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Rescue of defective mitogenic signaling by D-type cyclins
M F Roussel1, A M Theodoras, M Pagano
1Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Abstract:
Three gene products, including Myc and the D- and E-type G1 cyclins, are rate limiting for G1 progression in mammalian fibroblasts. Quiescent mouse NIH 3T3 fibroblasts engineered to express a mutant colony-stimulating factor (CSF-1) receptor (CSF-1R 809F) fail to synthesize c-myc and cyclin D1 mRNAs upon CSF-1 stimulation and remain arrested in early G1 phase. Ectopic expression of c-myc or either of three D-type cyclin genes, but not cyclin E, resensitized these cells to the mitogenic effects of CSF-1, enabling them to proliferate continuously in liquid culture and to form colonies in agar in response to the growth factor. Rescue by cyclin D1 was enhanced by c-myc but not by cyclin E and was reversed by infecting cyclin D1-reconstituted cells with a retroviral vector encoding catalytically inactive cyclin-dependent kinase 4. Induction of cyclin D1 mRNA by CSF-1 was restored in cells forced to express c-myc, and vice versa, suggesting that expression of the two genes is interdependent. Cells reconstituted with c-myc were prevented from entering S phase when microinjected with a monoclonal antibody to cyclin D1, and conversely, those rescued by cyclin D1 were inhibited from forming CSF-1-dependent colonies when challenged with a dominant-negative c-myc mutant. Cyclin D mutants defective in binding to the retinoblastoma protein were impaired in rescuing mitogenic signaling. Therefore, Myc and D-type cyclins collaborate during the mitogenic response to CSF-1, whereas cyclin E functions in a separate pathway.
Insights
Myc and D-type cyclins collaborate to drive cell cycle progression in response to growth factors. Cyclin E functions independently, highlighting distinct pathways in fibroblast proliferation.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Cell cycle progression is tightly regulated by key proteins.
- Myc and D-type cyclins are critical regulators of the G1 phase in mammalian cells.
- Disruptions in these pathways can lead to uncontrolled cell proliferation.
Purpose of the Study:
- To investigate the roles of Myc and D-type cyclins in fibroblast response to colony-stimulating factor (CSF-1).
- To elucidate the collaborative or independent functions of Myc, cyclin D, and cyclin E in mitogenesis.
- To understand the molecular mechanisms underlying G1 phase progression.
Main Methods:
- Utilized NIH 3T3 fibroblasts engineered with a mutant colony-stimulating factor receptor (CSF-1R 809F).
- Employed ectopic gene expression of c-myc, D-type cyclins, and cyclin E.
- Used retroviral vectors for gene delivery and dominant-negative mutants to probe protein function.
- Microinjection techniques were used to assess protein interactions and pathway dependencies.
Main Results:
- Ectopic expression of c-myc or cyclin D1 restored CSF-1-induced proliferation in arrested fibroblasts.
- Cyclin D1-mediated rescue was enhanced by c-myc but not cyclin E.
- Myc and cyclin D1 expression were found to be interdependent.
- Cyclin D mutants defective in retinoblastoma protein binding failed to rescue mitogenic signaling.
Conclusions:
- Myc and D-type cyclins cooperate in CSF-1-mediated mitogenic signaling.
- Cyclin E operates in a distinct pathway, separate from the Myc-cyclin D axis.
- These findings provide insights into the coordinated regulation of cell cycle entry.