SV40 T-antigen is required for maintenance of immortal growth in SV40-transformed human fibroblasts.

Tsuyama, Naohiro; Miura, Masami; Kitahira, Mayumi; Ishibashi, Shun; Ide, Toshinori

doi:10.1247/csf.16.55

Cited by 29 publications

(13 citation statements)

References 18 publications

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“…1A). As was previously reported (41), much higher levels of p16 RNA were observed in VA13 cells, an immortalized line derived from WI-38 by transformation with SV40, and in SVts8 cells, an immortalized line derived by introducing a temperature-sensitive mutant of T-Ag into TIG-3 cells (61). Phosphorimage analysis indicated that the difference in expression levels in the T-Ag transformants was approximately 50-fold.…”

Section: Resultssupporting

confidence: 83%

“…Restoring pRB activity by shifting the cells to the nonpermissive temperature reduced the levels of p16 RNA to those seen in senescent TIG-3 cells. However, by inactivating pRB and p53, SV40 T-Ag has extended the life span of SVts8 cells beyond the maximum number of PDs for TIG-3 cells so that inactivation of T-Ag causes them to undergo senescence (58,61). SVts8 cells shifted to the nonpermissive temperature therefore display levels of p16 RNA typical of senescent TIG-3 cells, as observed in Fig.…”

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confidence: 85%

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Regulation of p16^CDKN2 Expression and Its Implications for Cell Immortalization and Senescence

Hara

Smith

Parry

et al. 1996

Molecular and Cellular Biology

685

519

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p16CDKN2 specifically binds to and inhibits the cyclin-dependent kinases CDK4 and CDK6, which function as regulators of cell cycle progression in G 1 by contributing to the phosphorylation of the retinoblastoma protein (pRB). Human cell lines lacking functional pRB contain high levels of p16 RNA and protein, suggesting a negative feedback loop by which pRB might regulate p16 expression in late G 1 . By a combination of nuclear run-on assays and promoter analyses in human fibroblasts expressing a temperature-sensitive simian virus 40 T antigen, we show that p16 transcription is affected by the status of pRB and define a region in the p16 promoter that is required for this response. However, the effect is not sufficient to account for the differences in p16 RNA levels between pRB-positive and -negative cells. Moreover, p16 RNA is extremely stable, and the levels do not change appreciably during the cell cycle. Primary human fibroblasts express very low levels of p16, but the RNA and protein accumulate in late-passage, senescent cells. The apparent overexpression of p16 in pRB-negative cell lines is therefore caused by at least two factors: loss of repression by pRB and an increase in the number of population doublings.CDKN2, also referred to as INK4A, CDK4I, and MTS1 (23,36,49), is a putative tumor suppressor gene on human chromosome 9p21 that is genetically linked with familial inheritance of malignant melanoma and is inactivated by a variety of mechanisms in a broad spectrum of human cancers (reviewed in references 22 and 51). CDKN2 encodes a 156-amino-acid protein, designated p16, that specifically binds to and inactivates the cyclin-dependent kinases (CDKs) CDK4 and CDK6 (16,19,41,49). These kinases are the major catalytic partners for cyclins D1, D2, and D3 and collaborate with cyclin E-CDK2 in controlling the G 1 /S transition in mammalian cells (reviewed in references 9, 43, and 53).One of the critical substrates of the G 1 -specific cyclin-dependent kinases is the product of the retinoblastoma gene (pRB) which, in its hypophosphorylated state, exerts a negative influence on G 1 progression (42,63). Since phosphorylation of pRB equates with its functional inactivation, a relatively robust model has emerged in which the role of the cyclin D-dependent kinases is to initiate the phosphorylation of pRB (9,52,63). Consistent with such a scheme, antibody neutralization studies have indicated that D-type cyclin function is required up to a point in G 1 coincident with the block imposed by pRB (3,44). This requirement is lost in cells lacking functional pRB (29,30). Ectopic expression of p16, which specifically interferes with the cyclin D-dependent kinases CDK4 and CDK6, imposes an analogous G 1 block that is also dependent on functional pRB (14,24,31,34,48). Conversely, ectopic expression of D cyclins can accelerate G 1 progression, particularly when coupled with elevated expression of cyclin E (2,20,35,(44)(45)(46). The model can readily explain the role of p16 as a tumor suppressor, since loss of function will...

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Section: Resultssupporting

confidence: 83%

mentioning

confidence: 85%

Regulation of p16^CDKN2 Expression and Its Implications for Cell Immortalization and Senescence

Hara

Smith

Parry

et al. 1996

Molecular and Cellular Biology

685

519

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show abstract

“…A clonal association between virus and tumor cell is usually established via viral integration, and continued expression of viral oncogenes is necessary for maintenance of the malignant phenotype. [28][29][30] The absence of viral sequences is therefore strong evidence against a role for these viruses in the etiology of retinoblastoma. These findings therefore contradict previous reports in which HPV genomic DNA was detected in a subset of bilateral (presumed germline RB1 mutation) and unilateral nonfamilial (presumed nonhereditary) retinoblastoma tumors in Mexico and South America.…”

Section: Discussionmentioning

confidence: 99%

Human retinoblastoma is not caused by known pRb‐inactivating human DNA tumor viruses

Gillison¹,

Chen²,

Goshu³

et al. 2007

Intl Journal of Cancer

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Retinoblastomas occur as the consequence of inactivation of the tumor suppressor retinoblastoma protein (pRb), classically upon biallelic inactivation of the RB1 gene locus. Recently, human papillomavirus (HPV) genomic DNA has been detected in retinoblastomas. To investigate the possibility that oncoproteins encoded by pRb-inactivating DNA tumor viruses play a role in the pathogenesis of human retinoblastoma, 40 fresh-frozen tumors were analyzed for the presence of HPV, adenovirus (HAdV) and polyomavirus (BKV, JCV and SV40) genomic DNA sequences by real-time polymerase chain reaction (PCR). Tumors were screened for genetic and epigenetic alterations in all 27 exons of the RB1 gene locus and promoter by exonic copy number detection, sequencing and methylation-specific PCR of the promoter region. Retinoblastoma tumors from children with bilateral familial (n 5 1), bilateral nonfamilial (n 5 1) and unilateral nonfamilial (n 5 38) disease were analyzed. Inactivating modifications to the RB1 gene locus were identified on both the alleles in 27 tumors, one allele in 8, and neither allele in 5 cases. A median of over 107,000 tumor cells were analyzed for viral genomic DNA in each PCR reaction. All tumor samples were negative for 37 HPV types, 51 HAdV types, BKV and JCV genomic sequences. Very low copy number (0.2-260 copies per 100,000 tumor cells) SV40 genomic DNA detected in 8 of 39 samples was demonstrated to be consistent with an artifact of plasmid-derived SV40. In contrast to recent reports, we obtained substantial quantitative evidence indicating that neither HPV nor any other pRb-inactivating human DNA tumor viruses play a role in the development of retinoblastoma, regardless of RB1 genotype. ' 2006 Wiley-Liss, Inc.Key words: human papillomavirus; retinoblastoma; virus; causation Retinoblastoma is a rare childhood cancer of the retina, classically initiated by loss or mutation of both alleles of the retinoblastoma gene (RB1) during retinal development. Children with an inherited or de novo germline mutation in the RB1 gene develop bilateral or unilateral retinoblastoma with high penetrance, accounting for 40% of cases. In the majority of remaining cases, unilateral tumors occur as a consequence of somatic mutations that inactivate both RB1 alleles. Upon detailed analysis, mutations of the RB1 promoter or coding region can be identified in 92% of suspected RB1 alleles either as heterozygous germline mutations in heritable cases or as biallelic mutations in retinoblastoma tumors. 1 However, no RB1 inactivating event has been identified in 8% of RB1 alleles, leaving open the possibility that some retinoblastomas may be caused by alternative genetic or epigenetic events that modify retinoblastoma protein (pRB) function.The RB1 gene on chromosome 13q14 encodes a nuclear phosphoprotein (the tumor suppressor pRb) that plays a key regulatory role in the cell cycle check point between G1 and entry into Sphase. 2 In addition to mutations in the RB1 gene, pRb function can be abrogated by oncoproteins from several DNA tum...

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“…In the experiments to examine the eect of PMA, B cells were incubated with 30 ng/ml PMA for 0, 5, 16 and 40 h. In the cell transformation experiment with SV40 temperature sensitive (ts) T-antigen, human fetal lung ®broblasts TIG-3 (Matsuo et al, 1982) were transformed with pMT-1ODtsA that codes for an origindefective SV40tsT gene from which were established cell lines SVts9-3, SVts9-4 and SVts8 (Tahara et al, 1995). SVts9-3 and SVts9-4 cells had a prolonged lifespan, but senesced after serial passage around 100 population doubling levels (PDLs): SVts8 was immortalized (Tsuyama et al, 1991). SVOD1-2 ®broblasts were transformed by normal SV40 T-antigen.…”

Section: Cells and Cell Culturementioning

confidence: 99%

Differential regulation of human RecQ family helicases in cell transformation and cell cycle

et al. 2000

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SV40 T-antigen is required for maintenance of immortal growth in SV40-transformed human fibroblasts.

Cited by 29 publications

References 18 publications

Regulation of p16^CDKN2 Expression and Its Implications for Cell Immortalization and Senescence

Regulation of p16^CDKN2 Expression and Its Implications for Cell Immortalization and Senescence

Human retinoblastoma is not caused by known pRb‐inactivating human DNA tumor viruses

Differential regulation of human RecQ family helicases in cell transformation and cell cycle

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SV40 T-antigen is required for maintenance of immortal growth in SV40-transformed human fibroblasts.

Cited by 29 publications

References 18 publications

Regulation of p16CDKN2 Expression and Its Implications for Cell Immortalization and Senescence

Regulation of p16CDKN2 Expression and Its Implications for Cell Immortalization and Senescence

Human retinoblastoma is not caused by known pRb‐inactivating human DNA tumor viruses

Differential regulation of human RecQ family helicases in cell transformation and cell cycle

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Product

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Regulation of p16^CDKN2 Expression and Its Implications for Cell Immortalization and Senescence

Regulation of p16^CDKN2 Expression and Its Implications for Cell Immortalization and Senescence