Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity.
To elucidate the regulator-versus-target relationship in the cyclin D1/cdk4/retinoblastoma protein (pRB) pathway, we examined fibroblasts from RB-1 gene-deficient and RB-1 wild-type littermate mouse embryos (ME) and in human tumor cell lines that differed in the status of the RB-1 gene. The RB+/+ and RB-/- ME fibroblasts expressed similar protein levels of D-type cyclins, cdk4, and cdk6, showed analogous spectra and abundance of cellular proteins complexed with cdk4 and/or cyclins D1 and D2, and exhibited comparable associated kinase activities. Of the two human cell lines established from the same sarcoma biopsy, the RB-positive SKUT1B cells contained cdk4 that was mainly associated with D-type cyclins, contrary to a predominant cdk4-p16INK4 complex in the RB-deficient SKUT1A cells. Antibody-mediated neutralization of cyclin D1 arrested the RB-positive ME and SKUT1B cells in G1, whereas this cyclin appeared dispensable in the RB-deficient ME and SKUT1A cells. Lack of requirement for cyclin D1 therefore correlated with absence of functional pRB, regardless of whether active cyclin D1/cdk4 holoenzyme was present in the cells under study. Consistent with a potential role of cyclin D/cdk4 in phosphorylation of pRB, monoclonal anti-cyclin D1 antibodies supporting the associated kinase activity failed to significantly affect proliferation of RB-positive cells, whereas the antibody DCS-6, unable to coprecipitate cdk4, efficiently inhibited G1 progression and prevented pRB phosphorylation in vivo. These data provide evidence for an upstream control function of cyclin D1/cdk4, and a downstream role for pRB, in the order of events regulating transition through late G1 phase of the mammalian cell division cycle.
Lukas
J
J
Division of Cancer Biology, Danish Cancer Society, Copenhagen.
Bartkova
J
J
Rohde
M
M
Strauss
M
M
Bartek
J
J
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D019938:Cyclin D1; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D016193:G1 Phase / Q000235:genetics* / Q000502:physiology*; D016161:Genes, Retinoblastoma; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D015513:Oncogene Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D016213:Cyclins; D017931:DNA Primers; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D019938:Cyclin D1; D017346:Protein Serine-Threonine Kinases; C495900:CDK4 protein, human; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases