ATP-dependent transport of glutathione and glucuronate conjugates from hepatocytes into bile is mediated by a distinct member of the ATP-binding cassette superfamily. We have cloned and sequenced the canalicular isoform of the multidrug resistance protein from rat liver, and termed it cMrp. This membrane glycoprotein is composed of 1541 amino acids with an identity of 47.8% with the human multidrug resistance protein (MRP) and of 41.9% with the yeast cadmium factor (YCF1). The carboxyl-terminal 130 amino acids of the human hepatocyte canalicular isoform of MRP (cMRP) were 80.2% identical with rat cMrp. cMrp was not expressed in the liver of two mutant rat strains, the Eisai hyperbilirubinemic rat and the GY/TR- mutant, which are deficient in the ATP-dependent transport of conjugates across the canalicular membrane. Immunoblotting using an antibody raised against the carboxyl terminus of cMrp detected the glycoprotein of about 190 kDa only in the canalicular membrane from normal liver. Double immunofluorescence and confocal laser scanning microscopy localized cMrp exclusively to the canalicular membrane domain of hepatocytes and demonstrated its loss in the hyperbilirubinemic mutant rat. The results identify cMrp as a canalicular transport protein with a novel sequence and with a function similar to the one of the MRP.

Tumor neovasculature substantially derives from sprouting of existing vessels, whereas the functional contribution of bone marrow-derived progenitors to neovessels remains controversial. We used transgenic mouse models of multistep carcinogenesis to monitor incorporation of bone marrow-derived cells into the neovasculature and to elucidate mechanisms of endothelial precursor cell (EPC) recruitment into the tumor microenvironment. We unequivocally demonstrate integration of bone marrow cells into the tumor vasculature as a late event in carcinogenesis that temporally correlates with VEGF release by the tumor and mobilization of circulating EPC in the periphery. Moreover, we demonstrate a chemokine-dependent mechanism of EPC homing into tumor, whereby neovessels of late-stage tumors release a battery of CC chemokines, which direct CCR2 ؉ and CCR5 ؉ progenitors into the vasculature. Thus, we show that tumor vessels promote their own growth and development in a self-amplifying fashion.cancer ͉ neovascularization

In nontumorigenic mammary epithelial cells (EpH4), transforming growth factor-b (TGFb1) causes cell cycle arrest/apoptosis, but induces epitheliomesenchymal transition (EMT) in Ha-Ras-transformed EpH4 cells (EpRas). EMT is closely correlated with late-stage tumor progression and results in fibroblastic, migratory cells displaying a mesenchymal gene expression program (FibRas). EpRas and FibRas cells showed strongly increased cell substrate adhesion to fibronectin, collagens I/IV and laminin 1. Furthermore, Ras transformation caused enhanced or de-novo expression of the integrin subunits b1, a2 and a3, or a5 and a6, respectively, the latter subunits being even more strongly expressed in FibRas cells. Importantly, polarized EpRas cells expressed integrin subunits b1 and a6 at distinct (apical and lateral) membrane domains, while FibRas cells coexpressed these integrins and a5 at the entire plasma membrane. During EMT, EpRas cells formed an a5b1 complex and deposited its ligand fibronectin into the extracellular matrix. Function-blocking a5 antibodies attenuated migration, and caused massive apoptosis in EpRas cells undergoing TGFb1-induced EMT in collagen gels, but failed to affect EpRas-or FibRas-derived structures. We conclude that functional a5b1 integrin is centrally implicated in EMT induction. Importantly, FibRas cells also failed to deposit the a6b4 ligand laminin 5, suggesting that a6b4 is no longer functional after EMT and replaced by mesenchymal integrins such as a5b1.