HPV-16 E6 and E7 Genes, like SV40 Early Region Genes, Are Insufficient for Immortalization of Human Mesothelial and Bronchial Epithelial Cells

doi:10.1006/excr.1994.1218

Experimental Cell Research

Volume 213, Issue 2, August 1994, Pages 418-427

https://doi.org/10.1006/excr.1994.1218 Get rights and content

Abstract

Simian virus 40 (SV40)-transformed human cells usually have a period of increased proliferative potential that terminates in culture crisis, after which immortalization may or may not occur. This study addresses the question of whether human papillomavirus (HPV)-transformed cells may also undergo crisis. Two types of human epithelial cells were transfected with either HPV-16 E6 and E7 genes or SV40 early region genes, and all transfectants underwent at least 20 more population doublings than the normal cells from which they were derived. Mesothelial cells expressing SV40 or HPV-16 genes mostly entered crisis (4/4 SV40- and 3/4 HPV-transfected clones). Similarly, 1/3 SV40- and 2/3 HPV-transfected bronchial epithelial cell cultures entered crisis. For these two types of epithelial cells, we therefore conclude that HPV-16 E6 and E7 genes, like the SV40 T proteins, are insufficient for immortalization.

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Cited by (27)

Telomerase activity in pleural malignant mesotheliomas
2011, Lung Cancer
Citation Excerpt :
It remains a possibility that activation of ALT in MPMs is a rare occurrence; investigation of a larger panel of MPMs would be required to confirm this. We have previously reported that normal mesothelial cells from pleura or peritoneum have an extended, but finite, proliferative capacity following transformation with either the SV40 early region genes, the whole SV40 genome, or the HPV-16 E6 and E7 genes, and most cultures did not escape from the population crisis that ensued [22,30]. Moreover, SV40- and HPV-transformed cells had no detectable ALT or telomerase activity during the extended phase of proliferation prior to crisis, but activated one or other of these TMMs after escape from crisis [29].
New treatments are needed for malignant pleural mesothelioma (MPM), which currently has a poor prognosis. Cellular immortalisation, one of the hallmarks of cancer, depends on the activity of a telomere length maintenance mechanism (TMM) – either telomerase or alternative lengthening of telomeres (ALT). The TMMs are widely regarded as potential targets for cancer therapies and telomerase inhibitors have entered clinical trials. The aim of this study was to determine what proportion of MPMs use ALT and/or telomerase. Forty-three MPMs from 42 patients were examined for telomerase and ALT activity. Telomerase activity was detected by immunoaffinity purification followed by the telomere repeat amplification protocol (TRAP), and ALT activity was determined by the C-circle assay and by assessing telomere lengths using terminal restriction fragment analyses. We found that 43 of 43 MPMs were telomerase-positive[+] and ALT-negative[−]. Therefore, to investigate whether pleural mesothelial cells are unusually susceptible to activation of telomerase, we examined activation of the TMMs in an in vitro model of cellular immortalisation, in which normal pleural mesothelial cells were transduced with simian virus 40 (SV40) oncogenes. We found that normal mesothelial cells were TMM-negative, and that expression of the SV40 oncogenes did not directly activate telomerase or ALT. Immortalisation, which in this experimental system results from additional genetic changes that have not yet been identified, was accompanied by activation of either TMM. Therefore, pleural mesothelial cells are capable of activating either TMM in vitro, and the observation that 100% of MPMs were telomerase[+] suggests that there are factors in vivo that select for telomerase activity during oncogenesis of this tumour type. We conclude that MPM is a tumour that could be considered for anti-telomerase therapy.
Critical role for SV40 small-t antigen in human cell transformation
2001, Virology
Defining the ability of simian virus 40 (SV40) to transform human cells has become of even greater importance with the increased understanding that this virus may play a role in some human malignancies. This report documents the requirement for viral small-t (ST) antigen in large-T (LT)-driven transformation of primary fibroblasts, a requirement that cannot be met by a well-known oncogene, c-Ha-ras (EJ-ras), which can cooperate with LT in rodent systems. The cellular gene telomerase is not essential for transformation, although transformed clones are not immortal without it. Similarly, an immortal mesothelial cell line has been developed using LT and telomerase. Immortalized mesothelial cells are morphologically normal, but can be transformed by introduction of ST, or ST + ras, but not by ras alone. It is likely that ST will be required along with LT for transformation of most human cell types.
Cell transformation by the E7 oncoprotein of human papillomavirus type 16: Interactions with nuclear and cytoplasmic target proteins
1999, Advances in Cancer Research
The E7 oncoprotein of human papillomavirus type 16 (HPV-16) has long been known as a potent immortalizing and transforming agent. However, the molecular mechanisms underlying cell transformation and immortalization by E7 remain largely unknown. It is believed that E7 exerts its oncogenic function at least in part by modulating cellular growth regulatory pathways. Increasing experimental evidence suggests that cell transformation by E7 is mediated by the physical association of E7 with cellular regulatory proteins, whose functions are specifically altered by E7, as exemplified by the well-known interaction of E7 with the retinoblastoma protein. In this review, we summarize the available data on the interaction of E7 with cellular regulatory factors and functional consequences of these interactions. We will focus the review on a set of recently identified new target proteins for the E7 oncoprotein, which sheds new light on E7 functions required for cell transformation and immortalization. Similar to the case of the E6 protein of HPV-16, whose interaction with p53 was long considered its major activity, it now appears that the interactipn of E7 with the retinoblastoma protein represents just one of many distinct interactions that are relevant for cell transformation.
Genomic instability and telomerase activity in human bronchial epithelial cells during immortalization by human papillomavirus-16 E6 and E7 genes
1997, Experimental Cell Research
Human papilloma virus types 16 and 18 contribute to the development of cervical carcinomas in which the E6 and E7 genes are frequently retained and expressed in the tumors. Our study explored the ability of the E6 and/or E7 genes to immortalize normal human bronchial epithelial (NHBE) cells and to reactivate telomerase expression in these cells. We have introduced the human papillomavirus type 16 E6 or E7 genes alone or in combination (E6/E7) into NHBE cells using the retroviral construct pLXSN. Cells expressing either the E6 or the E7 oncoproteins alone displayed an increased colony-forming efficiency and a slightly extendedin vitrolife span before entering a crisis, from which immortalized cell lines were not obtained. Telomerase activity was not detected in cells expressing either E6 or E7 individually. Cells expressing the E6/E7 oncoproteins in combination had a substantially increased life span before entering crisis. A subpopulation of these cells escaped from crisis and achieved 130 population doublings, suggesting immortalization. Telomerase activity was detected in these postcrisis cells, but was not detected prior to crisis. In addition, karyotypic analysis showed evidence of genomic instability in mass cultures as well as clones expressing E6, E7, or E6/E7. Abnormalities included numerous monosomies and trisomies, chromatid gaps and breaks, double minutes, and aberrant chromosomes. These results demonstrate that expression of E6 and/or E7 is sufficient to induce genomic instability and an extended life span to NHBE cells, but the presence of both E6 and E7, along with at least one additional genetic or epigenetic event achieved during crisis, was required for reactivation of telomerase and the immortalization in this human cell type.
Role of human papillomaviruses in cancer of the respiratory and upper digestive tract
1997, Clinics in Dermatology
Correlation of mutagenesis level with expression of reparative enzyme O<sup>6</sup>-methylguanine DNA methyltransferase during establishment of cell lines in vitro
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Regular ArticleHPV-16 E6 and E7 Genes, like SV40 Early Region Genes, Are Insufficient for Immortalization of Human Mesothelial and Bronchial Epithelial Cells

Abstract

Regular Article
HPV-16 E6 and E7 Genes, like SV40 Early Region Genes, Are Insufficient for Immortalization of Human Mesothelial and Bronchial Epithelial Cells