Senescence                        is blocked in human cervical keratinocytes infected with high risk human                        papillomavirus (e.g. HPV type16). Viral oncoproteins HPV E6 and HPV E7                        access the cell cycle via cellular p53 and retinoblastoma proteins                        respectively. Previously we have shown that HPV E7, not HPV E6, is also                        responsible for cervical cancer cell survival . We                        now present evidence that SIRT1, an aging-related NAD-dependent                        deacetylase, mediates HPV E7 survival function in SiHa cervical cancer                        cells. Moreover, HPV E7 up-regulates SIRT1 protein when expressed in                        primary human keratinocytes. Conversely, SIRT1 levels decrease following                        RNAi-mediated silencing of HPV E7 in SiHa cells. Silencing HPV E6 has no                        effect on SIRT1 but, as expected, causes marked accumulation of p53 protein                        accompanied by p53-mediated up-regulation of p21. However, p53 acetylation                        (K382Ac) was barely detectable. Since p53 is a known SIRT1 substrate we                        propose that elevated SIRT1 levels (induced by HPV E7) attenuate p53                        pro-apoptotic capacity via its de-acetylation. Our discovery that HPV E7 up-regulates                        SIRT1 links a clinically important oncogenic virus with the                        multi-functional SIRT1 protein. This link may open the way for a more                        in-depth understanding of the process of HPV-induced malignant                        transformation and also of the inter-relationships between aging and                        cancer.                 The processes of aging and cancer share                        many characteristics despite their opposing phenotypes of senescence versus                        immortalisation . In bothThe yeast model provided                        the initial evidence linking SIRT1 with aging. Thus Sir2, the ancestral                        homologue of SIRT1, is a yeast NAD-dependent deacetylase and longevity factor ,17. In Much of our understanding                        of the molecular biology of cancer derives from early studies in which DNA                        tumour viruses were employed and the mechanisms by which they induce cell                        transformation from normal to cancerous growth were elucidated. Indeed studies                        with DNA tumour viruses led to the initial discovery of p53, the major tumour                        suppressor in humans . One ofin vivo).                                HPV E6 and E7 are expressed                        as bicistronic mRNA. In this current work we have exploited a previous                        unexpected observation, namely that in HPV16-positive SiHa cells it is possible                        to selectively and individually silence E6 and E7 by RNA interference (RNAi)                        despite the contiguous nature of their mRNAs . Here weIt is already established that HPV16 E6                        targets the p53 protein for rapid degradation with consequential loss of p53                        tumour suppressor functions, including maintenance of chromosomal integrity. We                        have now discovered that the second HPV viral oncogene, HPV16 E7, targets                        SIRT1. Specifically we demonstrate (i) that exogenous expression of HPV E7 (but                        not HPV E6) in primary human keratinocytes induces abnormally high levels of                        the SIRT1 protein, similar to those observed in human cervical cancer cells,                        and (ii) that HPV E7 (but not HPV E6) is required to maintain the abnormally                        high levels of SIRT1 protein expressed in cervical cancer cells. The ability of                        HPV E7 to up-regulate SIRT1 appears to be linked with HPV E7-mediated                        suppression of apoptosis in cervical cancer cells  since ouOur discovery that HPV E7                        influences the expression of SIRT1 provides the first link between an oncogenic                        virus and the aging-related SIRT1 protein, and enables access to diverse                        SIRT1-dependent cellular regulatory systems. This link may open the way for a                        more in-depth understanding of the inter-relationships between aging and                        cancer. It may also provide insight into the mechanism of chromosomal                        re-distribution during the switch from latent to malignant HPV viral infection,                        and also for the maintenance of HPV-induced malignancy.                                For                            this study we exploited the ability of RNAi to separately knockdown the viral                            oncogenes HPV16 E6 and HPV16 E7 in HPV16-positive SiHa cervical cancer cells.                            Individual knockdown of HPV E6 and E7 by their respective siRNAs was verified                            by quantitative RT-PCR .                            Treatment with trichostatin A, a class I and II histone deacetylase inhibitor,                            induced a dramatic increase in both K9Ac and K14Ac of histone H3 (data not                            shown) indicating high deacetylase activity at these sites in SiHa cells.                            Neither E6 nor E7 significantly affected K9 methylation of histone H3 in SiHa                            cells Figure .       To explore how E7 silencing might decrease S10P we examined levels of aurora B and                            survivin. Aurora B is the principal histone H3 S10 kinase in human cells  and itsIn                            the mouse model for BRCA1 tumor suppressor function there is evidence that wild                            type BRCA1 suppresses survivin expression via SIRT1-dependent epigenetic                            modification of histone H3 . Our ownE6                            silencing caused a delayed reduction in survivin protein levels Figure  39-41].-41.39-41HPV E7 silencing also resulted in reduced mRNA levels of survivin Figure         1 cells from ~60% to ~11% was                            observed 72h post-transfection  were unaffected by HPV E7 knock-down (data not shown)                            demonstrating that the reduction in SIRT1 protein levels reflects a selective effect                            of HPV E7 depletion. In contrast to HPV E7 depletion, the depletion of HPV E6                            had no effect on levels of SIRT1 protein . The                            HPV E7 effect on SIRT1 could be a late event in the process of malignant cell                            transformation by the HPV virus. Alternatively HPV E7-mediated up-regulation of                            SIRT1 might play a causative role in malignant cell transformation. In favour                            of the latter alternative we show that SIRT1 suppresses apoptosis in SiHa human                            cervical cancer cells . Primary human keratinocytes                            were transfected with HPV E6 or HPV E7 expression vectors and exogenously                            expressed E6 and E7 proteins were detected by immunoblotting  formulated into liposomes as described [                escribed ,30,45,46escribed .       escribed ,30,45,46E6                                and E7 cDNA cloning and exogenous expression.                 Mammalian                        expression vectors for HPV16 E6 and HPV16 E7 were generated by subcloning HPV16                        E6 cDNA or E7 cDNA, freshly cloned from low passage SiHa cells, into the                        multiple cloning site of pcDNA3.1. Generated constructs were verified by                        sequencing. DNA transfection was performed using lipofectamine (Invitrogen)                        according to the manufacturer's instructions as previously described [                escribed .       mRNA                                quantification.                 Total cellular RNA was isolated using RNeasy kit                        (Qiagen) [0C                        for 30min; 950C for 15 min; 30 cycles of 940C for 30 sec,                        580C for 45 sec, 720C for 1 min; followed by 720C                        for 5 min. For aurora B, primers 5'-ACAGACGGCTCCATCTGGCCT -3' and 5'-TCAGGCGACAGATTGAAGGGCA-3' were                        used in the thermal cycle: 500C for 30min; 950C for 15                        min; 50 cycles of 940C for 30 sec, 580C for 45 sec, 720C                        for 1 min; and then 720C for 5 min. Cycle parameters and primers for                        HPV E6, HPV E7, SIRT1 and lamin A/C mRNAs were as previously described [                (Qiagen) . 100ng RImmunoblotting.                 Total cell                        extracts were prepared from transfected cells [                ed cells  and    ed cells . ComparaCell                                cycle analysis and quantitation of apoptosis.                 Cell cycle                        analysis, and cell aggregate discrimination and histogram deconvolution using                        Cylchred software, were as previously described [                escribed ,20,45,46escribed .


Vaccination of mice with influenza matrix protein 2 induced cross-reactive antibody responses. Changes in influenza viruses require regular reformulation of strain-specific influenza vaccines. Vaccines based on conserved antigens provide broader protection. Influenza matrix protein 2 (M2) is highly conserved across influenza A subtypes. To evaluate its efficacy as a vaccine candidate, we vaccinated mice with M2 peptide of a widely shared consensus sequence. This vaccination induced antibodies that cross-reacted with divergent M2 peptide from an H5N1 subtype. A DNA vaccine expressing full-length consensus-sequence M2 (M2-DNA) induced M2-specific antibody responses and protected against challenge with lethal influenza. Mice primed with M2-DNA and then boosted with recombinant adenovirus expressing M2 (M2-Ad) had enhanced antibody responses that cross-reacted with human and avian M2 sequences and induced T-cell responses. This M2 prime-boost vaccination conferred broad protection against challenge with lethal influenza A, including an H5N1 strain. Vaccination with M2, with key sequences represented, may provide broad protection against influenza A. Yearly development of influenza vaccines that are antigenically matched to circulating strains pose extraordinary challenges. A rapidly developing pandemic would shorten the time for strain identification and vaccine preparation; meanwhile, antigenic changes would continue. Moreover, the need to immunize an entirely naive population would exacerbate problems with vaccine production and supply.,Vaccines based on conserved antigens would not require prediction of which strains would circulate during an approaching season and could avoid hurried manufacturing in response to outbreaks. Test vaccination with DNA constructs that express conserved influenza A nucleoprotein (NP) or NP plus matrix (M) induced antibody and T-cell responses and protected against heterosubtypic viruses  (www.hhs.gov/pandemicflu/plan/). We therefore evaluated M2-based vaccine efficacy against divergent challenge viruses.Priority is being given to developing vaccines that offer broad protection against multiple influenza subtypes, including H5N1. Indeed, development of conserved-antigen vaccines, and specifically M2-based vaccines, is part of the US Department of Health and Human Services Pandemic Influenza Plan   were synthesized with COOH-terminal cystine residue and conjugated to maleimide-activated keyhole limpet hemocyanin (KLH) for vaccines. The same peptides were also synthesized without COOH-terminal cystine and used for antibody and T-cell assays. Influenza A NP147–155 and M2e peptides were synthesized in the core facility of the Center for Biologics Evaluation and Research, US Food and Drug Administration. Severe acute respiratory syndrome (SARS) matrix peptide (209–221) was provided by the National Institutes of Health.12 particles/mL and confirmed as negative for replication-competent adenovirus by passage on nonpermissive cells.Plasmid and recombinant adenoviral (rAd) vectors that express B/NP and A/NP have been described . Three weeks later, the mice were given an intraperitoneal booster injection with peptide-KLH in incomplete Freund’s adjuvant; 13 days later blood was collected. Injections were started at 8–10 weeks of age for peptide and 6 weeks of age for DNA. DNA vaccination at doses of 50 μg/mouse (unless noted otherwise in a figure legend) in low-endotoxin PBS  was given intramuscularly in the quadriceps, half to each leg, in 3 doses 2 weeks apart. In some experiments, mice were given a booster injection of rAd intramuscularly at a dose of 1050) for H1N1 subtype was determined for 8-week-old naive BALB/c mice for each anesthetic . Subtype H5N1 was administered intranasally to mice anesthetized with 2,2,2-tribromoethanol in tert-amyl alcohol . Some mice were humanely killed so their lungs could be harvested; others were monitored for body weight and death. Monitoring continued until all animals died or were recovering, as indicated by body weight.Challenge virus in 50 μL of PBS was administered intranasally to anesthetized mice. Isoflurane or ketamine/xylazine was used for mice challenged with H1N1 subtype. Reported 50% lethal dose  and used MAbs GK1.5, specific for mouse CD4; 2.43, specific for mouse CD8; and SFR3-DR5, specific for a human leukocyte antigen as a negative control. Splenocytes were analyzed 2 days after challenge (before the next injection) by flow cytometry to confirm completeness of in vivo T-cell depletion, as described  peptide-KLH conjugate, or A/PR/8 virus). Mice were challenged with a moderate dose of A/PR/8 virus the day after serum transfer; antibody levels in recipients were not measured.T cells were enriched by negative selection that used magnetic beads. Briefly, splenocytes were depleted of erythrocytes and labeled with biotinylated antimouse B220, CD11b, and PanNK antibodies . After labeling, cells were incubated with Streptavidin MicroBeads . Unlabeled T cells and labeled non–T cells were separated through the Miltenyi AutoMACS system according to the manufacturer’s instructions.This assay detected T-cell responses to M2 peptides. ELISPOT IP plates  were coated with 50 µL of Hank’s balanced salt solution  containing 5 µg/mL of anti–interferon-γ (IFN-γ) MAb AN18 (BD Pharmingen) and incubated overnight at 4°C. The membrane was washed and then blocked with medium containing 10% fetal bovine serum for 60–90 min at room temperature. Splenocytes depleted of erythrocytes were added to wells in 2-fold dilutions, starting at 250,000 cells/well in 50 µL. Peptides  were added at a final concentration of 1 µg/mL. After incubation for 36–48 h at 37°C, bound IFN-γ was detected with 50 µL of biotinylated MAb R4–6A2 (BD Pharmingen) at 1 µg/mL. Spots were developed by using alkaline phosphatase–labeled streptavidin and 5-bromo, 4-chloro, 3-indolylphosphate/nitroblue tetrazolium substrate  and counted with an ELISPOT reader .50) assay as described ; those performed once used numbers of animals per group adequate for statistical significance. Lung virus titers were compared by using 1-way analysis of variance on log-transformed data, followed by pairwise multiple comparison (Holm-Sidak method). Weight loss after challenge was compared for survivors on each day by also using 1-way analysis of variance followed by pairwise multiple comparison (Holm-Sidak). This method overestimates body weight in groups with deaths because the animals that died would have had very low body weights, affecting the average, especially in the negative control groups. Nonetheless, differences between vaccinated groups were significant in the instances stated. Comparison of cumulative survival rates used the log-rank test, followed by pairwise multiple comparison, again by using the Holm-Sidak method. Overall significance level for Holm-Sidak tests was p = 0.05. All statistical analyses were performed with SigmaStat Software v3.11 .For proof-of-concept studies, peptides representing M2e-con  were conM2e-vaccinated mice were then challenged with either A/PR/8 or A/FM and monitored for weight loss (as a measure of illness) and death. Weight losses were consistent with a hierarchy of protection based on sequence similarity (data not shown), and differences from control mice were statistically significant. In the same groups, 100% of mice vaccinated with M2e-con or M2e-FM survived challenge with A/PR/8 and A/FM, but M2e-H5(HK)/KLH vaccination provided incomplete protection .To investigate whether, like M2e peptide, DNA vaccination could protect against viruses with quite divergent M2e sequence, we tested M- and M2-DNA for efficacy. M-DNA with the A/PR/8 sequence protected against challenge with A/PR/8 . M2-DNA +) responses than does DNA vaccination alone and can protect against challenge with highly pathogenic H5N1 subtype (18). We investigated whether boosting with rAd would also enhance immunity to M2. The A/M2 gene with the consensus sequence was cloned into a replication-deficient Ad construct (M2-Ad). Mice were primed with DNA as before and boosted with M2-Ad or control B/NP-Ad. Two weeks later, serum samples were collected and assayed for M2-specific immunoglobulin (Ig) G by ELISA on peptides as above  or A/FM (moderate dose) virus. Of mice vaccinated with M2-DNA+M2-Ad, 100% survived challenge with A/PR/8 and A/FM virus; of mice vaccinated with B/NP-DNA+B/NP-Ad, 20% survived challenge with A/PR/8 and none survived challenge with A/FM . Thus, t+ or CD8+ cells were <1% (data not shown). Of the B/NP control mice, 100% died of A/PR/8 infection by day 8 after challenge, while 100% of M2-immune mice treated with a control MAb (SFR) survived , we tested the ability of antibodies induced by DNA prime–Ad boost to passively transfer protection. All (100%) mice given serum from M2-immune or A/PR/8-infected mice survived, while only 50% given control B/NP-immune serum survived .Because protection against challenge with A/FM virus that has an M2e sequence quite divergent from that of the immunizing sequence was encouraging, we tested whether M2-DNA+M2-Ad vaccination could protect against challenge with H5N1 subtype. Mice were immunized 3× with B/NP-DNA (negative control), A/NP-DNA (positive control), or consensus M2-DNA, and boosted with matched rAd. Mice were challenged with a lethal dose of A/Thailand/SP-83/2004 (H5N1) virus in which M2e differed from the consensus by 3 amino acids. On day 5 after infection, a random subset of animals was killed and their lung virus titers were measured. Virus titers of mice vaccinated with A/NP or with M2 were significantly reduced compared with those of control mice . The remOur results indicate that M2 vaccination can induce cross-reactive antibody responses, virus-specific T-cell responses, and protection against challenge with lethal heterologous virus. As has been found in previous studies of M2-based vaccines, we found strong antibody responses to the conserved M2e region. Anti-M2 antibodies in the serum of mice immunized with M DNA suggest expression of M2 from this plasmid (not shown). However, reactivity could be due to the 9 amino acid portion shared with the M1 sequence, and we did not explore this possibility. Fan et al. used A/PR/8 and Aichi M2e-carrier conjugates for immunization, and the resulting antibodies did not cross-react with the avian M2e sequences tested  but also a 1947 subtype H1N1 virus (A/FM). The latter virus, which is virulent in mice, has an M2e sequence with 3 amino acid differences from the consensus and thus is as divergent from the consensus sequence as some M2-H5 sequences. Encouraged by this broad cross-reactivity and cross-protection, we expanded the study to DNA vaccination and DNA prime–Ad boost regimens. These approaches have the advantage of providing more epitopes than peptide immunization and relevant T-cell immunity.Using M2 consensus DNA vaccination with or without Ad boost, we again saw cross-reactivity on avian peptides M2e-H5(SP-83) and M2e-H5(HK), although cross-reactivity was low on the HK peptide. T-cell responses to M2 peptides were detected by ELISPOT.+ and CD8+ T cells during the challenge period reduced protection against a higher challenge dose. This could reflect M2e-specific memory T cells, which we have demonstrated in spleen and peripheral blood by ELISPOT, or a concurrent T-cell response to challenge virus supplementing the protective effects of antibodies.Several studies have shown that M2e-specific antibodies can mediate protection against influenza infection in vivo  isolated from a fatal human case, at a dose lethal to control mice. Virus replication in lungs and illness reflected by loss of body weight were also reduced by M2 immunization. Protection against challenge with other H5N1 subtypes remains to be explored, and serologic results on M2e-H5(HK) peptide suggest results of such studies might differ on the basis of sequence variations.M2 expression constructs with various M2e sequences could be used as vaccines. Our observation of protection across substantial sequence divergence means that H5-derived vaccines might also protect against circulating H1N1 and H3N2 subtypes. An additional advantage of protection across substantial divergence is potential protection by an M2 vaccine against an unexpected subtype that could cause a pandemic.One concern about M2 vaccines is the possibility of escape mutants. A study of forced escape mutants found limited diversity  have led to drug resistance, the mutations that confer resistance are within the transmembrane region (An M2 prime-boost regimen is intended to be combined with vaccination against additional antigens rather than acting as a standalone vaccine. For example, prime-boost vaccination against conserved NP is highly protective ([Vaccines based on conserved antigens are not intended to replace strain-matched vaccines that induce neutralizing antibodies and thus prevent infection. However, strain-matched vaccines may be difficult to produce in adequate quantities in short time periods, and continued antigenic drift may render them ineffective. Vaccinations as described here, based on M2, might reduce deaths and severity of disease while strain-matched vaccines were being prepared and could enhance protection afforded by inactivated vaccines. Immunogenicity and safety studies in people are needed to evaluate this approach.


A growing concern has raised regarding the pandemic potential of the highly pathogenic avian influenza (HPAI) H5N1 viruses. Consequently, there is an urgent need to develop an effective and safe vaccine against the divergent H5N1 influenza viruses. In the present study, we designed a tetra-branched multiple antigenic peptide (MAP)-based vaccine, designated M2e-MAP, which contains the sequence overlapping the highly conserved extracellular domain of matrix protein 2 (M2e) of a HPAI H5N1 virus, and investigated its immune responses and cross-protection against different clades of H5N1 viruses.Our results showed that M2e-MAP vaccine induced strong M2e-specific IgG antibody responses following 3-dose immunization of mice with M2e-MAP in the presence of Freunds' or aluminium  adjuvant. M2e-MAP vaccination limited viral replication and attenuated histopathological damage in the challenged mouse lungs. The M2e-MAP-based vaccine protected immunized mice against both clade1: VN/1194 and clade2.3.4: SZ/406H H5N1 virus challenge, being able to counteract weight lost and elevate survival rate following lethal challenge of H5N1 viruses.These results suggest that M2e-MAP presenting M2e of H5N1 virus has a great potential to be developed into an effective subunit vaccine for the prevention of infection by a broad spectrum of HPAI H5N1 viruses. The re-emergence of H5N1 highly pathogenic avian influenza (HPAI) in 2003 has caused 262 fatal cases among a total of 442 infected individuals Female BALB/c mice at 4-6 weeks were purchased from the Laboratory Animal Unit and housed in the animal facility of The University of Hong Kong following the approved animal care protocols. Mice were rested for 2 weeks before immunization. The animal study was approved by the Department of Health of Government of Hong Kong Special Administration Region, and University Animal Ethics Committee of The University of Hong Kong.50 of each virus stain was determined in mice after serial dilutions of the virus stock. All infectious experiments related to H5N1 viruses were performed in an approved biosafety level 3 (BSL-3) facility at The University of Hong Kong.HPAI H5N1 virus isolates used in this study were clade1: VN/1194 and clade2.3.4: SZ/406H. H5N1 viruses were grown in the allantoic cavities of 10-day-old embryonated chicken eggs. Virus-containing allantoic fluid was harvested and stored in aliquots at -80°C until use. The LDMice were subcutaneously (s.c.) prime vaccinated with M2e-MAP (10 μg/mouse) plus Freund's complete adjuvant  and boosted twice with the same amount of immunogen plus Freund's incomplete adjuvant  at 3-week intervals. For parallel experiments, mice were also intramuscularly (i.m.) vaccinated with the same amount of M2e-MAP immunogens plus alum adjuvant (Sigma) at the same condition of the above. Mice injected with Freund's or alum adjuvant were used as the respective control. Mouse sera were collected before immunization and 10 days post-each vaccination for detection of antibody responses.50 of clade1: VN/1194 or clade2.3.4: SZ/406H H5N1 virus stain. Infected mice were observed and weighed daily for 2 weeks. Lung tissues were collected from euthanized mice 5 days post-challenge for further virological test and histopathological analysis.Two weeks post-last vaccination, mice were intraperitoneally (i.p.) anesthetized with ketamine-xylazine (75/5 mg/kg), and intranasally (i.n.) challenged with 10LD2SO4. The absorbance at 450 nm was measured by an ELISA plate reader .The M2-specific IgG antibody in the vaccinated mouse sera was detected by ELISA as previously described  with som10TCID50/g of lung tissues.Lung tissues from euthanized mice were aseptically removed and homogenized in minimal essential medium (MEM) plus antibiotics to achieve 10% (w/v) suspensions of lungs. Ten-fold serial dilutions of samples were added in quadruplicate to the monolayer of Madin-Darby canine kidney (MDCK) cells seeded at 96-well cell culture plates 12 h before infection, and allowed to absorb for 2 h at 37°C. Fresh medium was then added to the cells and continued to incubate for 72 h. Virus cytopathic effect (CPE) was observed daily and the viral titer was determined by the hemagglutinin (HA) test indicated as follows. Fifty microlitre of 0.5% turkey red blood cells  was added to 50 μl of cell culture supernatant and incubated at room temperature for 30 min. Wells containing HA were scored as positive. The virus titer was calculated by the Reed and Muench method and expressed as LogThe lung tissues of challenged mice were immediately fixed in 10% neutral buffered formalin and embedded in paraffin wax. Sections were made at 4-6 μm thickness and mounted on slides. Histopathological changes were examined by H&E staining and observed under light microscopy as previously described .P < 0.0001 was considered significant. All analyses were performed in Graphpad Prism software.The significance between survival curves was analyzed by Kaplan-Meier survival analysis with log-rank test. Other data were analyzed using the 2-tailed Student's t test. 50: 50% lethal dose; Log10TCID50/g: Log10 50% tissue-culture infectious dose per gram.HPAI: highly pathogenic avian influenza; M2e-MAP: M2e-based multiple antigenic peptide; FCA: Freunds' complete adjuvant; FIA: Freunds' incomplete adjuvant; VN/1194: A/Vietnam/1194/2004(H5N1); SZ/406H: A/Shenzhen/406H/2006(H5N1); LDThe authors declare that they have no competing interests.GZ, BZ and YZ designed research. GZ, YL, JG, SS, HS and CC performed research. GZ, SS, ZK and YG analyzed data. GZ, LD, SJ, BZ and YZ wrote and modified the paper. All authors read and approved the final manuscript.


Slc39a4) is important for proper mammalian development and is an essential gene in mice. Recent studies suggest that this gene may also play a role in pancreatic cancer.The zinc transporter ZIP4 (Zip4 mRNA and protein were dramatically elevated in hepatocytes in the majority of human hepatocellular carcinomas relative to noncancerous surrounding tissues, as well as in hepatocytes in hepatocellular carcinomas occurring in farnesoid X receptor-knockout mice. Interestingly, meta-analysis of microarray data in the Geo and Oncomine databases suggests that Zip4 mRNA may also be elevated in many types of cancer. Potential mechanisms of action of ZIP4 were examined in cultured cell lines. RNAi knockdown of Zip4 in mouse Hepa cells significantly increased apoptosis and modestly slowed progression from G0/G1 to S phase when cells were released from hydroxyurea block into zinc-deficient medium. Cell migration assays revealed that RNAi knockdown of Zip4 in Hepa cells depressed in vitro migration whereas forced over-expression in Hepa cells and MCF-7 cells enhanced in vitro migration.Herein, we present evidence that this essential zinc transporter is expressed in hepatocellular carcinomas. ZIP4 may play a role in the acquisition of zinc by hepatocellular carcinomas, and potentially many different cancerous cell-types, leading to repressed apoptosis, enhanced growth rate and enhanced invasive behavior. Metallothionein genes), efflux  and uptake  of this metal  superfamily Zip4 gene is essential for proper early morphogenesis of the mouse embryo Zip4 is the only mammalian ZIP superfamily member which has been shown to be an essential gene; one that plays a fundamental role in adaptation to zinc deficiency. However, the fear-of-intimacy gene in Drosophila encodes a ZIP6-like zinc transporter that is essential during early development and plays a role in migration of germ cells zLIV-1) is essential during early morphogenesis and plays a role in the epithelial-mesenchymal transition (EMT) during gastrulation Given the diversity of functions of zinc it is perhaps not surprising that zinc transporters have recently been found to play roles in carcinogenesis Zip4 gene is also actively expressed in hepatocytes in human and mouse hepatocellular carcinomas (HCC), and meta-analyses of the Geo and Oncomine databases further suggested that enhanced expression of the Zip4 gene may occur in many different cancers. We present evidence that ZIP4 can repress apoptosis and enhance cell-cycle and cell migration. Therefore, ZIP4 can exert profound effects on cancer growth and metastasis.Given the fundamental role of ZIP4 in mammalian development and adaptation to zinc deficiency, and the recent report of its role in pancreatic cancer, we examined whether this gene may play a fundamental role in carcinogenesis. Herein, we report that the Zip4 in human hepatocellular carcinomas (HCC). RNA extracted from a panel of biopsies of human HCC and surrounding control tissue was analyzed by real-time quantitative RT-PCR for Zip4 mRNA and values were normalized to GAPDH mRNA . Similar results were obtained by normalizing Zip4 mRNA values with those of another internal control HPRT1 mRNA values (data not shown). The presence of ZIP4 protein in human HCC was confirmed by immunofluorescence detection in frozen sections  revealed high levels of  samples  as repor samples . Groups  samples , but no out mice . The majout mice .Zip4 is aberrantly expressed in human and mouse HCC, as well as in pancreatic adenocarcinoma, suggested that Zip4 expression might also be increased in other carcinomas. A meta-analysis of microarray data deposited in the Geo and Oncomine databases for Zip4 (Slc39a4) expression supported this hypothesis . In the Oncomine database Zip4 was found to be among the top 10% of genes expressed in many human cancer types and was often associated with an aggressive stage of the cancer. Expression in human HCC was also confirmed. This meta-analysis suggests, but does not prove, that active expression of Zip4 may occur in many different carcinomas. However, these analyses did confirm and extend our independent finding of active Zip4 expression in HCC from humans and mice.The finding that pothesis . In the Zip4 knockdown on apoptosis and progression through the cell cycle in mouse Hepa cells. These cells actively express the Zip4 gene and regulate the stability of this mRNA and the endocytosis and processing or turn-over of this protein in response to zinc Zip4 mRNA or a scrambled control RNAi, and infected cells were purified by FACS using GFP expression from the viral vector. Western blot analysis of membrane proteins from these cells revealed that Zip4 RNAi effectively reduced ZIP4 protein levels and largely prevented induction in response to zinc-deficient culture conditions, but had no effect on ZIP1 abundance . In contrast, when released into zinc-deficient medium, cells expressing Zip4 RNAi reentered the cell cycle more slowly than did those expressing control RNAi. At every time point examined the percentage of cells in S phase after release from the block was lower in the Zip4 RNAi cells compared with the control RNAi cells .t medium . The numt medium . After rAi cells  while thAi cells . These dZip4 RNAi, as well as these cells transiently transfected with a human Zip4 cDNA expression vector encoding human ZIP4 with a carboxyl-terminal HA tag  and may well be expressed in many other types of cancer. HCC is the fifth most common cancer in the world and third leading cause of cancer deaths resulting in over 600,000 deaths globally per year. The 5 year survival rate is reported to be below 5%  Zip4 mRNA occurs when zinc is deficient. The finding that ZIP4 localizes to what appears to be the apical surface of hepatocytes in the liver tumors from FXR-knockout mice suggests that either this protein is not zinc-regulated in those cells or that these cells are zinc deficient. Several studies have reported that HCCs have significantly lower levels of zinc than the surrounding tissues .HCC specimens were obtained through the LTCDS (Liver Tissue Cell Distribution Service) under the NIH Contract – #N01-DK-7-0004/HHSN267200700004C  GAPDH primers were included in every sample to control for sample variation. Zip4 mRNA product in each sample was normalized to that of the GAPDH mRNA. The amount of PCR products was measured by threshold cycle (Ct) values and relative mRNA levels are presented as unit values of 2̂[Ct(GAPDH) -Ct(Zip4)]. The primer sequences for human Zip4 mRNA (Slc39a4) were: sense 5′ AAGCACTGCTGCTGAACCTGGCCT 3′; and antisense 5′ GATGTCATCCTCGTACAGGGACAGCAGC 3′. The primer sequences for the human GAPDH mRNA were: sense 5′ ACATCATCCCTGCCTCTACTGG 3′; and antisense 5′ TCCGACGCCTGCTTCACC 3′.Total RNA was isolated from tumor and surrounding normal tissue by Trizol extraction. Real-time PCR was performed using total RNA and products were detected using the SYBR Green PCR master mix (appliedbiosystems.com). PCR included the following components: 100 nM of each primer, diluted cDNA templates and SYBR green PCR master mix. Samples were denatured at 95°C for 10 min and amplified for 40 cycles at 95°C for 15 sec and 60°C for 1 min. The melting-curve data were collected to confirm PCR specificity. Each cDNA sample was run in triplicate, and the corresponding no-reverse transcriptase (RT) mRNA sample was included as a negative control. The Frozen sections of human HCC and normal liver were thawed directly into phosphate-buffered saline (PBS) containing freshly prepared 2% paraformaldehyde and 0.1% Triton-X-100 for 30 min at room temperature. Sections were washed with PBS and incubated in PBS containing 50 mM lysine for 30 min to quench autofluorescence. Sections were washed in PBS, incubated in PBS containing 0.1% Triton-X-100/0.1% Tween 20 for 20 min, blocked with 10% normal goat serum in PBS for 1 hr and ZIP4 was detected, as described previously, using an antipeptide antibody against mouse ZIP4 FXR-knockout mouse livers and normal liver using the Zymed Laboratories Inc. Histostain-SP kit (invitrogen.com) for rabbit primary antibody and diaminobenzidine substrate staining, as described previously Immunohistochemistry was employed to detect ZIP4 in FXR-knockout mice were generated and back-crossed into the C57BL/6 genetic background, as described previously The Zip4 and actin mRNAs were as described 6 cpm/ml of hybridization solution.Total RNA (3–6 µg) was isolated, size-fractionated by agarose-formaldehyde gel electrophoresis and transferred and UV cross-linked to nylon membranes, as described in detail previously 2 incubator in Dulbecco's modified Eagle medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 100 units penicillin/ml, and 100 µg streptomycin/ml. To generate zinc-deficient conditions in culture, heat-inactivated fetal bovine serum (FBS) was treated with Chelex-100 resin, as described 4 cells per 10 cm plate in either zinc-adequate  or zinc-deficient (Chelex-treated FBS) medium.Mouse Hepa cells were maintained at 37°C in a humidified 5% COZip4 RNAi and control RNAi lentiviral constructs were created in third generation lentiviral vectors, amplified in HEK293 cells, purified by centrifugation, and titered in the Washington University Core Facility as described env, vpr, vpu, vif and nef. This vector has a self-inactivating LTR and envelope, VsVg, is expressed on a separate plasmid Zip4 and the scrambled RNAi control were expressed from the U6 promoter; this vector also expressed green fluorescence protein (GFP) driven by the PGK promoter. The oligos listed below were annealed, phosphorylated, and ligated into this vector just downstream of the U6 promoter at the AarI site.ACCGGGAGAAGACCTGTGTAGATCTCTTCCTGTCAAGATCTACACAGGTCTTCTCC 3′5′ CCTCTTCTGGACACATCTAGAGAAGGACAGTTCTAGATGTGTCCAGAAGAGGAAAA 5′3′ ACCGGGACCAGGATTCTGAGAAAGACTTCCTGTCATCTTTCTCAGAATCCTGGTCC 3′5′ CCTGGTCCTAAGACTCTTTCTGAAGGACAGTAGAAAGAGTCTTAGGACCAGGAAAA 5′3′ ACCGGCGCCTAATTGTCTGGACTATCTTCCTGTCAATAGTCCAGACAATTAGGCGC 3′5′ AAAAGCGCCTAATTGTCTGGACTATTGACAGGAAGATAGTCCAGACAATTAGGCGC 3′5′ Zip4 RNAi and control RNAi lentiviruses at a multiplicity of infection of 5∶1 overnight. Cells were washed extensively and then incubated for 24 hr in DMEM containing 10% FBS. A single cell suspension of these cells was prepared in PBS containing 20% FBS and analyzed by fluorescence activated cell sorting (FACS) for GFP expressed from the lentivirus construct. Fluorescence in the GFP channel was monitored and the intensity of autofluorescence in the wild-type cells was used to define the base line fluorescence. Cells with signal intensities clearly greater than that value were collected and used for experiments. At least 95% of the flow sorted cells were GFP positive.Mouse Hepa cells were incubated with Membrane proteins were prepared from Hepa cells and analyzed as described Zip4 RNAi or control RNAi were split 1∶10 from confluent cultures into zinc-adequate or zinc-deficient medium and then blocked in G0/G1 by incubating for 16 hr with 100 mM hydroxyurea  or blocked in G2/M by incubating for 16 hr with 0.1 µg/ml nocodazole . The blocking medium was then replaced with zinc-adequate or zinc-deficient medium to allow cells to progress in the cell cycle, and cells were harvested at different times thereafter. Cell cycle parameters were monitored by propidium iodide (PI) staining followed by FACS using an Epics XL Flow Cytometer (beckman.com) Hepa cells expressing Zip4 RNAi or control RNAi were plated onto 60 mm dishes and transfected with 4 µg of a human Zip4-HA expression vector or pcDNA3.1 control vector using Lipofectamine 2000 (invitrogen.com) in antibiotic free media according to the manufacturer's instructions. After 6 h the cells were serum starved for 24 h and 5×104cells were seeded onto Transwell inserts . Cells were allowed to migrate for 16 h after which the non-migratory cells were removed from inside the chamber with a cotton swab. Cells that had migrated through the membrane pores were fixed for 10 min , stained with 0.1% crystal violet for 15 min, washed with PBS, and photographed using a Microfire/Qcam CCD Olympus 1×81 microscope. Crystal violet stained cells were lysed in 1% SDS for 30 min and absorbance of the lysate was measured at 595 nm in a plate reader . Results were plotted as the mean absorbance ± S.D.Hepa cells expressing 5/cm2 and grown until 80% confluent in phenol-red-free RPMI supplemented with 5% fetal calf serum, 200 mM L-glutamine, 10 IU/ml penicillin, 10 µg/ml streptomycin, and 2.5 µg/ml fungizone. Cells in 60 mm dishes were transfected with 8.8 µg of an expression vector for mouse Zip4-HA 4 cells were seeded onto fibronectin-coated (1 µg/ml) Transwell inserts . Cells were allowed to migrate for a period of 16 h after which the non-migratory cells were removed from inside the chamber with a cotton swab. Cells that had migrated through the membrane pores were fixed  and stained with crystal violet. Cell migration was quantified by counting the numbers of cells in 5 randomly-chosen fields of view per membrane (n≥3 for each treatment) and results were plotted as the mean cell migration (percent of untreated cells ± S.D.).MCF-7 cells were seeded at 2×10http://www.ncbi.nlm.nih.gov/geo. This database was queried for Gene Profile of Slc39a4 and cancer, at the time of submission of this manuscript, which yielded 106 hits; Slc39a4 and hepatocellular carcinoma which yielded 5 hits; Slc39a4 and lymphoma which yielded 21 hits, Slc39a4 and colon cancer which yielded 5 hits; and Slc39a4 and melanoma which yielded 18 hits. Among these data sets several cancers which showed increased expression of Zip4 mRNA were noted. The Oncomine database was accessed at the following URL: http://www.oncomine.org. This database was queried for Slc39a4 among the top ten percent of genes expressed in tumors.The Geo microarray database at the NCBI was accessed at the following URL: Data were analyzed using the Students'-T-Test to determine P values. Data are expressed as the mean ± Standard Deviation (S.D.)


Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog  and two human receptor analogs . Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type (α2-3) receptor binding profile, with only moderate binding to human-type (α2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential. th Century (H2 in 1957 and H3 1968) both adapted successfully to human-type receptor specificity through only two amino acid mutations in the receptor binding pocket (Glutamine226→Leucine and Glycine228→Serine). The recent human infections reported with other avian subtypes such as H5, H7 and H9 have raised public health concerns and focused efforts on identifying potential subtypes from which a future pandemic strain may emerge. Since 1996, H7 viruses of the North American lineage have been circulating in regional live bird markets, containing an eight amino acid deletion in the receptor-binding site of HA. Here we report a detailed structural analysis of the receptor binding site of a hemagglutinin from the North American lineage of H7N2 viruses, in complex with avian and human receptor analogs, to understand how these viruses have adapted to such a dramatic structural change in the binding site while remaining one of the predominant circulating viral strains.Influenza virus adaptation to different hosts usually results in a switch in receptor specificity of the viral surface coat protein, hemagglutinin. Indeed, the hemagglutinin subtypes from the last two human influenza pandemics of the 20 Influenza is an acute respiratory virus that infects up to 20% of the population in the United States, resulting in ∼36,000 deaths annually Early cases of human infection by H7 influenza viruses are reported as far back as 1979 Since 1996, H7 viruses of the North American lineage have been circulating in regional live bird markets Here we report a detailed molecular analysis of the RBS of the HA from North American lineage H7N2 virus, NY107, including glycan microarray analyses and structural analyses of the HA in complex with an avian receptor analog  and two human receptor analogs . These results provide important insight into the interaction of H7 HAs with both avian and human hosts.By using x-ray crystallography, the structure of H7 HA from the NY107 virus was determined to 2.6 Å resolution . In addihttp://merops.sanger.ac.uk) suggests it to be a possible thrombin cleavage site.During viral replication, HA is synthesized as a single chain precursor (HA0) and cleaved by specific host proteases into the infectious HA1/HA2 form. In baculovirus expression systems, highly pathogenic HAs, with a polybasic cleavage site, are expressed as an HA1/HA2 form Based on their molecular phylogenies, HAs are divided into two groups and five clades: group 1 includes H8, H9, and H12; H1, H2, H5, and H6; H11, H13 and H16; group 2 includes H3, H4, and H14; H7, H10 and H15 The RBS is at the membrane distal end of each HA monomer and its specificity for sialic acid and the nature of its linkage to a vicinal galactose residue is a major determinant of host range-restriction. The consensus RBS for all current HAs is composed of three major structural elements: a 190-helix (residues 188–194), a 220-loop (residues 221–228), and a 130-loop (residues 134–138). In addition, highly conserved residues  form the base of the pocket.Although the NY107 RBS is similar to other subtypes , a previously observed specific feature of H7 HAs, is also observed in the NY107 150-loop region: two residues inserted at position 158 result in this loop protruding more than 6Å towards the binding site compared to other subtype HAs  [27]. MoPreviously, mutations in the HA receptor binding domains of H1N1 (Glu190Asp/Gly225Asp) and H2N2/H3N2 (Gln226Leu and Gly228Ser) subtypes were responsible for adaptation of these viruses to pandemic strains Glycan microarray analysis of recombinant NY107  revealedTo further assess the effect of the missing 220 loop on HA structural stability and receptor specificity it was essential to evaluate these functions on the ancestral HA containing the full length 220-loop. To this end, we engineered an HA with an avian H7 consensus (PQVNGQSG) 220-loop re-introduced (NY107-220ins) into the NY107 HA and recovered this virus by reverse genetics. Compared to the NY107 virus  glycan mWhen viruses containing this 220-loop deletion emerged in North America in the mid 90's, four additional amino acid substitutions, Gly114Arg, Asp119Gly, Gly186Glu and Gly205Arg, in the HA1 as well as an Asp19Asn in the HA2 chain were also introduced to most of the circulating isolates. Of these, Gly186Glu and Gly205Arg in the HA1 are close to the RBS, at the monomer interface, and could potentially modulate its structure and/or function. NY107 viruses with a restored consensus 220-loop and a single Glu186Gly (NY107-ins-186) or Arg205Gly (NY107-ins-205) substitution as well as the Glu186Gly/Arg205Gly double substitution (NY107-ins-186/205) were derived by reverse genetics and evaluated. Glycan microarray analysis for the three resulting viruses revealed similar glycan binding profiles with increased binding to α2-3 sialosides, including mixed α2-3/α2-6 branched sialosides (#60–64), α2-3 Neu5Gc (#66–70), but limited binding to the α2,6 sialosides , resultiTo understand from a structural perspective how NY107 interacts with host receptors, we solved the structure of NY107 in complex with an avian and two human receptor analogs. For the avian receptor analog, 3′SLN, the electron density maps revealed well-ordered features for the Sia-1, Gal-2, and GlcNAc-3 in the NY107 HA complex structure . Structutrans conformation of α2-3 linkages is essential and perhaps only the first two saccharides are required. Indeed, due to the absence of 220-loop in the NY107 HA structure, the “aperture” of the RBS formed by 220-loop and 130-loop in regular HAs is increased by ∼10 Å, so that the branched, internal, and perhaps more complicated glycans might be accommodated more efficiently.Published avian HA structures with an intact 220-loop form very close interactions with Gal-2 of 3′SLN via residue Gln226 which is important in receptor specificity and host adaptation. For example, in the avian H7/3′SLN HA structure it interacts with Gal-2 O4 cis conformation of α2-6 linkages in 6′SLN trisaccharides show a reduced binding affinity with NY107.In the NY107/6′SLN complex, only Sia-1 and Gal-2 are ordered . The SiaGlycan array results with NY107 revealed a strong binding signal for the internal α2-6 sialoside, LSTb. To further investigate this interaction, we solved the structure of the NY107/LSTb complex. The final model contained Sia-1, NAG-2, Gal-3, and Gal-5 in the RBS. Although glycan microarray data indicated NY107 to have a specific affinity for LSTb, few interactions were apparent from the crystal structure. Sia-1 still forms multiple hydrogen bonds with residues in the RBS . The braHuman infections by avian influenza viruses, including H7 subtypes, continue to pose a major public health threat. Although the species barrier prevents avian influenza viruses from widespread infection of the human population, the molecular determinants of efficient interspecies transmission and pathogenicity are still poorly understood. The viral coat protein HA however, is perhaps a critical molecule since previous pandemic viruses modified their receptor specificity and overcame the interspecies barrier to spread in the human population. Although HA structures alone and in complex with receptor analogs provide considerable insight into receptor binding, it is clear that HAs from different species and subtypes have significant structural variation. Indeed, low-pathogenic H7N2 avian influenza viruses with an 8 amino acid deletion within its RBS started to circulate in live-bird markets in the northeast United States in 1996. Despite what one would consider a debilitating mutation, these viruses have been reported as the predominant isolate The crystal structures of NY107 and its complexes with both avian and human receptor analogs describe a mechanism as to how an influenza virus might adapt by dramatically altering its RBS, and still be functional. Arg220 of the HA1 chain of NY107 compensates for the loss of the 220-loop, by forming hydrogen bonds with Gal-2 from the avian analog . However, in the LSTb complex, branched Gal-5 forms extra interactions with the 130-loop, thus improving the binding preference for this particular glycan. Consistent with the structural evidence, glycan microarray analyses of NY107 revealed a strong binding preference for the branched α2-6 sialoside, LSTb. Except for the absence of the 220-loop, other key residues within the RBS are conserved in NY107 and thus, direct interactions with sialic acid are maintained.The 220-loop is recognized as one of the three crucial structural elements in the RBS. Aside from the North American lineage H7N2 viruses, which have been circulating with a deletion (221–228) in this loop, there has been one other report describing a seven amino acid deletion (224–230) in a laboratory generated H3N2 escape mutant which was reported to have a slightly increased affinity for α2-3-linked glycans by hemagglutination assay i.e. glycan profiling data from human lung tissue on the Consortium for Functional Glycomics (CFG) web site. In addition, the internal sialoside, LSTb, was observed in both virus and recHA microarray data, suggesting this type of glycan has good affinity for this HA. The significance of this is unknown since LSTb has only been described in human milk When compared to NL219, another co-circulating H7 avian virus HA , overallet al. using a competitive solid-phase binding assay Interestingly, NY107 and NL219 virus receptor binding and specificity has been addressed previously using glycan microarray analysis that reported a significantly increased preference for α2-6 and decreased preference for α2-3-linked sialosides The 220-loop is an integral feature of the receptor binding site, and thus one would predict that such a deletion might have compromised this strain to be deleted from the population of circulating viruses. However, this was not the case The NY107 RBS with its more restricted α2-3 glycan binding preference and weak/moderate increase in α2-6 binding may have given the virus a selective advantage to be maintained in poultry at live bird markets and supplying farms. Certain terrestrial birds, such as quails and chickens, have recently been shown to present both human and avian types of receptors in the trachea and intestine Based on H3 numbering Soluble NY107 was recovered from the cell supernatant by metal affinity chromatography using Ni-NTA resin (Qiagen Inc.). Fractions containing NY107 were pooled and dialyzed against 10 mM Tris-HCl, 50 mM NaCl, pH 8.0, then subjected to ion-exchange chromatography (IEX) using a Mono-Q HR 10/10 column . IEX purified NY107 was subjected to thrombin digest  and purified by gel filtration chromatography using a Superdex-200 16/60 column  and 50 mM Tris-HCl, 100 mM NaCl, pH 8.0 as running buffer. Protein eluting as a trimer was buffer exchanged into 10 mM Tris-HCl, 50 mM NaCl, pH 8.0 and concentrated to 14.5 mg/ml for crystallization trials. At this stage, the protein sample still contained the additional plasmid-encoded residues at both the N (ADPG) and C terminus (SGRLVPR).Initial crystallization trials were set up using a Topaz Free Interface Diffusion (FID) Crystallizer system . Crystals were observed in several conditions containing PEG 3350 or PEG 4000. Following optimization, diffraction quality crystals for NY107 were obtained at room temperature using a modified method for microbath under oil Vm) of 2.9 Å3/Da. Rigid body refinement of the trimer led to an overall R/Rfree of 28.6%/37.4%. The model was then “mutated” to the correct sequence and rebuilt by Coot The structure of NY107 was determined by molecular replacement with Phaser Wild type and mutant viruses of NY107 (H7N2) and A/Netherland/219/2003 (H7N7) were generated from plasmids by a reverse genetics approach Glycan microarray printing and recHA analyses have been described previously The atomic coordinates and structure factors of NY107 are available from the RCSB PDB under accession codes 3M5G for the unliganded NY107, 3M5H for the NY107 with 3′-SLN and 3M5I and 3M5J for NY107 with 6′SLN and LSTb, respectively.A/New York/107/03 (H7N2), Genbank: ACC55270; A/Netherlands/219/03 (H7N7), Genbank: AAR02640; A/Hong Kong/1-9/68 (H3N2), 2HMG; A/Duck/Ukraine/1/63 (H3N8), PDB: 1MQL; A/South Carolina/1/18 (H1N1), PDB: 1RD8; A/Puerto Rico/8/34 (H1N1), PDB: 1RU7; A/Swine/Iowa/15/30 (H1N1), PDB: 1RUY; A/Singapore/1/1957 (H2N2), PDB: 2WRC; A/Viet Nam/1203/04 (H5N1), PDB: 2FK0; A/Duck/Singapore/3/97 (H5N3), PDB: 1JSM; A/Swine/Hong Kong/9/98 (H9N2), PDB: 1JSD; A/Turkey/Italy/8000/02 (H7N3), PDB: 1TI8; C/Johannesburg/1/66, 1FLC.Figure S1Sequence alignment of selected structurally available HAs. Human H3 (PDB: 2HMG), Avian H3 (PDB: 1MQL), 1918-Human H1 (PDB: 1RD8), 1934-Human H1 (PDB: 1RU7), Swine H1 (PDB: 1RUY), 1957-Huamn H2 (PDB: 2WRC), Human H5 (PDB: 2FK0), Avian H5 (PDB: 1JSM), Swine H9 (PDB: 1JSD), and Avian H7 (PDB: 1TI8) were used in the alignments. The fusion domain of HA1 is highlighted in magenta, the vestigial esterase domain is highlighted in green, the receptor binding domain is highlighted in blue, and the fusion domain of HA2 is highlighted in red. Residue numbering is based on the H3 HA sequence.(2.84 MB TIF)Click here for additional data file.Figure S2Expression and purification of NY107. SDS-PAGE reveals that NY107 was expressed as the HA0 form with a mass approximately 60kDa (middle lane). Thrombin cleavage resulted in an unexpected reduction in band size to a HA1/HA2 profile (right lane) with possible multiple glycoforms for the HA2 clearly present.(0.23 MB TIF)Click here for additional data file.Figure S3Comparison of glycan binding to NY107 with other HAs. A. Overlap of α2-3 ligands binding in the receptor binding site from NY-H7 (green), Av-H3 (orange), 1930-Hu-H1 (magenta), 1957-Hu-H2 (cyan), Av-H5 (yellow), and Sw-H9 (grey). B. Overlap of α2-6 linkage ligands binding in the receptor binding site from NY-H7 (green), Av-H3 (orange), 1930-Hu-H1 (magenta), 1957-Hu-H2 (cyan), Av-H5 (yellow), and Sw-H9 (grey).(2.55 MB TIF)Click here for additional data file.Figure S4Simulated annealing omit maps of the receptor binding site (contoured at 1σ). A. NY107 (blue), B. NY107-3′SLN (orange), C. NY107-6′SLN (red), and D. NY107-LSTb (magenta). The protein model is shown in cartoon, and the residues involved in the binding to receptor analogs were shown in sticks. Maps were generated using version 1.2 of the Crystallography and NMR System (CNS) software.(1.93 MB TIF)Click here for additional data file.Figure S5Comparison of NY107 RBS to HEF. Overlap of RBS from NY107 (green), Av-H7 (marine) and HEF (magenta).(1.12 MB TIF)Click here for additional data file.Table S1Comparison of r.m.s.d. (Å) for different HA domains. For analyzing differences in the overall structure, r.m.s.d. values were calculated between monomers or domains of different HA's, after the Cα atoms of the HA2 domains were superposed by sequence and structural alignment onto the equivalent domains of NY107.(0.04 MB DOC)Click here for additional data file.Table S2Comparison of r.m.s.d. (Å) for individual domains. Each domain was superimposed separately to determine how the individual NY107 domains compared to equivalent domains in the other structures.(0.04 MB DOC)Click here for additional data file.Table S3Molecular interactions between NY107 and receptor analogs. The hydrogen bond cutoff is 3.8 Å for the listing interactions.(0.07 MB DOC)Click here for additional data file.Table S4Glycan array differences between NY107, the fully restored NY107-ins, and NL219 (virus and rHA). The color coding in the left hand column reflects the same coloring scheme used in (0.19 MB DOC)Click here for additional data file.


To understand the molecular mechanism of host responses to highly pathogenic avian influenza virus infection and to get an insight into the means through which virus overcomes host defense mechanism, we studied global gene expression response of human lung carcinoma cells (A549) at early and late stages of infection with highly pathogenic avian Influenza A (H5N1) virus and compared it with a reverse genetics modified recombinant A (H5N1) vaccine virus using microarray platform.The response was studied at time points 4, 8, 16 and 24 hours post infection (hpi). Gene ontology analysis revealed that the genes affected by both the viruses were qualitatively similar but quantitatively different. Significant differences were observed in the expression of genes involved in apoptosis and immune responses, specifically at 16 hpi.We conclude that subtle differences in the ability to induce specific host responses like apoptotic mechanism and immune responses make the highly pathogenic viruses more virulent. Outbreaks of avian influenza A H5N1) virus, a highly pathogenic avian influenza (HPAI), are considered as a public health risk with pandemic potential  virus, a,3. The mIn vitro, in vivo and clinical studies have suggested that H5N1 viruses are very strong inducers of various cytokines and chemokines , in both humans and animals [It is well recognized that host immunological and genetic factors also play an important role in the pathogenesis of H5N1 viruses in humans . Recent  animals . However animals . Hence, Alveolar epithelial cells and macrophages are the key targets for H5N1 virus in the lungs . Using iA/Chicken/India/WB-NIV2664/2008 is a recent strain of H5N1 of clade 2.2 circulating in chicken population in India  and A/InAvian Influenza A (H5N1) virus, A/Chicken/India/WB-NIV2664/2008 (WB-NIV2664) isolated from West Bengal (India) outbreak in 2008  and reve2 incubator.Human lung carcinoma (A549) cells were maintained in Dulbecco's modified Eagle's tissue culture medium  containing 10% fetal calf serum, 100units/ml penicillin, 100 units/ml streptomycin in tissue culture flasks  at 37°C in a CO6 cells/ml were infected with the viruses at a multiplicity of infection (MOI) of 3. After 1 hour the inoculum was removed; the cells were washed twice with phosphate buffer saline (PBS) and supplemented with growth media. For each virus four different sets of tissue culture flask were infected and harvested at four different time points. Mock infected cells at each time point served as controls. Infection of H5N1 viruses was performed in BSL-3+ facility.Monolayers of A549 cells at a concentration of 3 × 10Total RNA was extracted from the control and infected cells at 4, 8, 16 and 24 hpi using Trizol reagent  and purified by the RNeasy kit  following standard methodology. Amplification of RNA and indirect labeling of Cy-dye was done by Amino Allyl MessageAmp II aRNA amplification kit  using manufacturer's instruction. One hundred nanograms of RNA from control and infected cells were used for the experiments. The RNA was reverse transcribed and amplified according to the manufacturer's protocol. The purified amino allyl aRNA was labeled with Cy3 and Cy5  for control and experimental samples respectively. Purified samples were lyophilized, resuspended in hybridization buffer  and hybridized on the Discover human chip . Hybridization was carried out in a Hybstation  and the conditions used were 55°C for 6 h, 50°C for 6 h, and 42°C for 6 h. Scanning was performed at 5-mm resolutions with the Scan array express . Grid alignment was done using gene annotation files and raw data were extracted into MS EXCEL.Data was analyzed using GENOWIZ Microarray and Pathway analysis tool . Replicated values for genes were merged and median values of the expression ratios were considered for the dataset (2 slides per time point were used). Empty spots were removed by filtering. Dye bias was dealt with by applying loess normalization. Log transformation (log2) was done to stabilize the variation in dataset and median centering was performed to bring down data distribution of dataset close to zero. In order to detect highly expressed genes, fold change analysis was done. Genes with 1.5 folds up/down-regulation were considered as differentially expressed at a p-value < 0.05, Student's t-test. Functional classification of the genes was performed using gene ontology and pathway analysis.-ΔΔCT. Primer sequences for the genes of interest were designed using Primer Express 2.0 software . The primer sequences used in this study are as follows: JUN FP 5' TCGACATGGAGTCCCAGGA 3'; JUN RP 5' GGCGATTCTCTCCAGCTTCC 3'; STAT1 FP 5' CCATCCTTTGGTACAACATGC 3'; STAT1 RP 5'TGCACATGGTGGAGTCAGG 3'; CXCL10 FP 5' TTCAAGGAGTACCTCTCTCTAG 3'; CXCL10 RP 5' CTGGATTCAGACATCTCTTCTC 3'; CCL5 FP 5'TACCATGAAGGTCTCCGC 3'; CCL5 RP 5'GACAAAGACGACTGCTGG 3'; β-ACTIN FP 5' CATGAAGTGTGACGTGGACATCC 3'; β- ACTIN RP 5' GCTGATCCACATCTGGAAGG 3'; BCL2 FP 5' GATGTCCAGCCAGCTGCACCTG 3'; BCL2 RP 5' CACAAAGGCATCCCAGCCTCC 3'.The differential expression data was validated by quantitative RT-PCR. One hundred nanograms of total RNA from control and infected A549 cells were used for quantitative RT-PCR analysis. Reaction was performed using the QuantiTect SYBR Green RT-PCR kit  according to the manufacturer's instructions. Reaction efficiency was calculated by using serial 10-fold dilutions of the housekeeping gene- β-actin and sample genes. Reactions were carried out on an ABI 7300 real-time PCR system  and the thermal profile used was Stage 1: 50°C for 30 min; Stage 2: 95°C for 15 min; Stage 3: 94°C for 15 sec, 55°C for 30 sec; and 72°C for 30 sec, repeated for 30 cycles. Melting curve analysis was performed to verify product specificity. Reactions were performed in triplicates. All quantitations  were normalized to that of β-Actin to generate ΔCT, and the difference between the ΔCT value of the sample and that of the reference (uninfected sample) was calculated as ΔΔCT. The relative level of gene expression was expressed as 2The number of differentially expressed genes at different time-points after WB-NIV2664 infection is given in Table Cluster analysis of the differentially expressed genes was carried out using GENOWIZ software. K-means clustering and hierarchical clustering methods resulted in identification of 5 distinct patterns of gene expression at different time-points Figure . A totalThe differentially expressed genes in cells infected with RG modified H5N1 (IBCDC-RG7) virus were involved in similar biological processes  as in response to H5N1 WB-NIV2664) virus  virus infection but decrease in the expression of same genes in infection with RG modified H5N1 (IBCDC-RG7) at 16 hpi. These cytokines which mainly included IL2, IL1, CXCL10 and RANTES were reported to be involved in cytokine-storm in response to viral infection in humans ,19 and tIn spite of overall similar cellular responses in both the infections, WB-NIV2664 was found to be a compelling inducer of cytokines CXCL10 and RANTES than IBCDC-RG-7. This differential expression of cytokines could result in a totally different host cellular response to both the virus infections. A hypothetical model showing probable cellular response to HPAI-H5N1 infection has been shown in figure Differential expression of STAT1 in the two virus infections observed in our study also indicate higher cytokine mediated inflammatory responses in WB-NIV2664 infection than the modified strain . IRF-1 hCytokine dysregulation plays a major role in pathogenesis of influenza A H5N1) viruses N1 viruse,24 as weThe differences in the constitution of the internal genes between the subtypes of influenza viruses may possibly play an important role in differential host gene expression responses. Internal genes of H5N1 viruses, like non structural (NS1) and polymerase basic protein 2 (PB2) have been correlated with host immune responses and high pathogenicity ,25. MoreUp-regulation of apoptotic genes like BAX, BAK1 in H5N1 WB-NIV2664) but not in IBCDC-RG7 infection could be a part of cytokine mediated response 64 but no. NS1 proThus, our findings show that HPAI-H5N1 is a better inducer of inflammation and cytokine mediated apoptosis compared to the RG modified H5N1 at a very specific stage of infection (16 hpi) which could explain its high pathogenicity. This study highlights the role played by the viral factors in inducing host defense mechanism by modulating host gene expression response.HPAI: (highly pathogenic avian influenza virus); hpi: (hours post infection); aRNA: ; RG modified: (Reverse genetics modified); GO: (gene ontology)The authors declare that they have no competing interests.AKC and ACM conceived and designed the experiments. AKC, VCV, SM, RS and SDP performed the experiments. AKC, VCV, SM performed data analysis and bioinformatics studies. AKC, SM and ACM wrote the paper. All authors read and approved the final manuscript.Table S1. List of significantly up-regulated and down-regulated genes in A549 cells infected with HPAI-H5N1 at different post-infection time points. Genes showing increase or decrease in expression by ≥ 1.5 folds  compared to controls at different post infection time points studied with HPAI-H5N1 have been enlisted.Click here for fileTable S2. List of significantly up- and down-regulated genes in A549 cell lines infected with RG modified H5N1 at different post-infection time points. Genes showing increase or decrease in expression by ≥ 1.5 folds  compared to controls at different post infection time points studied with RG modified H5N1 have been enlisted.Click here for fileFigure S1. Genes involved in chemokine & cytokine mediated signaling in highly-pathogenic and RG modified H5N1 infected A549 cell lines (16 h post infection time point). Red arrow indicates expression in highly-pathogenic H5N1 infected A549 cells and Blue arrow indicates expression in RG modified H5N1 infected A549 cells. Up- arrow indicates up-regulation and down-arrow indicates down-regulation.Click here for fileFigure S2. Genes involved in p53 signaling pathway in highly-pathogenic and RG modified H5N1 infected A549 cell lines (16 h post infection time point). Red arrow indicates expression in highly-pathogenic H5N1 infected A549 cells and Blue arrow indicates expression in RG modified H5N1 infected A549 cells. Up- arrow indicates up-regulation and down-arrow indicates down-regulation.Click here for file


Purpose: A comprehensive molecular analysis was conducted to identify prognostic and predictive markers for adjuvant S-1 chemotherapy in stage II/III Japanese gastric cancer (GC) patients and to evaluate their potential suitability for alternative cytotoxic or targeted drugs.Experimental Design: We investigated genetic polymorphisms of enzymes potentially involved in 5-fluoruracil (5-FU) metabolism as well as platinum resistance, previously identified genomic subtypes potentially predicting 5-FU benefit, and mRNA expression levels of receptor tyrosine kinases and KRAS as potential treatment targets in a single institution cohort of 252 stage II/III GC patients treated with or without S-1 after D2 gastrectomy.Results: 88% and 62% GC had a potentially 5-FU sensitive phenotype by SNP analyses of TS 3'UTR, and TS 5'UTR, respectively. 24%, 46%, 40%, 5%, and 44% GC had a potentially platinum sensitive phenotype by SNP analyses of GSTP1, ERCC1 rs11615, ERCC1 rs3212986, ERCC2, and XRCC1, respectively. High HER2, EGFR, FGFR2, or MET mRNA expression was observed in 49%, 66%, 72%, and 54% GC, respectively. High HER2 expression was the only significant prognosticator . High HER2 (p=0.031), low EGFR (p=0.124), high MET (p=0.165) RNA expression, and TS 5'UTR subtype 2R/2R, 2R/3C, or 3C (p=0.058) were significant independent predictors for S-1 resistance.Conclusions: The present study suggests that platinum-based or RTK targeted agents could be alternative treatment options for a substantial subgroup of Japanese GC patients currently treated with S-1. HER2, EGFR, MET, and TS 5'UTR SNP appear to be promising predictive markers for S-1 resistance warranting validation in an independent GC series. Gastric cancer (GC) is the fourth most prevalent cancer and the third leading cause of cancer-related death worldwide However, adjuvant S-1 chemotherapy does not seem to benefit all patients with stage II/III GC and severe or serious S-1 toxicity including anorexia, nausea, and diarrhea has been reported TS) DPYD) OPRT) ERCC1) Several previous GC studies have attempted to identify predictive or prognostic markers for S-1 chemotherapy. These studies included the investigation of protein expression of metabolic enzymes of 5-FU or folate such as thymidylate synthase  targeting therapy.The aim of the current study was (a) to conduct a comprehensive molecular analysis of potential prognostic and predictive factors for adjuvant S-1 chemotherapy and (b) to evaluate the potential applicability of alternative cytotoxic or targeted drugs in a series of Japanese stage II/III gastric cancer patients treated with or without adjuvant S-1. Our analyses covered a wide range of biomarkers previously suggested to be related to benefit from S-1, 5-FU or platinum based chemotherapy, such as gene polymorphisms of th edition of the TNM classification This study was approved by the institutional review board of the Kanagawa Cancer Center, Yokohama, Japan. Gastric cancer patients were selected from a prospective database of the Kanagawa Cancer Center, Department of Gastrointestinal Surgery, Yokohama, Japan, according to the following criteria: (i) histologically proven gastric adenocarcinoma, (ii) curative gastrectomy as primary treatment between June 2002 and March 2010 following Japanese gastric cancer treatment guidelines published in 2010 A total of 252 patients were selected for this study. A flow diagram of patients involved in each analysis step is presented in Figure Gastric cancer resection specimens were worked up according to the Japanese guidelines for gastric cancer DNA for polymorphism analyses was extracted from normal tissue  using the QIAamp DNA Micro kit  and quality controlled as described previously DPYD (A1627G7&IVS14+1G>A), OPRT (G638C), ERCC1 (C118T&C8092A), ERCC2 (K751Q) and XRCC1 (A399G) was designed using MassARRAY Online Design Tools (Sequenom) as described previously Genotype analysis was performed using the MassARRAY iPLEX system  according to manufacturer's instructions. A 7-plex assay interrogating GSTP1 (I105V) and TS 3'UTR (agttcat variant) genotyping was performed by Sanger sequencing. Purified pellets were dissolved in Hi-Di Formamide (Life Technologies) and analyzed using an ABI PRISM 3730 Genetic Analyzer (Life Technologies). Chromatograms were analyzed by SeqScape V2.5 (Life Technologies) and manual review.TS 5'UTR were investigated as described previously TS 5'UTR, the predominant alleles expected at TS 5'UTR are the 2R and 3R alleles. PCR products from 2R and 3R alleles differ by a single 28 bp repeat, which can be resolved using gel electrophoresis. To determine the TS 5' UTR 2R/3R genotypes, 10 µl of PCR product was purified by 4 µl of Exonuclease I (New England Biolabs) and Thermosensitive Shrimp Alkaline Phosphatase (Promega) mixture treatment. After Exo-TSAP purification, PCR products were digested with 1 µl HaeIII  followed by electrophoreses in 4% agarose/1xTBE gel.et alA set of 203 genes selected based upon the molecular gastric cancer signature published by Tan Tumors were classified based on their expression signature as G-INT/G-DIF using the nearest template prediction algorithm as described previously Associations between each biomarker and clinicopathological factors such as age, gender, location, tumor depth (pT), lymph node status (pN), distant metastasis (M), lymphatic channel invasion, blood vessel invasion, tumor size, macroscopic tumor type were examined in all patients. The analyses of the association between biomarker and histological subtype was restricted to those cases which were classified as either intestinal type or diffuse type GC. GC which were classified as mixed/unclassifiable according to Lauren (n=13) were excluded. Continuous factors  were analyzed by student t-test, and categorical factors were analyzed by chi-square test. For analyses using distinct SNP subtypes, we excluded minor subtypes where the number of patients associated with a particular genotype (GG of GSTP1 in 5 patients and TT of ERCC1 rs3212986-C8092A in 10 patients) was felt to be too small for reliable statistical analysis. ERCC2 SNP was not analyzed for statistical analysis because most patients showed uniformal SNP type of TT (94.9%).For survival analyses, the gene expression level cut off (high expression versus low expression) was determined by the maximum chi-square method For the test of predictive factors, the interaction of each biomarker with S-1 treatment in the Cox's proportional hazard model was analyzed in the surgery alone and the surgery+S-1 arm. As this was a post hoc analysis that may not have enough power to detect an interaction effect, a raised significance level of p<0.2 was used to classify an interaction as being significant Table TS 3'UTR (+6bp/-6bp and -6bp/-6bp) and TS 5'UTR  were found in 88% and 62% GC, respectively. SNP phenotypes suggested to predict 5-FU toxicity e.g. OPRT (GC+CC) and DPYD (AG+GG) were present in 36% and 48% GC, respectively, while those suggested to predict platinum sensitivity e.g. GSTP1 (AG+GG), ERCC1 rs11615 (CT+TT), ERCC1 rs3212986 (GT+TT), ERCC2 variant (GT), and XRCC1 rs25487 (AG+GG) were present in 24%, 46%, 40%, 5%, and 44% GC, respectively. High HER2, EGFR, FGFR2, or MET RNA expression identifying GC patients potentially benefitting from targeted therapy, was observed in 49%, 66%, 72%, and 54% GC, respectively.SNP genotypes suggested to predict 5-FU resistance e.g. DPYD2A was associated with the presence of distant metastasis (p=0.040). GT type of ERCC1 and AA type of XRCC1 were associated with distal location of the cancer (p=0.018) and female gender (p=0.043), respectively. The [+6bp/-6bp] type of TS 3'UTR and [2R/3C] or [3C/3C] type of TS 5'UTR were associated with younger age . No other associations between individual SNP biomarker status and clinicopathological factors were seen.EGFR and high FGFR2 RNA expression were associated with younger age . High EGFR, low HER2 and high FGFR2 RNA expression were associated with diffuse type histology . High FGFR2 was associated with increased frequency of vascular invasion (p=0.047), high MET with distal location of the cancer (p=0.012) and low KRAS with presence of distant metastasis (p=0.022).Second, we examined associations between clinicopathological factors and gene expression, either at the single gene or gene signature level. High 125 patients (51%) were classified as G-INT and 120 patients (49%) as G-DIFF when the two-category classification was employed. When the three-category classification was used, 88 (35.9%) patients were classified as G-INT, 71 (29%) patients as G-DIFF, and 86 (35.1%) patients as G-ambiguous. G-DIFF was associated with higher pT category (e.g. deeper invasion of the primary tumor) compared to G-INT or G-ambiguous type.HER2 RNA expression was associated with poor survival and was the only significant independent prognosticator . Patients with TS 5'UTR 2R/3G or 3C/3G or 3G/3G tended to have poorer survival than those with 2R/2R, 2R/3C, or 3C/3C  but the difference did not reach statistical significance.In order to distinguish between predictive and prognostic value of a biomarker, associations with survival were analysed separately by treatment e.g. the prognostic value of a marker was determined using data from the surgery alone treated patient group. For multivariate overall survival analyses, TNM stage, tumor size and macroscopic tumor type were included in the model together with the biomarker of interest. The results of the survival analyses are illustrated using a Forest plot of hazard ratios Figure . High HETS 5'UTR subtype 2R/2R, 2R/3C, or 3C/3C (p=0.058), high RNA expression of HER2 (p=0.031), low EGFR expression (p=0.124) and high MET expression (p=0.165), were significant independent predictors for S-1 resistance.To identify biomarkers that might predict which GC patient subgroup will benefit from adjuvant S-1 therapy, we determined the interaction of each biomarker with S-1 treatment using a Cox's proportional model by including S-1 treatment, TNM stage, tumor size, macroscopical tumor type, and biomarker in the model. The results are illustrated in a Forest plot of hazard ratios Figure . We founOPRTDPYDTSGSTP-1, ERCC1, and XRCC1, (iii) previously identified GC genomic subtypes  genetic polymorphisms of enzymes involved in 5-FU metabolism such as OPRT and DPYD SNPs and S-1 treatment benefit, only the TS 5'UTR SNP predicted benefit from S-1 adjuvant chemotherapy. This is in contrast to previous studies which suggested that protein, mRNA, or genetic polymorphism of TS Our study showed no relationship between TS gene is polymorphic, containing a double (2R) or triple (3R) tandem repeat known to be involved in the translational auto-regulation mechanism of TS expression TS polymorphisms were related with better survival in colorectal and gastric cancer patients receiving fluoropyrimidine TS polymorphism and S-1 efficacy. The present study was able to clarify that TS 5'UTR polymorphisms have no prognostic value in Japanese gastric cancer patients  but does predict benefit from adjuvant S-1 treatment depending on the SNP subtype of TS 5'UTR. This finding, if replicated in a second independent series, could indicate that determination of the TS 5'UTR SNP status which would be possible in any normal tissue including a routine blood sample might be a useful marker to stratify patients for adjuvant S-1 therapy and/or other therapy options.TS is an enzyme which catalyzes the methylation of fluoro-dUMP to dTMP, an essential precursor for DNA synthesis It has been shown recently that classifying gastric cancer based on their RNA expression signature into G-INT and G-DIFF identifies groups of patients with different prognosis GSTP-1, ERCC1 and XRCC1 have been previously identified as potential predictive biomarker for platinum based chemotherapy in colon cancer patients ERCC1 SNPs have shown to have prognostic and/or predictive value in the advanced (metastatic) gastric cancer setting GSTP-1, ERCC1, and XRCC1 were neither a prognosticator nor a predictor for S-1 adjuvant chemotherapy suggesting that adding platinum drugs to S-1 might improve patient's prognosis.In addition to the investigations into prognostic and predictive marker for S-1 benefit, we also used this gastric cancer cohort to explore whether there is any indication that patients might benefit from potential other cytotoxic drugs such as platinum based chemotherapy. Polymorphisms for HER2 RNA expression was associated with poor prognosis and low EGFR, high HER2 and high MET RNA expression were important predictor for S-1 resistance.Similarly, we explored whether patients in our series would potentially be eligible for RTK targeting drug therapy such as HER2 targeting trastuzumab, pertuzumab or T-DM1; EGFR targeting nimotuzumab, FGFR2 targeting AZD4547 or MET targeting rilotumumab or onartuzumab or MEK inhibitors which may be attractive therapies for KRAS-driven tumors. The present study demonstrated that high et al reported a low correlation between HER2 protein expression and RNA expression and suggested that HER2 protein and RNA may have a different clinical role in gastric cancer patients Our data seem to indicate that whereas therapy targeting HER2 or MET should not be combined with S-1 chemotherapy, a combination of EGFR targeting drugs with S-1 chemotherapy might be of added value. However, a recent study in a subset of patients from the ACTS-GC trial The present study has some limitations. This is a retrospective, single center case-control study with no treatment randomization. Thus, in contrast to the results from the ACTS-GC trial, the univariate overall survival curves were similar between the S-1 group and surgery alone group in the current study. However, the hazard ratio was almost identical to that observed in S-1 arm of ACTS phase III study when the survival was adjusted by Cox's multivariate analysis. In addition, the present study was only analyzed the Eastern cohort. Therefore, the further study should focus on the Western cohort. Furthermore, our study results require validation in a second independent cohort of gastric cancer patients treated with adjuvant S-1 which was not achievable within the current study cohort due to its sample size.HER2, EGFR, MET RNA expression and TS 5'UTR SNP which are able to identify patients with a high risk of recurrence and/or no potential benefit from adjuvant S-1 therapy. The findings of this study may potentially inform future trial design to test platinum drugs and targeting agents after biomarker stratification in order to improve survival of Japanese stage II/III gastric cancer. The promising results from this comprehensive but exploratory biomarker analyses require validation in a second series before clinical application.In conclusion, this is the first comprehensive biomarker analysis comparing biomarker status in stage II/III gastric cancer patients treated with surgery alone to those treated with surgery plus adjuvant S-1 chemotherapy. We identified several candidate markers such as


UBE2A (RAD6A) and UBE2B (RAD6B), in humans, we compared their expressions in melanomas and normal melanocytes. While both genes are weakly expressed in normal melanocytes, Rad6B is more robustly expressed in melanoma lines and patient-derived metastatic melanomas than RAD6A. The characterization of RAD6B transcripts revealed coexpression of various splice variants representing truncated or modified functional versions of wild-type RAD6B in melanomas, but not in normal melanocytes. Notably, two RAD6B isoforms with intact catalytic domains, RAD6BΔexon4 and RAD6Bintron5ins, were identified. We confirmed that RAD6BΔexon4 and RAD6Bintron5ins variants are expressed as 14 and 15 kDa proteins, respectively, with functional in vivo ubiquitin conjugating activity. Whole exome sequence analysis of 30 patient-derived melanomas showed RAD6B variants coexpressed with wild-type RAD6B in all samples analyzed, and RAD6Bintron5ins variants were found in half the cases. These variants constitute the majority of the RAD6B transcriptome in contrast to RAD6A, which was predominantly wild-type. The expression of functional RAD6B variants only in melanomas reveals RAD6B’s molecular heterogeneity and its association with melanoma pathogenesis.Rad6B, a principal component of the translesion synthesis pathway, and activator of canonical Wnt signaling, plays an essential role in cutaneous melanoma development and progression. As Rad6 is encoded by two genes, namely,  B-RAF, NF1 and N-RAS, and melanomas associated with chronically sun-exposed skin show a high UV signature bearing mutation load [Cutaneous melanoma is the most predominant and aggressive form of skin cancer and has the highest burden of somatic genetic alterations. The main genetic drivers are ion load ,3,4. Repion load . Inactivion load . Activation load ,9,10,11.ion load .UBE2A (RAD6A) and UBE2B (RAD6B), which are located on chromosomes Xq24-q25 and 5q23-q34, respectively [RAD6B transcription [In humans, RAD6 is encoded by two genes, ectively ,13,14, aectively ,16,17 anectively ,19 wherecription ,19. Inducription . RAD6 excription . RAD6 isChanges in transcriptome frequently arise from alternative splicing abnormalities in tumors . Dysregu2. Malignant melanoma 14–089 was negative for BRAF V600E and V600K, and 14–108 was positive for BRAF V600E. Acquisition and use of clinical samples were approved by the Wayne State University Institutional Review Board and written informed consent was obtained from each patient prior to enrollment . Patient-derived xenografts were established from 30 primary and/or metastatic melanomas by Champions Oncology, Inc.  with written informed consent and approval by the Institutional Animal Care and Use Committee [Normal human epidermal melanocytes HEMa-LP, and also human melanoma A375 and A2058 cells, were purchased from American Type Culture Collection . Human melanoma M14 cells were obtained from the National Cancer Institute, Bethesda, MD 20892, USA. HEMa-LP cells were maintained in dermal cell basal medium (ATCC) supplemented with the melanocyte growth supplements insulin (5 μg/mL), ascorbic acid (50 μg/mL), L-glutamine (6 mmol/L), epinephrine (1.0 μmol/L), calcium chloride (0.2 mmol/L) and M8 supplement (ATCC). The authenticated cell lines were used within 5–10 passages. Patient-derived metastatic melanoma cell lines 14-089 and 14-108 were generated by dissociation of metastatic brain tumors into single cell suspensions using the GentleMACs Dissociator and Human Tumor Kit  according to the manufacturer’s protocol. The resulting cultures were grown in Dulbecco’s Modified Eagle Medium (DMEM)/F12 media supplemented with 10% fetal bovine serum, non-essential amino acids and gentamicin  at 37 °C, 5% COommittee . DetailsTotal RNA was isolated from normal melanocytes and melanoma lines using Trizol reagent . cDNAs were synthesized from 0.75–2 μg of total RNA using Superscript III (Invitrogen), and full-length RAD6B was PCR amplified using forward 5′-TTCAGACTGACCGCGGGGCA-3′ and reverse 5′-AGATTAACAGACCAGTTGTC-3′ primers (Accession # NM_003337). RAD6A was PCR amplified using forward 5′-GGATGGAACATTTAAACTTAC-3′ and reverse 5′-TGCTGGACTATTGGGATTG-3′ primers (Accession # NM_003336), and GAPDH with forward 5′-AAATATGATGACACCAAGAAGG-3′ and reverse 5′-TGAAGTCGGAGGAGACCAC-3′ primers (Accession # NM_002046). RAD6B PCR products were subcloned into the pCR2.1-TOPO vector , and plasmid DNAs purified from transformed colonies were subjected to EcoRI digestion to release the insert. Two to four clones displaying the correct wild-type RAD6B size and all clones showing size variations from the wild-type RAD6B transcript were sequenced in both directions using vector-specific primers.RAD6B splice mutants (RAD6BΔexon4 and RAD6Bintron5ins) were subcloned into the EcoRI site of pCIneo vector , and sequences were confirmed by DNA sequencing. Full-length wild-type (WT) RAD6B-pCMVneo (1–2.5 μg)  and 3–12Whole cell lysates of normal melanocytes, melanoma lines, patient-derived melanoma brain metastases, and RAD6B-transfected cells were prepared as previously described . AliquotHomology models of the RAD6BΔexon4 and RAD6Bintron5ins variants were created using Swiss-Model from PDB file 2YB6 for RAD6. The models for each protein were exported as PDB files and imported into the Molecular Operating Environment (MOE) for additional analysis. Each model was treated with the QuickPrep function, which automatically corrects for common problems associated with structures in the PDB including missing atoms, geometry conflicts, inappropriate protonation and crystallographic artifacts. Each model was also aligned and superposed with the crystal structure of RAD18 (the cognate E3 ubiquitin ligase for RAD6) bound to RAD6B (2YBF) to examine the binding site of the E3 enzyme.RAD6A (UBE2A) and RAD6B (UBE2B) were extracted from TCGA cutaneous melanoma data through the UCSC Xena Functional Genomics Explorer  along with clinical and pathological information [Gene expression levels and copy number variations (gain) of ormation . Gene exUBE2A (RAD6A) and UBE2B (RAD6B) genes and isoform quantifications. The percentages of wild-type RAD6A and RAD6B relative to their corresponding variants were compared using the Wilcoxon test for paired samples.Whole exome sequencing of early passage patient-derived xenografts (PDX) established from 30 primary/metastatic melanomas was performed by Champions Oncology . WES anaRAD6A and RAD6B localized on chromosomes Xq24–25 and 5q23–31, respectively, encode for RAD6 proteins with ~95% amino acid sequence identity, we performed TCGA analysis to compare RAD6A and RAD6B expression levels and copy number alterations in melanoma patients. No significant differences in expression levels and copy number alterations for RAD6A and RAD6B were observed in a sample population of 474 and 472 melanoma patients, respectively  and encompasses six relatively small exons that are 464, 81, 26, 90, 89, and 1884 bp, separated by introns of 2743, 2205, 4025, 7518 and 1811 bp, respectively, and it transcribes a full-length mRNA of 2634 bases (Accession No. NM_003337) (The _003337) Da. The w_003337) Da. To de_003337) Db–g. Seq_003337) Dc. Many _003337) Db–e. Int_003337) Db–g. Our_003337) Df. This _003337) Dg. Altho_003337) Dg.Translation of this RAD6Bintron5ins splice variant is predicted to produce a 138 amino acid catalytically active mutant RAD6B protein lacking exon 6 but containing an in-frame intron 5-derived 25 amino acid peptide at the C-terminus. Although transcripts for the RAD6BΔexon4 and RAD6Bintron5ins splice variants were not detectable in Mel-Met-14-108 and Mel-Met-14-089 patient-derived melanomas, besides the wild-type RAD6 protein, a 14 kDa RAD6 band corresponding to the size of the translated RAD6BΔexon4 transcript (based on its amino acid sequence) was detected by Western blotting in Mel-Met-14-108 patient samples C. The RATo determine whether RAD6BΔexon4 and RAD6Bintron5ins variants are indeed translated, we inserted them into pCI-neo expression vector under the control of the constitutive CMV promoter. RAD6BΔexon4, RAD6Bintron5ins, and WT RAD6B vectors were transiently transfected into 293T or COS7 cells, and 48 h later they were processed for RNA and protein analysis. RT-PCR analysis confirmed the capability and size of the RAD6BΔexon4-derived 423 base transcript in transfected cells as compared to the 513 base endogenous wild-type RAD6B mRNA in untransfected or control 293T or COS7 cells A. WesterRT-PCR analysis of RAD6Bintron5ins variant transfected COS7 cells confirmed the expression of the RAD6Bintron5ins-derived 589 base transcript A. Since The 3D structures of RAD6BΔexon4 and RAD6Bintron5ins variants were created by homology/template modeling using the PDB file 2YB6 for RAD6. In both cases, 50 templates were generated. For both proteins, the top five models were ubiquitin conjugating enzymes with sequence identities ranging from 96% to 100% for RAD6BΔexon4 and from 84% to 88% for RAD6Bintron5ins. The Global Model Quality Estimation (GMQE) was 0.98 for RAD6BΔexon4 and 0.85 for RAD6Bintron5ins, indicating that both models are predicted to be highly reliable. The deletion of exon 4 in RAD6B removes amino acids 51–80 A, which The binding site for RAD18, the cognate E3 ligase for RAD6, located at the carboxyl terminus of RAD6B is intact , althougThe intron 5 insertion replaces amino acids 111–151 from the wild-type RAD6B protein with CLALLPRLECNGTILAHRNLCLPGSISAG. A homology model was created, which ended at R128 with the last C-terminal 11 residues not modeled C. Numerop < 0.001; To determine whether RAD6B variant expressions reflect a general property of melanomas, we performed variant analysis of human RAD6A and RAD6B transcripts uncovered by WES analysis of 30 patient-derived melanoma xenografts . The perRAD6B than RAD6A in melanomas. These data are corroborated by WES analysis of patient-derived primary and metastatic melanoma xenografts, which not only showed co-expression of RAD6B variants with wild-type RAD6B but also showed that these variants constitute the majority of the RAD6B transcriptome in contrast to RAD6A, which was predominantly represented by the wild-type form. Genomic analysis carried out in tumors and their matched patient-derived xenografts showed a tight correlation, as 88% of the mutations identified in the parental tumors were found to be present in the corresponding xenografts, establishing the validity of the WES data [We have previously shown that RAD6 is an early marker of cutaneous melanoma development, as increases in its expression correlate with increases in melanocytic proliferation and the disruption of the normal melanocyte to keratinocyte ratio in the skin . RAD6 isWES data . The ideAlternative splicing of pre-mRNA allows a gene to encode many more products, thus increasing the heterogeneity of the transcript and potential modification of the wild-type protein. Our data show that the major mechanism of RAD6B splice variant generation utilized in melanoma involves exon skipping that is localized to exons 2, 3 or 4. Interestingly, splice variants involving exons 5 and 6 were not observed in any of the melanoma samples analyzed. The fact that the RAD6B alternative transcripts identified in melanoma cells are not unique to melanomas suggests that these variants result from nontrivial splicing events, some of which are nonfunctional at the mRNA or protein level, resulting in nonsense mediated mRNA decay, a translation-coupled mechanism that eliminates transcripts containing premature translation termination codons. In mammalian cells, nonsense mediated decay has also been linked to pre-mRNA splicing . The RADSpliceosomal errors resulting in the production of aberrantly spliced transcripts are rare in normal cells, whereas they appear to be an intrinsic property of tumor cells . AlteratRAD6B knockout male mice are sterile , indicatMutations in splice sites, polypyrimidine tract, branch point, and intronic or exonic enhancers and silencers can affect splicing . Our datWe have identified recurrent RAD6B isoforms in melanoma samples having at least two of them with intact catalytic function. Since these splice variants were not detected in normal melanocytes, we posit that mis-splicing may be a consequence of altered events during tumorigenesis and that the variant isoforms may directly contribute to melanoma development and/or progression. It is also possible that the splice variants are not detected in normal melanocytes because of the weak transcriptions of wild-type RAD6B, as high transcription rates have been reported to affect splicing fidelity . Since o