Promoter hypermethylation analysis in myelodysplastic syndromes: diagnostic & prognostic implication.

BACKGROUND & OBJECTIVE: Myelodysplastic syndromes (MDS) are a heterogenous group of haematopoietic stem cell disorders that are multifactorial in their aetiology. Unique genetic alterations in combinations or in isolation account for a small fraction of MDS suggesting the epigenetic hypermethylation as a possible leading cause for MDS and its transformation to acute myelocytic leukaemia (AML). Therefore, in this study, promoter hypermethylation status of key cell cycle regulators was assessed as markers in MDS patients and association of hypermethylation with clinical progression of disease was also studied. METHODS: Promoter hypermethylation analysis of five tumour associated genes namely p16, p15, MGMT, hMLH1 and E-cadherin were done for 41 MDS patient samples with its various subtype. The hypermethylation analysis was done by using semi-nested multiplex PCR. RESULTS: Eighty per cent of (33/41) of the MDS samples were found to be methylated in any one of the four genes (p16, p15, MGMT and E-cadherin). The p15 methylation was found to be the most frequent 61 per cent (25/41), E-cadherin was methylated in 39 per cent (16/41) and p16 in 37 per cent (15/41) of the cases. MGMT gene showed a low 5 per cent (2/41) methylation whereas hMLH1 gene was not methylated in any one of the samples analysed. INTERPRETATION & CONCLUSION: Differential rate of methylation of the four genes (p16, p15, MGMT and E-cadherin) was observed in MDS samples. All the samples analysed showed the absence of a methylator phenotype in MDS. The methylation frequency of all these genes increased with the clinical severity of the MDS subtypes. Therefore, hypermethylation may be used as a diagnostic and prognostic tool in ascertaining the clinical severity of MDS.

Methylation of E-cadherin and hMLH1 genes in Indian sporadic breast carcinomas.

Hypermethylation of promoter regions leading to inactivation of tumor suppressor genes is a common event in the progression of several tumor types. We have employed a novel restriction digestion based multiplex PCR assay to analyse the methylation status of promoter regions of tumor suppressor genes (p16, hMLH1, MGMT and E-cadherin) in sporadic breast carcinomas of Indian women. The present results indicated the absence of hypermethylation in promoter region of p16 and MGMT genes. However, 6 of the 19 (31.6%) sporadic breast carcinomas showed hypermethylation in the promoters of two of the genes analysed; three in hMLH1 and another three in E-cad. Since our earlier studies have shown lack of genetic alterations such as missense mutations and deletions in the tumor associated genes-p16, ras and p14ARF in sporadic breast tumors, the epigenetic alterations of the two genes reported in the present study could be of interest and might be among the events in the genesis/progression of sporadic breast carcinomas.

Butachlor is cytotoxic and clastogenic and induces apoptosis in mammalian cells.

The ability of butachlor to induce cytotoxicity, clastogenicity and DNA damage was assessed using Chinese hamster ovary cells (CHO), Swiss mouse embryo fibroblasts (MEF) and human peripheral blood lymphocytes. A dose and time dependent loss of viability was evident upon treatment of CHO cells with butachlor. Cell killing to an extent of 50% was observed when cells were treated with 16.2 micrograms/ml of butachlor for 24 hr or with 11.5 micrograms/ml for 48 hr. The herbicide induced micronuclei significantly in cultured lymphocytes at 24 and 48 hr of treatment suggesting that it is clastogenic. To understand the mechanism of cell death caused by butachlor, its effect on DNA strand breaks was studied in MEF. A concomitant decrease in cell viability was observed with increase in DNA strand breaks. Agarose gel electrophoresis of DNA from herbicide treated CHO cells and cytochemical staining indicate the induction of apoptosis by butachlor.

Secreted proteins of normal and myc-ras oncogene transformed rat embryo fibroblasts.

Quiescent cultures of rat embryo fibroblasts synthesize and secrete several proteins in response to mitogenic stimulation. Two of these proteins have been characterized in this study and the effect of oncogenic transformation on these proteins was monitored. A serum induced 48,000 protein was shown to be related to plasminogen activator inhibitor while another serum-induced protein of Mr 45,000 was found to be an inhibitor of DNA synthesis. Transformation of rat embryo fibroblasts with oncogenes myc and ras resulted in drastic reduction in the level of these proteins. The reduced levels of protease inhibitor may be responsible for the loss of anchorage dependence of the transformed cells. The DNA synthesis inhibitor protein may act as a negative growth regulator and reduced levels of this protein in myc-ras transformed cells may accelerate the proliferation of these cells.

Purification and characterization of a DNA synthesis inhibitor protein from mouse embryo fibroblasts.

Α DNA synthesis inhibitor protein was purified from the conditioned medium of cycloheximide treated mouse embryo fibroblasts. This protein has a molecular weight of 45,000 as determined by gel filtration and Polyacrylamide gel electrophoresis. The levels of the [35S] methionine la belled 45 kDa protein in the medium and matrix were monitored across two cell cycles in synchronized cultures. The 45 kDa protein was present in higher levels in the medium of non-S-phase cells depicting a peak between the two S- phases. The DNA synthesis inhibitor protein was immunologically related to a chicken DNA-binding protein which showed similar cell cycle specific variations at the intracellular level. The purified 45 kDa protein inhibited DNA synthesis in murine and human cells. In mouse embryo fibroblasts, the DNA synthesis was inhibited to an extent of 86% by 0·25 μg/ml of the inhibitor, while higher amounts of the inhibitor were required to arrest DNA synthesis in human skin fibroblasts: in these cells, 4 μg/ml of the inhibitor inhibited DNA synthesis to an extent of 50%. The high levels of the 45 kDa protein in the medium of non-S phase cells and its DNA synthesis inhibitory potential suggest that this protein may be involved in the regulation of DNA synthesis during the cell cycle.

Translocation of plasminogen activator inhibitor-1 during serum stimulated growth of mouse embryo fibroblasts.

Serum-stimulated mouse embryo fibroblasts specifically secrete two proteins of molecular weights 48,000 and 26,000. The 48 kDa protein showed affinity to concanavalin A and was precipitated by antibody to plasminogen activator inhibitor. Immunoflowcytometry using anti plasminogen activator inhibitor-1 serum indicate the presence of the 48 kDa protein in quiescent cells; this protein was virtually absent in serum-stimulated cells. The presence of the plasminogen activator inhibitor-1 related protein in quiescent cells and its absence in serum-stimulated cells in combination with the observation on the absence of this protein, in the medium of quiescent cells and its presence in the medium of stimulated cells indicate that the 48 kDa protein was transferred from the cells into the medium upon serum-stimulation. The serum-mediated transfer of plasminogen activator inhibitor-1 from the cells into the medium was inhibited by actinomycin-D suggesting that the transfer process required actinomycin-D sensitive events. Treatment of pre-labelled quiescent cells with medium containing 20% fetal calf serum resulted in the gradual transfer of the labelled 48 kDa protein to the extra cellular matrix. These studies indicate that exposure of quiescent cells to fetal calf serum results in the transfer of plasminogen activator inhibitor-1 from the cells to the growth medium via extracellular matrix. The translocation of the protease inhibitor from the cells to the matrix and medium may enable the cellular and possibly the membrane proteases to act on growth factors or their receptors thereby initiating the mitogenic response.

Secreted proteins of quiescent, serum-stimulated and over-confluent mouse embryo fibroblasts.

Quiescent and proliferating cultures of Swiss mouse embryo fibroblasts were pulse labelled with [14C]-amino acids and the newly synthesized proteins that were secreted into the medium were resolved by electrophoresis on Polyacrylafde gradient gels. Conditioned media obtained from quiescent cultures that were stimulated to grow by the addition of 20% fetal calf serum showed the presence of two unique polypeptides of molecular weights 48000 and 26000. A polypeptide of molecular weight 45000 was present in increased amounts in serum-stimulated cells than in quiescent cells. This protein was also superinduced in quiescent cells by cycloheximide treatment. Mouse embryo fibroblasts grown under over-crowded conditions secreted two proteins of molecular weights 35000 and 11000. The 35 Κ polypeptide was shown to be related to the major excreted protein of transformed cells, since it was immunoprecipitated by an antiserum to major excreted protein. These results indicate that the 48 Κ and 26 Κ proteins may be proliferation specific proteins, while the 35 Κ protein present in the conditioned media of over-confluent cells may be a marker of morphological transformation.

DNA binding proteins of rat thigh muscle: Purification and characterization of an endonuclease.

Two major DNA binding proteins of molecular weights 34,000 and 38,000 have been identified in the 30,000 g supernatant (S-30) fraction of rat thigh muscle extracts. The presence of 38 KD DNA binding protein in the muscle S-30 could be demonstrated only if Triton X-100 treated extracts were used for Afinity chromatography suggesting that this protein may be a membrane associated DNA binding protein. The 38 KD DNA binding protein differed from the 34 KD DNA binding protein also in its chromatographic behaviour in DE-52 columns in which the 38 KD protein was retained, while the 34 KD protein came out in the flow-through in an electrophoretically pure form. The 34 KD DNA binding protein can also be purified by precipitation with MgCl2. Incubation of 0·15 Μ NaCl eluates (containing the 38 KD and/or 34 KD DNA binding protein) in the presence of 100 mM Mg2+ resulted in the specific precipitation of the 34 KD protein. Prolonged incubation (30 days) of the 0·15 Μ NaCl eluates containing the two DNA binding proteins at 4°C led to the preferential degradation of the 34 KD DNA binding protein. Nitrocellulose filter binding assays indicated selective binding of purified 34 KD protein to ss DNA. Purified 34 KD DNA binding protein cleaved pBR 322 supercoiled DNA, and electrophoresis of the cleavage products in agarose gels revealed a major DNA band corresponding to the circular form of DNA.