Biomedical subjects

Samuel W French

Publications and source records attributed to Samuel W French.

At least 19 recordsLinked to original sources

PubMed2006

CYP2E1 induced by ethanol causes oxidative stress, proteasome inhibition and cytokeratin aggresome (Mallory body-like) formation.

The role of oxidative stress in alcoholic liver disease and cytokeratin aggresome formation is the focus of this in vitro study. HepG2 cells transduced to over express CYP2E1 (E47) and control HepG2 cells (C34) were first treated with arachidonic acid, then Fe-NAT, and finally with ethanol. In the E47 ethanol-treated cells, CYP2E1 was induced and a higher level of reactive oxygen species and carbonyl proteins were generated. The proteasome activity decreased significantly in the E47 ethanol-treated cells. This inhibition was prevented when CYP2E1 was inhibited by DAS. Microarray analysis showed gene expression down regulation of the proteasome subunit, as well as ubiquitin pathway proteins in the E47 ethanol-treated cells. 4-Hydroxynonenal (4-HNE) adducts were increased in the E47 cells treated with ethanol. Furthermore, the immunoprecipitated 4-HNE modified proteins from these cells stained positive with antibodies to the proteasome subunit alpha 6. These results indicate that the ethanol induced CYP2E1 generates oxidative stress that is responsible for the decrease in proteasome activity. Cytokeratin 8 and 18 were induced by ethanol treatment of E47 cells and polyubiquitinated forms of these proteins were found in the polyubiquitin smear upon Western blots analysis. Cytokeratin aggresomes and Mallory body-like inclusions formed in the ethanol-treated E47 cells, indicating that the ubiquitinated cytokeratins accumulated as a result of the inhibition of the proteasome by ethanol treatment when oxidation of ethanol induced oxidative stress. This is the first report where ethanol caused Mallory body-like cytokeratin inclusions in transformed human liver cells in vitro.

Aldehydes↗

PubMed2006

Mammalian polynucleotide phosphorylase is an intermembrane space RNase that maintains mitochondrial homeostasis.

We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-beta-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in FoF1-ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.

Adenosine Triphosphate↗

PubMed2006

A new function in translocation for the mitochondrial i-AAA protease Yme1: import of polynucleotide phosphorylase into the intermembrane space.

Polynucleotide phosphorylase (PNPase) is an exoribonuclease and poly(A) polymerase postulated to function in the cytosol and mitochondrial matrix. Prior overexpression studies resulted in PNPase localization to both the cytosol and mitochondria, concurrent with cytosolic RNA degradation and pleiotropic cellular effects, including growth inhibition and apoptosis, that may not reflect a physiologic role for endogenous PNPase. We therefore conducted a mechanistic study of PNPase biogenesis in the mitochondrion. Interestingly, PNPase is localized to the intermembrane space by a novel import pathway. PNPase has a typical N-terminal targeting sequence that is cleaved by the matrix processing peptidase when PNPase engaged the TIM23 translocon at the inner membrane. The i-AAA protease Yme1 mediated translocation of PNPase into the intermembrane space but did not degrade PNPase. In a yeast strain deleted for Yme1 and expressing PNPase, nonimported PNPase accumulated in the cytosol, confirming an in vivo role for Yme1 in PNPase maturation. PNPase localization to the mitochondrial intermembrane space suggests a unique role distinct from its highly conserved function in RNA processing in chloroplasts and bacteria. Furthermore, Yme1 has a new function in protein translocation, indicating that the intermembrane space harbors diverse pathways for protein translocation.

ATP-Dependent Proteases↗

PubMed2006

The TCL1 oncoprotein binds the RNase PH domains of the PNPase exoribonuclease without affecting its RNA degrading activity.

TCL1 is an AKT kinase coactivator that, when dysregulated, initiates mature lymphocyte malignancies in humans and transgenic mice. While TCL1 augments AKT pathway signaling, additional TCL1 interacting proteins that may contribute to cellular homeostasis or transformation are lacking. Here, an exoribonuclease, PNPase, was identified in a complex with TCL1. The AKT interaction domain on TCL1 bound either RNase PH repeat domain of PNPase without influencing its RNA degrading activity, which was compatible with predicted docking models for a TCL1-PNPase complex. Our data provide a novel protein interaction for mammalian PNPase that may impact TCL1 mediated transformation.

Blotting, Western↗

PubMed2006

Histogenesis of solid pseudopapillary tumor of the pancreas: the case for the centroacinar cell of origin.

Solid pseudopapillary tumor (SPT) is an unusual pancreatic neoplasm of low malignant potential that most frequently occurs in young women. The tumor is indolent, with long patient survival, even in the presence of extension into adjacent organs and metastases. Histologically, it is a solid and cystic tumor with a prominent vascular network and degenerative pseudopapillae formation. Despite its distinctive morphology and cytological features, its histogenesis is unclear. Herein, we report a case of solid pseudopapillary tumor in a 41-year-old female in which the tumor cells immunohistochemically and ultrastructurally suggest a centroacinar cell origin. The tumor cells and the normal centroacinar cells stained positive for alpha-antitrypsin (alpha-AT), CD10, cyclin D1 and NSE. Ultrastructural examination shows similarities in nuclear shape, nucleoli location and cytoplasmic contents between neoplastic cells and normal centroacinar cells of the pancreas. Based on both immunohistochemical and ultrastructural features, we propose that the centroacinar cell is the origin of SPT.

Adult↗

PubMed2006

Regulation of murine cardiac 20S proteasomes: role of associating partners.

Our recent studies have provided a proteomic blueprint of the 26S proteasome complexes in the heart, among which 20S proteasomes were found to contain cylinder-shaped structures consisting of both alpha and beta subunits. These proteasomes exhibit a number of features unique to the myocardium, including striking differences in post-translational modifications (PTMs) of individual subunits and novel PTMs that have not been previously reported. To date, mechanisms contributing to the regulation of this myocardial proteolytic core system remain largely undefined; in particular, little is known regarding PTM-dependent regulation of cardiac proteasomes. In this investigation, we seek to elucidate the function and regulation of 20S proteasome complexes in the heart. Functionally viable murine cardiac 20S proteasomes were purified. Tandem mass spectrometry analyses, combined with native gel electrophoresis, immunoprecipitation, and immunoblotting, revealed the identification of 2 previously unrecognized functional partners in the endogenous intact cardiac 20S complexes: protein phosphatase 2A (PP2A), and protein kinase A (PKA). Furthermore, our results demonstrated that PP2A and PKA profoundly impact the proteolytic function of 20S proteasomes: phosphorylation of 20S complexes enhances the peptidase activity of individual subunits in a substrate-specific fashion. Moreover, inhibition of PP2A or the addition of PKA significantly modified both the serine- and threonine-phosphorylation profile of proteasomes; multiple individual subunits of 20S (eg, alpha1 and beta2) were targets of PP2A and PKA. Taken together, these studies provide the first demonstration that the function of cardiac 20S proteasomes is modulated by associating partners and that phosphorylation may serve as a key mechanism for regulation.

Animals↗

PubMed2006

Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/AkT signaling pathways.

Keloid, a chronic fibro-proliferative disease, exhibits distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells (MCs), and a milieu of enriched cytokines. Previous studies have demonstrated that co-culture with MCs stimulate type I collagen synthesis in fibroblasts, but the signaling mechanisms remain largely unknown. In this study, we investigated the signaling pathways involved in MC-stimulated type I collagen synthesis and the effects of green tea extract (GTE) and its major catechin, (-)-epigallocatechin-3-gallate (EGCG), on collagen homeostasis in keloid fibroblasts. Our results showed that MCs significantly stimulated type I collagen expression in keloid fibroblasts, and the upregulation of type I collagen was significantly attenuated by blockade of phosphatidylinositol-3-kinase (PI-3K), mammalian target of rapamycin (mTOR), and p38 MAPK signaling pathways, but not by blockade of ERK1/2 pathway. Furthermore, GTE and EGCG dramatically inhibited type I collagen production possibly by interfering with the PI-3K/Akt/mTOR signaling pathway. Our findings suggest that interaction between MCs and keloid fibroblasts may contribute to excessive collagen accumulation in keloids and imply a therapeutic potential of green tea for the intervention and prevention of keloids and other fibrotic diseases.

Camellia sinensis↗

PubMed2006

Preneoplastic liver cell foci expansion induced by thioacetamide toxicity in drug-primed mice.

Mice primed by feeding griseofulvin or diethyl 1,4-dihydro 1,4,6-trimethyl 3,5-pyridine decarboxylate for 5 months followed by drug withdrawal for 1 month (drug-primed mice) were given thioacetamide intraperitoneally, and the livers were subsequently studied at intervals up to 7 days. The hepatocellular proliferative response was measured by immunostaining for proliferative cell nuclear antigen. Necrosis was followed by measuring ALT. Mallory bodies were identified by immunoperoxidase stains for ubiquitin and cytokeratin. Preneoplastic foci were localized using immunofluorescence stain for glutathione S-transferase (GST mu) and histochemical stain for gamma glutamyl transpeptidase (GGT). The results showed that the preneoplastic foci selectively proliferated and expanded and formed nodules as indicated by quantitation of nuclei stained positive for proliferating cell nuclear antigen after thioacetamide treatment. Data support the hypothesis that the preneoplastic foci consisted of clones of hepatocytes which preferentially express GST mu, GGT and Mallory bodies. These preneoplastic cells selectively proliferate in response to the promoter effects of necrosis-induced liver cell regeneration ("chemical partial hepatectomy").

Animals↗

PubMed2006

Mallory body (cytokeratin aggresomes) formation is prevented in vitro by p38 inhibitor.

Microarray analysis of livers from mice fed diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) to induce Mallory body (MB) cytokeratin aggresome formation showed that gene expression for cellular adhesion molecules, cytokeratins, kinases and aggresome forming proteins were upregulated, when MBs were formed in vivo. This response was enhanced when the DDC was refed (mice fed DDC for 10 weeks followed by DDC withdrawal for 1 month, then refed DDC for 7 days). Immunofluorescent antibody staining of the MBs that formed showed that MAPK p38 was colocalized with ubiquitin and p62 in the MBs. To investigate further the mechanisms of MB formation, primary cultures derived from DDC primed mice and their controls were incubated for 6 days. Liver cells cultured for 3 h and 6 days were used for microarray analysis. At 3 h, there were no MBs formed, but MBs were numerous after 6 days of culture. At 3 h, the expression of a large number of genes was different when the control, and the DDC primed hepatocytes were compared, which indicates that the primed hepatocytes were phenotypically changed. The gene expression of many kinases including p38 was upregulated after 6 days where the gene expression of cytokeratins, adhesion molecules and aggresome forming proteins were upregulated when MBs formed. An inhibitor of p38 phosphorylation (SB202190) completely prevented MB formation. Western blot showed that phosphorylated p38 MAPK and total p38 were absent in vitro after the p38 inhibitor treatment. Immunostaining of 6-day DDC-primed hepatocyte cultures stained with antibodies to p62 and phospho-p38 MAPK showed that phosphorylated p38 MAPK was concentrated within the MBs. Antibodies to specific serine phosphorylated sites 73 and 431, located in cytokeratin 8, localized to Mallory bodies in vivo, indicating that cytokeratin 8 was hyperphosphorylated. The data supported the concept that MBs form as the result of hyperphosphorylation of cytokeratin 8 by p38.

Animals↗

PubMed2006

Mallory body forming cells express the preneoplastic hepatocyte phenotype.

The livers of mice fed diethyl 1,4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks formed Mallory bodies (MBs) in clusters of hepatocytes. Mice withdrawn from DDC for 9 months developed liver tumors. In the present study, the phenotype of the hepatocytes that formed MBs and tumors was characterized. Immunoperoxidase and immunofluorescent stains were done on the DDC-treated mouse livers, as well as mouse liver tumors and a human hepatocellular carcinoma that formed MBs. Antibodies to markers of hepatocellular neoplasms such as alpha-fetoprotein (AFP), ubiquitin B (UbB) fatty acid synthase (FAS) and alpha2 macroglobulin (A2m) stained the MB forming cells positive. Quantitative real-time RT-PCR assay was used to measure AFP, UbB, FAS and GCP-3 A2m mRNA levels in the livers of DDC fed mice and the DDC-induced mouse liver tumors. The FAS, UbB, GPC-3 and AFP mRNA levels were significantly increased in the MB forming liver cells. The in vitro model of MB formation was used to correlate MB formation with gene and protein expression. Primary cultures of DDC-primed hepatocytes were compared with the controls. A2m and UbB expression increased in the primary cultures of DDC-primed hepatocytes when MBs formed. Thus, the tumor markers used to identify hepatocellular carcinoma were upregulated in cells forming MBs in vivo and in vitro, suggesting that MB forming cells express preneoplastic phenotypic features.

Animals↗

PubMed2006

Gene expression patterns of the liver in response to alcohol: in vivo and in vitro models compared.

Two basic models of alcoholic liver disease pathogenesis exist, one in vivo and one in vitro. To justify the in vitro model, evidence is needed to show that it stimulates the in vivo model. Therefore, changes in gene expression caused by high ethanol level were compared using the two models. Many functional pathways were upregulated in both models. These included the insulin signaling pathway, TGFbeta signaling pathway, apoptosis, MAPK signaling pathway, wnt signaling pathway and apoptosis. Differences were found in the fatty acids synthesis pathway, which was upregulated in vivo; and glycosylation enzymes which were downregulated in vivo. Also, downregulated in vitro were beta oxidation by mitochondria and translation factors. Catalase and superoxide dismutase in mitochondria were upregulated in vitro. These two enzymes have antioxidant effects. In summary, remarkably similar responses to high alcohol levels in the form of changes in gene expression pathways were found in the in vivo and in vitro models tested.

Animals↗

PubMed2006

Pancreas recovery following cerulein-induced pancreatitis is impaired in plasminogen-deficient mice.

BACKGROUND & AIMS: The plasminogen (plg) system participates in tissue repair in several organs, but its role in pancreas repair remains poorly characterized. To understand better the role of plg in pancreas recovery following injury, we examined the course of cerulein-induced pancreatitis in plg-deficient and -sufficient mice. METHODS: Pancreatitis was induced by cerulein administration (50 microg/kg, 7 intraperitoneal injections). Mice were killed either at the acute phase (7 hours after the first cerulein injection) or during recovery (at 2, 4, and 7 days). In pancreatic sections, we examined pancreatic morphology, trypsin activation, inflammatory cell infiltration, acinar cell death, cell proliferation, extracellular matrix deposition, activation of stellate cells (PSCs), and components of the plg and metalloproteinase systems. RESULTS: In plg-sufficient mice, pancreatic plg levels and plasmin activity increased during the acute phase and remained elevated during recovery. Pancreatitis resolved in plg-sufficient mice within 7 days. Pancreas recovery involved reorganization of the parenchyma structure, removal of necrotic debris, cell proliferation, transient activation of PSCs, and moderate deposition of extracellular matrix proteins. Acute pancreatitis (7 hours) was indistinguishable between plg-deficient and -sufficient mice. In contrast, pancreas recovery was impaired in plg-deficient mice. Plg deficiency led to disorganized parenchyma, extensive acinar cell loss, poor removal of necrotic debris, reduced cell proliferation, and fibrosis. Fibrosis was characterized by deposition of collagens and fibronectin, persistent activation of PSCs, and up-regulation of pancreatic transforming growth factor beta1. CONCLUSIONS: Plg/plasmin deficiency leads to features similar to those found in chronic pancreatitis such as parenchymal atrophy and fibrosis.

Animals↗

PubMed2005

Modifications in P62 occur due to proteasome inhibition in alcoholic liver disease.

P62 is capable of binding the polyubiquitin chain that targets proteins for degradation by the proteasome through its ubiquitin associated domain (UBA). Immunostaining of hepatocytes from human liver with alcoholic hepatitis showed colocalization of ubiquitin and P62 in Mallory bodies. Rats fed ethanol chronically and their controls showed that P62 is colocalized with the proteasome in hepatocytes as shown by confocal microscopy. P62 cosedimented with 26S proteasomes isolated from livers of control and alcohol fed rats. P62 was increased in the 26S proteasome fraction when the proteasome chymotrypsin-like (ChT-L) activity decreased in rats fed ethanol. PS-341, a potent proteasome inhibitor was used to compare the inhibition of the proteasome with the inhibition which occurs with ethanol feeding. P62 protein levels were also increased in the purified proteasome fraction of rats given PS-341. This data indicates that modifications in P62 occur due to proteasome inhibition in experimental alcoholic liver disease.

Animals↗

PubMed2005

T cell leukemia-1 modulates TCR signal strength and IFN-gamma levels through phosphatidylinositol 3-kinase and protein kinase C pathway activation.

A signaling role for T cell leukemia-1 (TCL1) during T cell development or in premalignant T cell expansions and mature T cell tumors is unknown. In this study, TCL1 is shown to regulate the growth and survival of peripheral T cells but not precursor thymocytes. Proliferation is increased by TCL1-induced lowering of the TCR threshold for CD4(+) and CD8(+) T cell activation through both PI3K-Akt and protein kinase C-MAPK-ERK signaling pathways. This effect is submaximal as CD28 costimulation coupled to TCL1 expression additively accelerates dose-dependent T cell growth. In addition to its role in T cell proliferation, TCL1 also increases IFN-gamma levels from Th1-differentiated T cells, an effect that may provide a survival advantage during premalignant T cell expansions and in clonal T cell tumors. Combined, these data indicate a role for TCL1 control of growth and effector T cell functions, paralleling features provided by TCR-CD28 costimulation. These results also provide a more detailed mechanism for TCL1-augmented signaling and help explain the delayed occurrence of mature T cell expansions and leukemias despite tumorigenic TCL1 dysregulation that begins in early thymocytes.

Animals↗

PubMed2005

A phagocytic cell line markedly improves survival of infected neutropenic mice.

Disseminated candidiasis is a frequent infection in neutropenic patients, in whom it causes 50% mortality, despite antifungal therapy. As the duration of neutropenia is the strongest predictor of survival in neutropenic patients with invasive fungal infections, neutrophil transfusions are a logical, therapeutic option. However, significant technical barriers have prevented the clinical use of neutrophil transfusions. To overcome these barriers, we identified a human phagocytic cell line that could be administered to candidemic hosts in lieu of freshly harvested neutrophils. HL-60 cells killed Candida albicans in vitro. Activation of HL-60 cells with dimethyl sulfoxide and retinoic acid abrogated the cells' proliferation and augmented their killing of C. albicans. Administration of activated HL-60 cells to candidemic, neutropenic mice significantly improved survival (53% vs. 0%). Live HL-60 cells chemotaxed to sites of infection, phagocytized C. albicans, and reduced the fungal burden in key target organs. Although unactivated HL-60 cells also reduced tissue fungal burden in vivo, they did not improve survival as a result of their toxicity in infected mice. In contrast, no toxicity as a result of activated HL-60 cells was observed at up to 2 months of follow-up. To our knowledge, this is the first description of a cell line-based immunotherapy for an infectious disease. With further refinements, activated HL-60 cells have the potential to overcome the technical barriers to neutrophil transfusions.

Analgesics, Non-Narcotic↗

PubMed2005

CYP2E1 inhibition enhances mallory body formation.

Mallory body (MB) formation is a complex phenomenon seen in chronic liver disease. CYP2E1 may play a role in preventing MB formation since it is involved in the elimination of toxic drugs and chemicals. When mice were fed with diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks, Mallory bodies (MBs) developed in the liver at the end of this period. When DDC feeding was combined with CMZ (an efficient in vivo CYP2E1 inhibitor), more MBs formed compared to DDC feeding alone. DDC was shown to be a suicide inhibitor of CYP2E1. The level of CYP2E1 protein in the liver was further reduced by the DDC and CMZ treatment when measured by Western blot. To test whether CYP2E1 reduced MB formation, CYP2E1 knockout mice and CYP2E1 overexpressed mice were fed with DDC or DDC and CMZ for 10 weeks. MB formation increased markedly in the liver of CYP2E1 knockout mice when fed with DDC only. CYP2E1 overexpressed mice showed an increase in MB formation when the mice were fed with the combination of DDC and CMZ where the amount of CYP2E1 was reduced to levels seen in wild type mice. It was concluded that CYP2E1 inhibits MB formation by increasing the rate of elimination of DDC and/or its toxic intermediates.

Animals↗

PubMed2005

Chronic ethanol feeding and folate deficiency activate hepatic endoplasmic reticulum stress pathway in micropigs.

Previously, we showed that feeding micropigs ethanol with a folate-deficient diet promoted the development of hepatic injury while increasing hepatic levels of homocysteine and S-adenosylhomocysteine (SAH) and reducing the level of S-adenosylmethionine (SAM) and the SAM-to-SAH ratio. Our present goals were to evaluate mechanisms for hepatic injury using liver specimens from the same micropigs. The effects of ethanol feeding or folate-deficient diets, singly or in combination, on cytochrome P-450 2E1 (CYP2E1) and signal pathways for apoptosis and steatosis were analyzed using microarray, real-time PCR, and immunoblotting techniques. Apoptosis was increased maximally by the combination of ethanol feeding and folate deficiency and was correlated positively to liver homocysteine and SAH. Liver CYP2E1 and the endoplasmic reticulum stress signals glucose-regulated protein 78 (GRP78), caspase 12, and sterol regulatory element binding protein-1c (SREBP-1c) were each activated in pigs fed folate-deficient or ethanol diets singly or in combination. Liver mRNA levels of CYP2E1, GRP78, and SREBP-1c, and protein levels of CYP2E1, GRP78, nuclear SREBP, and activated caspase 12 each correlated positively to liver levels of SAH and/or homocysteine and negatively to the SAM-to-SAH ratio. The transcripts of the lipogenic enzymes fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase were elevated in the ethanol-fed groups, and each was positively correlated to liver homocysteine levels. The induction of abnormal hepatic methionine metabolism through the combination of ethanol feeding with folate deficiency is associated with the activation of CYP2E1 and enhances endoplasmic reticulum stress signals that promote steatosis and apoptosis.

Animals↗

PubMed2005

The p105/50 NF-kappaB pathway is essential for Mallory body formation.

To determine if nuclear factor-kappaB (NF-kB) plays a role in Mallory body (MB) formation, quantitative real-time RT-PCR assay was used to measure liver NF-kappaB1/p105 mRNA levels in 4 different groups of mice. Group 1: mice given IP saline for 15 weeks; group 2: mice fed diethyl 1,4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks when MBs were formed; group3: mice fed DDC 10 weeks, then withdrawn 5 weeks when MBs disappeared; group 4: mice fed DDC 10 weeks, withdrawn 4 weeks, then fed DDC+chlormethiazole (CMZ) for 1 week when MBs again formed. The mRNA for p105 NF-kappaB expression was significantly increased in the livers of mice treated with DDC (group 2) and DDC+CMZ (group 4) compared with the control livers (group 1) as well as the drug-withdrawal livers (group 3). Primary cultures of hepatocytes from drug-primed mice (the group 4 mice were withdrawn for another 4 weeks when the MBs had disappeared) were studied. The hepatocytes from drug-primed mice were MB free when isolated and used for primary culture. MBs began to form spontaneously within their cytoplasm after 2-3 days of culture. The NF-kappaB inhibitor (NF-kappaBi), a cell-permeable quinazoline compound that acts as a potent inhibitor of NF-kappaB transcriptional activation, was added to the medium 3 h after planting the cultures of liver cells. No MBs formed in the cells treated with 10 microM, 1 microM, and 0.1 microM NF-kappaBi for 6 days. MBs still formed in the cells treated with 10 nM NF-kappaBi for 6 days. Both DDC-primed and normal control liver cells began to enlarge and elongate after a few hours of culture. In contrast, the cells treated with NF-kappaBi stayed polyhedral in shape just as they appeared prior to culturing. The level of NF-kappaB1/p105 mRNA significantly increased in DDC-primed hepatocytes after 24 h of culture and in normal control hepatocytes after 48 h of culture. In DDC-primed hepatocytes, NF-kappaBi 0.1 muM treatment for 6 days significantly decreased mRNA expression of Src, p105/NF-kappaB1, ERK1, MEKK1, and JNK1/2. In normal control liver cells, NF-kappaBi treatment decreased mRNA expression of Src and JNK1 and stimulated the mRNA expression of p105/NF-kappaB1 and Junk2. NF-kappaBi treatment significantly decreased the total ERK1/2 protein and further decreased the phosphorylated (activated) form of ERK1/2 in the cultured hepatocytes. The results indicate that the p105 NF-kappaB pathway which putatively regulates ERK at both the transcriptional and post-translational levels regulates MB formation by way of changes in gene expression.

Animals↗