ATRA enhances the bystander effect of suicide gene therapy driven by the specific promoter LEP 503 in human lens epithelial cells

To establish a novel, targeted lentivirus-mediated LEP503-HSV-tk/GCV suicide gene therapy system combined with all trans-retinoic acid (ATRA) for the inhibition of human lens epithelial cell (HLEC) proliferation and treatment of posterior capsular opacification (PCO) after cataract surgery; to estim...

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Bibliographic Details
Published in:Molecular vision 2012-07, Vol.18, p.2053-2066
Main Authors: Yang, Jin, Liu, Tian-Jin, Jiang, Yong-Xiang, Lu, Yi
Format: Article
Language:English
Subjects:
Adjuvants
Bystander Effect - drug effects
Bystander Effect - genetics
Cell Proliferation
Cell Survival - drug effects
Connexin 43 - genetics
Connexin 43 - metabolism
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Dose-Response Relationship, Drug
Epithelial Cells - cytology
Epithelial Cells - drug effects
Epithelial Cells - metabolism
Gap Junctions - drug effects
Gap Junctions - genetics
Gap Junctions - metabolism
Gene Expression - drug effects
Genes, Reporter
Genes, Transgenic, Suicide
Genetic Vectors
Green Fluorescent Proteins - genetics
HeLa Cells
Herpes simplex virus
Humans
Lens, Crystalline - cytology
Lens, Crystalline - drug effects
Lens, Crystalline - metabolism
Lentivirus - genetics
Promoter Regions, Genetic
Retinal Pigment Epithelium - cytology
Retinal Pigment Epithelium - drug effects
Retinal Pigment Epithelium - metabolism
Tretinoin - pharmacology
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Summary:To establish a novel, targeted lentivirus-mediated LEP503-HSV-tk/GCV suicide gene therapy system combined with all trans-retinoic acid (ATRA) for the inhibition of human lens epithelial cell (HLEC) proliferation and treatment of posterior capsular opacification (PCO) after cataract surgery; to estimate the enhancement of the bystander effect by ATRA; and to explore the role of Connexin43 (Cx43) mediated gap junctional intercellular communication (GJIC) in the bystander effect of the HSV-K/GCV system. A Lenti-LEP503-HSV-tk-EGFP vector was generated by cloning the lens-specific promoter LEP503 (lens specific promoter 503) from genomic DNA of HLECs by PCR. The vector was then inserted into the promoter-less vector from lentivirus-based (CMV)-HSV-tk-EGFP. The expressional specificity of the LEP503 promoter was assessed by investigating the expression of EGFP (enhanced green fluorescent protein) and HSV-tk (herpes simplex virus thymidine kinase) mRNA, both driven by Lenti-LEP503-HSV-tk-EGFP vector, by fluorescence microscopy, RT-PCR, flow cytometry, and western blot assays in HLECs, human adult retinal pigment epithelium cells (RPECs), human adult skin fibroblast cells (ASFCs), and Hela cells. Morphological changes were observed by fluorescence microscopy and cell viability was determined using the Cell Counting kit-8 Cell Proliferation (CCK-8) and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays after Lenti-LEP503-HSV-tk/GCV system combined with ATRA treatment on HLECs. Flow cytometry, DNA fragmentation, and western blot assays were employed to analyze the mechanisms of bystander effects. The promoter LEP503-mediated HSV-tk was specifically expressed in HLECs, and ATRA dose-dependently strengthened the bystander effect following LEP503-mediated HSV-tk/GCV gene therapy against lens cells by upregulating the expression of the gap junction protein Cx43. The Lenti-LEP503-HSV-tk/GCV suicide gene therapy system, combined with ATRA as an adjuvant, may be a feasible supplementary method for PCO treatment that targets residual lens cells.
ISSN:1090-0535
1090-0535