Silvia Bacchetti

A survey of telomerase activity in human cancer.

Research on the association of the ribonucleoprotein enzyme, telomerase, with human cancer has expanded rapidly in recent years. Essentially all major types of cancer have been screened and the presence of telomerase activity has been detected in the vast majority of cases. In this article we provide a summary, in table form, of the current data.

Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.

hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization

Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumor progression. We report the cloning of a human gene, hEST2, that shares significant sequence similarity with the telomerase catalytic subunit genes of lower eukaryotes. hEST2 is expressed at high levels in primary tumors, cancer cell lines, and telomerase-positive tissues but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. Moreover, the message is up-regulated concomitant with the activation of telomerase during the immortalization of cultured cells and down-regulated during in vitro cellular differentiation. Taken together, these observations suggest that the induction of hEST2 mRNA expression is required for the telomerase activation that occurs during cellular immortalization and tumor progression.

Telomere elongation in immortal human cells without detectable telomerase activity.

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus‐transformed cell cultures were also negative in the pre‐crisis (i.e. non‐immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase‐negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase‐negative and telomerase‐positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.

Induction of hTERT Expression and Telomerase Activity by Estrogens in Human Ovary Epithelium Cells

ABSTRACT In mammals, molecular mechanisms and factors involved in the tight regulation of telomerase expression and activity are still largely undefined. In this study, we provide evidence for a role of estrogens and their receptors in the transcriptional regulation of hTERT, the catalytic subunit of human telomerase and, consequently, in the activation of the enzyme. Through a computer analysis of the hTERT 5′-flanking sequences, we identified a putative estrogen response element (ERE) which was capable of binding in vitro human estrogen receptor α (ERα). In vivo DNA footprinting revealed specific modifications of the ERE region in ERα-positive but not ERα-negative cells upon treatment with 17β-estradiol (E2), indicative of estrogen-dependent chromatin remodelling. In the presence of E2, transient expression of ERα but not ERβ remarkably increased hTERT promoter activity, and mutation of the ERE significantly reduced this effect. No telomerase activity was detected in human ovary epithelial cells grown in the absence of E2, but the addition of the hormone induced the enzyme within 3 h of treatment. The expression of hTERT mRNA and protein was induced in parallel with enzymatic activity. This prompt estrogen modulation of telomerase activity substantiates estrogen-dependent transcriptional regulation of the hTERT gene. The identification of hTERT as a target of estrogens represents a novel finding which advances the understanding of telomerase regulation in hormone-dependent cells and has implications for a potential role of hormones in their senescence and malignant conversion.

Alternative Lengthening of Telomeres Is Characterized by High Rates of Telomeric Exchange

Telomere maintenance activity is a hallmark of cancer. In some telomerase-negative tumors, telomeres become lengthened by alternative lengthening of telomeres (ALT), a recombination-mediated DNA replication process in which telomeres use other telomeric DNA as a copy template. Using chromosome orientation fluorescence in situ hybridization, we found that postreplicative exchange events involving a telomere and another TTAGGG-repeat tract occur at remarkably high frequencies in ALT cells (range 28–280/100 metaphases) and rarely or never in non-ALT cells, including cell lines with very long telomeres. Like the ALT phenotype itself, the telomeric exchanges were not suppressed when telomerase was activated in ALT cells. These exchanges are telomere specific because there was no correlation with sister chromatid exchange rates at interstitial locations, and they were not observed in non-ALT Bloom syndrome cells with very high sister chromatid exchange rates.

Histone Deacetylation Is Involved in the Transcriptional Repression of hTERT in Normal Human Cells

Trancriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic protein of human telomerase, plays a critical role in the activation of the enzyme during cell immortalization and tumorigenesis. However, the molecular mechanisms involved in the regulation of hTERTexpression are still not fully understood. We have previously cloned and characterized the genomic sequences and promoter of thehTERT gene. Here, we provide evidence that histone deacetylation is involved in the repression of hTERT in human cells. Inhibition of histone deacetylases by trichostatin A in telomerase-negative cells resulted in activation of telomerase activity and up-regulation of hTERT mRNA. Transient transfection experiments with a reporter under control of thehTERT promoter indicated that this promoter can be activated by trichostatin A. Finally, our results show that repression of the hTERT promoter by the Mad protein requires histone deacetylase activity, whereas de-repression by trichostatin A is independent of the E-boxes located in its core region.

Expression of SV40 large T antigen, but not small t antigen, is required for the induction of chromosomal aberrations in transformed human cells

Expression of the Simian virus 40 (SV40) early region in human cells results in the induction of chromosomal aberrations and polyploidy, and in transformation. To understand how genetic damage occurs and what role it plays in transformation, human diploid fibroblasts and embryonic kidney cells were transfected with plasmids encoding wild type or mutant forms of the viral early region, and the neo gene. Clones selected for G418 resistance and expressing viral genes were initially analyzed within 20 cell divisions. Our results demonstrate that expression of the SV40 large T antigen is sufficient for the induction of chromosomal damage and ploidy changes, and that small t does not contribute to these processes. Mutant plasmids lacking the SV40 origin of DNA replication were as proficient as wild type plasmids, indicating that viral DNA replication is not required for cytogenetic damage. We have also shown that chromosome aberrations, but not necessarily polyploidy, increase in frequency and complexity upon subculturing of the clones regardless of whether such populations arrest at crisis or yield immortal lines. Our results are compatible with the hypothesis that large T antigen destabilizes the cellular genome, and that specific mutations arising from this process may contribute to cell immortalization.

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.

Endothelial NOS, estrogen receptor β, and HIFs cooperate in the activation of a prognostic transcriptional pattern in aggressive human prostate cancer

The identification of biomarkers that distinguish between aggressive and indolent forms of prostate cancer (PCa) is crucial for diagnosis and treatment. In this study, we used cultured cells derived from prostate tissue from patients with PCa to define a molecular mechanism underlying the most aggressive form of PCa that involves the functional activation of eNOS and HIFs in association with estrogen receptor beta (ERbeta). Cells from patients with poor prognosis exhibited a constitutively hypoxic phenotype and increased NO production. Upon estrogen treatment, formation of ERbeta/eNOS, ERbeta/HIF-1alpha, or ERbeta/HIF-2alpha combinatorial complexes led to chromatin remodeling and transcriptional induction of prognostic genes. Tissue microarray analysis, using an independent cohort of patients, established a hierarchical predictive power for these proteins, with expression of eNOS plus ERbeta and nuclear eNOS plus HIF-2alpha being the most relevant indicators of adverse clinical outcome. Genetic or pharmacologic modulation of eNOS expression and activity resulted in reciprocal conversion of the transcriptional signature in cells from patients with bad or good outcome, respectively, highlighting the relevance of eNOS in PCa progression. Our work has considerable clinical relevance, since it may enable the earlier diagnosis of aggressive PCa through routine biopsy assessment of eNOS, ERbeta, and HIF-2alpha expression. Furthermore, proposing eNOS as a therapeutic target fosters innovative therapies for PCa with NO inhibitors, which are employed in preclinical trials in non-oncological diseases.