Role of epigenetic deregulation in hematogenous dissemination of malignant uveal melanoma.

It has become increasingly clear that epigenetic deregulation plays a fundamental role in cancer. Although the understanding of molecular, genetic and transcriptional alterations involved in the initiation and progression of uveal melanoma (UM) has grown significantly in recent years, little attention has been paid to the role of epigenetic changes. In cancer, epithelial-to-mesenchymal transition (EMT) enables trans-differentiation of epithelial tumor cells, endowing them with migratory and invasive properties. EMT-inducing transcription factors have been shown to interact with multiple epigenetic modifiers, thus reflecting the reversible nature of EMT. Therefore, the epigenetic therapy targeting these interactions may provide a promising therapeutic option, especially since no improvement in survival of patients with metastatic UM has been achieved using traditional approaches. This review summarizes current knowledge of epigenetic regulation of EMT in UM and emphasizes the need for deeper understanding of these highly dynamic and reversible processes. The potential for targeting individual members of the epigenetic machinery is also addressed.

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.

Identification of the deletions in the UGT1A1 gene of the patients with Crigler-Najjar syndrome type I from Slovakia.

Crigler-Najjar syndrome type I (CN I) is a rare autosomal recessive disorder due to hepatic dysfunction of uridine diphospho-glucuronosyltransferase (UGT) activity toward bilirubin. Complete inactivation of this enzyme causing CN I lead to accumulation of unconjugated bilirubin in serum and bile. Here we report the results of the molecular characterization of the uridine diphospho-glucuronosyltransferase 1A1 (UGT1A1) gene in a consanguineous family of Slovak Roms and an unrelated non-Romany family with CN I. Sequence analysis of UGT1A1 gene in all four Romany patients showed mutation in exon 4, a deletion of an A at codon 407 (1220delA), not yet described in homozygous status. All analysed patients were homozygous for 1220delA mutation and their 3 healthy sibs were heterozygous. The non-Romany patient was a compound heterozygote for two different deletions, 1220delA and 717-718delAG at codon 239. In the family of his cousin a son was born affected with CN I, who was homozygote for 717-718delAG mutation. His other niece affected with CN II was heterozygote for mutation 717-718delAG but homozygote for TA insertion and enhancer substitution T-3279G. Haplotype analysis suggests that the 1220delA mutation is identical by descent in both families, though they originate from two ethnically different populations (Slovaks vs. Roms).

Long distance PCR in detection of inversion mutations of F8C gene in hemophilia A patients.

In the present paper, the experience with detection of intron 22 inversion of F8C gene in severe hemophilia A patients using a recently described long-distance PCR (LD-PCR) method was reported. To test the sensitivity and the specifity of the LD-PCR, analysis of 46 DNA samples of patients and their family members, previously tested by Southern hybridization, was performed. In addition, 16 DNA samples of severe hemophilia A patients in which causative mutation was unknown, were included in analysis. Four-primers, P, Q, A&B, which are able to differentiate between the affected males with or without the inversion, and in female carriers, were used in LD-PCR. Two primers, P&Q, are located within the F8C gene flanking int22h1. Two further primers, A&B, flank int22h2 and int22h3, extragene homologs of int22h1. Nine combinations of four primers were used to identify the optimal one. Four-primers (P, Q, A&B), three-primers (P,Q & B;P, A & B; A, B & Q;P, Q & A) and two-primers (A & B; P & Q; A & Q; P & B) PCR amplifications were performed in the hemophilia A patients as well as in obligate carriers DNA samples. Successful amplification required introduction of some modifications of the original protocol. The most reproducible and uniform results were obtained using two-primers PCR, performed in four single reactions. Thus, a total of 46 DNA samples, 22 were hemizygous for inversion, 6 without the inversion, 14 carriers and 4 non-carriers of inversion. Perfect correlation between genotypes determined using Southern hybridization and LD-PCR was achieved. The optimalized two-primers LD-PCR protocol was used for analysis of 16 DNA samples of severe hemophilia A patients with unknown mutation. Ten cases of inversions and six cases without them were detected. Thus in additional 10 severe hemophilic patients DNA diagnosis was completed. The most successful and reproducible results were obtained performing four single LD-PCR reactions with combinations of two-primers A & B; P & Q; A&Q, and P&B in each DNA sample and this approach is recommended for routine using in clinical practice.