Cell immortalization: The role of viral genes and carcinogens

Khoobyarian, Newton; Marczynska, Barbara

doi:10.1016/0168-1702(93)90001-4

Cited by 11 publications

(11 citation statements)

References 91 publications

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“…13 E6 and E7 proteins inactivate p53 and Rb proteins, respectively, and thus inhibit the downstream tumor suppressor genes. 37 The hPi cells have been utilized to study the stage-specific chondrogenesis of MSCs and the regenerative potential of PRP/collagen scaffold. 12,15 The signaling pathways, stages, and molecular markers involved in chondrogenesis have been extensively investigated in our previous studies.…”

Section: Discussionmentioning

confidence: 99%

Establishment of a Promising Human Nucleus Pulposus Cell Line for Intervertebral Disc Tissue Engineering

Liu

Chen

et al. 2014

Tissue Engineering Part C: Methods

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Low-back pain caused by intervertebral disc degeneration could be recovered by the regeneration of the nucleus pulposus (NP). This study aimed to establish a chondrogenic recovery model with promising a human NP (hNP) cell line, an immortalized hNP (ihNP), which could be a screening platform to identify regenerative drugs. The ihNP cells were created from primary human NP cells transfected with a retroviral vector-driven HPV16 E6/E7. Growth properties and characteristics of ihNP were evaluated by comparing with parental NP cells. Successful immortalization of ihNP cells stably expressed HPV 16 E6/E7 mRNA. The doubling time of ihNP was shortened to 53.16±2.63 h compared with parental hNP-P1. Cell cycle regulators, including p53, p21, and pRB were downregulated compared to parental hNP-P1. The in vivo neoplastic forming assay also demonstrated that the ihNP was nontumorigenic. After 25 generations of cell cultures, the ihNP cells, yet stably expressed chondrogenic genes, including (SOX9), type II collagen (Col II), aggrecan, decorin, biglycan, and versican. Higher expressions of chondrogenic proteins, including Col II, phosphorylated SOX9 (p-SOX9), and CD44 were also determined. Under the stressful inflammatory conditions induced by lipopolysaccharides (LPS), the regenerative and anti-inflammatory potentials of ihNP in two-dimensional culture with the presence of platelet-rich plasma (PRP) were evaluated by reverse transcriptase polymerase chain reaction. PRP showed significant effects on restoring diminished chondrogenic markers and deleterious inflammatory responses induced by LPS in ihNP. The therapeutic potentials of ihNP in three-dimensional neocartilage model could also be exerted by PRP using histological evaluation and immunological staining. Hence, the established ihNP cells can provide a chondrogenic recovery model as a regenerative drug screening tool for further regenerative drug discovery and development.

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Section: Discussionmentioning

confidence: 99%

Establishment of a Promising Human Nucleus Pulposus Cell Line for Intervertebral Disc Tissue Engineering

Liu

Chen

et al. 2014

Tissue Engineering Part C: Methods

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“…Normal cells may be transformed in culture to the neoplastic state by any of a number of retroviral or activated oncogenes alone or in combination (1-4), by transforming growth factors (5, 6), or by chemical mutagens (7,8). Transformation of fibroblasts by many of these modalities is commonly accompanied by reduced synthesis of various actin-binding proteins, of which tropomyosins (TMs) (9-14), vinculin (15), gelsolin (16), and a-actinin (17) are examples.…”

Section: Introductionmentioning

confidence: 99%

“…These observations support the conclusion that tropomyosin suppression is a necessary event for the expression of components of the transformed phenotype, particularly with respect to anchorage-independent growth and tumorigenesis, which correlate closely with neoplastic potential. This potentially reversible requirement may link different initial events produced by a variety of oncogenic modalities to a common pathway leading to neoplastic growth.Normal cells may be transformed in culture to the neoplastic state by any of a number of retroviral or activated oncogenes alone or in combination (1-4), by transforming growth factors (5, 6), or by chemical mutagens (7,8). Transformation of fibroblasts by many of these modalities is commonly accompanied by reduced synthesis of various actin-binding proteins, of which tropomyosins (TMs) (9-14), vinculin (15), gelsolin (16), and a-actinin (17) are examples.…”

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confidence: 99%

Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene.

Prasad

Fuldner

Cooper

1993

Proc. Natl. Acad. Sci. U.S.A.

161

132

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Synthesis of certain members of the tropomyosin family of microfilament-associated proteins is suppressed in fibroblasts neoplastically transformed by a number of retroviral oncogenes, by transforming growth factor a, and by chemical mutagens. To test whether tropomyosin suppression is a required event in neoplastic transformation, expression of one of two suppressed tropomyosins in NIH 3T3 mouse cells transformed by the ras oncogene was restored by retrovirally mediated cDNA transfer. Cells expressing the inserted cDNA showed partial restoration of microfflament bundle formation (which is typicafly deranged in transformed cells) together with increased cytoplasmic spreading. More importantly, they lost anchorage-independent growth capability, and the onset of tumor growth in athymic mice was delayed. When tumors arose they no longer expressed the inserted cDNA. These observations support the conclusion that tropomyosin suppression is a necessary event for the expression of components of the transformed phenotype, particularly with respect to anchorage-independent growth and tumorigenesis, which correlate closely with neoplastic potential. This potentially reversible requirement may link different initial events produced by a variety of oncogenic modalities to a common pathway leading to neoplastic growth.Normal cells may be transformed in culture to the neoplastic state by any of a number of retroviral or activated oncogenes alone or in combination (1-4), by transforming growth factors (5, 6), or by chemical mutagens (7,8). Transformation of fibroblasts by many of these modalities is commonly accompanied by reduced synthesis of various actin-binding proteins, of which tropomyosins (TMs) (9-14), vinculin (15), gelsolin (16), and a-actinin (17) are examples. Consistent loss of expression of specific TM isoforms has also been found in cells cultured from human breast cancer (18). TMs and the other proteins named are essential structural and functional components of the actin-microfilament system of the cell (19-24), which has long been known to be severely disrupted in transformed cells (25,26).In the present study, to clarify whether TM suppression is a necessary event in the causal chain leading to neoplastic transformation, we have examined the effect of overcoming the deficiency of TM synthesis in retrovirally transformed cells (DT cells) through expression of a cDNA encoding the TM1 isoform. DT cells are NIH 3T3 mouse fibroblasts transformed by the v-Ki-ras oncogene and contain two integrated copies of the oncogene (27). They are highly tumorigenic in athymic mice, manifest anchorage-independent growth, and rarely produce spontaneous revertants. Of the five major TM isoforms expressed in NIH 3T3 cells, TM1 is suppressed by 50% and TM2 is almost absent in DT cells, whereas expression of the low molecular weight isoforms TM4 and TM5 is unaltered (9, 10). Nontransformed revertants of DT induced by chemical mutagens reexpress TM1The publication costs of this article were defrayed in part by page charge pa...

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“…Cultured human cells are much more refractory to immortalization than rodent cells; each human cell type studied has been found to require transformation by specific viral genes and additional mutations to as-yet-unknown cellular genes (17). Karyotypic instability is a diagnostic criterion of cancer cells and is also characteristic of all immortal (but not mortal) cells in culture (28,40).…”

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confidence: 99%

Elevated Recombination in Immortal Human Cells Is Mediated by HsRAD51 Recombinase

Xia

Shammas

Reis

1997

Molecular and Cellular Biology

123

108

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Normal diploid cells have a limited replicative potential in culture, with progressively increasing interdivision time. Rarely, cell lines arise which can divide indefinitely; like tumor cells, such "immortal" lines display frequent chromosomal aberrations which may reflect high rates of recombination. Recombination frequencies within a plasmid substrate were 3.5-fold higher in nine immortal human cell lines than in six untransformed cell strains. Expression of HsRAD51, a human homolog of the yeast RAD51 and Escherichia coli recA recombinase genes, was 4.5-fold higher in immortal cell lines than in mortal cells. Stable transformation of human fibroblasts with simian virus 40 large T antigen prior to cell immortalization increased both chromosomal recombination and the level of HsRAD51 transcripts by two-to fivefold. T-antigen induction of recombination was efficiently blocked by introduction of HsRAD51 antisense (but not control) oligonucleotides spanning the initiation codon, implying that HsRAD51 expression mediates augmented recombination. Since p53 binds and inactivates HsRAD51, T-antigen-p53 association may block such inactivation and liberate HsRAD51. Upregulation of HsRAD51 transcripts in T-antigen-transformed and other immortal cells suggests that recombinase activation can also occur at the RNA level and may facilitate cell transformation to immortality.Cultured human cells are much more refractory to immortalization than rodent cells; each human cell type studied has been found to require transformation by specific viral genes and additional mutations to as-yet-unknown cellular genes (17). Karyotypic instability is a diagnostic criterion of cancer cells and is also characteristic of all immortal (but not mortal) cells in culture (28,40). Homologous chromosomal recombination increases two-to eightfold, prior to any immortalization, in human cells transformed stably (6) or transiently (35) by simian virus 40 (SV40) large T antigen (SV-Tag). This suggests that recombination may be a common mechanism for genetic alterations leading to both immortalization and chromosomal instability.Homologous recombination is a multistep process involving many components (47). In Escherichia coli, the RecA recombinase protein plays a central role, coating DNA strands and mediating their alignment over areas of sequence identity to form triplex DNA intermediates, which are subsequently extended by ATP-dependent branch migration (5, 30). The RAD51 protein of Saccharomyces cerevisiae, which shows structural and functional homology to the E. coli RecA protein, is required for DNA repair and mitotic recombination in yeast (38,42). RAD51 transcript levels are strongly cell cycle regulated in S. cerevisiae (2). A RAD51-like gene, HsRAD51, which encodes a 339-amino-acid protein with a predicted molecular mass of 37 kDa, has been isolated from human cells (49). The deduced amino acid sequence is 83% similar (67% identical) to that of the yeast RAD51 protein and 56% similar (30% identical) to that of E. coli RecA protein. The HsRAD...

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Cell immortalization: The role of viral genes and carcinogens

Cited by 11 publications

References 91 publications

Establishment of a Promising Human Nucleus Pulposus Cell Line for Intervertebral Disc Tissue Engineering

Establishment of a Promising Human Nucleus Pulposus Cell Line for Intervertebral Disc Tissue Engineering

Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene.

Elevated Recombination in Immortal Human Cells Is Mediated by HsRAD51 Recombinase

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