Sabita N. Saldanha

Differential maintenance and de novo methylating activity by three DNA methyltransferases in aging and immortalized fibroblasts

Genomic methylation, which influences many cellular processes such as gene expression and chromatin organization, generally declines with cellular senescence although some genes undergo paradoxical hypermethylation during cellular aging and immortalization. To explore potential mechanisms for this process, we analyzed the methylating activity of three DNA methyltransferases (Dnmts) in aging and immortalized WI‐38 fibroblasts. Overall maintenance methylating activity by the Dnmts greatly decreased during cellular senescence. In immortalized WI‐38 cells, maintenance methylating activity was similar to that of normal young cells. Combined de novo methylation activity of the Dnmts initially decreased but later increased as WI‐38 cells aged and was strikingly elevated in immortalized cells. To further elucidate the mechanisms for changes in DNA methylation in aging and immortalized cells, the individual Dnmts were separated and individually assessed for maintenance and de novo methylating activity. We resolved three Dnmt fractions, one of which was the major maintenance methyltransferase, Dnmt1, which declined steadily in activity with cellular senescence and immortalization. However, a more basic Dnmt, which has significant de novo methylating activity, increased markedly in activity in aging and immortalized cells. We have identified this methyltransferase as Dnmt3b which has an important role in neoplastic transformation but its role in cellular senescence and immortalization has not previously been reported. An acidic Dnmt we isolated also had increased de novo methylating activity in senescent and immortalized WI‐38 cells. These studies indicate that reduced genome‐wide methylation in aging cells may be attributed to attenuated Dnmt1 activity but that regional or gene‐localized hypermethylation in aging and immortalized cells may be linked to increased de novo methylation by Dnmts other than the maintenance methyltransferase. J. Cell. Biochem. 84: 324–334, 2002.

Control mechanisms in the regulation of telomerase reverse transcriptase expression in differentiating human teratocarcinoma cells

Telomerase is active in about 90% of cancers and contributes to the immortality of cancer cells by maintaining the lengths of the ends of chromosomes. Undifferentiated embryonic human teratocarcinoma (HT) cells were found to express high levels of hTERT, the catalytic subunit of telomerase, and the hTERT promoter was unmethylated in these cells. Retinoic acid (RA)-induced differentiation led to hTERT gene silencing and increased methylation of the hTERT promoter. Treatment with trichostatin A, a histone deacetylase inhibitor, resulted in hTERT reactivation only in very early differentiating HT cells. After methylation patterns had been established within the hTERT promoter region in late differentiating cells, 5-azacytidine, a common demethylating agent, activated the hTERT gene but trichostatin A had no effect on hTERT transcription. These studies suggest that histone deacetylation is involved in early hTERT gene down-regulation and that DNA methylation may maintain silencing of the hTERT gene in these cells.

Analysis of telomerase activity and detection of its catalytic subunit, hTERT.

The discovery of the enzyme telomerase and its subunits has led to major advances in understanding the mechanisms of cellular proliferation, immortalization, aging, and neoplastic transformation. The expression of telomerase in more than 85% of tumors provides an excellent tool for the diagnosis, prognosis, and treatment of cancer. However, the techniques employed in its detection appear to play a significant role in the interpretation of the results. The telomeric repeat amplification protocol (TRAP assay) has been the standard assay in the detection of telomerase activity and many variations of this technique have been reported. Recent advances in the development of the TRAP assay and the incorporation of techniques that provide a quantitative and qualitative estimate of telomerase activity are assessed in this review. In addition to histological and cytological examination of tissues, distribution patterns of the catalytic subunit of telomerase, hTERT, are frequently used in the prognosis of tumors. The methods involved in the detection of hTERT as a biomarker of cellular transformation are also analyzed.

Epigenetic regulation of human telomerase reverse transcriptase promoter activity during cellular differentiation

The human telomerase reverse transcriptase (TERT) gene is transcriptionally inactivated in most differentiated cells but is reactivated in the majority of cancer cells. To elucidate how TERT is inactivated during differentiation, we applied all‐trans retinoic acid (ATRA) to induce the differentiation of human teratocarcinoma (HT) cells and human acute myeloid leukemia (HL60) cells. We first showed that TERT promoter activity decreased rapidly, which preceded a gradual loss of endogenous telomerase activity following ATRA induction. To elucidate the underlying mechanisms of the reduced TERT promoter activity during differentiation, we performed epigenetic studies on the TERT promoter and found a progressive histone hypoacetylation coupled with a gradual accumulation of methylated cytosines in the TERT promoter. We also observed that the TERT promoter was less methylated in pluripotent HT cells than in multipotent HL60 cells throughout a 12‐day differentiation process. This origin‐dependent epigenetic change was also confirmed in histone acetylation studies, indicating that the TERT promoter was more resistant to deacetylation in HT cells than in HL60 cells. Taken together, our results demonstrate synergistic involvement of DNA methylation and histone deacetylation in the down‐regulation of TERT promoter activity that may be dependent on the origin of the cell types, and they add new insight into the way telomerase activity may be regulated during differentiation.

The Role of Nutraceuticals in Chemoprevention and Chemotherapy and Their Clinical Outcomes

The genesis of cancer is often a slow process and the risk of developing cancer increases with age. Altering a diet that includes consumption of beneficial phytochemicals can influence the balance and availability of dietary chemopreventive agents. In chemopreventive approaches, foods containing chemicals that have anticancer properties can be supplemented in diets to prevent precancerous lesions from occurring. This necessitates further understanding of how phytochemicals can potently maintain healthy cells. Fortunately there is a plethora of plant-based phytochemicals although few of them are well studied in terms of their application as cancer chemopreventive and therapeutic agents. In this analysis we will examine phytochemicals that have strong chemopreventive and therapeutic properties in vitro as well as the design and modification of these bioactive compounds for preclinical and clinical applications. The increasing potential of combinational approaches using more than one bioactive dietary compound in chemoprevention or cancer therapy will also be evaluated. Many novel approaches to cancer prevention are on the horizon, several of which are showing great promise in saving lives in a cost-effective manner.

Epigenetic Regulation of Epidermal Stem Cell Biomarkers and Their Role in Wound Healing.

As an actively renewable tissue, changes in skin architecture are subjected to the regulation of stem cells that maintain the population of cells responsible for the formation of epidermal layers. Stems cells retain their self-renewal property and express biomarkers that are unique to this population. However, differential regulation of the biomarkers can initiate the pathway of terminal cell differentiation. Although, pockets of non-clarity in stem cell maintenance and differentiation in skin still exist, the influence of epigenetics in epidermal stem cell functions and differentiation in skin homeostasis and wound healing is clearly evident. The focus of this review is to discuss the epigenetic regulation of confirmed and probable epidermal stem cell biomarkers in epidermal stratification of normal skin and in diseased states. The role of epigenetics in wound healing, especially in diseased states of diabetes and cancer, will also be conveyed.

Epigenetic Approaches to Cancer Therapy

The dysregulation of genes is in many instances responsible for the initiation and development of cancers of various origins. However advanced analysis of cancerous cell genomes provides insights to possibilities other than mutations that favor a tumorigenic phenotype. Epigenomic studies have shown that gene expression can be regulated independently of the DNA sequence by modifying specific bases or chromatin residues and the changes are central to tumorigenesis. The epigenetic marks are heritable and reversible and consequently enzymes that regulate these changes are potential targets for cancer therapy. Inhibitors of epigenetic enzymes can restore the gene expression imbalances set by the epigenetic marks, restoring pathways that initiate cellular differentiation and apoptosis necessary for tumor inhibition. Here we review current approaches to targeting enzymes such as the DNA methyltransferases (DNMTs), histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), and histone demethylases (HDMTs) that regulate epigenetic pathways through drug-based inhibitors.