH 4 . The cells generating NO after pKecNOS transfection released significantly higher amounts of VEGF than cells transfected with control plasmids. Inhibition of NO generation by L-NAME decreased VEGF synthesis. In contrast to the effect of endogenous NO, we observed the inhibition of VEGF synthesis in the presence of high (10 or 100 μmol/L) concentrations of SNP. This effect was mimicked by chemically related ferricyanide and ferrocyanide compounds, suggesting that the inhibitory effect of sodium nitroprusside may be mediated by an NO-independent mechanism. The results indicate that endogenous NO enhances VEGF synthesis. The positive interaction between endogenous NO and VEGF may have implications for endothelial regeneration after balloon angioplasty and for angiogenesis.

To elucidate the mechanisms of the mammalian cell defense against cross-linking agents, we studied previously cellular responses to mitomycin C (MMC) treatment in two MMC-hypersensitive hamster cell mutants' V-H4 and V-C8, as well as their parental cell line V79. In the present report, we investigated whether alterations in cell cycle checkpoints and induction of apoptosis could be responsible for the MMC hypersensitivity of the V-H4 and V-C8 mutant cell lines. First, we found that parental and mutant cells exhibited similar cell cycle responses to MMC concentrations of equivalent cytotoxicity, arguing against a defective cell cycle checkpoint in hypersensitive cell lines. In contrast, we showed that mutant cells underwent greater levels of apoptosis following MMC treatment than parental cells. These findings indicate that increased induction of apoptosis contributes to the hypersensitivity of V-H4 and V-C8 cells to the growth inhibitory effect of MMC. This differential apoptotic response was observed with both equimolar and equitoxic MMC doses and was specific to the cross-linking agent MMC, suggesting that control of the apoptotic process is altered in both MMC-hypersensitive mutants. The defective genes in V-H4 and V-C8 cells would then function in the regulation of an apoptotic pathway triggered by MMC-induced damage and independent of p53-mediated transcription.