The ectopic expression of telomerase in normal human cells results in an extended lifespan, indicating that telomere shortening regulates the timing of cellular senescence. As telomerase expression is a hallmark of cancer, we investigated the long-term effects of forced expression of human telomerase catalytic component (hTERT) in normal human fibroblasts. In vitro growth requirements, cell-cycle checkpoints and karyotypic stability in telomerase-expressing cells are similar to those of untransfected controls. In addition, co-expression of telomerase, the viral oncoproteins HPV16 E6/E7 (which inactivate p53 and pRB) and oncogenic HRAS does not result in growth in soft agar. Thus, although ectopic expression of telomerase in human fibroblasts is sufficient for immortalization, it does not result in changes typically associated with malignant transformation.

Absence of cancer-associated changes in human fibroblasts immortalized with telomerase

Telomerase and telomere length have received a lot of recent attention from the biomedical research community. The cancer field started to take an interest in telomerase and telomere length approximately 7 years ago. Early experiments showed that telomeres shorten in primary fibroblasts with increased divisions in culture (1, 2) and that telomerase activity was not detected in these primary cultures. In contrast, after immortalization, telomere lengths are stabilized and telomerase is active (3). This suggested that telomerase might be required for the indefinite growth of immortal cells in culture. Around the same time, other experiments showed that tumors have shorter telomeres than normal tissue (2, 4) and that telomerase is active in many tumors and not in many normal samples (5, 6). Thus, the idea emerged that telomerase might be required for tumor growth in vivo. Testing this idea has fed the exponential increase in telomerase papers in the past 5 years.

https://www.pnas.org/content/pnas/95/1/90.full.pdf

Telomerase activity, cell proliferation, and cancer

It has previously been shown that the E7 protein from the cutaneous human papillomavirus type 1 (HPV1), which is associated with benign skin lesions, binds the product of the tumor suppressor gene retinoblastoma (pRb) with an efficiency similar to that of the E7 protein from the oncogenic HPV type 16. Despite this ability, HPV1 E7 does not display any activity in transforming primary cells. In addition, the two viral proteins differ in their mechanisms of targeting pRb. HPV16 E7 promotes pRb destabilization, while cells expressing HPV1 E7 do not show any decrease in pRb levels. In this study, we show that HPV1 E7, in contrast to HPV16 E7, has only a weak activity to neutralize the effect of cyclin-dependent kinase inhibitor p16 INK4a . By generation of HPV1/16 E7 chimeric proteins, we have identified a central motif in the two E7 proteins, which determines their different abilities to overcome the p16 INK4a -mediated cell cycle arrest. This motif is located downstream of the pRb-binding domain and comprises only three amino acids in HPV16 E7. Swapping this central motif in the two viral proteins causes an exchange of their activities involved in circumventing the inhibitory function of p16 INK4a . Most importantly, our data show that the efficiency of the E7 proteins in neutralizing the inhibitory effect of p16 INK4a correlates with their ability to promote pRb degradation.

Induction of pRb Degradation by the Human Papillomavirus Type 16 E7 Protein Is Essential To Efficiently Overcome p16INK4a-Imposed G1 Cell Cycle Arrest

Cellular transformation by human papillomaviruses: Lessons learned by comparing high- and low-risk viruses

Telomeres cap and protect the ends of chromosomes from degradation and illegitimate recombination. The termini of a linear template cannot, however, be completely replicated by conventional DNA-dependent DNA polymerases, and thus in the absence of a mechanisms to counter this effect, telomeres of eukaryotic cells shorten every round of DNA replication. In humans and possibly other higher eukaryotes, telomere shortening may have been adopted to limit the life span of somatic cells. Human somatic cells have a finite proliferative capacity and enter a viable growth arrested state called senescence. Life span appears to be governed by cell division, not time. The regular loss of telomeric DNA could therefore serve as a mitotic clock in the senescence programme, counting cell divisions. In most eukaryotic organisms, however, telomere shortening can be countered by the de novo addition of telomeric repeats by the enzyme telomerase. Cells which are "immortal' such as the human germ line or tumour cell lines, established mouse cells, yeast and ciliates, all maintain a stable telomere length through the action of telomerase. Abolition of telomerase activity in such cells nevertheless results in telomere shortening, a process that eventually destabilizes the ends of chromosomes, leading to genomic instability and cell growth arrest or death. Therefore, loss of terminal DNA sequences may limit cell life span by two mechanisms: by acting as a mitotic clock and by denuding chromosomes of protective telomeric DNA necessary for cell viability.

The roles of telomeres and telomerase in cell life span.

The two-hybrid system was used to detect interactions in vivo between HPV E7 and three 'Rb-like proteins', pRb, p107 and p130. The association between pE7 and pRb parallel the oncogenic potential of the specific HPV types. In contrast, the interaction between pE7 and p107 or p130 differ. While the HPV 16 E7 protein associates with the 'Rb-like' proteins strongly, both HPV 18 and 6b E7 proteins bind more weakly. We tested several HPV 6 E7 mutants carrying single amino acid mutations. Substitution of the glycine at position 22 with an aspartate was the only mutation capable of increasing the ability of HPV 6 E7 protein to bind pRb. However, association with p107 and p130 by the HPV 6 E7 protein was also increased by mutation of the arginine at position 4 with an aspartate. These data suggest that pRb, p107 and p130 interact with similar but non-identical domains of pE7. In addition, we used amphotropic retroviruses encoding the HPV 18 E6 and the different E7 genes to analyze their immortalizing activity. The wild-type HPV Is and 16 E7 genes complemented the HPV 18 E6 gene to immortalize human keratinocytes. In comparison, none of the cells infected with HPV 6 E7, wildtype or mutant- encoding retroviruses, became immortal. Thus, our data suggest that HPV 6 E7 lacks a property independent of pRb-association which is required for immortalization of human keratinocytes.

In-vivo analysis of hpv e7 protein association with prb, p107 and p130.

Human fibroblast cells must overcome both the M1 and the M2 stages of cellular senescence to immortalize, at which point cells almost always express telomerase activity. The human papillomavirus (HPV) oncoproteins, HPV-16 E6 and E7, can block the progression to senescence in fibroblasts by associations with p53 and pRb, respectively. Human mammary epithelial (HME) cells require only HPV-16 E6 to bypass M1, suggesting that pRb may not have a direct role in HME cells senescence. In the present report, we show that only wild-type HPV-16 E6 allows complete degradation of p53, immortalization and reactivation of telomerase activity in HME cells. These results suggest that the ability of HPV-16 wild-type and mutant E6 proteins to degrade p53 in intact HME cells and keratinocytes does not completely correlate with their ability to degrade p53 in a cell-free system. This discrepancy between in vitro and in vivo p53 degradation may be biologically significant and may provide insight into the susceptibility of certain human cells and tissues for reactivation of telomerase and immortalization.

p53 levels in human mammary epithelial cells expressing wild-type and mutant human papillomavirus type 16 (HPV-16) E6 proteins

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In-vivo analysis of hpv e7 protein association with prb, p107 and p130.

p53 levels in human mammary epithelial cells expressing wild-type and mutant human papillomavirus type 16 (HPV-16) E6 proteins