Global and gene specific DNA methylation in breast cancer cells was not affected during epithelial-to-mesenchymal transition in vitro.

Epithelial-to-mesenchymal transition (EMT) significantly affects the risk of metastasising in breast cancer. Plasticity and reversibility of EMT suggest that epigenetic mechanisms could be the key drivers of these processes, but little is known about the dynamics of EMT-related epigenetic alterations. We hypothesised that EMT, mediated by autocrine and paracrine signals, will be accompanied by changes in DNA methylation profiles. Therefore, conditioned medium from adipose tissue-derived mesenchymal stromal cells was used for induction of EMT in human breast cancer SK-BR-3 cell line. EMT-related morphological alterations and changes in gene expression of EMT-associated markers were assessed. To reverse EMT, 20 nm size gold nanoparticles (AuNPs) synthesized by the citrate reduction method were applied. Finally, DNA methylation of LINE-1 sequences and promoter methylation of TIMP3, ADAM23 and BRMS1 genes were quantitatively evaluated by pyrosequencing. Despite the presence of EMT-associated morphological and gene expression changes in tumour cells, EMT induced by adipose tissue-derived mesenchymal stromal cells had almost no effect on LINE-1 and gene-specific DNA methylation patterns of TIMP3, ADAM23 and BRMS1 genes. Although treatment for 24, 48 or 72 hours with 20 nm AuNPs at a concentration of 3 µg/ml slightly decreased gene expression of EMT-associated markers in SK-BR-3 cells, it did not alter global or gene-specific DNA methylation. Our results suggest that changes in DNA methylation are not detectable in vitro in early phases of EMT. Previously published positive findings could represent rather the sustained presence of potent EMT-inducing signals or the synergistic effect of various epigenetic mechanisms. Treatment with AuNPs slightly attenuated EMT, and their therapeutic potential needs to be further investigated.

Evaluation of protein expression and DNA methylation profiles detected by pyrosequencing in invasive breast cancer.

Breast carcinoma is the most common cancer with high mortality caused by metastatic disease. New molecular biomarkers predicting the tumour's metastatic potential would therefore improve metastasis prevention and personalised care. The aim of the study was to investigate the relationship between DNA methylation levels in invasivity and metastasising associated genes with aberrant protein expression and also to evaluate whether a similar DNA methylation level is present in the tumour and circulating cell-free DNA for utilising plasma DNA methylation as prognostic biomarker. By using pyrosequencing, we analysed DNA methylation levels of 11 genes, namely APC, ADAM23, CXCL12, ESR1, PGR B, CDH1, RASSF1A, SYK, TIMP3, BRMS1 and SOCS1 in tumour, plasma and peripheral blood cells from 34 patients with primary breast cancer, as well as plasma and peripheral blood cells from 50 healthy controls. Simultaneously, the expression of related proteins in paraffin-embedded tumour samples was evaluated by immunohistochemistry. Statistical analysis was performed by SPSS statistics 15.0 software. Tumour DNA hypermethylation was found in most commonly methylated RASSF1A (71.9%), APC (55.9%), ADAM23 (38%) and CXCL12 (34.4%) genes with methylation levels up to 86, 86, 53 and 64 %, respectively. In tumours, significantly higher methylation levels were found in nine genes, compared with the patients´ peripheral blood cell DNA. Furthermore, in patients methylation levels in peripheral blood cell DNA were significantly higher than in controls in CXCL12, ESR1 and TIMP3 genes, but the values did not exceed 15%. On the other hand, no correlations were observed in patients between DNA methylation in tumours and cell-free plasma DNA. Moreover, in patients and controls nearly identical values of cumulative DNA methylation (43.6 % ± 20.1 vs. 43.7 % ± 15.0) were observed in plasma samples. A variable spectrum from high to none expressions presented in tumour tissues in all of the proteins evaluated, however in APC and CXCL12 genes a visible decreasing trend of mean DNA methylation level with increasing expression of the corresponding protein was observed. The DNA methylation profiles manifested in our group of breast carcinomas are cancer specific, but they are not the only cause that affects the silencing of evaluated genes and the decrease of relevant protein products. The clinical utility of DNA methylation testing in peripheral blood cell DNA for cancer diagnosis and therapy need to be further investigated.

The role of clinical criteria, genetic and epigenetic alterations in Lynch-syndrome diagnosis.

Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) represents 1-3% of all diagnosed colorectal cancers (CRCs). This study aimed to evaluate the benefit of clinical criteria and several molecular assays for diagnosis of this syndrome. We examined tumors of 104 unrelated clinically characterized colorectal cancer patients for causal mismatch repair (MMR) deficiency by several methods: microsatellite instability (MSI) and loss of heterozygosity (LOH) presence, MMR protein absence, hypermethylation of MLH1 promoter and germline mutation presence. Twenty-five (24%) patients developed CRCs with a high level of MSI (MSI-H). Almost all (96%) had at least one affected relative, while this simple criterion was satisfied in only 22% (17/79) of individuals with low level MSI or stable cancers (MSI-L, MSS). Using strict Amsterdam criteria, the relative proportion of complying individuals in both sets of patients (MSI-H vs. MSI-L and MSS) decreased to 68% and 9%, respectively. The right-sided tumors were located in 54% of MSI-H persons when compared to 14% of cancers found in MSI-L or MSS patients. In 16 MSI positive patients with identified germline mutation by DNA sequencing, the gene localization of mutation could be indicated beforehand by LOH and/or immunohistochemistry (IHC) in four (25%) and 14 cases (88%), respectively. The IHC findings in MSI-H cancers with methylation in distal or both regions of MLH1 promoter have not confirmed the epigenetic silencing of the MLH1 gene. None of the patients with MSIL or MSS tumors was a carrier of the MLH1 del616 mutation, despite seven of them meeting Amsterdam criteria. The effective screening algorithm of Lynch-syndrome-suspected patients consists of evaluation of Bethesda or Revised Bethesda Guidelines fulfilling simultaneous MSI, LOH and IHC analyses before DNA sequencing. Variable methylation background in MLH1 promoter does not affect gene silencing and its role in Lynch-syndrome tumorigenesis is insignificant.

Novel and recurrent germline alterations in the MLH1 and MSH2 genes identified in hereditary nonpolyposis colorectal cancer patients in Slovakia.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in DNA mismatch repair genes, predominantly MSH2 and MLH1. Mutation carriers develop cancers in the colorectum, endometrium, ovary, stomach, small intestine and the upper urinary tract. We describe here the results of a mutational analysis of 11 unrelated HNPCC patients by direct genomic sequencing of MLH1 and MSH2. The alterations found include 7 novel changes and 4 different pathogenic mutations described previously in Poland, Moldavia, Finland, Germany, France and USA. Four novel pathogenic mutations in the MLH1 gene include two frameshift mutations (c.1150delG and c.1210_1211delCT), one missense mutation (c.793C>A) and one intron-exon border mutation (c.546- 2A>C). The last change resulted in the skipping of exon 7, as shown by sequencing of RT-PCR products. The only novel MSH2 pathogenic change was a nonsense mutation c.1129C>T. The novel intronic change c.381-41A>G in MLH1 was found in a patient carrying a previously-described mutation in the MSH2 gene. Interestingly, two unrelated patients carried also a novel change in the promoter region of MLH1 in one of the CpG islands (c.-269C>G). However, this alteration does not abrogate transcription, as shown by RT-PCR analysis. In summary, most (approximately 80%) pathogenic germline mutations detected in the studied group of patients by direct genomic sequencing of MLH1 and MSH2 were located in the MLH1 gene. These and previous data indicate that the majority of germline point mutations and small deletions/insertions in HNPCC families in Slovakia affect the MLH1 locus.

Increased DNA double strand breakage is responsible for sensitivity of the pso3-1 mutant of Saccharomyces cerevisiae to hydrogen peroxide.

Escherichia coli endonuclease III (endo III) is the key repair enzyme essential for removal of oxidized pyrimidines and abasic sites. Although two homologues of endo III, Ntgl and Ntg2, were found in Saccharomyces cerevisiae, they do not significantly contribute to repair of oxidative DNA damage in vivo. This suggests that an additional activity(ies) or a regulatory pathway(s) involved in cellular response to oxidative DNA damage may exist in yeast. The pso3-1 mutant of S. cerevisiae was previously shown to be specifically sensitive to toxic effects of hydrogen peroxide (H2O2) and paraquat. Here, we show that increased DNA double strand breakage is very likely the basis of sensitivity of the pso3-1 mutant cells to H2O2. Our results, thus, indicate an involvement of the Pso3 protein in protection of yeast cells from oxidative stress presumably through its ability to prevent DNA double strand breakage. Furthermore, complementation of the repair defects of the pso3-1 mutant cells by E. coli endo III has been examined. It has been found that expression of the nth gene in the pso3-1 mutant cells recovers survival, decreases mutability and protects yeast genomic DNA from breakage following H2O2 treatment. This might suggest some degree of functional similarity between Pso3 and Nth.

Approaches to identification of HNPCC suspected patients in Slovak population.

Patients with hereditary non-polyposis colorectal cancer (HNPCC) have a DNA mismatch repair defect (MMR) in their tumor tissue that results in instability of microsatellite DNA sequences (MSI). Thus, MSI analysis may effectively indicate this form of cancer that should be then proved by analysis of germline mutations in MMR genes. The aim of this study was to identify HNPCC suspected patients in the Slovak population by investigating microsatellite instability in colorectal tumor tissues. MSI was studied at 5-11 loci in matched tumor and normal DNA using radioactively labeled PCR products separated on sequencing gels. High microsatellite instability (MSI-H) was present only in patients younger than 50 years, in 100% of patients having two affected relatives by colorectal cancer and in 67% of patients with only one affected relative. In both groups of patients colorectal cancer was present in two successive generations. No MSI-H was found in the group of patients older than 50 years, even if they had positive family history for colorectal cancer. Among all markers used, the BAT26 mononucleotide repeat (100%), DI0S197 and D13S175 (62.5%) dinucleotide repeats were the most frequently altered in the tumor tissues. Retrospective analysis revealed that some of the patients having MSI-H tumors have had clinicopathological characteristics frequently reported to HNPCC. The family members of those patients with MSI-H are enrolled in preventive health care program until mutational analyses will enable to select carriers from non-carriers of mutated MMR genes.

Searching for a functional analogy between yeast Pso4 and bacterial RecA proteins in induced mitotic recombination.

The pso4-1 mutant of S. cerevisiae is phenotypically similar to the recA mutant of E. coli; it is sensitive to DNA cross-linking agents and defective in both recombination and mutagenesis. In this paper we have measured the effect of the recA gene expression on the frequency of mitotic crossing-over and mitotic gene conversion in response to DNA damage induced by photoactivated 8-methoxypsoralen (8-MOP + UVA), ultraviolet radiation (UV) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The diploid pso4-1 mutant and the repair wild type strain were transformed with the multicopy plasmid carrying the recA gene placed under the control of the ADH1 promoter. The results showed that RecA is not able to restore block in induced mitotic recombination in pso4-1 cells after DNA damaging agents used. Thus RecA protein is not able to substitute Pso4 protein in homologous mitotic recombination indicating that they have probably different functions in this process.

Constitutive increase of RecA protein: its influence on pyrimidine dimer excision and survival of UV-irradiated Escherichia coli.

Transformation of E. coli with the plasmid pRA containing recA gene increased the constitutive level of RecA protein 50-67 fold. This slightly inhibited pyrimidine dimer excision and reduced cell survival in three investigated, UV-irradiated E. coli strains. Our data support the view that RecA protein prematurely present at a high level may mask the dimers. The masking subsequently reduces the dimer excision and switches off the inducing signal.

DNA repair in Escherichia coli: the dual function of uvr genes.

It has been shown earlier that the starvation of E. coli for both amino-acids and thymine applied prior to UV irradiation inhibits pyrimidine dimer excision without affecting cell survival after UV irradiation. In such cells pyrimidine dimers are tolerated by a rather error-free process that depends on the activity of uvrB, recA and lexA genes. Data presented here show: (a) that the efficient toleration of unexcised dimers requires also the uvrA gene; (b) that the starvation increases the level of RecA protein about 4.7 times; (c) that the effect of starvation on subsequent pyrimidine dimer excision is reversed by a 2 h incubation in complete medium before the cells are UV irradiated. The data suggest that the uvrA, uvrB, recA, lexA dependent nonexcisional repair may be a pathway temporarily functioning in repeatedly damaged cells.

Inhibition of dimer excision in repeatedly UV-irradiated Escherichia coli: its requirement for RecA protein and de novo protein synthesis.

In UV-irradiated Escherichia coli dimer excision was found to be inhibited by predamage (M. Sedliaková, F. Masek and J. Brozmanová, FEBS Lett., 23 (1972) 325-326) or overproduction of RecA protein, which suggests that the coating of the dimers by this protein may make them inaccessible to the excision nuclease (M. Sedliaková, K. Kleibl and F. Masek, Mutat. Res., 191 (1987) 13-16). We measured the levels of RecA protein and dimer excision in cells irradiated with (i) a single dose of 50 J m-2, (ii) two separate doses of 30 and 50 J m-2, post-incubated with chloramphenicol; (iii) two separate doses of 30 and 50 J m-2, post-incubated without chloramphenicol. Dimer excision was complete in the first two cases, but in the latter it was inhibited by 40%. At the time of active dimer excision, there were marked differences in RecA protein content between the cells irradiated with a single dose and cells irradiated with two separate doses (both post-incubated without chloramphenicol), which might account for the differences in dimer excision. However, relatively small differences in RecA protein content were found in cells irradiated with two doses and post-incubated with or without chloramphenicol, which could therefore not account for the differences in dimer excision. The data suggest that the inhibition of dimer excision involves some short-lived component(s) other than RecA protein.

Immunological quantification of RecA protein in cell extracts of E. coli after exposure to chemical mutagens or UV radiation.

Increased synthesis of RecA protein is induced in E. coli cells after their damage, the rate of synthesis being dependent on the extent of DNA alterations. The level of the RecA protein was determined in E. coli cell extracts after damage induced by NQO, MNNG, MMC, NAL or UV radiation, using competitive enzyme-linked immunosorbent assay (ELISA). Purified E. coli RecA protein and rabbit monospecific polyclonal antibodies against it were prepared for the quantitative assay. The level of the RecA protein was increased after treatment with all mutagens. Contrary to other induced proteins, the synthesis of the RecA protein increased within 30 min after damage with UV radiation at a relatively slow rate. The ELISA method made it possible to determine 0.5-50 ng of the RecA protein in bacterial extracts. The method can be employed as an auxiliary test for DNA damage determination and also in studied concerning the role of the RecA protein in repair processes.

The Escherichia coli recA gene increases resistance of the yeast Saccharomyces cerevisiae to ionizing and ultraviolet radiation.

The Escherichia coli recA protein coding region was ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase promoter region to produce plasmid pADHrecA. Transformation of the wild-type yeast strains YNN-27 and 7799-4B, as well as the recombination-deficient rad52-1 C5-6 mutant, with this shuttle plasmid resulted in the expression of the bacterial 38 kDa RecA protein in exponential phase cells. The wild-type YNN27 and 7799-4B transformants expressing the bacterial recA gene showed increased resistance to the toxic effects of both ionizing and ultraviolet radiation. RecA moderately stimulated the UV-induced mutagenic response of 7799-4B cells. Transformation of the rad52-1 mutant with plasmid pADHrecA did not result in the complementation of sensitivity to ionizing radiation. Thus, the RecA protein endows the yeast cells with additional activities, which were shown to be error-prone and dependent on the RAD52 gene.

Expression of the Escherichia coli recA gene in the yeast Saccharomyces cerevisiae.

The isolation of the protein coding region of the recA gene from Escherichia coli by extensive Bal31 digestion is described. The structural recA gene was ligated into an extrachromosomally replicating yeast expression vector, downstream of the yeast alcohol-dehydrogenase gene promoter region, to produce pADHrecA plasmid. The pADHrecA plasmid was transformed into the wild-type and the repair deficient strains of Saccharomyces cerevisiae. The crude protein samples were extracted from the individual yeast transformants. A 38 kDa protein was present in all transformants containing the recA gene on plasmid. Thus the recA gene from E coli was successfully expressed in cells from a lower eukaryote.

Cytogenetic study of blood in women who had used oral contraceptives.

Changes in the number and structure of chromosomes in peripheral lymphocytes of 31 healthy women using oral contraceptives (OC) were studied. Blood samples were taken prior to and after one, two and three months of using oral contraceptives. No significant differences were found in evaluating numerical anomalies but highly significant differences were detected in structural aberrations and in structural aberrations without gaps.