E2F-1 binding affinity for pRb is not the only determinant of the E2F-1 activity

E2F-1 is the major cellular target of pRB and is regulated by pRB during cell proliferation. Interaction between pRB and E2F-1 is dependent on the phosphorylation status of pRB. Despite the fact that E2F-1 and pRB have antagonistic activities when they are overexpressed, the role of the E2F-1-pRB interaction in cell growth largely remains unknown. Ideally, it would be better to study the properties of a pRB mutant that fails to bind to E2F, but retains all other activities. To date, no pRB mutation has been characterized in sufficient detail to show that it specifically eliminates E2F binding but leaves other interactions intact. An alternative approach to this issue is to ask whether mutations that change E2F proteins binding affinity to pRB are sufficient to change cell growth in aspect of cell cycle and tumor formation. Therefore, we used the E2F-1 mutants including E2F-1/S332-7A, E2F-1/S375A, E2F-1/S403A, E2F-1/Y411A and E2F-1/L132Q that have different binding affinities for pRB to better understand the roles of the E2F-1 phosphorylation and E2F-1-pRB interaction in the cell cycle, as well as in transformation and gene expression. Data presented in this study suggests that in vivo phosphorylation at amino acids 332-337, 375 and 403 is important for the E2F-1 and pRB interaction in vivo. However, although E2F-1 mutants 332-7, 375 and 403 showed similar binding affinity to pRB, they showed different characteristics in transformation efficiency, G₀ accumulation, and target gene experiments.     

Tumor suppression by a severely truncated species of retinoblastoma protein

Rb(+/+):Rb(-/-) chimeric mice are healthy until early in adulthood when they develop lethal pituitary tumors composed solely of Rb(-/-) cells. In an effort to delineate the minimal structures of the retinoblastoma protein necessary for RB tumor suppression function, chimeric animals derived from stably transfected RB(-/-) embryonic stem (ES) cells were generated. One such ES cell transfectant expressed a human RB allele encoding a stable, truncated nuclear derivative lacking residues 1 to 378 (Delta 1-378). Others encoded either wild-type human RB or an internally deleted derivative of the Delta 1-378 mutant. All gave rise to viable chimeric animals with comparable degrees of chimerism. However, unlike control mice derived, in part, from naive Rb(-/-) ES cells or from ES cells transformed by the double RB mutant, Delta 1-378/Delta exon22, animals derived from either wild-type RB- or Delta 1-378 RB-producing ES cells failed to develop pituitary tumors. Thus, in this setting, a substantial fraction of the RB sequence is unnecessary for RB-mediated tumor suppression.     

Mutation of the LXCXE Binding Cleft of pRb Facilitates Transformation by ras In Vitro but Does Not Promote Tumorigenesis In Vivo

Background

The Retinoblastoma protein (pRB) is a key tumor suppressor that is functionally inactivated in most cancers. pRB regulates the cell division cycle and cell cycle exit through protein–protein interactions mediated by its multiple binding interfaces. The LXCXE binding cleft region of pRB mediates interactions with cellular proteins that have chromatin regulatory functions. Chromatin regulation mediated by pRB is required for a stress responsive cell cycle arrest, including oncogene induced senescence. The in vivo role of chromatin regulation by pRB during senescence, and its relevance to cancer is not clear.

Methodology/Principal Findings

Using gene-targeted mice, uniquely defective for pRB mediated chromatin regulation, we investigated its role during transformation and tumor progression in response to activation of oncogenic ras. We report that the pRB^∆L mutation confers susceptibility to escape from HrasV12 induced senescence and allows transformation in vitro, although these cells possess high levels of DNA damage. Intriguingly, LSL-Kras, Rb1 ^∆L/∆L mice show delayed lung tumor formation compared to controls. This is likely due to the increased apoptosis seen in the early hyperplastic lesions shortly following ras activation that inhibits tumor progression. Furthermore, DMBA treatment to induce sporadic ras mutations in other tissues also failed to reveal greater susceptibility to cancer in Rb1 ^∆L/∆L mice.

Conclusions/Significance

Our data suggests that chromatin regulation by pRB can function to limit proliferation, but its loss fails to contribute to cancer susceptibility in ras driven tumor models because of elevated levels of DNA damage and apoptosis.     

Neural precursor cells differentiating in the absence of Rb exhibit delayed terminal mitosis and deregulated E2F 1 and 3 activity

The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis we examined the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural stem cells derived from Rb-deficient mice. Although Rb -/- neural precursor cells are able to differentiate in vitro we show that these cells exhibit a significant delay in terminal mitosis relative to wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts with the putative Rb target E2F3 in neural precursor cells, our results indicate that it clearly cannot restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3 activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In these studies we show that Rb is essential for the regulation of E2F 1 and 3 activity as well as the onset of terminal mitosis in neural precursor cells.     

Visualizing dynamic E2F-mediated repression in vivo

Although many E2F target genes have been identified recently, very little is known about how any single E2F site controls the expression of an E2F target gene in vivo. To test the requirement for a single E2F site in vivo and to learn how E2F-mediated repression is regulated during development and tumorigenesis, we have constructed a novel series of wild-type and mutant Rb promoter-LacZ transgenic reporter lines that allow us to visualize the activity of a crucial E2F target in vivo, the retinoblastoma tumor suppressor gene (Rb). Two mutant Rb promoter-LacZ constructs were used to evaluate the importance of a single E2F site or a nearby activator (Sp1/Ets) site that is found mutated in low-penetrance retinoblastomas. The activity of the wild-type Rb promoter is dynamic, varying spatially and temporally within the developing nervous system. While loss of the activator site silences the Rb promoter, loss of the E2F site stimulates its activity in the neocortex, retina, and trigeminal ganglion. Surprisingly, E2F-mediated repression of Rb does not act globally or in a static manner but, instead, is a highly dynamic process in vivo. Using neocortical extracts, we detected GA-binding protein alpha (GABPalpha, an Ets family member) bound to the activator site and both E2F1 and E2F4 bound to the repressor site of the Rb promoter in vitro. Additionally, we detected binding of both E2F1 and E2F4 to the Rb promoter in vivo using chromatin immunoprecipitation analysis on embryonic day 13.5 brain. Unexpectedly, we detect no evidence for Rb promoter autoregulation in neuroendocrine tumors from Rb+/-; RbP-LacZ mice that undergo loss of heterozygosity at the Rb locus, in contrast to the situation in human retinoblastomas where high RB mRNA levels are found. In summary, this study provides the first demonstration that loss of an E2F site is critical for target gene repression in vivo and underscores the complexity of the Rb and E2F family network in vivo.