Ground glass hepatocytes (GGHs) are the historic hallmarks for the hepatocytes in the late and non-replicative stages of hepatitis B virus (HBV) infection. We have identified type I and type II GGHs that contain two mutant types of large HBV surface antigens (HBsAg) with deletions over the pre-S1 and pre-S2 regions, respectively. These pre-S mutant HBVsAg accumulate in endoplasmic reticulum (ER), resulting in strong ER stress. Type II GGHs often appear in hepatic nodules in the late phases of HBV infection and proliferate in clusters, suggesting that these mutant pre-S1/S2 HBsAg may be involved in HBV-related hepatocarcinogenesis, associated with ER stress. In this study, we investigated the potential genomic instability imposed by pre-S mutant HBsAg. Based on the analysis of comet assays, we found that the pre-S1 and pre-S2 mutant HBsAg caused oxidative stress and DNA damage. The DNA repair gene ogg1 was greatly induced by over-expression of pre-S mutant HBsAg. Induction of the DNA repair gene ogg1 was also detected in the pre-S2 HBsAg transgenic mice, as well as the type II GGHs from patients with hepatocellular carcinoma, strongly suggesting that the pre-S mutant HBsAg contributes to the oxidative DNA damage to hepatocytes. In addition, the mutation rates in the X-linked hprt gene were enhanced in mouse hepatoma ML1-4a cells, which constitutively expressed the pre-S1/S2 HBsAg. These results indicate that pre-S1/S2 mutant HBsAg, which make up GGHs, induce oxidative DNA damage and mutations in hepatocytes in the late stages of HBV infection.

Expression of mutant proteins or viral infection mayinterfere with proper protein folding activity in the endoplasmic reticulum (ER). Several pathways that maintain cellular homeostasis were activated in response to these ER disturbances. Here we investigated which of these ER stress-activated pathways induce COX-2 and potentially oncogenesis. Tunicamycin and brefeldin A, two ER stress inducers, increased the expression of COX-2 in ML-1 or MCF-7 cells. Nuclear translocation of NF-B and activation of pp38 MAPK were observed during ER stress. I B␣ kinase inhibitor Bay 11-7082 or I B␣ kinase dominant negative mutant significantly inhibited the induction of COX-2. pp38 MAPK inhibitor SB203580 or eIF2␣ phosphorylation inhibitor 2-aminopurine attenuated the nuclear NF-B DNA binding activity and COX-2 induction. Expression of mutant hepatitis B virus (HBV) large surface proteins, inducers of ER stress, enhanced the expression of COX-2 in ML-1 and HuH-7 cells. Transgenic mice showed higher expression of COX-2 protein in liver and kidney tissue expressing mutant HBV large surface protein in vivo. Similarly, increased expression of COX-2 mRNA was observed in human hepatocellular carcinoma tissue expressing mutant HBV large surface proteins. In ML-1 cells expressing mutant HBV large surface protein, anchorage-independent growth was enhanced, and the enhancement was abolished by the addition of specific COX-2 inhibitors. Thus, ER stress due either to expression of viral surface proteins or drugs can stimulate the expression of COX-2 through the NF-B and pp38 kinase pathways. Our results provide important insights into cellular carcinogenesis associated with latent endoplasmic reticulum stress.

No abstract

Substantial evidence suggests that inflammatory cytokines, immune cells, and angiogenesis are important for endometriosis. In this study, we investigated the role of the sequential events in the development of endometriosis in a mouse model. Uterine tissue was transplanted into the peritoneum of ovariectomized mice and then supplemented with estrogen or vehicle. On different days after transplantation, cell proliferation, angiogenesis, and infiltrated immune cells in ectopic tissue were examined using immunochemical staining. Many disintegrated blood vessels but no bromodeoxyuridine-positive cells in ectopic tissue were observed in the estrogen-treated group on posttransplantation d 1 and 2. On d 4-7, bromodeoxyuridine-positive cells were detected in the blood vessels of ectopic tissue, indicating that angiogenesis was initiated in this stage. Angiogenesis also occurred in ectopic tissue in the vehicle-treated group. Profound infiltration of neutrophils in ectopic tissue occurred on d 1-4, when the number of neutrophils and levels of macrophage inflammatory protein (MIP)-1alpha and MIP-2 chemokines in peritoneal fluids also reached their peak. Peritoneal macrophage numbers did not change, but secretions of TNFalpha, IL-6, MIP-1alpha, and MIP-2 from macrophages isolated on d 2 were higher than on d 0. In vitro studies showed that peritoneal neutrophils and macrophages secreted vascular endothelial growth factor, which was up-regulated by TNFalpha and IL-6. Our results suggest that neutrophils and macrophages may promote angiogenesis in the early stage of endometriosis and that chemokines and cytokines amplify the angiogenic signal for the growth of endometriotic tissue.

Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL) 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL family in individual type of cancers.

Mounting evidence supports the involvement of HBV and its gene products in the multistep progression of liver tumorigenesis. 2 A protein-designated HBx has been extensively studied; the data reveal a role of the protein as a transactivator involved in cell growth, apoptosis, DNA damage signals, mitogen-activated protein kinase, and JAK/STAT signaling pathways. 3 Recently, the large surface protein (LHBs) and a C-terminally truncated middle surface protein (MHBs t ) have likewise been recognized as transactivators that share the same mechanism for transcriptional activation. 4,5 This group of activators may trigger a protein kinase C-dependent activation of the c-Raf-1/mitogen-activated protein kinase 2 signal transduction cascade, resulting in the activation of transcription factors such as activator protein 1 and nuclear factor B. The functional activity of these activators is dependent on the cytoplasmic orientation of the pre-S2 region of MHBs t and LHBs that is also related to their intracellular retention. 6,7 Besides the MHBs t , we have previously identified a mutant Abbreviations: ⌬S2-LHBs, mutant with a deletion in the pre-S2 region of the large surface protein; HBV, hepatitis B virus; ER, endoplasmic reticulum; HCC, hepatocellular carcinoma; GGH, ground glass hepatocyte; cDNA, complementary DNA; HH4, nontransformed human hepatocyte cell line; BFA, brefeldin A; VT, vomitoxin; PCNA, proliferating cell nuclear antigen; CDK, From the

Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH 2 terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD 313 , which is at the junction of the NH 2 -terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis.

MST3 is a member of the sterile-20 protein kinase family with a unique preference for manganese ion as a cofactor in vitro; however, its biological function is largely unknown. Suppression of endogenous MST3 by small interference RNA enhanced cellular migration in MCF-7 cells with reduced expression of E-cadherin at the edge of migrating cells. The alteration of cellular migration and protruding can be rescued by RNA interference-resistant MST3. The expression of surface integrin and Golgi apparatus was not altered, but phosphorylation on tyrosine 118 and tyrosine 31 of paxillin was attenuated by MST3 small interfering RNA (siRNA). Threonine 178 was determined to be one of the two main autophosphorylation sites of MST3 in vitro. Mutant T178A MST3, containing alanine instead of threonine at codon 178, lost autophosphorylation and kinase activities. Overexpression of wild type MST3, but not the T178A mutant MST3, inhibited migration and spreading in Madin-Darby canine kidney cells. MST3 could phosphorylate the protein-tyrosine phosphatase (PTP)-PEST and inhibit the tyrosine phosphatase activity of PTP-PEST. We conclude that MST3 inhibits cell migration in a fashion dependent on autophosphorylation and may regulate paxillin phosphorylation through tyrosine phosphatase PTP-PEST.