Inhibition of rabbit keratocyte and human fetal lens epithelial cell proliferation by retrovirus-mediated transfer of antisense cyclin G1 and antisense MAT1 constructs

Juergen Kampmeier, Ashley Behrens, Yan Wang, Ann Yee, W. French Anderson, Frederick L. Hall, Erlinda M. Gordon, Peter J. McDonnell

Research output: Contribution to journalArticle

Abstract

The aim of this study is to evaluate the potential of gene transfer of cell cycle control genes as treatment of corneal haze or secondary cataract formation. The guiding hypothesis is that strategic modulation of the cyclin G1 or MAT1 gene by retrovirus-mediated gene transfer will inhibit proliferation of rabbit keratocytes (RabK) and fetal human lens epithelial (FHLEpi) cells in vitro. RabK and FHLEpi cell cultures were transduced in triplicate with retroviral vectors bearing either a nuclear-targeted β- galactosidase, an antisense cyclin G1 (aG1), an antisense MAT1 (aMAT1) construct, or the neo(r) gene. The presence of β-galactosidase activity in the transduced cultures was detected by immunohistochemical X-Gal staining, while cyclin G1 and MAT1 protein expression levels were evaluated by Western analysis. Proliferation of RabKs and FHLEpi cells was analyzed by counting the number of cells in the aG1 and aMAT1 vector-transduced cultures over 5 days. The mean transduction efficiency was 34.4% (SD 1.41) for RabKs and 19.7% (SD 1.83) for FHLEpi cells. Downregulation of cyclin G1 and MAT1 protein expression was noted 24 hr after transduction of RabK cultures with the respective vectors. Cytostatic effects of the aG1 and aMAT1 vectors in both RabKs and FHLEpi cells were most pronounced on the fifth day (RabKs, p < 0.0007; FHEpi cells, p < 0.001). An increased incidence of apoptosis was identified in both aG1 and MAT1-transduced FHLEpi cells. Taken together, these data suggest the potential utility of developing aG1 and aMAT1 retroviral vectors in gene therapy protocols for corneal haze and secondary cataract formation.

Original languageEnglish (US)
Pages (from-to)1-8
Number of pages8
JournalHuman gene therapy
Volume11
Issue number1
DOIs
StatePublished - 2000

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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