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

D.А. Vasina(1), D.D. Zhdanov(1, 2), E.V. Orlova(3), V.S. Orlova(1), M.V. Pokrovskaya(2), S.S. Aleksandrova(2), N.N. Sokolov(2)
1-Peoples` Friendship University of Russia, Miklukho-Макlay str., 6, Moscow, Russian Federation, 117198;
2-Institute of Biomedical Chemistry, Pogodinskayast, 10, building 8, Moscow, Russian Federation, 119121;
3-Institute of Theoretical and Experimental Biophysics, Institutskaya st. 3, Pushchino, Moscow region, Russian Federation, 142290
E-mail: zhdanovdd@mail.ru

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