Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor beta growth inhibition in p16INK4A(-) human mammary epithelial cells.

Failures to arrest growth in response to senescence or transforming growth factor beta (TGF-beta) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16(INK4A) resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-beta. The ability to maintain growth in TGF-beta was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-beta resistance may explain our previously described gain of TGF-beta resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-beta resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.

The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters.

In human cells, telomerase activity is regulated by transcriptional control of the telomerase reverse transcriptase gene (hTERT) whose product is the catalytic subunit of the enzyme. The hTERT promoter is active in virtually all types of tumors and immortal cells, but is silent in most adult somatic tissues. In this study, we placed the herpes simplex virus thymidine kinase gene under the control of the hTERT promoter with the aim of restricting its expression to tumor cells. In transfection experiments, the hTERT promoter driven thymidine kinase gene (hTERTp/TK) conferred ganciclovir sensitivity to all tumor and immortal cell lines tested, whereas normal somatic cells remained largely unaffected. Human hTERTp/TK-positive cancer cells implanted in nude mice developed into tumors that could be eradicated by ganciclovir treatment. The hTERTp/TK cassette was inserted into an adenovirus vector and its efficacy in reducing tumor growth was compared with that of an adenovirus carrying the thymidine kinase gene under the control of the cytomegalovirus immediate-early promoter (CMVp/TK). In a xenograft model using the human 143B osteosarcoma cell line, a single injection of either virus resulted in equivalent tumor regression and survival upon ganciclovir treatment. In animals injected intratumorally with the CMVp/TK adenovirus, expression of the thymidine kinase gene was detected in tumors, as well as in liver samples. Expression of the suicide gene in combination with ganciclovir resulted in severe liver histopathology and in an elevation of hepatic enzymes. In sharp contrast, when the hTERT promoter controlled the thymidine kinase gene, transgene expression was observed in tumors, but not in liver samples. Normal liver function in these animals was confirmed by serum levels of hepatic enzymes that were indistinguishable from those of control healthy mice. These results indicate that by restricting thymidine kinase expression to tumor cells, the hTERT promoter allows the tumoricidal effect of the suicidal gene to be exerted without detrimental consequences on healthy tissues and vital organs. The tight specificity of expression imparted by the hTERT promoter will assist the development of novel approaches to the treatment of a broad array of cancer types.

Expression of the hTERT gene is regulated at the level of transcriptional initiation and repressed by Mad1.

Telomerase, an enzymatic activity responsible for the replication of chromosome end structures, is strongly upregulated in most human cancers. In contrast, most differentiated tissues are telomerase negative. The rate-limiting step for telomerase activity seems to be the expression of the catalytic subunit of the enzyme, encoded by the human telomerase reverse transcriptase (hTERT) gene. The precise mechanism of how hTERT is regulated has not been elucidated yet. We show here that the down-regulation of hTERT mRNA during 12-O-tetradecanoylphorbol-13-acetate-induced differentiation of human U937 cells is a consequence of a fast decrease in the rate of transcription rather than changes in its half-life. The only transcription factor that has so far been implicated in the regulation of hTERT expression is the c-Myc oncoprotein. Our analysis shows that another member of the myc/marx/mad network, mad1, encoding a transcriptional repressor that is significantly increased by 12-O-tetra-decanoylphorbol-13-acetate treatment, represses hTERT promoter-driven reporter gene activity in transient transfection assays. This effect is dependent on the NH2 terminal domain of Madl, which mediates the association with the transcriptional corepressor mSin3. Our findings suggest the involvement of an additional transcription factor in the regulation of hTERT expression and may provide a model for how hTERT activity is controlled during the differentiation process in human somatic tissues.

Telomerase. A target for anticancer therapy.

Telomerase is absent in most normal tissues, but is abnormally reactivated in all major cancer types. Telomerase enables tumor cells to maintain telomere length, allowing indefinite replicative capacity. Albeit not sufficient in itself to induce neoplasia, telomerase is believed to be necessary for cancer cells to grow without limit. The presence of telomerase has been detected in virtually all cancer types including the most prevalent cancers of the prostate, breast, lung, colon, bladder, uterus, ovary, and pancreas as well as in lymphomas, leukemias, and melanomas. In addition, data from cancer patients indicate that telomerase levels correlate with clinical outcome in neuroblastomas, leukemias, and prostate, gastric, and breast cancers. Studies using an antisense to the human telomerase RNA component demonstrate that telomerase in human tumor lines can be blocked ex vivo. In these experiments, telomerase inhibition led to telomere shortening and cancer cell death, validating telomerase as a target for anticancer therapy. Telomerase is a uniquely appealing target for drug discovery because its dichotomic expression in normal versus cancer cells suggests that no serious side effects would result from a treatment abrogating telomerase activity. A variety of approaches to telomerase inhibition are being investigated and are discussed.