Several Distinct Polycomb Complexes Regulate and Co-Localize on the INK4a Tumor Suppressor Locus

Misexpression of Polycomb repressive complex 1 (PRC1) components in human cells profoundly influences the onset of cellular senescence by modulating transcription of the INK4a tumor suppressor gene. Using tandem affinity purification, we find that CBX7 and CBX8, two Polycomb (Pc) homologs that repress INK4a, both participate in PRC1-like complexes with at least two Posterior sex combs (Psc) proteins, MEL18 and BMI1. Each complex contains a single representative of the Pc and Psc families. In primary human fibroblasts, CBX7, CBX8, MEL18 and BMI1 are present at the INK4a locus and shRNA-mediated knockdown of any one of these components results in de-repression of INK4a and proliferative arrest. Sequential chromatin immunoprecipitation (ChIP) reveals that CBX7 and CBX8 bind simultaneously to the same region of chromatin and knockdown of one of the Pc or Psc proteins results in release of the other, suggesting that the binding of PRC1 complexes is interdependent. Our findings provide the first evidence that a single gene can be regulated by several distinct PRC1 complexes and raise important questions about their configuration and relative functions

Molecular Mechanisms of Cardiovascular Damage Induced by Anti-HER-2 Therapies

In the last two decades, newer biological drugs have been designed in order to \u201ctarget\u201d specific proteins involved in cancer proliferation and overcome the increased risk of cardiovascular toxicity associated with \u201cbroad-spectrum\u201d classic chemotherapeutics. Unfortunately, these proteins are also important for the maintenance of cardiovascular homeostasis. The humanized anti-ErbB2 antibody, trastuzumab, is the prototypical biological drug first introduced in antineoplastic protocols for the treatment of ErbB2+ breast cancer. Indeed, not only is this protein overexpressed in several breast cancers, but also it plays a major role in the cardiovascular system in cell growth, including myocyte growth, and inhibition of apoptosis and can modulate the oxidative damage induced by anthracyclines. Hence, patients treated with trastuzumab developed systolic dysfunction, especially when administered with or shortly after doxorubicin

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

Cytotoxic lymphocytes (CLs) contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Among these cytotoxins is granzyme B (GrB), a protease that has also been implicated in activation (restimulation)-induced cell death of natural killer (NK) and T cells, but the underlying mechanism and its regulation are unclear. Here we show that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol. The model lysosomal stressors sphingosine and Leu-Leu-methyl-ester, and CLs from gene-targeted mice were used to show that LMP releases GrB in both a time- and concentration-dependent manner, and that the liberated GrB is responsible for cell death. The endogenous GrB inhibitor Serpinb9 (Sb9) protects CLs against LMP-induced death but is decreasingly effective as the extent of LMP increases. We also used these model stressors to show that GrB is the major effector of LMP-mediated death in T cells, but that in NK cells additional effectors are released, making GrB redundant. We found that limited LMP and GrB release occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to GrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the lymphocyte cytoplasm and its ensuing interaction with Sb9 dictates whether or not the cell survives. The GrB-Sb9 nexus may therefore represent an additional mechanism of limiting lymphocyte lifespan and populations

Individual CREB-target genes dictate usage of distinct cAMP-responsive coactivation mechanisms

CREB is a key mediator of cAMP- and calcium-inducible transcription, where phosphorylation of serine 133 in its Kinase-Inducible Domain (KID) is often equated with transactivation. Phospho-Ser133 is required for CREB to bind the KIX domain of the coactivators CBP and p300 (CBP/p300) in vitro, although the importance of this archetype coactivator interaction for endogenous gene expression is unclear. Here, we show that the CREB interaction with KIX is necessary for only a part of cAMP-inducible transcription and CBP/p300 recruitment. Surprisingly, individual cAMP-inducible genes with CREB bound at their promoters differed in their reliance on KIX and none examined showed complete dependence. Alternatively, we found that arginine 314 (Arg314) in the CREB basic-leucine zipper (bZIP) domain contributed to CBP/p300 recruitment and KIX-independent CREB transactivation function. This implicates Transducer Of Regulated CREB (TORC), an unrelated cAMP-responsive coactivator that binds via Arg314, and which can bind CBP/p300, in these functions. Interestingly, KIX was also required for the full cAMP induction of a gene that did not require CREB. Thus, individual CREB-target gene context dictates the relative contribution of at least two different cAMP-responsive coactivation mechanisms