pX Expression Research Articles

The Effects Of Aerobic Exercise On Free Radical Expression In Hippocampus Of Aging Rats

PURPOSE: The effects of aerobic exercise on free radical expression in hippocampus of aging rats. METHODS: Eighty male SD rats were divided into 4 groups(n=20) at random: control group(C), D-galactose aging model group(A), pre-aging aerobic exercise intervention group (S1), aerobic exercise intervention on aging group (S2). Following a 1 week acclimation to laboratory conditions, D-gal were injected in D, S1 and S2 groups rat’s peritoneal to make aging model, the injection dosage via body weight of the rats, 100mg / kg, once a day for 6 weeks. Group C were injected the same dosage of saline. We did 1 hour per day, six times per week’s swimming training seprately before and during the injection of D-gal administration in group S1 and S2. At the end of modeling and swimming, Morris water maze was used to evaluate the spatial learning and memory function of rats; the activities of SOD, GSH PX and MDA expression in hippocampus of rats in each group were measured. All data were processed with one-way ANOVA, level of significance was set at α=0.05. RESULTS: (1) Compared with group C, group A had obvious symptoms of aging. (2)Water maze navigation trial showed that group C, S1 and S2 formed stable spatial learning and memory function on day 3 but that of group A formed on day 4; in the space exploration experiment group C reached the destination for most times, the percentage of the original site quadrant of group C was the highest, and there were significant differences between other groups (P<0.01); group S2 followed but group A and S1 were relatively low. (3) Morphological observation showed that the hippocampal neurons of rats in group A appeared derangement, deepening of cell staining and cytoplasmic edema. (4) The activities of SOD and GSH-Px in the hippocampus of group S2 were similar to group C, and the activities of group A and S1 were significantly lower than group C (P < 0.01); the expression levels of MDA were significantly down-regulated in group C, group S2 followed, and all significantly lower than those in group A and S1(P<0.05). CONCLUSIONS:Swimming in the process of aging can improve and maintain the spatial learning and memory function of brain and delay brain aging. The mechanism may be related to reduce free radical expression’s regulatio.

Polo-like Kinase 1 Activated by the Hepatitis B Virus X Protein Attenuates Both the DNA Damage Checkpoint and DNA Repair Resulting in Partial Polyploidy

Hepatitis B virus X protein (pX), implicated in hepatocarcinogenesis, induces DNA damage because of re-replication and allows propagation of damaged DNA, resulting in partial polyploidy and oncogenic transformation. The mechanism by which pX allows cells with DNA damage to continue proliferating is unknown. Herein, we show pX activates Polo-like kinase 1 (Plk1) in the G(2) phase, thereby attenuating the DNA damage checkpoint. Specifically, in the G(2) phase of pX-expressing cells, the checkpoint kinase Chk1 was inactive despite DNA damage, and protein levels of claspin, an adaptor of ataxia telangiectasia-mutated and Rad3-related protein-mediated Chk1 phosphorylation, were reduced. Pharmacologic inhibition or knockdown of Plk1 restored claspin protein levels, Chk1 activation, and p53 stabilization. Also, protein levels of DNA repair protein Mre11 were decreased in the G(2) phase of pX-expressing cells but not with Plk1 knockdown. Interestingly, in pX-expressing cells, Mre11 co-immunoprecipitated with transfected Plk1 Polo-box domain, and inhibition of Plk1 increased Mre11 stability in cycloheximide-treated cells. These results suggest that pX-activated Plk1 by down-regulating Mre11 attenuates DNA repair. Importantly, concurrent inhibition of Plk1, p53, and Mre11 increased the number of pX-expressing cells with DNA damage entering mitosis, relative to Plk1 inhibition alone. By contrast, inhibition or knockdown of Plk1 reduced pX-induced polyploidy while increasing apoptosis. We conclude Plk1, activated by pX, allows propagation of DNA damage by concurrently attenuating the DNA damage checkpoint and DNA repair, resulting in polyploidy. We propose this novel Plk1 mechanism initiates pX-mediated hepatocyte transformation.

Hepatitis B Virus X Protein Increases the Cdt1-to-Geminin Ratio Inducing DNA Re-replication and Polyploidy

Hepatitis B virus X protein (pX) is implicated in hepatocellular carcinoma pathogenesis by an unknown mechanism. Employing the tetracycline-regulated pX-expressing 4pX-1 cell line, derived from the murine AML12 hepatocyte cell line, we demonstrate that pX induces partial polyploidy (>4N DNA). Depletion of p53 in 4pX-1 cells increases by 5-fold the polyploid cells in response to pX expression, indicating that p53 antagonizes pX-induced polyploidy. Dual-parameter flow cytometric analyses show pX-dependent bromodeoxyuridine (BrdUrd) incorporation in 4pX-1 cells containing 4N and >4N DNA, suggesting pX induces DNA re-replication. Interestingly, pX increases expression of endogenous replication initiation factors Cdc6 and Cdtl while suppressing geminin expression, a negative regulator of rereplication. In comparison to a geminin knockdown 4pX-1 cell line used as DNA re-replication control, the Cdt1/geminin ratio is greater in 4pX-1 cells expressing pX, indicating that pX promotes DNA re-replication. In support of this conclusion, pX-expressing 4pX-1 cells, similar to the geminin knockdown 4pX-1 cells, continue to incorporate BrdUrd in the G2 phase and exhibit nuclear Cdc6 and MCM5 co-localization and the absence of geminin. In addition, pX expression activates the ATR kinase, the sensor of DNA re-replication, which in turn phosphorylates RAD17 and H2AX. Interestingly, phospho-H2AX-positive and BrdUrd -positive cells progress through mitosis, demonstrating a link between pX-induced DNA re-replication and polyploidy. Our studies high-light a novel function of pX that likely contributes to hepatocellular carcinoma pathogenesis.

Hepatitis B Virus X Protein via the p38MAPK Pathway Induces E2F1 Release and ATR Kinase Activation Mediating p53 Apoptosis

Hepatitis B virus (HBV) X protein (pX) is implicated in hepatocellular carcinoma (HCC) pathogenesis by an unknown mechanism. Deletions or mutations of genes involved in the p53 pathway are often associated with HBV-mediated HCC, indicating rescue from p53 apoptosis is a likely mechanism in HBV-HCC pathogenesis. Herein, we determined the mechanism by which pX sensitizes hepatocytes to p53-mediated apoptosis. Although it is well established that the Rb/E2F/ARF pathway stabilizes p53, and the DNA damage-activated ATM/ATR kinases activate p53, the mechanism that coordinates these two pathways has not been determined. We demonstrate that the p38MAPK pathway activated by pX serves this role in p53 apoptosis. Specifically, the activated p38MAPK pathway stabilizes p53 via E2F1-mediated ARF expression, and also activates the transcriptional function of p53 by activating ATR. Knockdown of p53, E2F1, ATR, or p38MAPKalpha abrogates pX-mediated apoptosis, demonstrating that E2F1, ATR, and p38MAPKalpha are all essential in p53 apoptosis in response to pX. Specifically, in response to pX expression, the p38MAPK pathway activates Cdk4 and Cdk2, leading to phosphorylation of Rb, release of E2F1, and transcription of ARF. The p38MAPK pathway also activates ATR, leading to phosphorylation of p53 on Ser-18 and Ser-23, transcription of pro-apoptotic genes Bax, Fas, and Noxa, and apoptosis. In conclusion, pX sensitizes hepatocytes to p53 apoptosis via activation of the p38MAPK pathway, which couples p53 stabilization and p53 activation, by E2F1 induction and ATR activation, respectively.

Role of Neuronal Interferon-γ in the Development of Myelopathy in Rats Infected with Human T-Cell Leukemia Virus Type 1

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of not only adult T-cell leukemia but also HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Among the rat strains infected with HTLV-1, chronic progressive myelopathy, named HAM rat disease, occurs exclusively in WKAH rats. In the present study, we found that HTLV-1 infection induces interferon (IFN)-gamma production in the spinal cords of HAM-resistant strains but not in those of WKAH rats. Neurons were the major cells that produced IFN-gamma in HTLV-1-infected, HAM-resistant strains. Administration of IFN-gamma suppressed expression of pX, the gene critically involved in the onset of HAM rat disease, in an HTLV-1-immortalized rat T-cell line, indicating that IFN-gamma protects against the development of HAM rat disease. The inability of WKAH spinal cord neurons to produce IFN-gamma after infection appeared to stem from defects in signaling through the interleukin (IL)-12 receptor. Specifically, WKAH-derived spinal cord cells were unable to up-regulate the IL-12 receptor beta2 gene in response to IL-12 stimulation. We suggest that the failure of spinal cord neurons to produce IFN-gamma through the IL-12 pathway is involved in the development of HAM rat disease.

Regulation of p21 and p27 expression by the hepatitis B virus X protein and the alternate initiation site X proteins, AUG2 and AUG3.

The hepatitis B virus X gene has three in-frame start codons encoding the pX, AUG2 and AUG3 proteins. The AUG2 and AUG3 genes are 5'-truncated in respect to the full-length pX gene; however, all three genes terminate at the same stop codon. The activity of pX as an oncogene is well characterized; however, less is known about the AUG2 and AUG3 proteins. The effects of pX, AUG2 and AUG3 on p21Cip,1/WAF,1/MDA6 and p27Kip-1 cyclin kinase inhibitor (CKI) protein expression, and the impact they have on proliferation, were investigated in CHO K-1 cells. CHO K-1 cells were chosen because they can be transfected at 100% efficiency. p21- and p27-luciferase reporter expression is modulated by increasing doses of the hepatitis B X proteins. At low concentrations of pX or AUG2, p21- and p27-luciferase activity was increased, and at high concentrations, p21- and p27-luciferase activity was decreased. Expression of the AUG3 gene showed a different profile: it was increasingly stimulatory with dose for both promoters. Western blot analyses demonstrated that p21 and p27 protein levels were modulated as predicted based on data generated in the promoter-luciferase experiments. Tritiated thymidine labeling of DNA showed biphasic kinetics of incorporation in the presence of varying pX and AUG2 concentrations, whereas labeling decreased with AUG3 concentration. The growth inhibitory effect of pX expression was reduced by antisense ablation of either p21 or p27. The relative expression level of pX, AUG2, and AUG3 impacts on CKI expression and cell proliferation. Our findings may explain why divergent effects of pX expression on growth have been observed by different groups, which may be related to relative pX expression levels.

Provirus expansion and deregulation of apoptosis-related genes in the spinal cord of a rat model for human T-lymphocyte virus type I-associated myeloneuropathy.

Apoptosis of the spinal oligodendrocytes is the main factor linked to the pathogenesis of human T-lymphocyte virus type I (HTLV-I)-induced myeloneuropathy in rats (HAM rat). To clarify apoptosis-related mechanisms, expression of apoptosis-related genes in the spinal cord of these rats was chronologically examined by means of a semiquantitative reverse transcriptase-polymerase chain reaction. Provirus expansion and increment of HTLV-I pX mRNA were evident at 7 months after the induced infection. Tumor necrosis factor-alpha increased gradually soon after pX expression. The expression of a major apoptosis-resistant gene, bcl-2, was markedly suppressed at a period of the provirus expansion and bax was also down-regulated. p53 was consistently expressed at high levels. These findings were never observed in spinal cords of HAM-resistant strains with HTLV-I infection even throughout their entire life. Collective evidence suggests that the local provirus expansion and deregulation of apoptosis-related genes, especially down-regulation of bcl-2, may lead to apoptosis of oligodendrocytes, thus being a major pathogenetic pathway in the HTLV-I-induced myeloneuropathy.

Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes.

Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca(2+) and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G(2)/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.

Hepatitis B virus X protein increases expression of p21Cip-1/WAF1/MDA6 and p27Kip-1 in primary mouse hepatocytes, leading to reduced cell cycle progression

Previously, we have linked prolonged intense mitogen-activated protein kinase (MAP kinase; MAPK) signaling in hepatocytes to increased expression of p21Cip-1/WAF1/MDA6 (p21) and p16INK4a (p16), that leads to a p21-dependent growth arrest. In this study, we investigated the impact of hepatitis B virus X protein (pX) expression on MAPK-modulated cell cycle progression in primary mouse hepatocytes. In hepatocytes, expression of pX enhanced protein levels of p21 and p27, but not of p16. The elevated levels of p21 and p27 correlated with reduced DNA synthesis in wild-type (+/+) hepatocytes and with a weak stimulation of DNA synthesis in p21 null (−/−) cells. Antisense p27 messenger RNA (mRNA) (p27as) increased DNA synthesis in +/+ and p21 −/− cells, and pX blunted this effect in +/+ cells. In p21 −/− cells, however, p27as permitted pX to further stimulate DNA synthesis. These data argue that a reduced ability to enhance expression of both p21 and p27 is required to fully reveal the growth-potentiating properties of pX. This finding also implies that depending on the functional status of the p21 and p27 genes, expression of pX can have 2 very different effects on hepatocyte proliferation. Prolonged intense MAPK signaling reduced DNA synthesis in +/+ cells and enhanced DNA synthesis in p21 −/− cells. The enhancement of DNA synthesis in p21 −/− cells was blocked by pX, and the effect of pX was abrogated by p27as. Furthermore in p21 −/− cells, overexpression of p16 blocked MAPK-stimulated DNA synthesis, and this effect was partially reversed by p27as. These data argue that p27 can also cooperatively interact with p16 to inhibit DNA synthesis in hepatocytes. Collectively, our findings show that reduced expression of p16, p21, and p27, which can occur during hepatocellular carcinoma, enhances the ability of MAPK signaling and pX to cause proliferation in hepatocytes. Thus loss of cyclin kinase inhibitor function may play an important role in the process of tumor progression after chronic hepatitis B virus infection. (HEPATOLOGY 2001;34:906-917.)

Hepatitis B Virus X Protein Differentially Activates RAS-RAF-MAPK and JNK Pathways in X-transforming Versus Non-transforming AML12 Hepatocytes

The hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis of chronic HBV patients by an unknown mechanism. Activities of pX likely relevant to hepatocyte transformation include activation of the mitogenic RAS-RAF-MAPK and JNK pathways. To assess the importance of mitogenic pathway activation by pX in transformation, we employed a cellular model system composed of two tetracycline-regulated, pX-expressing cell lines, constructed in AML12-immortalized hepatocytes. This system includes the differentiated 3pX-1 and the de-differentiated 4pX-1 hepatocytes. Our studies have demonstrated that conditional pX expression transforms only 3pX-1 cells. Here, comparative in vitro kinase assays and various in vivo analyses demonstrate that pX affects an inverse activation of RAS-RAF-MAPK and JNK pathways in 3pX-1 versus 4pX-1 cells. Sustained pX-dependent RAS-RAF-MAPK pathway activation is observed in pX-transforming 3pX-1 cells, whereas sustained pX-dependent JNK pathway activation is observed in pX non-transforming 4pX-1 cells. This differential, pX-dependent mitogenic pathway activation affects differential activation of cAMP-response element-binding protein and c-Jun and determines the proliferative response of 3pX-1 and 4pX-1 cells. Furthermore, tetracycline-regulated, pX-NLS-expressing cell lines demonstrate that expression of the nuclear pX-NLS variant minimally activates the RAS-RAF-MAPK pathway and results in markedly reduced transformation. These results link sustained, pX-mediated activation of RAS-RAF-MAPK pathway to hepatocyte transformation.

Activation of NF-kappaB by hepatitis B virus X protein through an IkappaB kinase-independent mechanism.

pX, the hepatitis B virus-encoded transcription coactivator, is involved in viral infection in vivo. pX stimulates the activity of several transcription factors including nuclear factor-kappaB (NF-kappaB), but the mechanism of activation is poorly understood. The IkappaB kinase complex (IKK) mediates activation of NF-kappaB in response to various extracellular stimuli, including inflammatory cytokines like tumor necrosis factor and interleukin 1, human T cell lymphoma virus 1 Tax protein, and tumor promoters like phorbol esters. It is not known whether IKK also mediates activation of NF-kappaB by pX. Here we report that IKK was not essential for activation of NF-kappaB by pX. Expression of pX resulted in the degradation of IkappaBalpha in the absence of its phosphorylation at Ser(32) and Ser(36) residues. Although pX stimulated the activity of cotransfected IKK-beta when it was overexpressed, it failed to activate endogenous IKK. Furthermore, expression of pX stimulated NF-kappaB nuclear translocation and transcriptional activity in IKK-gamma-null fibroblast 5R cells. Our data indicate that pX stimulates NF-kappaB activity through a mechanism that is dependent on IkappaBalpha degradation but not on IKK activation.

HBV Protein X Potentiates TGF Beta Signaling

Hepatitis B virus (HBV) infection leads to several potentially life-threatening hepatic diseases. Similarly, dysregulated, chronic TGF-β signaling can also result in liver damage by extracellular matrix proteins whose accumulation has been implicated in several liver diseases. Lee et al. have uncovered a link between HBV infection and the potentiation of TGF-β signaling. The HBV protein X (pX) is required for HBV replication and stimulates transcription through viral and cellular promoters. Transfectin of pX into cells treated with TGF-β, activin, or bone morphogenetic protein-2 (BMP-2), resulted in greatly increased Smad-dependent transcription, in vitro and in vivo. pX associated with Smad4 constitutively, and with Smad3 in a TGF-β-dependent manner, and expression of pX resulted in great increases of Smad3 and Smad4 transport into the nucleus. pX also associated with TFIIB, a component of the basal transcription complex, and stabilized the interaction of Smad3 and Smad4 with TFIIB. pX also relieved the E1A-dependent interference of Smad3-p300 interactions which resulted in increased transcription. Thus, because TGF-β has an established role in chronic hepatitis and cirrhosis, these data suggest that pX potentiates TGF-β-directed pathogenesis and may explain how HBV causes such delibitating liver diseases. These studies also reveal pX as a possible target for drug therapy to lessen disease symptoms caused by HBV. D. K. Lee, S. H. Park, Y. Yi, S.-G. Choi, C. Lee, W. T. Parks, H. S. Cho, M. P. de Caestecker, Y. Shaul, A. B. Roberts, S.-J. Kim, The hepatitis B virus encoded oncoprotein pX amplifies TGF-β family signaling through direct interaction with Smad4: potential mechanism of hepatitis B virus-induced liver fibrosis. Genes Dev. 15 , 455-466 (2001). [Abstract] [Full Text]

Inverse expression of peroxisomes and connexin-43 in the granulosa cells of the quail follicle.

Studying the regulation of peroxisome (Px) expression could improve our understanding of human peroxisomal disorders. The granulosa of the largest preovulatory quail follicles proved to be a relevant model because (a) Px expression changes according to the follicular maturation stage and (b) Px expression varies regionally according to the distance of the granulosa relative to the germinal disc region containing the female gamete (oocyte). The question was asked whether Px expression is related to the extent of metabolic cell coupling and whether zonal Px variation is causally related to oocytal factors. This was evaluated by the presence of catalase and Cx-43 (marker proteins for peroxisomes and gap junctions, respectively) and by in vitro experiments with granulosa explants. The data obtained show that the expression of Cx-43 and Px is inversely correlated both temporally and spatially. Uncoupling of gap junctions results in an upregulation of alpha-catalase immunofluorescence. This is in agreement with reports that gap junctions are often negatively affected by Px proliferators. The zonal gradient in Px expression appears to be imposed by the oocyte, as is the case for steroidogenesis and proliferative capacity in the granulosa epithelium. (J Histochem Cytochem 48:167-177, 2000)

Hepatitis B virus X protein transactivates the inducible nitric oxide synthase promoter.

The capability of hepatitis B virus (HBV) to increase the transcription of the human hepatic inducible nitric oxide synthase (iNOS) by transactivating its promoter has been studied. We have observed by reverse-transcription polymerase chain reaction (RT-PCR) that although the mRNA for the iNOS was almost undetectable in the human hepatoblastoma cell line, HepG2, it was constitutively expressed in the 2.2.15 cell line (a derivative of the HepG2 that produces complete HBV particles). Transfection of HepG2 and 2.2.15 cells with the p1iNOS-CAT plasmid (containing a 1.1-kb fragment of the iNOS promoter) resulted in an increase in chloramphenicol acetyl transferase (CAT) activity in 2.2.15 cells. Similar results were observed when HepG2 and Chang liver cell lines were cotransfected with the p1iNOS-CAT plasmid and the complete HBV genome. It was shown that pX was responsible for the transactivation by cotransfection of HepG2 cells with the p1iNOS-CAT and plasmids expressing the HBV-encoded pX protein, core antigen, and e antigen. Cotransfection of HepG2 cells with the pX expression plasmids and a series of deletion mutants of the 1.1-kb iNOS promoter fragments established that transactivation by pX depends on the presence of at least one nuclear factor-kappaB (NF-kappaB) binding site. This was further confirmed by cotransfecting cells with a plasmid expressing the NF-kappaB inhibitor, IkappaB.

Differential Immediate Early Gene Expression in Conditional Hepatitis B Virus pX-transforming VersusNontransforming Hepatocyte Cell Lines

We report construction and characterization of tetracycline-controlled hepatitis B virus pX-expressing hepatocyte (AML12) cell lines. These cell lines were constructed in AML12 clonal isolates (clones 3 and 4), which express constitutively the tetracycline-controlled transactivator. Since pX is implicated in HCC, this immortalized hepatocyte model system was used to investigate the mechanism of pX in transformation. Clonal isolates of 3pX and 4pX lineages display conditional synthesis of pX mRNA and protein and a 2-fold increase in growth saturation density following tetracycline removal, implicating pX in monolayer overgrowth. Interestingly, only 3pX clones display pX-dependent anchorage independence. Clone 3 lineages express hepatocyte nuclear factor-1alpha and hepatocyte-specific marker genes; clone 4 lineages express hepatocyte nuclear factor-1beta and reduced levels of hepatocyte-specific marker genes, suggesting the importance of the differentiated hepatocyte in pX-mediated oncogenic transformation. Importantly, 3pX and 4pX lineages display differential expression of immediate early genes c-fos and ATF3. The pX-transforming 3pX lineage displays early, pX-dependent induction of ATF3 and prolonged induction of c-fos. The nontransforming 4pX cells display an absence of pX-dependent ATF3 induction and transient induction of c-fos. Our results support the direct link of pX expression to oncogenic transformation in 3pX lineage clones and underscore the advantage of this conditional cellular model system for studying mechanisms of pX-mediated oncogenesis.

Transactivation of the human MDR1 gene by hepatitis B virus X gene product

Background/Aims: Persistent hepatitis B virus (HBV) infection may cause hepatocellular carcinoma. Patients with hepatocellular carcinoma are characterized by nonresponsiveness to chemotherapeutic agents. While many studies have been devoted to understanding the hepatocarcinogenesis mechanism of HBV, the possible relationship between HBV and the drug sensitivity phenotype of cancer cells has rarely been addressed. The hepatitis B virus X gene encodes a transcription transactivator which has been suggested to be a potential factor in viral hepatocarcinogenesis. The role of HBV pX in mediating the drug resistance phenotype of hepatoma cell lines was examined in this study. Methods: Standard transfection and chloramphenicol acetyltransferase assay was utilized to examine the effect of HBV pX transactivator on a reporter gene under the control of the human multidrug resistance (MDR) 1 upstream regulatory elements. Selected Hep G2 clones with or without HBV pX expression were tested for their sensitivity towards various anti-cancer agents by utilization of MTT assay. Results: The expression of HBV pX in both Hep G2 (p53+) and Hep 3B (p53−) cells resulted in transactivation of the reporter gene under control of the human MDR1 upstream regulatory elements. Northern blot analysis indicated that expression of the endogenous MDR1 gene was also elevated in Hep G2 clones with HBV pX expression. Decreased drug sensitivity towards adriamycin, vinblastine, and VP-16 was observed in Hep G2 clones with HBV pX expression. Conclusions: HBV pX can transactivate the MDR1 gene. Drug sensitivity was altered in Hep G2 cells with HBV pX expression.

PX, the HBV-encoded coactivator, suppresses the phenotypes of TBP and TAFII250 mutants.

Hepatitis B virus (HBV) infects humans and causes a wide range of clinical manifestations, from acute hepatitis to hepatocellular carcinoma (HCC). The HBV genome contains multiple promoters with gene expression regulated predominantly by the cellular transcription initiation machinery. Accordingly, the HBV-encoded pX, the only known viral regulator, is a potent transcription coactivator. We investigated the relationship between pX and cellular coactivators. We show that pX restores wild-type activity to inactive TBPAS mutants with poor TAFII250 and activator-binding activity. This pX-mediated recovery, however, is not obtained with inactive TBPAS mutants in binding of other general transcription factors. Remarkably, ts13, a cell line temperature sensitive for TAFII250 function, exhibiting growth arrest and apoptosis at the restrictive temperature, is rescued partially by pX expression, thus generating a pX-dependent cell growth. Collectively, our results suggest that pX suppresses some of the phenotypes of TBP and TAFII250 mutations, implying that pX circumvents the need for a holo-TFIID complex for transcription activation to proceed.

The hepatitis B virus X gene induces p53-mediated programmed cell death.

The human hepatitis B virus (HBV) protein pX is a multifunctional regulatory protein that is known to affect both transcription and cell growth. Here we describe induction of apoptosis in NIH 3T3 polyclonal cell lines upon stimulation of pX expression from a dexamethasone inducible mouse mammary tumor virus (MMTV)-X expression vector. The effect of long-term pX expression on the cell survival of mouse fibroblasts was confirmed in colony generation assays. This effect is not shared either by the other HBV products and it is c-myc mediated, as shown by the use of a dominant negative deletion mutant of c-myc. pX also sensitize cells to programmed cell death after exposure to DNA damaging agents. Taking advantage of stable transfectants carrying the p53val135 temperature-sensitive allele, we directly demonstrate that induction of apoptosis by pX requires p53. In p53 null mouse embryo fibroblasts pX activates transcription and confers an evident growth advantage without loss of cell viability. Although pX protein was not detectable in the experimental conditions we used, our results indicate that its expression affects both cell growth and cell death control.

Constitutive expression of the HTLV-I pX and env regions in Jurkat T-cells induces differential activation of SRE, CRE and NF kappa B pathways.

Human T-cell leukemia virus type I (HTLV-I) causes adult T-cell leukemia/lymphoma (ATLL). HTLV Tax, the viral transcriptional activator, can activate a variety of cellular genes. HTLV-mediated T-cell transformation, however, may involve additional viral proteins expressed from singly- as well as doubly-spliced viral mRNA. To determine the combined effect of these viral proteins on cellular gene expression in Jurkat T-cells, we derived stable transfectants that constitutively express the HTLV-I pX and env regions (J3.9). J3.9 cells show substantially increased mRNA levels of egr-1 and c-jun but no induction of either CD25 or GM-CSF by Northern blotting. This pattern corresponded to the activation of an egr-1 but not a GM-CSF promoter-driven reporter construct in transient gene expression assays. In DNA electrophoretic mobility shift assays (EMSA), nuclear extract from J3.9 cells has significantly increased binding to CRE and SRE but not nuclear factor kappa B (NF kappa B) DNA oligos, as compared to J-Neo cell extract. These results suggest that low level expression of pX and env region gene products in Jurkat T-cells stimulates persistent activation of CRE- and SRE- but not NF kappa B-induced cellular genes.

Hepatitis B virus pX activates NF-kappa B-dependent transcription through a Raf-independent pathway

In this study, we characterized the molecular events involved in the activation of the ubiquitous transcription factor NF-kappa B by the viral transactivator pX. pX expression in HeLa cells determines a manyfold increase in NF-kappa B-dependent transcription, which is associated with an increase in p50/p65 heterodimer DNA-binding activity. Since the I kappa B-alpha inhibitory subunit proteolytic degradation, which follows its phosphorylation/modification, is a key event in NF-kappa B activation by different stimuli (such as growth factors, phorbol esters, tumor necrosis factor, UV irradiation, and oxygen radicals), we investigated pX effects on I kappa B-alpha, as well as the possible involvement of known signalling pathways in pX-induced NF-kappa B-dependent transcription. We observed that although pX had no direct effect on p50 or p65, it was able to restore the I kappa B-alpha-suppressed p50/p65 activity. More directly, the stable expression of pX in HeLa cells resulted in reduced levels of I kappa B-alpha in the cytoplasm. Pretreatment of the cells with H7, calphostin C, tyrphostin 25, or N-acetylcysteine did not impair the effects of pX on NF-kappa B, thus ruling out the involvement of protein kinase C, tyrosine kinases, and oxygen radicals. Finally, while most of the known NF-kappa B-activating agents converge on Raf-1 protein kinase, when Raf-1 activity is blocked by overexpression of a dominant negative mutant, the effects of pX on NF-kappa B are not impaired. Thus, we suggest that although pX is able to activate the Ras/Raf-1-signalling pathway, it triggers NF-kappa B activation by an as yet unidentified Raf-1-independent pathway.