The long term goal of this project are to: 1) determine the importance of specific protooncogenes in the regulation of malignant hematopoiesis, and 2) ascertain if blockade of oncogene encoded proteins will result in death of the malignant cell or reversion of the malignant phenotype. Therefore our aims are to: 1. Block the synthesis of proteins encoded by the protooncogenes c-myc, c- myb, c-abl, and c-kit, using anti-senseoligodeoxynucleotides. Oligomers will be introduced into cells by direct incubation, electroporation, or transfection. Protein synthetic blockade will be immunochemically verified. Subsequent effects on leukemia/lymphoma cell growth, maturation, and malignant behavior will then be examined. Specifically cells exposed to anti-sense oligomers will be studied for changes in a) morphology, b) cell cycle activity, c) ploidy, d) change in surface antigen profiles, e) clonogenicity in semi-solid culture media, and f) ability to form tumors in nude mice. Similar studies will be carried out on clinical specimens derived from patients with acute and chronic leukemias where possible. 2. Investigate the cellular and molecular consequences of blocking protooncogene encoded protein synthesis. We hypothesize that inhibition of protooncogenes that encode nuclear binding proteins or putative parts of the signal transducing apparatus will have both immediate, and remote adverse effects on cell proliferation and development. the relative importance of these lesions vis a vis cell function is unknown. To investigate the chain of events which occur when a targeted gene is inhibited we will use immunochemistry, RT-PCR, and standard molecular techniques. In the case of inhibiting MYC and MYB we will focus on molecular events which regulate passage through the cell cycle; expression of genes both upstream (e.g. p53) and downstream (e.g. DNA polymerase alpha) of the targeted gene will be examined. We will also use RT-PCR or standard run-on techniques to examine effects on transcription of the targeted gene, or effects on transcription of alternatively spliced forms of the targeted gene. In the case of abl and kit, we will examine the ability of cells to bind and respond to growth factors, the fate of the proteins/receptors, and effects on phosphoprotein synthesis. Duration of induced cell function lesions will be determined, and the those which appear most likely to be lethal will be identified. 3. Develop an in vitro system for bone marrow purging using anti-sense oligodeoxynucleotides. If malignant and normal hematopoietic cells have differential sensitivity to anti-sense oligomers this might prove to be of therapeutic utility. Accordingly, dose response toxicity curves for normal and malignant cells to each oligomer, or oligomer combination will be generated. Malignant and normal cells will then be mixed together, exposed to oligomers in a variety of culture conditions, and then evaluated for their ability to clone in semi-solid culture media, and form tumors in nude mice will be assessed.
Showing the most recent 10 out of 13 publications
