[Abstract unavailable

SummaryMicroglia play key roles in brain development, homeostasis, and function, and it is widely assumed that the adult population is long lived and maintained by self-renewal. However, the precise temporal and spatial dynamics of the microglial population are unknown. We show in mice and humans that the turnover of microglia is remarkably fast, allowing the whole population to be renewed several times during a lifetime. The number of microglial cells remains steady from late postnatal stages until aging and is maintained by the spatial and temporal coupling of proliferation and apoptosis, as shown by pulse-chase studies, chronic in vivo imaging of microglia, and the use of mouse models of dysregulated apoptosis. Our results reveal that the microglial population is constantly and rapidly remodeled, expanding our understanding of its role in the maintenance of brain homeostasis.

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Cell killing is a critical pharmacological activity of imatinib to eradicate Bcr/Abl + leukemias. We found that imatinib kills Bcr/Abl + leukemic cells by triggering the Bcl-2-regulated apoptotic pathway. Imatinib activated several proapoptotic BH3-only proteins: bim and bmf transcription was increased, and both Bim and Bad were activated posttranslationally. Studies using RNAi and cells from gene-targeted mice revealed that Bim plays a major role in imatinib-induced apoptosis of Bcr/Abl + leukemic cells and that the combined loss of Bim and Bad abrogates this killing. Loss of Bmf or Puma had no effect. Resistance to imatinib caused by Bcl-2 overexpression or loss of Bim (plus Bad) could be overcome by cotreatment with the BH3 mimetic ABT-737. These results demonstrate that Bim and Bad account for most, perhaps all, imatinib-induced killing of Bcr/Abl + leukemic cells and suggest previously undescribed drug combination strategies for cancer therapy.

BackgroundThe epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.Methods and FindingsWe performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.ConclusionsInhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy.

Despite the clinical success of anti-CD20 monoclonal antibody (mAb) in the treatment of lymphoma, there remains considerable uncertainty about its mechanism of action. Here we show that the ability of mAbs to translocate CD20 into low-density, detergentinsoluble membrane rafts appears to control how effectively they mediate complement lysis of lymphoma cells. In vitro studies using a panel of anti-B-cell mAbs revealed that the anti-CD20 mAbs, with one exception (B1), are unusually effective at recruiting human complement. Differences in complement recruitment could not be explained by the level of mAb binding or isotype but did correlate with the redistribution of CD20 in the cell membrane following mAb ligation. Membrane fractionation confirmed that B1, unlike 1F5 and rituximab, was unable to translocate CD20 into lipid rafts. In addition, we were able to drive B1 and a range of other anti-B-cell mAbs into a detergent-insoluble fraction of the cell by hyper-cross-linking with an F(ab) 2 anti-Ig Ab, a treatment that also conferred the ability to activate lytic complement. Thus, we have shown that an important mAb effector function appears to be controlled by movement of the target molecule into membrane rafts, either because a raft location favors complement activation by mAbs or because rafts are more sensitive to complement

The anti-CD20 mAb rituximab is central to the treatment of B-cell malignancies, but resistance remains a significant problem. We recently reported that resistance could be explained, in part, by internalization of rituximab (type I anti-CD20) from the surface of certain B-cell malignancies, thus limiting engagement of natural effectors and increasing mAb consumption. Internalization of rituximab was most evident in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but the extent of internalization was heteroge- IntroductionThe anti-CD20 mAb rituximab has improved the overall survival of patients with follicular (FL) and diffuse large B-cell lymphoma (DLBCL). [1][2][3][4] However, in MCL, only modest responses are seen 5 and in CLL, fludarabine, cyclophosphamide and rituximab (FCR) therapy delivers improved responses but has yet to show a similar improvement in overall survival, 6 albeit the current follow-up is relatively short. Interestingly, those responses seen in CLL have often been achieved with high doses of rituximab, 6 suggesting that more mAb is needed to coat the targets or that it is consumed in some way. Even within rituximab-responsive lymphomas, a proportion of cases show resistance on first treatment with rituximab or eventually become resistant to rituximab-containing combination therapy (reviewed in Stolz et al 7 ). The molecular basis of this resistance and the observed sensitivity of different lymphoma subtypes is unclear (reviewed in Lim et al 8 ), but is highly relevant to improving outcomes.In addition to understanding target resistance, many groups are working to deliver anti-CD20 mAb reagents with improved affinity and more potent engagement of cytotoxic effectors. Anti-CD20 mAb can be defined as type I (eg, rituximab, ofatumumab) or type II (eg tositumumab, GA101), according to their ability to redistribute CD20 into lipid rafts in the plasma membrane and function in various effector assays. 9-11 It is still not clear what characteristics are required for the optimal reagent, but it is generally accepted that Fc:Fc ␥ receptor (Fc␥R) interactions are crucial to the efficacy of anti-CD20 mAb. [12][13][14][15] In particular, Fc␥RIIIa on myeloid effectors appears critical in controlling Ab potency and in keeping with this, lymphoma patients bearing the higher affinity 158V allele in Fc␥RIIIa respond better to rituximab compared with those with the low affinity 158F allotype, 16 leading many investigators to focus on augmenting the interaction of mAb with Fc␥RIIIa, for example via defucosylation. 17 Less attention has been given to the potential effects of the ITIM-containing inhibitory Fc␥R, Fc␥RIIb. Fc␥RIIb is a negative regulator of ITAM-containing receptors, such as the B-cell receptor (BCR) and the activatory Fc␥R. 18 Most hematopoietic cells coexpress inhibitory and activatory Fc␥R, and tumors are reported to be more sensitive to mAb immunotherapy in Fc␥RII Ϫ/Ϫ mice because of the removal of the inhibitory restraint of this receptor from myeloid effectors such as macro...