Date Published: July 13, 2017
Publisher: Public Library of Science
Author(s): Rishi Kumar Jaiswal, Pramod Kumar, Amod Sharma, Deepak Kumar Mishra, Pramod Kumar Yadava, Gabriele Saretzki.
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.
Cancer cells have unlimited proliferation potential. One way of acquiring this involves reactivation of a specialized reverse transcriptase called telomerase which solves the end replication problem by adding telomeric repeats on to the 3’ ends of template strands so as to minimize on attrition of the lagging strands at their terminal 5’ ends. Telomerase activity is found to be high in nearly 90% of cancerous cells as compared to normal differentiated somatic cells which do not have detectable telomerase activity. The telomerase basically consists of six main subunits viz. hTERT (human telomerase reverse transcriptase), dyskerin, p23, Hsp90, hTERC (human telomerase RNA component) and TEP1 (telomerase-associated protein 1) . Out of these six subunits, hTERT and hTERC can reconstitute the classical telomere lengthening in vitro and also perform many extracurricular functions of regulatory nature in vivo . Stabilization of telomere length of fibroblast and other cell types is achieved by ectopic expression of hTERT in these cell lines which thus acquire infinite replicative potential . Immortalization of both cancer cells and normal stem cells can be achieved by overexpression of telomerase [4–6]. Moreover, knowing the main roles of telomerase in cancer cells would be helpful in the development of exact therapeutic strategies on the basis of telomerase inhibition [7,8]. Here, we have studied proteomic profile of cells following hTERT overexpression in two different cell lines viz., the human osteosarcoma cell line U2OS, which is telomerase negative and HeLa, a cervical cancer cell line that has its own telomerase activity.
Telomerase components perform many functions apart from its canonical role in telomere lengthening. One such extracurricular function of hTERT is the regulation of cell’s signaling pathways. Many types of cancer do not express telomerase and being telomerase negative cannot manifest extracurricular activity of telomerase. They have ALT pathway to maintain telomere length. Remarkably, hTERT could be expressed at very high levels even in cells with a basal endogenous level of expression and allowed normal viability. Telomerase negative cells nevertheless offer a good experimental system to study the effect of hTERT overexpression on expression of other genes. Human osteosarcoma is a primary malignant tumor of the bone and the U2OS cell line derived from it is telomerase negative. In the present study, we document, for the first time significant effect of hTERT overexpression on the proteomic profile of U2OS cells. Here, we found Hsp90, Hsp70 and Hsp60 upregulated after hTERT overexpression in this cell line. Heat shock proteins usually act as molecular chaperones and are expressed at high levels in many cancers, although Hsp overexpression is only a poor prognosis in terms of survival and response to therapy in specific cancer types . Heat shock protein 90 (Hsp90) is an abundant molecular chaperone that helps in conformational maturation and stabilization of various oncogenic proteins leading to tumor cell survival and disease progression . Hsp90 interacts with a variety of intracellular proteins and is involved in differentiation, survival and cell growth . It is demonstrated that inhibiting Hsp90 in osteosarcoma cells induces apoptosis . Jennifer McCleese (2009) reported that inhibitioin of Hsp90 results in loss of cell viability, induction of apoptosis, and inhibition of cell proliferation in osteosarcoma cells . It has also been shown that blocking HSP90 addiction inhibits tumor cell proliferation, metastasis and development . Moreover, Hsp90 is also a subunit of telomerase complex and it stabilizes telomerase and helps in loading of telomerase complex to telomere . Though there is no hTERT in U2OS cells, its overexpression upregulated Hsp90 (Fig 4d) suggesting intactness of the circuitry of intercommunication between hTERT and Hsps in telomerase negative cells.