Arnab Pal

Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia

Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine‐phosphate‐guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O2 for 6 weeks and compared with cells cultured in 21% O2 for the same duration. Real‐time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter‐alu PCR profile of DNA from cells cultured under 6‐week hypoxia, its 4‐week revert back to normoxia and 6‐week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells.

Fluorescence acquisition during hybridization phase in quantitative real-time PCR improves specificity and signal-to-noise ratio.

Quantitative real-time PCR (qPCR) is a standard method used for quantification of specific gene expression. This utilizes either dsDNA binding dyes or probe based chemistry. While dsDNA binding dyes have the advantage of low cost and flexibility, fluorescence due to primer dimers also interferes with the fluorescence of the specific product. Sometimes it is difficult, if not impossible, to standardize conditions and redesign primers in such a way that only specific fluorescence of the products of test and reference genes are acquired. Normally, the fluorescence acquisition in qPCR using dsDNA binding dyes is done during the melting phase of the PCR at a temperature between the melting points of primer dimers and the specific product. We have modified the protocol to acquire fluorescence during the hybridization phase. This significantly increased the signal-to-noise ratio and enabled the use of dsDNA binding dyes for mRNA quantification in situations where it was not possible when measurement was done in the melting phase. We have demonstrated it for three mRNAs, E6, E7, and DNMT1 with beta-actin as the reference gene, and for two miRNAs. This modification broadens the scope of qPCR using dsDNA binding dyes.

CpG Hypermethylation of the C-myc Promoter by dsRNA Results in Growth Suppression

Deregulation of the c-myc proto-oncogene plays an important role in carcinogenesis. It is, therefore, commonly found to be overexpressed in various types of tumors. Downregulation of c-myc expression assumes great importance in tumor therapy because of its ability to promote and maintain cancer stem cells. Apart from post-transcriptional gene silencing (PTGS), siRNAs have also been shown to cause transcriptional gene silencing (TGS) through epigenetic modifications of a gene locus. This approach can potentially be used to silence genes for longer periods and at a much lesser dosage than PTGS. In this study, we have examined the effect of transfection of a novel siRNA directed against a CpG island encompassing the CT-I(2) region in the P2 promoter of c-myc in U87MG and other cell lines. Transient transfection with this siRNA resulted in c-myc promoter CpG hypermethylation and decreased expression of c-myc (both mRNA and protein) and its downstream targets. A decrease was also observed in the expression of some stemness markers (oct-4 and nanog). Stable transfection also confirmed the promoter CpG hypermethylation and reduced c-myc expression along with reduced cell proliferation and an increase in apoptosis and senescence. A significant decrease in c-myc levels was also observed in three other cancer cell lines after transient transfection under similar conditions. Thus this novel siRNA has the capability of becoming an effective therapeutic tool in malignancies with overexpression of c-myc and may be of particular use in the eradication of recalcitrant cancer stem cells.