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Stable expression of antisense hTR inhibits in vitro pancreatic cancer cell growth
Lisong Teng1, Shimei Chen, Fahey Thomas J
1Department of Oncological Surgery, Zhejiang University School of Medicine, Hangzhou, 310003, China. lsteng@hos.zju.edu.cn
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.
Insights
Downregulating the RNA component (hTR) of telomerase significantly inhibited pancreatic cancer cell growth and proliferation. This suggests targeting hTR is a potential anticancer strategy for pancreatic cancer.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Telomerase is a key enzyme in cancer cell immortalization.
- The human telomerase RNA component (hTR) is essential for telomerase activity.
- Pancreatic cancer cells exhibit high telomerase activity, contributing to their proliferation.
Purpose of the Study:
- To investigate the effect of inhibiting the hTR component of telomerase on pancreatic cancer cell growth.
- To determine if targeting hTR can serve as an anticancer strategy.
Main Methods:
- Constructed an antisense hTR expression plasmid.
- Introduced the plasmid into human pancreatic carcinoma cells (Panc1).
- Analyzed cell growth, hTR expression, telomerase activity, and anchorage-independent growth.
Main Results:
- Antisense hTR expression led to significant downregulation of endogenous hTR.
- Telomerase activity was markedly decreased in transformed cells.
- Cell proliferation and anchorage-independent growth were significantly reduced without inducing senescence.
Conclusions:
- hTR is a critical component of human telomerase activity.
- Downregulating hTR is a potential therapeutic target for pancreatic cancer treatment.
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