Corneal toxicity of cell-penetrating peptides that inhibit Herpes simplex virus entry.

Cell-penetrating peptides (CPPs) inhibit Herpes simplex virus entry at low micromolar concentrations and may be useful either as prophylactic or therapeutic agents for herpetic keratitis. The aim of this study was to assess the in vitro and in vivo toxicity of three CPPs-EB, TAT-C, and HOM (penetratin)-for the cornea. Incubation of primary (HK320) or immortalized (THK320) human keratocytes with the EB peptide (up to 100 microM), bHOMd (up to 200 microM), or TAT-C (up to 400 microM) resulted in no evidence of toxicity using a formazan dye-reduction assay. Similar results were obtained with a human trabecular meshwork cell line (TM-1), primary human foreskin fibroblasts (DP-9), Vero, and HeLa cells with EB and TATC. The bHOMd peptide showed some toxicity in Vero and HeLa cells, with CC50 values of 70 and 93 microM, respectively. The EB peptide did not inhibit macromolecular synthesis in Vero cells at concentrations below 150 microM, although cell proliferation was blocked at concentrations of EB above 50 microM. In vivo toxicity was assessed by applying peptides in Dulbecco's modified Eagle's medium to the cornea 4 times daily for 7 d. At concentrations 1000 times the IC50 values, the EB and bHOM peptides showed no toxicity, whereas TAT-C caused some mild eyelid swelling. Some slight epithelial cell sloughing was seen with the bKLA peptide in vivo. These results suggest that these CPPs-and EB in particular-have a favorable toxicity profile, and that further development is warranted.

Cytokine induced apoptosis in human retinoblastoma cells.

PURPOSE: To determine potential anti-proliferative properties of interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF-alpha) on human retinoblastoma cells. METHODS: Fluorescent antibody staining was used to detect IFN-gamma and TNF-alpha receptors on the cells. Y79 and Weri Rb-1 cells were exposed to IFN-gamma alone, TNF-alpha alone, or a combination of IFN-gamma and TNF-alpha, and apoptosis was measured by caspase 3 activation and annexin V staining. Cell cycle arrest was measured by BrdU incorporation and FACS analysis. RESULTS: Both cell lines expressed receptors for IFN-gamma and TNF-alpha. There appeared to be two populations of both receptors in the Weri Rb-1 cell line. Apoptosis was induced in Y79 cells by IFN-gamma, but not TNF-alpha, and the combination of the cytokines did not increase apoptosis above IFN-gamma alone in Y79 cells. Apoptosis was induced in Weri Rb-1 cells only upon exposure to both cytokines. The cell cycle was not significantly altered in either cell line. CONCLUSIONS: Human retinoblastoma cells respond to IFN-gamma or a combination of IFN-gamma and TNF-alpha by becoming apoptotic, but Y79 and Weri Rb-1 cells behave differently. The differential response of the two cell lines is not due to a lack of expression of IFN-gamma or TNF-alpha receptors. The data raise the possibility that differences in apoptotic pathways exist between the two cell lines with interesting implications for the induction of apoptosis as a therapy for retinoblastoma.

Evaluation of the antitumor effects of Herpes simplex virus lacking ribonucleotide reductase in a murine retinoblastoma model.

PURPOSE: To determine if an attenuated herpes simplex virus (HSV) lacking the large subunit of ribonucleotide reductase has antitumor effects in a transgenic mouse model of retinoblastoma (LHbetaTAg). METHODS: LHbetaTAg mice were injected ocularly with 1 x 10(6) pfu of the hrR3 virus and tumor sizes were measured 3 weeks later. Replication of the virus in the eye and cultured murine retinoblastoma cells was tested by titration. Distribution of the virus in tumor was measured by X-gal staining. RESULTS: Intraocular injection of mice with hrR3 (n = 24) did not result in a significant reduction in tumor size compared to uninjected (n = 24) or PBS injected controls (n = 16). Neither the hrR3, nor the HSV RE6 mutant, which was previously shown to have antitumor effects in vivo, replicated in cultured murine tumor cells in vitro, compared to wild-type HSV. The hrR3 virus also did not replicate significantly in tumor cells in vivo, compared to normal eye tissue. CONCLUSIONS: These results suggest that mutant HSV lacking ribonucleotide reductase do not display oncolytic activity in the LHbetaTAg mouse and that this model may not be suitable for studying viral oncolysis as a therapy for retinoblastoma.