Investigation Of The Mechanism Of Tumor Cell Apoptosis Induced By HDAC Inhibitor

Telomerase activity is involved in cellular immortality, and tomerase is composed primarily of the catalytic subunit (hTERT) and the RNA template (hTERC), hTERT is the catalytic subunit of telomerase holoenzyme as well as the rate-limiting component of the telomerase enzyme complex. However, the role of the hTERT in apoptosis induced by histone deacetylase (HDAC) inhibitor has been marginally addressed. The present study was designed to evaluate the change of hTERT expression in apoptosis of cervical cancer cell lines induced by histone deacetylase inhibitor trichostatin A (TSA), and determine the role and mechanism of hTERT in this procession. Treated cervical cancer cell line HeLa and SiHa with 1.5μmol/L TSA, for the first time, we found that TSA transiently activated cervical cancer cell lines HeLa cells and Siha cells growth within 12hrs, but inhibited them after that time point, which were consistent with hTERT expression, telomerase activity and telomere length change, These observations indicated that the telomerase activity was essential to the apoptosis induced by HDAC inhibitor. Respectively Transfected the wild type and dominant negative of hTERT into these cell lines and treated them with 1.5μmol/L TSA, We showed here that the apoptosis rate of cells containing wild type hTERT was significant lower than cells containing dominant negative hTERT. Furthermore, we found that hTERT could downregultaed the expression of p21waf1, but had no effect on p53, so we suggested that the anti-apoptosis effect of hTERT might be through p21waf1 dependant pathway and p53 independent pathway. 【Objective】To establish a new technique (SMART) to screen some novel functional proapoptosis genes associated with HDAC inhibitors.【Methods】Different cancer cell lines MCF-7 and HeLa were treated with Trichostatin A. Apoptosis was determined by various of methods; 4.5×107 MCF-7 cells treated with 250nM Trichostatin A for 0～48h were collected to isolate mRNA. 5μg mRNA was then reverse transcripted into cDNA. After end modification, linker addition and enzyme digestion, cDNA was inserted into plasmid pCEP4 in an antisense orientation to construct an antisense cDNA library; 40μg of the antisense cDNA library was transfected into HeLa cells with empty vector as control. 48h later selection began with 150nmol/L Trichostatin A and 200μg/ml Hygromycin B. Selective medium were replaced every two days and lasted for 12 days until there were no colonies survived in the plate transfected with control vector while several colonies survived in the plate transfected with antisense cDNA library. The surviving colonies were amplified and Hirt DNA were extracted and digested.【Results】Trichostatin A selectively induced MCF-7 and HeLa cells apoptosis, which was determined by MTT and flow cytometry .10μg mRNA isolated from 4.5×107 MCF-7 treated with 250nM Trichostatin A. An antisense cDNA library containing 2×105 colonies were constructed and the mean size of inserts was approximately 1.5kb. There was a difference between cells transfected with antisense cDNA library and cells transfected with control vector after double drugs selection for 12 days. There were several colonies surviving in the plate transfected with antisense cDNA library while none in the plate transfected with control vector. Surviving colonies were pooled and Hirt DNA was extracted. We identified 76 effective colonies in cDNA library. Employing the biology information analysis, we chose Liv-1, Smad4, and Ubiquitin B to make further mechanical research.【Conclusion】1. SMART cDNA library based on HDAC inhibitor inducing apoptosis was successfully constructed.2. Employing SMART library and biological information analysis,we got 76 effective colonies3. We have made further mechanical research on Liv-1, Smad4, and Ubiquitin B.