title: P21-driven multifusion gene system for evaluating the efficacy of histone deacetylase inhibitors by in vivo molecular imaging and for transcription targeting therapy of cancer mediated by histone deacetylase inhibitor.
abstract: Overexpressed histone deacetylase (HDAC) activity has been linked with tumor initiation and progression that prompt the development of histone deacetylase inhibitors (HDACIs) as anticancer agents. HDACI was reported to be able to activate p21 promoter through the SP1 binding sites in the proximal region of p21(WAF1/CIP1) promoter. In this study, we established a p21(WAF1/CIP1) promoter-driven triple-fused reporter gene system (p21-3H) to evaluate the efficacy of HDACI and the ganciclovir (GCV)-mediated anticancer effect contributed by HDACI-induced and p21-driven truncated herpes simplex virus-1 thymidine kinase sr39 mutant (ttksr39) in vitro and in vivo.
  
  
  METHODS
  The p21-3H construct was generated and stably or transiently transfected into H1299 cell lines. These cells were treated with trichostatin A or vorinostat (suberoylanilide hydroxamic acid [SAHA]) to evaluate the activation of p21 promoter-driven reporter gene expression by in vitro confocal fluorescence microscopy, luciferase assay, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil ((3)H-FEAU) cellular uptake, in vivo bioluminescence imaging, and 9-(4-(18)F-fluoro-3-hydroxymethylbutyl) guanine ((18)F-FHBG) small-animal PET imaging. The therapeutic efficacy on p21-3H-expressing tumor xenografts was assessed by daily administration with SAHA (100 mg/kg intraperitoneally) or GCV (20 mg/kg) for 9 d, followed by tumor volume measurement.
  
  
  RESULTS
  On treatment with trichostatin A or SAHA, H1299 cells carrying p21-3H showed a significant increase of luciferase activity, cellular uptake of (3)H-FEAU (Moravek), and DsRed expression. In vivo tumor xenografts carrying p21-3H also showed increased luciferase activity by luminescent imaging and enhanced accumulation of (18)F-FHBG by small-animal PET imaging. Furthermore, when cells transfected with p21-3H or p21/PstI-3H (which lacks p53-binding sites) were treated, the increase of luciferase activity was similar in both groups, indicating that HDACI-induced p21 promoter activation is independent of p53. Both in vitro and in vivo results showed improved therapeutic effect by combined treatment of GCV and HDACI.
  
  
  CONCLUSIONS
  We have established an HDACI-inducible, p21-driven reporter system that has the potential for evaluating the anticancer effect of HDACIs on cancer cells by multiple molecular imaging modalities. Furthermore, ttksr39 in a p21-3H reporter construct provides a potential combination with thymidine kinase-mediated gene therapy to optimize the therapeutic benefit of HDACI.
authors.affiliation: Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Ya-Ju
authors.initials: YJ
authors.lastname: Hsieh
authors.forename: Luen
authors.initials: L
authors.lastname: Hwu
authors.forename: Yi-Chieh
authors.initials: YC
authors.lastname: Chen
authors.forename: Chien-Chih
authors.initials: CC
authors.lastname: Ke
authors.forename: Fu-Du
authors.initials: FD
authors.lastname: Chen
authors.forename: Hsin-Ell
authors.initials: HE
authors.lastname: Wang
authors.forename: Kang-Ping
authors.initials: KP
authors.lastname: Lin
authors.forename: Hsin-Hsien
authors.initials: HH
authors.lastname: Yeh
authors.forename: Chi-Wei
authors.initials: CW
authors.lastname: Chang
authors.forename: Ren-Shyan
authors.initials: RS
authors.lastname: Liu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D004396:Coloring Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D015536:Down-Regulation / Q000187:drug effects; D005260:Female; D050939:Gene Fusion; D056572:Histone Deacetylase Inhibitors / Q000627:therapeutic use*; D006801:Humans; D007202:Indicators and Reagents; D008156:Luciferases / Q000235:genetics; D049449:Luminescence; D051379:Mice; D008819:Mice, Nude; D018613:Microscopy, Confocal; D057054:Molecular Imaging; D009369:Neoplasms / Q000000981:diagnostic imaging* / Q000188:drug therapy*; D010957:Plasmids / Q000235:genetics; D049268:Positron-Emission Tomography; D013778:Tetrazolium Salts; D013844:Thiazoles; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D004396:Coloring Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D056572:Histone Deacetylase Inhibitors; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; D013844:Thiazoles; D008156:Luciferases; C022616:thiazolyl blue
keywords: 18F-FHBG; HSV1-tk; PET; histone deacetylase inhibitor; p21