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 investigate the telomerase activity and to document biological behaviors of HeLa cells upon treatment with specific PI3K/AKT signaling pathway inhibitor, LY294002.

METHODSCCK-8 assay was used to determine IC50 of LY294002. The expressions of total AKT and phosphorylation AKT (P-AKT) were determined using Western blot. Telomerase activity of cell was measured by TRAP-ELISA assay. Cell growth curve, flow cytometry technique and Hoechst33258 stain were used to evaluate the cell growth, cell cycle and apoptosis respectively. Cell migration was determined by cell wound healing assay.

RESULTSIC50 value of LY294002 of treated HeLa cells was 1.73 mg/L. Western blot showed that LY294002 enabled to decrease P-AKT activity in the presence of same total AKT protein. The cell telomerase activity was decreased to 36.72% in contrast to 98.61% of the control. LY294002 decreased the telomerase activity in HeLa cells, and the growth capacity of the cells was significantly suppressed. The number of cells at G0/G1 phases increased to 66.88% compared with that of the control cells (47.36%). The apoptosis rate also increased from 2.4% to 14.9%. The relative migration distance decreased to 24.6% compared with that of control (62.57%).

CONCLUSIONLY294002 inhibition of PI3K/AKT signaling pathway leads to alteration of telomerase activity along with changes of the biological behaviors of the HeLa cells suggesting that regulation of telomerase activity may be closely related to PI3K/AKT signaling pathway.

Apoptosis ; drug effects ; Blotting, Western ; Cell Line ; Cell Movement ; drug effects ; physiology ; Cell Proliferation ; drug effects ; Chromones ; pharmacology ; Enzyme Inhibitors ; pharmacology ; G1 Phase ; drug effects ; HeLa Cells ; Humans ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; Signal Transduction ; drug effects ; physiology ; Telomerase

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS: To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS: hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS: These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.
Amino Acid Chloromethyl Ketones/pharmacology ; Antineoplastic Agents/*pharmacology ; Caspases/antagonists & inhibitors/metabolism ; Cell Line, Tumor ; Cisplatin/*pharmacology ; Cysteine Proteinase Inhibitors/pharmacology ; Cytochrome c Group/metabolism ; Drug Resistance, Neoplasm/genetics ; Humans ; Neoplasms/therapy ; RNA, Small Interfering ; Telomerase/*antagonists & inhibitors/genetics/metabolism ; bcl-2-Associated X Protein/metabolism

A trans-splicing ribozyme which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA was previously suggested as a useful agent for tumor-targeted gene therapy. In this study, we evaluated in vivo function of the hTERT-targeting trans-splicing ribozymes by employing the molecular analysis of expression level of genes affected by the ribozyme delivery into peritoneal carcinomatosis mice model. To this effect, we constructed adenoviral vector encoding the specific ribozyme. Noticeably, more than four-fold reduction in the level of hTERT RNA was observed in tumor nodules by the systemic infection of the ribozyme-encoding virus. Such hTERT RNA knockdown in vivo induced changes in the global gene expression profile, including the suppression of specific genes associated with anti-apoptosis including bcl2, and genes for angiogenesis and metastasis. In addition, specific trans-splicing reaction with the targeted hTERT RNA took place in the tumors established as peritoneal carcinomatosis in mice by systemic delivery of the ribozyme. In conclusion, this study demonstrates that an hTERT-specific RNA replacement approach using trans-splicing ribozyme represents a potential modality to treat cancer.
Animals ; Cell Line ; Gene Expression/*physiology ; Genetic Vectors ; Humans ; Mice ; Neoplasm Metastasis ; Neoplasms/genetics/pathology ; RNA, Catalytic/genetics/*metabolism ; RNA, Messenger/genetics/metabolism ; RNA, Neoplasm/genetics/metabolism ; Telomerase/antagonists & inhibitors/genetics/*metabolism ; Trans-Splicing/*genetics

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 investigate the inhibiting effect of telomerase with hTERT antisense on TNF-alpha-induced apoptosis in prostate cancer cells PC3.

METHODSAntisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by telomeric repeat amplification protocol (TRAP) and telomerase PCR-ELISA Kit, cell viability was determined by MTT assay, and cell apoptosis was observed by morphological method and determined by flow cytometry.

RESULTSAS PS-ODN could significantly inhibit the telomerase activity and increase the susceptibility of TNF-alpha-induced apoptosis of PC3 cells.

CONCLUSIONInhibition of telomerase with hTERT antisense can enhance TNF-alpha-induced apoptosis in prostate cancer cells.

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Enzyme-Linked Immunosorbent Assay ; Humans ; Male ; Oligodeoxyribonucleotides, Antisense ; genetics ; pharmacology ; Polymerase Chain Reaction ; Prostatic Neoplasms ; enzymology ; genetics ; pathology ; Telomerase ; antagonists & inhibitors ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo study the impact of arsenic trioxide (As2O3) on human colorectal carcinoma LS-174T cells and their activity of telomerase.

METHODSLS-174T cells and xenograft model of nude mice were treated with As2O3. The inhibitory effect of As2O3 on survival of LS-174T cells was determined by MTT assay. Apoptosis was determined by electron microscopy and fluorescence microscopy. Cell cycle was assessed by flow cytometry. Telomerase activity in LS-174T cells was determined by PCR-ELISA kit.

RESULTSWith the increasing concentration of As2O3, the ratio of living cells to dead cells decreased significantly, and the IC50 value was 5.23 micromol/L. Apoptosis curve appeared after 24 h and cells turned to apoptosis in a time-dependent manner. As2O3 inhibited the telomerase activity in cell extraction, obviously in a concentration-dependent and time-dependent manner. Inhibitiory effect of As2O3 on xenograft model of nude mice was observed by tumor volume and weight measurement, showing a significant difference between As2O3 and control groups (P < 0.05).

CONCLUSIONBoth the experiments in vitro and in vivo showed an inhibitory effect of As2O3 on colonrectal cancer S-174T cell growth, probably by induction of apoptosis and inhibition of telomerase activity.

Animals ; Apoptosis ; drug effects ; Arsenicals ; administration & dosage ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Colonic Neoplasms ; pathology ; prevention & control ; ultrastructure ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Inhibitory Concentration 50 ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microscopy, Electron ; Microscopy, Fluorescence ; Oxides ; administration & dosage ; pharmacology ; Polymerase Chain Reaction ; methods ; Random Allocation ; Telomerase ; antagonists & inhibitors ; genetics ; metabolism ; Time Factors ; Tumor Burden ; drug effects ; Xenograft Model Antitumor Assays

BACKGROUNDTelomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells. Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis.

METHODSAntisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry.

RESULTSAS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-alpha while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group.

CONCLUSIONSAS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase activity.

Apoptosis ; Cell Line, Tumor ; Flow Cytometry ; Humans ; Oligonucleotides, Antisense ; therapeutic use ; RNA, Messenger ; analysis ; Telomerase ; analysis ; antagonists & inhibitors ; genetics ; Thionucleotides ; therapeutic use ; Tumor Necrosis Factor-alpha ; physiology ; Urinary Bladder Neoplasms ; enzymology ; pathology ; therapy

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

AIMTo investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in prostate cancer cells (PC3).

METHODSAntisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) and polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA was measured by reverse transcription PCR (RT-PCR) assay and gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by morphological method and determined by flow cytometry.

RESULTSThe telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide (S PS-ODN) and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-alphatreatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-alpha treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group.

CONCLUSIONhTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-alpha-induced apoptosis of PC3 cells.

Actins ; metabolism ; Apoptosis ; drug effects ; Cell Line, Tumor ; DNA Primers ; Humans ; Male ; Oligodeoxyribonucleotides ; pharmacology ; Prostatic Neoplasms ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; antagonists & inhibitors ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology