OBJECTIVE: To clarify growth inhibition in pancreatic cancer cells by interference with the hTR component of the telomerase reverse transcriptase enzymatic complex. METHODS: A 593 bp full length hTR cDNA was subcloned...OBJECTIVE: To clarify growth inhibition in pancreatic cancer cells by interference with the hTR component of the telomerase reverse transcriptase enzymatic complex. METHODS: A 593 bp full length hTR cDNA was subcloned into a mammalian expression vector pcDNA3.1(-) in the antisense orientation to construct an antisense hTR expression plasmid. These were introduced into panc1 cells, a human pancreatic carcinoma cell line, by lipofectin and G418-resistant stable transformants were expanded. Resulting stable clones were screened for the presence of the hTR insert by PCR with T7 and BGH reverse primers located on the flanks of the multiclonal site of the pcDNA3.1 vector. Cell growth rate, hTR expression, telomerase activity and anchorage-independent growth properties were analyzed. RESULTS: Significant downregulation of endogenous hTR was evident in the antisense-hTR transformed cells and telomerase activity was markedly decreased compared to control cells in standard TRAP assays. Furthermore, cell proliferation and the anchorage-independent growth ability in antisense-hTR expressing cells were significantly decreased compared with control parental cells. However, no crisis or senescence phenomena were observed. CONCLUSIONS: These data indicate that hTR may be a critical component of human telomerase activity and suggest that downregulation of the RNA component of human telomerase is a possible target for anticancer strategies.展开更多
文摘OBJECTIVE: To clarify growth inhibition in pancreatic cancer cells by interference with the hTR component of the telomerase reverse transcriptase enzymatic complex. METHODS: A 593 bp full length hTR cDNA was subcloned into a mammalian expression vector pcDNA3.1(-) in the antisense orientation to construct an antisense hTR expression plasmid. These were introduced into panc1 cells, a human pancreatic carcinoma cell line, by lipofectin and G418-resistant stable transformants were expanded. Resulting stable clones were screened for the presence of the hTR insert by PCR with T7 and BGH reverse primers located on the flanks of the multiclonal site of the pcDNA3.1 vector. Cell growth rate, hTR expression, telomerase activity and anchorage-independent growth properties were analyzed. RESULTS: Significant downregulation of endogenous hTR was evident in the antisense-hTR transformed cells and telomerase activity was markedly decreased compared to control cells in standard TRAP assays. Furthermore, cell proliferation and the anchorage-independent growth ability in antisense-hTR expressing cells were significantly decreased compared with control parental cells. However, no crisis or senescence phenomena were observed. CONCLUSIONS: These data indicate that hTR may be a critical component of human telomerase activity and suggest that downregulation of the RNA component of human telomerase is a possible target for anticancer strategies.
