Gwo-Tarng Sheu

Template Requirement and Initiation Site Selection by Hepatitis C Virus Polymerase on a Minimal Viral RNA Template

RNA-dependent RNA polymerase, NS5B protein, catalyzes replication of viral genomic RNA, which presumably initiates from the 3′-end. We have previously shown that NS5B can utilize the 3′-end 98-nucleotide (nt) X region of the hepatitis C virus (HCV) genome as a minimal authentic template. In this study, we used this RNA to characterize the mechanism of RNA synthesis by the recombinant NS5B. We first showed that NS5B formed a complex with the 3′-end of HCV RNA by binding to both the poly(U-U/C)-rich and X regions of the 3′-untranslated region as well as part of the NS5B-coding sequences. Within the X region, NS5B bound stem II and the single-stranded region connecting stem-loops I and II. Truncation of 40 nt or more from the 3′-end of the X region abolished its template activity, whereas X RNA lacking 35 nt or less from the 3′-end retained template activity, consistent with the NS5B-binding site mapped. Furthermore, NS5B initiated RNA synthesis from a specific site within the single-stranded loop I. All of the RNA templates that have a double-stranded stem at the 3′-end had the same RNA initiation site. However, the addition of single-stranded nucleotides to the 3′-end of X RNA or removal of double-stranded structure in stem I generated RNA products of template size. These results indicate that HCV NS5B initiates RNA synthesis from a single-stranded region closest to the 3′-end of the X region. These results have implications for the mechanism of HCV RNA replication and the nature of HCV RNA templates in the infected cells.

Sustained activation of ERK and Cdk2/cyclin-A signaling pathway by pemetrexed leading to S-phase arrest and apoptosis in human non-small cell lung cancer A549 cells

Pemetrexed, a multitargeted antifolate with the ability to inhibit several enzymes involved in purine and pyrimidine syntheses, has demonstrated clinical activity in non-small cell lung cancer cells, as well as in a broad array of other solid tumors. In this study, we show that inducing cell cycle S-phase arrest and apoptosis in human lung adenocarcinoma A549 cells with pemetrexed is associated with increased cyclin-A and cyclin-dependent kinase 2 (Cdk2) protein and Cdk2/cyclin-A kinase activity. Knockdown of cyclin-A using small interfering RNA (siRNA), and inhibiting Cdk2 activity with flavopiridol, strikingly reduced S-phase arrest and apoptosis. Moreover, pemetrexed induced sustained activation of extracellular signal-regulated kinase1/2 (ERK1/2). Knockdown of ERK1/2 using specific siRNA, as well as known inhibitors (PD98059 and U0126), effectively suppressed the expression of cyclin-A and Cdk2, and reduced S-phase arrest and apoptosis induced by pemetrexed. These data provide the first evidence that pemetrexed-induced S-phase arrest and apoptosis is associated with an increase in Cdk2 and cyclin-A expression and activation, which is ERK-dependent and upstream of caspase-3. Our findings suggest that the ERK-mediated Cdk2/cyclin-A signaling pathway is an important regulator of pemetrexed-induced S-phase arrest and apoptotic cell death.

N-acetyl cysteine mitigates curcumin-mediated telomerase inhibition through rescuing of Sp1 reduction in A549 cells

Curcumin is a natural compound that has been extensively observed due to its potential as an anticancer drug. Curcumin restrains cancer cell progression via telomerase activity suppression. However, the exact mechanism is still unknown. In this study, we demonstrate that the effects of curcumin on cell viability and telomerase activity can be blunted by reactive oxygen species (ROS) inhibitor N-acetyl cysteine (NAC). The ROS induced by curcumin in A549 cells was detected by flow cytometry. Using Western blot and RT-PCR, human telomerase reverse transcriptase (hTERT) decreased in the presence of curcumin. Sp1 is one of the important transcription factors in hTERT expression. Our data showed that curcumin decreases the expression of Sp1 through proteasome pathway. In addition, NAC blunted the Sp1 reduction and hTERT downregulation by curcumin. Further, reporter assay and DNA affinity precipitation assay confirmed the influence of curcumin on Sp1 in hTERT regulation. This is the first study to demonstrate that curcumin induces ROS production resulting in Sp1 binding activity inhibition and hTERT downregulation.

MnSOD promotes tumor invasion via upregulation of FoxM1-MMP2 axis and related with poor survival and relapse in lung adenocarcinomas

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme responsible for the elimination of superoxide radical. The role of MnSOD in tumor progression in different human cancers is still controversial. In the present study, MnSOD expression in lung cancer cells was explored by knockdown or overexpression using transfection of a short hairpin RNA (shRNA) or an expression vector, respectively, to determine whether MnSOD expression mediates lung cancer cell migration, invasion, and oncogenic potential by increasing FoxM1 and MMP2 expression. Western blotting showed that FoxM1 and MMP2 expression was dependent on MnSOD expression, suggesting that FoxM1 could be upregulated by MnSOD. Three FoxM1 promoters were constructed to verify this activation of FoxM1 by MnSOD and to determine the transcription factors responsible. Luciferase reporter and chromatin immunoprecipitation assays indicated that MnSOD overexpression in lung cancer cells promoted binding of E2F1 and Sp1 to their putative FoxM1 promoter-binding sites and activated FoxM1 reporter activity. MnSOD also enhanced the potential for cell migration, invasion, and anchorage-independent colony growth on soft-agar plates, again via upregulation of FoxM1 and MMP2 expression. In patients with lung cancer, evaluation of MnSOD expression in lung tumors by immunohistochemistry indicated a positive correlation between FoxM1 and MMP2 mRNA expressions. Kaplan–Meier and Cox regression analysis revealed a poorer overall survival (OS) and relapse-free survival (RFS) in patients with MnSOD-positive tumors than with MnSOD-negative tumors. We conclude that MnSOD may promote tumor aggressiveness via upregulation of the FoxM1–MMP2 axis, and that MnSOD expression can independently predict survival and relapse in patients with resected lung adenocarcinoma. Mol Cancer Res; 11(3); 261–71. ©2012 AACR.

L-type calcium channel blockers reverse docetaxel and vincristine-induced multidrug resistance independent of ABCB1 expression in human lung cancer cell lines

Multidrug resistance (MDR) of cancer cells to cytotoxic drugs significantly impedes chemotherapeutic treatment. The purpose of this study is to characterize docetaxel (DOC) or vincristine (VCR) selected A549 and H1299 non-small cell lung cancer (NSCLC) sublines that exhibit MDR phenotypes and followed by re-sensitization study. Although all drug resistant sublines showed cross-resistance to DOC, VCR, and doxorubicin (DXR), the expression of ATP-binding cassette (ABC) transporter B1 (ABCB1) gene was found to be strongly induced in DOC but not in VCR resistant A549 sublines by quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR). In DOC and VCR resistant H1299 sublines, moderate expression of ABCB1 was detected. The levels of ABCB1 protein and efflux activities were further examined by immunoblotting and rhodamin-123 staining assay. The results showed that both ABC and non-ABC mediated MDR are existed. Furthermore, verapamil (VER), an inhibitor of ABCB1 and an L-type calcium channel blocker, is capable of reversing the resistance in all drug-resistant sublines independent of ABCB1 expression. Importantly, VER only sensitizes resistant sublines but has no effect on parental cancer cells. Other L-type calcium channel blockers, such as diltiazem (DIL) and nifedipine (NIF), also sensitize MDR sublines without interfering with ABCB1 activity but with lower efficacy than VER. Our data showed that in addition to ABCB1, calcium channel activity may play a crucial role in DOC- and VCR-acquired MDR. Therefore, inhibition of calcium influx may provide a new target to modulate MDR in chemotherapy.

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1α binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the βγδ complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant β, γ, and δ subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the β and δ subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the β subunit and reduced the Km, while addition of α to β or the βδ complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of δ. Thus, the β and δ subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of β was altered by association with other subunits, while the site on δ was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (α2βγ2δ)2 is proposed and discussed. (Mol Cell Biochem 191: 181–186, 1999)

A novel p53 mutant retained functional activity in lung carcinomas

The gene p53 is a critical tumor suppressor that can respond to multiple signals of cellular gatekeepers for growth and division. The mdm2 gene is one of the downstream target genes for transcriptional activation by the product of p53 tumor suppressor gene. Transactivation of mdm2 gene is represented by the presence of a functional P53 protein. To understand the biological function of mutant p53 in tumorigenesis, we constructed a number of p53 mutants by site-directed mutagenesis (H179Y, L194R, S240R, R249S, A276D, E286Q), followed by characterization of each P53 mutants ability to transactivate mdm2, bax and p21waf. The transactivation properties of p53 mutants were compared by co-transfection with pGL-3-mdm2, pGL-3-bax and pGL-3-p21waf into the P53 null cell line H1299 derived from a non-small cell lung carcinoma. Among them mt p53 S240R and E286Q were shown to have enhanced transactivating activity of pGL3-mdm2, at about 43.2 and 28.2% of the wt p53 vector, respectively, while the remaining four had nearly the same level of activity as the negative control did. Furthermore, data indicated that mt p53 S240R had as high an ability to suppress the growth of the p53 null cell line H1299 as wild type p53. Therefore, mutant p53 alone is an insufficient indicator of poor prognosis. Instead, functional p53 may affect lung cancer prognosis.

Recombinant Subunits of Mammalian Elongation Factor 1 Expressed in Escherichia coli SUBUNIT INTERACTIONS, ELONGATION ACTIVITY, AND PHOSPHORYLATION BY PROTEIN KINASE CKII

The first step in elongation requires two different activities; elongation factor (EF)-1α transfers aminoacyl-tRNA to the ribosome and is released upon hydrolysis of GTP, EF-1βγδ catalyzes exchange of GDP on EF-1α with GTP. To analyze the role of the individual subunits of EF-1 in elongation, the cDNAs for the β, γ, and δ subunits of EF-1 from rabbit were cloned, and proteins of 225, 437, and 280 amino acids, respectively, were expressed in Escherichia coli. The purified recombinant β subunit migrates as a dimer and the γ subunit as a trimer upon gel filtration, whereas the δ subunit forms a large aggregate. Complexes of βγ, γδ and βγδ were formed by self-association and eluted with a molecular mass of approximately 160, 530, and 670 kDa, respectively; no interaction was observed between β and δ. The activity of the recombinant subunits was determined with native EF-1α by measuring stimulation of the rate of elongation by poly(U)-directed polyphenylalanine synthesis. Recombinant β and δ alone stimulated the rate of elongation by 10-fold, with a ratio of 5α:2β or δ. The βγδ complex stimulated EF-1α activity up to 10-fold with a ratio of 20α to 1βγδ. Phosphorylation of the β and δ subunits alone or in βγδ by protein kinase CKII had no effect on the rate of elongation.

Pemetrexed induces both intrinsic and extrinsic apoptosis through ataxia telangiectasia mutated/p53-dependent and -independent signaling pathways

Pemetrexed, a new‐generation antifolate, has demonstrated promising single‐agent activity in front‐ and second‐line treatments of non‐small cell lung cancer. However, the molecular mechanism of pemetrexed‐mediated antitumor activity remains unclear. The current study shows that pemetrexed induced DNA damage and caspase‐2, ‐3, ‐8, and ‐9 activation in A549 cells and that treatment with caspase inhibitors significantly abolished cell death, suggesting a caspase‐dependent apoptotic mechanism. The molecular events of pemetrexed‐mediated apoptosis was associated with the activation of ataxia telangiectasia mutated (ATM)/p53‐dependent and ‐independent signaling pathways, which promoted intrinsic and extrinsic apoptosis by upregulating Bax, PUMA, Fas, DR4, and DR5 and activating the caspase signaling cascade. Supplementation with dTTP allowed normal S‐phase progression and rescued apoptotic death in response to pemetrexed. Overall, our findings reveal that the decrease of thymidylate synthase and the increase of Bax, PUMA, Fas, DR4, and DR5 genes may serve as biomarkers for predicting responsiveness to pemetrexed.

Hepatitis delta virus epigenetically enhances clusterin expression via histone acetylation in human hepatocellular carcinoma cells.

Both isoforms of the hepatitis delta antigen (HDAg) of hepatitis delta virus (HDV) are highly associated with virus proliferation and may act as co-activators of cellular gene expression. Human hepatocellular carcinoma (HCC) cell line Huh7, which stably expresses HDAgs, was differentially screened and the results showed that clusterin gene expression was enhanced. The mechanisms for HDAg-mediated clusterin gene upregulation were investigated. Expression of HDAgs was associated with enhanced histone H3 acetylation within the clusterin promoter in a chromatin immunoprecipitation assay. Transient transfection of HDAg-expressing plasmids into Huh7 cells also enhanced clusterin expression and histone acetylation. Furthermore, HDV replication was associated with histone hyperacetylation and clusterin induction. The effect of increased clusterin expression was determined by a chemosensitivity assay with adriamycin treatment. These data indicated that HDV-induced clusterin protein increases cell survival potential. Thus, it is possible that epigenetic regulation by HDV contributes to a pathological outcome of hepatitis D/hepatitis B viral hepatitis and HCC.