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Apoptosis is a physiological mechanism of cell death that plays an important role in the regulation of tissue homeostasis. The regulation of apoptosis is a complex process and involves a number of gene products including the survival factor Bcl-2, which has been found to be frequently deregulated in human cancers. In addition to deregulation of apoptosis, the process of neoplasia is also believed to be driven by the activation of telomerase, a ribonucleoprotein complex that adds telomeric repeats (hexanucleotide 5′-TTAGGG-3′) to the ends of replicating chromosomes. Activation of telomerase has been detected in a vast majority of human cancer cells. Although recent studies have demonstrated the wide occurrence of telomerase activation and Bcl-2 deregulation in human cancer cells, it remains unclear whether there is any linkage between the deregulation of Bcl-2 and telomerase activity in cancer cells. In the studies presented here, we report that the stable overexpression of Bcl-2 in human cancer cells with low Bcl-2 expression was accompanied by increased levels of telomerase activity. In addition, using an IL-2-dependent cytotoxic T-cell line, CTLL-2, we demonstrated that IL-2 deprivation (8 h), which is known to down-regulate Bcl-2 expression, also resulted in concurrent inhibition of telomerase activity in the absence of any detectable apoptosis and accumulation of cells in the G0/G1phase of the cell cycle. Re-exposure of IL-2-deprived CTLL-2 cells to the recombinant IL-2 led to the up-regulation of both Bcl-2 expression and telomerase activity. Taken together, these findings establish a close linkage between the modulation of telomerase activity by survival factor Bcl-2, and provide a model to study regulation of telomerase activity by an anti-apoptotic pathway that is widely deregulated in cancer cells. Apoptosis is a physiological mechanism of cell death that plays an important role in the regulation of tissue homeostasis. The regulation of apoptosis is a complex process and involves a number of gene products including the survival factor Bcl-2, which has been found to be frequently deregulated in human cancers. In addition to deregulation of apoptosis, the process of neoplasia is also believed to be driven by the activation of telomerase, a ribonucleoprotein complex that adds telomeric repeats (hexanucleotide 5′-TTAGGG-3′) to the ends of replicating chromosomes. Activation of telomerase has been detected in a vast majority of human cancer cells. Although recent studies have demonstrated the wide occurrence of telomerase activation and Bcl-2 deregulation in human cancer cells, it remains unclear whether there is any linkage between the deregulation of Bcl-2 and telomerase activity in cancer cells. In the studies presented here, we report that the stable overexpression of Bcl-2 in human cancer cells with low Bcl-2 expression was accompanied by increased levels of telomerase activity. In addition, using an IL-2-dependent cytotoxic T-cell line, CTLL-2, we demonstrated that IL-2 deprivation (8 h), which is known to down-regulate Bcl-2 expression, also resulted in concurrent inhibition of telomerase activity in the absence of any detectable apoptosis and accumulation of cells in the G0/G1phase of the cell cycle. Re-exposure of IL-2-deprived CTLL-2 cells to the recombinant IL-2 led to the up-regulation of both Bcl-2 expression and telomerase activity. Taken together, these findings establish a close linkage between the modulation of telomerase activity by survival factor Bcl-2, and provide a model to study regulation of telomerase activity by an anti-apoptotic pathway that is widely deregulated in cancer cells. In recent years, it has become accepted that apoptosis or programmed cell death plays an important role in the regulation of tissue development and homeostasis (1Korsmeyer S.J. Trends Genet. 1995; 11: 101-105Abstract Full Text PDF PubMed Scopus (615) Google Scholar, 2Williams G.T. Smith C.A. Cell. 1993; 74: 777-779Abstract Full Text PDF PubMed Scopus (903) Google Scholar). Deregulation of apoptosis has been shown to contribute to the pathogenesis of a number of human diseases including cancer (3Carson D.A. Ribeiro J.M. Lancet. 1993; 341: 1251-1254Abstract PubMed Scopus (692) Google Scholar, 4Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6179) Google Scholar). Regulation of apoptosis is a complex process, which involves a number of cellular genes including that for B cell leukemia/lymphoma 2 (Bcl-2) 1The abbreviations used are:Bcl-2B cell leukemia/lymphoma 2 gene productIL-2interleukin-2rIL-2recombinant interleukin-2CTLL-2cytotoxic T-cell linemAbmonoclonal antibodyPCRpolymerase chain reactionRT-PCRreverse transcription-PCRCHAPS3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acidTRAPtelomeric repeat amplification protocolbpbase pair(s)TRFterminal restriction fragment and related family members (5Boise L.H. Gottschalk A.R. Quaintans J. Thompson C.B. Curr. Top. Microbiol. Immunol. 1995; 200: 107-121Crossref PubMed Scopus (201) Google Scholar). The bcl-2 gene was first identified at the breakpoint of a chromosomal translocation t(14:18) in B follicular lymphoma (1Korsmeyer S.J. Trends Genet. 1995; 11: 101-105Abstract Full Text PDF PubMed Scopus (615) Google Scholar). A bcl-2 gene encodes a protein of 26 kDa that protects cells against apoptosis in a variety of experimental systems. Overexpression of Bcl-2 has been shown to suppress the initiation of apoptosis in response to a number of stimuli including anticancer drugs (1Korsmeyer S.J. Trends Genet. 1995; 11: 101-105Abstract Full Text PDF PubMed Scopus (615) Google Scholar, 3Carson D.A. Ribeiro J.M. Lancet. 1993; 341: 1251-1254Abstract PubMed Scopus (692) Google Scholar, 5Boise L.H. Gottschalk A.R. Quaintans J. Thompson C.B. Curr. Top. Microbiol. Immunol. 1995; 200: 107-121Crossref PubMed Scopus (201) Google Scholar, 6Mandal M. Kumar R. Cell Growth Diff. 1996; 7: 311-318PubMed Google Scholar, 7Kumar R. Mandal M. Lipton A. Harvey H. Thompson C.B. Clin. Cancer Res. 1996; 2: 115-118Google Scholar, 8Mandal M. Maggirwar S.B. Sharma N. Kaufmann S.H Sun S.-C. Kumar R. J. Biol. Chem. 1996; 271: 30354-30359Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 9Mandal M. Xipu W. Kumar R. Carcinogenesis. 1997; 18: 229-232Crossref PubMed Scopus (40) Google Scholar). Recent studies have indicated that cells from a variety of human cancer may have a decreased ability to undergo apoptosis in response to some physiologic stimuli (4Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6179) Google Scholar), and a defect in apoptosis may be involved in the aberrant survival and/or development of cancer. B cell leukemia/lymphoma 2 gene product interleukin-2 recombinant interleukin-2 cytotoxic T-cell line monoclonal antibody polymerase chain reaction reverse transcription-PCR 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid telomeric repeat amplification protocol base pair(s) terminal restriction fragment In addition to deregulation of apoptosis, it is increasingly clear that the process of neoplasia is characterized by the activation of telomerase, a ribonucleoprotein enzyme complex that adds telomeric repeats (hexanucleotide 5′-TTAGGG-3′) to the ends of replicating chromosomes, telomeres (10Blackburn E.T. Szotstak J.W. Annu. Rev. Biochem. 1984; 53: 163-194Crossref PubMed Scopus (506) Google Scholar, 11Morin G.B. Cell. 1989; 59: 521-529Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). Telomeres play an important role in chromosome structural integrity and functions including protection against the activation of DNA-damage checkpoints, and to counter the loss of terminal DNA segments that occurs when linear DNA is replicated (12Blackburn E.H. Nature. 1991; 350: 569-571Crossref PubMed Scopus (3041) Google Scholar). Recent reports have shown the involvement of telomerase function in acquisition of immortality in cancer cells as telomerase activity has been detected in the vast majority of human cancer cell lines and tumors tested, but is either absent or expressed at very low levels in most (but not all) somatic cells (13Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6514) Google Scholar, 14de Lange T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2882-2886Crossref PubMed Scopus (294) Google Scholar, 15Bacchetti S. Counter C.M. Int. J. Oncol. 1995; 7: 423-432PubMed Google Scholar). The mechanism of telomerase activation during immortalization is not known at the moment. It has been proposed (16Wright W.E. Shay J.W. Trends Cell Biol. 1995; 5 (, 1995): 293-297Abstract Full Text PDF PubMed Scopus (127) Google Scholar) that telomerase activity is repressed in somatic cells; during cell division, telomeres continue to shorten until they reach a critical point at which some cellular factor(s) detect the shortened telomere, resulting in the exit from the cell cycle (M1 crisis) and the cell's senescence. Mutations in genes detecting genetic damage allow a clonal population to continue to divide and escape senescence, resulting in further telomeric loss. Further cell division leads to M2 crisis, and most cells die. However, a rare cell somehow activates its telomerase activity, resulting in stabilization of telomere length and immortalization, which may represent an essential requirement for the expansion of human cancer cells. Thus, activation of telomerase during immortalization may be also linked with the proliferation of cancer cells (16Wright W.E. Shay J.W. Trends Cell Biol. 1995; 5 (, 1995): 293-297Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 17Shay J.W. Wright W.E. Curr. Opin. Oncol. 1996; 8: 66-71Crossref PubMed Scopus (389) Google Scholar). Although both activation of telomerase activity and Bcl-2 deregulation have been widely detected in human tumor cells, it remains unclear whether there is any linkage between the deregulation of Bcl-2 and telomerase activity in cancer cells. In the studies presented here, we investigated the influence of Bcl-2 expression on the levels of telomerase activity. We report that the overexpression of Bcl-2 in human cancer cells with low levels of Bcl-2 such as cervical carcinoma HeLa cells and colorectal carcinoma DiFi cells was accompanied by enhanced levels of telomerase activity compared with the levels of a telomerase activity present in control cells. In contrast, down-regulation of Bcl-2 expression in an IL-2-dependent cytotoxic T-cell line, CTLL-2, by IL-2 deprivation (8 h) resulted in concurrent inhibition of telomerase activity, and both of these phenotypes (down-regulation of Bcl-2 and telomerase activity in IL-2-deprived CTLL-2 cells) could be effectively reversed by the addition of rIL-2. Taken together, these results demonstrate a close linkage between the deregulation of survival factor Bcl-2 and the telomerase activity in human cancer cells. The cell lines used were human cervical carcinoma HeLa cells (8Mandal M. Maggirwar S.B. Sharma N. Kaufmann S.H Sun S.-C. Kumar R. J. Biol. Chem. 1996; 271: 30354-30359Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), human colorectal carcinoma DiFi cells (9Mandal M. Xipu W. Kumar R. Carcinogenesis. 1997; 18: 229-232Crossref PubMed Scopus (40) Google Scholar,19Wu X. Fan Z. Masui H. Rosen N. Mendelsohn J. J. Clin. Invest. 1995; 95: 1897-1905Crossref PubMed Scopus (367) Google Scholar), and mouse cytotoxic T-cell line CTLL-2 (20Jennings S.R. Bonneau R.H. Smith P.M. Wolcott R.M. Chervenak R. Cell. Immunol. 1991; 133: 234-252Crossref PubMed Scopus (111) Google Scholar). HeLa cells were cultured in modified Eagle's medium. DiFi cells were maintained in Dulbecco's modified Eagle's medium/F-12 (1:1), and CTLL-2 cells were cultured in RPMI 1640 supplemented with 0.5 ng/ml rIL-2 (Immunex). All complete culture medium contained 10% fetal bovine serum. Cells at a density of 106 cells/100-mm diameter plate were transfected with plasmid DNA containing the full-length human Bcl-2 cDNA and a selectable marker, a neomycin phosphotransferase gene (21Seto M. Jaeger U. Hockett R.D. Graminger W. Bennetts S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (456) Google Scholar), by calcium phosphate precipitation procedures as described (6Mandal M. Kumar R. Cell Growth Diff. 1996; 7: 311-318PubMed Google Scholar, 7Kumar R. Mandal M. Lipton A. Harvey H. Thompson C.B. Clin. Cancer Res. 1996; 2: 115-118Google Scholar, 8Mandal M. Maggirwar S.B. Sharma N. Kaufmann S.H Sun S.-C. Kumar R. J. Biol. Chem. 1996; 271: 30354-30359Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 9Mandal M. Xipu W. Kumar R. Carcinogenesis. 1997; 18: 229-232Crossref PubMed Scopus (40) Google Scholar). Expression of Bcl-2 in individually isolated clones was determined by immunoblotting with Bcl-2 mAb. Once a stable cell line from each clone had been established, the drug was removed from the culture medium. The clonal lines have been maintained in drug-free medium since then, and expression of Bcl-2 was periodically examined. As a control, cells were transfected either with an unrelated PKR-plasmid DNA containing neomycin marker or with plasmid DNA containing neomycin phosphotransferase gene. All experiments were performed with cells in a logarithmic phase by controlling the plating density. Cell extracts were prepared as described (26Kumar R. Atlas I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6599-6603Crossref PubMed Scopus (92) Google Scholar). Cell lysate containing equal amounts of total protein (15–25 μg) were resolved on a 10% SDS-polyacrylamide gel electrophoresis, followed by probing with an anti-Bcl-2 mAb (Neomarkers Inc.) using alkaline phosphatase-conjugated second antibody (7Kumar R. Mandal M. Lipton A. Harvey H. Thompson C.B. Clin. Cancer Res. 1996; 2: 115-118Google Scholar, 8Mandal M. Maggirwar S.B. Sharma N. Kaufmann S.H Sun S.-C. Kumar R. J. Biol. Chem. 1996; 271: 30354-30359Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). As an internal control, the same blot was cut into two pieces, and the upper portion was probed with an unrelated heat shock protein (Hsp)-70 mAb (Neomarkers Inc.) or actin antibody (Sigma). Low molecular mass colored markers (Amersham Corp.) were used as molecular weight standards. Quantitation of specific protein bands was performed by using a protein data base scanner (Molecular Dynamics). Subconfluent cultures were used to prepare the detergent CHAPS extracts (13Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6514) Google Scholar). Telomerase enzyme activity was measured by using a PCR-based telomeric repeat amplification protocol (TRAP) kit from the Oncor Inc. as described (22Olson D.J. Gibo D.M. Saggers G. Debinski W. Kumar R. Cell Growth Diff. 1997; 8: 417-423PubMed Google Scholar, 23Engelhardt M. Kumar R. Albanell J. Pettengell R. Han W. Moore M.A.S. Blood. 1997; Google Scholar). Each reaction product was amplified in the presence of an internal TRAP assay standard (36 bp). The TRAP reaction products were separated by 10% polyacrylamide gel electrophoresis, dried, and autoradiographed. The basal level of telomerase activity (ladder formation) was measured by serial dilution of the protein extracts, and an appropriate range of protein concentration was selected that produced a linear response. Each set of TRAP assay included control reaction tubes without any extract, and with extracts treated with RNase A (200 μg/ml). To quantitate the levels of telomerase activity, the average densitometric optical density of first six TRAP bands after a primer band was presented as a ratio to the internal TRAP assay standard band. Total RNA was prepared from subconfluent cells by TRIzol reagent (Life Technologies, Inc.), and 1 μg of RNA was subjected to RT-PCR using a RNA PCR core kit (Perkin Elmer) as per C.B. Cancer Res. 1996; Google Scholar). was for of reaction product were by containing for were using the C.B. Cancer Res. 1996; Google Scholar, J. S. Weinrich S.L. J. S. West M.D. Harley C.B. Science. 1995; PubMed Scopus Google Scholar). As PCR reaction product we used product product and product from control cells. Cell cycle was determined by with as described (26Kumar R. Atlas I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6599-6603Crossref PubMed Scopus (92) Google Scholar). cells were with and the of an to The resulting was in the and in the of a DNA by using a with the To a between Bcl-2 expression and telomerase activity, we investigated whether deregulation of Bcl-2 in HeLa cells the levels of telomerase activity. these HeLa cells were transfected with a Bcl-2 expression (21Seto M. Jaeger U. Hockett R.D. Graminger W. Bennetts S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (456) Google Scholar) and clonal cell lines Bcl-2 were (8Mandal M. Maggirwar S.B. Sharma N. Kaufmann S.H Sun S.-C. Kumar R. J. Biol. Chem. 1996; 271: 30354-30359Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). 1 A the levels of Bcl-2 protein in of such and 1 and levels of Bcl-2, compared with the levels detected in the HeLa cells or in the clone an gene in B the of Bcl-2 overexpression on the levels of telomerase activity. It was that overexpression of Bcl-2 in HeLa cells leads to a in the levels of telomerase activity. The in telomerase activity resulted from overexpression of Bcl-2, since it was detected in both clone 1 and clone 1 but not in control HeLa cells or HeLa cells an unrelated gene The in the levels of telomerase activity in cells was not to of telomere terminal restriction as determined by the of with telomeric not of between telomerase activity and length has been shown A. J. S. EMBO J. 1995; Scopus Google Scholar, J. Shay J.W. S. Cancer Res. 1996; Google Scholar). To whether the between Bcl-2 deregulation and enhanced telomerase activity is a to HeLa cells or whether they it with cells with low levels of Bcl-2, we the influence of Bcl-2 deregulation on the levels of telomerase activity in human colorectal carcinoma DiFi cells. 2 the levels of Bcl-2 expression in DiFi cells, its and control there was a overexpression of Bcl-2 in clone with in clone compared with DiFi cells or control in B that the clones have levels of telomerase activity compared with either control DiFi cells or The in the levels of telomerase activity were not to the presence of a telomerase in extracts as we results when the levels of telomerase activity in DiFi clones as a function of protein concentration The in the levels of telomerase activity in cells compared with the levels in control cells was not to enhanced expression of as determined by a RT-PCR assay 2 activity in DiFi and its clones as a function of protein extracts from DiFi cells and its clones were for telomerase activity by TRAP DiFi cells TRAP were performed using and μg of for its Bcl-2 clones TRAP were performed using and μg of cell μg) from clone was by with RNase A (200 μg/ml). shown of two We determined the of regulation of Bcl-2 expression on the of telomerase activity in IL-2-dependent CTLL-2 cells G. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). As from the data in G. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar), IL-2 deprivation of CTLL-2 cells for as as resulted in the down-regulation inhibition compared with control cells) of Bcl-2 expression compared with the levels present in control cells 2 with and was with down-regulation of telomerase activity at deprivation The down-regulation in the levels of telomerase activity was not to IL-2 cell cycle and/or apoptosis, as these at and not deprivation was further by the data in that there was of of IL-2 deprivation on the expression of such as and IL-2 is a known of Bcl-2 G. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar), we whether of IL-2-deprived CTLL-2 cells to rIL-2 telomerase activity. in 5 that the down-regulation of both Bcl-2 expression and telomerase activity could be reversed by the addition of rIL-2 to IL-2-deprived h) CTLL-2 cells. experiments indicated that apoptosis by IL-2 deprivation h) was by the addition of as from the data G. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). Taken together, these results that IL-2 regulation of Bcl-2 expression may be related with modulation of telomerase activity in CTLL-2 model of telomerase activity by IL-2 in CTLL-2 cells. CTLL-2 cells were cultured in IL-2-deprived medium for or CTLL-2 culture was supplemented by recombinant IL-2 for or CHAPS extracts were for the expression of Bcl-2, and B by CHAPS extracts from the were for the levels of telomerase activity. and μg of reaction without cell shown of It is accepted that apoptosis is a physiological that plays an important role in the of tissue homeostasis. Apoptosis is by specific cellular including Bcl-2, deregulation of apoptosis to the pathogenesis of a number of human diseases including and Bcl-2 deregulation is frequently with human cancer. lines of that in addition to deregulation of apoptosis, the process of neoplasia may be also driven by the activation of telomerase, a ribonucleoprotein enzyme complex that adds telomeric repeats to the ends of replicating chromosomes. Although activation of telomerase activity has been detected in human cancer cells, it remains unclear whether there is any linkage between the deregulation of Bcl-2 and telomerase activity. The present study was to the between the modulation of Bcl-2 expression and telomerase activity. We have demonstrated that the deregulation of Bcl-2 expression in human cancer cells with low levels of Bcl-2 is linked with the increased levels of telomerase activity as overexpression of Bcl-2 in HeLa and DiFi cells was accompanied by increased telomerase activity compared with control and/or cells. the activation of telomerase activity has been shown to be with the development of human cancer (16Wright W.E. Shay J.W. Trends Cell Biol. 1995; 5 (, 1995): 293-297Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 17Shay J.W. Wright W.E. Curr. Opin. Oncol. 1996; 8: 66-71Crossref PubMed Scopus (389) Google Scholar), of involvement of survival factor Bcl-2 in the deregulation of telomerase activity may provide an important into the molecular mechanism involved in the initiation of cellular The that Bcl-2 telomerase activity without of telomeric in HeLa cells is as it the that such as Bcl-2 deregulation that may influence telomerase activity in cancer cells. The that HeLa clones with levels of Bcl-2 expression 1 enhanced levels of telomerase activity that the deregulation of Bcl-2 the levels may not further telomerase activity, and level could from cell to that IL-2 deprivation of CTLL-2 cells for was to down-regulate both Bcl-2 expression and telomerase activity without any detectable apoptosis is as it a close between the levels of survival factor Bcl-2 and telomerase activity in cancer cells. IL-2 was to influence the levels of both Bcl-2 and telomerase activity in CTLL-2 cells, it remains to be resolved whether the modulation of telomerase activity by IL-2 is in Bcl-2 expression and/or a of IL-2 and/or both Bcl-2 expression and telomerase activity a Taken together, these findings provide in of between the deregulation of Bcl-2 and telomerase activity, and with the role of telomerase in the integrity of against DNA It remains to be whether the in the levels of telomerase activity is a to Bcl-2 or a with anti-apoptotic gene products such as Recent studies have indicated that the cells from a variety of human cancer may have a decreased ability to undergo apoptosis in response to physiologic and a defect in apoptosis has been proposed to be involved in the aberrant survival and/or development of cancer (4Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6179) Google Scholar). in recent years, such as of that Bcl-2 have become the of to control cancer cell In addition to apoptosis, telomerase has also a of as a in cancer The levels of telomerase activity in cells and its in human cancer cells have the that telomerase may also an important to control cell In of wide occurrence of deregulation of Bcl-2 and telomerase activity in cancer cells, to to the of close linkage between these two phenotypes has been findings of the modulation of telomerase activity by a widely deregulated survival Bcl-2, may an important model to study the regulation of telomerase activity by an pathway and could to to control cancer cell by both We J. Korsmeyer for Bcl-2 expression H. Bonneau CTLL-2 cells, Debinski for and J. and T. for primer and
Mandal et al. (Thu,) studied this question.