Novel retinoblastoma mutation abrogating the interaction to E2F2/3, but not E2F1, led to selective suppression of thyroid tumors.
Bottom Line: These observations show that pRb mediates the inactivation of E2F function and its contribution to tumorigenesis is highly dependent on the cell type.Last, by using a reconstitution assay of synthesized proteins, we showed that the D326V missense pRb bound to E2F1 but failed to interact with E2F2/3.These results reveal the effect of the pRb N-terminal domain on E2F function and the impact of the protein on tumorigenesis.
Affiliation: Team for Advanced Development and Evaluation of Human Disease Models, Riken BioResource Center, Tsukuba, Ibaraki, Japan.Show MeSH
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Mentions: To understand the mechanism of the D326V mutation on the tumor spectrum, we performed comparative analyses for Rb1D326V/+ and Rb1+/− mice. Because inactivation of the functions of E2F was considered to be the major mechanism by which pRb suppressed tumor development, we focused on the E2F proteins in the mutant mice. For this purpose, we initially tried to determine the protein level in multiple tissues, including thyroid tissues, using immunoblot analysis, but we did not detect any significant differences between the controls and the mutant mice. We hypothesized that this is because each tissue contained multiple types of cells, which respond differently to the inactivation of pRb function. In fact, previous reports indicate that protein levels of E2F are increased by the depletion of pRb; this occurs via the expression of shRNA in prostate cancer cells and by targeted mutation in mouse embryonic fibroblasts.15,16 In contrast, two reports have demonstrated that E2F proteins increased their stabilities by interacting with pRb17,18 (Fig. S2). These observations indicate that pRb deficiency might have a positive or negative influence on the abundance of E2F proteins, depending on the cell context. Hence, there is a need to examine the cell type-specific effect of pRb deficiency. For this purpose, we performed IHC to evaluate the level of E2F and found that E2F1, E2F2 and E2F3 were decreased in the thyroid C-cells of the mutant Rb1+/− mice (Fig.3a). These results indicate that, in the thyroid C-cells, “free” active E2F were unstable, while pRb-bound E2F were stable and accumulated.17,18
Affiliation: Team for Advanced Development and Evaluation of Human Disease Models, Riken BioResource Center, Tsukuba, Ibaraki, Japan.