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.

Key words: H19 gene; regulatory sequences; gene therapy; bladder cancer; DT-A gene; HVS-tk geneOne of the most serious problems in cancer therapy is the obvious necessity to limit the action of the drug(s) used in the therapy to tumor cells only, in order to prevent damage to healthy tissue. The approach used in our work on cancer therapy is based on the use of regulatory sequences of genes that are differentially expressed between normal and cancer cells, to drive the expression of a toxin gene in tumor cells only.The H19 gene is highly expressed in embryonic tissues, expressed at low levels (or not at all) in normal adult tissues and expressed in tumors derived from tissues (such as bladder) that exhibited the gene during embryonic development. 1-3 H19 RNA is not present in normal adult bladder tissue but can be detected in bladder cancer cells in over 75% of bladder tumors; however, the relative number of H19-expressing cells is reduced during tumor differentiation. 4 The change in H19 RNA level may be caused by a change either in the level of H19 transcription or in H19 RNA stability or both. 5 We have previously reported that transcription of the human H19 gene is under the control of regulatory sequences downstream to the coding sequence. 6 We demonstrated that luciferase activity could be selectively expressed in cells transfected with a plasmid carrying the luc gene linked to the 814 bp 5Ј-flanking region of the H19 gene. 6 Our results showed that elements responsible for basal promoter activity are contained within the 85 bp upstream region and that cell specificity is mainly conferred by the 814 bp upstream sequences. 7 .We established a murine in vivo model for bladder carcinoma in which the dynamics of H19 expression and tumor progression could be correlated. 8 . Mice develop solid invasive bladder cancer after 20 -30 weeks of N-butyl-N(4-hydroxybutyl)nitrosamine (BBN) treatment after hyperplasia and dysplasia of the urothelium. 8 We showed that H19 is expressed in the preneoplastic phase during BBN-induced tumor evolution. 8 Moreover, when tumor formation is inhibited by the anticancer drug halofuginone, H19 expression is extinguished. 9 Halofuginone also exerts anticancer effects on tumors formed by subcutaneous transplantation of MBT2-t50 murine bladder carcinoma cells (H19-positive cells) into syngeneic mice. 9 Tumor cell-specific gene therapy might be accomplished with a construct carrying H19 regulatory sequences that determine the tissue-specific expression of a toxin or suicide enzyme gene. We therefore constructed expression vectors containing "therapeutic genes" such as the gene for the A fragment of diphtheria toxin (DT-A) or herpes simplex virus thymidine kinase (HSV-tk), under the control of a 814 bp 5Ј-flanking region of the H19 gene.Here we present evidence that the constructs expressing either the toxin or the suicide gene driven by the H19 regulatory sequence could selectively exert their cytotoxic effects in H19-positive cells. The cell killing activity of these constructs was in ac...

The human H19 is paternally imprinted (maternally expressed). It is transcribed by RNA pol II, but has no protein product. Its function is unknown. We showed that the transcription of the human H19 gene is under the simultaneous control of both a 5P upstream (promoter) region and a 3P downstream region in cell lines derived from human choriocarcinomas. Moreover, the activation of the H19 promoter by retinoic acid in cells derived from human testicular germ cell tumors is dependent upon the 3P downstream region. The possibility that the action of retinoic acid on the H19 promoter is an indirect one and involves a member of the AP2 transcription factor family is discussed.z 1998 Federation of European Biochemical Societies.

H19 is expressed in a large percentage of bladder tumors, but not expressed in healthy bladder tissue. The aim of this study is to define H19 optimal transcriptional regulatory sequences in tumor cells, which can potentially be used to control expression of a toxin gene in constructs to be used in bladder cancer gene therapy trials in mice and human. Transient expression assays revealed that elements responsible for promoter activity are contained within the 85 bp upstream region. The transcriptional activity of this region was strongly inhibited by the methylation of the Hpa II sites. A modest cell specificity is conferred by the upstream sequences. The human and murine promoter activities were significantly increased by the human H19 4.1 kb enhancer sequence. The 85 bp H19 upstream region contains all the elements to interact with the enhancer. We showed that the human H19 promoter is highly active in a murine bladder carcinoma cell line, justifying its use to drive the expression of a cytotoxin gene in gene therapy trials in mice.

The human H19 gene is a paternally imprinted oncofetal gene, highly expressed in several fetal tissues, downregulated in nearly all adult tissues but re-expressed in carcinomas of tissues which express the gene in fetal life. It has no known protein product and till today, no function could be designated to H19 RNA. Cells derived from bladder carcinomas and hepatocellular carcinomas were transfected with plasmids carrying a luciferase reporter gene under the control of a 800 nucleotides long promoter region of the H19 gene either alone or together with different parts of a 5 kb downstream region, previously shown to possess enhancer activity. Our results provide evidence that three regions of the 3P P downstream sequence can independently stimulate the H19 promoter activity in a tissue and cell specific manner. The growth rate of two cell populations, both derived from the same bladder carcinoma cell line and which differ in their H19 RNA content, were compared. The cells with a high H19 RNA level stopped their proliferation after 48 h when cultivated in a low serum containing media while the cells lacking H19 RNA continued their proliferation for at least an additional 48 h period.z 1999 Federation of European Biochemical Societies.