title: BAX gene over-expression via nucleofection to induce apoptosis in human lens epithelial cells.
abstract: Despite significant advances in cataract surgery techniques, posterior capsule opacification (PCO) remains a common complication. In PCO, remaining epithelial cells cloud the lens capsule and impair postoperative vision. This in vitro study was designed to investigate the potential of a gene-based approach, specifically over-expression of the proapoptotic BAX gene, to prevent PCO. Human lens epithelial cells (HLECs) were transfected by nucleofection with a plasmid encoding a fusion protein of green fluorescent protein and human BAX. The expression levels of BAX and its antiapoptotic counterpart BCL2 were determined by realtime reverse transcription polymerase chain reaction, Western blotting and immunofluorescence. BAX over-expression-induced cell death was analyzed by fluorescence-activated cell sorting using the Annexin V antibody. Fluorescence microscopy and transmission electron microscopy were used to assess changes in morphology and ultrastructure. Differential expression of the downstream apoptosis-related factor, caspase 3, was detected by Western blotting. Nucleofection efficiency was high (nearly 80%). BAX-transfected HLECs showed remarkably enhanced BAX gene expression and BAX:BCL2 ratio, but relatively little change in endogenous BCL2 expression. BAX over-expression also led to significant cytotoxicity, induction of apoptosis-related characteristics and activation of caspase 3. In conclusion, our results indicate that BAX gene over-expression can trigger cell death in HLECs via an apoptotic pathway. Thus, BAX may be a promising candidate for human gene therapy to treat PCO.
authors.affiliation: Department of Ophthalmology, Eye and Ear, Nose and Throat Hospital, Fudan University, 83 Fenyang Road, Shanghai 200031,  China.
authors.forename: Yanwen
authors.initials: Y
authors.lastname: Fang
authors.forename: Xiaofen
authors.initials: X
authors.lastname: Mo
authors.forename: Yi
authors.initials: Y
authors.lastname: Luo
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
mesh_terms: D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002387:Cataract Extraction / Q000009:adverse effects; D002460:Cell Line; D018274:Electroporation; D004847:Epithelial Cells / Q000502:physiology*; D005434:Flow Cytometry; D015870:Gene Expression; D019254:Genes, bcl-2; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology*
chemical_list: D051017:Apoptosis Regulatory Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D053148:Caspase 3