The long term bone marrow culture system will be applied to investigate the mechanism by which oncogenic retroviruses can induce leukemias. This culture system provides an intermediate between the usual short term culture systems and the in vivo situation in that it maintains the hematopoietic stem cells. A stable balance is established between stem cell self-renewal and differentiation which permits continued hematopoiesis to continue in vitro for several months. Thus, both the hematopoietic stem cells and the committed progenitors to several hematopoietic lineages are continuously present in the cultures as stable populations and can serve as potential targets for leukemogenesis. This proposal focuses on the alterations induced at the stem cell level. We have demonstrated that infection of these cultures with a retrovirus which carries the v-src oncogene induced a stable alteration in the stem cells resulting in increased self-renewal potential and permitting serial recloning in vitro with continued maintenance of the multipotency of the stem cell. Remarkably, these altered stem cells were not leukemogenic. To investigate the mechanism of this phenomenon, a variety of new viral constructs will be produced which are designed to a) introduce a selectable marker, b) control the level of src expression in the infected cells, and c) direct the infection to particular sub-populations in the cultures. This will allow evaluation of which infected cells are involved, whether src is required transiently or continuously, and whether secondary events are involved. The results from these experiments will also provide a basis for the use of the culture system for other viral oncogenes, particularly in terms of possible stem cell effects. This information will also be useful for the investigation of some problems in stem cell development and in practical problems associated with gene therapy experiments.
