Telomerase-negative Human Cells Research Articles

Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes.

The human telomerase reverse transcriptase (hTERT) gene is repressed in most somatic cells, whereas the expression of the mouse mTert gene is widely detected. To understand the mechanisms of this human-specific repression, we constructed bacterial artificial chromosome (BAC) reporters using human and mouse genomic DNAs encompassing the TERT genes and neighboring loci. Upon chromosomal integration, the hTERT, but not the mTert, reporter was stringently repressed in telomerase-negative human cells in a histone deacetylase (HDAC)-dependent manner, replicating the expression of their respective endogenous genes. In chimeric BACs, the mTert promoter became strongly repressed in the human genomic context, but the hTERT promoter was highly active in the mouse genomic context. Furthermore, an unrelated herpes simplex virus-thymidine kinase (HSV-TK) promoter was strongly repressed in the human, but not in the mouse, genomic context. These results demonstrated that the repression of hTERT gene was dictated by distal elements and its chromatin environment. This repression depended on class I HDACs and involved multiple corepressor complexes, including HDAC1/2-containing Sin3B, nucleosome remodeling and histone deacetylase (NuRD), and corepressor of RE1 silencing transcription factor (CoREST) complexes. Together, our data indicate that the lack of telomerase expression in most human somatic cells results from its repressive genomic environment, providing new insight into the mechanism of long-recognized differential telomerase regulation in mammalian species.-Cheng, D., Zhao, Y., Wang, S., Zhang, F., Russo, M., McMahon, S. B., Zhu, J. Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes.

CDK1 differentially regulates G-overhang generation at leading- and lagging-strand telomeres in telomerase-negative cells in G2 phase

Human telomeres contain single-stranded 3' G-overhangs that function in telomere end protection and telomerase action. Previously we have demonstrated that multiple steps involving C-strand end resection, telomerase elongation and C-strand fill-in contribute to G-overhang generation in telomerase-positive cancer cells. However, how G-overhangs are generated in telomerase-negative human somatic cells is unknown. Here, we report that C-strand fill-in is present at lagging-strand telomeres in telomerase-negative human cells but not at leading-strand telomeres, suggesting that C-strand fill-in is independent of telomerase extension of G-strand. We further show that while cyclin-dependent kinase 1 (CDK1) positively regulates C-strand fill-in, CDK1 unlikely regulates G-overhang generation at leading-strand telomeres. In addition, DNA polymerase α (Polα) association with telomeres is not altered upon CDK1 inhibition, suggesting that CDK1 does not control the loading of Polα to telomeres during fill-in. In summary, our results reveal that G-overhang generation at leading- and lagging-strand telomeres are regulated by distinct mechanisms in human cells.

Dyskerin is required for tumor cell growth through mechanisms that are independent of its role in telomerase and only partially related to its function in precursor rRNA processing

Dyskerin is an essential nucleolar protein required for the biogenesis of ribonucleoproteins that incorporate H/ACA RNAs. Through binding to specific H/ACA RNAs, dyskerin exerts most of its influence in the cell. To that end, dyskerin is a core component of the telomerase complex and is required for normal telomere maintenance. Dyskerin is also required for post-transcriptional processing of precursor rRNA. Germline dyskerin mutations increase cancer susceptibility. Conversely, wild-type dyskerin is usually over-expressed and not mutated in sporadic cancers. However, the contributions of dyskerin to sporadic tumorigenesis are unknown. Described herein, we demonstrate that acute loss of dyskerin function by RNA interference significantly reduced steady-state levels of H/ACA RNAs, disrupted the morphology and inhibited anchorage-independent growth of telomerase-positive and telomerase-negative human cell lines. Unexpectedly, dyskerin depletion only transiently delayed rRNA maturation but with no appreciable effect on the levels of total 18S or 28S rRNA. Instead, while rRNA processing defects typically trigger p53-dependent G1 arrest, dyskerin-depleted cells accumulated in G2/M by a p53-independent mechanism, and this was associated with an accumulation of aberrant mitotic figures that were characterized by multi-polar spindles. Telomerase activity and the rate of rRNA processing are typically increased during neoplasia. However, our cumulative findings indicate that dyskerin contributes to tumor cell growth through mechanisms which do not require the presence of cellular telomerase activity, and which may be only partially dependent upon the protein's role in rRNA processing. These data also reinforce the notion that loss and gain of dyskerin function may play important roles in tumorigenesis.

Growth defects in mouse telomerase RNA-deficient cells expressing a template-mutated mouse telomerase RNA

Cellular viability requires telomere maintenance, which, in mammals, is mainly mediated by the reverse transcriptase telomerase. Telomerase core components are a catalytic subunit TERT and an RNA subunit TR (hTR in humans, mTR in mouse) that carries the template to generate telomeres de novo. Telomere dysfunction can lead to senescence or apoptosis and impairs the continued growth of immortal cancerous cell lines. The introduction of a template-mutated hTR in telomerase-positive and telomerase-negative human cell lines results in dramatic growth defects. No study has addressed the consequences of expressing a template-mutated mTR in mouse immortal cell lines. Therefore, we analyzed the effects of long-term expression of a template-mutated mTR in the telomerase-positive and telomerase-negative murine cell lines CB17 and DKO301, respectively. Whereas the CB17 clones expressing the template-mutated mTR did not demonstrate any growth impairment, many of the DKO301 clones expressing the template-mutated mTR underwent growth and cell cycle defects and eventual cell death. These results suggest that in the absence of wild-type telomerase, the expression of the template-mutated mTR likely perturbs telomere function, leading to decreased cellular viability. Furthermore, whereas the expression of template-mutated hTR in telomerase-negative human cell lines leads to immediate cellular toxicity, the expression of the template-mutated mTR in the telomerase-negative mouse cell line did not.

Telomerase-Independent Telomere Length Maintenance in the Absence of Alternative Lengthening of Telomeres–Associated Promyelocytic Leukemia Bodies

Immortal tumor cells and cell lines employ a telomere maintenance mechanism that allows them to escape the normal limits on proliferative potential. In the absence of telomerase, telomere length may be maintained by an alternative lengthening of telomeres (ALT) mechanism. All human ALT cell lines described thus far have nuclear domains of unknown function, termed ALT-associated promyelocytic leukemia bodies (APB), containing promyelocytic leukemia protein, telomeric DNA and telomere binding proteins. Here we describe telomerase-negative human cells with telomeres that contain a substantial proportion of nontelomeric DNA sequences (like telomerase-null Saccharomyces cerevisiae survivor type I cells) and that are maintained in the absence of APBs. In other respects, they resemble typical ALT cell lines: the telomeres are highly heterogeneous in length (ranging from very short to very long) and undergo rapid changes in length. In addition, these cells are capable of copying a targeted DNA tag from one telomere into other telomeres. These data show that APBs are not always essential for ALT-mediated telomere maintenance.

Expression of mutant telomerase in immortal telomerase-negative human cells results in cell cycle deregulation, nuclear and chromosomal abnormalities and rapid loss of viability.

We have reconstituted wild type or mutant telomerase activity in two human cell lines that lack constitutive expression of both core subunits of the enzyme and maintain telomeres by a telomerase-independent mechanism (ALT cells). Wild type telomerase RNA and four telomerase RNAs with single point mutations in their template domain were used to express enzymes specifying different telomeric DNA sequences. Expression of wild type telomerase for up to 32 days had no detectable effect on cell growth or viability. In contrast, cells expressing mutant telomerases had slower growth rate, abnormal cell cycle and reduced viability. Dramatically aberrant nuclei, typical of cells undergoing mitotic catastrophe, and large numbers of fused chromosomes were also characteristic of these populations. Notably, all phenotypes were apparent within the first few cell divisions after expression of the enzymes. Unlike wild type, mutant telomerase activity was progressively selected against with cell culturing, and this correlated with the disappearance of cells with aberrant phenotypes. Our results suggest that even very limited synthesis of mutated sequences can affect telomere structure in human cells, and that the toxicity of mutant telomerases is due to telomere malfunction.

Telomeres and telomerase: implications for cancer and aging.

Maintenance of telomere stability is required for cells to escape from replicative senescence and proliferate indefinitely. Telomere length is maintained by a balance between processes that lengthen telomeres (telomerase) and processes that shorten telomeres (the end-replication problem). Telomerase is a cellular ribonucleoprotein reverse transcriptase which stabilizes telomere length by adding hexameric (TTAGGG) repeats to the telomeric ends of the chromosomes, thus compensating for the continued erosion of telomeres. Introduction of the telomerase catalytic protein component into normal telomerase-negative human cells results in restoration of telomerase activity and extension of cellular life span. Human cells with introduced telomerase maintain a normal chromosome complement and continue to grow in a normal manner. Telomerase-induced manipulations of telomere length may thus be important not only for cell and tissue engineering but also for dissecting the molecular mechanisms underlying inherited genetic diseases, as well as defining the genetic pathways leading to cancer. Because almost all human tumors express telomerase activity, inhibition of telomerase may result in gradual erosion of telomeres and eventual cessation of cell proliferation or induction of apoptosis. Thus telomerase may also be a promising target for cancer therapy.

Telomerase activity as a measure for monitoring radiocurability of tumor cells.

Radiotherapy plays a key role in the treatment of many tumors. It is difficult to determine what fraction of tumor cells survives after treatment with ionizing radiation. A convenient and sensitive biochemical assay could be efficacious in determining the potential success of radiotherapy. Since telomerase activity is frequently associated with the malignant phenotype, we sought to determine whether a correlation existed between ionizing radiation-induced cell killing and telomerase activity. We evaluated telomerase activity in two telomerase-positive and one telomerase-negative human cell line exposed to ionizing radiation. Telomerase activity was determined using a PCR-based telomeric repeat amplification protocol coupled with ELISA. We found ionizing radiation treatment to decrease the telomerase activity (in plateau phase cells of RKO, HeLa; and growing cells of RKO) in a dose-dependent manner, which correlated with cell death in in vitro tests as well as during tumor regression in nude mice. In contrast, growing HeLa cells after 24 h postradiation treatment showed an increase in telomerase activity, but there was no increase in the levels of mRNA of hTERT. To assess the sensitivity of the telomerase activity assay, we performed mixing experiments of HeLa and AG1522 cell extracts. These studies showed that telomerase activity could be detected in lysate equal to a single HeLa cell when mixed with 10,000 AG1522 cells. Our results indicate that even a few surviving neoplastic cells can be detected by telomerase activity assay. Therefore, detection of telomerase activity may be a useful monitor of radiotherapeutic efficacy and an early predictor of outcome.