Global DNA methylation and increased DNMT3A expression in multiple myeloma patients.

AIMS: The aim of this study was to compare the expression profile of selected DNA methyltransferases and global DNA methylation status in patients with different phases of multiple myeloma (MM) . For the analysis, different cellular populations including unsorted myeloma cells and a set of plasma cells purified by relevant antibodies were used. Consequently, laboratory data were compared to patients' clinical data. PATIENTS AND METHODS: For the analysis, unsorted bone marrow cell population of 44 MM patients (30 newly diagnosed, 9 relapsed and 5 patients in remission) and a set of 8 patients' samples of sorted plasma cells were used. We used commercially available RNA isolated from BM of 3 healthy individuals as control samples. Expression analysis of three DNA methyltransferases - DNMT1, DNMT3A, and DNMT3B was performed by quantitative RT-PCR and the patient global DNA methylation profiles were detected by colorimetric assay. RESULTS: Unchanged DNMT1 expression was detected in the selected cohort of patients. Normalized DNMT3A gene expression was globally higher in comparison with controls in unsorted and sorted cell populations. Low (0.08-1.81%) global DNA methylation status in unsorted samples of multiple myeloma patients did not correlate either with expression profiles of monitored DNA methyltransferases or with the stages of MM based on Durie-Salmon and International Staging System. CONCLUSION: This is the first comparative study between DNA methyltransferases expression profiles and global DNA methylation status in different phases of multiple myeloma patients. No significant correlation between the level of global methylation and the clinical stage of the unsorted cell population of patients with multiple myeloma was registered. Overexpression of the DNMT3A gene occurred in both sorted and unsorted cell populations of patients with multiple myeloma. This fact highlights the DNMT3A as a potential marker of multiple myeloma tumor progression. Moreover, we demonstrated comparable results in the expression of DNA methyltransferases in both sorted and unsorted cell populations. This is a promising result from the methodical point of view because when compared to samples of unsorted multiple myeloma cells, samples of sorted cells bring reduction of the number of possible analyses performed.

Possible Therapeutic Potential of Disulfiram for Multiple Myeloma.

Multiple myeloma (MM) is a malignant disease of the plasma cells representing approximately 10% of all hemato-oncological diseases. Detection of the disease is most probable at around 65 years of age, and the average survival of patients is estimated to be 5-10 years, specifically due to frequent relapses and resistance to the therapy used. Thus, the search for new therapeutic approaches is becoming a big challenge. Disulfiram (DSF), a substance primarily known as a medication against alcoholism, has often been mentioned in recent years in relation to cancer treatment for its secondary anti-cancer effects. Recent studies performed on myeloma cell lines confirm high inhibition of the cell growth activity if a complex of disulfiram and copper is used. Its significant potential is now being seen in the cure of haematological malignities.

Different effect of sodium butyrate on cancer and normal prostate cells.

Sodium butyrate, as a naturally occurring inhibitor of histone deacetylases (HDACI), is a non-toxic agent, with an ability to change histone acetylation and expression of large number genes. This study shows different effects of sodium butyrate on expression and transcription activity of the androgen receptor in cancer (LNCaP, C4-2) and normal (RWPE-1) prostate cells. Moreover, we studied the coregulator expressions and histone acetylation alteration in cancer and normal cells. Coregulators, coactivators as well as corepressors, play an important role in AR-mediated growth and progression of prostate cancer. There is a competition between coactivators and corepressors for binding on the AR and therefore the changes in coregulators expression and ratio could be important for prostate cancer survival. Our study was focused on two coregulators, SMRT and p300, which interact with AR in multiprotein complex and affect the AR transcription activity. Our data indicate that sodium butyrate has an effect on AR coregulators expression, transcription activity and histone acetylation in cancer cells, but there is only minimal effect in normal cells. In addition, the results of changes in acetylation level on lysine residues of histone H4 after sodium butyrate treatment confirm its epigenetic effect on prostate cancer cells.

Effect of histone deacetylase and DNA methyltransferase inhibitors on the expression of the androgen receptor gene in androgen-independent prostate cancer cell lines.

Androgen receptor (AR) expression in prostate cancer (CaP) cells varies due to the multiple changes including epigenetic modifications such as DNA methylation and histone deacetylation. DNA methyltransferase and histone deacetylase inhibitors are promising for the treatment of CaP. The aim of our study was to analyze the 5-aza-2'-deoxycytidine (Aza­dC) and sodium butyrate (NaB) effects on CaP cells with modified AR gene expression. The androgen-independent human prostate cancer cell lines PC3 (lacking a functional AR) and DU145 (strongly limited expression due to methylations in the AR gene) were used. PCR of bisulfite-modified DNA and RT-PCR with bisulfite-sequencing were used for AR gene analysis of DU145 and PC3 cells following their treatment with Aza-dC and/or NaB. Re-acetylated histones around the AR gene were detected by conventional PCR of immunoprecipitated DNA obtained from treated cells. In both cell lines without the AR expression, the combined treatment was followed with significant decrease of cell viability. The co-treatment of DU145 cells caused site-specific demethylation in the AR promoter region followed by gene re-expression and increased acetylation in histones H3 and H4. The co-treatment with Aza-dC and NaB was the most effective in demethylation and re-expression of the AR gene. In the AR gene promoter, the location and density of deme-thylated CpGs indicated the existence of distinct promoter hot spot that could be a target of AR gene inactivation therapy of CaP patients during androgen deprivation.