Immortalization of human fibroblasts by SV40 large T antigen results in the reduction of cyclin D1 expression and subunit association with proliferating cell nuclear antigen and Waf1.
Protein complexes containing cyclins and cyclin-dependent protein kinases (cdks) have been shown to be rearranged in both spontaneous and viral tumor antigen-transformed cells. We have examined G1- and S-phase cyclin/cdk complexes as a function of the neoplastic progression of human diploid fibroblasts transfected with the SV40 large T antigen. We find that the expression of cyclin D1 and its association with proliferating cell nuclear antigen (PCNA) and Waf1 remain unchanged in precrisis human fibroblasts transfected with SV40 large T antigen. However, in these same cells the association of cdk4 with cyclin D1, PCNA, and Waf1 is disrupted. Upon immortalization, cyclin D1 protein expression is decreased, and binding of both PCNA and Waf1 with the remaining cyclin D1 is reduced. In contrast, large T antigen increased the expression of cyclin A and cyclin E proteins in both precrisis and immortal cells and did not reduce the binding of PCNA or Waf1 to either cdk2 or cyclin A proteins. These results show that large T-antigen expression in human fibroblasts selectively uncouples cyclin D1 from cdk4, and subsequent immortalization of these cells results in additional changes to the cyclin D1-dependent cell cycle regulatory pathways.
Peterson
S R
SR
Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545, USA.
Gadbois
D M
DM
Bradbury
E M
EM
Kraemer
P M
PM
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D019938:Cyclin D1; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism*; D005347:Fibroblasts; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D015513:Oncogene Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D046911:Macromolecular Substances; D015513:Oncogene Proteins; D018809:Proliferating Cell Nuclear Antigen; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D019938:Cyclin D1; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases