Korean J Lab Med.  2006 Aug;26(4):287-293. 10.3343/kjlm.2006.26.4.287.

Modulation of Telomerase Activity and Human Telomerase Reverse Transcriptase Expression by Caspases and Bcl-2 Family Proteins in Cisplatin-Induced Cell Death

• Park YP
• Choi SC
• Cho MY
• Song EY
• Kim JW
• Paik SG
• Kim YK
• Kim JW
• Lee HG

• KMID: 2238837
• DOI: http://doi.org/10.3343/kjlm.2006.26.4.287

Abstract

BACKGROUND: Human telomerase is a ribonucleoprotein polymerase, which synthesizes telomeric repeat sequences, and human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit, as well as the rate-limiting component, of telomerase. In this study, we attempted to identify the modulators of telomerase, and to determine the molecular mechanisms underlying cisplatin-induced apoptosis.
METHODS
To determine the role of telomerase in cisplatin-induced apoptosis, we measured telomerase activity and analyzed apoptosis using PI and trypan blue staining. Also, we inhibited the caspase activations using Z-VAD-fmk to analyze the effects on expression of hTERT protein. Finally, we induced the transient co-expression of the Bcl-2 and Bak genes in HEK293 cells, and then, the telomerase activity and expression of hTERT were evaluated.
RESULTS
In the Bcl-2-overexpressing HeLa cells, telomerase activity was more enhanced, and cell death was reduced to 40-50% that of the mock controls. This finding suggests that Bcl-2-induced telomerase activity exerts an antiapoptotic effect in cisplatin-induced death. As caspase activation was inhibited via Z-VAD-fmk, the hTERT protein was recovered in the mock controls, but not in the Bcl-2-overexpressing cells. This suggests that the expression of hTERT can be regulated by caspases, but Bcl-2 was located within the upstream pathway. Moreover, when the Bcl-2 and Bak genes were co-transfected into the HEK293, both telomerase activity and hTERT protein were prominently reduced.
CONCLUSIONS
Bcl-2-induced telomerase activity inhibits cisplatin-induced apoptosis in HeLa cells, and can be regulated via both caspases and the interaction of Bcl-2 and Bak.

Keyword

Apoptosis; Cisplatin; hTERT; Bcl-2; Bak

MeSH Terms

Apoptosis
Caspases*
Catalytic Domain
Cell Death*
Cisplatin
HEK293 Cells
HeLa Cells
Humans*
Ribonucleoproteins
Telomerase*
Trypan Blue
Caspases
Cisplatin
Ribonucleoproteins
Telomerase
Trypan Blue

Figure

• 

  Fig. 1. Telomerase activity in cisplatin-induced cell death. (A) Viability of HeLa cells were decreased by cisplatin dose-dependently. The listed values of optical density (OD) are expressed as the means ±SD of triplicates. (B) Bcl-2 wt expression enhanced the telomerase activity contrast to mock controls. Heat-inactivated extract was used as a control for specificity. (C, D) Fifty percents of apoptotic cells by cisplatin were reduced by Bcl-2 wt expression. Apoptotic cell death was evaluated via propidium iodide staining (C) and trypan blue staining (D).

• 

  Fig. 2. hTERT expression by Z-VAD-fmk inhibition in cisplatin-induced cell death. Bcl-2 and hTERT expression were evaluated via Western blot analysis. hTERT expression was changed by caspase inhibition. hTERT protein level was more increased by pretreatment of Z-VAD-fmk in mock control cells but not in Bcl-2 wt expressing cells.

• 

  Fig. 3. Telomerase activity and hTERT expression by interaction of Bcl-2with Bak. (A) HEK293 cells were co-transfected with a Bcl-2 wt (0.5 μg) and Bak (1–0.125 μg), or empty vectors. After 24 hr of incubation, telomerase activity was measured. Telomerase activity in Bcl-2and 0.5 μg of Bak co-expressing cells was reduced in contrast to other Bcl-2wt expressing cells. (B) Western blot analysis for the detection of Bcl-2and Bak or hTERT. Reduced expression of hTERT appeared in Bcl-2 wt and 0.25–0.5 μg of Bak co-expressing cells. Alpha tubulin was utilized to ensure equal loading. *This study was supported by grants of FG05–40–01, NTM 0020213 of the 21C frontier function human genome project from ministry of science & technology of Korea.

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