Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells

We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8-hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetra -carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 10(5) dG, whereas levels in the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 10(5) dG, respectively. The 8OHdG induced by 20 microM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was approximately 6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 10(6) cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 microM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79.     

A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells

It has been estimated that over three million workers in the USA are potentially exposed to silica or other mineral dusts. Results of epidemiological studies evaluating whether silica or glass fibers increase lung cancer risk to the exposed workers are inconclusive. Detection of DNA damage in cells exposed to genotoxic agents is being used to assess the carcinogenic potential of environmental agents. The alkaline (pH > 13) single cell gel/comet (SCG) assay was used to determine and compare DNA damage in cultured Chinese hamster lung fibroblasts (V79 cells) and human embryonic lung fibroblasts (Hel 299 cells) exposed to crystalline silica (Min-U-Sil 5), amorphous silica (Spherisorb), carbon black, and glass fibers (AAA-10). V79 or Hel 299 cells were exposed to these mineral dusts for 3 h at various concentrations. Min-U-Sil 5 and AAA-10, at almost all concentrations tested, caused a significant increase in DNA migration measured as tail length in both V79 and Hel 299 exposed cells. However, the increase was much higher in V79 then in Hel 299 cells for Min-U-Sil 5. Tail length was also increased relative to controls after amorphous silica treatment, but not to the same extent as that induced by crystalline silica. Exposure to carbon black did not induce DNA migration at any of the concentrations tested. These results indicate that silica and glass fibers, but not carbon black, can induce DNA damage in mammalian cells, and that crystalline silica has a higher DNA-damaging activity than amorphous silica. For glass fibers, induction of DNA damage in both V79 and Hel 299 cells was observed even at a concentration 10 times lower than silica and the response was similar in both cell lines. These results suggest that the SCG/comet assay is useful for the detection of DNA damage caused by occupationally related dusts/particles.     

Weekly doxorubicin in the treatment of patients with AIDS-related Kaposi''s sarcoma. AIDS Clinical Trials Group

It has been shown that the X-ray-sensitive Chinese hamster V79 mutants (V-E5, V-C4 and V-G8) are similar to ataxia-telangiectasia (A-T) cells. To determine whether the AT-like rodent cell mutants are defective in the gene homologous to A-T (group A, C or D), human chromosome 11 was introduced to the V-E5 and V-G8 mutant cells by microcell-mediated chromosome transfer. Forty independent hybrid clones were obtained in which the presence of chromosome 11 was determined by in situ hybridization. The presence of the region of chromosome 11q22-23 was shown by molecular analysis using polymorphic DNA markers specific for the ATA, ATC and ATD loci. Seventeen of the obtained monochromosomal Chinese hamster hybrids contained a cytogenetically normal human chromosome 11, but only twelve hybrid cell lines were shown to contain an intact 11q22-23 region. Despite the complementation of the X-ray sensitivity by a normal chromosome 11 introduced to A-T cells (complementation group D), these twelve Chinese hamster hybrid clones showed lack of complementation of X-ray and streptonigrin hypersensitivity. The observed lack of complementation does not seem to be attributable to hypermethylation of the human chromosome 11 in the rodent cell background, since 5-azacytidine treatment had no effect on the streptonigrin hypersensitivity of the hybrid cell lines. These results indicate that the gene defective in the AT-like rodent cell mutants is not homologous to the ATA, ATC or ATD genes and that the human gene complementing the defect in the AT-like mutants seems not to be located on human chromosome 11.     

Contribution of known mitogenic signaling pathways to induction of DNA synthesis in quiescent Chinese hamster fibroblasts

The mitogenic pathways so far identified in mammalian cells fall into three main categories: tyrosine kinase, kinase C, and the cAMP-dependent pathways. In quiescent murine 3T3 fibroblasts, all three signaling pathways synergize with each other to restart DNA synthesis. In order to establish if the same was true in other rodent fibroblast lines we studied the effects of factors, known to modulate the above-mentioned pathways, on DNA synthesis in Chinese hamster embryo fibroblasts (CHEF/18). The factors examined were: (1) EGF and insulin representative of tyrosine kinase-activating growth factors, (2) TPA as specific activator of protein kinase C, (3) cholera toxin, dibutyryl cyclic AMP, and theophylline as compounds increasing cAMP levels. We found that EGF alone is a strong mitogen in CHEF/18 cells, probably because it can modulate by itself all three pathways. Although cAMP acts as a growth enhancer in 3T3 cells, in CHEF/18 where high levels of cAMP were found, increased concentrations of this second messenger produce strong DNA synthesis inhibition and temporal disturbance of ribosomal protein S6 phosphorylation. Possible interpretations of these findings are presented.     

Interaction between radiation and drug damage in mammalian cells. II. The effect of actinomycin-D on the repair of the sublethal radiation damage in plateau phase cells

The effect of actinomycin-D (AMD) on radiation damage repair was studied in plateau phase V79 Chinese hamster cells. Sublethal radiation damage repair, as demonstrated by survival fluctuations following two x-ray exposures separted by time, was observed in our plateau phase cells. Plateau phase cells exposed to 0.01-0.04 mug/ml AMD (a nontoxic regimen to 8 hours) between x-ray exposures were less able to repair sublethal damage. If plateau phase cells were plated at low dilutions into fresh medium (conditions for resuming exponential growth) immediately after the first x-ray dose, and exposed to 0.01--0.04 mug/ml AMD until the second dose, inhibition of sublethal damage repair and additional cell killing were observed particularly at 0.04 mug/ml AMD. It is suggested that radiation-drug damage interactions should be studied in plateau phase cells and in cells resuming exponential growth after plateau phase (possibly analogous to "recruitment"), as well as in exponential phase cultures.     

Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis

This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing.     

Inhibition of the Mr 70,000 S6 kinase pathway by rapamycin results in chromosome malsegregation in yeast and mammalian cells

The antifungal and immunosuppressive drug rapamycin arrests the cell cycle in G1-phase in both yeast and mammalian cells. In mammalian cells, rapamycin selectively inhibits phosphorylation and activation of p70 S6 kinase (p70(S6K)), a protein involved in the translation of a subset of mRNAs, without affecting other known kinases. We now report that rapamycin causes chromosome malsegregation in mammalian and yeast cells. Chromosome malsegregation was determined by metaphase chromosome analysis of human lymphocytes and lymphoblasts, detection of CREST-positive micronuclei in human lymphoblasts and Chinese hamster embryonic fibroblast (CHEF) cells, and selection of doubly prototrophic cells in a specially constructed yeast strain. The number of ana-telophases with displaced chromosomes and interphase and mitotic cells with an irregular number of centrosomes was also determined in CHEF cells. In quiescent mammalian cells (human lymphocytes and CHEF cells) induced with growth factor to re-enter the cell cycle, rapamycin was effective when cells were exposed at the time of p70(S6K) activation. In yeast, rapamycin was more effective when treatment was started in G1- than in G2-synchronized cells. Cells from ataxia telangiectasia (A-T) patients are characterized by chromosome instability and have recently been found to be resistant to the growth-inhibiting effect of rapamycin. We found that an A-T lymphoblastoid cell line was also resistant to the induction of chromosome malsegregation by rapamycin, but the level of spontaneous aneuploidy was higher than in normal cells. In yeast, the induction of chromosome malsegregation was dependent on the presence of a wild-type TUB2 gene, encoding the beta-subunit of tubulin. The finding that rapamycin acts in different cell types and organisms suggests that the drug affects a conserved step important for proper segregation of chromosomes. One or more proteins required for chromosome segregation could be under the control of the rapamycin-sensitive pathway.     

Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen

GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.     

Enhanced expressions of glucose-6-phosphate dehydrogenase and cytosolic aldehyde dehydrogenase and elevation of reduced glutathione level in cyclophosphamide-resistant human leukemia cells

PURPOSE: To determine the involvement of p53 in ionizing radiation-induced excision and recombination repair. MATERIALS AND METHODS: Shuttle vector pZ189 containing radiation-induced single strand breaks plus base damage (ocDNA), ultraviolet-radiation damage (uvDNA), or a restriction enzyme-produced double strand break (linDNA) were processed in unirradiated or irradiated p53wt and p53mut lymphoblasts. Mutation frequencies in the supF-tRNA target gene and survival of plasmids processed in p53wt and p53mut hosts were compared. RESULTS: Mutation frequencies of oc-, uv- or linDNA were similar after processing in unirradiated p53wt and p53mut hosts. However, the mutation frequency of ocDNA and uvDNA decreased 50% when processed in irradiated p53wt hosts but was unaltered in irradiated p53mut hosts. In contrast, linDNA mutation frequencies varied similarly whether processed in irradiated p53wt or p53mut hosts: mutation frequency decreased twofold when linDNA was transfected immediately after host irradiation but increased twofold when transfection was delayed by 2h. Double strand break rejoining capacity, determined by the ratio of the number of progenies from linDNA to that from undamaged pZ189, differed both qualitatively and quantitatively in irradiated p53wt and p53mut hosts. CONCLUSIONS: These studies show induction of DNA repair in mammalian cells by ionizing radiation and indicate the involvement of p53 in the modulation of excision repair fidelity and double strand break rejoining capacity.     

Genetic alterations and molecular mechanisms underlying colorectal tumorigenesis

Tumor cells are cells that have acquired damage to genes that directly regulate their cell cycles. In the multistep process leading to colorectal carcinoma, the adenoma-carcinoma sequence is characterized by progressive accumulation of genetic abnormalities (K-ras oncogene mutation, allelic deletion on chromosome 5q, 18q, 17p). In a hereditary non-polyposis syndrome (Lynch syndrome II) and in about a quarter of the cases of sporadic colorectal cancer there is a DNA micro-instability which contributes to the acquisition of mutations that cause loss of tumor-suppressor function. The p53 tumor-suppressor gene is the most frequently mutant gene in human cancer. In colorectal cancer cells missense p53 mutations and allelic deletion on chromosomal locus 17p13.1 are found with very high frequency. One of biological roles of p53 gene is to ensure that, in response to genotoxic damage, cells arrest in G1 and attempt to repair their DNA before it is replicated. In addition, p53 is required for apoptosis in response to severe DNA damage, included the damage induced by chemotherapeutics drugs and ionizing radiation. The loss of p53 function results in genomic instability and has been implicated in the evolution of normal cells into cancer cells.     

Illegitimate recombination leading to allelic loss and unbalanced translocation in p53-mutated human lymphoblastoid cells

Allelic loss and translocation are critical mutational events in human tumorigenesis. Allelic loss, which is usually identified as loss of heterozygosity (LOH), is frequently observed at tumor suppressor loci in various kinds of human tumors. It is generally thought to result from deletion or mitotic recombination between homologous chromosomes. In this report, we demonstrate that illegitimate (nonhomologous) recombination strongly contributes to the generation of allelic loss in p53-mutated cells. Spontaneous and X-ray-induced LOH mutations at the heterozygous thymidine kinase (tk) gene, which is located on the long arm of chromosome 17, from normal (TK6) and p53-mutated (WTK-1) human lymphoblastoid cells were cytogenetically analyzed by chromosome 17 painting. We observed unbalanced translocations in 53% of LOH mutants spontaneously arising from WTK-1 cells but none spontaneously arising from TK6 cells. We postulate that illegitimate recombination was occurring between nonhomologous chromosomes after DNA replication, leading to allelic loss and unbalanced translocations in p53-mutated WTK-1 cells. X-ray irradiation, which induces DNA double-strand breaks (DSBs), enhanced the generation of unbalanced translocation more efficiently in WTK-1 than in TK6 cells. This observation implicates the wild-type p53 protein in the regulation of homologous recombination and recombinational DNA repair of DSBs and suggests a possible mechanism by which loss of p53 function may cause genomic instability.     

Hypersensitivity to DNA cross-linking agents associated with up-regulation of glucose-regulated stress protein GRP78

We have shown previously that NAD/poly(ADP-ribose) polymerase-deficient cells that overexpress Mr 78,000 glucose-regulated stress protein (GRP78) are resistant to topoisomerase II inhibitors, such as etoposide, m-amsacrine, and doxorubicin. However, these cells have been found to be hypersensitive to DNA cross-linking agents, including melphalan, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These observations prompted us to examine whether overexpression of GRP78 is associated with modulation of cytotoxicity of clinically useful DNA-cross-linking agents such as melphalan, BCNU, and cisplatin. We up-regulated GRP78 in V79 Chinese hamster cells by 2-5-fold using two independent approaches that include exposure to 6-aminonicotinamide, or 2-deoxyglucose. Subsequently, these GRP78-overexpressing cells were trypsinized, plated in regular medium without GRP78-inducing agents, and allowed a 5-h attachment time before being treated with melphalan, BCNU, or cisplatin for 1 h to determine clonogenic survivals. In addition, repair of DNA cross-links induced by those agents were determined by alkaline elution assay. Our results show that the GRP78-overexpressing V79 cells are hypersensitive to DNA cross-linking agents compared to the control V79 cells. Furthermore, repair of drug-induced DNA cross-links appears to be considerably slower in these cells relative to that found in control V79 cells. Thus, our results suggest that (a) up-regulation of GRP78 is associated with an impairment of DNA cross-link repair, (b) up-regulation of GRP78 is associated with potentiation of cytotoxicity induced by alkylating and platinating agents, and (c) up-regulation of GRP78 can be considered as a potentially useful tool to modulate the cytotoxicity of clinically useful alkylating and platinating agents.     

Restoration of X-ray and etoposide resistance, Ku-end binding activity and V(D) J recombination to the Chinese hamster sxi-3 mutant by a hamster Ku86 cDNA

Ku is a heterodimeric protein composed of 86 and 70 kDa subunits that binds preferentially to the double-stranded ends of DNA. Recent molecular characterization of ionizing-radiation sensitive (IRs) mutants belonging to the XRCC5 complementation group demonstrated the involvement of Ku in DNA double-strand break (DSB) repair and lymphoid V(D)J recombination. Here, we describe the isolation of a full-length hamster cDNA encoding the large subunit of the Ku heterodimer and demonstrate that the stable expression of this cDNA can functionally restore IR, Ku DNA end-binding activity and V(D)J recombination proficiency in the Chinese hamster IRs sxi-3 mutant. Moreover, we also demonstrate that sxi-3 cells are hypersensitive to etoposide, a DNA topoisomerase II inhibitor, and that resistance to this drug was restored by the Ku86 cDNA. These experiments suggest that a defect in the large subunit of the heterodimeric Ku protein is the sole factor responsible for the known defects of sxi-3 cells and our data of further support the role of Ku in DNA DSB repair and V(D)J recombination.     

Cancer progression and p53

In a complex organism, somatic cells are under intermittent selection pressure for the emergence of mutants that can survive environmental insults and that can grow autonomously despite adverse conditions. Repeated rounds of mutation, selection, and proliferation may lead to cancer. The organism prevents malignant transformation by assuring accurate DNA repair before cell division, by forcing the death of cells with excessive DNA damage, and by placing limits on the replicative lifespans of most somatic cells. The p53 gene is a "guardian of the genome"--it regulates multiple components of the DNA damage control response and promotes cellular senescence. Disabling mutations and deletions of p53 occur in 50% of human tumours. p53-deficient cancers are often unstable, aggressive, and resistant to therapy.     

Impact of preoperative symptoms on survival after surgical correction of organic mitral regurgitation: rationale for optimizing surgical indications

The effect of serum starvation on the expression and phosphorylation of PKC-alpha and p53 in Chinese hamster V79 cells was investigated. Serum starvation led to growth arrest, rounding up of cells and the appearance of new PKC-alpha and p53 bands on Western blots. Prolonged incubation (> or = 48 hr) in serum-deprived medium led to cell detachment and death. Moving cells to fresh medium containing 10% serum before, but not after, cell detachment reversed the changes observed in PKC-alpha and p53, and also prevented later cell detachment. Radiolabelling studies showed that the higher-molecular-weight PKC-alpha and p53 bands result from increased phosphorylation, while a lower-molecular-weight PKC-alpha band reflects newly synthesized protein. Immunocomplex kinase assays have shown that the increased phosphorylation of PKC-alpha is associated with its increased activity. To study the relationship between PKC-alpha, p53 and cell death, cells were treated either with TPA, to down-regulate PKC or with staurosporine, to inhibit PKC activity. Staurosporine, a potent PKC inhibitor and inducer of programmed cell death, caused the appearance of new PKC-alpha and p53 bands similar to those induced by serum starvation. If serum starvation was preceded by prolonged (48 hr) TPA treatment to down-regulate PKC-alpha, cell detachment and death did not take place within the same time frame. Intracellular fractionation of cells demonstrated that increased expression of PKC-alpha and the appearance of the associated higher and lower molecular-weight bands occurred in the nucleus. These data highlight the association of PKC-alpha and p53 with cellular events leading to cell death.     

Apoptosis and cancer chemotherapy

Cytotoxic drugs currently remain as the basis for the chemotherapy of metastatic cancer. Why they fail to kill sufficient tumour cells in the major human solid cancers, such as the carcinomas, is suggested in this review to be due to the inherent inability of these cells to engage apoptosis after drug-induced damage. As a paradigm for drug resistant cancers, the resistance of bladder carcinoma cell lines to DNA damaging drugs is described here in terms of their response to the topoisomerase II poison etoposide. 60%-70% of bladder carcinomas have mutant p53; this can prevent the detection of and response to DNA damage. In vitro studies with a bladder carcinoma cell line containing a wild type p53 showed that it underwent a G1 checkpoint after etoposide, potentially allowing DNA damage repair, as well as apoptosis. In lines with mutant or non-functional p53 there is no checkpoint and no apoptosis. All lines showed constitutive expression of bcl-2 and bcl-XL (the suppressors of apoptosis) with low and non-inducible levels of bax (a promoter of apoptosis). Taken together, this menu of gene expression is more favourable to survival than apoptosis after the imposition of drug-induced DNA damage and may contribute to their inherent drug resistance.     

S-oxygenation of thiourea results in the formation of genotoxic products

Thiourea (TU) is a thyroid carcinogen which has previously been shown to cause genotoxicity in various test systems in vitro and in vivo. The mechanism underlying these effects has not yet been elucidated. The present study addressed the question of whether the formation of oxidized products of TU might be involved in genotoxicity. Chemical oxidation of [14C]TU with hydrogen peroxide in the presence of calf thymus DNA resulted in the formation of [14C]formamidine sulfinate ([14C]FASA), [14C]cyanomide, and [14C]urea and in covalent binding of radioactivity to the DNA. Incubation of V79 Chinese hamster cells with 10-20 mM TU for 18 hr but not for 3 hr, increased the frequency of micronuclei to a slight extent. In cells depleted of glutathione, which can prevent the oxidation of TU, micronucleus induction by TU was more pronounced and detectable both after 3 and 18 hr of incubation. Exposure of the cells to 1.25 to 10 mM FASA for 3-5 hr induced micronuclei, DNA repair synthesis, and gene mutations in the cells. Flavin-containing monooxygenase (FMO], an enzyme known to catalyze the S-oxygenation of TU in liver, could not be detected in the postmitochondrial supernatant (S-9) of the V79 cells. There is evidence, however, that TU can easily autoxidize to S-oxygenated products. Both FASA and TU caused a slight induction of DNA repair synthesis in cultured rat hepatocytes, but FASA was active at lower concentrations than TU. Cyanamide did not elicit repair. The finding that FASA, a product of both the nonenzymatic and the enzymatic S-oxygenation of TU, is genotoxic in cultured mammalian cells provides for the first time a hypothesis to explain the genotoxicity of TU.