Glutathione Regulates Telomerase Activity in 3T3 Fibroblasts

Changes in telomerase activity have been associated either with cancer, when activity is increased, or with cell cycle arrest when it is decreased. We report that glutathione, a physiological antioxidant present at high intracellular concentrations, regulates telomerase activity in cells in culture. Telomerase activity increases in 3T3 fibroblasts before exponential cell growth. The peak of telomerase activity takes place 24 h after plating and coincides with the maximum levels of glutathione in the cells. When cells are treated with buthionine sulfoximine, which decreases glutathione levels in cells, telomerase activity decreases by 60%, and cell growth is delayed. Glutathione depletion inhibits expression of E2F4 and Id2, which regulate the cell cycle. When glutathione levels are restored after incubation with glutathione monoethylester, telomerase activity and the cell cycle-related proteins return to control values. To discover the effect of glutathione redox status on the telomerase multicomplex structure, we incubated protein extracts from fibroblasts with different glutathione redox buffers. Telomerase activity is maximal under reduced conditions i.e. when the reduced/oxidized glutathione ratio is high. Consequently glutathione concentration parallels telomerase activity. These results underscore the main role of glutathione in the control of telomerase activity and of the cell cycle. Changes in telomerase activity have been associated either with cancer, when activity is increased, or with cell cycle arrest when it is decreased. We report that glutathione, a physiological antioxidant present at high intracellular concentrations, regulates telomerase activity in cells in culture. Telomerase activity increases in 3T3 fibroblasts before exponential cell growth. The peak of telomerase activity takes place 24 h after plating and coincides with the maximum levels of glutathione in the cells. When cells are treated with buthionine sulfoximine, which decreases glutathione levels in cells, telomerase activity decreases by 60%, and cell growth is delayed. Glutathione depletion inhibits expression of E2F4 and Id2, which regulate the cell cycle. When glutathione levels are restored after incubation with glutathione monoethylester, telomerase activity and the cell cycle-related proteins return to control values. To discover the effect of glutathione redox status on the telomerase multicomplex structure, we incubated protein extracts from fibroblasts with different glutathione redox buffers. Telomerase activity is maximal under reduced conditions i.e. when the reduced/oxidized glutathione ratio is high. Consequently glutathione concentration parallels telomerase activity. These results underscore the main role of glutathione in the control of telomerase activity and of the cell cycle. The eukaryotic chromosomes are capped by telomeres, which consist of telomeric DNA repeated in tandem, associated with several proteins. These structures play an important role in the stability and the complete replication of the chromosomes. Conventional DNA polymerases cannot fully replicate the 3′-end of the lagging strand of linear molecules, and therefore in every cell division telomeric sequences are lost (1Kornberg A. Science. 1969; 163: 1410-1418Crossref PubMed Scopus (223) Google Scholar). Telomerase is an important enzyme that ensures the maintenance of normal telomere(definition) length. This activity is high in human cancers (2Kim 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 (6550) Google Scholar) but virtually absent in normal tissues, except germinal cells (3Harley C.B. Futcher A.B. Greider C.W. Nature. 1990; 345: 458-460Crossref PubMed Scopus (4632) Google Scholar). Telomerase regulation is not well understood, but its changes are related to both cancer and aging (4Sharpless N.E. DePinho R.A. J. Clin. Investig. 2004; 113: 160-168Crossref PubMed Scopus (418) Google Scholar). Glutathione (GSH) 1The abbreviations used are: GSH, glutathione; RTA, relative telomerase activity; BSO, buthionine sulfoximine; GSSG, Oxidized Glutathione. is the most abundant non-protein thiol in cells. Its activity ranges in the level of 5 μmol/gram of tissue (5Vina J. Hems R. Krebs H.A. Biochem. J. 1978; 170: 711-713Crossref PubMed Scopus (6) Google Scholar), i.e. higher than that of glucose. It has a critical role in the maintenance thiol redox status in cells. Changes in the redox ratio (GSH/GSSG) of GSH are relevant to the maintenance of enzyme activities (6Eggleston L.V. Krebs H.A. Biochem. J. 1974; 138: 425-435Crossref PubMed Scopus (332) Google Scholar). The aim of this work was to study the role of glutathione in the regulation of the telomerase activity in cells. We found that cellular glutathione levels correlate with telomerase activity in 3T3 fibroblasts. The peak of telomerase activity coincides with the GSH peak. Depletion of GSH with buthionine sulfoximine (BSO) reduces the telomerase activity and that of regulators of the cell cycle such as Id2 and E2F4 after 24 and 48 h of treatment. Furthermore, changes in the GSH/GSSG redox potential modulate telomerase activity. We concluded that the GSH levels in cells and subsequently oxidative stress regulate, at least in part, the activity of telomerase as well as that of other relevant cell cycle-related proteins. Cell Culture—3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (25 units/ml penicillin, 25 μg/ml streptomycin, and 0.3 μg/ml amphotericin B) in 5% CO2 in air at 37 °C in 25 or 75 cm2 flasks. In experiments using BSO to deplete cellular GSH, fibroblasts were cultured with and without BSO (10 μm) for 24 or 48 h. To replenish intracellular GSH levels, we cultured fibroblasts with and without glutathione monoethylester (0.2 mm) for 24 or 48 h. Flow Cytometric Analysis—Flow cytometric