Gluconeogenesis, lipogenesis, and HBV replication are commonly regulated by PGC-1α-dependent pathway.

PGC-1α, a major metabolic regulator of gluconeogenesis and lipogenesis, is strongly induced to coactivate Hepatitis B virus (HBV) gene expression in the liver of fasting mice. We found that 8-Br-cAMP and glucocorticoids synergistically induce PGC-1α and its downstream targets, including PEPCK and G6Pase. Also, HBV core promoter activity was synergistically enhanced by 8-Br-cAMP and glucocorticoids. Graptopetalum paraguayense (GP), a herbal medicine, is commonly used in Taiwan to treat liver disorders. Partially purified fraction of GP (named HH-F3) suppressed 8-Br-cAMP/glucocorticoid-induced G6Pase, PEPCK and PGC-1α expression and suppressed HBV core promoter activity. HH-F3 blocked HBV core promoter activity via inhibition of PGC-1α expression. Ectopically expressed PGC-1α rescued HH-F3-inhibited HBV surface antigen expression, HBV mRNA production, core protein levels, and HBV replication. HH-F3 also inhibited fatty acid synthase (FASN) expression and decreased lipid accumulation by down-regulating PGC-1α. Thus, HH-F3 can inhibit HBV replication, gluconeogenesis and lipogenesis by down-regulating PGC-1α. Our study indicates that targeting PGC-1α may be a therapeutic strategy for treatment of HBV infections. HH-F3 may have potential use for the treatment of chronic hepatitis B patients with associated metabolic syndrome.

Role of JNK and p38 MAPK in Taiwanin A-induced cell death.

AIM: The lignan compound Taiwanin A is cytotoxic for human cancer cells. Taiwanin A has been previously shown to damage microtubules, induce mitotic arrest and cause apoptosis in cancer cells. The goal of the current study is to identify intracellular signaling pathways that are involved in Taiwanin A-mediated apoptosis. MAIN METHODS: We examined the activation of three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK), in HepG2 cells after Taiwanin A treatment. The role of MAPK activation in Taiwanin A-induced apoptosis was examined using Western blotting, caspase activity assays combined with specific MAPK inhibitors and shRNA treatment to knockdown JNK. KEY FINDINGS: Taiwanin A activated all three MAPKs (ERK, p38 and JNK). Cytotoxicity was blocked by the p38 MAPK inhibitor SB203580 and the JNK inhibitor SP600125 but not by the ERK inhibitor PD98059. A combined treatment of SB203580 and SP600125 showed increased effects on the inhibition of Taiwanin A cytotoxicity, suggesting that both JNK and p38 play a role in Taiwanin A-induced apoptosis. Inhibition of p38 activity reduced Taiwanin A-induced p53 phosphorylation on Ser15. Direct interaction of Taiwanin A-activated p38 and p53 was demonstrated by immunoprecipitation. In addition, inhibition of JNK by SP600125 or silencing of the JNK scaffold protein JIP2 reduced phosphorylation of Bcl-2, which may help to promote anti-apoptotic pathways. SIGNIFICANCE: We demonstrated for the first time that two distinct apoptotic pathways, the p38-p53 and JNK-Bcl-2 pathways, were triggered by the anti-microtubule compound Taiwanin A.

Identification of a two-layer regulatory network of proliferation-related microRNAs in hepatoma cells.

To elucidate how microRNA (miRNA)-regulated networks contribute to the uncontrolled growth of hepatoma cells (HCCs), we identified several proliferation-related miRNAs by comparing miRNA expression patterns in clinical HCC samples and growth-arrested HepG2 cells. To explore the molecular functions targeted by these miRNAs, we classified genes differentially expressed in clinical HCC samples into six functional clusters based on their functional similarity. Using target enrichment analysis, we discovered that targets of three proliferation-related miRNAs-miR-101, miR-199a-3p and miR-139-5p-were significantly enriched in the 'transcription regulation' functional cluster. An interactome network consisting of these three miRNAs and genes in the 'transcriptional control' cluster revealed that all three miRNAs were highly connected hubs in the network. All three miRNA-centered subnetworks displayed characteristics of a two-layer regulatory architecture, with transcription factors and epigenetic modulators as the first neighbors and genes involved in cell-cycle progression as second neighbors. The overexpression of miR-101 in HepG2 cells reduced the expression of transcription regulators and genes in cell-cycle progression and suppressed the proliferation and colony formation of HepG2 cells. This study not only provides direct experimental data to support the 'miRNA-centered two-layer regulatory network' model, but our results also suggest that such a combinatorial network model may be widely used by miRNAs to regulate critical biological processes.

Knockdown of HURP inhibits the proliferation of hepacellular carcinoma cells via downregulation of gankyrin and accumulation of p53.

We determined earlier that the hepatoma upregulated protein (HURP) is overexpressed in hepatocellular carcinoma (HCC), but the role of this protein during cancer development and progression remains unknown. Here, we observed that the overexpression of HURP in HEK293 cells promoted the ubiquitination of p53 and its degradation by the proteasome. In contrast, HURP knockdown using short-hairpin RNA reversed these effects. Knockdown of HURP promoted the accumulation of p53 in SK-Hep-1 cells (p53+/-), and these cells showed reduced proliferation, while the p53-mutant Mahlavu cells were not affected. HURP knockdown did not affect the proliferation of H1299 lung carcinoma cells and Hep3B HCC cells which lack p53. Knockdown of HURP also sensitized SK-Hep-1 cells to cisplatin. On the other hand, the expression of exogenous p53 in H1299 and Hep3B cells was decreased following overexpression of HURP, and these cells showed decreased sensitivity to cisplatin-induced apoptosis. Importantly, overexpression of HURP promoted the proliferation of HEK293 cells in an anchorage-independent manner, and inoculation of SK-Hep-1 cancer cells that expressed short-hairpin RNA to knockdown HURP resulted in smaller tumors in nude mice. Gankyrin, a positive regulator of the E3 ubiquitin ligase MDM2, was found to be upregulated following HURP expression, and gankyrin knockdown decreased the HURP-mediated downregulation of p53. Notably, we detected a positive correlation between elevated HURP and gankyrin protein levels in HCC patients (r(2) = 0.778; N = 9). Taken together, these results indicate that HURP represents an oncogene that may play a role in HCC progression and chemoresistance.

Receptor for hyaluronan-mediated motility isoform B promotes liver metastasis in a mouse model of multistep tumorigenesis and a tail vein assay for metastasis.

The gene encoding the receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in many human cancers. However, it is unclear whether RHAMM plays a causal role in tumor initiation or progression. Using somatic gene transfer in a mouse model of islet cell tumorigenesis, we demonstrate that RHAMM isoform B (RHAMM(B)) promotes tumor growth and metastases to lymph nodes and the liver. The propensity of RHAMM(B)-expressing cells to metastasize to the liver was confirmed using an experimental metastasis assay in which cells were injected into the tail vein of immunodeficient mice. However, RHAMM(B) did not increase cell migration or proliferation in culture. In initial efforts to identify signaling pathways activated by RHAMM(B), we found that RHAMM(B) induced phosphorylation of epidermal growth factor receptor (EGFR), Erk1/2, and STAT3 and conferred susceptibility to apoptosis after treatment with an EGFR inhibitor, gefitinib. Taken together, the results indicate that RHAMM(B) promotes hepatic metastasis by islet tumor cells, perhaps through growth factor receptor-mediated signaling.

A translational regulator, PUM2, promotes both protein stability and kinase activity of Aurora-A.

Aurora-A, a centrosomal serine-threonine kinase, orchestrates several key aspects of cell division. However, the regulatory pathways for the protein stability and kinase activity of Aurora-A are still not completely understood. In this study, PUM2, an RNA-binding protein, is identified as a novel substrate and interacting protein of Aurora-A. Overexpression of the PUM2 mutant which fails to interact with Aurora-A, and depletion of PUM2 result in a decrease in the amount of Aurora-A. PUM2 physically binds to the D-box of Aurora-A, which is recognized by APC/C(Cdh1). Overexpression of PUM2 prevents ubiquitination and enhances the protein stability of Aurora-A, suggesting that PUM2 protects Aurora-A from APC/C(Cdh1)-mediated degradation. Moreover, association of PUM2 with Aurora-A not only makes Aurora-A more stable but also enhances the kinase activity of Aurora-A. Our study suggests that PUM2 plays two different but important roles during cell cycle progression. In interphase, PUM2 localizes in cytoplasm and plays as translational repressor through its RNA binding domain. However, in mitosis, PUM2 physically associates with Aurora-A to ensure enough active Aurora-A at centrosomes for mitotic entry. This is the first time to reveal the moonlight role of PUM2 in mitosis.

Phosphorylation-dependent association of the G4-1/G5PR regulatory subunit with IKKß negatively modulates NF-κB activation through recruitment of protein phosphatase 5.

The transcription factor NF-κB (nuclear factor κB) co-ordinates various gene expressions in response to diverse signals and is a critical regulator of inflammation and innate immunity. Several negative regulators of NF-κB have been identified as downstream targets of NF-κB and function as a feedback control of NF-κB activation. A few protein phosphatases have also been shown to inactivate NF-κB activation. However, little is known about how protein phosphatases detect and respond to NF-κB activation. In the present study, we report a regulatory subunit of PP5 (protein phosphatase 5), G4-1, that physically interacts with IKKß [IκB (inhibitor of NF-κB) kinase ß] and negatively regulates NF-κB activation. The association of G4-1 with IKKß depends on the kinase activity of IKKß. Mapping of the G4-1-binding domain of IKKß reveals that the serine-rich domain in the C-terminus of IKKß is required for G4-1 binding. When seven autophosphorylated serine residues in this domain were mutated to alanine, the mutant form of IKKß lost its ability to bind G4-1 and was more potent than the wild-type kinase to activate NF-κB. Knockdown of G4-1 enhanced TNFα (tumour necrosis factor α)-induced NF-κB activity, and knockdown of PP5 totally abolished the inhibitory activity of G4-1 on NF-κB activation. The results of the present study suggest that G4-1 functions as an adaptor to recruit PP5 to the phosphorylated C-terminus of activated IKKß and to down-regulate the activation of IKKß.

Scutellariae radix suppresses hepatitis B virus production in human hepatoma cells.

The traditional Chinese medicine Xiao-Chai-Hu-Tang (HD-7) has been widely used to treat liver diseases in China and Japan. HD-7 consists of seven different ingredients, but which one provides the therapeutic benefits is still unknown. Here, we identified the "Minister herb" Scutellariae radix (HD-1S), but not the "King herb" Bupleuri radix (HD-1B) in HD-7, as the active component that suppresses HBV gene expression and virus production in human hepatoma cells. We have found that an aqueous extract of HD-1S not only suppressed wild-type virus but also lamivudine-resistant HBV mutant in human hepatoma cells. We show that HD-1S selectively suppresses HBV core promoter activity. Electrophoretic mobility shift assay analysis has revealed that HD-1S treatment decreases the DNA-binding activity of nuclear extract of HepA2 cells to a specific cis-element of the HBV core promoter, which includes the peroxisome proliferator-activated receptor binding site and HNF4. Furthermore, ectopic expression of PGC-1 abolished the suppression of HD-1S on HBV core promoter activity suggesting that HD-1S may act through modulating hepatic transcriptional machinery to suppress HBV viral gene expression and virus production.

PKCalpha mediated induction of miR-101 in human hepatoma HepG2 cells.

BACKGROUND: Protein Kinase C (PKC) is a serine/threonine kinase that involved in controlling of many cellular processes such as cell proliferation and differentiation. We have observed previously that TPA (12-O-tetradecanoylphorbol 13-acetate) induces cell cycle arrest in G0/G1 phase in human hepatoma HepG2 cells. However, is there any miRNA involved in PKCalpha mediated cell growth arrest is still unknown. METHODS: We first surveyed 270 miRNA expression profiles in 20 pairs of human hepatoma tissues. We identified 11 up-regulated and 23 down-regulated miRNAs (FDR < = 0.01; fold-change > = 2) in human hepatoma tissue after Student's T-test and Mann-Whitney rank test. We then examined miRNAs expression profile in TPA treated HepG2 cells. Two miRNAs, miR-101, and miR-29c, were shown to be significantly down regulated in human hepatoma tissues and induced over 4-fold in HepG2 cells under TPA treatment. RESULTS: In this study, we examined TPA regulated miRNA expression profile in human hepatoma HepG2 cells. We identified two miRNAs, 101 and 29c, were induced by TPA and down regulated in human hepatoma tissues suggest that they might play as tumor suppressor gene and in tumor formation of HCC. Since induction kinetics of miR-101 by TPA was much faster than miR-29c suggests that the induction of miR-101 may be the primary response of TPA treatment. We then further investigated how miR-101 was regulated by TPA. MiR-101 targets two subunits of PRC2 complex, enhancer of zeste homolog 2 (EZH2) and EED, and was shown to play as a tumor suppressor gene in human prostate, breast and liver cancers. The target sequence of miR-101 located in the 3' UTR of both EZH2 and EED's mRNA was identified by bioinformatic analysis and was validated by reporter luciferase activity assay. Then we showed that TPA not only up regulated miR-101 expression, but also reduced protein level of EZH2, EED and H3K27me3 in HepG2 cells. Using lenti-virus-mediated shRNA to knockdown endogenous PKCalpha expression, we observed that TPA induced growth arrest, elevation of miR-101 and reduction of EZH2, EED and H3K27me3 proteins were all PKCalpha dependent. Specific inhibitor of ERK completely blocked TPA induced miR-101 expression. CONCLUSIONS: Therefore, this is the first time to show that PKCalpha and ERK pathway play important role to activate miR-101 expression, reduce PRC2 complex and H3K27me3 level. This epigenetic regulatory pathway may represent a novel mechanism of carcinogenesis and deserve further investigation.

Transcriptional up-regulation of SOD1 by CEBPD: a potential target for cisplatin resistant human urothelial carcinoma cells.

Bladder cancer is the fourth most common type of cancer in men (ninth in women) in the United States. Cisplatin is an effective agent against the most common subtype, urothelial carcinoma. However, the development of chemotherapy resistance is a severe clinical problem for the successful treatment of this and other cancers. A better understanding of the cellular and molecular events in response to cisplatin treatment and the development of resistance are critical to improve the therapeutic options for patients. Here, we report that expression of the CCAAT/enhancer binding protein delta (CEBPD, C/EBPdelta, NF-IL6beta) is induced by cisplatin in the human bladder urothelial carcinoma NTUB1 cell line and is specifically elevated in a cisplatin resistant subline. Expression of CEBPD reduced cisplatin-induced reactive oxygen species (ROS) and apoptosis in NTUB1 cells by inducing the expression of Cu/Zn-superoxide dismutase (SOD1) via direct promoter transactivation. Several reports have implicated CEBPD as a tumor suppressor gene. This study reveals a novel role for CEBPD in conferring drug resistance, suggesting that it can also be pro-oncogenic. Furthermore, our data suggest that SOD inhibitors, which are already used as anti-angiogenic agents, may be suitable for combinatorial chemotherapy to prevent or treat cisplatin resistance in bladder and possibly other cancers.

The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities.

The existence of multifunctional enzymes in the nucleotide biosynthesis pathways is believed to be one of the important mechanisms to facilitate the synthesis and the efficient supply of deoxyribonucleotides for DNA replication. Here, we used the bacterially expressed yeast thymidylate kinase (encoded by the CDC8 gene) to demonstrate that the purified Cdc8 protein possessed thymidylate-specific nucleoside diphosphate kinase activity in addition to thymidylate kinase activity. The yeast endogenous nucleoside diphosphate kinase is encoded by YNK1, which appears to be non-essential. Our results suggest that Cdc8 has dual enzyme activities and could duplicate the function of Ynk1 in thymidylate synthesis. We also discuss the importance of the coordinated expression of CDC8 during the cell cycle progression in yeast.

Aurora-A overexpression in mouse liver causes p53-dependent premitotic arrest during liver regeneration.

Aurora-A, a serine-threonine kinase, is frequently overexpressed in human cancers, including hepatocellular carcinoma. To study the phenotypic effects of Aurora-A overexpression on liver regeneration and tumorigenesis, we generated transgenic mice overexpressing human Aurora-A in the liver. The overexpression of Aurora-A after hepatectomy caused an earlier entry into S phase, a sustaining of DNA synthesis, and premitotic arrest in the regenerating liver. These regenerating transgenic livers show a relative increase in binuclear hepatocytes compared with regenerating wild-type livers; in addition, multipolar segregation and trinucleation could be observed only in the transgenic hepatocytes after hepatectomy. These results together suggest that defects accumulated after first round of the hepatocyte cell cycle and that there was a failure to some degree of cytokinesis. Interestingly, the p53-dependent checkpoint was activated by these abnormalities, indicating that p53 plays a crucial role during liver regeneration. Indeed, the premitotic arrest and abnormal cell death, mainly necrosis, caused by Aurora-A overexpression were genetically rescued by p53 knockout. However, trinucleation of hepatocytes remained in the regenerating livers of the transgenic mice with a p53 knockout background, indicating that the abnormal mitotic segregation and cytokinesis failure were p53 independent. Moreover, overexpression of Aurora-A in transgenic liver led to a low incidence (3.8%) of hepatic tumor formation after a long latency period. This transgenic mouse model provides a useful system that allows the study of the physiologic effects of Aurora-A on liver regeneration and the genetic pathways of Aurora-A-mediated tumorigenesis in liver.

VEGFA upregulates FLJ10540 and modulates migration and invasion of lung cancer via PI3K/AKT pathway.

BACKGROUND: Lung adenocarcinoma is the leading cause of cancer-related deaths among both men and women in the world. Despite recent advances in diagnosis and treatment, the mortality rates with an overall 5-year survival of only 15%. This high mortality is probably attributable to early metastasis. Although several well-known markers correlated with poor/metastasis prognosis in lung adenocarcinoma patients by immunohistochemistry was reported, the molecular mechanisms of lung adenocarcinoma development are still not clear. To explore novel molecular markers and their signaling pathways will be crucial for aiding in treatment of lung adenocarcinoma patients. METHODOLOGY/PRINCIPAL FINDINGS: To identify novel lung adenocarcinoma-associated /metastasis genes and to clarify the underlying molecular mechanisms of these targets in lung cancer progression, we created a bioinformatics scheme consisting of integrating three gene expression profile datasets, including pairwise lung adenocarcinoma, secondary metastatic tumors vs. benign tumors, and a series of invasive cell lines. Among the novel targets identified, FLJ10540 was overexpressed in lung cancer tissues and is associated with cell migration and invasion. Furthermore, we employed two co-expression strategies to identify in which pathway FLJ10540 was involved. Lung adenocarcinoma array profiles and tissue microarray IHC staining data showed that FLJ10540 and VEGF-A, as well as FLJ10540 and phospho-AKT exhibit positive correlations, respectively. Stimulation of lung cancer cells with VEGF-A results in an increase in FLJ10540 protein expression and enhances complex formation with PI3K. Treatment with VEGFR2 and PI3K inhibitors affects cell migration and invasion by activating the PI3K/AKT pathway. Moreover, knockdown of FLJ10540 destabilizes formation of the P110-alpha/P85-alpha-(PI3K) complex, further supporting the participation of FLJ10540 in the VEGF-A/PI3K/AKT pathway. CONCLUSIONS/SIGNIFICANCE: This finding set the stage for further testing of FLJ10540 as a new therapeutic target for treating lung cancer and may contribute to the development of new therapeutic strategies that are able to block the PI3K/AKT pathway in lung cancer cells.

Ung1p-mediated uracil-base excision repair in mitochondria is responsible for the petite formation in thymidylate deficient yeast.

The budding yeast CDC21 gene, which encodes thymidylate synthase, is crucial in the thymidylate biosynthetic pathway. Early studies revealed that high frequency of petites were formed in heat-sensitive cdc21 mutants grown at the permissive temperature. However, the molecular mechanism involved in such petite formation is largely unknown. Here we used a yeast cdc21-1 mutant to demonstrate that the mutant cells accumulated dUMP in the mitochondrial genome. When UNG1 (encoding uracil-DNA glycosylase) was deleted from cdc21-1, we found that the ung1Delta cdc21-1 double mutant reduced frequency of petite formation to the level found in wild-type cells. We propose that the initiation of Ung1p-mediated base excision repair in the uracil-laden mitochondrial genome in a cdc21-1 mutant is responsible for the mitochondrial petite mutations.

Helioxanthin inhibits interleukin-1 beta-induced MIP-1 beta production by reduction of c-jun expression and binding of the c-jun/CREB1 complex to the AP-1/CRE site of the MIP-1 beta promoter in Huh7 cells.

An elevated level of macrophage inflammatory protein-1beta (MIP-1beta) induced by IL-1beta has been correlated with chronic hepatic inflammatory disease. However, molecular mechanism of IL-1beta-induced MIP-1beta expression in hepatic cells is obscure. Previously, we reported the mechanism of the anti-hepatitis B virus (HBV) activity of helioxanthin (HE-145). Here, we demonstrated that HE-145 inhibited IL-1beta-induced MIP-1beta expression in a dose-dependent manner in Huh7 cells. To understand the mode of action of HE-145, we first examined how IL-1beta induced MIP-1beta expression at the molecular level. Using selective inhibitors, we found that JNK and p38 pathways participated in IL-1beta-induced MIP-1beta expression. HE-145 specifically suppressed IL-1beta-induced c-jun mRNA and protein expression and prevented c-jun-mediated AP-1 DNA-binding activity, whereas it had no effect on IL-1beta-induced activation of JNK, p38 and ATF2. Further studies indicated that HE-145 may downregulate c-jun mRNA expression directly at transcriptional level without requirement of de novo protein synthesis. Mutational analysis and supershift assays indicated that IL-1beta stimulated c-jun and CREB1 binding to the essential AP-1/CRE site of the MIP-1beta promoter. The inhibitory effect of HE-145 on IL-1beta-induced MIP-1beta promoter activity was completely reversed by overexpressing c-jun. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay consistently revealed that HE-145 reduced c-jun binding to the AP-1/CRE site in vitro and in vivo. Our results established a major role for c-jun in IL-1beta-induced MIP-1beta expression in hepatic cells. The reduction in IL-1beta-induced c-jun expression and subsequent binding of the c-jun/CREB1 complex to AP-1/CRE site mainly contributed to the inhibitory action of HE-145 on IL-1beta-induced MIP-1beta production.

Hurp deficiency in mice leads to female infertility caused by an implantation defect.

First revealed in cancer studies, HURP (hepatoma up-regulated protein) is a cell cycle-associated gene with elevated expression in the G(2)/M phase. Cell culture studies have revealed that HURP is an essential factor required for spindle formation and chromosome congression during mitosis. However, the function of HURP in an in vivo context has not been explored. We generated a Hurp knock-out (Hurp(-/-)) mouse to investigate the role of HURP in development under normal physiological conditions. Hurp(-/-) mice develop normally and are indistinguishable from their wild-type littermates. Interestingly, breeding experiments revealed that Hurp(-/-) females are completely infertile, whereas the males reproduce normally. Ovulation, fertilization, and pre-implantation embryo development are normal; however, the Hurp(-/-) females are unable to form implantation sites due to an inability to undergo the decidual reaction. This is caused by a defect in endometrial stromal proliferation that leads to implantation failure. Additionally, HURP expression in the uterus coincides with the implantation stage and can be induced by estrogen treatment. Our results demonstrate for the first time that HURP affects endometrial stromal proliferation during implantation but is dispensable during normal development in mice; specifically, HURP has an essential function in uterine biology.

The role of helioxanthin in inhibiting human hepatitis B viral replication and gene expression by interfering with the host transcriptional machinery of viral promoters.

A non-nucleosidic compound, Helioxanthin (HE-145), was found to suppress HBV gene expression and replication in HCC cells. To understand the molecular mode of action of HE-145 on HBV gene expression, the effects of HE-145 on four viral promoter activities using luciferase as a reporter were examined. It was found that HE-145 selectively suppresses surface antigen promoter II (SPII) and core promoter (CP) but has no effect on surface antigen promoter I (SPI) or promoter for X gene (Xp). The suppressive effects of HE-145 on either SPII or CP activity is liver-specific, since no suppressive activity of HE-145 was observed when CP or SPII promoter activity was assayed in non-liver cells such as HeLa or 293T. To examine the mode of action of HE-145, EMSA analysis revealed that HE-145 decreased the DNA-binding activity of nuclear extract of HepA2 cells to specific cis element of HBV promoter for core antigen, including peroxisome proliferator-activated receptors (PPARs), PPARs binding site (PPRE), alpha-fetoprotein transcription factor (FTF), and Sp1. Ectopic expression of PPAR gamma or HNF4 alpha partially reversed the HE-145-mediated suppression of HBV RNA. Therefore, HE-145 may represent a novel class of anti-HBV agents which selectively modulate transcriptional machinery of human liver cells to suppress HBV gene expression and replication.

Rate of evolution in brain-expressed genes in humans and other primates.

Brain-expressed genes are known to evolve slowly in mammals. Nevertheless, since brains of higher primates have evolved rapidly, one might expect acceleration in DNA sequence evolution in their brain-expressed genes. In this study, we carried out full-length cDNA sequencing on the brain transcriptome of an Old World monkey (OWM) and then conducted three-way comparisons among (i) mouse, OWM, and human, and (ii) OWM, chimpanzee, and human. Although brain-expressed genes indeed appear to evolve more rapidly in species with more advanced brains (apes > OWM > mouse), a similar lineage effect is observable for most other genes. The broad inclusion of genes in the reference set to represent the genomic average is therefore critical to this type of analysis. Calibrated against the genomic average, the rate of evolution among brain-expressed genes is probably lower (or at most equal) in humans than in chimpanzee and OWM. Interestingly, the trend of slow evolution in coding sequence is no less pronounced among brain-specific genes, vis-à-vis brain-expressed genes in general. The human brain may thus differ from those of our close relatives in two opposite directions: (i) faster evolution in gene expression, and (ii) a likely slowdown in the evolution of protein sequences. Possible explanations and hypotheses are discussed.

Taiwanin A induced cell cycle arrest and p53-dependent apoptosis in human hepatocellular carcinoma HepG2 cells.

Taiwanin A, a lignan isolated from Taiwania cryptomerioides Hayata, has previously been reported to have cytotoxicity against human tumor cells, but the mechanisms are unclear. In this study, we examined the molecular mechanism of cell death of human hepatocellular carcinoma HepG2 cells induced by Taiwanin A. Taiwanin A has been found to induce cell cycle arrest at G2/M phase as well as caspase-3-dependent apoptosis within 24 h. We performed both in vitro turbidity assay and immunofluorescence staining of tubulin to show that Taiwanin A can inhibit microtubule assembly. Moreover, the tumor suppressor protein p53 in HepG2 cells was activated by Taiwanin A within 12 h. Inhibition of p53 by either pifithrin-alpha or by short hairpin RNA which blocks p53 expression attenuates Taiwanin A cytotoxicity. Our results demonstrate that Taiwanin A can act as a new class of microtubule damaging agent, arresting cell cycle progression at mitotic phase and inducing apoptosis through the activation of p53.

GSKIP is homologous to the Axin GSK3beta interaction domain and functions as a negative regulator of GSK3beta.

Although prominent FRAT/GBP exhibits a limited degree of homology to Axin, the binding sites on GSK3 for FRAT/GBP and Axin may overlap to prevent the effect of FRAT/GBP in stabilizing beta-catenin in the Wnt pathway. Using a yeast two-hybrid screen, we identified a novel protein, GSK3beta interaction protein (GSKIP), which binds to GSK3beta. We have defined a 25-amino acid region in the C-terminus of GSKIP that is highly similar to the GSK3beta interaction domain (GID) of Axin. Using an in vitro kinase assay, our results indicate that GSKIP is a good GSK3beta substrate, and both the full-length protein and a C-terminal fragment of GSKIP can block phosphorylation of primed and nonprimed substrates in different fashions. Similar to Axin GID(381-405) and FRATtide, synthesized GSKIPtide is also shown to compete with and/or block the phosphorylation of Axin and beta-catenin by GSK3beta. Furthermore, our data indicate that overexpression of GSKIP induces beta-catenin accumulation in the cytoplasm and nucleus as visualized by immunofluorescence. A functional assay also demonstrates that GSKIP-transfected cells have a significant effect on the transactivity of Tcf-4. Collectively, we define GSKIP as a naturally occurring protein that is homologous with the GSK3beta interaction domain of Axin and is able to negatively regulate GSK3beta of the Wnt signaling pathway.