Most human tumours have genetic mutations in their Rb and p53 pathways, but retinoblastoma is thought to be an exception. Studies suggest that retinoblastomas, which initiate with mutations in the gene retinoblastoma 1 (RB1), bypass the p53 pathway because they arise from intrinsically death-resistant cells during retinal development. In contrast to this prevailing theory, here we show that the tumour surveillance pathway mediated by Arf, MDM2, MDMX and p53 is activated after loss of RB1 during retinogenesis. RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumour progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells. Our data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought. In addition, they support the idea that MDMX is a specific chemotherapeutic target for treating retinoblastoma.

The function of the p53 tumor suppressor protein must be highly regulated because p53 can cause cell death and prevent tumorigenesis. In cultured cells, the p90 MDM2 protein blocks the transcriptional activation domain of p53 and also stimulates the degradation of p53. Here we provide the first conclusive demonstration that p90 MDM2 constitutively regulates p53 activity in homeostatic tissues. Mice with a hypomorphic allele of mdm2 revealed a heretofore unknown role for mdm2 in lymphopoiesis and epithelial cell survival. Phenotypic analyses revealed that both the transcriptional activation and apoptotic functions of p53 were increased in these mice. However, the level of p53 protein was not coordinately increased, suggesting that p90 MDM2 can inhibit the transcriptional activation and apoptotic functions of p53 in a manner independent of degradation. Cremediated deletion of mdm2 caused a greater accumulation of p53, demonstrating that p90 MDM2 constitutively regulates both the activity and the level of p53 in homeostatic tissues. The observation that only a subset of tissues with activated p53 underwent apoptosis indicates that factors other than p90 MDM2 determine the physiological consequences of p53 activation. Furthermore, reduction of mdm2 in vivo resulted in radiosensitivity, highlighting the importance of mdm2 as a potential target for adjuvant cancer therapies.

The p53 inhibitor murine double-minute gene 2 (Mdm2) is a target for potential cancer therapies, however increased p53 function can be lethal. To directly address whether reduced Mdm2 function can inhibit tumorigenesis without causing detrimental side effects, we exploited a hypomorphic murine allele of mdm2 to compare the effects of decreased levels of Mdm2 and hence increased p53 activity on tumorigenesis and life span in mice. Here we report that mice with decreased levels of Mdm2 are resistant to tumor formation yet do not age prematurely, supporting the notion that Mdm2 is a promising target for cancer therapeutics.

The corepressor mSin3A is the core component of a chromatin-modifying complex that is recruited by multiple gene-specific transcriptional repressors. In order to understand the role of mSin3A during development, we generated constitutive germ line as well as conditional msin3A deletions. msin3A deletion in the developing mouse embryo results in lethality at the postimplantation stage, demonstrating that it is an essential gene. Blastocysts derived from preimplantation msin3A null embryos and mouse embryo fibroblasts (MEFs) lacking msin3A display a significant reduction in cell division. msin3A null MEFs also show mislocalization of the heterochromatin protein, HP1␣, without alterations in global histone acetylation. Heterozygous msin3A ؉/؊ mice with a systemic twofold decrease in mSin3A protein develop splenomegaly as well as kidney disease indicative of a disruption of lymphocyte homeostasis. Conditional deletion of msin3A from developing T cells results in reduced thymic cellularity and a fivefold decrease in the number of cytotoxic (CD8) T cells, while helper (CD4) T cells are unaffected. We show that CD8 development is dependent on mSin3A at a step downstream of T-cell receptor signaling and that loss of mSin3A specifically decreases survival of double-positive and CD8 T cells. Thus, msin3A is a pleiotropic gene which, in addition to its role in cell cycle progression, is required for the development and homeostasis of cells in the lymphoid lineage.mSin3A functions as a platform that mediates the association of histone deacetylase (HDAC) enzymes and other chromatin-modifying activities with a large number of DNA-binding transcriptional repressors (for reviews, see references 4 and 29). In order to interact with a wide range of repressors, Sin3 proteins from yeast to mammals have evolved and maintained four imperfect repeats known as paired amphipathic helix (PAH) domains (6, 49, 63), each of which appear to possess innate specificity for recruiting a defined repertoire of transcriptional repressors or corepressors. Recent structural studies have begun to define the basis for specific interactions between the second PAH domain of mSin3A (PAH2) and the transcriptional repressors Mad1 and HBP1 (8,11,19,57,58). The association of Sin3 with sequence-specific transcription factors serves to bring the complex in the vicinity of the transcription factor target genes, thereby permitting the complex to modulate transcriptional output by altering chromatin structure.Sin3 is thought to predominantly influence chromatin structure by mediating histone deacetylation through its recruitment of class I HDACs (HDAC1 and HDAC2) (3,21,22,34,41). Another Sin3 complex protein, SDS3, acts as a bridging protein between the HDACs and the Sin3 HDAC interaction domain (HID), a highly conserved region lying between PAH3 and PAH4 (2,13,15,16,36). The RbAp46 and RbAp48 proteins also associate with HDACs in the Sin3 complex and appear to target HDAC activity to histone tails. Other components of the complex may serve to couple Sin3 to spe...

Baculovirus p35 prevents programmed cell death in diverse organisms and encodes a protein inhibitor (P35) of the CED-3/interleukin-1␤-converting enzyme (ICE)-related proteases. By using site-directed mutagenesis, we have identified P35 domains necessary for suppression of virus-induced apoptosis in insect cells, the context in which P35 evolved. During infection, P35 was cleaved within an essential domain at or near the site DQMD-87G required for cleavage by CED-3/ICE family proteases. Cleavage site substitution of alanine for aspartic acid at position 87 (D87A) of the P 1 residue abolished P35 cleavage and antiapoptotic activity. Although the P 4 residue substitution D84A also caused loss of apoptotic suppression, it did not eliminate cleavage and suggested that P35 cleavage is not sufficient for antiapoptotic activity. Apoptotic insect cells contained a CED-3/ICE-like activity that cleaved in vitro-translated P35 and was inhibited by recombinant wild-type P35 but not P 1-or P 4-mutated P35. Thus, baculovirus infection directly or indirectly activates a novel CED-3/ICE-like protease that is inhibited by P35, thereby preventing virus-induced apoptosis. Our findings confirmed the inhibitory activity of P35 towards the CED-3/ICE proteases, including recombinant mammalian enzymes, and were consistent with a mechanism involving P35 stoichiometric interaction and cleavage. P35's inhibition of phylogenetically diverse proteases accounts for its general effectiveness as an apoptotic suppressor.