Neutrophils respond to invading bacteria by adopting a polarized morphology, migrating in the correct direction, and engulfing the bacteria. How neutrophils establish and precisely orient this polarity toward pathogens remains unclear. Here we report that in resting neutrophils, the ERM (ezrin, radixin, and moesin) protein moesin in its active form (phosphorylated and membrane bound) prevented cell polarization by inhibiting the small GTPases Rac, Rho, and Cdc42. Attractant-induced activation of myosin phosphatase deactivated moesin at the prospective leading edge to break symmetry and establish polarity. Subsequent translocation of moesin to the trailing edge confined the formation of a prominent pseudopod directed toward pathogens and prevented secondary pseudopod formation in other directions. Therefore, both moesin-mediated inhibition and its localized deactivation by myosin phosphatase are essential for neutrophil polarization and effective neutrophil tracking of pathogens.

The promyelocytic leukemia (PML) gene encodes a putative tumor suppressor gene involved in the control of apoptosis, which is fused to the retinoic acid receptor α (RARα) gene in the vast majority of acute promyelocytic leukemia (APL) patients as a consequence of chromosomal translocations. The PMLRARα oncoprotein is thought to antagonize the function of PML through its ability to heterodimerize with and delocalize PML from the nuclear body. In APL, this may be facilitated by the reduction to heterozygosity of the normal PML allele. To determine whether PML acts as a tumor suppressor in vivo and what the consequences of deregulated programmed cell death in leukemia and epithelial cancer pathogenesis are, we crossed PML −/ − mice with human cathepsin G (hCG)-PMLRARα or mammary tumor virus (MMTV)/neu transgenic mice (TM), models of leukemia and breast cancer, respectively. The progressive reduction of the dose of PML resulted in a dramatic increase in the incidence of leukemia, and in an acceleration of leukemia onset in PMLRARα TM. By contrast, PML inactivation did not affect neu-induced tumorigenesis. In hemopoietic cells from PMLRARα TM, PML inactivation resulted in impaired response to differentiating agents such as RA and vitamin D 3 as well as in a marked survival advantage upon proapoptotic stimuli. These results demonstrate that: (a) PML acts in vivo as a tumor suppressor by rendering the cells resistant to proapoptotic and differentiating stimuli; (b) PML haploinsufficiency and the functional impairment of PML by PMLRARα are critical events in APL pathogenesis; and (c) aberrant control of programmed cell death plays a differential role in solid tumor and leukemia pathogenesis.

Dendritic cells (DCs) are bone marrow–derived leukocytes that function as potent antigen presenting cells capable of initiating T cell–dependent responses from quiescent lymphocytes. DC pulsed with tumor-associated antigen (TAA) peptide or protein have recently been demonstrated to elicit antigen-specific protective antitumor immunity in a number of murine models. Transduction of DCs with TAA genes may allow stable, prolonged antigen expression as well as the potential for presentation of multiple, or unidentified, epitopes in association with major histocompatibility complex class I and/or class II molecules. To evaluate the potential efficacy of retrovirally transduced DCs, bone marrow cells harvested from BALB/c mice were transduced with either a model antigen gene encoding β-galactosidase (β-gal) or a control gene encoding rat HER-2/neu (Neu) by coculture with irradiated ecotropic retroviral producer lines. Bone marrow cells were differentiated into DC in vitro using granulocyte/macrophage colony-stimulating factor and interleukin-4. After 7 d in culture, cells were 45–78% double positive for DC phenotypic cell surface markers by FACS ® analysis, and DC transduced with β-gal were 41–72% positive for β-gal expression by X-gal staining. In addition, coculture of β-gal transduced DC with a β-gal–specific T cell line (CTLx) resulted in the production of large amounts of interferon-γ, demonstrating that transduced DCs could process and present endogenously expressed β-gal. DC transduced with β-gal and control rat HER-2/neu were then used to treat 3-d lung metastases in mice bearing an experimental murine tumor CT26.CL25, expressing the model antigen, β-gal. Treatment with β-gal–transduced DC significantly reduced the number of pulmonary metastatic nodules compared with treatment with Hank's balanced salt solution or DCs transduced with rat HER-2/neu. In addition, immunization with β-gal–transduced DCs resulted in the generation of antigen-specific cytotoxic T lymphocytes (CTLs), which were significantly more reactive against relevant tumor targets than CTLs generated from mice immunized with DCs pulsed with the L d -restricted β-gal peptide. The results observed in this rapidly lethal tumor model suggest that DCs transduced with TAA may be a useful treatment modality in tumor immunotherapy.

According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1β converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (ΔΨ m ). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the ΔΨ m is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the ΔΨ m collapse and subsequent apoptosis. Cytosols from anti-Fas–treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their ΔΨ m . In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + ΔΨ m collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.