Lucy G. Andrews

Epigenetic and Genetic Mechanisms Contribute to Telomerase Inhibition by EGCG

The ends of human chromosomes are protected from the degradation associated with cell division by 15–20 kb long segments of hexameric repeats of 5′‐TTAGGG‐3′ termed telomeres. In normal cells telomeres lose up to 300 bp of DNA per cell division that ultimately leads to senescence; however, most cancer cells bypass this lifespan restriction through the expression of telomerase. hTERT, the catalytic subunit essential for the proper function of telomerase, has been shown to be expressed in approximately 90% of all cancers. In this study we investigated the hTERT inhibiting effects of (−)‐epigallocatechin‐3‐gallate (EGCG), the major polyphenol found in green tea catechins, in MCF‐7 breast cancers cells and HL60 promyelocytic leukemia cells. Exposure to EGCG reduced cellular proliferation and induced apoptosis in both MCF‐7 and HL60 cells in vitro, although hTERT mRNA expression was decreased only in MCF‐7 cells when treated with EGCG. Furthermore, down‐regulation of hTERT gene expression in MCF‐7 cells appeared to be largely due to epigenetic alterations. Treatment of MCF‐7 cells with EGCG resulted in a time‐dependent decrease in hTERT promoter methylation and ablated histone H3 Lys9 acetylation. In conjunction with demethylation, further analysis showed an increase in hTERT repressor E2F‐1 binding at the promoter. From these findings, we propose that EGCG is effective in causing cell death in both MCF‐7 and HL60 cancer cell lines and may work through different pathways involving both anti‐oxidant effects and epigenetic modulation. J. Cell. Biochem. 103: 509–519, 2008.

Genistein depletes telomerase activity through cross-talk between genetic and epigenetic mechanisms

Genistein, a natural isoflavone found in soybean products, has been reported to down‐regulate telomerase activity and that this prevents cancer and contributes to the apoptosis of cancer cells. However, the precise molecular mechanism by which genistein represses telomerase is not clear. Here, we show that genistein inhibits the transcription of hTERT (human telomerase reverse transcriptase), the catalytic subunit of the human telomerase enzyme, in breast MCF10AT benign cells and MCF‐7 cancer cells in a time‐ and dose‐dependent manner. Three major DNA methyltransferases (DNMT1, 3a and 3b) were also decreased in genistein‐treated breast cancer cells suggesting that genistein may repress hTERT by impacting epigenetic pathways. Sequential depletion of the hTERT promoter revealed that the hTERT core promoter region is responsible for the genistein‐induced repression of hTERT transcription. Using a newly developed technique of chromatin immunoprecipitation (ChIP)‐related bifulfite sequencing analysis, we found an increased binding of E2F‐1 to the hTERT promoter is due to the site‐specific hypomethylation of the E2F‐1 recognition site. In addition, we found that genistein can remodel chromatin structures of the hTERT promoter by increasing trimethyl‐H3K9 but decreasing dimethyl‐H3K4 in the hTERT promoter. The repression of hTERT was enhanced by combination with genistein and the DNMT inhibitor, 5‐aza‐2′‐deoxycytidine (5‐aza‐dCyd). These findings collectively show that genistein is working, at least in part, through epigenetic mechanisms of telomerase inhibition in breast benign and cancer cells and may facilitate approaches to breast cancer prevention and treatment using an epigenetic modulator combined with genistein.

Aging, cancer and nutrition: the DNA methylation connection.

Cancer and aging are two coupled developmental processes as reflected by the higher incidence of cancer in the elderly human population group. Genetic mutations accumulate in somatic cells with age, which may explain in part the association of age with cancer. Epigenetic mechanisms are also frequently involved in controlling gene functions during development and tumorigenesis. A common molecular feature associated with both aging and tumorigenesis is global hypomethylation of the genomic DNA. The contributing mechanisms underlying this hypomethylation are not yet well understood. Epigenetic investigation of cancer and aging has recently emerged as a fruitful area of study and has added exciting insights into some of the mysteries surrounding aging and cancer. Recent studies have also shown that dietary factors can modulate DNA methylation and thereby contribute to aging and tumorigenesis. Thus, DNA methylation provides an important common link between aging, cancer and nutrition.

Transcriptional control of the DNA methyltransferases is altered in aging and neoplastically-transformed human fibroblasts

Although it has been known for quite some time that genomic methylation is significantly altered in aging and neoplastic tissues and cells, the underlying mechanisms responsible for these alterations are not yet known. Since DNA methylation affects many different cellular processes including, most significantly, gene expression, elucidation of the basis for aberrations in DNA methylation in aging and cancer is of high priority. To address this problem, we sought to analyze changes in gene expression, protein production and enzyme activity of the three major DNA methyltransferases (Dnmt1, 3a, and 3b) in aging and neoplastically-transformed WI-38 human fetal lung fibroblasts. We have found that the gene expression of each of the three Dnmts parallels changes in protein production and enzyme activity of the Dnmts not only in aging cells, but also in WI-38 fibroblasts induced to undergo neoplastic transformation using defined genetic elements. These findings strongly implicate change in gene expression as an underlying mechanism in the altered genomic methylation of these cells. Striking changes in the gene expression of the Dnmts were observed in aging cells with the mRNA of Dnmt1 becoming reduced while the mRNA of Dnmt3b increased steadily in aging cells consistent with our observations in protein production and activity of these enzymes. Surprisingly, Dnmt3a actually decreased in gene expression in aging cells. We therefore propose that the transcriptional control of Dnmt1, the predominant maintenance methyltransferase, is significantly suppressed in aging cells and contributes to the reduced genomic methylation of these cells. The paradoxical sporadic gene hypermethylation in aging cells appears to be related to transcriptional up-regulation of the Dnmt3b gene. In addition, we sought to explore the coordinated changes in gene expression, protein production, and enzyme activity of these Dnmts in early cellular transformation. In these cells, the gene expression of all the three major Dnmts were up-regulated followed by marked increases in Dnmt protein and enzyme activity. These results therefore collectively indicate that changes in transcriptional control of the Dnmts are the likely cause for the known alterations in DNA methylation in aging cells and in cells undergoing tumorigenesis. They also show that changes in transcription of Dnmt1 and Dnmt3b are probably most important in affecting the generalized hypomethylation and specific hypermethylation seen in aging cells while gene expression of all the Dnmts is significantly increased in cancer cells. These findings should have broad implications in elucidating the underlying causes of changes in DNA methylation in aging and tumorigenesis and point to variations in gene expression of the individual Dnmts as a likely mechanism involved in these processes.

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.

Induction of endogenous telomerase (hTERT) by c-Myc in WI-38 fibroblasts transformed with specific genetic elements

Elucidation of the mechanisms governing expression of the human telomerase reverse transcriptase (hTERT) is important for understanding cancer pathogenesis. Approximately 90% of tumors express hTERT, the major catalytic component of telomerase. Activation of telomerase is an early event, and high levels of this activity correlate with poor prognosis. Recent studies have shown that the transcription factors c-Myc and Mad1 activate and repress hTERT, respectively. It is not clear how these transcription factors compete for the same recognition sequence in the hTERT core promoter region. Studies have shown that the combined expression of SV40 large T antigen (T-Ag), hTERT, and H-Ras is able to transform human cells. In this study, we used a distinct human cell type, WI-38 fetal lung fibroblasts used extensively for senescence studies. We transduced cells with amphotropic retroviral constructs containing SV40 T antigen, hTERT, and activated H-ras. Transduced cells exhibited anchorage independence in soft agar and expressed increased levels of c-Myc and endogenous hTERT. These effects were observed by 25 population doublings (PDs) following the establishment of the neoplastic cell line. During the process of transformation, we observed a switch from Mad1/Max to c-Myc/Max binding to oligonucleotide sequences containing the hTERT promoter distal and proximal E-boxes. c-Myc can bind specifically to the hTERT promoter in vitro, indicating that c-Myc expression in tumors may account for the increased expression of hTERT observed in vivo. These findings indicate that the widely used model system of WI-38 fibroblasts can be employed for transformation studies using defined genetic elements and that the endogenous hTERT and c-Myc are induced in these cells during early tumorigenesis. Such studies should have important implications in the mechanisms of hTERT and c-Myc induction in the beginning stages of tumorigenesis and facilitate extension of these studies to novel models of tumorigenesis in cellular senescence.

Telomerase Inhibition in Cancer Therapeutics: Molecular-Based Approaches

Current standard cancer therapies (chemotherapy and radiation) often cause serious adverse off-target effects. Drug design strategies are therefore being developed that will more precisely target cancer cells for destruction while leaving surrounding normal cells relatively unaffected. Telomerase, widely expressed in most human cancers but almost undetectable in normal somatic cells, provides an exciting drug target. This review focuses on recent pharmacogenomic approaches to telomerase inhibition. Antisense oligonucleotides, RNA interference, ribozymes, mutant expression, and the exploitation of differential telomerase expression as a strategy for targeted oncolysis are discussed here in the context of cancer therapeutics. Reports of synergism between telomerase inhibitors and traditional cancer therapeutic agents are also analyzed.