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ABSTRACT: Tamoxifen (TAM) is a non-steroidal anti-estrogen used widely in the treatment and chemoprevention of breast cancer. TAM treatment can lead to DNA damage, but the mechanism of this process is not fully understood and the experimental data are often inconclusive. We compared the DNA-damaging potential of TAM in normal human peripheral blood lymphocytes and MCF-7 breast cancer cells by using the comet assay. In order to assess whether oxidative DNA damage may contribute to TAM-induced lesions, we employed two DNA repair enzymes: endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg). The kinetics of repair of DNA damage was also measured. In order to evaluate the involvement of free radicals in the genotoxicity of TAM we pre-treated the cells with nitrone spin traps: DMPO and POBN. The use of common antioxidants: vitamin C, amifostine and genistein, helped to assess the contribution of free radicals. TAM damaged DNA in both normal and cancer cells, inducing mainly DNA strand breaks but not alkali-labile sites. The drug at 5 and 10 microM induced DNA double strand breaks (DSBs) in lymphocytes and at 10 microM in MCF-7 cells. We observed complete repair of DSBs in cancer cells by contrast with incomplete repair of these lesions in lymphocytes. In both types of cells TAM induced oxidized purines and pyrimidines. Incubation of the cells with nitrone spin traps and antioxidants decreased, with exception of amifostine in MCF-7 cells, the extents of DNA damage in both kinds of cells, but the results were more distinct in cancer cells. Our results indicate that TAM can be genotoxic for normal and cancer cells by free radicals generation. It seems to have a higher genotoxic potential for normal cells, which can be the result of incomplete repair of DNA DSBs. Free radicals scavengers can modulate TAM-induced DNA damage interfering with its antitumour activity in cancer cells.
Archive für Toxikologie 08/2007; 81(7):519-27. · 4.67 Impact Factor
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ABSTRACT: The response of the cell to DNA damage and its ability to maintain genomic stability by DNA repair are crucial in preventing cancer initiation and progression. Therefore, polymorphism of DNA repair genes may affect the process of carcinogenesis. The importance of genetic variability of the components of mismatch repair (MMR) genes is well documented in colorectal cancer, but little is known about its role in breast cancer. hMSH2 is one of the crucial proteins of MMR. We performed a case-control study to test the association between two polymorphisms in the hMSH2 gene: an A --> G transition at 127 position producing an Asn --> Ser substitution at codon 127 (the Asn127Ser polymorphism) and a G --> A transition at 1032 position resulting in a Gly --> Asp change at codon 322 (the Gly322Asp polymorphism) and breast cancer risk and cancer progression. Genotypes were determined in DNA from peripheral blood lymphocytes of 150 breast cancer patients and 150 age-matched women (controls) by restriction fragment length polymorphism and allele-specific PCR. We did not observe any correlation between studied polymorphisms and breast cancer progression evaluated by node-metastasis, tumor size and Bloom-Richardson grading. A strong association between breast cancer occurrence and the Gly/Gly phenotype of the Gly322Asp polymorphism (odds ratio 8.39; 95% confidence interval 1.44-48.8) was found. Therefore, MMR may play a role in the breast carcinogenesis and the Gly322Asp polymorphism of the hMSH2 gene may be considered as a potential marker in breast cancer.
Breast Cancer Research and Treatment 01/2006; 94(3):199-204. · 4.43 Impact Factor
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ABSTRACT: Matrix metalloproteinases play a crucial role in the cancer invasion and metastasis, angiogenesis and tumorigenicity. A single guanine insertion--the 1G/2G polymorphism in the promoter of the matrix metalloproteinase 1 (MMP-1) gene creates a binding site for the transcription factor AP-1 and thus may affect the transcription level of MMP-1. The C-->T substitution at the polymorphic site of the MMP-9 gene promoter results in a higher transcription activity of the T-allelic promoter trough the loss of binding site for a repressor protein. The aim of this work was to investigate the influence of 1G/2G and C-->T polymorphisms on the MMP-1 and MMP-9 level and therefore on the occurrence and progression of breast cancer.
We investigated the distribution of genotypes and frequency of alleles of the 1G/2G and C-->T polymorphisms for 270 patients with breast cancer and 300 healthy women served as control. The genotypes were determined by RFLP-PCR. Additionally, we estimated the level of MMP-1 and MMP-9 antigens in tumor samples and normal breast tissue using ELISA.
The levels of MMP-1 in tumor samples of node positive patients ware significantly higher than in samples of node negative patients (p<0.05). Increased level of MMP-9 correlates with Bloom-Richardson grading III (p<0.05), increased tumor size (p<0.05) and absence of estrogen and progesterone receptors (p<0.01). Additionally, both MMP-1 and MMP-9 levels were higher in tumor than in the normal breast tissue. We showed the higher risk of metastasis development in lymph node for the 2G/2G genotype (OR=2.14; CI 95% 1.24;3.69) and the 2G allele carriers (OR=1.68; CI 95% 1.19;2.39). We found correlation between the T allele (OR=2.61; CI 95% 1.33;4.87), 2G (OR=2.58; CI 95% 1.35;4.91) and malignance.
The results suggest that MMP-1 is responsible for the local invasion and MMP-9 is associated with the malignance and the growth of the tumor. We suggest that the 2G allele of the 1G/2G MMP-1 gene polymorphism may be associated with the lymph node metastasis in patients with breast cancer and therefore it can be considered as a progression marker in this disease.
Breast Cancer Research and Treatment 01/2006; 95(1):65-72. · 4.43 Impact Factor
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ABSTRACT: We performed a case-control study (150 cases and 150 controls) to test the association between three polymorphisms in BRCA2 and RAD51 genes and breast cancer risk. Genotypes were determined in DNA from blood cells by PCR-RFLP. Cancer occurrence was strongly associated with the BRCA2 Met/1915Thr homozygous polymorphic variants, whereas heterozygous variant was associated with significant reduction in breast cancer risk. Gene-gene interaction between the BRCA2-Met1915Thr Thr/Thr and BRCA2-Met784Val Met/Met homozygous variants increased the risk. Therefore, the Met1915Thr polymorphism in the BRCA2 gene may be considered as an independent marker of breast cancer.
Breast Cancer Research and Treatment 12/2005; 94(2):105-9. · 4.43 Impact Factor
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ABSTRACT: Impaired DNA repair may fuel up malignant transformation of breast cells due to the accumulation of spontaneous mutations in target genes and increasing susceptibility to exogenous carcinogens. Moreover, the effectiveness of DNA repair may contribute to failure of chemotherapy and resistance of breast cancer cells to drugs and radiation. The breast cancer susceptibility genes BRCA1 and BRCA2 are involved in DNA repair. To evaluate further the role of DNA repair in breast cancer we determined: (1) the kinetics of removal of DNA damage induced by hydrogen peroxide and the anticancer drug doxorubicin, and (2) the level of basal, oxidative and alkylative DNA damage before and during/after chemotherapy in the peripheral blood lymphocytes of breast cancer patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. We observed slower kinetics of DNA repair after treatment with hydrogen peroxide and doxorubicin in lymphocytes of breast cancer patients compared to control individuals. The level of basal, oxidative and alkylative DNA damage was higher in breast cancer patients than in the control and the difference was more pronounced when patients after chemotherapy were engaged, but usually the level of DNA damage in these patients was too high to be measured with our system. Our results indicate that peripheral blood lymphocytes of breast cancer patients have more damaged DNA and display decreased DNA repair efficacy. Therefore, these features can be considered as risk markers for breast cancer, but the question whether they are the cause or a consequence of the illness remains open. Nevertheless, our results suggest that research on the mutagen sensitivity and efficacy of DNA repair could impact the development of new diagnostic and screening strategies as well as indicate new targets to prevent and cure cancer. Moreover, the comet assay may be applied to evaluate the suitability of a particular mode of chemotherapy to a particular cancer patient.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 11/2004; 554(1-2):139-48. · 2.85 Impact Factor
