WILD-TYPE AND MUTANT RETINOBLASTOMA PROTEIN IN PARAFFIN SECTIONS

Inactivation of the retinoblastoma susceptibility (RB) gene plays a ro le in the pathogenesis of a variety of human malignancies, Recently, i t has become feasible to study RE expression in archival tissues, and it is expected that immunohistochemical studies on routinely processed tumors will further elucidate the biologic and clinical significance of RE mutations. Our study was designed to address two issues that are critical for the interpretation of such studies, i.e., whether mutant RE protein (pRB) can reliably be distinguished from normal pRB and wh ether there are significant differences in the performance characteris tics of various anti-RE antibodies, We studied cell blocks of 26 mutan t RE cell lines (11 lines without any RE expression, nine lines expres sing truncated mRNA/pRB, six lines carrying missense mutations) with f ive different anti-RE monoclonal antibodies, using a recently describe d procedure that includes an antigen retrieval step, The specific stai ning pattern for pRB was nuclear, Cytoplasmic staining was found to be nonspecific and could be strong, Some truncated and all full-length m utant pRBs localized to the nucleus, creating positive nuclear stainin g that might be indistinguishable from the staining pattern of cells c arrying wild-type RE. The five antibodies tested showed significant di fferences in sensitivity, specificity, and background reactivity. Our data suggest that a significant subset of mutant pRB has preserved nuc lear translocation capacity, that not all anti-RE antibodies are equal ly suitable for immunohistochemical analysis of RE expression, and tha t any such analysis is bound to include a certain, albeit probably sma ll, number of positive stains, despite the absence of functional pRB.