Antiapoptotic B cell leukemia/lymphoma 2 (BCL2) family proteins are expressed in many cancers, but the circumstances under which these proteins are necessary for tumor maintenance are poorly understood. We exploited a novel functional assay that uses BCL2 homology domain 3 (BH3) peptides to predict dependence on antiapoptotic proteins, a strategy we call BH3 profiling. BH3 profiling accurately predicts sensitivity to BCL2 antagonist ABT-737 in primary chronic lymphocytic leukemia (CLL) cells. BH3 profiling also accurately distinguishes myeloid cell leukemia sequence 1 (MCL1) from BCL2 dependence in myeloma cell lines. We show that the special sensitivity of CLL cells to BCL2 antagonism arises from the requirement that BCL2 tonically sequester proapoptotic BIM in CLL. ABT-737 displaced BIM from BCL2's BH3-binding pocket, allowing BIM to activate BAX, induce mitochondrial permeabilization, and rapidly commit the CLL cell to death. Our experiments demonstrate that BCL2 expression alone does not dictate sensitivity to ABT-737. Instead, BCL2 complexed to BIM is the critical target for ABT-737 in CLL. An important implication is that in cancer, BCL2 may not effectively buffer chemotherapy death signals if it is already sequestering proapoptotic BH3-only proteins. Indeed, activator BH3-only occupation of BCL2 may prime cancer cells for death, offering a potential explanation for the marked chemosensitivity of certain cancers that express abundant BCL2, such as CLL and follicular lymphoma.

microRNAs (miRNAs) are a large class of endogenous short RNAs that repress gene expression. Many miRNAs are conserved throughout evolution, and dysregulation of miRNA pathways has been correlated with an increasing number of human diseases. In animals, miRNAs typically bind to the 3' untranslated region (3'UTR) of target mRNAs with imperfect sequence complementarity and repress translation. Despite their importance in regulating biological processes in numerous organisms, the mechanisms of miRNA function are largely unknown. Here, we report in vitro reactions for miRNA-directed translational gene silencing. These reactions faithfully recapitulate known in vivo hallmarks of mammalian miRNA function, including a requirement for a 5' phosphate and perfect complementarity to the mRNA target in the 5' seed region. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a polyA tail, whereas increasing polyA tail length alone can increase miRNA silencing activity.

The ability of polyomavirus large T antigen (LT) to promote cell cycling, to immortalize primary cells, and to block differentiation has been linked to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family. Our previous studies have shown that LT requires an intact N-terminal DnaJ domain, in addition to an Rb binding site, for activation of simple E2F-containing promoters and stimulation of cell cycle progression. Here we show that some LT effects dependent on interaction with the Rb family are largely DnaJ independent. In differentiating C2C12 myoblasts, overexpression of LT caused apoptosis. Although this activity of LT completely depended on Rb binding, LTs with mutations in the J domain remained able to kill. Comparisons of Rb؊ and J ؊ LTs revealed additional differences. Wild-type but not Rb ؊ LT activated the cyclin A promoter under serum starvation conditions. Genetic analysis of the promoter linked the Rb requirement to an E2F site in the promoter. LTs with mutations in the J domain were still able to activate the promoter. Finally, J mutant LTs caused changes in phosphorylation of both pRb and p130. In the case of p130, Thr-986 was shown to be a site that is regulated by J mutant LT. Taken together, these observations reveal that LT regulation of Rb function can be separated into both DnaJ-dependent and DnaJ-independent pathways.Polyomaviruses (Pys) have provided valuable models because the cellular pathways they use are generally important for cell function. The multifunctional large T antigens (LTs) represent one example. Their most obvious role is in DNA replication. Murine PyLT functions in initiation (21) of viral DNA synthesis, but it is also responsible for integration (15) and excision (4) of the viral genome. Biochemical studies on simian virus 40 (SV40) LT have provided important clues about basic cellular processes such as DNA replication (9, 78). LT's broad effects on the host cell led to the discovery of p53 (40,46) and to considerable insight into the Rb tumor suppressor and E2F transcription factor families (47).The effects of murine PyLT on the host cell can be viewed from a biological or a mechanistic perspective. LT induces cellular DNA synthesis (24, 66) and can immortalize primary cells (3,58). LT can also block the differentiation of either myoblasts (51) or preadipocytes (14). It has the ability to promote apoptosis (20). Finally, LT is a transactivator of cellular genes (39,54,55).Much of what LT does to cells depends upon its interactions with the Rb tumor suppressor family through an LXCXE motif starting at residue 141 (17, 32). Immortalization (22,34,41), induction of cell DNA synthesis (70), overcoming p53-induced cell cycle arrest (16), and prevention of differentiation (52) can all depend on Rb family binding. Transactivation of the thymidine kinase, polymerase ␣, PCNA, dihydrofolate reductase, and thymidylate synthase genes (54) as well as regulation of interferon-inducible gene expression (80) also requires an intact pRb/p107 binding site o...

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