OBJECTIVETo explore the anti-cancer effects of siRNAs targeting hTERT in SMMC-7721 cells.

METHODSTwo siRNAs targeting hTERT mRNA were designed and synthesized by T7 transcription system in vitro. MMT, RT-PCR and Western blot were applied to evaluate effects on inhibiting cell growth, hTERT mRNA and protein expression in SMMC-7721 cells.

RESULTSsiRNAs decreased cell proliferation in a dose-dependent manner. At a concentration of 100 nmol/L, siRNAs exhibited obvious effects on inhibiting hTERT mRNA and protein expression in SMMC-7721 cells.

CONCLUSIONsiRNAs targeting hTERT have significant inhibitory effects on hTERT gene expression in SMMC-7721 cells. siRNA has the possibility to become a new anti-cancer agent

DNA-Binding Proteins ; antagonists & inhibitors ; genetics ; Gene Targeting ; Genetic Therapy ; Humans ; Liver Neoplasms ; pathology ; therapy ; RNA, Small Interfering ; genetics ; Telomerase ; antagonists & inhibitors ; genetics

OBJECTIVESTo study the inhibitory effects of mouse telomerase RNA (mTR) antisense oligodeoxynucleotide(ASODN) on telomerase activity in rat spermatogonia.

METHODS9-mer phosphorothioate mTR-ASODN was encapsulated by Lipofect AMINE 2000 (LF 2000) and transfected to type A spermatogonia in Snrague Dawley (SD) rat. Telomerase activity was detected by aid of TRAP-SYBR-Green staining and Bioluminescence technique in type A spermatogonia treated or untreated with ASODN.

RESULTSmTR-ASODN conjugated with LF 2000 could significantly inhibit telomerase activity of spermatogonia(P < 0.01). mTR mRNA level also decreased while the spermatogonia were treated with ASODN for 24 h. No change of telomerase activity and apoptosis were observed when SODN, RODN or single LF 2000 was used.

CONCLUSIONSAntisense oligodeoxynucleotide of mTR conjugated with LF 2000 could significantly inhibit telomerase activity of spermatogonia. mTR-ASODN might inhibit telomerase activity of spermatogonia at transcription level.

Animals ; Male ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA ; antagonists & inhibitors ; genetics ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Spermatogonia ; cytology ; enzymology ; ultrastructure ; Telomerase ; antagonists & inhibitors ; genetics ; metabolism

This study was aimed to construct a plasmid expressing siRNA specific for the human telomerase reverse transcriptase (hTERT) gene and to evaluate the ability of small interference RNA(siRNA) for inhibiting telomerase activity in HeLa cells. 64 nucleotides, in which 19 nt were homologous with hTERT gene, were chemically synthesized, annealed and linked into pSUPER to get pSUP-hTE. Then pSUP-hTE was digested with enzyme. We obtained its fragmant concluding promoter and 64nt. So we cloned it into pEGFP-C1 for constructing pEGFP-hTE which contains neo gene and the enhanced green fluorescent protein (EGFP). Recombinant pEGFP-hTE was transfected to HeLa cells. These cells were screened with medium containing G418. When stable colonies appeared, G418-resistant cells were harvested and propagated. At the different cell generations, hTERT mRNA and protein expression, telomerase activity and cell growth activity were analyzed. Compared with control cells, HeLa cells transfected with pEGFP-hTE showed that hTERT mRNA level and hTERT protein expression decreased and telomerase activity reduced by 38%, but the cells growth activity displayed no changes. So pEGFP-hTE could specifically inhibit expression of hTERT and telomerase activity. These results suggested that siRNA targeting hTERT gene might provide a new strategy for cancer biotherapy.
Base Sequence ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; HeLa Cells ; Humans ; Molecular Sequence Data ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; pharmacology ; Telomerase ; antagonists & inhibitors ; genetics ; Transfection

OBJECTIVETo investigate the effects of sodium selenite on telomerase activity and expression of hTERT mRNA in cadmium-transformed 16HBE cells.

METHODSTelomerase activity and expression of genes were measured after cultured cadmium-transformed 16HBE cells were exposed to sodium selenite at different doses (0.625, 1.25, 2.50, 5.00 micromol/L) for 24 hours.

RESULTSSelenium decreased telomerase activity in cadmium-transformed 16HBE cells. There existed an obvious dose-effect relationship between the selenium concentration and these changes. The expression of hTERT and c-myc mRNA also decreased but the expression of mad1 mRNA increased after exposure to selenium for 24 hours. No difference was found in expression of hTRF1 and hTRF2 mRNA after incubated with sodium selenite for 24 hours, compared with control group.

CONCLUSIONSelenium inhibits telomerase activity by decreasing hTERT and c-myc mRNA expression and increasing mad1 mRNA expression in cadmium-transformed 16HBE cells and selenium concentration is significantly correlated with these changes.

Base Sequence ; Cadmium ; pharmacology ; Cell Line, Transformed ; DNA Primers ; Humans ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sodium Selenite ; pharmacology ; Telomerase ; antagonists & inhibitors ; genetics

OBJECTIVETo evaluate antisense technology for human telomerase inhibition in the treatment of endometrial cancer.

METHODSAn antisense oligodeoxynucleotides (AODN) directed against the human telomerase transcriptase (hTERT), designed and synthesized to serve as a telomerase inhibitor, was transfected into endometrial carcinoma cell line HEC-1A by lipofectin. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to test the expression of hTERT mRNA and hTERT protein before and after transfection. Telomerase activity was tested by telomeric repeat amplification protocol. The proliferation and growth of HEC-1A were also studied by methyl thiazolyl tetrazolium and cell growth curve before and after transfection.

RESULTSAODN could down-regulate the expression of hTERT mRNA and protein, inhibiting telomerase activity and proliferation of endometrial cancer cell line in a dose- and period-dependent manner.

CONCLUSIONAntisense oligodeoxynucleotides of human telomerase transcriptase definitely inhibits the proliferation of endometrial cancer cell line. Telomerase inhibitor may thus become a new gene therapeutic agent for endometrial carcinoma.

Caspases ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Endometrial Neoplasms ; pathology ; therapy ; Female ; Genetic Therapy ; Humans ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; analysis ; Telomerase ; antagonists & inhibitors ; genetics

This study was purposed to investigate the effect of 3'-azido-2', 3'-dideoxythymidine (AZT)on the proliferation and telomerase activity of human acute myeloid leukemia cell line KG-1a. The effect of proliferation was detected by MTT assay after the KG-1a cell were stimulated for 24, 48 and 72 h with different concentrations of AZT; telomerase activity was detected with TRAP-PCR-ELISA assay; RT-PCR was used to detect telomerase hTERT mRNA expression. The results showed that the proliferation of KG-1a cells was inhibited in a time and concentration dependent manner after exposure to AZT for 24, 48 and 72 h; the KG-1a cells decreased in S phase and increased in G(2)/M phase with the increasing of the concentration of AZT; telomerase activity and hTERT-mRNA expression in the experimental groups decreased after treated with AZT, which was positively correlated with concentration of AZT. It is concluded that AZT inhibits KG-1a cell proliferation and induces apoptosis, which maybe related with its decreasing the telomerase activity and hTERT mRNA expression.
Apoptosis ; drug effects ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Leukemia ; metabolism ; pathology ; Telomerase ; antagonists & inhibitors ; metabolism ; Zidovudine ; pharmacology

OBJECTIVETo clarify growth inhibition in pancreatic cancer cells by interference with the hTR component of the telomerase reverse transcriptase enzymatic complex.

METHODSA 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.

RESULTSSignificant 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.

CONCLUSIONSThese 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.

DNA-Binding Proteins ; Humans ; Pancreatic Neoplasms ; pathology ; therapy ; RNA, Antisense ; therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; antagonists & inhibitors ; genetics ; Tumor Cells, Cultured

The gene for LPTS is originally cloned as a human liver-related putative tumor suppressor (LPTS) gene that encodes a full length protein of 328 amino acids (LPTS-L). LPTS-L is also identified as a telomerase inhibitor to regulate telomere length in the cells. To facilitate the functional and structural studies of LPTS-L protein, the cDNA for LPTS-L was cloned into the expression vector pET-24 in frame to generate a recombinant plasmid pET-24-LPTS. The LPTS-L protein was expressed in E. coli BL21 solublely, and purified by Ni Sepharose affinity chromatography which, however, is not fit for large scale protein purification. The gene of LITS-L was then PCR amplified to remove the 6 x His tag, and cloned into pET-24a. The non-fusion protein of LPTS-L was expressed in E. coli B21, and purified by phosphocellulose P11 chromatography. The purity of LPTS-L protein was about 55% after that procedure,and arrived at 80% after second purification by Sephadex G-100 chromatography. Western Blotting analysis showed that the band reflects the specific binding of anti-LPTS antiserum against the purified LPTS-L protein. The TRAP assay was performed to detect the telomerase inhibitory activity of LPTS-L protein in vitro. It was observed that the purified LPTS-L inhibited the activity of telomerase greatly, similarly with that of LPTS-L protein purified by Ni Sepharose 4B. Our results suggest that phosphocellulose P11 plus Sephadex G-100 chromatography could substitute for Ni Sepharose 4B affinity chromatography for preparation of purified LPTS-L protein. Through this study, a technique for preparation of LPTS-L protein in a large scale is established.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Recombination, Genetic ; Telomerase ; antagonists & inhibitors ; Tumor Suppressor Proteins ; biosynthesis ; genetics

Telomerase activity is usually detected in most tumor tissues but not in normal tissues. Recently, there is increasing evidence that telomerase activity is associated with cell proliferation without malignancy, whereas there is little information about telomerase activity and its relationship with cell proliferation in chronic hyperproliferative skin diseases. Thus, we studied telomerase activity in skins from 10 patients with psoriasis and compared telomerase activity with the expression of Ki-67, a proliferation marker, using immunohistochemical staining. The effect of retinoic acid on the telomerase activity in HaCaT cells was also evaluated. Telomerase activity was detected in 7 (70%) of 10 lesional skins of psoriasis and none of the nonlesional skin. Telomerase activity in lesional skin was significantly associated with Ki-67 labelling index. Retinoic acid treatment on HaCaT cells inhibited telomerase activity, which correlated with inhibition of cell proliferation by the agent. The results of our study represent another example that shows telomerase activity correlates with cellular proliferation. Further studies on the regulation of the telomerase are needed to understand the cellular factors involved in controlling telomerase activity.
Cell Division/drug effects ; Cell Line ; Enzyme Inhibitors/*pharmacology ; Human ; Ki-67 Antigen/*analysis ; Psoriasis/*enzymology ; Skin/*enzymology ; Telomerase/antagonists & inhibitors/*metabolism ; Tretinoin/*pharmacology

OBJECTIVETo assess the telomerase activity inhibitory effect of ham merhead ribozyme targeting the 5'-end of human telomerase reverse transcriptase mRNA (hTERT-5'RZ), to compare it with the effect of another ribozyme teloRZ, and the combine the applications of the two ribozymes.

METHODShTERT-5'RZ gene was synthesized and cloned into pcDNA3.1(+); the ribozyme was produced by in vitro transcription. The teloRZ ribozyme was produced in the same way by in vitro transcription of p(SPT19-teloRZ) which had been constructed by the present authors. The ribozymes were transiently transfected into HeLa cells by liposome every 24 hours. After 72 hours, the cells were collected and their telomerase activities were assayed.

RESULTSThe ribozyme targeting the 5'-end of hTERT mRNA exhibited a very strong telomerase-inhibitory activity, the combined use of hTERT-5'RZ and teloRZ also showed clear inhibitory activity, but the inhibitory effect of teloRZ used alone was not so strong.

CONCLUSIONThese observations suggest that the use of hTERT-5'RZ and the combined use of hTERT-5'RZ and teloRZ are more effective in telomerase inhibition as compare with the use of teloRZ alone. They may find applications in cancer therapy.

5' Untranslated Regions ; Cloning, Molecular ; DNA-Binding Proteins ; HeLa Cells ; Humans ; RNA, Catalytic ; metabolism ; RNA, Messenger ; Telomerase ; antagonists & inhibitors ; genetics ; Transcription, Genetic