The retinoblastoma (RB) gene has a key role in normal cellular growth and human tumor development. LOSS of RB function is a critical event in the initiation or progression of many human cancers both rare and common. Although studies to date have emphasized the C-terminal RB protein region, recent findings from our laboratory indicate that the N-terminal region of the RB protein, as well as utilization of the second AUG start codon, may be of unexpected importance. RB protein translated from the second in-frame AUG codon (pRB94) was a markedly stronger inhibitor of cell growth and cell cycle progression than the full-length RB protein (pRB). To date pRB94 has inhibited the tumor cell growth of all human tumor types studied, including both RB-positive and RB-negative cancer cell lines, whereas pRB has not inhibited growth of RB-positive tumor cells and has much less effect on RB -negative tumor cells. Moreover, pRB94 does not appear to inhibit the growth of normal cells. In addition, significant expression of pRB94 has also been found in senescing progeria fibroblasts and cell lines after the induction of differentiation, and thus may be a component of the aging or the differentiation pathway in various cell types. The similarities and differences between pRB94 and pRB will be examined utilizing inducible RB expression constructs which are designed for production of only pRB94 or pRB. This will include comparison of binding to E2F and other transcription factors. Isolation of specific pRB94-associated proteins will be attempted and to further define N-terminal functional domains of the RB protein. Standard molecular and cellular biological methodologies will be used for these studies, including plasmid vector and cDNA library construction, screening cDNA libraries, subcloning, site directed mutagenesis, DNA sequencing, transfection, protein purification, polyclonal and monoclonal antibody production and microinjection. The results of these studies should provide an in-depth characterization of the N-terminal truncated RB proteins (particularly pRB94) and their role in tumor cell growth suppression, differentiation and senescence as well as their ability to overcome RB human tumor suppressor resistance. These studies will also help elucidate additional functions of the RB in general and provide important basic information regarding normal cell growth regulation as well as the malignant process.
