A Retinoblastoma Allele That Is Mutated at Its Common E2F Interaction Site Inhibits Cell Proliferation in Gene-Targeted Mice

Abstract

The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1^ΔG) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1^ΔG/ΔG mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1^ΔG/ΔG MEFs display a magnitude of E2F target gene derepression similar to that of Rb1^−/− cells, even though Rb1^ΔG/ΔG cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1^ΔG/ΔG MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G₁ to arrest proliferation. Some Rb1^ΔG/ΔG mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.

ACKNOWLEDGMENTS

We thank numerous colleagues for advice and reagents. We specifically thank Kristen Kernohan and Nathalie Bérubé for their assistance with developing and analyzing our ChIP experiments.

M.J.C. is the recipient of a CIHR M.D./Ph.D. studentship. M.J.C., M.J.T., S.T., and S.M.F. are/were members of the CIHR-Strategic Training Program in Cancer Research and Technology Transfer. F.A.D. is the Wolfe Senior Fellow in Tumor Suppressor Genes. This work was funded by an operating grant from the CIHR (MOP-89765) to F.A.D. and a grant from the NIH (RO1 CA98956) to J.M.P. and G.L.