Proteomic identification of proteins differentially expressed following overexpression of hTERT (human telomerase reverse transcriptase) in cancer cells

Abstract

Reverse transcriptase activity of telomerase adds telomeric repeat sequences at extreme ends of the newly replicated chromosome in actively dividing cells. Telomerase expression is not detected in terminally differentiated cells but is noticeable in 90% of the cancer cells. hTERT (human telomerase reverse transcriptase) expression seems to promote invasiveness of cancer cells. We here present proteomic profiles of cells overexpressing or knocked down for hTERT. This study also attempts to find out the potential interacting partners of hTERT in cancer cell lines. Two-dimensional gel electrophoresis (2-DE) of two different cell lines U2OS (a naturally hTERT negative cell line) and HeLa revealed differential expression of proteins in hTERT over-expressing cells. In U2OS cell line 28 spots were picked among which 23 spots represented upregulated and 5 represented down regulated proteins. In HeLa cells 21 were upregulated and 2 were down regulated out of 23 selected spots under otherwise identical experimental conditions. Some heat shock proteins viz. Hsp60 and Hsp70 and GAPDH, which is a housekeeping gene, were found similarly upregulated in both the cell lines. The upregulation of these proteins were further confirmed at RNA and protein level by real-time PCR and western blotting respectively.

hTERT promotes tumor progression by enhancing TSPAN13 expression in osteosarcoma cells

“…We have further observed that, viability of the cells was enhanced following hTERT overexpression but knocking down of TSPAN13 in this hTERT overexpressing cells repressed the viability of the cells. We have already published that hTERT overexpression in U2OS cells enhanced the migratory potential of U2OS cells but knocking down TSPAN13 decreases the migration rate of hTERT overexpressing U2OS cells. Cell cycle assay in hTERT overexpressing U2OS cells revealed that hTERT overexpression inhibits the apoptosis of the cancer cells but knocking down of TSPAN13 enhanced hTERT overexpressing U2OS cell apoptosis.…”

“…Western blotting was performed as previously described . Stable U2OS carrying pBABE‐puro empty vector and pBABE‐hTERT were lysed in RIPA buffer, (GCC biotech, West Bengal, India).…”

TGF‐β‐mediated regulation of plasminogen activators is human telomerase reverse transcriptase dependent in cancer cells

“…Western blotting was performed as previously described . Stable cells were lysed in radio immunoprecipitation assay buffer (GCC Biotech).…”

“…Western blotting was performed as previously described. 11,12 Stable cells were lysed in radio immunoprecipitation assay buffer (GCC Biotech). Cell lysates were quantified by Bradford assay, and 40 μg of total protein was separated by SDS polyacrylamide gel electrophoresis.…”

Measles Virus N and P Proteins Find Interacting Partners in Hela Cells.

“…The eluate was analyzed by SDS PAGE after removing salt by gel filtration. The corresponding bands were trypnized and submitted for MALDI-TOF Analysis Semi-Quantitative Real Time PCR:-Semi-quantitative real time PCR was carried out according to the protocol as reported earlier 12 . For this study the oligonucleotides used are listed below in Table 1.…”