Telomerase confers resistance to caspase-mediated apoptosis

Abstract

There is growing evidence that accelerated telomeric attrition and/or aberrant telomerase activity contributes to pathogenesis in a number of diseases. Likewise, there is increasing interest to develop new therapies to restore or replace dysfunctional cells characterized by short telomeric length using telomerase-positive counterparts or stem cells. While telomerase adds telomeric repeats de novo contributing to enhanced proliferative capacity and lifespan, it may also increase cellular survival by conferring resistance to apoptosis. Consequently, we sought to determine the involvement of telomerase for reduced apoptosis using ovarian surface epithelial cells. We found that expression of hTERT, the catalytic component of telomerase, was sufficient and specific to reduce caspase-mediated cellular apoptosis. Further, hTERT expression reduced activation of caspases 3, 8, and 9, reduced expression of pro-apoptotic mitochondrial proteins t-BID, BAD, and BAX and increased expression of the anti-apoptotic mitochondrial protein, Bcl-2. The ability of telomerase to suppress caspase-mediated apoptosis was p-jnk dependent since abrogation of jnk expression with jip abolished resistance to apoptosis. Consequently, these findings indicate that telomerase may promote cellular survival in epithelial cells by suppressing jnk-dependent caspase-mediated apoptosis.

Figure 1

hTERT expression extends mean telomeric length and increases cell growth. IOSE686 and IOSE80 cells were transfected ± hTERT cDNA and assayed for telomerase activity by PCR-ELISA (A), mean telomeric length by Southern blot analysis (B), and short term growth in cell culture by MTS assay (C). Telomerase activity and cell growth are expressed as the absorbance at 450 nm and 490 nm, respectively, ± SE while mean telomeric length is expressed as average kb ± SE. Abbreviations: cDNA, single-stranded complementary deoxyribonucleic acid; hTERT, catalytic component of telomerase; IOSE, SV-40 large T-antigen transfected ovarian surface epithelial cell; PCR-ELISA, telomerase polymerase chain reaction-enzyme-linked immunosorbent assay; SE, standard error of mean.

Figure 2

Telomerase enhances cell survival. IMCC3 and IOSE80 cells were transfected ± hTERT cDNA, treated with 25 μM CP for 2 hr and then assayed for cell growth by MTS (A) or apoptosis by DNA laddering (B). Cell growth is expressed as the absorbance at 490 nm ± SE. Abbreviations: cDNA, single-stranded complementary deoxyribonucleic acid; CP, cisplatin; hTERT, catalytic component of telomerase; IOSE, SV-40 large T-antigen transfected ovarian surface epithelial cell; SE, standard error of mean.

Figure 3

Telomerase confers resistance to caspase 3-dependent apoptosis. IOSE cells transfected ± hTERT cDNA were treated with 25 μM CP for 2 hours, 20J/m² (A), or 1 μM STS for 4 hours (B), and assayed for activated caspase 3 (17 kDa and 19 kDa bands) or DFF45 cleavage (45 kDa band) by Western blot analysis. (C) Control IOSE cells, IOSE cells transfected with hTERT, and IOSE cells transfected with both hTERT and DN hTERT cDNA were treated ± 1 μM STS and examined for telomerase activity by PCR-ELISA. Telomerase activity was expressed as the absorbance at 450 nm ± SE. Samples from (C) were analyzed by Western immunoblot for activated caspase 3 (D). Actin served as a loading control for all Western immunoblots. Abbreviations: cDNA, single-stranded complementary deoxyribonucleic acid; CP, cisplatin; DFF, DNA fragmentation factor; DN, dominant negative; hTERT, catalytic component of telomerase; IOSE, SV-40 large T-antigen transfected ovarian surface epithelial cell; MTS, ; PCR-ELISA, telomerase polymerase chain reaction-enzyme-linked immunosorbent assay; SE, standard error of mean, STS, staurosporine.

Figure 4

Telomerase-mediated anti-apoptosis is caspase-dependent. IOSE cells transfected ± hTERT cDNA were treated with 1ng/ml TNF-α for 24 hours and assayed for telomerase activity by PCR-ELISA for up to 72 hours following treatment (A). Telomerase activity was expressed as the absorbance at 450 nm ± SE. Samples from (A) were also analyzed by Western immunoblot for procaspase 3, activated caspase 3 (B), cleaved caspase 9 (C), and cleaved caspase 8 (D). Actin served as a loading control for all Western immunoblots. Densitometric analysis of Western blots is provided in graphical presentation below respective blots. Abbreviations: cDNA, single-stranded complementary deoxyribonucleic acid; hTERT, catalytic component of telomerase; IOSE, SV-40 large T-antigen transfected ovarian surface epithelial cell; PCR-ELISA, telomerase polymerase chain reaction-enzyme-linked immunosorbent assay; SE, standard error of mean, STS, staurosporine; TNF-α, tumor necrosis factor-α.

Figure 5

Telomerase alters pro- and anti-apoptotic mitochondrial protein expression. IOSE cells transfected ± hTERT cDNA were treated with 1 mg/ml TNF-α for 24 hours and analyzed by Western immunoblot for Bcl-2 (A), tBID and BID (B), BAX (C), and XIAP (D). Actin served as a loading control for all Western immunoblots. Densitometric analysis of Western 2blots for the ratios of t-BID/Bcl-2, BAX/Bcl-2 and BAD/Bcl-2 are provided in graphical representation in (E–G), respectively. Abbreviations: cDNA, single-stranded complementary deoxyribonucleic acid; hTERT, catalytic component of telomerase; IOSE, SV-40 large T-antigen transfected ovarian surface epithelial cell; TNF-α, tumor necrosis factor-α.

Figure 6

Telomerase-mediated anti-apoptosis is jnk-dependent. (A) IOSE cells transfected ± hTERT cDNA were treated with 1 ng/ml TNF-α and analyzed by Western immunoblot for p-jnk and jnk. Densitometric analysis of Western blots for the levels of p-jnk is provided in graphical representation below the Western immunoblot. IOSE cells, transfected with hTERT cDNA ± jip cDNA, were treated with 1 ng/ml TNF-α for 24 hours and analyzed by Western immunoblot for p-jnk (B), cleaved caspase 8 (C), cleaved caspase 3 (D), and levels of tBID/Bcl-2 (E). The results are presented as densitometric analyses of Western blots in graphical presentation. Actin served as a loading control for all Western immunoblots. Abbreviations: cDNA, single-stranded complementary deoxyribonucleic acid; hTERT, catalytic component of telomerase; IOSE, SV-40 large T-antigen transfected ovarian surface epithelial cell; TNF-α, tumor necrosis factor-α.