Benjamin T. Vroman

Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver

To study directly the functions of the cells that line the bile ducts inside the liver, we developed a new technique for isolating intrahepatic bile duct epithelial cells (IBDECs) from normal rat liver. Parenchymal and nonparenchymal cells were separated from whole liver by enzymatic digestion and mechanical disruption; subpopulations of individual nonparenchymal cells then were isolated by serial counter-flow elutriation, isopycnic centrifugation, and immunoaffinity separation with a specific monoclonal antibody against an antigen on the plasma membrane of IBDECs. Using this approach, we isolated 1.2 +/- 0.2 x 10(6) (mean +/- SE) viable (greater than 95% trypan blue exclusion) cells, greater than 95% of which were identified as IBDECs by morphologic appearance and specific cytochemical markers. The IBDECs averaged 7.4 +/- 0.16 microM in diameter and retained their in situ appearance, including morphologic polarity. They appeared as single cells or as cell doublets attached by tight junctions that excluded ruthenium red. Microvilli were abundant and were restricted to the apical (i.e., luminal) domain of the plasma membrane. Coated pits were observed on both apical and basolateral cell surfaces. Internally, IBDECs contained a well-developed system of organelles, including mitochondria, Golgi, and discrete types of vesicles, such as coated vesicles, multivesicular bodies, and lysosomes. These results indicate that a highly purified suspension of viable, morphologically intact, and polar IBDECs can be prepared from normal rat liver using a novel approach that separates liver cells on the basis of size, density, and specific membrane components. The availability of such a model will allow experimental studies to be performed directly on IBDECs, an approach that has not previously been possible.

Hsp90 Inhibition Depletes Chk1 and Sensitizes Tumor Cells to Replication Stress

DNA damage and replication stress activate the Chk1 signaling pathway, which blocks S phase progression, stabilizes stalled replication forks, and participates in G2 arrest. In this study, we show that Chk1 interacts with Hsp90, a molecular chaperone that participates in the folding, assembly, maturation, and stabilization of specific proteins known as clients. Consistent with Chk1 being an Hsp90 client, we also found that Chk1 but not Chk2 is destabilized in cells treated with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG-mediated Chk1 loss blocked the ability of Chk1 to target Cdc25A for proteolytic destruction, demonstrating that the Chk1 signaling pathway was disrupted in the 17-AAG-treated cells. Finally, 17-AAG-mediated disruption of Chk1 activation dramatically sensitized various tumor cells to gemcitabine, an S phase-active chemotherapeutic agent. Collectively, our studies identify Chk1 as a novel Hsp90 client and suggest that pharmacologic inhibition of Hsp90 may sensitize tumor cells to chemotherapeutic agents by disrupting Chk1 function during replication stress.

Morphologic Demonstration of Receptor-Mediated Endocytosis of Epidermal Growth Factor by Isolated Bile Duct Epithelial Cells

It was recently shown that intrahepatic bile duct epithelial cells in situ or after isolation from rat liver have coated pits and vesicles, suggesting that they participate in receptor-mediated endocytosis. Therefore, using a morphologic approach and epidermal growth factor coupled to horseradish peroxidase or colloidal gold as probes, we studied freshly isolated or short-term cultured intrahepatic bile duct epithelial cells prepared from normal rat liver to determine if they participate in receptor-mediated endocytosis. Immunoelectron microscopy using a monoclonal antibody against the epidermal growth factor receptor was also used to examine for the presence of the growth factor receptor on the cells. Immediately after isolation, the cells did not internalize either epidermal growth factor-horseradish peroxidase or epidermal growth factor-colloidal gold; no growth factor receptor could be shown on these cells by immunocytochemistry, either. In contrast, cells cultured for 24 h bound and internalized both epidermal growth factor-horseradish peroxidase and epidermal growth factor-colloidal gold at 37 degrees C and showed growth factor receptors diffusely distributed on the plasma membrane. When cultured cells exposed to epidermal growth factor-colloidal gold were fixed with glutaraldehyde containing saponin and tannic acid, colloidal gold particles were observed in coated pits and in coated and uncoated vesicles. Preincubation of cultured cells with native epidermal growth factor completely blocked the internalization of both epidermal growth factor-horseradish peroxidase and epidermal growth factor-colloidal gold. When rat liver was stained in situ for epidermal growth factor receptor, reaction product was observed by immunoelectron microscopy exclusively on the basal surface of the plasma membrane of the intrahepatic bile duct epithelial cells. These results indicate that bile duct epithelial cells internalize epidermal growth factor by endocytosis via coated pits containing receptors localized in situ exclusively to the basal domain of their plasma membranes. The data demonstrate for the first time that intrahepatic bile duct epithelial cells participate in receptor-mediated endocytosis and raise the possibility that they are a target for epidermal growth factor.

Ionizing radiation–induced MEK and Erk activation does not enhance survival of irradiated human squamous carcinoma cells

PURPOSE Ionizing radiation (IR) triggers several intracellular signaling cascades that have commonly been regarded as mitogenic, including the Raf-MEK-Erk kinase cascade. In addition to promoting proliferation, activated MEK and Erk may also prevent cell death induced by cytotoxic stimuli. Because Raf, MEK, and Erk are activated by IR in some tumor cell lines, this suggests that IR-induced activation of the kinase cascade may enhance the survival of irradiated cells. METHODS AND MATERIALS IR-induced activation of MEK and Erk was assessed in irradiated UM-SCC-6 cells, a human squamous carcinoma cell line. Activation of MEK and Erk was blocked with the pharmacological inhibitor of MEK activation, PD098059. Clonogenic survival was assessed in irradiated UM-SCC-6 cells that were pretreated with nothing or with the MEK inhibitor. RESULTS In UM-SCC-6 cells, IR doses as low as 2 Gy rapidly activated MEK and Erk. Pretreatment of the cells with the pharmacological inhibitor of MEK activation, PD098059, effectively blocked IR-induced activation of MEK and Erk. However, inhibition of the kinase cascade did not affect the clonogenic survival of irradiated cells in either early or delayed-plating experiments. CONCLUSION Taken together, these results suggest that although MEK and Erk are rapidly activated by IR treatment, these protein kinases do not affect the clonogenic survival of irradiated UM-SCC6 cells.

Fluid-phase endocytosis by intrahepatic bile duct epithelial cells isolated from normal rat liver.

Although recent data from our laboratory have established the occurrence of receptor-mediated endocytosis in intrahepatic bile duct epithelial cells (IBDEC) isolated from normal rat liver, no studies have assessed the role of isolated IBDEC in fluid-phase endocytosis. Therefore, to determine if IBDEC participate in fluid-phase endocytosis, we incubated morphologically polar doublets of IBDEC isolated from normal rat liver with horseradish peroxidase (HRP, 5 mg/ml), a protein internalized by fluid-phase endocytosis, and determined its intracellular distribution by electron microscopic cytochemistry. Pulse-chase studies using quantitative morphometry were also performed to assess the fate of HRP after internalization. After incubation at 37 degrees C, IBDEC internalized HRP exclusively at the apical (i.e., luminal) domain of their plasma membrane; internalization was completely blocked at 4 degrees C. After internalization, HRP was seen in acid phosphatase-negative vesicles and in acid phosphatase-positive multivesicular bodies (i.e., secondary lysosomes). Small acid phosphatase-negative vesicles containing HRP moved progressively from the apical to the basal domain of IBDEC. Pulse-chase studies showed that HRP was then discharged by exocytosis at the basolateral cell surface. These results demonstrate that IBDEC prepared from normal rat liver participate in fluid-phase endocytosis. After internalization, HRP either is routed to secondary lysosomes or undergoes exocytosis after transcytosis from the luminal to the basolateral cell surface. Our results suggest that IBDEC modify the composition of bile by internalizing both biliary proteins and fluid via endocytic mechanisms.

Identification and characterization of RAD9B, a paralog of the RAD9 checkpoint gene

RAD9 is an integral element of the PCNA-like HUS1-RAD1-RAD9 (9-1-1) complex that participates in genotoxin-induced CHK1 activation. We have identified a novel RAD9 paralog, dubbed RAD9B, in humans and mice. RAD9 and RAD9B share extensive amino acid homology throughout their entire sequences (36% identity, 48% similarity). Northern blotting revealed that RAD9B transcripts are highly expressed in human testes, with lower levels found in skeletal muscle. In contrast, RT-PCR analysis and immunoprecipitation demonstrated that RAD9B is also expressed in tumor cells. Like RAD9, RAD9B associates with HUS1, RAD1, and RAD17, suggesting that it is a RAD9 paralog that engages in similar biochemical reactions. In addition, we have also shown that RAD9 and RAD9B interact with the HUS1 paralog, HUS1B. Taken together, these results suggest that these proteins can combinatorially assemble into distinct 9-1-1 clamps that may have distinct biological functions.