Expression of HBX, an oncoprotein of hepatitis B virus, blocks reoviral oncolysis of hepatocellular carcinoma cells.

Although reovirus has been used in tests as a potential cancer therapeutic agent against a variety of cancer cells, its application to hepatocellular carcinoma cells, in which the hepatitis B virus (HBV) X (HBX) protein of HBV plays a primary role, has not yet been explored. Here, we describe experiments in which we use reovirus to treat Chang liver carcinoma cells expressing either a vector only (Chang-vec) or a vector encoding HBX protein (Chang-HBX). Although Chang-vec cells readily support reoviral proliferation and undergo apoptosis, Chang-HBX cells are highly resistant to reoviral infection and virus-induced apoptosis, even though HBX protein induces activation of Ras and inactivation of PKR, which are normally thought to enhance reoviral oncolysis. The resistance of Chang-HBX cells to reovirus may instead be explained by HBX-induced downregulation of death receptor 5 and activation of Stat1. Phosphorylated Stat1 activates interferon (IFN)-stimulated regulatory element (ISRE)- and IFN-gamma-activated sequence (GAS)-mediated transcription, leading to the production of IFN-beta, whereas the reduced expression of Stat1 with its siRNA results in a decrease in IFN-beta production, by which Chang-HBX cells eventually succumb to reovirus infection. This result further indicates that HBX induces the establishment of an antiviral state through Stat1 activation. Thus, it appears that active Ras does not override the antiviral effect mediated by the activation of Stat1. Accordingly, we report that HBX, an oncoprotein of HBV, can prevent reoviral oncolysis of hepatocellular carcinoma. This suggests there may be limits to the practical application of reovirus in the treatment of human cancers already expressing other oncoviral proteins.

Dual effects of hepatitis C virus Core protein on the transcription of cyclin-dependent kinase inhibitor p21 gene.

Transcription of p21 was activated in hepatitis C virus (HCV) Core-expressing HepG2 cells where its upstream p53 was stabilized. However, this effect was not absolutely required for the activation of p21 by Core, as demonstrated in Hep3B cells. In addition, an opposite effect on the transcription of p21 was observed in NIH3T3 and primary hepatocytes, where p53 was not decreased by Core. To explain the p53-independent regulation of p21 by Core, we identified a Core-responsive element between positions -74 and -83 of the p21 promoter, exactly overlapped with a tumour growth factor beta (TGF-beta)/butyrate responsive element. Furthermore, we demonstrated that Core could activate the p21 through the element by stimulating a butyrate pathway, whereas this was inhibited through a TGF-beta pathway. The opposing effects of Core protein on the transcription of p21 might be important in understanding the progression of hepatic disease in HCV-positive patients.

Bringing up bashful baby. Developmental pathways to social phobia.

Shyness is a risk factor for, or an early manifestation of, more enduring problems with social anxiety. But the majority of shy children do not develop social phobia, and factors that further increase risk are poorly understood, underscoring the complexity of this relationship. Studies uniformly show that social phobia (particularly the generalized subtype) runs in families, and twin studies suggest that a moderate component of this familial tendency is genetic in origin. Understanding the genetic etiology of other neuropsychiatric disorders characterized by abnormal social interest, social communication (e.g., autism), or both may prove informative for social phobia. The contribution of unique experiences to the development of social phobia is clear from genetic studies, but studies to date have failed to elucidate what kinds of experiences might be involved. Given patient reports that socially traumatic conditioning experiences have often occurred, detailed evaluation of these kinds of experiences in monozygotic twins discordant for social phobia would be a particularly informative research strategy. Nongenetic familial factors probably have more limited effects on the development of social phobia, although the impact of parental modeling of, and acquiescence to, childhood social fears deserves to be further investigated. These factors may be particularly salient for the expression of social phobia in children whose genes render them susceptible. If so, it should be possible to design early interventions to prevent the progression from phobia proneness (e.g., designated on the basis of family history) to phobic disorder.

Intra- and extra-familial influences on alcohol and drug misuse: a twin study of gene-environment correlation.

AIMS: Genotype-environment correlation refers to the extent to which individuals are exposed to environments as a function of their genetic propensities. These correlations are important in the study of psychopathology because they identify environments that may maintain the expression of underlying genetic liabilities for a disorder. The present study examined the correlation between genetic liabilities for alcohol and drug misuse with perceptions of the social environments of the family of origin and the classroom. DESIGN: Postal survey data were collected from monozygotic and dizygotic twin pairs. SETTING: Twin pairs were recruited from Vancouver, British Columbia, Canada using newspaper advertisements and media stories. PARTICIPANTS: Eighty-five monozygotic and 77 dizygotic twin pairs were recruited from the general population. MEASUREMENTS: Twin pairs completed self-report measures of alcohol and drug misuse contained in the Dimensional Assessment of Personality Pathology, the Family Environment Scale, the Classroom Environment Scale, and the Traumatic Events Questionnaire. FINDINGS: Genetically indexed alcohol and drug misuse scores were regressed on the environmentally indexed FES and CES scales. Genetic liabilities for alcohol and drug misuse were associated with decreased perceived family moral-religious emphases, family cohesion and classroom task orientation and increased perceptions of classroom order and organization (strictness). CONCLUSIONS: Genotype-environment correlations, in particular, moral-religious emphases in the home, appear to be important in the development of substance misuse.

Transcriptional repression of p21(waf1) promoter by hepatitis B virus X protein via a p53-independent pathway.

The X-gene product of hepatitis B virus (HBx) has been implicated in hepatitis B virus (HBV)-mediated hepatocellular carcinoma through its ability to induce liver cancer in some transgenic mice and to transactivate a variety of viral and cellular promoters. In this study, we demonstrated that the level of p21(waf1) RNA was decreased in the HBx-expressing cells and this effect was due to the transcriptional repression of the p21(waf1) gene by HBx via a p53-independent pathway. As the Sp1 binding sites of the p21(waf1) promoter were sufficient to confer HBx responsiveness to a previously non-responsive promoter, we suggested that HBx represses the transcription of p21(waf1) by downregulating the activity of Sp1. Because the tumor repressor p21(waf1) protein is a universal inhibitor of cyclin-CDK complexes and DNA replication that induces cell cycle arrest at the G1-S checkpoint, the repression of p21(waf1) by HBx might play an important role in a HBV-mediated pathogenesis.

The repressive activity of hepatitis C virus core protein on the transcription of p21(waf1) is regulated by protein kinase A-mediated phosphorylation.

Hepatitis C virus (HCV) core protein is known to repress the transcription of p21(waf1) directly in a p53-independent manner. In this study, the region of HCV core protein responsible for the transcriptional repression of p21 promoter was determined. N-terminal half of core protein almost completely lost the ability to repress p21 promoter, indicating that the domain required for the majority of p21 repression is located between amino acid positions 84 and 191. The trans-repression activity of HCV core mutant S99L on p21 gene expression was similar to that of wild type core protein whereas mutation of the 116th amino acid Ser into either Ile or Ala completely abolished the repressive ability of HCV core protein. In addition, the trans-repression activity of HCV core mutant S116D was similar to that of wild type core protein, suggesting that an acidic aspartate residue can mimic the effect of phosphorylation. When treated with a protein kinase A (PKA) inhibitor, H-89, the inhibitory activity of wild-type HCV core protein was dose-dependently decreased and was completely lost at the concentration of 5 microM. On the contrary, the repression activity of HCV core protein was increased by treatment with a PKA activator, dibutyryl-cAMP, indicating that the p21 repressive activity of HCV core protein is regulated by phosphorylation at S-116 by protein PKA

Sources of structure: genetic, environmental, and artifactual influences on the covariation of personality traits.

The phenotypic structure of personality traits has been well described, but it has not yet been explained causally. Behavior genetic covariance analyses can identify the underlying causes of phenotypic structure; previous behavior genetic research has suggested that the effects from both genetic and nonshared environmental influences mirror the phenotype. However, nonshared environmental effects are usually estimated as a residualterm that may also include systematic bias, such as that introduced by implicit personality theory. To reduce that bias, we supplemented data from Canadian and German twin studies with cross-observer correlations on the Revised NEO Personality Inventory. The hypothesized five-factor structure was found in both the phenotypic and genetic/familial covariances. When the residual covariance was decomposed into true nonshared environmental influences and method bias, only the latter showed the five-factor structure. True nonshared environmental influences are not structured as genetic influences are, although there was some suggestion that they do affect two personality dimensions, Conscientiousness and Love. These data reaffirm the value of behavior genetic analyses for research on the underlying causes of personality traits.

Covariance structure of neuroticism and agreeableness: a twin and molecular genetic analysis of the role of the serotonin transporter gene.

The Revised NEO Personality Inventory domains of Neuroticism and Agreeableness are considered factorially distinct despite several intercorrelations between these domains. The genetic correlation, an index of the degree to which these intercorrelations are caused by genetic influences, was estimated using data from 913 monozygotic and 562 dizygotic volunteer twin pairs from Canada, Germany, and Japan. The serotonin transporter gene, 5-HTTLPR, was assayed in a sample of 388 nontwin sibling pairs from the United States to determine the contribution of the serotonin transporter locus to the covariation between the Neuroticism and Agreeableness scales. In all four samples, genetic influences contributed to the covariance of Neuroticism and Agreeableness, with the serotonin transporter gene accounting for 10% of the relationship between these domains.

The heritability of attitudes: a study of twins.

The genetic basis of individual differences in attitudes was examined in a survey of 195 pairs of monozygotic twins and 141 pairs of same-sex dizygotic twins. A principal components analysis of the 30 attitude items in the survey identified 9 attitude factors, of which 6 yielded significant heritability coefficients. Nonshared environmental factors accounted for the most variance in the attitude factors. Possible mediators of attitude heritability were also assessed, including personality traits, physical characteristics, and academic achievement. Analyses showed that several of these possible mediators correlated at a genetic level with the heritable attitude factors, suggesting that the heritability of the mediator variables might account for part of the heritable components of some attitudes. There was also some evidence that highly heritable attitudes were psychologically "stronger" than less heritable attitudes.

Familial aggregation of anxiety-related quantitative traits in generalized social phobia: clues to understanding "disorder" heritability?

Social phobia, particularly the generalized form, is strongly familial. Given the probable continuity from extremes of normative personality (e.g., shyness) to social phobia to personality disorder (e.g., avoidant personality disorder), it is unlikely that social phobia itself, at the level of an Axis I disorder, is transmitted. Rather, it seems more parsimonious, and in keeping with current notions about the structure and heritability of mental disorders, that one or more temperamental risk factors for social phobia is transmitted. The goal of this study was to explore this possibility by examining relevant quantitative traits in a family study of generalized social phobia (GSP). First-degree (n = 103) relatives of patients with DSM-IV GSP and 65 first-degree relatives of not socially phobic comparison subjects (NC) completed a panel of self-report questionnaires that included measures of trait anxiety, social anxiety, and personality. Regression analysis was used to examine associations between group membership (i.e., whether that family member was a first-degree relative of a GSP proband) and these measures. First-degree relatives of GSP probands scored significantly higher than first-degree relatives of not socially phobic probands on measures of trait anxiety and social anxiety and on the Harm Avoidance subscale of the TPQ. One large factor, accounting for 84% of the variance, was strongly associated with being a first-degree relative of a GSP proband. Quantitative traits elevated in probands with generalized social phobia are also elevated in their first-degree relatives. Future family and genetic studies of social phobia should consider the possibility that one or more traits (or some aggregation thereof) may better approximate the phenotype of interest. More extensive efforts at phenotype refinement should be undertaken before such studies proceed.

Behavioural-genetic perspectives on personality function.

In the wake of the recent announcements that the human genome has been mapped, efforts to identify the genetic loci underlying personality function will grow and intensify. Much research has already been done in this area, but it has for the most part been limited to classical biometrical approaches designed to determine if personality has a heritable basis. These so-called "heritability" studies estimate how much of the individual differences in personality are attributable to genetic differences among people. Molecular-genetic approaches, on the other hand, are designed to identify specific putative loci, but have yielded mixed results. The inconsistency in research findings can be attributed in part to the lack of sufficient numbers of genetic markers in the chromosomal regions of interest--a problem that the creation of a map of the human genome will help to rectify. This map and its inevitable refinements, however, can only advance the search for the genes for personality to a limited degree. Serious unresolved problems in the conceptualization and definition of personality and its dysfunction remain, which will hamper the search for personality genes.

Heritability of personality disorders in childhood: a preliminary investigation.

The heritability of personality disorder features was investigated in 112 child (ages 4-15 years) twin pairs (70 monozygotic and 42 dizygotic pairs). Parents assessed personality disorder features using the Coolidge Personality and Neuropsychological Inventory for Children (CPNI; Coolidge, 1998) that measures 12 personality disorders according to the criteria in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) (American Psychiatric Association, 1994). Structural equation model-fitting methods indicated that the median heritability coefficient for the 12 scales was .75 (ranging from .81 for the Dependent and Schizotypal Personality Disorder scales to .50 for the Paranoid and Passive-Aggressive Personality Disorder scales). These results suggest that childhood personality disorders have a substantial genetic component and that they are similar to heritability estimates of personality disorder traits in adults and counter hypotheses that only temperaments and higher-order personality disorder traits have significant genetic components (Paris, 1997).

Causal modeling of relations among learning history, anxiety sensitivity, and panic attacks.

We used structural equation modeling (SEM) to test the hypothesis that childhood instrumental and vicarious learning experiences influence frequency of panic attacks in young adulthood both directly, and indirectly through their effects on anxiety sensitivity (AS). A total of 478 university students participated in a retrospective assessment of their childhood learning experiences for arousal-reactive sensations (e.g., nausea, racing heart, shortness of breath, dizziness) and arousal-non-reactive sensations (i.e., colds, aches and pains, and rashes). SEM revealed that learning history for arousal-reactive somatic symptoms directly influenced both AS levels and panic frequency; AS directly influenced panic frequency; and learning history for arousal-non-reactive symptoms directly influenced AS but did not directly influence panic frequency. These results are consistent with the findings of previous retrospective studies on the learning history origins of AS and panic attacks, and provide the first empirical evidence of a partial mediation effect of AS in explaining the relation between childhood learning experiences and panic attacks in young adulthood. Implications for understanding the etiology of panic disorder are discussed.

Personality disorder traits, family environment, and alcohol misuse: a multivariate behavioural genetic analysis.

AIMS: This study seeks to estimate the extent to which a common genetic and environmental basis is shared between (i) traits delineating specific aspects of antisocial personality and alcohol misuse, and (ii) childhood family environments, traits delineating broad domains of personality pathology and alcohol misuse. DESIGN: Postal survey data were collected from monozygotic and dizygotic twin pairs. SETTING: Twin pairs were recruited from Vancouver, British Columbia and London, Ontario, Canada using newspaper advertisements, media stories and twin clubs. PARTICIPANTS: Data obtained from 324 monozygotic and 335 dizygotic twin pairs were used to estimate the extent to which traits delineating specific antisocial personality traits and alcohol misuse shared a common genetic and environmental aetiology. Data from 81 monozygotic and 74 dizygotic twin pairs were used to estimate the degree to which traits delineating personality pathology, childhood family environment and alcohol misuse shared a common aetiology. MEASUREMENTS: Current alcohol misuse and personality pathology were measured using scales contained in the self-report Dimensional Assessment of Personality Pathology. Perceptions of childhood family environment were measured using the self-report Family Environment Scale. FINDINGS: Multivariate genetic analyses showed that a subset of traits delineating components of antisocial personality (i.e. grandiosity, attention-seeking, failure to adopt social norms, interpersonal violence and juvenile antisocial behaviours) are influenced by genetic factors in common to alcohol misuse. Genetically based perceptions of childhood family environment had little relationship with alcohol misuse. CONCLUSIONS: Heritable personality factors that influence the perception of childhood family environment play only a small role in the liability to alcohol misuse. Instead, liability to alcohol misuse is related to genetic factors common a specific subset of antisocial personality traits describing conduct problems, narcissistic and stimulus-seeking behaviour.

Toward an empirically based classification of personality disorder.

A framework for an empirically based classification of personality disorder is proposed that has two components: (a) a definition of personality disorder, and (b) a scheme for describing individual differences in personality disorder traits. It is suggested that the diagnosis process should begin by establishing the presence of personality disorder and then proceed to a description of the personality on a set of trait dimensions. It is argued that a definition of personality disorder should reflect an understanding of the nature of the "harmful dysfunction" implied by a diagnosis of personality disorder. With this approach, personality disorder is defined as the failure to solve life tasks involving the development of integrated representations of self and others, and the capacity for adaptive kinship and societal relationships. The second component of a classification is a system to describe individual differences. It is suggested that these should be based on taxonomies of normal and disordered traits, and that the classification incorporates both higher-order patterns and more specific basic traits. Given that personality appears to be inherited as a large number of genetic dimensions, it is suggested that the primary level for describing individual differences is that of the basic or lower-level traits rather than broader or higher-level traits used in descriptions of normal personality.

Transcriptional regulation of herpes simplex virus type 1 ICP0 promoter by virion protein 16.

HSV regulatory proteins ICP0 and VP16 independently regulate transcription of the ICP0 gene during virus infection. In this study, we tried to determine the possible regulatory mechanism of ICP0 expression during virus infection. Among eight putative VP16 binding sites present in the ICP0 regulatory sequence, the most upstream one alone was sufficiently responsive to VP16-mediated activation. When the G/C-rich sequence present in front of the last TAATGARAT sequence of the ICP0 promoter was either deleted or point mutated, the activational effect of VP16 on the promoter was completely abolished. Furthermore, according to the gel mobility shift assay using a labeled double-stranded oligonucleotide derived from the G/C-rich sequence in the ICP0 promoter, specific protein binding to the probe was clearly demonstrated and was approximately fivefold upregulated by HSV-1 infection. Therefore, the G/C-rich sequence might play a critical role in VP16-mediated activation of the ICP0 promoter and the effect may be a result of the enhanced binding of a protein to the G/C-rich sequence during virus infection.

Nucleotide sequence and phylogenetic analysis of long terminal repeats of human endogenous retrovirus K family (HERV-K) on human chromosomes.

It has been suggested that human endogenous retroviruses K family (HERV-K) has a role in disease, and solitary long terminal repeats (LTRs) of HERV-K have been potentially capable of affecting the expression of closely located genes. Using the human monochromosomes 8, 9, 17, and 18, with specific PCR primers, we identified thirty-four sequences of new HERV-K LTRs. Those LTR elements were analyzed phylogenetically with the human-specific HERV-K LTRs using neighbor-joining and maximum parsimony methods. Clones HKL8-5, HKL9-5, and HKL9-8 are related by more than 99% homology with the human-specific HERV-K LTRs. The HKL9-5 clone on chromosome 9 was 100% identical with the sequences of human-specific LTR, AC002400, on chromosome 16. The findings suggest that there has been recent proliferation, transposition, or chromosomal translocation of HERV-K LTR elements on human chromosomes.

A novel function of IL-12p40 as a chemotactic molecule for macrophages.

IL-12p70 plays a pivotal role in regulating the Th1/Th2 balance in the initial stage of immune responses. In contrast, IL-12p40, which is produced excess over IL-12p70, has been known to down-regulate IL-12p70-mediated responses by acting as an antagonist. To investigate in vivo function of IL-12p40, RH7777 rat hepatoma cells were engineered to inducibly express mouse IL-12p40 under the tight control of doxycycline (dox). In the absence of dox, s.c. injection of these cells into syngeneic rat was shown to generate tumors. However, the induction of IL-12p40 by dox was sufficient for inhibiting tumor formation, as well as for tumor regression. Immunohistochemical analysis showed that macrophages, but not CD4+ T, CD8+ T, and NK cells, were predominantly recruited into tumor sites as early as 3 days after IL-12p40 induction. These results were further supported by the observation that IL-12p40, but not C-terminal deletion mutants by more than 5 amino acids, was able to chemoattract peritoneal macrophages in vitro, suggesting that IL-12p40, when produced in a large excess over IL-12p70 in vivo, can initially amplify the immune responses against tumors by directly recruiting macrophages. Our findings indicate that IL-12p40 may function as an effector molecule as well as an antagonist of IL-12p70.

Activation of the thymidine kinase promoter by herpes simplex virus type 1 immediate early proteins.

The herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene promoter contains binding sites for the cellular transcription factors such as Spl, CTF, and TFIID, each of which affects basal level expression of the TK gene. The transcription of the TK gene was induced by viral immediate early proteins, ICP0 and ICP4 in an additive manner, but was repressed by ICP22 and ICP27. To gain further insights into the role of ICP0 and ICP4 for expression of the TK gene during virus infection, several mutants with deletions or point mutations in each of the transcriptional regulatory elements were generated starting at -109 and progressing toward +1. According to the CAT assay involving these mutants, the cellular transcription factor (CTF) binding site was necessary for efficient expression in the presence of transfected ICP0 and ICP4 or during virus infection, whereas the Sp1 binding site had a minor effect on ICP0-mediated TK expression. These results indicate that the immediate early proteins of HSV-1 regulate expression of the TK gene during virus infection by modulating activities of cellular transcription factors such as CTF.