ing apoptosis but are as active as wild-type p53 in inducing G1 arrest (Friedlander et al., 1996;Ludwig et al., 1996;Smith et al., 1999). They are also less able to

One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaf let of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB͞c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two-to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.Programmed cell death (apoptosis) plays a crucial role in the pathogenesis of a number of disorders including AIDS and other viral illnesses, cerebral and myocardial ischemia, autoimmune and neurodegenerative diseases, organ and bone marrow transplant rejection, and tumor response to chemotherapy and radiation (1-3). Since the original description of apoptosis by Wyllie in 1972, its assessment in vivo has required direct examination of biopsied or aspirated material (4). An imaging technique capable of localizing and quantifying apoptosis in vivo would permit assessment of disease progression or regression and similarly define the efficacy of therapy designed to inhibit or induce cell death (5-6).Cells undergoing apoptosis redistribute phosphatidylserine (PS) from the inner leaflet of the plasma membrane lipid bilayer to the outer leaflet (7,8). The externalization of PS is a general feature of apoptosis occurring before membrane bleb formation and DNA degradation (7,8). Annexin V, a human protein with a molecular weight of 36,000 has a high affinity for cell or platelet membranes with exposed PS in vitro and in vivo (9-13). This observation has led to testing radiolabeled annexin V in animal models of acute thrombosis and imaging of atrial thrombi in patients with atrial fibrillation (14, 15). In the current study, annexin V was derivatized with hydrazinonicotinamide (HYNIC) and coupled to technetium 99m ...

Using the yeast two-hybrid system, we have isolated a cDNA (designated BBP, for Bcl2-binding protein) for a protein (Bbp) that interacts with Bcl2. Bbp is identical to 53BP2, a partial clone of which was previously isolated in a two-hybrid screen for proteins that interact with p53. In this study, we show that specific interactions of Bbp/53BP2 with either Bcl2 or p53 require its ankyrin repeats and SH3 domain.

The tumor suppressor protein p53, once activated, can cause either cell cycle arrest or apoptosis through transactivation of target genes with p53 DNA binding sites (DBS). To investigate the role of p53 DBS in the regulation of this profound, yet poorly understood decision of life versus death, we systematically studied all known and potential p53 DBS. We analysed the DBS separated from surrounding promoter regions in yeast and mammalian assays with and without DNA damage. p53 efficiently utilized the DBS of MDM2 and of genes connected to cell cycle arrest, DNA repair and the death receptor pathway of apoptosis. However, p53 was unable to utilize two-thirds of the isolated DBS, a subset that included almost all DBS of apoptosis-related genes. Neither ASPP2, a p53-interacting protein reported to specifically stimulate p53 transcriptional activity on apoptosis-related promoters, nor DNA damage resulted in p53 utilization of isolated DBS of apoptosis-related genes. Thus, a major regulation of p53 activity occurs at the level of p53 DBS themselves by posing additional requirements for the successful utilization of apoptosisrelated DBS.