AZT on telomerase activity and cell proliferation in HS 839.T melanoma cells / AZT na atividade da telomerase e na proliferação de células de melanoma HS 839.T

PURPOSE: To evaluate telomerase activity and proliferation of HS839.T melanoma cells, subjected to the action of AZT. METHODS: Cells were grown in triplicate, AZT at different concentrations: 50, 100 and 200μM, was added and left for 24 and 48 hours, and its effects were compared with the control group. Telomerase activity was detected by PCR and cell proliferation was evaluated by MTT. RESULTS: After 24 hours, there was no inhibition of cell proliferation or telomerase activity when compared to the control group. After 48 hours, there was a momentary decrease, suggesting that the cell lines used in this study are sensitive to AZT, but quickly recover both the enzyme activity and cell proliferation. CONCLUSION: The action of AZT on the melanoma cells studied, at the concentrations and times tested, did not inhibit telomerase activity nor affect cell proliferation.

OBJETIVO: Avaliar a atividade da telomerase e da proliferação de células de melanoma HS839.T submetidas à ação do AZT. MÉTODOS: As células foram cultivadas, em triplicata, com diferentes concentrações de AZT: 50, 100 e 200µM, por 24h e 48h, seus efeitos comparados com o grupo controle. A atividade da telomerase foi detectada por PCR e a proliferação celular avaliada por MTT. RESULTADOS: No tempo de 24 horas, não houve inibição da proliferação celular e da atividade da telomerase em comparação com o grupo controle. No período de 48 horas, houve uma diminuição momentânea, sugerindo que as células das linhagens utilizadas neste estudo são sensíveis ao AZT, mas que recuperam a atividade enzimática e proliferativa. CONCLUSÃO: Nas células de melanoma HS839.T estudadas e nas concentrações e tempos propostos, a ação do AZT não inibiu a atividade da telomerase e não afetou a proliferação celular.

Human Telomerase Reverse Transcriptase (hTERT): A Target Molecule for the Treatment of Cisplatin-resistant Tumors / 대한진단검사의학회지

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.

Gene expression responses in vivo by human telomerase reverse transcriptase (hTERT)-targeting trans-splicing ribozyme

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.

Differentiation of human nasopharyngeal carcinoma xenografts and repression of telomerase activity induced by arsenic trioxide.

BACKGROUND: Arsenic trioxide (As2O3) induced apoptosis and differentiation of acute promyelocytic leukaemia. A few in vivo experimental investigations of its efficacy in solid tumours have been done. This study was designed to explore the differentiation-inducing effect, and the possible mechanisms involved, of As2O3 on human nasopharyngeal carcinoma CSNE-1 xenografts. METHODS: Nasopharyngeal carcinoma cell CSNE-1 was established as a xenograft in nude mice. The tumour-bearing mice were treated with As2O3 at a dose of 5 mg/kg/day. To assess tumour differentiation, tumour growth was observed and histological changes were analysed under light and electron microscopy. Expression of latent membrane protein 1 (LMP1) and cytokeratin 4 (CK4) was determined by immunohistochemistry. A PCR-based telomeric repeat amplification protocol assay (TRAP-ELISA) was used to measure telomerase activity. RESULTS: The xenografts underwent differentiation. LMP 1 of the cells decreased significantly and there was a pronounced decline in telomerase activity. CONCLUSION: As2O3 can inhibit xenograft growth and induce morphological and functional differentiation of CSNE-1 cells. The As2O3-induced differentiation was associated with downregulation of telomerase activity.

In vitro and in vivo inhibition of telomerase activity from Chinese hamster V79 cells.

While studying the inhibition of telomerase activity in Chinese hamster V79 cells using polymerase chain reaction (PCR) based telomeric repeat amplification protocol (TRAP) assay, we had earlier observed that 7-deaza deoxy guanosine triphosphate (7-deaza dGTP) and oligonucleotide (TTAGGG)4 inhibited telomerase activity in vitro. In the present study, we report inhibition of telomerase activity by modified base 7-deaza deoxy adenosine triphosphate (7-deaza dATP) and phosphorothioate TTAGGG (PS-TTAGGG). Both the compounds inhibited telomerase activity in a concentration dependent manner; 8.5 microM of 7-deaza dATP and 0.1 microM of PS-TTAGGG being the concentration for 50% of the maximum inhibition. This observation supports our earlier hypothesis that incorporation of a modified nucleotide into telomere possibly interferes with the recognition of the telomerase and TTAGGG interferes with the RNA component of telomerase. We have further shown that treatment of cells with nicotinamide (NA) and benzamide (BA), well known inhibitors of poly (ADP-ribose) polymerase, reduced telomerase activity. We speculate that modification of the telomeric binding proteins or other components by poly (ADP-ribosyl)ation may be involved in such inhibition.

Protein kinase C modulates telomerase activity in human cervical cancer cells

Telomerase, a ribonucleoprotein reverse transcriptase that extends telomeres of eukaryotic chromosomes is repressed in normal somatic cells but is activated during development and neoplasia. The regulation mechanism of telomerase activity in cancer cells is not clearly known. In this report, a possible affect of PKC on telomerase activity was examined using HeLa and CUMC-6 cervical cancer cell lines. Exposure of cells to PKC inhibitor, bisindolylmaleimide I and Go6976, and high levels of PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in the inhibition of PKC activity in both cells. Telomerase activities were also inhibited by bisindolyl-maleimide I and Go6976, respectively, in a time-dependent manner. As PKC activity changes in TPA-treated cervical cancer cells, telomerase activities were increased at low dose of TPA and decreased at high dose. The expression levels of human telomerase subunits, human telomerase RNA (hTR) were not influenced by PKC modulating drugs. In contrast, the expression of full-length human telomerase reverse transcriptase (hTERT) was decreased after exposure to bisindolylmaleimide I and Go6976 in a time-dependent manner. hTERT expression was not affected by low dose of TPA. In contrast, high dose of TPA inhibited hTERT expression level. But the expression patterns of beta-deletion transcript of hTERT after 72 h of treatment with PKC inhibitors or high dose of TPA exposure were not discernable as compared with those of full-length hTERT transcripts to PKC modulating drugs. These results suggest that PKC-modulating drugs altered telomerase activities by affecting full-length hTERT expression profile in human cervical cancers.

Detection of Telomerase Activity in Psoriasis Lesional Skin and Correlaton with Ki-67 Expression and Suppression by Retinoic Acid

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.