Role of base-excision repair in the treatment of childhood acute lymphoblastic leukaemia with 6-mercaptopurine and high doses of methotrexate.

Methotrexate (MTX) and 6-mercaptopurine (6MP) are the most commonly used drugs in the therapy of childhood acute lymphoblastic leukaemia (ALL). The main genotoxic effect of MTX resulting from inhibition of thymidylate synthase is mis-incorporation of uracil into DNA, which is considered essential for the effectiveness of the Protocol M in ALL IC BFM 2002/EURO LB 2002 regimens. In this study, we investigated the level of basal and induced DNA damage as well as the effectiveness of DNA repair in lymphocytes of children with ALL at four time-points during therapy with MTX and 6MP. To assess DNA damage and the efficacy of DNA repair we used the modified alkaline comet assay with uracil DNA glycosylase (Udg) and endonuclease III (EndoIII). In addition, we examined the induction of apoptosis in the lymphocytes of the patients during treatment. Finally, we compared the activity of base-excision repair (BER), involved in removal of both uracil and oxidized bases from DNA in lymphocytes of children with ALL and lymphocytes of healthy children. BER efficiency was estimated in an in vitro assay with cellular extracts and plasmid substrates of heteroduplex DNA with an AP-site. Our results indicate that there is a significant decrease in the efficacy of DNA repair associated with an increased level of uracil in DNA and induction of apoptosis during therapy. Moreover, it was found that the BER capacity was decreased in the lymphocytes of ALL patients in contrast to that in lymphocytes of healthy children. Thus, we suggest that an impairment of the BER pathway may play a role in the pathogenesis and therapy of childhood ALL.

Impaired nucleotide excision repair pathway as a possible factor in pathogenesis of head and neck cancer.

Tobacco smoking is one of the major risk factors in pathogenesis of head and neck squamous cell carcinomas (HNSCC). Many of the chemical compounds present in tobacco are well-known carcinogens which form adducts with DNA. Cells remove these adducts mainly by the nucleotide excision repair pathway (NER). NER also eliminates a broad spectrum of pyrimidine dimers (CPD) and photo-products (6-4PP) induced by UV-radiation or DNA cross-links after cisplatin anti-cancer treatment. In this study DNA damage and repair was examined in peripheral blood lymphocytes obtained from 20 HNSCC patients and 20 healthy controls as well as HTB-43 larynx and SSC-25 tongue cancer cell lines. DNA repair kinetics in the examined cells after cisplatin or UV-radiation treatment were investigated using alkaline comet assay during 240min of post-treatment incubation. MTT assay was used to analyse cell viability and the Annexin V-FITC kit specific for kinase-3 was employed to determine apoptosis after treating the cells with UV-radiation at dose range from 0.5 to 60J/m(2). NER capability was assessed in vitro with cell extracts by the use of a bacterial plasmid irradiated with UV-light as a substrate for the repair. The results show that lymphocytes from HNSCC patients and HTB-43 or SSC-25 cancer cells were more sensitive to genotoxic treatment with UV-radiation and displayed impaired DNA repair. Also evidenced was a higher rate of apoptosis induction after UV-radiation treatment of lymphocytes from the HNSCC patients and the HTB-43 cancer cells than after treatment of those from healthy donors. Finally, our results showed that there was a significant decrease in NER capacity in HTB-43 or SSC-25 cancer cells as well as in peripheral blood lymphocytes of HNSCC patients compared to controls. In conclusion, we suggest that the impaired NER pathway might be a critical factor in pathogenesis of head and neck cancer.

The association of polymorphisms in DNA base excision repair genes XRCC1, OGG1 and MUTYH with the risk of childhood acute lymphoblastic leukemia.

The aim of this study was to evaluate the association of polymorphisms in genes encoding three key proteins of DNA base excision repair (BER): the OGG1 Ser326Cys, the MUTYH Tyr165Cys and the XRCC1 Arg399Gln with the risk of childhood acute lymphoblastic leukemia (ALL). Our study included 97 children patients with ALL (mean age 5.4±2.5) and 131 healthy children (mean age 6.2±2.8) used as controls. Genetic polymorphisms in BER pathway genes were examined using PCR and restriction fragment length polymorphism (RFLP). We have demonstrated that the OGG1 Cys/Cys genotype increases the risk of ALL (OR 5.36) whereas the Ser/Ser genotype variant strongly reduces the risk of this cancer among Polish children (OR 0.45). Although we did not observe the differences in single nucleotide polymorphisms (SNPs) in MUTYH and XRCC1 genes between control group and children with ALL, we have shown that the combined genotypes of examined genes can modulate the risk of childhood ALL in Polish population. We found that the combined genotype Arg/Gln-Cys/Cys of XRCC1/OGG1 (OR 3.83) as well as the Cys/Cys-Tyr/Tyr of OGG1/MUTYH (OR 6.75) increases the risk of ALL. In contrast, the combined genotype Arg/Arg-Ser/Ser of XRCC1/OGG1 (OR 0.40) as well as the Ser/Ser-Tyr/Tyr of OGG1/MUTYH (OR 0.43) played a protective role against this malignant disease. In conclusion, we suggest that polymorphisms of BER genes may be used as an important predictive factor for acute lymphoblastic leukemia in children.

Effect of gliclazide on nucleotide excision repair (NER) and non-homologous DNA end joining (NHEJ) in normal and cancer cells.

The results of clinical studies revealed that gliclazide may reduce the risk of cancer in type 2 diabetic patients (T2DM), although the mechanism of possible protective effect is not sufficiently explored. The increased level of DNA damage and impaired DNA repair system in diabetes mellitus may play a substantial role in neoplastic transformation. Recently, we have demonstrated that gliclazide protected DNA against damage introduced by the oxidative stress, but its action on the DNA repair mechanisms is unclear. Therefore, the aim of this study was to assess whether gliclazide has any effect on the DNA repair pathways, e.g. nucleotide excision repair (NER) and non-homologous end joining (NHEJ). NER activity was assessed in the extract of human lymphocytes and pancreatic cancer cells (PANC-1) treated or not with gliclazide by use of an UV-irradiated plasmid as a substrate and by quantitative PCR performed to evaluate the efficacy of the removal of UV-induced lesions from the p53 gene by intact cells. The efficacy of NHEJ pathway was examined by a simple and rapid in vitro assay based on fluorescent detection of repair products. We did not observe significant differences between the efficiency of NER and NHEJ for extracts of lymphocytes alone and lymphocytes treated with gliclazide. Contrary, gliclazide increased the efficacy of NER (46.0% vs. 84.0%, p<0.01) and NHEJ (58.0% vs. 66.0%, p<0.05) in PANC-1 cells. In conclusion, the present study showed that gliclazide did not affect NER and NHEJ in human normal cells, but it may stimulate DNA repair in cancer cells.

Association between polymorphisms of the BRCA2 gene and clinical parameters in breast cancer.

BACKGROUND: A C/T transition - rs4987117 (the Thr1915Met polymorphism) and an A/G transition - rs11571653 (the Met784Val polymorphism) in the BRCA2 gene were linked to breast cancer risk in Polish and Japanese populations, respectively. AIM: To study the association between polymorphisms of the BRCA2 gene and clinical parameters in breast cancer. METHODS: Both polymorphisms were evaluated by RFLP - PCR in blood samples obtained from 117 women with sporadic breast cancer. Patients were stratified by genotype, Bloom - Richardson grade, TNM stage, estrogene and progesterone receptors (PR) status and the linkages of each genotype with each stratum were calculated by logistic regression. RESULTS: Variant genotypes and alleles of both polymorphisms of the BRCA2 gene were inversely related to hormone receptor status for a group of patients with at least one positive receptor status as compared to a group with both receptors negative status (OR 0.27, 95% CI 0.07 - 0.95, p = 0.043 and OR 0.39, 95% CI 0.19 - 0.82, p = 0.013 for Met1915Met homozygote and 1915Met allele, respectively and OR 0.02, 95% CI 0.00 - 0.13, p = 0.0005 and OR 0.43, 95% CI 0.21 - 0.88, p = 0.021, for Val784Val homozygote and the 784Val allele. No association was found between both polymorphisms and Bloom - Richardson grading and TNM staging. CONCLUSIONS: Our results suggest that variant genotypes of the Thr1915Met and Met784Val polymorphisms of the BRCA2 gene may be indicative factors in therapy of ductal breast cancer.

Polymorphisms of the DNA base excision repair gene MUTYH in head and neck cancer.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCC) comprise about 6% of all malignant neoplasms. The major risk factors of -HNSCC are smoking and alcohol consumption. Genetic polymorphisms of DNA repair enzymes may lead to genetic instability and carcinogenesis. MUTYH gene encodes a DNA glycosylase that can initiate the base excision repair (BER) pathway and prevent G:C > T:A transversion by excising adenine mispaired with 8-hydroxyguanine produced by reactive oxygen species (ROS). AIM: to perform a case-control study to test the association between polymorphism in the MUTYH gene: Tyr165Cys and head and neck cancer risk progression. METHODS: Genotypes were determined in DNA from peripheral blood lymphocytes of 193 patients (among them 97 subjects with precancerous hyperplastic laryngeal lesions and 96 subjects with head and neck cancer) and 140 age, sex and ethnic-matched cancer-free controls by tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR). RESULTS: We found an association between head and neck cancer risk and the Tyr165Tyr variant of the MUTYH gene (OR 2.18; 95% CI 1.19-3.97). For Tyr165Tyr genotype we also observed positive correlation with cancer progression assessed by tumor size (OR 4.56; 95% CI 1.60-12.95). We did not observe any correlation between Tyr165Cys polymorphism of MUTYH gene and precancerous hyperplastic laryngeal lesions risk. CONCLUSION: The Tyr165Tyr polymorphic variant of the MUTYH gene may be associated with head and neck cancer in Polish population.

No association between the Arg194Trp and Arg399Gln polymorphisms of the XRCC1 gene and colorectal cancer risk and progression in a Polish population.

BACKGROUND: The risk of sporadic colorectal cancer can be associated with environmental and lifestyle factors that may be sources of physical and chemical carcinogens, modulated by products of many low penetrance genes. Polymorphisms of DNA repair genes may influence variation in individual DNA repair capacity, which is crucial for preventing genomic instability, which, in turn, may be associated with risk of cancer. XRCC1 is an essential protein for the base excision repair pathway which primarily deals with DNA base modifications, arisen spontaneously or as a consequence of the action of environmental factors. AIM: To perform a case-control study and test the association between two polymorphisms in the XRCC1 gene: Arg194Trp and Arg399Gln and colorectal cancer risk and progression. METHODS: Genotypes were determined in DNA from peripheral blood lymphocytes of 100 colorectal cancer patients and 100 age, sex and ethnic-matched cancer-free controls by PCR RFLP. RESULTS: We found that both polymorphisms of the XRCC1 gene were not associated with risk and progession of colorectal cancer in a Polish population. Moreover, there was not such association form the Arg194Trp/Arg399Gln haplotypes. CONCLUSION: The Arg194Trp and Arg399Gln polymorphisms of the XRCC1 gene may not be associated with colorectal cancer in Polish population.

Molecular characterisation of streptomycin-resistant Mycobacterium tuberculosis strains isolated in Poland.

Primary drug resistance of Mycobacterium tuberculosis strains in Poland increased two-fold between 1997 and 2000. Among 3705 drug-resistant strains investigated in 2000, 169 were resistant to streptomycin alone or in combination with isoniazid, rifampicin and/or ethambutol. The molecular basis of streptomycin resistance for 88 (52%) of these strains in comparison with 15 susceptible controls was determined. The most prevalent mutation was the single substitution Lys43Arg in the rpsL gene, found in 30.7% of the strains analysed. However, as many as 51% of the strains investigated carried no mutation in the rpsL or rrs genes. The multiple mutations present in two Beijing family strains were also identified.