Using flow cytometric techniques, transplantation bioassays, and Southern analysis of DNA we have defined several discrete stages in development of MCF virus-accelerated leukemia in AKR mice. These stages represent (i) spread of infection to the major subpopulations of thymocytes with concommitant alteration of cell-cycle kinetics, (ii) emergence of clonal populations of proliferating cells, and (iii) progression to the explantable, transplantable phenotype of primary leukemia cells. Our data indicate that thymic leukemogenesis induced by MCF viruses is a multistep process which can be dissected temporally. In this proposal we plan to determine the significance of these stages during leukemogenesis and mechanisms of their induction. Attention will be focused on thymic lymphoblasts, the level of thymocyte differentiation at which transformation is most likely to occur. Effects of virus infection will be studied in thymus and in organ cultures of 13d old fetal thymus. Subpopulations of infected cells will be identified by correlated 2-parameter analysis of light scatter and immunofluorescent staining of T-lymphocyte markers or viral gp70. Subpopulations will be fractionated by centrifugal elutriation or by cell sorting and their cell cycle kinetics will be determined by flow cytometric analysis of acridine orange staining. Cells will be examined at different stages of leukemogenesis for activation of the c-myc oncogene. Altered gene expression will be measured by Northern blot analysis of c-myc transcription. Preferential proviral integration on chromosome 15 as a possible mechanism of c-myc activation will be studied by in situ hybridization of thymocytes infected by a specially constructed MCF virus containing an insert of bacteriophage Lambda. DNA rearrangements at the c-myc locus as a result of proviral integration will be determined by Southern blotting. We will determine if trisomy 15 is associated with early events of transformation at stage II or with progression to the stage of autonomous growth found in primary leukemias. Progressive changes in transplantation properties and in vitro proliferation capacity of fractionated stage II cells will be monitored kinetically.
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