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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 (Dnmtl, 3a, and 3b) in aging and neoplastically-transformed WI-38 human fetal lung fibroblasts.

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.

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.