Comprehensive monitoring of genotypic and phenotypic antiviral resistance was performed on 673 entecavir (ETV)-treated nucleoside naïve hepatitis B virus (HBV) patients. ETV reduced HBV DNA levels to undetectable by PCR (<300 copies/mL, <57 IU/mL) in 91% of hepatitis B e antigen (HBeAg)-positive and -negative patients by Week 96. Thirteen percent (n ؍ 88) of the comparator lamivudine (LVD)-treated patients experienced a virologic rebound (>1 log increase from nadir by PCR) in the first year, with 74% of these having LVD resistance (LVDr) substitutions evident. In contrast, only 3% (n ؍ 22) of ETV-treated patients exhibited virologic rebound by Week 96. Three ETV rebounds were attributable to LVDr virus present at baseline, with one having a S202G ETV resistance (ETVr) substitution emerge at Week 48. None of the other rebounding patients had emerging genotypic resistance or loss of ETV susceptibility. Genotyping all additional ETV patients with PCR-detectable HBV DNA at Weeks 48, 96, or end of dosing identified seven additional patients with LVDr substitutions, including one with simultaneous emergence of LVDr/ETVr. Generally, ETV patients with LVDr were detectable at baseline (8/10) and most subsequently achieved undetectable HBV DNA levels on ETV therapy (7/10). No other emerging substitutions identified decreased ETV susceptibility. In conclusion, ETVr emergence in ETV-treated nucleoside naïve patients over a 2-year period is rare, occurring in two patients with LVDr variants. These findings suggest that the rapid, sustained suppression of HBV replication, combined with a requirement for multiple substitutions, creates a high genetic barrier to ETVr in nucleoside naïve patients.
Hepatocyte growth factor (HGF) modulates cell adhesion, migration, and branching morphogenesis in cultured epithelial cells, events that require regulation of cell-matrix interactions. Using mIMCD-3 epithelial cells, we studied the effect of HGF on the focal adhesion proteins, focal adhesion kinase (FAK) and paxillin and their association. HGF was found to increase the tyrosine phosphorylation of paxillin and to a lesser degree FAK. In addition, HGF induced association of paxillin and activated ERK, correlating with a gel retardation of paxillin that was prevented with the ERK inhibitor U0126. The ability of activated ERK to phosphorylate and induce gel retardation of paxillin was confirmed in vitro in both full-length and amino-terminal paxillin. Several potential ERK phosphorylation sites in paxillin flank the paxillin-FAK association domains, so the ability of HGF to regulate paxillin-FAK association was examined. HGF induced an increase in paxillin-FAK association that was inhibited by pretreatment with U0126 and reproduced by in vitro phosphorylation of paxillin with ERK. The prevention of the FAK-paxillin association with U0126 correlated with an inhibition of the HGFmediated FAK tyrosine phosphorylation and inhibition of HGF-dependent cell spreading and adhesion. An examination of cellular localization of FAK and paxillin demonstrated that HGF caused a condensation of focal adhesion complexes at the leading edges of cell processes and FAK-paxillin co-localization in these large complexes. Thus, these data suggest that HGF can induce serine/threonine phosphorylation of paxillin most probably mediated directly by ERK, resulting in the recruitment and activation of FAK and subsequent enhancement of cell spreading and adhesion.Hepatocyte growth factor (HGF) 1 is a heparin-binding protein that is produced primarily by fibroblasts and peritubular mesenchymal cells (1, 2) and that can stimulate the c-Met receptor to initiate at least three distinct responses, mitogenesis, scattering/migration, and branching tubule formation (3, 4). The ability of HGF to induce cell migration and morphogenesis implies that it must initiate intracellular signaling events that regulate cell-matrix interactions. In support of this finding, HGF has recently been shown to directly stimulate integrindependent tumor cell adhesion to laminin, fibronectin, and vitronectin (5-7). In studies using immortalized renal murine inner medullary collecting duct (mIMCD-3) epithelial cells, we have demonstrated that HGF induces increased adhesion to fibronectin and type 1 collagen, and that this increase in adhesion is dependent on ERK activation (8).Presently, there is little known about the mechanism for the HGF-dependent increase in epithelial cell adhesion. In two studies (5, 6), the stimulation with HGF has been found to cause tyrosine phosphorylation of FAK and to mediate an increased linkage between the integrin complexes and the actin cytoskeleton. In breast carcinoma cells, Beviglia and Kramer (6) found that HGF induced tyrosine phosphoryla...
Virologic resistance emerging during entecavir (ETV) therapy for hepatitis B virus (HBV)requiresT he genetic changes that impart resistance to the four approved nucleoside analogs used for the treatment of chronic hepatitis B virus (HBV) have been described (Table 1). Lamivudine (LVD) resistance (LVDr) arises from substitutions of the methionine (rtM204) to valine (rtM204V), isoleucine (rtM204I), or rarely serine (rtM204S) in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the HBV reverse transcriptase (RT). The rtM204V change is always accompanied by an rtL180M growth adaptive substitution, 1 whereas the rtM204I substitution is found with or without rtL180M. Additionally, the rtV173L substitution has been found in some LVDr isolates and increases HBV replication capacity in vitro. 2 Resistance to telbivudine also arises from changes of the YMDD motif rtM204. 3 In contrast, resistance to adefovir (ADV) arises through rtN236T or rtA181V changes, whereas recent studies indicate that the rtA181T change often associated with ADV is more likely related to LVD therapy. 4,5 Entecavir (ETV), a novel deoxyguanosine analog, has demonstrated efficacy for treating HBV patients infected with wild-type or LVDr HBV. [6][7][8][9] Comprehensive resistance monitoring during clinical studies has revealed less than 1% viral resistance in nucleoside-treatment-naive (nucleoside-naive) patients through four years of therapy, reflecting a high genetic barrier to resistance that likely
We have examined the ability of epidermal growth factor (EGF)-stimulated ERK activation to regulate Grb2-associated binder-1 (Gab1)/phosphatidylinositol 3-kinase (PI3K) interactions. Inhibiting ERK activation with the MEK inhibitor U0126 increased the EGF-stimulated association of Gab1 with either full-length glutathione S-transferase-p85 or the p85 C-terminal Src homology 2 (SH2) domain, a result reproduced by coimmunoprecipitation of the native proteins from intact cells. This increased association of Gab1 and the PI3K correlates with an increase in PI3K activity and greater phosphorylation of Akt. This result is in direct contrast to what we have previously reported following HGF stimulation where MEK inhibition decreased the HGFstimulated association of Gab1 and p85. In support of this divergent effect of ERK on Gab1/PI3K association following HGF and EGF stimulation, U0126 decreased the HGF-stimulated association of p85 and the Gab1 c-Met binding domain but did not alter the EGF-stimulated association of p85 and the c-Met binding domain. An examination of the mechanism of this effect revealed that the treatment of cells with EGF ؉ U0126 increased the tyrosine phosphorylation of Gab1 as well as its association with another SH2-containing protein, SHP2. Furthermore, overexpression of a catalytically inactive form of SHP2 or pretreatment with pervanadate markedly increased EGF-stimulated Gab1 tyrosine phosphorylation. These experiments demonstrate that EGF and HGF-mediated ERK activation result in divergent effects on Gab1/PI3K signaling. HGF-stimulated ERK activation increases the Gab1/PI3K association, whereas EGF-stimulated ERK activation results in a decrease in the tyrosine phosphorylation of Gab1 and a decreased association with the PI3K. SHP2 is shown to associate with and dephosphorylate Gab1, suggesting that EGFstimulated ERK might act through the regulation of SHP2.
Forces such as strain modulate intestinal epithelial biology. Shear and pressure influence other cells. The effects of pressure on human colon cancer cells are poorly understood. Increasing ambient pressure for 30 min by 15 mm Hg over atmospheric stimulated adhesion to matrix proteins of four human colon cancer cell lines and primary cells from three human colon cancers, but not bovine aortic smooth-muscle cells. This effect was energy dependent and cation dependent (blocked by azide and chelation), accompanied by tyrosine phosphorylation of intracellular proteins including focal adhesion kinase, and blocked by tyrosine kinase inhibition (genistein, tyrphostin, and erbstatin) and a functional antibody to the beta1 integrin subunit. Although pressure stimulated adhesion even in a balanced salt solution, baseline and pressure-stimulated adhesion were each substantially diminished in the absence of serum. These data suggest that relatively low levels of increased pressure may stimulate malignant colonocyte adhesion by a cation-dependent beta1-integrin-mediated mechanism, perhaps via focal adhesion kinase-related tyrosine phosphorylation. In addition to elucidating another aspect of physical force regulation of colonocyte biology, these findings may be relevant to the effects of increased pressure engendered by colonic peristalsis, surgical manipulation, or laparoscopic surgery on colon cancer cell adhesion.
The signals involved in restitution during mucosal healing are poorly understood. We compared focal adhesion kinase (FAK) and paxillin protein and phosphorylation, extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 activation, as well as FAK and paxillin organization in static and migrating human intestinal Caco-2 cells on matrix proteins and anionically derivatized polystyrene dishes (tissue culture plastic). We also studied effects of FAK, ERK, and p38 blockade in a monolayer-wounding model. Compared with static cells, cells migrating across matrix proteins matrix-dependently decreased membrane/cytoskeletal FAK and paxillin and cytosolic FAK. Tyrosine phosphorylated FAK and paxillin changed proportionately to FAK and paxillin protein. Conversely, cells migrating on plastic increased FAK and paxillin protein and phosphorylation. Migration matrix-dependently activated p38 and inactivated ERK1 and ERK2. Total p38, ERK1, and ERK2 did not change. Caco-2 motility was inhibited by transfection of FRNK (the COOH-terminal region of FAK) and PD-98059, a mitogen-activated protein kinase-ERK kinase inhibitor, but not by SB-203580, a p38 inhibitor, suggesting that FAK and ERK modulate Caco-2 migration. In contrast to adhesion-induced phosphorylation, matrix may regulate motile intestinal epithelial cells by altering amounts and distribution of focal adhesion plaque proteins available for phosphorylation as well as by p38 activation and ERK inactivation. Motility across plastic differs from migration across matrix.
Massage-like stroking induces acute antinociceptive effects that can be reversed by an oxytocin antagonist, indicating activation of oxytocin on endogenous pain controlling systems. We now demonstrate an increase in hindpaw withdrawal latencies (HWLs), in response to thermal and mechanical stimuli, which was present after six treatments of massage-like stroking every other day and which continued to increase through the remaining seven treatments. Repeated massage-like stroking also resulted in increased oxytocin-like immunoreactivity (oxytocin-LI) levels in plasma and periaquaductal grey matter (PAG). Furthermore, increases in HWLs were also present after injections of oxytocin into the PAG (0.1, 0.5 and 1.0 nmol). Intra-PAG oxytocin injection of 1 nmol followed by 1 or 20 nmol of naloxone attenuated the increments in HWL. Also, there was a dose-dependent attenuation of the oxytocin-induced antinociceptive effects following intra-PAG injection of the mu-opioid antagonist beta-funaltrexamine (beta-FNA) and the kappa-opioid antagonist nor-binaltorphimine (nor-BNI) but not the delta-antagonist naltrindole. The long-term antinociceptive effects of massage-like stroking may be attributed, at least partly, to the oxytocinergic system and its interaction with the opioid system, especially the mu- and the kappa-receptors in the PAG.
Based on our previous observations that active ERK associates with and phosphorylates Gab1 in response to HGF, and the prediction that the ERK phosphorylation site is adjacent to one of the phosphatidylinositol 3-kinase (PI3K) SH2 binding motifs, we examined the possibility that ERK phosphorylation can regulate the Gab1/ PI3K association. The HGF-mediated association of Gab1 with either full-length GST-p85 or its isolated N-or C-terminal SH2 domains was inhibited by ϳ50% in the setting of ERK inhibition, a result confirmed by coimmunoprecipitation of the native proteins. A 14-amino acid peptide encoding 472 YVPMTP 477 (one of the major p85 binding sites in Gab1 and the predicted ERK phosphorylation site) was synthesized with either phosphotyrosine alone (pY), or phosphotyrosine ؉ phosphothreonine (pYT). In both pull-down assays and competition assays, pYT demonstrated a higher affinity for p85 than did pY alone. Finally, examination of the phosphorylation state of Akt after HGF stimulation revealed that ERK inhibition resulted in a decrease in Akt activation at both 5 and 10 min. These results suggest that activated ERK can phosphorylate Gab1 in response to HGF stimulation and thereby potentiate the Gab1/PI3K association and subsequent PI3K activation.The docking protein Gab1 associates with several receptor tyrosine kinases known to induce cell morphogenesis, including the hepatocyte growth factor (HGF) 1 receptor, c-Met, and the epidermal growth factor (EGF) receptor. Following receptor activation, Gab1 associates and is tyrosine-phosphorylated, in turn recruiting multiple SH2 domain containing signal transducing proteins such as SHC, SHP2, phosphoinositide 3-kinase (PI3K), Crk/CRKL, and phospholipase C␥ (1-6). These proteins form an intricate signaling complex that regulates cell migration, morphology, and tubule formation. The critical nature of Gab1 signaling in normal cell biology has been demonstrated by the finding that Gab1-deficient mice die in utero (7) and display developmental defects in myotomes, placenta, and liver that recapitulate the loss of HGF and c-Met (8).The phenotypic effects of Gab1 signaling have been found to be critically dependent on Gab1 interactions with the PI3K. The PI3K is a heterodimer composed of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110). Two SH2 domains are located in the C-terminal region of the p85 subunit and have been shown to bind to membrane-associated tyrosinephosphorylated proteins, resulting in recruitment of the PI3K to the membrane and activation of the lipid kinase activity of the 110-kDa subunit. This activity results in the formation of phosphoinositide 3,4,5-trisphosphate (PI 3,4,5 P 3 ) at the membrane, which in turn serves to recruit and help activate such proteins as protein kinase C (PKC) and Akt (9, 10). The activated PI3K has been demonstrated to result in the regulation of various cellular activities including proliferation (11), differentiation (12), and prevention of apoptosis (13). We have found that PI3K activity is cr...
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