Apoptosis, a morphologically distinct form of cell death that has been observed during development, after withdrawal of trophic hormones, and after treatment with ionizing radiation or chemotherapeutic agents, in characterized by active participation of endogenous cellular enzymes in the disassembly of the cell. The present proposal seek funds for collaborative studies by two laboratories with a long-standing interest in the mechanism of chemotherapy-induced apoptosis. Previous studies from these laboratories have demonstrated that 1) topoiso-merase- directed drugs and other clinically useful antineoplastic agents trigger apoptosis in a variety of cell types; 2) the morphologic changes of apoptosis are accompanied by the early, quantitative cleavage of a number of nuclear polypeptides; 3) inhibition of this proteolytic cleavage prevents many of the other events associated with apoptosis; 4) the proteolytic cleavage is mediated by multiple cysteine-dependent aspartate-directed proteases (caspases) with differing substrate preferences; 5) multiple members of the caspase family become active in cytosol and nuclei of cells before the morphological manifestations of apoptotic death are evident; 6) the pathways leading to activation of the caspases after treatment with etoposide, topotecan, cisplatin, methotrexate and ionizing radiation are distinguishable from the pathways of activation after Fas ligation; and 7) the morphological and biochemical events of apoptosis can be recapitulated with synchrony and high fidelity in a unique cell-free assay developed using cytosol from cells committed to apoptosis. We now propose to 1) utilize a recently developed affinity labeling procedure to more fully characterize the caspases that are activated after treatment with etoposide and other chemotherapeutic agents in several leukemia and solid tumor cell lines; 2) exploit the affinity labeling procedure as well as high resolution four-dimensional time lapse video microscopy of cells containing fluorescent caspase substrates to determine the temporal order of caspase activation in situ and the subcellular distribution of the active caspases; 3) utilize the cell-free system of apoptosis to identify additional caspase substrates, especially substrates that are cleaved early in the course of apoptosis; and 4) identify and characterize a pair of abundant nucleotide analogs that are elevated an hour before the earliest detectable caspase activity in apoptotic HL-60 cells so that the role of these compounds in apoptotic signaling can be examined. These studies will provide improved understanding of the biochemistry of chemotherapy-induced apoptosis in anticipation that the insights gained will be useful in designing more effective cancer chemotherapeutic treatment in the future. RANT=R01CA77421 Ionizing radiation or hydroxyurea (HU - an inhibitor of DNA synthesis) induce cell cycle delays. The delays are mediated by cell cycle checkpoint controls, blocking entry to mitosis until the DNA is repaired or fully replicated. Caffeine treatment of radiation- or HU-treated cells over-rides checkpoint controls, re-establishing cell cycle progress into mitosis. This effect increases radio- and HU sensitivity and in particular increases preferentially the radiosensitivity of p53- defective cells. Roughly half of all human tumors are p53-defective, offering the possibility of tumor-specific radiosensitization. We are therefore investigating the mechanism by which caffeine over-rides checkpoint controls, using a genetically tractable model system - the fission yeast (Schizosaccharomyces pombe). To that end, mutants have been isolated which cannot be sensitized to HU or radiation by caffeine. Two are point mutants of rhp6 (rad6 homolog pombe) and these retain checkpoint functions in the presence of caffeine. Rhp6 is therefore required for caffeine-induced checkpoint over-ride and radiosensitization. The mutant phenotype supports a model in which Rhp6 functions either to promote mitosis (in the absence of DNA damage) or to participate in DNA (post-replication) repair (in the presence of DNA damage). Based on comparison with the budding yeast system, we suggest that Rhp6 requires, respectively, fission yeast homologs of Ubr1 and Rad18 to perform these functions.
Aim 1 of this investigation is to validate or refute this model by identification of fission yeast RAD18 and UBR1 homologs, construction of mutants for these genes and comparison of the mutant phenotypes with those predicted by the model.
Aim 2 continues the phenotypic and genetic characterization of the remaining (five) mutants identified as resistant to caffeine sensitization. Experiments are proposed to test for roles in repair and progression, the results of which will be used to support or modify the proposed model.
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