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Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity
1Division of Cancer Biology, Danish Cancer Society, Copenhagen.
Abstract:
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.
Insights
Cyclin D1/cdk4 acts upstream of the retinoblastoma protein (pRB), regulating cell cycle progression. Its absence is compensated in pRB-deficient cells, highlighting pRB
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The cyclin D1/cdk4/retinoblastoma protein (pRB) pathway is crucial for cell cycle regulation.
- Understanding the precise roles of cyclin D1 and pRB in this pathway is essential for comprehending cell division control.
- The RB-1 gene status significantly impacts cellular responses to cell cycle regulators.
Purpose of the Study:
- To clarify the regulatory relationship between cyclin D1/cdk4 and pRB.
- To investigate the functional consequences of RB-1 gene deficiency on cell cycle progression.
- To determine the role of cyclin D1 in G1 phase transition in the presence or absence of functional pRB.
Main Methods:
- Comparative analysis of RB-1 gene-deficient and wild-type mouse embryo fibroblasts.
- Examination of human tumor cell lines with differing RB-1 gene status (RB-positive and RB-deficient).
- Assessment of cyclin D1/cdk4 complex formation, kinase activity, and effects of antibody-mediated neutralization on cell proliferation and pRB phosphorylation.
Main Results:
- RB-1 deficient and wild-type cells showed similar levels of D-type cyclins, cdk4, and cdk6, with comparable kinase activities.
- Cyclin D1 neutralization arrested RB-positive cells in G1 but was dispensable for RB-deficient cells, indicating a pRB-dependent requirement.
- Antibodies targeting active cyclin D1/cdk4 complexes inhibited proliferation and pRB phosphorylation in RB-positive cells.
Conclusions:
- Cyclin D1/cdk4 functions upstream of pRB in regulating the late G1 phase of the cell cycle.
- The requirement for cyclin D1 in G1 arrest is dependent on the presence of functional pRB.
- These findings establish a sequential regulatory model where cyclin D1/cdk4 controls pRB activity for cell cycle progression.