FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1969297400> ?p ?o ?g. }

• W1969297400 endingPage "2041" @default.
• W1969297400 startingPage "2029" @default.
• W1969297400 abstract "Background & Aims: RNA interference (RNAi) is a powerful tool to silence gene expression. The adenoviral vector expressing small interfering RNA (siRNA) is highly effective in mammalian cells. However, its potential use as a therapeutic tool to target an oncogene specifically remains to be seen. We applied the adenovirus-delivered siRNA (AdSiRNA) to inhibit a hepatocellular carcinoma (HCC) oncogene, p28GANK, in HCC cell lines and investigated its antitumor effects. Methods: The T7-RNA polymerase system was used to screen the specific target site. Double-strand oligonucleotide for transcription of short hairpin RNA was constructed into the adenoviral vector. Four HCC cell lines were infected with the RNAi-containing adenovirus. The RNAi effects on HCC were studied in cultured cells as well as in animal models. Results: p28GANK expression was suppressed by up to 80% in HCC cells. Depletion of p28GANK inhibited HCC cell growth and tumorigenesis, enhanced dephosphorylation of RB1, and decreased transcription activity of E2F-1 in HuH-7 cells. Furthermore, depletion of p28GANK induced caspase-8- and caspase-9-mediated apoptosis of HCC cells. Finally, targeting p28GANK by adenovirus injection inhibited the growth of established tumors in nude mice. Conclusions: This study shows that the T7-system screening-based AdSiRNA can be used successfully to silence an oncogene. We proved the therapeutic potential of AdSiRNA on the treatment of HCC by targeting p28GANK. Our results indicate that p28GANK may serve as a novel therapeutic target for treating HCC. Background & Aims: RNA interference (RNAi) is a powerful tool to silence gene expression. The adenoviral vector expressing small interfering RNA (siRNA) is highly effective in mammalian cells. However, its potential use as a therapeutic tool to target an oncogene specifically remains to be seen. We applied the adenovirus-delivered siRNA (AdSiRNA) to inhibit a hepatocellular carcinoma (HCC) oncogene, p28GANK, in HCC cell lines and investigated its antitumor effects. Methods: The T7-RNA polymerase system was used to screen the specific target site. Double-strand oligonucleotide for transcription of short hairpin RNA was constructed into the adenoviral vector. Four HCC cell lines were infected with the RNAi-containing adenovirus. The RNAi effects on HCC were studied in cultured cells as well as in animal models. Results: p28GANK expression was suppressed by up to 80% in HCC cells. Depletion of p28GANK inhibited HCC cell growth and tumorigenesis, enhanced dephosphorylation of RB1, and decreased transcription activity of E2F-1 in HuH-7 cells. Furthermore, depletion of p28GANK induced caspase-8- and caspase-9-mediated apoptosis of HCC cells. Finally, targeting p28GANK by adenovirus injection inhibited the growth of established tumors in nude mice. Conclusions: This study shows that the T7-system screening-based AdSiRNA can be used successfully to silence an oncogene. We proved the therapeutic potential of AdSiRNA on the treatment of HCC by targeting p28GANK. Our results indicate that p28GANK may serve as a novel therapeutic target for treating HCC. RNA interference (RNAi) is the process whereby double-stranded RNA results in rapid destruction of a specific messenger RNA (mRNA).1Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. How cells respond to interferons.Annu Rev Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3361) Google Scholar Because 21- to 23-nucleotide small interfering RNA (siRNA) generated by ribonuclease III cleavage from longer double-stranded RNA was shown to induce efficient RNAi in mammalian cells,2Hannon G.J. RNA interference.Nature. 2002; 418: 244-251Crossref PubMed Scopus (3486) Google Scholar, 3Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature. 2001; 411: 494-498Crossref PubMed Scopus (8081) Google Scholar siRNA has been used routinely in gene silencing by transfection of chemically synthesized siRNA.4Sharp P.A. RNA interference 2001.Genes Dev. 2001; 15: 485-490Crossref PubMed Scopus (655) Google Scholar To circumvent the high cost of synthetic siRNA and to establish stable gene knock-down cell lines by siRNA, several plasmid vector systems were designed to produce siRNA inside cells driven by RNA polymerase III-dependent promoters such as U6 and H1-RNA gene promoters.5Czauderna F. Santel A. Hinz M. Fechtner M. Durieux B. Fisch G. Leenders F. Arnold W. Giese K. Klippel A. Kaufmann J. Inducible shRNA expression for application in a prostate cancer mouse model.Nucleic Acids Res. 2003; 31: e127Crossref PubMed Scopus (151) Google Scholar, 6Paddison P.J. Caudy A.A. Bernstein E. Hannon G.J. Conklin D.S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.Genes Dev. 2002; 16: 948-958Crossref PubMed Scopus (1306) Google Scholar, 7Sui G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.Proc Natl Acad Sci U S A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google Scholar, 8Miyagishi M. Taira K. U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells.Nat Biotechnol. 2002; 20: 497-500Crossref PubMed Scopus (677) Google Scholar, 9Paul C.P. Good P.D. Winer I. Engelke D.R. Effective expression of small interfering RNA in human cells.Nat Biotechnol. 2002; 20: 505-508Crossref PubMed Scopus (749) Google Scholar Nevertheless, transient siRNA expression and low and variable transfection efficiency remain the problems for chemically synthesized and vector-derived siRNA. Recently, several virus vectors have been developed for efficient delivery of siRNA into mammalian cells.10Van den Haute C. Eggermont K. Nuttin B. Debyser Z. Baekelandt V. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in mouse brain.Hum Gene Ther. 2003; 14: 1799-1807Crossref PubMed Scopus (104) Google Scholar, 11Barton G.M. Medzhitov R. Retroviral delivery of small interfering RNA into primary cells.Proc Natl Acad Sci U S A. 2002; 99: 14943-14945Crossref PubMed Scopus (261) Google Scholar, 12Xia H. Mao Q. Paulson H.L. Davidson B.L. siRNA-mediated gene silencing in vitro and in vivo.Nat Biotechnol. 2002; 20: 1006-1010Crossref PubMed Scopus (793) Google ScholarFor high-level expression of exogenous gene products, as required in cancer treatment, adenovirus is an efficient tool13Verma I.M. Somia N. Gene therapy—promises, problems and prospects.Nature. 1997; 389: 239-242Crossref PubMed Scopus (1548) Google Scholar, 14Vile R. Cancer gene therapy—new approaches to tumour cell killing.J Gene Med. 2000; 2: 141-143Crossref PubMed Scopus (17) Google Scholar with a highly hepatotrophic character.15Li Q. Kay M.A. Finegold M. Stratford-Perricaudet L.D. Woo S.L. Assessment of recombinant adenoviral vectors for hepatic gene therapy.Hum Gene Ther. 1993; 4: 403-409Crossref PubMed Scopus (295) Google Scholar, 16Kay M.A. Graham F. Leland F. Woo S.L. Therapeutic serum concentration of human alpha-1-antitrypsin after adenoviral mediated gene transfer into mouse hepatocytes.Hepatology. 1995; 21: 815-819PubMed Google Scholar Furthermore, adenoviral vectors are relatively easy to produce and high titers of recombinant adenovirus particles may be obtained.17Anderson W.F. Human gene therapy.Nature. 1998; 392: 25-30Crossref PubMed Scopus (64) Google Scholar These vectors are valuable particularly for transgene expression in hard-to-transfect cells and have been used widely for the expression of transgenes under both clinical and experimental conditions. In this study we sought to use an adenoviral vector using modified RNA Pol II cytomegalovirus promoter to deliver siRNA targeting an oncogene, to determine whether this technique can be used to inhibit oncogene over-expression specifically, and whether this inhibition results in antitumor effects.Hepatocellular carcinoma (HCC) ranks among the most common malignancies in Asia and tropical Africa.18Chen C.J. Yu M.W. Liaw Y.F. Epidemiological characteristics and risk factors of hepatocellular carcinoma.J Gastroenterol Hepatol. 1997; 12: S294-S308Crossref PubMed Scopus (452) Google Scholar, 19Sithinamsuwan P. Piratvisuth T. Tanomkiat W. Apakupakul N. Tongyoo S. Review of 336 patients with hepatocellular carcinoma at Songklanagarind Hospital.World J Gastroenterol. 2000; 6: 339-343PubMed Google Scholar Although there are many modalities of treatment, the recurrence and metastasis rates are high, and the prognosis is unsatisfactory.20Tang Z.Y. Hepatocellular carcinoma.J Gastroenterol Hepatol. 2000; 15: G1-G7Crossref PubMed Scopus (57) Google Scholar Therefore, the understanding of the molecular mechanisms involved in HCC initiation and progression becomes critical to developing more effective treatments for HCC. Recently, a novel oncogene named gankyrin was cloned in HCC by complementary DNA subtractive hybridization.21Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Reduced stability of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas.Nat Med. 2000; 6: 96-99Crossref PubMed Scopus (278) Google Scholar Its sequence is identical to the p28 gene, whose product is one of the nonadenosine triphosphatase subunits of PA700 (19S), a regulatory complex of the human 26S proteasome.22Hori T. Kato S. Saeki M. DeMartino G.N. Slaughter C.A. Takeuchi J. Toh-e A. Tanaka K. cDNA cloning and functional analysis of p28(Nas6p) and p40.5(Nas7p), two novel regulatory subunits of the 26S proteasome.Gene. 1998; 216: 113-122Crossref PubMed Scopus (78) Google Scholar The product of this novel gene (p28GANK) containing 6 ankyrin repeats has been shown to form complexes with RB1, cyclin-dependent kinase 4, the S6 adenosine triphosphatase subunit of the 26S proteasome, and MAGE-A4.23Dawson S. Apcher S. Mee M. Higashitsuji H. Baker R. Uhle S. Dubiel W. Fujita J. Mayer R.J. Gankyrin an ankyrin-repeat oncoprotein interacts with CDK4 kinase and the S6 ATPase of the 26S proteasome.J Biol Chem. 2002; 277: 10893-10902Crossref PubMed Scopus (111) Google Scholar, 24Nagao T. Higashitsuji H. Nonoguchi K. Sakurai T. Dawson S. Mayer R.J. Itoh K. Fujita J. MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity.J Biol Chem. 2003; 278: 10668-10674Crossref PubMed Scopus (86) Google Scholar The p28GANK gene is one of the first genes to be over-expressed in a rodent model of hepatocarcinogenesis.25Park T.J. Kim H.S. Byun K.H. Jang J.J. Lee Y.S. Lim I.K. Sequential changes in hepatocarcinogenesis induced by diethylnitrosamine plus thioacetamide in Fischer 344 rats induction of gankyrin expression in liver fibrosis, pRB degradation in cirrhosis, and methylation of p16(INK4A) exon 1 in hepatocellular carcinoma.Mol Carcinog. 2001; 30: 138-150Crossref PubMed Scopus (55) Google Scholar Its overproduction leads to transformation of cultured NIH/3T3 cells and induces tumor formation in nude mice. p28GANK over-expression accelerates hyperphosphorylation and degradation of RB1, releasing the activity of the transcription factor E2F-1 from RB1 repressor complex. Furthermore, binding of p28GANK to cyclin-dependent kinase 4 prevents the latter from binding to p16INK4a.26Li J. Tsai M.D. Novel insights into the INK4-CDK4/6-Rb pathway counter action of gankyrin against INK4 proteins regulates the CDK4-mediated phosphorylation of Rb.Biochemistry. 2002; 41: 3977-3983Crossref PubMed Scopus (82) Google Scholar p16INK4a is a member of the INK4 family of proteins, which inhibit cyclin-dependent kinase 4/6 and thus themselves have tumor-suppressor functions. As a p16INK4a antagonist, p28GANK functions as an accelerator for cell-cycle progression. Our previous study showed the over-expression of p28GANK mRNA was found in ∼97% of HCC patients.27Fu X.Y. Wang H.Y. Tan L. Liu S.Q. Cao H.F. Wu M.C. Over-expression of p28/gankyrin in human hepatocellular carcinoma and its clinical significance.World J Gastroenterol. 2002; 8: 638-643Crossref PubMed Scopus (89) Google Scholar So we hypothesize that p28GANK can be used as a promising target for drug therapy in HCC. Here, we discuss our study of the applicability of the adenovirus-delivered siRNA (AdSiRNA) as therapy against p28GANK over-expression in HCC by ex vivo and in vivo experiments.Materials and MethodsCell CultureHepG2, Hep3B, and 293T cell lines were obtained from American Type Culture Collection (Manassas, VA) and the HuH-7 cell line was kindly provided by Dr. Axel Ullrich (Max-Planck-Institute of Biochemistry, Martinsried, Germany). The SMMC-7721 cell line was from the Cell Research Institute of the Chinese Academy of Sciences (Shanghai, China). The HEK293 cell line was a generous gift of Dr. Nan Li (Department of Immunology, Second Military Medical University [SMMU], China). All of the cells were grown in Dulbecco’s modified Eagle medium (Gibco BRL, Life Technologies, NY) with 10% fetal bovine serum supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. Primary human hepatocytes (PHHs) were isolated from the fresh surgical specimens of patients undergoing partial hepatectomy for hemangioma. Informed consent was obtained from each patient and the procedure was approved by the local medical committee. Healthy liver tissue (5–20 g) was used to isolate normal PHHs by a 2-step collagenase perfusion and PHHs were cultured according to the established methods.28Gripon P. Diot C. Theze N. Fourel I. Loreal O. Brechot C. Guguen-Guillouzo C. Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide.J Virol. 1988; 62: 4136-4143Crossref PubMed Google Scholar, 29Berry M.N. Halls H.J. Grivell M.B. Techniques for pharmacological and toxicological studies with isolated hepatocyte suspensions.Life Sci. 1992; 51: 1-16Crossref PubMed Scopus (81) Google Scholar All cells were maintained in a humidified 37°C incubator with 5% CO2.Identification of Effective Target Site in the p28GANK Coding RegionTo select the specific siRNA sequence that can suppress p28GANK gene expression effectively, we used T7-RNA polymerase-directed in vitro transcription to obtain 3 siRNAs double strands corresponding to different coding regions of the p28GANK gene. In vitro transcription using T7-RNA polymerase requires the first nucleotide of the RNA transcript to be G and to have a C at the end to allow annealing with the complementary RNA, which also starts with a G.30Milligan J.F. Groebe D.R. Witherell G.W. Uhlenbeck O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.Nucleic Acids Res. 1987; 15: 8783-8798Crossref PubMed Scopus (1878) Google Scholar For siRNA studies in mammalian cells, two 21 (22)-mer RNAs with 19 (20) complementary nucleotides and 3′ terminal noncomplementary dimers of uridine necessitates the target sequences for an siRNA should start with AA.31Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature. 2001; 411: 494-498Crossref PubMed Scopus (6051) Google Scholar Thus, under the AAG-N17 (N18)-C rule, 3 target sites were selected. Desalted DNA oligonucleotides were ordered from Sangon Corporation (Shanghai, China) (Table 1). The T7-RNA polymerase-directed production of small RNA transcripts has been described previously.34Donze O. Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase.Nucleic Acids Res. 2002; 30: e46Crossref PubMed Scopus (240) Google Scholar The annealed T7-RNA polymerase-synthesized siRNAs (T7-siRNAs) were stored at −20°C before use.Table 1Oligonucleotide Templates, Real-Time PCR Primers, and Probes Used in This StudyUsageNameSequence (5′→3′)T7-siRNA transcriptionT7 primerTAATACGACTCACTATAGGFP-SaGFP-RNAi target sequence was chosen according to Caplen et al.32ATGAACTTCAGGGTCAGCTTGCTATAGTGAGTCGTATTAGFP-ACGGCAAGCTGACCCTGAAGTTCTATAGTGAGTCGTATTANucleotide-1-SAAGAATACTCTCCTTCAACTCTATAGTGAGTCGTATTANucleotide-1-AAAGAGTTGAAGGAGAGTATTCTATAGTGAGTCGTATTANucleotide-2-SAAGACACTGAGGGTAACACTCCTATAGTGAGTCGTATTANucleotide-2-AAAGGAGTGTTACCCTCAGTGTCTATAGTGAGTCGTATTANucleotide-3-SAAGGTGGCCTGGGTTTAATACTATAGTGAGTCGTATTANucleotide-3-AAAGTATTAAACCCAGGCCACCTATAGTGAGTCGTATTAReal-time RT-PCRp28GANK-SATGCTAAGGACCATTATGAGGCTAp28GANK-ATCTTGGATGTTTGTGGATGCTTTGp28GANK-PAGCAATGCACCGGGCAGCAGCβ-actin-Sbβ-actin real-time RT-PCR used primers and probe as described as Kreuzer et al.33AGCCTCGCCTTTGCCGAβ-actin-ACTGGTGCCTGGGGCGβ-actin-PCCGCCGCCCGTCCACACCCGCCNOTE. Nucleotide-1, -2, and -3 are target sites in the p28GANK coding region. S, sense primer; A, antisense primer; P, probe.a GFP-RNAi target sequence was chosen according to Caplen et al.32Caplen N.J. Parrish S. Imani F. Fire A. Morgan R.A. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.Proc Natl Acad Sci U S A. 2001; 98: 9742-9747Crossref PubMed Scopus (923) Google Scholarb β-actin real-time RT-PCR used primers and probe as described as Kreuzer et al.33Kreuzer K.A. Lass U. Landt O. Nitsche A. Laser J. Ellerbrok H. Pauli G. Huhn D. Schmidt C.A. Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of beta-actin transcripts as quantitative reference.Clin Chem. 1999; 45: 297-300Crossref PubMed Scopus (170) Google Scholar Open table in a new tab Cotransfection of pEGFP-C1 and influenza hemagglutin (HA)-tagged p28GANK plasmids along with T7-siRNAs into 293T cells was performed using Lipofectamine (Invitrogen, Life Technologies, Carlsbad, CA). Cells were harvested at 48 hours after transfection, washed once with cold phosphate-buffered saline (PBS), and lysed in lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.4, 2 mmol/L ethylenediaminetetraacetic acid, 1% NP-40) containing protease inhibitors (Boehringer Mannheim, Mannheim, Germany). The protein concentration was determined with the bicinchoninic acid (BCA) protein assay reagent in accordance with the manufacturer’s protocol (Pierce, Rockford, IL). Extracts (40 μg per lane) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane. The proteins were revealed with anti-green fluorescence protein (GFP) (NeoMarkers, Fremont, CA), anti-HA (Roche Diagnostics, Indianapolis, IN), and anti-actin (Sigma, St. Louis, MO) antibodies, followed by incubation with horseradish-peroxidase-conjugated anti-rabbit or mouse immunoglobulin G secondary antibodies (Sigma). The bands were visualized by using the enhanced chemiluminescence system (Santa Cruz Biotechnology, Santa Cruz, CA).Real-time reverse-transcription polymerase chain reaction (RT-PCR) was applied to detect the mRNA level in transfected cells. Briefly, mRNA in transfected and mock cells was extracted by using TRIzol reagent (Gibco BRL). The RT reaction was performed in a 25-μL volume with the avian myeloblastosis virus (AMV) reverse transcriptase (Invitrogen) under the recommended conditions in the presence of a random hexamer primer. The primers and probes used for real-time quantitative PCR are listed in Table 1. All PCR reactions were performed using an iCycle thermal cycling instrument (Bio-Rad Laboratories, Hercules, CA). For each PCR run, a master mix was prepared on ice with 1 × PCR buffer, 5 mmol/L MgCl2, 200 μmol/L deoxynucleoside triphosphate, 200 nmol/L of each primer, 200 nmol/L probe, 1 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Life Sciences), and 2 μL of complementary DNA solution in a total of 50 μL. The thermal cycling conditions were an initial denaturation step at 94°C for 5 minutes and 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. PCR reactions for β-actin were performed as the inner control. Results of the real-time PCR were processed according to the method described.35Johnson M.R. Wang K. Smith J.B. Heslin M.J. Diasio R.B. Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction.Analytical Biochem. 2000; 278: 175-184Crossref PubMed Scopus (322) Google Scholar The mRNA levels of transfected cells were standardized against the mock cells (set at 1).Production of Recombinant AdenovirusWe adopted the GeneSuppressor system (IMG-1200) purchased from Imgenex Corporation (San Diego, CA) to construct the AdSiRNA targeting oncoprotein p28GANK in HCC cell lines. This system is based on the expression of short-hairpin RNAs from modified Pol II cytomegalovirus promoter and a minimal polyA cassette.12Xia H. Mao Q. Paulson H.L. Davidson B.L. siRNA-mediated gene silencing in vitro and in vivo.Nat Biotechnol. 2002; 20: 1006-1010Crossref PubMed Scopus (793) Google Scholar The recombinant adenovirus is made in HEK293 cells by homologous recombination between a linearized shuttle plasmid (pSuppressor-Adeno) expressing the short-hairpin RNAs and the backbone plasmid (pacAD5). The adenoviral system allows virus-mediated delivery of siRNA (short-hairpin RNAs) into various cell types, particularly those not amenable to efficient transfection. According to the earlier-described screening of effective siRNA targeting p28GANK, nucleotide-2 was selected to be synthesized as an inserted sequence. The synthesized oligonucleotides containing Xho I and Xba I overhangs were as follows: Sip28GANK forward, 5′-TCGAAAGACACTGAGGGTAACACTCCttcaagagaGGAGTGTTACCCTCAGTGTCTT-3′; reverse, 5′-CTAGAAGACACTGAGGGTAACACTCCtctcttgaaGGAGTGTTACCCTCAGTGTCTT-3′. The nucleotides for the stem-loop structure are lower case.36Brummelkamp T.R. Bernards R. Agami R. A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 19: 550-553Crossref Scopus (3945) Google Scholar Production of recombinant adenovirus was performed following the manufacturer’s protocol. Briefly, 2 annealed complementary oligonucleotides were cloned into pSuppressorAdeno vector by Xho I and Xba I. Positive clones were selected and confirmed by DNA miniprep (Qiagens, Hilden, Germany) Sal I digestion, and DNA sequencing. Because the Sal I site would be lost during cloning, the recombinant plasmids containing inserts will not be linearized by Sal I. The shuttle plasmids pSuppressorAdeno-p28GANK and pSuppressorAdeno-GFP (provided in the kit) were linearized with Pac I and purified by ethanol precipitation. A total of 1.5 × 106 cells of packaging cell line HEK293 were plated in 60-mm plates the day before transfection. Cells were transfected with 15 μg of linearized shuttle plasmids and 4 μg pacAD5 backbone plasmid by the transfection reagent provided in the kit. The next day, the medium containing the transfection mix was replaced with 6 mL of growth medium. Transfected cells were incubated for an additional 7–10 days. Viruses (AdSip28GANK and AdSiGFP) were harvested and identified by PCR. The amplification and titering of viruses were according to the manufacturer’s instructions (Imgenex). To acquire the equal infection efficiency in different HCC cell lines, the adenovirus 5 (the same virus type as in the virus-mediated RNAi system) packaging of GFP (Ad5-GFP, from Stratagene, La Jolla, CA) was infected into 4 HCC cell lines and PHHs and showed the same as up to 90% infection efficiency in HepG2, Hep3B, HuH-7, and PHHs cells at a multiplicity of infection (MOI) of 10, in SMMC-7721 cells at a MOI of 20, at which concentrations no virus-toxicity effect on cells was found. Thus, the following experiments were performed using viruses at such MOIs except for special indications.Adenoviral Infection, RT-PCR, and Western BlotsFour HCC cells were plated at a density of 2.5 × 104/cm2. Viral infection was performed on the following day in a minimal volume of serum-free Dulbecco’s modified Eagle medium for 1.5 hours. Mock (without virus) cultures were used as additional controls. After infection for 1.5 hours, 2 mL of fresh growth medium was added and cells were placed in the incubator for an additional 2–4 days. Cells were lysed and mRNA was prepared by TRIzol reagent (Gibco BRL). Real-time RT-PCR was performed to determine the mRNA level in infected cells following the same procedure described earlier. For protein detection, the cell lysates were harvested and analyzed by Western blots. The antibodies used were anti-p28GANK (Santa Cruz), anti-RB1/phosph-Ser780-RB1/phosph-Ser807-RB1 (Cell Signaling Technology, Beverly, MA), anti-poly(ADP)ribose polymerase (PARP) (Cell Signaling Technology), anti-caspase-8/caspase-9 (NeoMarkers), and anti-actin (Sigma) antibodies. Densitometry analysis of protein levels was performed by using Scion-Image 4.0.2 software (Scion Corporation, Frederick, MD).MTT AssayMTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assays were performed according to the procedure described.37Carmichael J. DeGraff W.G. Gazdar A.F. Minna J.D. Mitchell J.B. Evaluation of a tetrazolium-based semiautomated colorimetric assay assessment of chemosensitivity testing.Cancer Res. 1987; 47: 936-942PubMed Google Scholar Briefly, the cells (5 × 103 cells/well) were seeded in quadruplicate in 96-well, flat-bottomed plates. The following day, cells were treated with 25 μL/well of AdSip28GANK, AdSiGFP, or were untreated (mock) for 1.5 hours before adding fresh growth medium for an additional 5 days of incubation. Each day 20 μL of MTT solution (5 mg/mL in PBS) (Amresco, Solon, OH) was added to the wells and incubated for 4 hours at 37°C. MTT crystals then were solubilized in isopropanol with 2% sodium dodecyl sulfate and .04 mol/L HCl overnight, and the plates were read in a Microplate Reader (Bio-Rad Model 550; Tokyo, Japan) at 570 nm. Four replicate wells were tested per assay condition, and each experiment was repeated twice.Colony Formation Assay and Ex Vivo Tumor InhibitionSMMC-7721 cells were infected with AdSip28GANK or AdSiGFP at an MOI of 20 for 4 days. Then the infected and mock cells (500 cells/well) were plated in each of 3 wells of a 6-well plate. At day 14 the plates were fixed in 70% methanol, stained with .1% crystal violet, and the numbers of colonies greater than 100 μm in diameter were counted.Four days after infection, 3 × 106 infected or mock SMMC-7721 cells were injected subcutaneously into male athymic nude mice (4 weeks of age, 5 mice/group; BiKai Co., Shanghai, China). The tumors were monitored with a caliper every week over a 5-week period after initial cells were injected. The tumor volume was calculated according to the formula: V = length × width2 × .5.38Huang X. Wong M.K. Zhao Q. Zhu Z. Wang K.Z. Huang N. Ye C. Gorelik E. Li M. Soluble recombinant endostatin purified from Escherichia coliantiangiogenic activity and antitumor effect.Cancer Res. 2001; 61: 478-481PubMed Google ScholarCell-Cycle AnalysisStandard fluorescence-activated cell-sorter analysis was used to determine the distribution of cells in cell cycle or apoptosis of the cells. Briefly, the cells were infected with AdSip28GANK or AdSiGFP virus for 2–4 days. Adherent cells then were collected by trypsinization and fixed with 70% ethanol overnight at 4°C. After washing with PBS, the cells were treated with 100 μg/mL RNase A (Roche Diagnostics), 50 μg/mL of propidium iodide (Sigma), and .05% (vol/vol) Triton X-100, and incubated for 45 minutes at room temperature. The samples were analyzed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). The cell-cycle distribution was established by plotting the intensity of the propidium iodide signal, which reflects the cellular DNA content. Apoptotic cells were identified as a hypodiploid DNA peak representing cells (sub-G1). Findings from at least 20,000 cells were collected and analyzed with CellQuest software (Becton Dickinson).E2F-1 Reporter Gene AssayHuH-7 cells (2.5 × 104/cm2) were plated in a 24-well plate the day before infection. After being infected with AdSip28GANK or AdSiGFP for 2 days, the mock and infected cells were transfected with a combination of plasmids encoding firefly luciferase under control of the human E2F-1 promoter (115-bp synthesized DNA fragment, gift of Dr. Stephen Safe, 200 ng of plasmid) and Renilla luciferase under the cytomegalovirus promoter (2 ng of plasmid) using Lipofectamine (Invitrogen). Cells were harvested 36 hours later. Luciferase assays were performed with a commercial dual-luciferase kit (Promega, Madison, WI), using a Lumat LB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany) as the protocol described. The values of luciferase activity driven by the E2F-1 promoter were normalized for Renilla luciferase readings in the same extract.4′,6-Diamidino-2-Phenylindole Staining and Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling AssayAfter infection for 4 days, virus-treated and untreated (mock) cells were fixed with 4% paraformaldehyde (wt/vol) in PBS, pH 7.4, at room temperature for 10 minutes. Then the cells were stained with 4′,6-diamidino-2-phenylindole (Sigma) at 1 μg/mL in PBS for 2 minutes, washed with PBS, and mounted using PBS:glycerol (3:1, vol/vol). Fluorescence was visualized with an Olympus standard fluorescence microscope (Olympus, Tokyo, Japan). Normal nuclei were identified as noncondensed chromatin dispersed over the entire nucleus. Apoptotic nuclei were identified by condensed chromatin, contiguous to the nuclear membrane, and nuclear fragmentation of condensed chromatin.Apoptotic cells were confirmed further by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay using the In Situ Cell Death Detection Kit from Roche (Mannheim, Germany). Briefly, another set of cells used as desc" @default.
• W1969297400 created "2016-06-24" @default.
• W1969297400 creator A5007694279 @default.
• W1969297400 creator A5008390139 @default.
• W1969297400 creator A5029097014 @default.
• W1969297400 creator A5051418301 @default.
• W1969297400 creator A5054780916 @default.
• W1969297400 creator A5059899312 @default.
• W1969297400 creator A5066098323 @default.
• W1969297400 creator A5080930329 @default.
• W1969297400 date "2005-06-01" @default.
• W1969297400 modified "2023-10-17" @default.
• W1969297400 title "Use of Adenovirus-Delivered siRNA to Target Oncoprotein p28GANK in Hepatocellular Carcinoma" @default.
• W1969297400 cites W1563075736 @default.
• W1969297400 cites W1578810612 @default.
• W1969297400 cites W1611419331 @default.
• W1969297400 cites W1616824889 @default.
• W1969297400 cites W1681143022 @default.
• W1969297400 cites W1975853638 @default.
• W1969297400 cites W1977674251 @default.
• W1969297400 cites W1985323089 @default.
• W1969297400 cites W1988967884 @default.
• W1969297400 cites W2002615250 @default.
• W1969297400 cites W2007618242 @default.
• W1969297400 cites W2016961672 @default.
• W1969297400 cites W2029079353 @default.
• W1969297400 cites W2035145745 @default.
• W1969297400 cites W2038783205 @default.
• W1969297400 cites W2041127974 @default.
• W1969297400 cites W2067928188 @default.
• W1969297400 cites W2072543946 @default.
• W1969297400 cites W2085272777 @default.
• W1969297400 cites W2094646089 @default.
• W1969297400 cites W2103565638 @default.
• W1969297400 cites W2112349562 @default.
• W1969297400 cites W2131796097 @default.
• W1969297400 cites W2136447541 @default.
• W1969297400 cites W2137060659 @default.
• W1969297400 cites W2146888510 @default.
• W1969297400 cites W2146900615 @default.
• W1969297400 cites W2152554398 @default.
• W1969297400 cites W2153747430 @default.
• W1969297400 cites W2153761135 @default.
• W1969297400 cites W2159670669 @default.
• W1969297400 cites W2166765502 @default.
• W1969297400 cites W2168353195 @default.
• W1969297400 cites W2171109371 @default.
• W1969297400 cites W2182871465 @default.
• W1969297400 cites W2405592905 @default.
• W1969297400 cites W4211039187 @default.
• W1969297400 cites W4253422011 @default.
• W1969297400 cites W4254660436 @default.
• W1969297400 cites W76685319 @default.
• W1969297400 doi "https://doi.org/10.1053/j.gastro.2005.03.001" @default.
• W1969297400 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15940635" @default.
• W1969297400 hasPublicationYear "2005" @default.
• W1969297400 type Work @default.
• W1969297400 sameAs 1969297400 @default.
• W1969297400 citedByCount "89" @default.
• W1969297400 countsByYear W19692974002012 @default.
• W1969297400 countsByYear W19692974002013 @default.
• W1969297400 countsByYear W19692974002014 @default.
• W1969297400 countsByYear W19692974002015 @default.
• W1969297400 countsByYear W19692974002016 @default.
• W1969297400 countsByYear W19692974002017 @default.
• W1969297400 countsByYear W19692974002018 @default.
• W1969297400 countsByYear W19692974002019 @default.
• W1969297400 countsByYear W19692974002020 @default.
• W1969297400 countsByYear W19692974002021 @default.
• W1969297400 crossrefType "journal-article" @default.
• W1969297400 hasAuthorship W1969297400A5007694279 @default.
• W1969297400 hasAuthorship W1969297400A5008390139 @default.
• W1969297400 hasAuthorship W1969297400A5029097014 @default.
• W1969297400 hasAuthorship W1969297400A5051418301 @default.
• W1969297400 hasAuthorship W1969297400A5054780916 @default.
• W1969297400 hasAuthorship W1969297400A5059899312 @default.
• W1969297400 hasAuthorship W1969297400A5066098323 @default.
• W1969297400 hasAuthorship W1969297400A5080930329 @default.
• W1969297400 hasBestOaLocation W19692974001 @default.
• W1969297400 hasConcept C104317684 @default.
• W1969297400 hasConcept C143998085 @default.
• W1969297400 hasConcept C159047783 @default.
• W1969297400 hasConcept C22615655 @default.
• W1969297400 hasConcept C2778019345 @default.
• W1969297400 hasConcept C502942594 @default.
• W1969297400 hasConcept C54009773 @default.
• W1969297400 hasConcept C54355233 @default.
• W1969297400 hasConcept C71924100 @default.
• W1969297400 hasConcept C86803240 @default.
• W1969297400 hasConceptScore W1969297400C104317684 @default.
• W1969297400 hasConceptScore W1969297400C143998085 @default.
• W1969297400 hasConceptScore W1969297400C159047783 @default.
• W1969297400 hasConceptScore W1969297400C22615655 @default.
• W1969297400 hasConceptScore W1969297400C2778019345 @default.
• W1969297400 hasConceptScore W1969297400C502942594 @default.
• W1969297400 hasConceptScore W1969297400C54009773 @default.
• W1969297400 hasConceptScore W1969297400C54355233 @default.
• W1969297400 hasConceptScore W1969297400C71924100 @default.

• next