Abstract
Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The functional telomerase complex contains a telomerase reverse transcriptase catalytic subunit and a telomerase template RNA. We have previously demonstrated that human telomerase reverse transcriptase (hTERT) catalytic subunit is functionally compatible with a telomerase template RNA from rabbit. In this study, we show that hTERT is also functionally compatible with a telomerase template RNA from bovine. Introduction of hTERT into bovine lens epithelial cells (BLECs) provides the transfected cells telomerase activity. The expressed hTERT in BLECs supports normal growth of the transfected cells for 108 population doublings so far, and these cells are still extremely healthy in both morphology and growth. In contrast, the vector-transfected cells display growth crisis after 20 population doublings. These cells run into cellular senescence due to shortening of the telomeres and also commit differentiation as indicated by the accumulation of the differentiation markers, beta-crystallin and filensin. hTERT prevents the occurrence of both events. By synthesizing new telomere, hTERT prevents replicative senescence, and through regulation of MEK/ERK, protein kinase C, and protein kinase A and eventual suppression of the MEK/ERK signaling pathway, hTERT inhibits differentiation of BLECs. Our finding that hTERT can suppress RAS/RAF/MEK/ERK signaling pathway to prevent differentiation provides a novel mechanism to explain how hTERT regulates cell differentiation.
Highlights
Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes
The Establishment of the hTERTExpression Bovine Lens Epithelial Cell Line—We have previously demonstrated that human telomerase reverse transcriptase catalytic subunit (hTERT) is functionally compatible with the telomerase RNA from rabbit [28]
To further confirm the functional compatibility between hTERT and other vertebrate template RNA (TR), we have introduced the hTERT into bovine lens epithelial cells (BLEC) derived from primary cultures
Summary
Juan Wang , Hao Feng , Xiao-Qin Huang , Hua Xiang , Ying-Wei Mao , Jin-Ping Liu, Qin Yan, Wen-Bin Liu, Yan Liu, Mi Deng, Lili Gong, Shuming Sun, Chen Luo, Shao-Jun Liu, Xuan-Jie Zhang Yun Liu and David Wan-Cheng Li 1, 2, 3. We have previously demonstrated that human telomerase reverse transcriptase catalytic subunit (hTERT) is functionally compatible with a telomerase template RNA from rabbit (rTR). The encoding sequence for this catalytic subunit has been cloned in human and other species [9,10,11,12,13,14] Telomerase from these different sources shares both sequence and functional similarity with the reverse transcriptase [15]. Cell Extract Preparation and Telomerase Activity Assay—Cellular extracts from parental BLECs, vector-transfected BLECs (pCI-BLEC) and hTERTtransfected BLECs (pCI-hTERT-BLEC) were prepared in 1 × CHAPS lysis buffer and cleared by centrifugation for 20 min at 4oC (12,000g) These extracts were assayed for telomerase activity with a PCR-based telomeric repeat amplification protocol (TRAP) [33] using a kit from Intergen. At the end of each reaction, 25 μl of the reaction products were analyzed in a 10% polyacrylamide gel
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