Catalytically active human telomerase mutants with allele-specific biological properties

Expression of the catalytic subunit of human telomerase, hTERT, extends human primary fibroblast life span. Such life span extension has generally been reported to be accompanied by net telomere lengthening, which led to the hypothesis that it is the telomere lengthening that causes the life span extension. Here we show that hTERT+C and hTERT-FlagC, mutant telomerase proteins with either 10 additional residues or a FLAG epitope added to the hTERT C-terminus, confer significant but limited life span extension to IMR90 human primary lung fibroblasts. However, as the cells continue to grow for >100 population doublings past their normal senescence point, bulk telomere length continues to erode to lengths much shorter than those seen at the senescence of control telomerase-negative cells. Expression of hTERT+C immortalized IMR90 cells transformed by three different oncogenes. Again, bulk telomeres became much shorter than those of the control cells at crisis. Additional hTERT mutants were constructed and analyzed similarly. Enzymatically active hTERT-N125A+T126A, like other previously reported conserved GQ domain mutants and C-terminally HA-tagged hTERT, failed to extend life span. Another GQ domain mutant, hTERT-E79A, was indistinguishable from wild-type hTERT in its cell growth effects, but there was no net telomere lengthening. These results uncover further hTERT allele-specific phenotypes that uncouple telomerase activity, net telomere lengthening and life span extension.     

Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.     

Telomerase can extend the proliferative capacity of human myoblasts,but does not lead to their immortalization

Normal cells in culture display a limited capacity to divide and reach a non-proliferative state called cellular senescence. Spontaneous escape from senescence resulting in an indefinite life span is an exceptionally rare event for normal human cells and viral oncoproteins have been shown to extend the replicative life span but not to immortalize them. Telomere shortening has been proposed as a mitotic clock that regulates cellular senescence. Telomerase is capable of synthesizing telomere repeats onto chromosome ends to block telomere shortening and to maintain human fibroblasts in proliferation beyond their usual life span. However, the consequence of telomerase expression on the life span of human myoblasts and on their differentiation is unknown. In this study, the telomerase gene and the puromycin resistance gene were introduced into human satellite cells, which are the natural muscle precursors (myoblasts) in the adult and therefore, a target for cell-mediated gene therapy. Satellite cells expressing telomerase were selected, and the effects of the expression of the telomerase gene on proliferation, telomere length, and differentiation were investigated. Our results show that the telomerase-expressing cells are able to differentiate and to form multinucleated myotubes expressing mature muscle markers and do not form tumors in vivo. We also demonstrated that the expression of hTERT can extend the replicative life of muscle cells although these failed to undergo immortalization.     

Mass cultured human fibroblasts overexpressing hTERT encounter a growth crisis following an extended period of proliferation

During the process of immortalization, at least two mortality checkpoints, M1 and M2, must be bypassed. Cells that have bypassed M1 (senescence) have an extended life span, but are not necessarily immortal. Recent studies have shown that ectopic expression of the catalytic subunit of telomerase (hTERT) enables normal human cells to bypass senescence (M1) and oncogene transformed cells to avert crisis (M2) and become immortal. However, it is unclear whether hTERT expression is sufficient for normal human fibroblasts to overcome both M1 and M2 and become immortal. We have investigated the role of telomerase in immortalization by maintaining mass cultures of hTERT-transduced primary human fetal lung fibroblasts (MRC-5 cells) for very long periods of time (more than 2 years). In the present studies, up to 70% of MRC-5 cells were transduced with retroviral vectors that express hTERT. hTERT-transduced cells exhibited high levels of telomerase activity, elongation of telomeres, and proliferation beyond senescence. However, after proliferating for more than 36 population doublings (PDLs) beyond senescence, the overall growth rate of hTERT-expressing cells declined. During theses periods of reduced growth, hTERT-transduced MRC-5 cells exhibited features typical of cells in crisis, including an increased rate of cell death and polyploidy. In some instances, very late passage cells acquired a senescence-like phenotype characterized by arrest in the G1 phase of the cell cycle and greatly reduced DNA synthesis. At the onset of crisis, hTERT-transduced cells expressed high levels of telomerase and had very long telomeres, ranging up to 30 kb. Not all cells succumbed to crisis and, consequently, some cultures have proliferated beyond 240 PDLs, while another culture appears to be permanently arrested at 160 PDLs. Late passage MRC-5 cells, including postcrisis cells, displayed no signs of malignant transformation. Our results are consistent with the model in which telomerase and telomere elongation greatly extends cellular life span without inducing malignant changes. However, these investigations also indicate that hTERT-expressing cells may undergo crisis following an extended life span and that immortality is not the universal outcome of hTERT expression in normal diploid fibroblasts.     

外源性端粒酶基因对人脐静脉内皮细胞的影响

为了观察外源性端粒酶逆转录酶基因(hTERT)在人脐静脉血管内皮细胞(HUVEC)的表达及对细胞功能和生长的影响。采用逆转录病毒载体转导的方法,将hTERT基因转入HUVEC,检测基因转导后内皮细胞端粒酶的活性和生物学特性。结果发现hTERT转导后细胞端粒酶表达阳性,未转导的亲代细胞为阴性;转导细胞的体外生存时间延长但未永生化,而内皮细胞黏附分子表达的功能未受影响。     

Transient expression of human telomerase extends the life span of normal human fibroblasts

We utilized the Cre/lox recombination system to transiently express the catalytic subunit of telomerase (hTERT) in normal diploid foreskin fibroblasts (BJ cells). A retroviral construct containing an hTERT cDNA, flanked by loxP-sites was introduced into near senescent BJ cells (population doubling 85). At population doubling (PD) 92, which exceeds the typical life span of these cells, we excised the gene via Cre-mediated recombination. All clones lost telomerase activity and showed telomere shortening over an additional 50 PDs. Interestingly, the average telomere length in these cells became shorter than in untreated BJ cells at senescence. This may be due to hTERT preferentially elongating the shortest telomeres, leading to greater length uniformity. In summary, transient telomerase expression and only a very small average telomere elongation by hTERT resulted in a 50% increase in life span of human fibroblasts. This suggests a potentially safe use of hTERT in tissue engineering.     

Critical and distinct roles of p16 and telomerase in regulating the proliferative life span of normal human prostate epithelial progenitor cells

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, alpha2beta1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.