Human telomerase accelerates growth of lens epithelial cells through regulation of the genes mediating RB/E2F pathway

Abstract

Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The catalytic core of human telomerase is composed of an RNA template known as hTER (human telomerase RNA) and a protein subunit named hTERT (human telomerase reverse transcriptase). We have been studying other functions of the telomerase besides its major role in telomere maintenance. In our previous work, we have demonstrated that the hTERT can functionally interact with a rabbit TER to regulate expression of other genes and also attenuate the induced apoptosis. Here we report that overexpression of hTERT in a human lens epithelial cell line accelerates growth of the transfected lens epithelial cells. Associated with the acceleration of cell growth, expression of p53, p21 and GCIP (Grap2 cyclin-D interacting protein) is downregulated in the hTERTtransfected cells. With the downregulation of p21 and GCIP, the retinoblastoma protein (RB) is completely hyperphosphorylated in the hTERT-transfected cells. As expected, in the presence of RB hyperphosphorylation, the E2F transactivity is upregulated. Inhibition of telomerase activity abolishes the observed growth acceleration and also the related molecular changes. Furthermore, expression of hTERT in telomerasenegative human lens epithelial cells derived from primary cultures also accelerates growth of the transfected cells. Taken together, our results suggest that hTERT, when overexpressed in human lens epithelial cells, accelerates cell growth rate through regulation of RB/E2F pathway and possibly other genes.

Actions of human telomerase beyond telomeres

“…(b) Similarly, several tumor suppressors including pRB and p53 negatively regulate hTERT expression and repress telomerase activity [6]. However, overexpression of hTERT in human lens epithelial cell lines results in down-regulation of p21, p53 expression, as well as hyperphosphorylation of pRB and up-regulation of E2F transcriptional activity [77]. And suppression of hTERT expression also results in elevated p53 and p21 transcription [78].…”

Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities

“…Both p53À/À MLECs (c) and p53À/À PC-3 cells (d) were transfected and harvested as described in (a) and (b). The harvested cells were used for extraction of enzymes for the detection of the b-galactosidase and CAT activities as described previously (Xiang et al, 2002). As control, the same reporter gene with the p53-binding site mutated, together with an efficacy plasmid, pRSVb-Gal, and the wild-type p53 expression construct (pCI-p53) or each of the mutant p53 expression constructs (pCI-p53-S15A, pCIp53-S37A, pCI-p53-S15A/S37A, pCI-p53-S15D, pCI-p53-S37D, pCI-p53-S15D/S37D) was also transfected into both p53À/À MLECs (c) and p53À/À PC-3 cells (d).…”

“…The HLECs were grown in Dulbecco's modified Eagle's minimal essential medium (DMEM) (Invitrogen) containing 10% fetal bovine serum as described previously (Xiang et al, 2002). The medium was prepared in ion-exchanged doubledistilled water to give an osmolarity of 30075 mosmol supplemented with 26 mM NaHCO 3 and 50 units/ml penicillin and streptomycin.…”

“…The transfected cells were plated in 100-mm culture dishes for growth and then harvested after 48 h for detection of expression of various forms of p53 and for assays of b-galactosidase and CAT activities as described previously (Xiang et al, 2002).…”

Human Bcl-2 activates ERK signaling pathway to regulate activating protein-1, lens epithelium-derived growth factor and downstream genes

“…The stable lines of vectortransfected cells (column 1), Bcl-2-transfected cells (columns 2 and 4) and both Bcl-2-and antisense-bcl-2-transfected cells (column 3) were further transfected with a CAT reporter gene (Figure 7a) driven by the bA3/A1-crystallin gene promoter (À382 to þ 22) carrying a native AP-1 site (columns 1-3) or a mutated AP-1 site (column 4) located at À264 to À258 together with a control plasmid pRSV-b-Gal. At 48 h after transfection, the cells were harvested for analysis of CAT activity as described (Xiang et al, 2002). Note that Bcl-2 positively regulated the chicken bA3/A1-crystallin gene promoter and this regulation is AP-1 dependent.…”