OVEREXPRESSION OF APOPTOTIC ENDONUCLEASE ENDOG INHIBITS TELOMERASE ACTIVITY IN HUMAN CD4+ T CELLS

DOI: https://doi.org/None

Vasina D.А., Zhdanov D.D., Orlova E.V., Orlova V.S., Pokrovskaya M.V., Aleksandrova S.S., Sokolov N.N.

Introduction. Alternative splicing of telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase) regulates the telomerase activity. The increased expression of non-active spliced hTERT results in inhibition of telomerase. However, very little is known about the mechanism of hTERT mRNA alternative splicing. Apoptotic endonucleases are known to participate in this process. The aim of the study was to determine the function of apoptotic endonuclease EndoG in the alternative splicing of hTERT and telomerase activity in normal human T lymphocytes. Methods. Human CD4+-Т-lymphocytes were transfected with plasmid containing EndoG gene pEndogG-GFP or control plasmid pGFP. The expression of EndoG and hTERT splice-variants was determined by real-time RT-PCR. Intracellular protein amounts were analyzed by Western blotting. Telomeric repeat amplification protocol was used to measure telomerase activity. Results. Correlation analysis revealed the strong correlation between the expression of EndoG and hTERT splice-variants in CD4+- and СD8+Т-lymphocytes from 12 healthy donors. This correlation is more obvious in CD4+-T-cells. The overexpression of EndoG in CD4+-T-cells downregulated the expression of the active full-length hTERT variant and upregulated the non-active spliced variant. Reduction of full-length hTERT causes downregulation of telomerase activity 16 hours later transfection. Conclusion. In this work we demonstrated the correlation between the expression of EndoG and hTERT splice variants in human T lymphocytes. The EndoG overexpression results in alternative splicing of hTERT mRNA and downregulation of telomerase activity in CD4+Т cells
Keywords: 
EndoG, telomerase, hTERT, transfection, alternative splicing
Список литературы: 
  1. Blackburn E.H. Telomere states and cell fates. Nature. 2000; 408 (6808): 53–6.
  2. Kim N.W., Piatyszek M.A., Prowse K.R., Harley C.B., West M.D., Ho P.L. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994; 266 (5193): 2011–5.
  3. Meyerson M., Counter C.M., Eaton E.N., Ellisen L.W., Steiner P., Caddle S.D. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell. 1997; 90 (4): 785–95.
  4. Saebøe-Larssen S., Fossberg E., Gaudernack G. Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT): analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues. BMC Mol. Biol. 2006; 7: 26.
  5. Ulaner G.A., Hu J.F., Vu T.H., Oruganti H., Giudice L.C., Hoffman A.R. Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium. Int. J. Cancer. 2000; 85 (3): 330–5.
  6. Ulaner G.A., Hu J.F., Vu T.H., Giudice L.C., Hoffman A.R. Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts. Cancer Res. 1998; 58 (18): 4168–72.
  7. Listerman I., Sun J., Gazzaniga F.S., Lukas J.L., Blackburn E.H. The major reverse transcriptase-incompetent splice variant of the human telomerase protein inhibits telomerase activity but protects from apoptosis. Cancer Res. 2013; 73 (9): 2817–28.
  8. Harley C.B., Futcher A.B., Greider C.W. Telomeres shorten during ageing of human fibroblasts. Nature. 1990; 345 (6274): 458–60.
  9. Oulton R., Harrington L. A human telomerase-associated nuclease. Mol Biol Cell. 2004; 15 (7): 3244–56.
  10. Lydeard J.R., Jain S., Yamaguchi M., Haber J.E. Break-induced replication and telomerase-independent telomere maintenance require Pol32. Nature. 2007; 448 (7155): 820–3.
  11. Nagata S., Nagase H., Kawane K., Mukae N., Fukuyama H. Degradation of chromosomal DNA during apoptosis. Cell Death Differ. 2003; 10 (1): 108–16.
  12. Ruiz-Carrillo A., Renaud J. Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes. EMBO J. 1987; 6 (2): 401–7.
  13. Diener T., Neuhaus M., Koziel R., Micutkova L., Jansen-Dürr P. Role of endonuclease G in senescence-associated cell death of human endothelial cells. Exp Gerontol. 2010; 45 (7–8): 638–44.
  14. Basnakian A.G., Apostolov E.O., Yin X., Abiri S.O., Stewart A.G., Singh A.B. Endonuclease G promotes cell death of non-invasive human breast cancer cells. Exp Cell Res. 2006; 312 (20): 4139–49.
  15. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227 (5259): 680–5.
  16. Hofnagel O., Luechtenborg B., Stolle K., Lorkowski S., Eschert H., Plenz G. Proinflammatory cytokines regulate LOX-1 expression in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004; 24 (10): 1789–95.
  17. Kovalenko N.A., Zhdanov D.D., Bibikova M.V., Gotovtseva V.I. The influence of compound aITEL1296 on telomerase activity and the growth of cancer cells Biomed Khim. 2011; 57 (5): 501–10.