During mammalian development, many cells are programmed to die most mediated by apoptosis. The Fas antigen coded by the structural gene for mouse lymphoproliferation mutation (lpr), is a cell surface protein belonging to the tumour necrosis factor/nerve growth factor receptor family, and mediates apoptosis. The Fas antigen messenger RNA is expressed in the thymus, liver, heart, lung and ovary. We prepared a monoclonal antibody against mouse Fas antigen, which immunoprecipitated the antigen (M(r) 45K) and had cytolytic activity against cell lines expressing mouse Fas antigen. We report here that staining of mouse thymocytes with the antibody indicated that thymocytes from the wild-type and lprcg mice expressed the Fas antigen, whereas little expression of the Fas antigen was found in lpr mice. Intraperitoneal administration of the anti-Fas antibody into mice rapidly killed the wild-type mice but neither lpr nor lprcg mice. Biochemical, histological and electron microscope analyses indicated severe damage of the liver by apoptosis. These findings suggest that the Fas antigen is important in programmed cell death in the liver, and may be involved in fulminant hepatitis in some cases.

Apoptosis-inducing Fas ligand (FasL) is a type II membrane protein, predominantly expressed in the activated T cells. FasL is cleaved by a putative metalloproteinase to produce a soluble form. Here, we blocked the shedding of human FasL by deleting its cleavage site. Although human Jurkat cells and mouse primary hepatocytes that express a low level of Fas were resistant to the soluble form of FasL, they were efficiently killed by membrane-bound FasL. Furthermore, soluble FasL inhibited cytotoxicity of the membrane-bound FasL. These results indicate that the membrane-bound form of FasL is the functional form and suggest that shedding of FasL is to prevent the killing of the healthy bystander cells by cytotoxic T cells.

The role of the multidrug resistance protein MRP4/ABCC4 in vivo remains undefined. To explore this role, we generated Mrp4-deficient mice. Unexpectedly, these mice showed enhanced accumulation of the anticancer agent topotecan in brain tissue and cerebrospinal fluid (CSF). Further studies demonstrated that topotecan was an Mrp4 substrate and that cells overexpressing Mrp4 were resistant to its cytotoxic effects. We then used new antibodies to discover that Mrp4 is unique among the anionic ATP-dependent transporters in its dual localization at the basolateral membrane of the choroid plexus epithelium and in the apical membrane The endothelial cells of the brain's capillaries are tightly joined to form a hydrophobic permeability barrier (32) termed the blood-brain barrier. Pgp expression in these cells limits the movement of hydrophobic cationic drugs from the blood into the brain (36,42,43). However, in vitro, these capillary endothelial cells also transport organic anions unidirectionally toward the capillary lumen in an energy-dependent fashion (5, 25, 41). Therefore, the capillary endothelial cells appear to express an unidentified anionic ABC transporter. Currently, it is unknown whether an anionic ABC transporter is expressed at functional levels in vivo in the endothelium of brain capillaries.The ABC transporter Mrp4, originally described as a nucleotide transporter (37), is known to transport a diverse array of compounds (2,7,34) and is capable of transporting organic anions as well as antiviral and antiretroviral compounds that do not easily penetrate the central nervous system (CNS) (2, 3, 9, 27, 37). Mrp4 expression was previously demonstrated on the basolateral membrane of the prostate gland and the apical membrane of the kidney (21, 44). Studies in cultured epithelial cells have demonstrated basolateral localization of Mrp4 (22). Transporters typically route to one surface in polarized cells. For instance, the Mrp (ABCC) subfamily members localize to either the basolateral or apical membrane, but not to both. MRP1 is restricted to the basolateral membrane of the choroid plexus and intestine, whereas MRP2 is found on the apical membrane in the intestine and liver (26,29). Mrp4 might be unique among the Mrp transporters in having cell-or tissuedependent polarized expression, but the biological importance of this unique ability to localize either apically or basolaterally remains unknown.We have developed Mrp4 knockout mice, and here we report their first use to show that Mrp4 is expressed in the lumen of brain capillaries and in the basolateral membrane in the choroid plexus epithelium. In vivo, Mrp4 restricts topotecan movement from the blood into the CSF and from the capillaries into the brain tissue by virtue of its unique ability to traffic to either the apical or basolateral membrane. We further show that Mrp4 overexpression confers resistance to the camptothecin topotecan. These studies have specific therapeutic implications for targeting the CNS that might harbor tumors but have more general impl...

Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA-mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1-mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.

The Fas ligand (FasL) is expressed in activated T cells and induces apoptosis in Fas-bearing cells. A cytotoxic T lymphocyte (CTL) clone specific for hepatitis B surface antigen (HBsAg) causes an acute liver disease in HBsAg transgenic mice. Here we observed that the CTL clone killed hepatocytes expressing HBsAg in a Fas-dependent manner. Administration of the soluble form of Fas into HBsAg transgenic mice prevented the CTL-induced liver disease. In the second model, mice were primed with Propionibacterium acnes. A subsequent challenge with lipopolysaccharide (LPS) killed the mice by inducing liver injury. Neutralization of FasL rescued the mice from LPS-induced mortality, and Fas-null mice were resistant to LPS-induced mortality. These results suggest that FasL has an essential role in the development of hepatitis.