Mice lacking p27(Kip1) have been created by gene targeting in embryonic stem cells. These mice are larger than the control animals, with thymus, pituitary, and adrenal glands and gonadal organs exhibiting striking enlargement. CDK2 activity is elevated about 10-fold in p27(-/-) thymocytes. Development of ovarian follicles seems to be impaired, resulting in female sterility. Similar to mice with the Rb mutation, the p27(-/-) mice often develop pituitary tumors spontaneously. The retinas of the mutant mice show a disturbed organization of the normal cellular layer pattern. These findings indicate that p27(Kip1) acts to regulate the growth of a variety of cells. Unexpectedly, the cell cycle arrest mediated by TGFbeta, rapamycin, or contact inhibition remained intact in p27(-/-) cells, suggesting that p27(Kip1) is not required in these pathways.

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-Some of the principal requisites of toxicity screening methods in drug discovery are their ease to perform and high throughput, as well as the possibility to predict the occurrence of clinical events. Phospholipidosis is one of the toxicities often induced by potential drugs. Several physicochemical methods for the prediction of phospholipidosis have been reported. The purpose of the present study was to examine the predictability of methods based on lipophilicity and charge parameters. We employed a test set of 33 compounds including 11 in-house compounds. The phospholipidosis-inducing potential (PLIP) of the test set compounds was determined by the fluorescence-labeled lipid accumulation assay using isolated rat hepatocytes. This assay was verified by transmission electron microscopy (EM). The usefulness of the ClogP -most basic pK a (pK a -MB) plot to the PLIP of compounds was examined. This plot was unable to predict the PLIP of zwitterions. In order to improve its predictability, the net charge of a given molecule (NC) was introduced instead of pK a -MB, since the NC corresponds directly to the ionization state of compounds in the organelles. Compounds with high ClogP (> 1) and high NC (1≤NC≤2) tended to be positive. This finding was also confirmed using 30 additional validation set compounds obtained from the literature. The ClogP -NC plot differentiated positive and negative compounds with more than 98% accuracy (62/63), indicating its usefulness in drug discovery.

-We investigated and compared the cytotoxicity of 16 reference compounds in four in vitro systems: primary cultured rat hepatocytes, hepatoma HepG2 cell line, non-hepatic HeLa and Balb/c 3T3 cell lines. After 24 hr of exposure to the test compounds, the water-soluble tetrazolium salts WST-1 assay was used as an endpoint to evaluate cytotoxicity. Acetaminophen, diclofenac sodium cyclophosphamide and disulfiram displayed from 2 to more than10 times higher IC 50 values in three cell lines than in rat primary cultured hepatocytes. The cytotoxic effects of aspirin, amiodarone, clorfibiric acid, chlorpromazine, erythomycin, lithocholic acid, cisplatin and quinidine in rat hepatocytes were similar or 2 times stronger than those observed in cell lines. Ketoconazole resulted in the lowest IC 50 value in the HeLa cell line. The data suggested that the compounds which are known to be metabolism-mediated liver toxicants have a differential hepatotoxicity in vitro and that primary cultured rat hepatocytes could represent a valuable tool for both screening and study of the effects of bio-transformation on the cytotoxicity of new chemical entities and xenobiotics in vitro.

The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of aand l2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 33FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of a-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. Conclusion: AP2 mediates the internalization and subsequent degradation of CMresident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis. (HEPATOLOGY 2012;55:1889-1900