Orotate phosphoribosyltransferase expression level in tumors is a potential determinant of the efficacy of 5-fluorouracil

Although the intratumoral expression levels of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are known to affect the antitumor activity of 5-fluorouracil (5-FU), the importance of orotate phosphoribosyltransferase (OPRT) has remained unclear. This study investigated the relationship between intratumoral OPRT expression and the antitumor activity of 5-FU using human NCI60 cell lines with similar levels of TS and DPD messenger RNAs, as well as 31 tumor xenografts. The OPRT mRNA level was positively correlated with the 5-FU efficacy in these cell lines. In vitro, the 50% growth-inhibitory concentrations of 5-FU were closely correlated with the OPRT mRNA levels in cancer cell lines with similar levels of TS mRNAs when combined with a DPD inhibitor. Moreover, downregulation of OPRT with small-interfering RNA decreased the sensitivities of the cultured tumor cells to 5-FU. These results suggest that the OPRT expression level in tumors is an additional determinant of the efficacy of 5-FU.     

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.     

The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells

The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.     

Involvement of p53 function in different magnitude of genotoxic and cytotoxic responses in in vitro micronucleus assays

In in vitro micronucleus (MN) assays the sensitivity to MN induction or cytotoxicity can vary depending on the kind of cells employed. This study was conducted to examine the involvement of the p53 function in the different sensitivities between Chinese hamster lung (CHL) cells and human lymphoblastoid TK6 cells in MN assays. MN induction and cytotoxicity were compared using MN-inducing chemicals reported as DNA reactive clastogens, non-DNA reactive clastogens or aneugens. The study revealed that the maximum levels of MN induction in p53-compromised CHL cells were higher than those in p53-competent TK6 cells, but MN were significantly induced in TK6 cells at lower concentrations than in CHL cells. Most of the test chemicals produced a more severe cytotoxicity in TK6 cells, suggesting TK6 cells are more sensitive for cytotoxicity than CHL cells. An additional experiment with 9 MN inducers revealed that the magnitude of MN induction and cytotoxicity were comparable between p53-competent TK6 cells and its p53-null mutant NH32 cells at the same concentrations. Furthermore, the MN frequencies induced by methylmethane sulfonate, aphidicolin and hydroxyurea in NH32 cells were identical to those in TK6 cells at different recovery times. From these results, it is suggested that the p53 abrogation does not explain the difference in sensitivity to MN induction or cytotoxicity between CHL and TK6 cells. In this regard, p53 abrogated NH32 cells can be an option for the in vitro MN assay.     

Pharmacogenetic studies on the prediction of efficacy and toxicity of fluoropyrimidine-based adjuvant therapy in colorectal cancer

The cytotoxic effect of 5-fluorouracil (5-FU) is mediated by the inhibition of thymidylate synthase (TS), however, at the same time 5-FU is catabolized by dihydropyrimidine dehydrogenase (DPD). Efficacy of 5-FU may therefore depend on the TS and DPD activity and on pharmacogenetic factors influencing these enzymes. Our aims were (1) to determine the distribution of DPD activity, the frequency of DPD deficiency and the DPD (IVS14+1G>A) mutation in the peripheral blood mononuclear cells of colorectal cancer (CRC) patients, and study the relationship between DPD deficiency and toxicity of 5-FU; (2) to investigate the influence of TS polymorphisms and DPD activity on the survival of CRC patients receiving 5-FU-based adjuvant therapy. The frequency of DPD deficiency was determined by radiochemical methods in the peripheral blood mononuclear cells (PBMCs) of 764 CRC patients treated with 5-FU. The relationship between the TS polymorphisms, DPD activity and the disease-free and overall survival was studied in 166 CRC patients receiving 5-FU-based adjuvant therapy. TS polymorphisms were determined in the DNA samples separated from the PBMCs, by PCR-PAGE and PCR-RFLP-PAGE (restriction fragment length polymorphism) methods. Low DPD values (<10 pmol/min/106 PBMCs) were demonstrated in 160/764 patients (20.9%), and of those DPD deficiency (<5 pmol/min/106 PBMCs) was verified in 38 patients (4.9%). In the latter group severe (>Gr 3) toxicity was found in 87%. The prevalence of the DPD IVS14+1G>A mutation among the 38 DPD-deficient patients was 7.8% (3/38) and was accompanied by severe Gr 4 toxic symptoms (neutropenia, mucositis, diarrhea). TS polymorphisms showed a relationship with the survival of CRC patients. It is important to mention that by combining the 3-3 genotypes of 5'-TSER and 3'-TSUTR polymorphisms the obtained 8 genotype combinations showed significantly different Kaplan-Meier survival curves. The evaluation of these curves with Cox regression analysis resulted in two prognostically different groups: "A" good prognosis (RR<1) and "B" bad prognosis (RR>1). The disease-free- and overall survival of these two groups were significantly different. DPD activity also showed correlation with the survival; patients with DPD activity <10 pmol/min/106 PBMCs showed significantly longer disease-free and overall survival. The determination of DPD activity proved to be a more valuable parameter in the evaluation of serious 5-FU-related toxicity compared to the IVS14+1G>A mutation analysis. According to the Cox multivariate analysis the combination of germline TS polymorphisms and DPD activity is/an independent prognostic marker of survival in CRC patients treated with adjuvant 5-FU therapy.     

Heterocyclic amine induced apoptotic response in the human lymphoblastoid cell line TK6 is linked to mismatch repair status.

The human lymphoblastoid cell, TK6, exhibited a dose-dependent cytotoxic and apoptotic response following treatment with the food borne heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Augmentation of the p53 protein and increases in p21-WAF1 levels were also observed. Comparison of the survival by clonogenic assays and the percentage of apoptotic cells (cells containing subG1 DNA or condensed nuclei) revealed that only 10-20% of the PhIP-induced cell death could be attributed to apoptosis that occurred in the first 24h after treatment. MT1, a derivative of TK6 that contains mutations in both alleles of its hMSH6 gene and is mismatch repair deficient, showed a decreased apoptotic response. A significant increase (P<0.05) in apoptosis was observed in TK6 and not in MT1 following treatment with 2.5microg/ml PhIP. A five- to six-fold increase and less than a two-fold increase in the fraction of apoptotic cells were observed in TK6 and MT1, respectively. Treatment with 5microg/ml PhIP resulted in significant increases in apoptosis (P<0.05) in TK6 and MT1. The percentages of apoptotic cells were, however, two- to three-fold higher in TK6 than in MT1. HCT116, a hMLH1 defective mismatch repair deficient colorectal carcinoma cell line, also exhibited lower PhIP-induced apoptosis than its mismatch repair proficient chromosome transfer cell line (HCT116+chr3) following PhIP treatment. These results show that PhIP-induced apoptosis is mediated through a mismatch repair dependent pathway. Accumulation of p53 in TK6 and MT1 were evident in samples taken 24h after PhIP treatment. Increases in p21-WAF1 were also observed in both cell lines confirming that the p53 was functional. The lower apoptotic response of MT1 but similar p53 accumulation in TK6 and MT1 suggest that the mismatch repair protein(s) are involved downstream of p53 or that PhIP-induced apoptosis is p53-independent.     

Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity.

The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.     

Suppression of apoptosis and clonogenic survival in irradiated human lymphoblasts with different TP53 status

The influence of radiation-induced apoptosis on radiosensitivity was studied in a set of closely related human lymphoblastoid cell lines differing in TP53 status. The clonogenic survival of irradiated TK6 cells (expressing wild-type TP53), WTK1 cells (overexpressing mutant TP53), and TK6E6 cells (negative for TP53 owing to transfection with HPV16 E6) was assessed in relation to the induction of apoptosis and its suppression by caspase inhibition or treatment with PMA as well as after treatment with caffeine. Measurements using the alkaline comet assay and pulsed-field electrophoresis of the induction and repair of DNA strand breaks showed similar kinetics of the processing of early DNA damage in these cell lines. The cytochalasin B micronucleus assay revealed identical levels of residual damage in the first postirradiation mitosis of these cells. Abrogation of TP53-dependent apoptosis in TK6E6 cells resulted in a distinct increase in radioresistance. Further suppression of apoptosis as observed in WTK1 cells overexpressing mutant TP53 apparently was not responsible for the high radioresistance of WTK1 cells, since other means of highly efficient suppression of apoptosis (caspase inhibition or PMA treatment) increased the clonogenic survival of irradiated TK6 cells only to levels similar to those of TK6E6 cells with abrogated TP53-dependent apoptosis. Considering the similar levels of residual chromosomal damage in TK6E6 cells and WTK1 cells, a hitherto unknown mechanism of tolerance needs to be inferred for these TP53 mutant cells. This residual damage tolerance, however, appears to require an intact G2/M-phase checkpoint function since the relative radioresistance of the WTK1 cells was completely lost upon caffeine treatment, which also resulted in a failure of the TK6 and TK6E6 cells to execute apoptosis. In this situation, the cellular response seems to be dominated entirely by TP53-independent mitotic failure.     

The prognostic significance of thymidine phosphorylase, thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expressions in breast carcinomas

Thymidine phosphorylase (TP), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) have been indicated as possible predictive markers for epithelial malignancies. All these three enzymes are actively involved in 5-FU metabolism. In this report, we investigated mRNA expression of these factors with real-time quantitative PCR in a series of 86 micro-selected breast carcinomas and 8 micro-selected tumour-adjacent normal breast epithelial specimens. Highly variable mRNA expressions of these factors were observed in both normal and cancerous samples. TP and TS mRNA expressions in breast carcinomas were elevated, but only TS mRNA expression showed a trend for statistical difference, compared with the expression in normal breast epithelial samples. Although the DPD mRNA expression range in tumours was also elevated, the average mean was reduced in tumours compared to that in normal samples. No association between mRNA expressions of TP, TS and DPD and clinicopathological features such as histological grade, tumour size, node status, S-phase fraction, ploidy, and clinical stage was found. A negative association between DPD mRNA expression and age was, however, revealed. Ten-year follow-up analysis showed no association between TP and DPD mRNA expression and clinical outcome. An high level of TS mRNA expression, however, was associated with a shorter clinical survival, indicating its potential role as a clinical marker in breast carcinoma.     

Reduction of misleading ("false") positive results in mammalian cell genotoxicity assays. I. Choice of cell type

Current in vitro mammalian cell genotoxicity assays show a high rate of positive results, many of which are misleading when compared with in vivo genotoxicity or rodent carcinogenicity data. P53-deficiency in many of the rodent cell lines may be a key factor in this poor predictivity. As part of an European Cosmetics Industry Association initiative for improvement of in vitro mammalian cell assays, we have compared several rodent cell lines (V79, CHL, CHO) with p53-competent human peripheral blood lymphocytes (HuLy), TK6 human lymphoblastoid cells, and the human liver cell line, HepG2. We have compared in vitro micronucleus (MN) induction following treatment with 19 compounds that were accepted as producing misleading or "false" positive results in in vitro mammalian cell assays [6]. Of these, six chemicals (2-ethyl-1,3-hexandiol, benzyl alcohol, urea, sodium saccharin, sulfisoxazole and isobutyraldehyde) were not toxic and did not induce any MN at concentrations up to 10mM. d,l-Menthol and ethionamide induced cytotoxicity, but did not induce MN. o-Anthranilic acid was not toxic and did not induce MN in V79, CHL, CHO, HuLy and HepG2 cells up to 10mM. Toxicity was induced in TK6 cells, although there were no increases in MN frequency up to and above the 55% toxicity level. The other 10 chemicals (1,3-dihydroxybenzene, curcumin, propyl gallate, p-nitrophenol, ethyl acrylate, eugenol, tert-butylhydroquinone, 2,4-dichlorophenol, sodium xylene sulfonate and phthalic anhydride) produced cytotoxicity in at least one cell type, and were evaluated further for MN induction in most or all of the cell types listed above. All these chemicals induced MN at concentrations <10mM, with levels of cytotoxicity below 60% (measured as the replication index) in at least one cell type. The rodent cell lines (V79, CHO and CHL) were consistently more susceptible to cytotoxicity and MN induction than p53-competent cells, and are therefore more susceptible to giving misleading positive results. These data suggest that a reduction in the frequency of misleading positive results can be achieved by careful selection of the mammalian cell type for genotoxicity testing.     

Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H2O2 in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells.     

Induction of telomerase activity by irradiation in human lymphoblasts

Neuhof, D., Ruess, A., Wenz, F. and Weber, K. J. Induction of Telomerase Activity by Irradiation in Human Lymphoblasts. Radiat. Res. 155, 693-697 (2001). Telomerase activity is a radiation-inducible function, which suggests a role of this enzyme in DNA damage processing. Since the tumor suppressor TP53 plays a central role in the regulation of the cellular response to DNA damage, our study explored the ability of ionizing radiation to change telomerase activity and telomere length in two closely related human lymphoblast cell lines with different TP53 status. TK6 cells (wild-type TP53) and WTK1 cells (mutated TP53) were exposed to different doses of X rays, and telomerase activity was measured by PCR ELISA at different times after irradiation. A dose-dependent increase in telomerase activity was observed. One hour after irradiation with 4 Gy, TK6 and WTK1 cells showed an approximately 2.5-fold increase; for lower doses (0.1 to 1 Gy), telomerase induction was seen only in TK6 cells. Telomerase induction was observed by 0.5 h after irradiation, with a further increase up to 24 h. Irradiated TK6 and WTK1 cells had longer telomeres (+1.3 kb) than unirradiated cells 14 days after exposure. Our data demonstrate a dose-dependent induction of telomerase activity and lengthening of telomeres by ionizing radiation in human lymphoblasts. Induction of telomerase activity by radiation does not generally appear to be controlled by the TP53-dependent DNA damage response pathway. However, for low doses, induction of telomerase requires wild-type TP53.     

miR-155-TP53INP1调节轴在结直肠癌化疗药物敏感性中的作用机制

摘要 目的：初步揭示miR-155通过靶向调节TP53INP1表达水平影响结直肠癌细胞对5-FU化疗敏感性。方法：将人结肠直肠癌细胞系HCT116进行培养,提取细胞总RNA后,采用miR-155逆转录特异性引物构建反转录体系进行PCR扩增,通过qRT-PCR检测miR-155在5-FU耐药细胞HCT116/FU及敏感细胞株HCT116中的表达情况;取对数生长期细胞,分别转染miR-155mimics、miR-155抑制剂、miR-155阴性对照后,采用CCK-8法检测miR-155对细胞5-FU药物敏感性的影响,双荧光素酶报告基因系统验证miR-155与TP53INP1的靶基因关系,Western blot检测miR-155对 TP53INP1表达的影响。结果：miR-155在HCT116 /Fu细胞中的表达量是HCT116细胞的7.25倍;在相同5-FU浓度时,HCT116+阴性对照的细胞生长抑制率均高于HCT116+mimics、半数抑制浓度显著低于HCT116+mimics,差异均具有统计学意义(P＜0.05);TP53INP1是miR-155的靶基因,能显著降低野生型TP53INP1 3''-UTR的荧光素酶活性;转染miR-155 mimics后,TP53INP1的相对表达量显著下降,转染miR-155抑制剂后,TP53INP1的相对表达量显著升高,差异均具有统计学意义(P＜0.05)。结论：miR-155水平升高使HCT116细胞对5-FU的敏感性降低,miR-155可能通过靶向调节TP53INP1的表达水平,从而影响结直肠癌细胞对5-FU的敏感性。     

Involvement of TP53 in apoptosis induced in human lymphoblastoid cells by fast neutrons

We investigated the involvement of TP53 in apoptosis induced by fast neutrons in cells of three human B-lymphoblast cell lines derived from the same donor and differing in TP53 status: TK6 (wild-type TP53), WTK1 (mutant TP53) and NH32 (knockout TP53). Cells were exposed to X rays or to fast neutrons at doses ranging from 0.5 to 8 Gy. Apoptosis was determined by measurements of the sub-G0 /G1-phase DNA content and by the externalization of phosphatidylserine. Fast neutrons induced extensive apoptosis in TK6 cells, as shown by the formation of hypodiploid particles, the externalization of phosphatidylserine, and the activation of caspases. In contrast, cell death was triggered at a significantly lower rate in cells lacking functional TP53. However, TP53-independent cell death also expressed the morphological and biochemical hallmarks of apoptosis. Proliferation tests and clonogenic assays showed that fast neutrons can nevertheless kill WTK1 and NH32 cells efficiently. The absence of functional TP53 only delays radiation-induced cell death, which is also mediated by caspases. These results indicate that fast-neutron irradiation activates two pathways to apoptosis and that the greater relative biological effectiveness of fast neutrons reflects mainly an increase in clonogenic cell death.     

Thymidylate synthase and dihydropyrimidine dehydrogenase levels are associated with response to 5-fluorouracil in Caenorhabditis elegans

5-Fluorouracil (5-FU), a pyrimidine antagonist, has a long history in cancer treatment. The targeted pyrimidine biosynthesis pathway includes dihydropyrimidine dehydrogenase (DPD), which converts 5-FU to an inactive metabolite, and thymidylate synthase (TS), which is a major target of 5-FU. Using Caenorhabditis elegans as a model system to study the functional and resistance mechanisms of anti-cancer drugs, we examined these two genes in order to determine the extent of molecular conservation between C. elegans and humans. Overexpression of the worm DPD and TS homologs (DPYD-1 and Y110A7A.4, respectively) suppressed germ cell death following 5-FU exposure. In addition, DPYD-1 depletion by RNAi resulted in 5-FU sensitivity, while treatment with Y110A7A.4 RNAi and 5-FU resulted in similar patterns of embryonic death. Thus, the pathway of 5-FU function appears to be highly conserved between C. elegans and humans at the molecular level.     

Thymidylate synthase predicts for clinical outcome in invasive breast cancer

Thymidylate synthase (TS) is a major target of 5-fluorouracil (5-FU) and dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the degradation of 5-FU. Whether TS or DPD could be used as valuable parameters for 5-FU sensitivity in clinical patients are largely unknown. We analyzed TS and DPD expression in breast carcinomas to evaluate the clinicopathological significance of these enzymes in patients with invasive breast cancer receiving 5-FU-based chemotherapy. A total of 197 patients with invasive ductal carcinoma were included in our study. Both the TS and DPD expression were analyzed using immunohistochemical method for all the surgical samples. Sixty-three out of 197 (31.97%) patients are positive for TS expression, and 77 out of 197 (39.09%) patients are positive for DPD expression. TS expression was not correlated with DPD expression. Patients with TS-positivity had aggressive phenotype including large tumor size, low differentiation and nodal metastasis. DPD expression is not related with phenotype or prognosis. Multivariate analysis demonstrated that TS expression was an independent prognostic factor for both disease-free and overall survival. The current study demonstrated that TS but not DPD expression was associated with both progression and prognosis in breast cancer receiving 5-FU-based chemotherapy. TS expression in the primary tumor might be useful as a predictive parameter for the efficacy of 5-FU-based chemotherapy for breast cancer.     

Involvement of mismatch repair proteins in adaptive responses induced by N-methyl-N'-nitro-N-nitrosoguanidine against γ-induced genotoxicity in human cells

As humans are exposed to a variety of chemical agents as well as radiation, health effects of radiation should be evaluated in combination with chemicals. To explore combined genotoxic effects of radiation and chemicals, we examined modulating effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a direct-acting methylating agent, against genotoxicity of γ-radiation. Human lymphoblastoid TK6 cells and its mismatch-deficient derivative, i.e., MT1 cells, were treated with MNNG for 24h before they were exposed to γ-irradiation at a dose of 1.0 Gy, and the resulting genotoxicity was examined. In TK6 cells, the pretreatments with MNNG at low doses suppressed frequencies of the thymidine kinase (TK) gene mutation and micronucleus (MN) formation induced by γ-irradiation and thus the dose responses of TK and MN assays were U-shaped along with the pretreatment doses of MNNG. In contrast, the genotoxic effects of MNNG and γ-irradiation were additive in MT1 cells and the frequencies of TK mutations and MN induction increased along with the doses of MNNG. Apoptosis induced by γ-radiation was suppressed by the pretreatments in TK6 cells, but not in MT1 cells. The expression of p53 was induced and cell cycle was delayed at G2/M phase in TK6, but not in MT1 cells, by the treatments with MNNG. These results suggest that pretreatments of MNNG at low doses suppress genotoxicity of γ-radiation in human cells and also that mismatch repair proteins are involved in the apparent adaptive responses.