Immortalization of primary cells by DNA tumor viruses

Linder, Stig; Marshall, Heather

doi:10.1016/0014-4827(90)90027-8

Cited by 45 publications

(14 citation statements)

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“…On the other hand, the increased activity of p53 was reported in senescing human fibroblasts (Atadja et al, 1995). In addition, the products of oncogenic DNA viruses such as SV40, papilloma and adenoviruses, which can occasionally immortalize human cells, bind to p53 and Rb proteins, rendering them inactive (for review, Linder and Marshall, 1990). These findings indicate that mutations and/or dysfunctions of the p53 gene may be linked to the immortalization of human cells.…”

mentioning

confidence: 94%

Transformation of normal human fibroblasts into immortalized cells with the mutant p53 gene and X‐rays

Fushimi¹,

Iijima²,

Gao³

et al. 1997

Int. J. Cancer

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In vitro cell transformation is a valuable approach for studying the mechanisms of multistep carcinogenesis of human cells. Since immortalization is an essential step for in vitro neoplastic transformation of human cells, this study addresses the question of whether mutant p53 contributes to the immortalization process of human cells. The mutant p53 gene (mp53: codon273 Arg-His ) was introduced into normal human fibroblasts (OUMS-24 line) and a G418-resistant clone, OUMS-24/P6 line, was obtained. This clone showed an extended life span and chromosome abnormalities, but senesced at the 79th population doubling level (PDL). When these cells were subjected to intermittent X-ray treatment, they became an immortalized cell line (OUMS-24/P6X). Although these immortalized cells showed chromosome abnormalities, they were not tumorigenic. On the other hand, normal OUMS-24 cells into which mp53 had not been introduced were not immortalized by the same X-ray treatment. These results indicate that introduction and expression of mp53 alone were not sufficient for immortalization of human cells, and that mutations of the remaining wild-type p53 or other genes may have been necessary for immortalization. In vitro cell transformation is a valuable model for studying the mechanisms of multistep carcinogenesis. The human cell system is useful for studying the various stages of in vitro transformation, because events in the transformation process, from normal to neoplastic phenotypes, are clearly defined by the successive stages of aging, immortalization, and neoplastic transformation (Namba et al., 1988). For normal human cells to undergo neoplastic transformation, they must first be immortalized. However, normal human cells have a finite capacity to replicate and eventually enter a state of irreversible growth arrest (for review, Vojta and Barrett, 1995). This feature has been termed cellular senescence or replicative senescence. Cellular senescence is thought to be a genetically programmed process rather than the result of a random accumulation of damage. In normal human cells, in contrast to rodent cells, cellular senescence is particularly stringent and spontaneous immortalization is an extremely rare event.The immortalization process of human cells is probably related to deregulation of cellular senescence. Serum-stimulated senescent and quiescent human fibroblasts express many of the same genes regulating the cell cycle such as c-myc, c-jun and c-Ha-ras (Rittling et al., 1986;Seshadri and Campisi, 1989). However, senescent cells have lost the ability to express c-fos and cdc2-cyclin A kinase activity and to phosphorylate Rb protein (Seshadri and Campisi, 1990;Stein et al., 1990Stein et al., , 1991. p53 regulates the cell-cycle machinery partially through the expression of p21, because p21 inhibits the action of multiple cyclin-dependent kinase (cdk) complexes and arrests growth (Noda et al., 1994).We obtained 3 human cell lines immortalized with either 4-nitroquinoline 1-oxide (4-NQO) or 60 Co gamma-rays (Namba et al., 1988...

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confidence: 94%

Transformation of normal human fibroblasts into immortalized cells with the mutant p53 gene and X‐rays

Fushimi¹,

Iijima²,

Gao³

et al. 1997

Int. J. Cancer

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“…Generally, this strategy requires abrogation of p53 and pRB-mediated terminal proliferation and/or activation of a telomerase reverse transcriptase ( hTERT ) maintenance mechanism [9]. Several methods have been developed for immortalizing cells in vitro [10]. Among these, the introduction of viral genes, such as SV40 [11] or human papilloma-virus (HPV) E6/E7 genes [12] and the hTERT gene [13-15] have been widely used.…”

Section: Introductionmentioning

confidence: 99%

Immortalization of human adipose-derived stromal cells: production of cell lines with high growth rate, mesenchymal marker expression and capability to secrete high levels of angiogenic factors

et al. 2014

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IntroductionHuman adipose-derived stromal cells (hASCs), due to their relative feasibility of isolation and ability to secrete large amounts of angiogenic factors, are being evaluated for regenerative medicine. However, their limited culture life span may represent an obstacle for both preclinical investigation and therapeutic use. To overcome this problem, hASCs immortalization was performed in order to obtain cells with in vitro prolonged life span but still maintain their mesenchymal marker expression and ability to secrete angiogenic factors.MethodshASCs were transduced with the human telomerase reverse transcriptase (hTERT) gene alone or in combination with either SV-40 or HPV E6/E7 genes. Mesenchymal marker expression on immortalized hASCs lines was confirmed by flow cytometry (FC), differentiation potential was evaluated by immunocytochemistry and ELISA kits were used for evaluation of angiogenic factors. Green fluorescent protein (GFP) gene transduction was used to obtain fluorescent cells.ResultsWe found that hTERT alone failed to immortalize hASCs (hASCs-T), while hTERT/SV40 (hASCs-TS) or hTERT/HPV E6/E7 (hASCs-TE) co-transductions successfully immortalized cells. Both hASCs-TS and hASCs-TE were cultured for up to one year with a population doubling level (PDL) up to 100. Comparative studies between parental not transduced (hASCs-M) and immortalized cell lines showed that both hASCs-TS and hASCs-TE maintained a mesenchymal phenotypic profile, whereas differentiation properties were reduced particularly in hASCs-TS. Interestingly, hASCs-TS and hASCs-TE showed a capability to secrete significant amount of HGF and VEGF. Furthermore, hASCs-TS and hASCs-TE did not show tumorigenic properties in vitro although some chromosomal aberrations were detected. Finally, hASCs-TS and hASCs-TE lines were stably fluorescent upon transduction with the GFP gene.ConclusionsHere we demonstrated, for the first time, that hASCs, upon immortalization, maintain a strong capacity to secrete potent angiogenic molecules. By combining hASCs immortalization and their paracrine characteristics, we have developed a “hybridoma-like model” of hASCs that could have potential applications for discovering and producing molecules to use in regenerative medicine (process scale-up).In addition, due to the versatility of these fluorescent-immortalized cells, they could be employed in in vivo cell-tracking experiments, expanding their potential use in laboratory practice.

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“…We and others have shown that spontaneous immortalization of human cells in vitro (a cell culture term for unlimited proliferative capacity of cells) is an extremely rare event (23,32,50) requiring alteration or mutations in several genes which are normally involved in the regulation of cellular senescence (16,42,57). It has been suggested that cellular immortalization is a critical and perhaps rate-limiting step in the development of most human cancers (18,50).…”

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confidence: 99%

Spontaneous In Vitro Immortalization of Breast Epithelial Cells from a Patient with Li-Fraumeni Syndrome

Shay

Tomlinson

Piatyszek

et al. 1995

Molecular and Cellular Biology

143

102

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Individuals with germ line mutations in the p53 gene, such as Li-Fraumeni syndrome (LFS), have an increased occurrence of many types of cancer, including an unusually high incidence of breast cancer. This report documents that normal breast epithelial cells obtained from a patient with LFS (with a mutation at codon 133 of the p53 gene) spontaneously immortalized in cell culture while the breast stromal fibroblasts from this same patient did not. Spontaneous immortalization of human cells in vitro is an extremely rare event. This is the first documented case of the spontaneous immortalization of breast epithelial cells from a patient with LFS in culture. LFS patient breast stromal fibroblasts infected with a retroviral vector containing human papillomavirus type 16 E7 alone were able to immortalize, whereas stromal cells obtained from patients with wild-type p53, similarly infected with human papillomavirus type 16 E7, did not. The present results indicate a protective role of normal pRb-like functions in breast stromal fibroblasts but not in breast epithelial cells and reinforces an important role of wild-type p53 in the regulation of the normal growth and development of breast epithelial tissue.Familial cancer syndromes with germ line mutations, such as the dominantly inherited p53 mutations present in Li-Fraumeni syndrome (LFS), have helped to illustrate the important role of tumor suppressor genes in the development of human cancers (3,30,31,35,36,37,52). The p53 gene is presently considered to be one of the most frequently mutated genes in human cancer (19,22,28,29,47,56,58), and the functional effects of mutations in evolutionarily conserved regions of the p53 phosphoprotein are currently a subject of intense study (24,25,28,43,46,(59)(60)(61)(62)(63). While a complete understanding of wild-type p53 function is not yet known, it is generally believed that perturbations of wild-type p53 function may lead to genomic instability and permit the expansion of the pool of proliferating cells, which leads to a cascade of additional mutations, increasing the probability of neoplastic transformation. The discovery of the importance of the tumor suppressor gene p53, and the identification of germ line mutations in p53 in LFS-affected families, has led to a growing awareness of the cancer risk to such families. Even though rare bone and soft tissue sarcomas are relatively common in families affected by LFS, other, more frequently occurring forms of cancer other than breast cancer (such as colorectal carcinoma) are not overrepresented. Among women in families affected by LFS, breast tumors are the most prevalent cancer (afflicting at least 50%), with 28% of the breast cancers diagnosed before age 30 and 89% diagnosed before age 50 (21, 31, 37). A molecular explanation for the specifically increased incidence of breast cancer, particularly early-onset breast cancer, in families affected by LFS relative to other forms of cancer has not yet been elucidated (20, 41).We and others have shown that spontaneous immortalization ...

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Immortalization of primary cells by DNA tumor viruses

Cited by 45 publications

References 75 publications

Transformation of normal human fibroblasts into immortalized cells with the mutant p53 gene and X‐rays

Transformation of normal human fibroblasts into immortalized cells with the mutant p53 gene and X‐rays

Immortalization of human adipose-derived stromal cells: production of cell lines with high growth rate, mesenchymal marker expression and capability to secrete high levels of angiogenic factors

Spontaneous In Vitro Immortalization of Breast Epithelial Cells from a Patient with Li-Fraumeni Syndrome

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