CYCLIN D1 IS DISPENSABLE FOR G(1) CONTROL IN RETINOBLASTOMA GENE-DEFICIENT CELLS INDEPENDENTLY OF CDK4 ACTIVITY

To elucidate the regulator-versus-target relationship in the cyclin D1 /cdk-4/retinoblastoma protein (pRB) pathway, we examined fibroblasts f rom RB-1 gene-deficient and RB-1 wild-type littermate mouse embryos (M E) and in human tumor cell lines that differed in the status of the RB -1 gene. The RB(+/+) and RB(-/-) ME fibroblasts expressed similar prot ein 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 RE-positive SKUT1B cells contained cdk4 that was mainly associated wi th D-type cyclins, contrary to a predominant cdk4-p16(INK4) complex in the RE-deficient SKUT1A cells. Antibody-mediated neutralization of cy clin D1 arrested the RB-positive ME and SKUT1B cells in G(1), whereas this cyclin appeared dispensable in the RB-deficient ME and SKUT1A cel ls. Lack of requirement for cyclin D1 therefore correlated with absenc e of functional pRB, regardless of whether active cyclin D1/cdk4 holoe nzyme was present in the cells under study. Consistent with a potentia l role of cyclin D/cdk4 in phosphorylation of pRB, monoclonal anti-cyc lin D1 antibodies supporting the associated kinase activity failed to significantly affect proliferation of RE-positive cells, whereas the a ntibody DCS-6, unable to coprecipitate cdk4, efficiently inhibited G(1 ) progression and prevented pRB phosphorylation in vivo. These data pr ovide evidence for an upstream control function of cyclin D1/cdk4, and a downstream role for pRB, in the order of events regulating transiti on through late G(1) phase of the mammalian cell division cycle.