A single mutation in the E2 glycoprotein of hepatitis C virus broadens the claudin specificity for its infection.

Entry of the hepatitis C virus (HCV) into host cells is a multistep process mediated by several host factors, including a tight junction protein claudin-1 (CLDN1). We repeatedly passaged HCV-JFH1-tau, an HCV substrain with higher infectivity, on Huh7.5.1-8 cells. A multi-passaged HCV-JFH1-tau lot was infectious to CLDN1-defective S7-A cells, non-permissive to original HCV-JFH1-tau infection. We identified a single mutation, M706L, in the E2 glycoprotein of the HCV-JFH1-tau lot as an essential mutation for infectivity to S7-A cells. The pseudovirus JFH1/M706L mutant could not infect human embryonic kidney 293 T (HEK293T) cells lacking CLDN family but infected HEK293T cells expressing CLDN1, CLDN6, or CLDN9. Thus, this mutant virus could utilize CLDN1, and other CLDN6 and CLDN9, making HCV possible to infect cells other than hepatocytes. iPS cells, one of the stem cells, do not express CLDN1 but express CLDN6 and other host factors required for HCV infection. We confirmed that the HCV-JFH1-tau-derived mutant with an M706L mutation infected iPS cells in a CLDN6-dependent manner. These results demonstrated that a missense mutation in E2 could broaden the CLDN member specificity for HCV infection. HCV may change its receptor requirement through a single amino acid mutation and infect non-hepatic cells.

Thermostable hepatitis C virus JFH1-derived variant isolated by adaptation to Huh7.5.1 cells.

Hepatitis C virus (HCV) infection and propagation in cultured cells have mainly been investigated using the infectious clinical clone JFH1. However, its infectivity is not high enough for infection to be detected easily. In this study, we attempted to isolate HCV-JFH1 variants adapted to human hepatoma Huh7.5.1 cells. By performing serial passages of the wild-type HCV-JFH1 in Huh7.5.1 cells, we obtained a variant that was capable of inducing severe cytopathic effects and showed approximately 700-fold higher infectivity than the wild-type HCV-JFH1. Further, when highly permissive Huh7.5.1-8 cells were infected with this variant, viral particles were produced at >1011 copies ml-1, making this variant one of the most efficient HCV production systems. Two adaptive mutations were noted in the variant genome: a1994c (K74T) in the core protein region and t3014c (I414T) in the E2 protein region. Both mutations contributed to enhanced infectivity and their combination showed synergistic effects in this regard. An examination of recombinant viruses carrying K74T, I414T and K74T/I414T mutations revealed that none of the mutations had an effect on the steps after viral entry (genome replication, particle assembly and egress), but led to the viral infection becoming less dependent on scavenger receptor class B type I, changes of the infectious particles to a broader and lower range of densities, and enhanced thermal stability of the infectious viruses. Thus, this Huh7.5.1-adapted HCV-JFH1 variant with higher and stable infectivity should be a valuable tool for studying the molecular mechanisms behind the life cycle of HCV and for antiviral screening.

p38MAPK and Rho-dependent kinase are involved in anoikis induced by anicequol or 25-hydroxycholesterol in DLD-1 colon cancer cells.

Anchorage-independent growth is evidence of the malignant transformation of cells. We previously reported the characterization of anicequol, a novel inhibitor of the anchorage-independent growth of tumor cells, and here we show that the effects of 25-hydroxycholesterol (25-HC) on colon cancer cells were very similar to those of anicequol. By analyzing the effects of inhibitors and performing RNA interference experiments, we found that p38 mitogen-activated protein kinase (p38MAPK) was involved in anicequol- and 25-HC-induced anoikis in DLD-1 cells. In addition, Rho-associated, coiled-coil containing protein kinase (ROCK) was also associated with anoikis induced by anicequol or 25-HC. Taken together, our findings suggest that activation of the p38MAPK and ROCK pathways might provide a new therapeutic strategy against cancer, and raise the possibility that tumor metastasis is influenced by 25-HC under physiological conditions.

Selective estrogen receptor modulators inhibit hepatitis C virus infection at multiple steps of the virus life cycle.

We screened for hepatitis C virus (HCV) inhibitors using the JFH-1 viral culture system and found that selective estrogen receptor modulators (SERMs), such as tamoxifen, clomifene, raloxifene, and other estrogen receptor α (ERα) antagonists, inhibited HCV infection. Treatment with SERMs for the first 2 h and treatment 2-24 h after viral inoculation reduced the production of HCV RNA. Treating persistently JFH-1 infected cells with SERMs resulted in a preferential inhibition of extracellular HCV RNA compared to intracellular HCV RNA. When we treated two subgenomic replicon cells, which harbor HCV genome genotype 2a (JFH-1) or genotype 1b, SERMs reduced HCV genome copies and viral protein NS5A. SERMs inhibited the entry of HCV pseudo-particle (HCVpp) genotypes 1a, 1b, 2a, 2b and 4 but did not inhibit vesicular stomatitis virus (VSV) entry. Further experiment using HCVpp indicated that tamoxifen affected both viral binding to cell and post-binding events including endocytosis. Taken together, SERMs seemed to target multiple steps of HCV viral life cycle: attachment, entry, replication, and post replication events. SERMs may be potential candidates for the treatment of HCV infection.

A cell-based, microplate colorimetric screen identifies 7,8-benzoflavone and green tea gallate catechins as inhibitors of the hepatitis C virus.

We describe a cell-based, microplate colorimetric screen for anti-hepatitis C virus (HCV) drugs that exploits the HCV-JFH1 viral culture system. Antiviral activity was assessed by measuring protection against the HCV-JFH1-induced cytopathic effect (CPE) in Huh7.5.1 cells using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay. The use of serum-free medium substantially sensitized Huh7.5.1 cells to HCV-induced CPE, causing sufficient cell death to perform colorimetric assays for anti-HCV activity in 96-well plates. As a proof of concept, we carried out a pilot screen of an inhibitor library and identified cyclosporin A and tamoxifen, two compounds with reported anti-HCV activity. Using the assay, we discovered the anti-HCV properties of the plant flavonoids epigallocatechin gallate (EGCG) and 7,8-benzoflavone (α-naphthoflavone). Other gallate-type catechins and flavones also displayed anti-HCV activity, but 5,6-benzoflavone (ß-naphthoflavone), flavanone, and non-gallate catechins were inactive. EGCG apparently acted mainly on HCV entry, although it may also block other steps. In contrast, 7,8-benzoflavone was presumed to inhibit later stages of the HCV life cycle. This assay is simple, reliable and cost-effective; does not require any specially engineered cell lines or viruses; and should be useful in the identification of compounds with anti-HCV activity.

The resorcylic acid lactone hypothemycin selectively inhibits the mitogen-activated protein kinase kinase-extracellular signal-regulated kinase pathway in cells.

The resorcylic acid lactone hypothemycin has been shown to inactivate protein kinases by binding to a cysteine conserved in 46 protein kinases, including mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK) and platelet-derived growth factor receptor (PDGFR). We assessed the selectivity of hypothemycin in cellular contexts. Hypothemycin normalized the morphology and inhibited anchorage-independent growth of Ki-ras transformed normal rat kidney (NRK) cells with selectivity and potency comparable to or greater than that of the MEK inhibitor U0126. In Ki-ras-transformed and phorbol 12-myristate 13-acetate (PMA)-treated NRK cells, hypothemycin blocked ERK activation but showed a minimal effect on autophosphorylation of protein kinase D1 (PKD1), another kinase containing the conserved cysteine. Hypothemycin potently inhibited PDGFR autophosphorylation and activation of the MEK-ERK pathway in platelet-derived growth factor (PDGF)-treated NRK cells. However, the phosphoinositide-3-kinase (PI3K) pathway was only modestly attenuated. Hypothemycin also inhibited growth factor- and anchorage-independent growth of human cancer cell lines with a constitutively active MEK-ERK pathway. Although hypothemycin has the potential to inactivate various protein kinases, the results indicate that in intracellular environments, hypothemycin can inhibit the MEK-ERK axis with sufficient selectivity to normalize transformed phenotypes of cells dependent on this pathway.

Bisebromoamide, a potent cytotoxic peptide from the marine cyanobacterium Lyngbya sp.: isolation, stereostructure, and biological activity.

A novel cytotoxic peptide, termed bisebromoamide (1), has been isolated from the marine cyanobacterium Lyngbya sp. Its planar structure was determined by 1D and 2D NMR spectroscopy. The absolute stereostructure of 1 was determined by chemical degradation followed by chiral HPLC analysis. Bisebromoamide (1) exhibited potent protein kinase inhibition: the phosphorylation of ERK in NRK cells by PDGF-stimulation was selectively inhibited by treatment with 10-0.1 microM of 1.

Gamma-herpesviruses and cellular signaling in AIDS-associated malignancies.

gamma-Herpesviruses, Epstein-Barr virus (EBV/HHV-4) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), are involved in human carcinogenesis, particularly in immunocompromised patients. Virus-associated malignancies are becoming of significant concern for the mortality of long-lived immunocompromised patients, and therefore, research of advanced strategies for AIDS-related malignancies is an important field in cancer chemotherapy. Detailed understanding of the EBV and KSHV lifecycle and related cancers at the molecular level is required for novel strategies of molecular-targeted cancer chemotherapy. The present review gives a simple outline of the functional interactions between KSHV- and EBV-viral gene products and host cell deregulated signaling pathways as possible targets of chemotherapy against AIDS-related malignancies.

Highly effective recombinant format of a humanized IgG-like bispecific antibody for cancer immunotherapy with retargeting of lymphocytes to tumor cells.

We previously reported the marked in vitro and in vivo antitumor activity of hEx3, a humanized diabody (small recombinant bispecific antibody) with epidermal growth factor receptor (EGFR) and CD3 retargeting. Here, we fabricated a tetravalent IgG-like bispecific antibody with two kinds of single-chain Fv (scFv), i.e. humanized anti-EGFR scFv and anti-CD3 scFv, that contains the same four variable domains as hEx3, on the platform of human IgG1 (hEx3-scFv-Fc). hEx3-scFv-Fc prepared from mammalian cells showed specific binding to both EGFR and CD3 target antigens. At one-thousandth (0.1-100 fmol/ml) of the dose of normal hEx3, hEx3-scFv-Fc showed intense cytotoxicity to an EGFR-positive cell line in a growth-inhibition assay using lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells). The enhanced antitumor effect was more clearly observed when peripheral blood mononuclear cells (PBMCs) were used as effector cells, indicating the utility of IgG-like fabrication. These results suggested that the intense antitumor activity is attributable to the multivalency and the presence of the fused human Fc, a hypothesis that was supported by the results of flow cytometry, PBMC proliferation assay, and protein kinase inhibition assay. Furthermore, the growth inhibition effects of hEx3-scFv-Fc were considerably superior to those of the approved therapeutic antibody, cetuximab, which recognizes the same EGFR antigen even when using PBMCs as effector cells. The high potency of hEx3-scFv-Fc may translate into improved antitumor therapy and lower costs of production because of the smaller doses needed.

Synthesis and biological evaluation of benzamides and benzamidines as selective inhibitors of VEGFR tyrosine kinases.

A series of benzamidines and benzamides was synthesized as selective inhibitors of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, and tested for inhibitory activity toward autophosphorylation by the enzyme assay. Selective inhibition of VEGFR-2 tyrosine kinase was observed in the salicylic amide 4e and the anthranilic amidine 5a, and their percent inhibitions of VEGFR-2 tyrosine kinase were 44-60% at a 10 microM concentration of compounds. The salicylic amide 4a showed inhibition of both VEGFR-1 and VEGFR-2 tyrosine kinases at a 10 microM concentration.

Ets-1-dependent expression of vascular endothelial growth factor receptors is activated by latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus through interaction with Daxx.

Vascular endothelial growth factor (VEGF) and its receptors are highly expressed in Kaposi's sarcoma (KS) lesion and play a key role in angiogenesis. Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) has multiple functions related to viral latency and KSHV-induced oncogenesis. In this report, we have identified Daxx as a LANA-binding protein by co-immunoprecipitation analysis of HeLa cells stably expressing LANA. LANA associated with Daxx in a PEL cell line infected with KSHV. LANA and Daxx also bound in vitro, suggesting direct interaction. From the results of binding assays, a region containing the Glu/Asp-rich domain within LANA, and a central region including the second paired amphipathic helix within Daxx contributed to the interaction. To address the physiological significance of this interaction, we focused on a Daxx-mediated VEGF receptor gene regulation. We found that Daxx repressed Ets-1-dependent Flt-1/VEGF receptor-1 gene expression, and that LANA inhibited the repression by Daxx in a reporter assay. Analyses of flow cytometry and real-time PCR revealed that expression of VEGF receptor-1 and -2 in LANA-expressing human umbilical vein endothelial cells (HUVECs) significantly increased. Co-immunoprecipitation and immunoblotting experiments suggested that LANA-bound Daxx to inhibit the interaction between Daxx and Ets-1. Chromatin immunoprecipitation assays showed that Daxx associated with VEGF receptor-1 promoter in HUVECs, and that LANA expression reduced this association. These results suggested that LANA contributes to a high expression of VEGF receptors in KS lesion by interfering with the interaction between Daxx and Ets-1.

Reduction of hepatitis C virus NS5A phosphorylation through its interaction with amphiphysin II.

Hepatitis C virus non-structural protein 5A (NS5A) is a pleiotropic protein with key roles in viral RNA replication, modulation of cellular-signaling pathways and interferon (IFN) responses. To search for possible host factors involved in mediating these functions of NS5A, we adopted an affinity purification approach coupled with mass spectrometry to examine protein-protein interactions, and found that human amphiphysin II (also referred to as Bin1) specifically interacts with NS5A in mammalian cells. Pull-down assays showed that the Src homology 3 (SH3) domain of amphiphysin II is required for NS5A interaction and that c-Src also interacts with NS5A in cells. IFN-alpha treatment reduced the interaction of NS5A with c-Src, but not amphiphysin II, suggesting that the latter is independent of the IFN-signaling pathway. NS5A is a phosphoprotein and its phosphorylation status is considered to have an effect on viral RNA replication. In vitro kinase assays demonstrated that its interaction with amphiphysin II inhibits phosphorylation of NS5A. These results suggest that amphiphysin II participates in the HCV life cycle by modulating the phosphorylation of NS5A.

BimEL is an important determinant for induction of anoikis sensitivity by mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitors.

Loss of contact with substratum triggers apoptosis in many normal cell types, a phenomenon termed anoikis. We reported previously that mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitors induced apoptosis in nonanchored MDA-MB231 and HBC4 human breast cancer cells, whereas anchored cells remained viable. Here, we report that activation of the BH3-only protein BimEL is the major mechanism for induction of anoikis sensitivity by MEK inhibitors in MDA-MB231 and HBC4 cells. On treatment with MEK inhibitors, BimEL in MDA-MB231 and HBC4 cells rapidly increased, irrespective of the state of anchorage. However, it translocated to mitochondria only in nonanchored cells, explaining why attached cells remain viable. MDA-MB231 and HBC4 cells had exceedingly low basal levels of BimEL compared with other breast cancer cells, suggesting that maintenance of low BimEL amount is important for survival of these cells. MEK inhibitors also induced the electrophoretic mobility shift of BimEL, indicative of reduced phosphorylation. In vitro, BimEL was phosphorylated by extracellular signal-regulated kinase on Ser(69), which resides in the BimEL-specific insert region. Using phosphospecific antibody against this site, we show that this residue is actually phosphorylated in cells. We also show that phosphorylation of Ser(69) promotes ubiquitination of BimEL. We conclude that MEK inhibitors sensitize MDA-MB231 and HBC4 cells to anoikis by blocking phosphorylation and hence degradation of BimEL, a mechanism that these cells depend on to escape anoikis.

Poly(ADP-ribose) polymerase-1 inhibits ATM kinase activity in DNA damage response.

DNA double-strand breaks (DSB) mobilize DNA-repair machinery and cell cycle checkpoint by activating the ataxia-telangiectasia (A-T) mutated (ATM). Here we show that ATM kinase activity is inhibited by poly(ADP-ribose) polymerase-1 (PARP-1) in vitro. It was shown by biochemical fractionation procedure that PARP-1 as well as ATM increases at chromatin level after induction of DSB with neocarzinostatin (NCS). Phosphorylation of histone H2AX on serine 139 and p53 on serine 15 in Parp-1 knockout (Parp-1(-/-)) mouse embryonic fibroblasts (MEF) was significantly induced by NCS treatment compared with MEF derived from wild-type (Parp-1(+/+)) mouse. NCS-induced phosphorylation of histone H2AX on serine 139 in Parp-1(-/-) embryonic stem cell (ES) clones was also higher than that in Parp-1(+/+) ES clone. Furthermore, in vitro, PARP-1 inhibited phosphorylation of p53 on serine 15 and (32)P-incorporation into p53 by ATM in a DNA-dependent manner. These results suggest that PARP-1 negatively regulates ATM kinase activity in response to DSB.

Nucleolar Nek11 is a novel target of Nek2A in G1/S-arrested cells.

We previously reported that Nek11, a member of the NIMA (never-in-mitosis A) family of kinases, is activated in G(1)/S-arrested cells. We provide herein several lines of evidence for a novel interaction between Nek11 and Nek2A. Both Nek11 and Nek2A, but not Nek2B, were detected at nucleoli, and the Nek2A-specific C-terminal end (amino acids 399-445) was responsible for nucleolar localization. Endogenous Nek11 coimmunoprecipitated with endogenous Nek2A, and non-catalytic regions of each kinase were involved in the complex formation. Nek11L interacted with phosphorylated Nek2A but barely with the kinase-inactive Nek2A (K37R) mutant. In addition, both Nek2A autophosphorylation activity and the Nek11L-Nek2A complex formation increased in G(1)/S-arrested cells. These results indicate that autophosphorylation of Nek2A could stimulate its interaction with Nek11L at the nucleolus. Moreover, Nek2 directly phosphorylated Nek11 in the C-terminal non-catalytic region and elevated Nek11 kinase activity. The non-catalytic region of Nek11 showed autoinhibitory activity through intramolecular interaction with its N-terminal catalytic domain. Nek2 dissociated this autoinhibitory interaction. Altogether, our studies demonstrate a unique mechanism of Nek11 activation by Nek2A in G(1)/S-arrested cells and suggest a novel possibility for nucleolar function of the NIMA family.

[Screening of protein kinase inhibitors].

To screen the compounds that inhibit protein kinases, we used an assay system in which activities of protein kinase A, protein kinase C, src family protein tyrosine kinases and eukaryotic elongation factor 2 kinase were simultaneously detected on a single gel for the cytoplasmic protein kinases. For the receptor type protein kinases, EGFR and Flt-1 tyrosine kinases were examined. Here, we briefly described some of the protein kinase inhibitors that have been approved or are under clinical development, and present some novel inhibitors that were found in our screening system.

Interferon regulatory factor-2 regulates cell growth through its acetylation.

We have previously shown that interferon regulatory factor-2 (IRF-2) is acetylated by p300 and PCAF in vivo and in vitro. In this study we identified, by mass spectrometry, two lysine residues in the DNA binding domain (DBD), Lys-75 and Lys-78, to be the major acetylation sites in IRF-2. Although acetylation of IRF-2 did not alter DNA binding activity in vitro, mutation of Lys-75 diminished the IRF-2-dependent activation of histone H4 promoter activity. Acetylation of IRF-2 and IRF-2-stimulated H4 promoter activity were inhibited by the adenovirus E1A, indicating the involvement of p300/CBP. Mutation of Lys-78, a residue conserved throughout the IRF family members, led to the abrogation of DNA binding activity independently of acetylation. H4 is transcribed only in rapidly growing cells and its promoter activity is dependent on cell growth. Consistent with a role for acetylated IRF-2 in cell growth control, IRF-2 was acetylated only in growing NIH 3T3 cells, but not in growth-arrested counterparts. Chromatin immunoprecipitation assays showed that IRF-2 interacted with p300 and bound to the endogenous H4 promoter only in growing cells, although the levels of total IRF-2 were comparable in both growing and growth-arrested cells. These results indicate that IRF-2 is acetylated in a cell growth-dependent manner, which enables it to contribute to transcription of cell growth-regulated promoters.

Mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitors restore anoikis sensitivity in human breast cancer cell lines with a constitutively activated extracellular-regulated kinase (ERK) pathway.

Anchorage-independent growth is a hallmark of oncogenic transformation. We reported that the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 inhibited anchorage-independent growth of Ki-ras-transformed rat fibroblasts by simultaneously blocking both extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR)-p70(S6K) pathways. Here, we examined the effects of U0126 on the growth of eight human breast cancer cell lines. U0126 selectively repressed anchorage-independent growth of MDA-MB231 and HBC4 cells, two lines with constitutively activated ERK. Loss of contact with substratum triggers apoptosis in many normal cell types, a phenomenon termed anoikis. U0126 sensitized MDA-MB231 and HBC4 to anoikis, i.e., upon treatment with U0126, cells deprived of anchorage entered apoptosis, whereas adherent cells remained viable. Another MEK inhibitor PD98059 also induced anoikis sensitivity in MDA-MB231 cells but not in HBC4 cells. However, HBC4 cells were sensitized to anoikis when PD98059 was combined with the mTOR inhibitor rapamycin. To study the biochemical basis for induction of anoikis sensitivity, we examined the effects of the MEK inhibitors on ERK and p70(S6K) pathways in anchored versus nonanchored cells. As in Ki-ras-transformed rat fibroblasts, U0126 reduced activation of both ERK and p70(S6K) in MDA-MB231 and HBC4 cells, irrespective of anchorage. PD98059, in anchored cells, was more selective for the ERK pathway and did not significantly block the p70(S6K) pathway. Removal of anchorage substantially sensitized p70(S6K) to PD98059 in MDA-MB231 cells, whereas p70(S6K) in suspended HBC4 cells remained fairly refractory. U0126 was either without effect or less inhibitory on p70(S6K) in MDA-MB453 and SKBR3, two cell lines in which anoikis sensitivity was not induced. Thus, susceptibility of the p70(S6K) pathway to MEK inhibitors appeared to be an important determinant of anoikis sensitivity. The results indicate that concurrent inhibition of MEK-ERK and mTOR-p70(S6K) pathways induces apoptosis in MDA-MB231 and HBC4 cells when cells are deprived of anchorage but not when anchored. Inhibitors of MEK-ERK and mTOR-p70(S6K) pathways may provide a therapeutic strategy to selectively target neoplasms proliferating at ectopic locations, with acceptable effects on normal cells in their proper tissue context.