The under- phosphorylated (p110/pRB), but not the phosphorylated (pI2O/ppRB), form of retinoblastoma protein (RB) serves as an endogenous inhibitor of cell proliferation, differentiation and senescence. Most recently, it has been reported that loss of Rb gene is also associated with inappropriate apoptosis (or programmed cell death) in lens fibre cells. However, how RB protein functions during apoptosis is still unknown. The investigators have found that during the process of apoptosis induced by various anticancer agents (e.g., VP-16) in human promyelocytic leukemia HL60 cells, the level of p110/pRB was high and not substantially changed. In contrast, p120/ppRB was rapidly reduced just at the onset of chemotherapy-induced apoptosis, which were followed first by induction of another phosphorylated form of RB, p115/ppRB, and then by one of several smaller phosphorylated, RB-related proteins, including p68 and p48. They have developed a hypothesis that a ppRB- specific protease(s) (ppRB-PTS) is/are induced by a variety of anticancer drugs at the onset of apoptosis, and that the activation of ppRB-PTS and consequent degradation of p12O/ppRB may play an active role in regulating cellular apoptosis. Therefore, the broad, long-term objective is to define the function of ppRB-PTS in apoptosis.
The Specific Aims of this proposal are the following: 1) to develop an in vitro assay for detection of the ppRB-PTS activity. The drug-induced ppRB-PTS activity will be detected in vitro by incubating a substrate protein, p115/ppRB, prepared from immunoprecipitates of metabolically [32P]orthophosphate-labeled, VP-16-treated HL60 cells, with a whole cell extract from unlabeled, VP-16-treated cells, followed by detecting production of [32P]-labeled p68 and p48; 2) to purify the ppRB-PTS protein and clone its gene. ppRB-PTS will be purified by using chromatographic columns of FPLC, RB antibody and the cleavage site- containing synthetic oligopeptides, followed by gel elution. The purified ppRB-PTS will be sequenced, and its cDNA probe and antibody will be generated. The gene encoding ppRB-PTS will be cloned by using its cDNA probe and/or antibody; 3) to sequence a major ppRB-PTS cleavage site located on RB. p68 and other ppRB fragments will be purified using RB antibody affinity columns and gel elution. Sequences of the purified fragments will be compared to that of the authentic human RB protein, and at least one ppRB-PTS cleavage site will be determined; 4) to study functions of the p120/ppRB degradation in chemotherapy-induced apoptosis. The ppRB-PTS gene will be transfected and expressed in a defined cell line, and the effects to cellular apoptosis will be studied. In addition, it will be tested whether the ppRB-PTS cleavage site-containing oligopeptides can be used as an inhibitor of cellular apoptosis, and whether overexpression of bcl-2 oncoprotein can block the apoptosis- associated ppRB degradation. These studies should provide insight into the molecular mechanisms of the ppRB degradation, and may provide a basis for the development of novel therapies aimed at activating the ppRB-PTS function in vivo.
