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.

Retinoid-induced histone deacetylation inhibits telomerase activity in estrogen receptor-negative breast cancer cells.

BACKGROUND: Multiple mechanisms regulate cancer-associated telomerase activity at the level of human telomerase reverse transcriptase (hTERT) transcription which may serve as novel targets for anticancer approaches. MATERIALS AND METHODS: The effects of prolonged all-trans retinoic acid (ATRA) exposure on hTERT regulation in estrogen receptor-negative SK-BR-3 breast cancer cells were examined. RESULTS: ATRA had a profound effect on the morphology and proliferation rate of the SK-BR-3 cells. ATRA also hindered the ability of these cancer cells to grow independently, rendering them more like normal somatic cells. The effect of ATRA on the decrease of telomerase activity was found to be associated with a rapid decrease in histone H3-lysine 9 acetylation (H3-K9-Ac) of the hTERT promoter. Extended-exposure to ATRA in these cells also caused the initiation of a putative compensatory mechanism, counteracting the induced surge in apoptosis. CONCLUSION: A rapid decrease of H3-K9 acetylation at the hTERT promoter could be an important mechanism by which ATRA shuts down telomerase activity and mediates its antitumor effects in estrogen receptor-negative breast cancer cells.

Differential expression of epigenetic modulators during human embryonic stem cell differentiation.

Although the progression of aging and the diseases associated with it are extensively studied, little is known about the initiation of the aging process. Telomerase is down-regulated early in embryonic differentiation, thereby contributing to telomeric attrition and aging. The mechanisms underlying this inhibition remain elusive, but epigenetic studies in differentiating human embryonic stem (hES) cells could give clues about how and when DNA methylation and histone deacetylation work together to contribute to the inactivation of hTERT, the catalytic subunit of telomerase, at the onset of the aging process. We have confirmed the differentiation status of cultured hES colonies with morphological assessment and immunohistochemical stainings for pluripotent stem cells. In hES cells with varying degrees of differentiation, we have shown a stronger association between hES differentiation and expression of the epigenetic regulators DNMT3A and DNMT3B than between genetic modulators of differentiation such as c-MYC. We also propose a new model system for analyses of stem cell regions, which are differentially down-regulating the expression of hTERT and the actions of epigenetic modulators such as the DNMTs and histone methyltransferases.

The Novel Retinoid, 9cUAB30, Inhibits Telomerase and Induces Apoptosis in HL60 Cells.

Telomerase, a ribonucleoprotein important to neoplastic immortality, is up-regulated in approximately 85% of cancers, including leukemias. In this study, 9cUAB30, a novel retinoic acid, resulted in differentiation of HL60 leukemia cells as indicated by morphologic changes characteristic of granulocytes. It also caused a down-regulation of hTERT gene expression and a decrease in telomerase activity. Telomerase inhibition was followed by loss of proliferative capacity, induction of apoptosis, and partial differentiation. These findings demonstrate the effectiveness of 9cUAB30 at inhibiting telomerase activity by down-regulating hTERT gene expression in human leukemic cells.

Epigenetic regulation of telomerase in retinoid-induced differentiation of human leukemia cells.

Changes in the promoter methylation of hTERT, the gene that encodes telomerase, a ribonucleoprotein responsible for replacing telomeric repeats, have been demonstrated in differentiating cells where hTERT is inhibited, suggesting epigenetic regulation of hTERT. All-trans retinoic acid (ATRA) induces differentiation in human leukemia cells and has had significant clinical success treating promyelocytic leukemia in what is termed 'differentiation therapy'. It is thought that the inhibition of telomerase is a target of retinoids and is closely tied to the differentiated phenotype. This study demonstrates the epigenetic changes associated with ATRA-induced inhibition of telomerase activity, including the hypoacetylation and hypermethylation of the hTERT promoter. Further, we have found changes in the differential expression of the three DNA methyltransferases during ATRA-induced differentiation of HL60 human leukemia cells. These results suggest that alteration of DNA methylation may play a role in the activation of telomerase in cancer cells and that epigenetic mechanisms may represent a target for differentiation therapy mechanisms. We propose that epigenetic changes in the hTERT promoter represent a stable locking mechanism in the retionoid-induced suppression of telomerase activity.

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.

Aging cell culture: methods and observations.

Culturing and subcultivation of normal human diploid fibroblasts have advanced our understanding of the molecular events involved in aging. This progress is leading to the development of therapies that slow or ablate the adverse physiological and pathological changes associated with aging. It has been established that normal human diploid fibroblasts can proliferate in culture for only finite periods of time. Hayflick and Moorhead and others have described numerous types of cells, ranging from fetal to adult, that were incapable of indefinite proliferation. There are many ways to study aging in vitro, and this chapter summarizes some laboratory procedures.

A method to detect DNA methyltransferase I gene transcription in vitro in aging systems.

Epigenetic alterations of DNA play key roles in determining gene structure and expression. Methylation of the 5-position of cytosine is thought to be the most common modification of the genome in mammals. Studies have generally shown that hypermethylation in gene regulatory regions is associated with inactivation and reduced transcription and that alteration in established methylation patterns during development can affect embryonic viability. Changes in methylation have also been associated with aging and cellular senescence as well as tumorogenesis. DNA methyltransferase 1 (DNMT1) is thought to play an important role in maintaining already established methylation patterns during DNA replication and catalyzes the transfer of a methyl moiety from S-adenosyl-L-methionine (SAM) to the 5-position of cytosines in the CpG dinucleotide. Several studies illustrate changes in activity and transcription of DNMT1 during aging and here we show a comprehensive method of detection of DNMT1 mRNA transcription from senescing cells in culture.

A method to study the expression of DNA methyltransferases in aging systems in vitro.

The methylation of CpG dinucleotides located in key protein binding sites within gene regulatory regions often leads to gene silencing. A mechanism of aging is proposed whereby an accumulation of methylation at gene regulatory sites contributes to cellular senescence. DNA methyltransferases (DNMTs) are enzymes that catalyze the transfer of a methyl moiety from S-adenosyl-L-methionine (SAM) to the cytosine of a CpG dinucleotide and are responsible for establishing and maintaining methylation patterns in the genome. It is important to study not only transcription of the DNMTs, but also their protein expression because studies illustrate that it is possible for the enzymes to undergo posttranslational physical changes in response to stimulation even though gene transcription remains unchanged. Here, we discuss an in vitro method to study protein expression of DNMTs in aging systems.

The low-toxicity 9-cis UAB30 novel retinoid down-regulates the DNA methyltransferases and has anti-telomerase activity in human breast cancer cells.

Retinoic acids and their derivatives potentiate anti-cancer effects in breast cancer cells. The aberrant expression of telomerase is critical to the continued proliferation of most cancer cells. Thus, telomerase is an attractive target for chemoprevention and treatment of breast cancer. 9cUAB30 is a novel synthetic retinoid X receptor-selective retinoic acid (RA) that effectively reduces the tumorigenic phenotype in mouse breast carcinoma with lower toxic effects than natural retinoid treatments. We have assessed 9cUAB30 retinoic acid treatment of human breast cancer cells to determine the potential of this drug as an effective telomerase inhibitor and its application to cancer therapy. 9cUAB30 was found to decrease DNA methyltransferase and telomerase expression in MDA-MB-361, T-47D, and MCF-7 human breast cancer cells and to inhibit the proliferation of these cells. This low-toxicity retinoid also reduced colony formation in soft agar assays in each of these cell types. Combination treatments of 9cUAB30 and all-trans RA proved to be synergistically more effective than either RA alone, further suggesting a possible general epigenetic mechanism that contributes to the anti-telomerase activity of the retinoids. Therefore, the novel retinoid, 9cUAB30, is effective in inhibiting the growth of human breast cancer cells, its anti-cancer effects appear to be related to telomerase inhibition and the mechanism for this process could be mediated through epigenetic modifications.

Evidence of extra-telomeric effects of hTERT and its regulation involving a feedback loop.

The human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the enzyme telomerase which is responsible for telomeric maintenance and extension. Using RNA interference to knock down hTERT mRNA expression, we provide evidence that hTERT exerts extra-telomeric effects on the cell cycle and on its own regulatory proteins, specifically: p53 and p21. We tested our hypothesis that hTERT regulates its own expression through effects on upstream regulatory genes using transformed human embryonic kidney (HEK 293) cells, p53 and p16(INK4a) null human ovarian cancer SKOV-3 cells, and p53-null MDA-MB-157 human mammary cancer cells. In HEK 293 cells, hTERT knockdown resulted in elevated p53 and p21 transcription and a decrease in cellular proliferation. Similar results were observed in the MDA-MB-157 cell line where p21 was upregulated, correlating with cell growth inhibition. In contrast, we observed a decrease in expression of p21 in SKOV-3 cells with hTERT knockdown and cell growth appeared to be unaffected. These findings suggest that hTERT may be involved in a feedback loop system, thereby playing a role in its own regulation.

Methods of telomerase inhibition.

Telomerase is central to cellular immortality and is a key component of most cancer cells although this enzyme is rarely expressed to significant levels in normal cells. Therefore, the inhibition of telomerase has garnered considerable attention as a possible anticancer approach. Many of the methods applied to telomerase inhibition focus on either of the two major components of the ribonucleoprotein holoenzyme, that is, the telomerase reverse transcriptase (TERT) catalytic subunit or the telomerase RNA (TR) component. Other protocols have been developed to target the proteins, such as tankyrase, that are associated with telomerase at the ends of chromosomes. This chapter summarizes some of these recent advances in telomerase inhibition.

hTERT knockdown in human embryonic kidney cells using double-stranded RNA.

The method of RNA interference (RNAi) is an easy means of knocking down a gene without having to generate knockout mutants, which may prove to be difficult and time consuming. RNAi is a naturally occurring process that involves targeting the mRNA of a gene by introducing RNAs that are complementary to the target mRNA. The foreign RNAs activate an endogenous enzyme, DICER, which degrades the target mRNA. There are many ways of eliciting the RNAi response in a cell. In this chapter, we describe the use of double-stranded RNA (dsRNA) to knockdown human telomerase reverse transcriptase (hTERT), the gene that codes for the catalytic subunit of the human telomerase enzyme. dsRNA can be used to generate the RNAi response in cells of embryonic origin, such as human embryonic kidney (HEK) cells. The RNAi effect is transient because the dsRNA eventually gets degraded in the cells, and it is useful to study the short-term effects of a gene knockdown.

RNA interference using a plasmid construct expressing short-hairpin RNA.

RNA interference (RNAi) is one of the most commonly used procedures for gene targeting in today's cutting edge technology and has great potential for use in clinical therapy. Using a plasmid construct that exogenously expresses short-hairpin RNAs (shRNAs) targeting a desired gene transcript not only helps to study the downstream effects of a gene product but also offers an alternative to viral vectors for gene therapy. Using a plasmid vector to knockdown a gene allows for long-term and permanent gene knockdown, without the need to generate knockout genotypes. Here, we detail the methodology for constructing a plasmid targeting the human telomerase reverse transcriptase (hTERT) gene through RNAi using the Ambion pSilencer system.

Retrovirus-mediated RNA interference. Targeting hTERT through stable expression of short-hairpin RNA.

RNA interference (RNAi) has recently emerged as a reliable tool for studying the effects of knocking down or ablating the expression of specific genes. It is hoped that progress made in the laboratory toward in vitro down regulation of gene expression may be carried over into the clinic for treatment of diseases in which the expression of a specific gene is associated with initiation or progression of that disease. Such is the case with telomerase, an exciting drug target that has been the focus of numerous investigations with a wide variety of inhibitors. This chapter describes the use of retrovirally introduced short-hairpin RNA as an effector of stable, long-term RNAi in human cells.

The impact of metabolism on DNA methylation.

Methylation of genomic cytosines is one of the best characterized epigenetic mechanisms, and investigation of its relationship with other biochemical pathways represents a critical stage in the elucidation of biological information processing. The field also has immense potential for the development of medical treatments for any number of conditions ranging from aging to neurological disorders. The DNA methylation status of genes is responsible for many heritable traits and varies more or less independently of the genetic code. This variation is often a result of cellular environmental factors including metabolism. A key metabolite in this regard is homocysteine. Knowledge of homocysteine metabolism continues to be amassed, yet the part played by aberrant DNA methylation in homocysteine-related pathologies is often, at best, conjectural. In this analysis, we will review recent insights and attempt to speculate meaningfully concerning the dynamics of the methionine cycle in relation to DNA methylation and disease.

Epigenetic control of telomerase and modes of telomere maintenance in aging and abnormal systems.

Epigenetic control provides a mechanism for the reversible silencing of telomerase expression that occurs as a natural consequence of differentiation. Significant overlap between indirect telomerase regulation pathways and cell cycle checkpoint pathways exist, suggesting that these discrete genetic elements (namely, p21, p53, and hTERT) synergistically cooperate to inhibit tumorigenesis. Mutations in these pathways have been known to contribute to cancer formation. However, the incorporation of epigenetic regulatory mechanisms provides another line of defense against these negative occurrences. These proteins are also implicated in the process of senescence, caused in eukaryotic cell lines by telomere shortening. Although the debate continues, there is significant evidence to classify the process of cellular senescence as an in vitro model for human aging. In addition, the study of stem cells gives information about the down-regulation of hTERT in the aging process. Diseases such as Werner S syndrome, ATM (ataxia telangiectasia mutated kinase), DKC (dyskeratosis congenita), and atherosclerosis have been linked to aberrant telomerase expression and other aging-related tissue malfunctions could be related to the presence of senescent cells changing the cellular microenvironment. Therefore, restoring telomerase activity as a putative therapeutic strategy necessitates further study to elucidate the intricacies linking genetic and epigenetic modulations of hTERT.

Genetic and epigenetic modulation of telomerase activity in development and disease.

Telomerase activity is one of the most important factors that have been linked to multiple developmental processes, including cell proliferation, differentiation, aging and senescence. Dysregulation of telomerase has often been found in developmental abnormalities, such as cancer, loss of function in the hematopoietic system, and low success rate of somatic cloning. A comprehensive network of transcription factors has been shown to be involved in the genetic control of telomerase expression and activity. Epigenetic mechanisms have recently been shown to provide an additional level of regulation, and may be responsible for the diverse expression status of telomerase that is manifested in a tissue and cell-type-dependent manner. This article summarizes the recent developments in the field of telomerase research with a focus on the coregulation of the telomerase gene by both genetic and epigenetic pathways. Developmental consequences of aberrant telomerase activity will also be summarized and discussed.

Telomerase inhibition by retinoids precedes cytodifferentiation of leukemia cells and may contribute to terminal differentiation.

Human promyelocytic leukemia HL60 cells display high telomerase activity, a phenotype related to their immortal status. All-trans retinoic acid (ATRA) is a clinically effective cytodifferentiating agent. To understand the mechanism underlying ATRA-induced cytodifferentiation, we did a kinetic analysis of the role of ATRA in inhibiting telomerase in HL60 cells. Our studies indicate that telomerase inhibition by ATRA occurred relatively early after treatment of HL60 cells due to a rapid decrease in hTERT gene expression. More importantly, however, we found through monitoring the expression of CD11b, a marker for granulocytic differentiation of HL60 cells, that down-regulation of telomerase preceded the differentiation of HL60 cells. These observations suggest that the hTERT gene may be a primary target of ATRA regulation of cellular differentiation and the antileukemia activity of ATRA may be mediated by its ability to induce the differentiation of the promyelocytic leukemia cells through down-regulation of the hTERT gene.

Profiling DNA methylation by bisulfite genomic sequencing: problems and solutions.

The surge of interest in DNA methylation during the last two decades has triggered an urgent need for an effective method to detect the methylation status of the cytosines in the genome. Bisulfite genomic sequencing is the most attractive choice so far for many laboratories. Various protocols have been established, but difficulties are often encountered, particularly by individuals who have limited experience in this field. This analysis presents a simple protocol that has consistently worked well in our laboratory. Discussions of potential technical problems and corresponding solutions are also included to facilitate the reproducibility of this protocol.