Characterization of Rb1 function in the absence of normal E2F binding
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Deregulation of the retinoblastoma susceptibility ( Rb1 ) gene has been observed in most human cancers, including prostate cancer. The molecular mechanisms underlying Rb1 protein (pRb) mediated tumor suppression are unclear, although its ability to bind and regulate E2F transcription factors is considered centrally important. However Rb1 utilizes E2F-independent mechanisms, which have not been well characterized in vivo, to influence cell fate. To assess the importance of such mechanisms in vivo, we generated a mutant mouse carrying an Rb1 allele (R654W) that encodes a protein deficient for binding and regulation of E2F transcription factors. R654W mutant embryos exhibit cell cycle defects similar to those observed in Rb1 null embryos, reinforcing the importance of pRb/E2F interaction for normal cell cycle control. However, R654W embryos survive at least 2 days longer than Rb1 null embryos. Increased life span is associated with improved erythrocyte and fetal liver macrophage differentiation. In contrast, R654W pRb does not rescue differentiation defects associated with pRb-deficient retinae. These data indicate that Rb1 makes important cell-type-specific contributions to cellular differentiation that are genetically separable from its ability to stably bind and regulate E2F transcription factors. The mutant Rb1 allele has no detectable tumor suppressor activity in the pituitary or thyroid glands relative to a null allele, suggesting normal pRb mediated E2F regulation is critical for tumor suppression in these tissues. Surprisingly, the R654W allele is able to suppress tumorigenesis in the prostate. Rb1 null prostate tissues treated with testostrone plus estradiol developed prostatic intraepithelial neoplastic lesions (PIN). In contrast, prostate tissue expressing wild type or mutant Rb1 did not. Immunohistochemical analysis showed increased proliferation in both null and mutant prostate comparing to wild type, indicating that the mutant Rb1 is deficient in controlling proliferation. Suppression of tumor progression in wild type or mutant prostate tissue is associated with induction of cellular senescence in the prostate epithelium as indicated by senescence-associated β-gal staining as well as other senescence markers, such as IL-6. The ability of wild type and mutant Rb1 to enforce a senescence checkpoint in response to hormone treatment is cell autonomous as prostate epithelial cells (PrE) cultured in vitro readily undergo senescence in the presence of wild type and mutant Rb1, but not in the absence of Rb1. Previously published work indicates that pRb can induce cell cycle arrest independent of E2F by stabilizing the CKI (cyclin-dependent kinase inhibitor) p27 Kip1 . We suggest the mutant Rb1 is able to enforce cellular senescence through this mechanism since the mutant pRb is still able to bind Skp2, thereby stabilizing p27 Kip1 . In this study, we utilized genetically engineered mice containing wild type, null, or mutant Rb1 alleles to assess the importance of the pRb/E2F binding interaction in Rb1 -mediated tumor suppression in-vivo. Our conclusion is that pRb can suppress tumorigenesis in some tissues using mechanisms that do not depend on normal E2F binding and regulation.