Bax exists in a dynamic equilibrium between the cytosol and mitochondria to control apoptotic priming.

The proapoptotic Bcl-2 protein Bax is predominantly found in the cytosol of nonapoptotic cells and is commonly thought to translocate to mitochondria following an apoptotic stimulus. The current model for Bax activation is that BH3 proteins bind to cytosolic Bax, initiating mitochondrial targeting and outer-membrane permeabilization. Here, we challenge this and show that Bax is constitutively targeted to mitochondria but in nonapoptotic cells is constantly translocated back to the cytosol. Using live-cell spinning-disk confocal imaging with a combination of FLIP, FRAP, and photoactivatable GFP-Bax, we demonstrate that disrupting adhesion-dependent survival signals slows the rate of Bax's dissociation from mitochondria, leading to its accumulation on the outer mitochondrial membrane. The overall accumulation of mitochondrial Bax following loss of survival signaling sensitizes cells to proapoptotic BH3 proteins. Our findings show that Bax is normally in a dynamic equilibrium between cytosol and mitochondria, enabling fluctuations in survival signals to finely adjust apoptotic sensitivity.

Analysis of inhibitor of apoptosis protein family expression during mammary gland development.

BACKGROUND: Inhibitors-of-Apoptosis-Proteins (IAPs) are an evolutionarily conserved family of proteins capable of regulating several facets of apoptosis. IAPs are frequently dysregulated in cancer, but their role in the regulation of apoptosis during developmental processes is not fully understood. Here we examined the expression of IAPs during the post-natal development of the mouse mammary gland, which is a tissue that exhibits a profound induction of apoptosis during involution. RESULTS: Six out of eight mammalian IAP family members are expressed in the mammary gland. Notably, quantitative PCR and immunoblotting revealed that XIAP, c-IAP1 and c-IAP2 are down-regulated in pregnancy and lactation, and prior to the onset of involution. In cultured mammary epithelial cells (MECs), XIAP levels decreased in response to inhibition of growth factor signalling. Maintaining XIAP levels in MECs by expressing exogenous XIAP protected them from all apoptotic stimuli tested. CONCLUSIONS: These data suggest that the developmental regulation of IAP expression in vivo contributes to naturally occurring programmes of cell death.

Targeting inhibitor of apoptosis proteins in combination with ErbB antagonists in breast cancer.

INTRODUCTION: Inhibitor of apoptosis (IAPs) proteins are a family of proteins that can block apoptosis in normal cells and have been suggested to cause resistance to apoptosis in cancer. Overexpression of oncogenic receptor tyrosine kinases is common in breast cancer; in particular 20% of all cases show elevated Her2. Despite clinical success with the use of targeted therapies, such as Trastuzumab, only up to 35% of Her2-positive patients initially respond. We reasoned that IAP-mediated apoptosis resistance might contribute to this insensitivity to receptor tyrosine kinase therapy, in particular ErbB antagonists. Here we examine the levels of IAPs in breast cancer and evaluate whether targeting IAPs can enhance apoptosis in response to growth factor receptor antagonists and TRAIL. METHODS: IAP levels were examined in a breast cancer cell line panel and in patient samples. IAPs were inhibited using siRNA or cell permeable mimetics of endogenous inhibitors. Cells were then exposed to TRAIL, Trastuzumab, Lapatinib, or Gefitinib for 48 hours. Examining nuclear morphology and staining for cleaved caspase 3 was used to score apoptosis. Proliferation was examined by Ki67 staining. RESULTS: Four members of the IAP family, Survivin, XIAP, cIAP1 and cIAP2, were all expressed to varying extents in breast cancer cell lines or tumours. MDAMB468, BT474 and BT20 cells all expressed XIAP to varying extents. Depleting the cells of XIAP overcame the intrinsic resistance of BT20 and MDAMB468 cells to TRAIL. Moreover, siRNA-based depletion of XIAP or use of a Smac mimetic to target multiple IAPs increased apoptosis in response to the ErbB antagonists, Trastuzumab, Lapatinib or Gefitinib in Her2-overexpressing BT474 cells, or Gefitinib in EGFR-overexpressing MDAMB468 cells. CONCLUSIONS: The novel findings of this study are that multiple IAPs are concomitantly expressed in breast cancers, and that, in combination with clinically relevant Her2 treatments, IAP antagonists promote apoptosis and reduce the cell turnover index of breast cancers. We also show that combination therapy of IAP antagonists with some pro-apoptotic agents (for example, TRAIL) enhances apoptosis of breast cancer cells. In some cases (for example, MDAMB468 cells), the enhanced apoptosis is profound.

Rapid induction of P-glycoprotein expression by high permeability compounds in colonic cells in vitro: a possible source of transporter mediated drug interactions?

P-glycoprotein (PGP) substrates with high membrane permeability, such as propranolol and verapamil, are considered to be essentially "transparent" to PGP since the transporter does not significantly limit their absorption or elimination. However, the question of whether such compounds can modulate PGP expression in epithelial cells following short-term exposure, with potential consequences for drug interactions, has not been addressed. LS180 colonic epithelial cells were exposed to propranolol or verapamil at concentrations (50-300 microM) consistent with those likely to be present in the gut lumen during oral dosing. Both compounds stimulated four to six-fold increases in MDR1 mRNA and PGP protein expression measured by quantitative real-time PCR and immunoblotting, respectively. These changes were accompanied by an induction in transporter activity measured by rhodamine 123 efflux. In contrast, metoprolol, a compound with similar permeability but no affinity for PGP had no effect on PGP expression. The induction of PGP by propranolol and verapamil was rapid with significant increases occurring within 3h with maximal stimulation after 6h exposure. Rifampicin, shown to cause clinical drug interactions via a PXR-mediated increase in PGP expression, exhibited a very similar time-course and extent of induction. In conclusion, verapamil and propranolol, whose trans-epithelial permeability are unaffected by PGP, appear to be effective inducers of PGP expression in gut epithelial cells in vitro. While the in vivo significance of these observations is unknown, this questions whether high permeability, "PGP-transparent" compounds, currently favoured in drug selection strategies, should be evaluated in terms of their potential for transporter-mediated drug interactions.

Interferon gamma induces translocation of commensal Escherichia coli across gut epithelial cells via a lipid raft-mediated process.

BACKGROUND & AIMS: The "leaky gut" hypothesis proposes that leakage of enteric bacteria into the body resulting from disruption of the epithelial barrier is a critical step in the pathophysiology of various disorders such as inflammatory bowel disease and sepsis. However, the pathways and underlying mechanisms by which commensal bacteria cross the epithelial barrier in inflammatory conditions remain unclear. This study investigated the mechanisms of interferon gamma-mediated bacterial translocation across human colonic epithelial monolayers. METHODS: Caco-2 and T84 monolayers were exposed to interferon gamma. Barrier function was assessed by transepithelial electrical resistance and lucifer yellow permeability. Internalization and translocation of Escherichia coli strain C25 were measured by quantitative bacterial culture. Expression and distribution of junctional proteins were assessed by immunoblotting and confocal imaging. RESULTS: Minimal apical to basolateral translocation of C25 was observed in untreated T84 and Caco-2 monolayers. Interferon gamma caused a dramatic, dose-dependent increase in C25 translocation, which was uncoupled from cytokine-induced increases in paracellular permeability and disruption of tight junction proteins at low interferon gamma concentrations. These effects were associated with increased internalization of viable bacteria into, but not adherence to, Caco-2 cells. Interferon gamma-mediated bacterial translocation was abolished by pretreatment with the cholesterol-disrupting drugs filipin and methyl-beta-cyclodextrin, whereas these agents had no effect on infection of Caco-2 by the enteric pathogen Shigella sonnei. CONCLUSIONS: Normally poorly invasive enteric bacteria may, in situations of inflammatory stress, exploit lipid raft-mediated transcytotic pathways to cross the intestinal epithelium, and these effects may precede cytokine-induced disruption of tight junctions.

Predicting P-glycoprotein effects on oral absorption: correlation of transport in Caco-2 with drug pharmacokinetics in wild-type and mdr1a(-/-) mice in vivo.

PURPOSE: Cell-based permeability screens are widely used to identify drug-P-glycoprotein (PGP) interaction in vitro. However, their reliability in predicting the impact of PGP on human drug pharmacokinetics is poorly defined. The aim was to determine whether a quantitative relationship exists between PGP-mediated alterations in Caco-2 permeability and oral pharmacokinetics in mice. METHODS: Two indicators of drug efflux were measured in Caco-2 for a group of 10 compounds, the ratio of A-B and B-A transport (R9B-A/A-B)) and the ratio of A-B transport in the presence and absence of a PGP inhibitor, GF120918 (R(GF)). These data were correlated with ratios of oral plasma levels in either mdr1a(-/-) or mdr1a/1b(-/-) and wild-type mice (R(KO/WT in vivo)) calculated from literature data on these compounds. RESULTS: A significant, positive correlation (r2 = 0.8, p < 0.01) was observed between RGF and R(KO/WT in vivo). In contrast, R(B-A/A-B), a more commonly used in vitro measure, showed a much weaker correlation with in vivo data (r2 = 0.33, p = 0.11). A strong correlation with R(GF) was also observed after correction of in vivo data for PGP effects on IV clearance. CONCLUSION: The increase in A-B drug permeability following inhibition of PGP in Caco-2 allows a reasonable prediction of the likely in vivo impact that PGP will have on plasma drug levels after oral administration.