Use of Transcriptional Regulatory Sequences of Telomerase (hTER and hTERT) for Selective Killing of Cancer Cells

Telomerase (hTER and hTERT) plays a crucial role in cellular immortalization and carcinogenesis. Telomerase activity can be detected in about 85% of different malignant tumors, but is absent in most normal cells. In situ hybridization analysis showed that high levels of hTER and hTERT expression are present in bladder cancer, while no signal was detected in normal tissue. Therefore, in this work we propose to use hTER and hTERT transcriptional regulatory sequences to control the expression of a cytotoxic gene in bladder tumor cells, resulting in the selective destruction of this cell population. Expression vectors containing the diphtheria toxin A-chain (DT-A) gene were linked to hTER and hTERT transcriptional regulatory sequences, respectively. Inhibition of protein synthesis occurred in bladder and hepatocellular carcinoma cells transfected with the plasmids containing the DT-A gene under the control of the hTER or hTERT promoters in correlation with their activity. These studies support the feasibility of using hTER and hTERT transcriptional regulatory sequences for targeted patient-oriented gene therapy of human cancer. Telomerase (hTER and hTERT) plays a crucial role in cellular immortalization and carcinogenesis. Telomerase activity can be detected in about 85% of different malignant tumors, but is absent in most normal cells. In situ hybridization analysis showed that high levels of hTER and hTERT expression are present in bladder cancer, while no signal was detected in normal tissue. Therefore, in this work we propose to use hTER and hTERT transcriptional regulatory sequences to control the expression of a cytotoxic gene in bladder tumor cells, resulting in the selective destruction of this cell population. Expression vectors containing the diphtheria toxin A-chain (DT-A) gene were linked to hTER and hTERT transcriptional regulatory sequences, respectively. Inhibition of protein synthesis occurred in bladder and hepatocellular carcinoma cells transfected with the plasmids containing the DT-A gene under the control of the hTER or hTERT promoters in correlation with their activity. These studies support the feasibility of using hTER and hTERT transcriptional regulatory sequences for targeted patient-oriented gene therapy of human cancer. IntroductionOne of the most crucial enzymes in cell immortality, cell aging, and cancer is telomerase. The telomerase enzyme is composed of three subunits, two of them are essential for its activity, the RNA component coded by the hTER gene and the catalytic subunit coded by the hTERT gene, both were cloned and sequenced (1Feng J. et al.The RNA component of human telomerase.Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2062) Google Scholar, 2Nakamura T.M. et al.Telomerase catalytic subunit homologs from fission yeast and human.Science. 1997; 277: 955-959Crossref PubMed Scopus (2052) Google Scholar, 3Cong Y.S. Wen J. Bacchetti S. The human telomerase catalytic subunit hTERT: Organization of the gene and characterization of the promoter.Hum. Mol. Genet. 1999; 8: 137-142Crossref PubMed Scopus (417) Google Scholar, 4Takakura et al.Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells.Clin. Cancer Res. 1999; 59: 551-557Google Scholar). It has been demonstrated that in many cell lines that express hTER, telomerase activity is not detected, in contrast to hTERT expression that is generally found in telomerase positive cell lines (5Soder A.I. Hoare S.F. Muir S. Going J.J. Parkinson E.K. Keith W.N. Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer.Oncogene. 1997; 14: 1013-1021Crossref PubMed Scopus (166) Google Scholar). However several studies showed clear differentials in hTER expression between certain cancerous and their adjacent normal tissue (5Soder A.I. Hoare S.F. Muir S. Going J.J. Parkinson E.K. Keith W.N. Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer.Oncogene. 1997; 14: 1013-1021Crossref PubMed Scopus (166) Google Scholar). This difference in expression levels suggests that hTER promoter may be an interesting candidate to be used in gene therapy. Expression of hTERT is observed at high levels in malignant tumors and cancer cell lines but not in normal tissues or telomerase-negative cell lines, indicating a strong correlation between hTERT expression and telomerase activity in a variety of tumors (6Ito H. Kyo S. Kanaya T. Takakura M. Inoue M. Namiki M. Expression of human telomerase subunits and correlation with telomerase activity in urothelial cancer.Clin. Cancer Res. 1998; 4: 1603-1608PubMed Google Scholar, 7Takakura M. Kyo S. Kanaya T. Tanaka M. Inoue M. Expression of human telomerase subunits and correlation with telomerase activity in cervical cancer.Cancer Res. 1998; 58: 1558-1561PubMed Google Scholar). These data suggest that hTERT expression, at least in some contexts, may be the limiting factor for telomerase activity. These findings and others strongly suggest an important role for telomerase in cell immortalization and tumorigenesis, making this enzyme a promising candidate for cancer diagnosis and therapy. Great efforts were invested to develop therapies by inhibiting telomerase activity in the cells of cancer tissues without causing serious harm to the healthy tissue of the patients (1Feng J. et al.The RNA component of human telomerase.Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2062) Google Scholar, 8Kent S. Telomerase: The immortalizing enzyme. Update on Geron Corporation.Life Extension Rep. 1995; 15Google Scholar).A 5’ flanking region of 341 bp in the hTER gene was identified as the minimal promoter region, containing elements responsible for activity (9Zhao J.Q. Hoare S.F. McFarlane R. Muir S. Parkinson E.K. Black D.M. Keith W.N. Cloning and characterization of human and mouse