Antibodies detecting abnormalities of the retinoblastoma susceptibility gene product (pp110RB) in osteosarcomas and synovial sarcomas

Abstract

The retinoblastoma (RB) susceptibility gene is one member of a putative "cancer suppressor gene" family in which loss of gene function is associated with tumor formation. Using antibodies generated against a trypE-RB fusion protein, we previously identified a nuclear phosphoprotein, pp110RB, as the RB gene product. Here we describe three additional polyclonal antibodies that were generated to separate epitopes of pp110RB with three synthetic peptides deduced from the RB cDNA sequence. All four antibodies could specifically recognize the same phosphoprotein in human cells. This protein was phosphorylated on serine and threonine, but not tyrosine, residues. RB homologous proteins with molecular masses of 105-128 kD were detected in other vertebrates, such as monkey, rodent, and chicken, by at least two antibodies, indicating evolutionary conservation of the RB gene. These antibodies were specific and sensitive for monitoring RB gene inactivation as demonstrated by screening several osteosarcoma and synovial sarcoma cell lines. Of nine cell lines examined, three expressed no immunoprecipitable normal RB protein. DNA rearrangement and abnormal RB mRNA were detected in two of these three cell lines, whereas RB protein was absent from one synovial sarcoma cell line in which normal-sized RB mRNA was clearly present. Therefore, direct immunoprecipitation of RB protein can reveal RB gene mutations that are undetectable by DNA and mRNA analysis. These results further support a crucial role for the RB gene in the oncogenesis of some mesenchymal tumors.