Ellagic Acid-Changed Epigenome of Ribosomal Genes and Condensed RPA194-Positive Regions of Nucleoli in Tumour Cells.

We studied the effect of ellagic acid (EA) on the morphology of nucleoli and on the pattern of major proteins of the nucleolus. After EA treatment of HeLa cells, we observed condensation of nucleoli as documented by the pattern of argyrophilic nucleolar organizer regions (AgNORs). EA also induced condensation of RPA194-positive nucleolar regions, but no morphological changes were observed in nucleolar compartments positive for UBF1/2 proteins or fibrillarin. Studied morphological changes induced by EA were compared with the morphology of control, non-treated cells and with pronounced condensation of all nucleolar domains caused by actinomycin D (ACT-D) treatment. Similarly as ACT-D, but in a lesser extent, EA induced an increased number of 53BP1-positive DNA lesions. However, the main marker of DNA lesions, γH2AX, was not accumulated in body-like nuclear structures. An increased level of γH2AX was found by immunofluorescence and Western blots only after EA treatment. Intriguingly, the levels of fibrillarin, UBF1/2 and γH2AX were increased at the promoters of ribosomal genes, while 53BP1 and CARM1 levels were decreased by EA treatment at these genomic regions. In the entire genome, EA reduced H3R17 dimethylation. Taken together, ellagic acid is capable of significantly changing the nucleolar morphology and protein levels inside the nucleolus.

CARM1 modulators affect epigenome of stem cells and change morphology of nucleoli.

CARM1 interacts with numerous transcription factors to mediate cellular processes, especially gene expression. This is important for the maintenance of ESC pluripotency or intervention to tumorigenesis. Here, we studied epigenomic effects of two potential CARM1 modulators: an activator (EML159) and an inhibitor (ellagic acid dihydrate, EA). We examined nuclear morphology in human and mouse embryonic stem cells (hESCs, mESCs), as well as in iPS cells. The CARM1 modulators did not function similarly in all cell types. EA decreased the levels of the pluripotency markers, OCT4 and NANOG, particularly in iPSCs, whereas the levels of these proteins increased after EML159 treatment. EML159 treatment of mouse ESCs led to decreased levels of OCT4 and NANOG, which was accompanied by an increased level of Endo-A. The same trend was observed for NANOG and Endo-A in hESCs affected by EML159. Interestingly, EA mainly changed epigenetic features of nucleoli because a high level of arginine asymmetric di-methylation in the nucleoli of hESCs was reduced after EA treatment. ChIP-PCR of ribosomal genes confirmed significantly reduced levels of H3R17me2a, in both the promoter region of ribosomal genes and rDNA encoding 28S rRNA, after EA addition. Moreover, EA treatment changed the nuclear pattern of AgNORs (silver-stained nucleolus organizer regions) in all cell types studied. In EA-treated ESCs, AgNOR pattern was similar to the pattern of AgNORs after inhibition of RNA pol I by actinomycin D. Together, inhibitory effect of EA on arginine methylation and effect on related morphological parameters was especially observed in compartment of nucleoli.

Advanced microscopy techniques used for comparison of UVA- and γ-irradiation-induced DNA damage in the cell nucleus and nucleolus.

Every day, genomes are affected by genotoxic factors that create multiple DNA lesions. Several DNA repair systems have evolved to counteract the deleterious effects of DNA damage. These systems include a set of DNA repair mechanisms, damage tolerance processes, and activation of cell-cycle checkpoints. This study describes selected confocal microscopy techniques that investigate DNA damage-related nuclear events after UVA- and γ-irradiation and compare the DNA damage response (DDR) induced by the two experimental approaches. In both cases, we observed induction of the nucleotide excision repair (NER) pathway and formation of localized double-strand breaks (DSBs). This was confirmed by analysis of cyclobutane pyrimidine dimers (CPDs) in the DNA lesions and by increased levels of γH2AX and 53BP1 proteins in the irradiated genome. DNA damage by UVA-lasers was potentiated by either BrdU or Hoechst 33342 pre-sensitization and compared to non-photosensitized cells. DSBs were also induced without BrdU or Hoechst 33342 pre-treatment. Interestingly, no cyclobutane pyrimidine dimers (CPDs) were detected after 405 nm UVA laser micro-irradiation in non-photosensitized cells. The effects of UVA and γ-irradiation were also studied by silver staining of nucleolar organizer regions (AgNORs). This experimental approach revealed changes in the morphology of nucleoli after genome injury. Additionally, to precisely characterize DDR in locally induced DNA lesions, we analysed the kinetics of the 53BP1 protein involved in DDR by fluorescence recovery after photobleaching (FRAP).

PRMT1 arginine methyltransferase accumulates in cytoplasmic bodies that respond to selective inhibition and DNA damage.

Protein arginine methyltransferases (PRMTs) are responsible for symmetric and asymmetric methylation of arginine residues of nuclear and cytoplasmic proteins. In the nucleus, PRMTs belong to important chromatin modifying enzymes of immense functional significance that affect gene expression, splicing and DNA repair. By time-lapse microscopy we have studied the sub-cellular localization and kinetics of PRMT1 after inhibition of PRMT1 and after irradiation. Both transiently expressed and endogenous PRMT1 accumulated in cytoplasmic bodies that were located in the proximity of the cell nucleus. The shape and number of these bodies were stable in untreated cells. However, when cell nuclei were microirradiated by UV-A, the mobility of PRMT1 cytoplasmic bodies increased, size was reduced, and disappeared within approximately 20 min. The same response occurred after γ-irradiation of the whole cell population, but with delayed kinetics. Treatment with PRMT1 inhibitors induced disintegration of these PRMT1 cytoplasmic bodies and prevented formation of 53BP1 nuclear bodies (NBs) that play a role during DNA damage repair. The formation of 53BP1 NBs was not influenced by PRMT1 overexpression. Taken together, we show that PRMT1 concentrates in cytoplasmic bodies, which respond to DNA injury in the cell nucleus, and to treatment with various PRMT1 inhibitors.

Tumor-specific histone signature and DNA methylation in multiple myeloma and leukemia cells.

Understanding the epigenetics of tumor cells is of clinical significance for the treatment of cancer, and thus, chemists have focused their efforts on the synthesis of new generation of inhibitors of histone deacetylases (HDACs) or methylation-specific enzymes as novel important anti-cancer drugs. Here, we tested whether the histone signature and DNA methylation in multiple myeloma (MM) and leukemia cells is tumor-specific as compared with that in non-malignant lymphoblastoid cells. We observed a distinct histone signature in c-myc, Mcl-1, and ribosomal gene loci in MOLP8 MM and K562 leukemia cells, when compared with lymphoblastoid cells. Histone and DNA methylation patterns in MOLP8 cells were partially modified by the clinically promising HDAC inhibitor, vorinostat. In comparison with lymphoblastoid WIL2NS cells, MOLP8 cells and K562 cells were characterized by an absence of the gene silencing marker H3K9me2 in the c-myc and ribosomal genes. However, high levels of H3K27me3 were detected in the promoters and coding regions of selected genomic regions in these cells. Treatment by vorinostat increased the level of DNA methylation at the c-myc promoter, and this alteration was accompanied by a decrease in c-MYC protein. In MOLP8 cells, vorinostat significantly increased the H3K9 acetylation in the Mcl-1 coding regions and promoter. Both MOLP8 and K562 leukemia cells were characterized by decreased levels of H3K9me2 in the Mcl-1 gene as compared with lymphoblastoid WIL2NS cells. Lower levels of H3K9me1 in the Mcl-1 promoter, however, were specific for MM cells as compared with the other cell types studied. In other MM and leukemia cell lines, COLO677, OPM2, and U937, the ribosomal genes were less prone to epigenetic heterogeneity as compared to the c-myc and Mcl-1 proto-oncogenes. Taken together, these data describe both tumor-specific and loci-specific histone signature and DNA methylation profiles.

Expression of RAN, ZHX-2, and CHC1L genes in multiple myeloma patients and in myeloma cell lines treated with HDAC and Dnmts inhibitors.

Real time PCR is a powerful tool for studying the expression of genes involved in the pathophysiology of human diseases. Recent studies of the RAN (6p21), ZHX-2 (8q24.3), CHC1L (13q14.3) loci highlight the importance of these genes in multiple myeloma (MM) prognosis and therapeutic applications. Here, we described a detailed Real-Time PCR method for the detection of RAN, ZHX-2, and CHC1L expression, which could be applied in clinical situations. The expression profiles of these genes were studied in peripheral blood lymphocytes of healthy individuals, patients suffering from MM, and in the myeloma cell line, MOLP-8. Low expression levels of RAN, ZHX-2, and CHC1L were observed in myeloma patients, compared with peripheral blood lymphocytes and MOLP-8 cells. An inhibitor of histone deacetylases (TSA) had the ability to decrease expression of CHC1L and ZHX-2 in MOLP-8 cells, while expression of RAN was relatively stable in peripheral blood lymphocytes, control MOLP-8, and TSA- or 5-azacytidine treated MOLP-8 cells. In myeloma patients, we observed significant decreases in the expression of selected genes, but it was patient-specific. Our experiments illustrate new methodological approaches and troubleshooting for conducting gene expression studies in clinical laboratories.

Nuclear topography of the 1q21 genomic region and Mcl-1 protein levels associated with pathophysiology of multiple myeloma.

Chromosomal rearrangements and copy number variation are frequently observed in cancer cells, including multiple myeloma (MM). Karyotypic abnormalities seen in MM cells correlate with the disease stage and drug responses. Here, we investigate the nuclear arrangement of the 1q21 region; amplification of this region is an important diagnostic and prognostic marker of MM. We examined the lymphoblastoid cell line CD138- ARH-77, multiple myeloma CD138+ MOLP-8 cells, and the CD138+ bone marrow fraction of patients diagnosed with MM. In this experimental system, we observed that gamma-radiation and selected cytostatic drugs such as melphalan and dexamethasone did not significantly alter the nuclear radial arrangement of the 1q21 region and other relevant regions of chromosome 1. Similarly, conserved nuclear radial positioning after cytostatic treatment was observed for the c-myc, TP53, CCND1, and IgH loci. When analyzed Mcl-1, a protein encoded by a gene mapped to the 1q21 region, we found that the variant Mcl1S is highly expressed in multiple myeloma MOLP-8 cells, but not in peripheral blood lymphocytes of healthy donors or lymphoblastoid ARH-77 cells; this is in contrast to the expression pattern of the Mcl-1L variant. On the basis of these observations we suggest that the 1q21 region is an important diagnostic marker of MM, particularly the gene encoding the Mcl-1S variant, which can be easily detected by western analysis.