The overall objective of this proposal is to provide research training in the basic sciences of immunology and molecular biology for Cheryl L. Willman, who is currently completing her clinical training in pathology. This training will be provided by the Department of Cell Biology at the University of New Mexico. The primary sponsor for the candidate will be Thomas B. Tomasi. The goals of this training period will be accomplished by a combination of didactic and laboratory training in PHASE I, followed by intense research efforts in PHASE II.
The specific aim of the proposed research experiments are to study gene expression in the development of a single eucaryotic cell lineage. The studies will specifically focus on proto-oncogene expression as this gene family is hypothesized to play a key role in normal cell differentiation and multiple probes are available. Distinct precursor subpopulations in the myeloid/macrophage cell lineage are currently being isolated in the sponsor's laboratory, from murine bone marrow, Murine precursor cells are isolated by an experimental protocol involving short-term culture and enrichment of bone marrow cells in the presence of myeloid/macrophage growth factors, analysis of surface phenotype, and separation by multiparameter cell sorting. These murine cell populations have been ordered into a stepwise differentiation scheme by the order of appearance of surface antigens detected by monoclonal antibodies. Similar studies are now underway in the human system. These precursor populations will be examined for expression of a variety of different protooncogenes. RNA from these cells will be isolated and hybridized to recombinant probes containing oncogene segment by """"""""dot-blot"""""""" and Northern hybridization techniques. Expression in each stage will be compared to determine if these genes display altered expression as a function of differentiation, thereby functioning as stage-specific markers of hematopoiesis. The presence of """"""""elevated"""""""" proto-oncogene expression in certain tumors is unclear because cohort normal cells are often not available for comparison. In a final set of experiments, we will attempt to compare proto-oncogene expression in uncultured human myelomonocytic leukemias with phenotypically matched control cells from our precursor populations to determine if proto-oncogen expression, by being activated or elevated, plays a role in malignant transformation.
