Retinoblastoma (RB) Gene Structure and Function in Primary Canine Osteosarcoma Cell Lines

Abstract

Tumor suppressor genes are an important factor in the transformation of normal to cancerous cells. The retinoblastoma susceptibility gene (Rb), when deleted, removes an important cell growth negative regulator and thus subjects the cell to continuous DNA replication. The objectives in this study are to establish canine osteosarcoma cell lines from primary explants and to compare their Rb gene expression to positive and negative human and canine cell line controls via immunoblotting and Northern analysis. The gene itself will also be examined using Southern analysis. Immunoblotting with two different anti-human Rb antibodies yielded no cross-reactivity with canine proteins. Attempts to optimize the heterologous recognition of canine proteins were not successful. Experiments are planned to develop specific anti-canine Rb reagents for characterization of canine samples. Initial Northern analyses of canine Rb expression utilizing a human cDNA were inconclusive and will require further optimization for proper interpretation. The human Rb cDNA has previously been used to describe canine Rb restriction fragment length polymorphisms (RFLPs) in genomic DNA extracted from canine tumors. Optimal Southern analysis of Rb RFLPs in primary canine osteosarcoma cell lines is in progress but has not been completed.