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Involvement of p63 in the herpes simplex virus-1-induced demise of corneal cells Research

The transcription factor p63 plays a pivotal role in the development and maintenance of epithelial tissues, including the ocular surface. In an effort to gain insight into the pathogenesis of keratitis caused by HSV-1, we determined the expression patterns of the p63 and Bax proteins in the Staatens Seruminstitute Rabbit Cornea cell line (SIRC). Methods: SIRC cells were infected with HSV-1 at various multiplicities and maintained for different periods of time. Virus replication was measured by indirect immunofluorescence assay and Western blot analysis. Cell viability was determined by MTT assay. The apoptotic response of the infected cells was quantified. | Orosz et al. Journal of Biomedical Science 2010 17 47 http www.jbiomedsci.eom content 17 1 47 a NSC Tha cost of publication In Journal of Blomodlcal Science Is boms by tlM National Science Council Taiwan JOURNAL OF BIOMEDICAL SCIENCE RESEARCH Open Access Involvement of p63 in the herpes simplex v rus-1-induced demise of corneal cells László Orosz 1 Éva Gallyas2 Lajos Kemény3 4 Yvette Mándi1 Andrea Facsko5 and Klára Megyeri 1 Abstract Background The transcription factor p63 plays a pivotal role in the development and maintenance of epithelial tissues including the ocular surface. In an effort to gain insight into the pathogenesis of keratitis caused by HSV-1 we determined the expression patterns of the p63 and Bax proteins in the Staatens Seruminstitute Rabbit Cornea cell line SIRC . Methods SIRC cells were infected with HSV-1 at various multiplicities and maintained for different periods of time. Virus replication was measured by indirect immunofluorescence assay and Western blot analysis. Cell viability was determined by MTT assay. The apoptotic response of the infected cells was quantified by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of p63 and Bax proteins. Results Indirect immunofluorescence assays and Western blot analyses demonstrated the presence of HSV-1 glycoprotein D gD in the infected SIRC cell line and the pattern of gD expression was consistent with efficient viral replication. The results of MTT and ELISA assays showed that HSV-1 elicited a strong cytopathic effect and apoptosis played an important role in the demise of the infected cells. Mock-infected SIRC cells displayed the constitutive expression of ANp63a. The expressions of the Bax-P and TAp63y isoforms were considerably increased whereas the level of ANp63a was decreased in the HSV-1-infected SIRC cells. Experiments involving the use of acyclovir showed that viral DNA replication was necessary for the accumulation of