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The BRAF gene, encoding a mitogen-activated protein kinase kinase kinase, is mutated in several human cancers, with the highest incidence occurring in cutaneous melanoma. The activating V599E mutation accounted for 80% of all mutations detected in cutaneous melanoma cell lines. Reconstitution experiments have shown that this mutation increases ectopically expressed B-Raf kinase activity and induces NIH3T3 cell transformation. Here we used tumor-derived cell lines to characterize the activity of endogenous mutated B-Raf protein and assess its specific role in transformation. We show that three cell lines (OCM-1, MKT-BR, and SP-6.5) derived from human choroidal melanoma, the most frequent primary ocular neoplasm in humans, express B-Raf containing the V599E mutation. These melanoma cells showed a 10-fold increase in endogenous B-RafV599E kinase activity and a constitutive activation of the MEK/ERK pathway that is independent of Ras. This, as well as melanoma cell proliferation, was strongly diminished by siRNA-mediated depletion of the mutant B-Raf protein. Moreover, blocking B-RafV599E-induced ERK activation by different experimental approaches significantly reduced cell proliferation and anchorage-independent growth of melanoma cells. Finally, quantitative immunoblot analysis allowed us to identify signaling and cell cycle proteins that are differentially expressed between normal melanocytes and melanoma cells. Although the expression of signaling molecules was not sensitive to U0126 in melanoma cells, the expression of a cluster of cell cycle proteins remained regulated by the B-RafV599E/MEK/ERK pathway. Our results pinpoint this pathway as an important component in choroidal melanoma cell lines.

The t(8;21) translocation, found in adult acute myelogenous leukemia, results in the formation of an AML1/ETO chimeric transcription factor. AML1/ETO expression leads to alterations in hematopoietic progenitor cell differentiation, although its role in leukemic transformation is not clear. The N-terminal portion of AML1, which is retained in AML1/ETO, contains a region of homology to the FAST proteins, which cooperate with Smads to regulate transforming growth factor beta1 (TGF-beta1) target genes. We have demonstrated the physical association of Smad proteins with AML1 and AML1/ETO by immunoprecipitation and have mapped the region of interaction to the runt homology domain in these AML1 proteins. Using confocal microscopy, we demonstrated that AML1, and ETO and/or AML1/ETO, colocalize with Smads in the nucleus of t(8;21)-positive Kasumi-1 cells, in the presence but not the absence of TGF-beta1. Using transient transfection assays and a reporter gene construct that contains both Smad and AML1 consensus binding sequences, we demonstrated that overexpression of AML1B cooperates with TGF-beta1 in stimulating reporter gene activity, whereas AML1/ETO represses basal promoter activity and blocks the response to TGF-beta1. Considering the critical role of TGF-beta1 in the growth and differentiation of hematopoietic cells, interference with TGF-beta1 signaling by AML1/ETO may contribute to leukemogenesis.

SMADs are transforming growth factor beta (TGF-beta) receptor substrates and mediators of TGF-beta transcriptional responses. Here we provide evidence that the coactivators p300 and CBP interact with Smads 1 through 4. The biological relevance of this interaction is shown in vivo by overexpression of the adenovirus E1A protein and mutant forms of E1A that lack p300-binding sites. Wild-type E1A, but not the mutants, inhibits SMAD-dependent transcriptional responses to TGF-beta. E1A also inhibits the intrinsic transactivating function of the Smad4 MH2 domain. In addition, overexpression of p300 enhances SMAD-dependent transactivation. Our results suggest a role for p300/CBP in SMAD-mediated transcriptional activation and provide an explanation for the observed ability of E1A to interfere with TGF-beta action.