We have recently observed that recombinant interferon gamma (rIFN-gamma) causes suppression of CML bone marrow progenitor colony growth and increases the proportion of Ph' negative metaphases recovered from culture. We postulate that this naturally occurring regulator of hematopoiesis selectively suppresses the growth of malignant Ph' positive CML progenitors while sparing or enhancing the growth of a coexistent, viable, possibly benign Ph' negative progenitor pool. We propose to explore the mechanisms underlying the observed effects of rIFN- gamma on CML progenitors. 1.) We will employ the bcr DNA probe as a sensitive method for detecting malignant cells in culture, and as a means for verifying that the Ph' negative cell populations found in culture have evidence at the molecular level of a benign nature. 2.) We will determine the role of the monocyte and of the NK cell in mediating rIFN-gamma effects on CML bone marrow progenitors: Monocytes will be isolated from CML bone marrow, and the effects of rIFN-gamma on the depleted bone marrow population and on the isolated monocyte population determined. The role of monocytes derived from normal syngeneic or normal allogeneic bone marrow in regulating CML bone marrow hematopoiesis will be studied. To follow up preliminary observations, we will assess the importance of tumor necrosis factor (TNF) in mediating the effects of rIFN-gamma- stimulated monocytes, as well as the direct effects of rTNF on CML bone marrow progenitors. Simultaneously, the importance of the FACS-purified NK cell population in regulation of CML bone marrow progenitor growth will be explored. We will employ cytogenetic and DNA techniques to determine the benign or malignant nature of the CML NK cell itself. The lytic activity of unstimulated and rIL2-stimulated CML NK cells will be determined and compared to the activity of normal NK cells. Effects of CML NK cells stimulated with rIL2, rIFN-gamma or combinations of the two cytokines on CML bone marrow progenitor growth, karyotype and presence of abnormal bcr will be tested. 3.) Finally, we will employ the long-term liquid culture (LTLC) hematopoietic progenitor assay to study the effects of rIFN-gamma on the behavior of primitive stem cells found in the adherent layer. These methods may be useful for the exploration of mechanisms underlying the effects of naturally occurring anti- tumor agents on the proliferation of human myeloid malignancies and may be directly applicable to the development of rational, individualized therapy for disorders of bone marrow hematopoiesis.
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