Separation of simian virus 40 large-T-antigen-transforming and origin-binding functions from the ability to block differentiation.

Cherington, Van; Brown, Melissa A.; Paucha, Eva; Louis, Joshua St; Spiegelman, Bruce M.; Roberts, Thomas M.

doi:10.1128/mcb.8.3.1380

Cited by 80 publications

(42 citation statements)

References 46 publications

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“…6 This inhibitory effect of SV40 T-Ag could explain the limited differentiation capacity of the SV40 T-Ag-transformed human white preadipocytes described previously. 9 In this study, we demonstrate, for the first time, that co-expression of hTERT and HPV-E7 in human preadipocytes allows cells not only to display an indefinite life span but also to retain their features of primary cells.…”

Section: Discussionmentioning

confidence: 99%

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Reconstitution of telomerase activity combined with HPV-E7 expression allow human preadipocytes to preserve their differentiation capacity after immortalization

et al. 2003

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Overexpression of SV40 T-antigen (SV40 T-Ag) has been widely used to overcome replicative senescence of human primary cells and to promote cell immortalization. However, in the case of certain cell types, such as preadipocytes, the differentiation process of immortalized cells is blocked by SV40 T-Ag expression. In this study, human telomerase reverse transcriptase (hTERT) and papillomavirus E7 oncoprotein (HPV-E7) genes were coexpressed in human preadipocytes to test whether this combination could maintain cell differentiation capacity after immortalization. We demonstrated that the HPV-E7/hTERT expressing preadipocytes displayed an indefinite life span. Interestingly, immortalized cells were diploid and presented no chromosomic alterations. These immortalized cells were able to accumulate and hydrolyze intracellular triglycerides and to express adipocyte markers. These data demonstrate, for the first time, that coexpression of hTERT and HPV-E7 in human preadipocytes allows cells not only to display an indefinite life span but also to retain their capacity to differentiate.

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Section: Discussionmentioning

confidence: 99%

“…4,5 Nevertheless, the differentiation process of certain cell types, such as preadipocytes, is clearly blocked by SV40 T-Ag expression. 6 Differentiation of preadipocytes into adipocytes (i.e. adipogenesis) is a well-described system regulated by various factors.…”

Section: Introductionmentioning

confidence: 99%

Reconstitution of telomerase activity combined with HPV-E7 expression allow human preadipocytes to preserve their differentiation capacity after immortalization

et al. 2003

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“…Thus, this approach has limitations for the study of differentiation in general and the function of white adipocytes in particular. Indeed, there is evidence that wild-type SV40 large T-antigen can block the differentiation of preadipocytes (11). A possible solution to this problem is to use modified conditionally regulated SV40 T-antigen.…”

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confidence: 99%

Conditionally immortalized white preadipocytes: a novel adipocyte model

Morganstein

Christian

Turner

et al. 2008

Journal of Lipid Research

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This study describes a novel approach to generate conditionally immortalized preadipocyte cell lines from white adipose tissue (IMWAT) that can be induced to differentiate into white adipocytes even after expansion in culture. Such adipocytes express markers of white fat such as peroxisome proliferator-activated receptor g and aP2 but not brown fat markers, have an intact insulin signaling pathway, and express proinflammatory cytokines. They can be readily transduced with adenoviral vectors, allowing them to be used to investigate the consequences of the depletion of specific adipocyte factors using short hairpin RNA. This approach has been used to study the effect of reduced expression of the nuclear receptor corepressor receptor interacting protein 140 (RIP140), a regulator of adipocyte function. The depletion of RIP140 results in changes in metabolic gene expression that resemble those in adipose tissue of the RIP140 null mouse. Thus, IMWAT cells provide a novel model for adipocytes that are derived from preadipocytes rather than fibroblasts and provide an alternative system to primary preadipocytes for the investigation of adipocyte function. The adipocyte plays a key role in the development of obesity and diabetes, functioning both as an energy store and as an endocrine tissue to regulate whole body energy balance. Key features of adipocytes include lipid storage and a rapid response to insulin stimulation that involves the activation of a number of signaling pathways, leading to diverse metabolic changes (reviewed in Refs. 1, 2). Recent evidence has also shown that adipocytes are an important source of inflammatory cytokines (3). The generation of genetically modified mice and the isolation of adipocytes from them provides an opportunity to study the signaling pathways involved in adipose biology and as models for the pathogenesis of obesity and diabetes. Although primary cultures from adipose tissue can be readily differentiated into adipocytes (4), they have a limited lifespan and are not always suitable for molecular studies.A number of cell lines that can be induced to differentiate into adipocytes (5) have been generated for use in such studies. The analysis of 3T3-L1 adipocytes (6) has provided many insights, but these cells are aneuploid and were derived from embryo fibroblasts rather than preadipocytes.Procedures for the isolation of immortalized adipose cell lines from the mouse have been described (7), including the use of SV40 transforming genes (7,8). However, SV40 T-antigen functions by deactivating proteins, including Retinoblastoma (Rb), and recent studies have shown that Rb null preadipocytes differentiate preferentially into brown adipocytes (9). It appears that members of the E2F family, themselves regulated by Rb, have roles in adipogenesis (10). Thus, this approach has limitations for the study of differentiation in general and the function of white adipocytes in particular. Indeed, there is evidence that wild-type SV40 large T-antigen can block the differentiation of preadipoc...

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“…p values are shown for significant differences. RbBS that impair transformation (20,37,38) (and hence p110…”

Section: Table 1 Pooled Data For Nuclear Import Kinetics For Gfp-t-agmentioning

confidence: 99%

Binding of p110 Retinoblastoma Protein Inhibits Nuclear Import of Simian Virus SV40 Large Tumor Antigen

Fulcher

Dias

Jans

2010

Journal of Biological Chemistry

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Nuclear import of the simian virus 40 large tumor antigen (T-ag) is dependent on its nuclear localization signal (NLS) within amino acids 126 -132 that is recognized by the importin ␣/␤1 heterodimer, as well as a protein kinase CK2 site at serine 112 upstream of the NLS, which enhances the interaction ϳ50-fold. Here we show for the first time that T-ag nuclear import is negatively regulated by N-terminal sequences (amino acids 102-110), which represent the binding site (BS) for the retinoblastoma (Rb) tumor suppressor protein (p110 Rb ). Quantitative confocal laser scanning microscopic analysis of the transport properties of T-ag constructs with or without Rb binding site mutations in living transfected cells or in a reconstituted nuclear transport system indicates that the presence of the RbBS significantly reduces nuclear accumulation of T-ag. A number of approaches, including the analysis of T-ag nuclear import in an isogenic cell pair with and without functional p110Rb implicate p110Rb binding as being responsible for the reduced nuclear accumulation, with the Ser 106 phosphorylation site within the RbBS appearing to enhance the inhibitory effect. Immunoprecipitation experiments confirmed association of T-ag and p110Rb and dependence thereof on negative charge at Ser 106 . The involvement of p110 Rb in modulating T-ag nuclear transport has implications for the regulation of nuclear import of other proteins from viruses of medical significance that interact with p110Rb , and how this may relate to transformation.Proteins that perform their respective roles in the nucleus generally require a nuclear localization signal (NLS) 2 recognized by members of the importin (IMP) family of transporters. In the case of Simian virus 40 (SV40) large tumor antigen (T-ag), the NLS (PKKKRKV 132 , single letter amino acid code) (1) is recognized by a heterodimer of IMP ␣ and ␤1, which mediates passage of T-ag through the nuclear envelope-localized nuclear pores and into the nucleus (2-4). Release within the nucleus occurs upon binding of the monomeric guanine nucleotide-binding protein Ran to IMP␤, which dissociates the trimeric IMP-NLS-containing protein complex (2,4,5).Phosphorylation has been shown to be able to regulate nuclear protein import through modulation of NLS-IMP interaction either positively or negatively (2, 6 -14). A well characterized example is that of T-ag, which has several phosphorylation residues N-terminal to its NLS, which are phosphorylated during SV40 infection (15, 16), including the CK2 site at Ser 111/112 , which enhances nuclear uptake by enhancing NLS recognition by IMP ␣/␤ (9, 17, 18), and the cyclin-dependent kinase site at Thr 124 , which effects cytoplasmic retention (6,19).The present study reports for the first time the regulatory role of amino acids 102-110, previously shown to represent the "transforming region" of T-ag, corresponding to the binding site (BS) for the tumor suppressor p110Rb protein (retinoblastoma susceptibility factor or Rb) (20 -22). Using quantitative confocal laser...

show abstract

Separation of simian virus 40 large-T-antigen-transforming and origin-binding functions from the ability to block differentiation.

Cited by 80 publications

References 46 publications

Reconstitution of telomerase activity combined with HPV-E7 expression allow human preadipocytes to preserve their differentiation capacity after immortalization

Reconstitution of telomerase activity combined with HPV-E7 expression allow human preadipocytes to preserve their differentiation capacity after immortalization

Conditionally immortalized white preadipocytes: a novel adipocyte model

Binding of p110 Retinoblastoma Protein Inhibits Nuclear Import of Simian Virus SV40 Large Tumor Antigen

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