p21Waf1/Cip1 and p53 Gene Therapy in a Mouse Model of Retinoblastoma

Abstract

Abstract: : Purpose: Evaluate the anti-neoplastic effect of gene therapy with human p21^Waf1/Cip1 or p53 in a mouse model of retinoblastoma. Methods: The anti-neoplastic effect of a replication deficient adenoviral construct, expressing human p21^Waf1/Cip1 (rAd.p21) or human p53 (rAd.p53), was studied in vitro on a cell line derived from tumors formed in transgenic mice that ectopically express the SV40 Large T-antigen in the inner nuclear layer of the retina (LHB-Tag). A recombinant adenovirus without an added transgene was used as a vector control (rAd.control). Three different particle concentrations were studied: 10⁷,10⁸, and 10⁹, and cell viability was assessed by trypan blue exclusion and FACS analysis after Annexin V labeling. Subsequent experiments using rAd.p21 were conducted in vivo using LHB-Tag mice, aged from 12 to 13 weeks. These mice received an intra-tumoral injection of 10⁸ particles of either rAd.p21 or rAd.control in the left eye. No treatment was administered in the right eye. Effects of treatment were assessed on parraffin sections of the eyes 2 weeks after injection by morphometric histology, BrdU incorporation and TUNEL. Results: In vitro, rAd.p21 reduced cell viability and increased Annexin V labeling relative to untreated and rAd.control cells at all three concentrations of virus tested. The rAd.p53 construct also increased cell death, but only at the highest concentration used. Preliminary results on in vivo-treated tumors indicate that rAd.p21 increases TUNEL relative to controls. Morphological changes, including the presence of voids, were detected in tumors treated with rAd.p21. These findings may have resulted from increased cell death within the tumor cores. Conclusions: Our data suggest that both p21 and p53 over-expression is able to induce cell death in a mouse model of retinoblastoma, but that p21 is a more potent anti-tumor agent. This may reflect the fact that these tumors originate from the overexpression of the SV40 Tag, which inactivates p53 function.

Keywords: retinoblastoma • gene transfer/gene therapy • cell death/apoptosis