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A leukemoid reaction occurs after inoculation of L1210 leukemic cells into recipient mice and the degree of granulocytosis is correlated with tumor progression. It was found that the sera of leukemic mice contained elevated levels of colony stimulating activity (CSA) when compared with normal mouse sera. Media conditioned by L-1210 cells in vitro (L-1210-CM) contained CSA which stimulated normal bone marrow myeloid colony growth and an auto-stimulatory activity (ASA) which stimulated L-1210 cell proliferation. We studied the effects of trans -retinoic acid (RA) and 1,25 dihydroxyvitamin D3 (VD3) on the production of growth substances by L1210 cells. When L1210-CM was prepared in the presence of RA and VD3, the CSA and ASA were markedly inhibited. A combination of the two agents was more effective than either agent. Mice inoculated with 1 × 10 5 L1210 suspension culture cells treated with either agent or both combined survived significantly longer than controls. Mice inoculated with L1210 cells treated with the two agents combined survived longest. By using antibodies, preliminary analysis of growth substances generated in L1210-CM showed that it contains primarily GM-CSF and M-CSF-like activities which were distinct from ASA. Combination antibody titer assays revealed that ASA was not significantly inhibited with anti-GM-CSF and anti-M-CSF antibodies, while CSA was inhibited by between 61 and 84%. We conclude that RA and VD3 synergistically inhibit the release of growth-enhancing substances by L1210 cells which may reduce the growth advantage of leukemic cells and the resulting leukocytosis in lymphocytic leukemia.

The human monoblast cell line, U937, was employed to elucidate early events associated with differentiation induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1,25-dihydroxy-Vitamin D3 (VD3). Exposure of cells to a combination of GM-CSF and VD3 resulted in an up-regulation of c-fos mRNA within 1 h and a marked down-regulation of c-myc mRNA by 24 h and this was associated with a shift of cell population from the S phase to the G0 + G1 phase of the cell cycle by 18%. This was followed by a marked enhancement of monocyte-associated cell surface antigens [OKM1 (CD11b), LeuM3 (CD14), M77.7], as determined by monoclonal antibodies and flow cytometry. Functional characteristics such as nitroblue-tetrazolium reduction, alpha-naphthyl butyrate esterase activity, and phagocytic capability occurred. Cells treated with GM-CSF or VD3 alone showed only minor changes. These results demonstrate a potent synergistic effect of GM-CSF and VD3 on induction of U937 differentiation. This differentiation was partially blocked by H7, a protein kinase C (PKC) inhibitor. Changes in c-myc and c-fos mRNA expressions and a shift in cell cycle were shown to be early events in this process.

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. IFN-alpha has been used in the management of leukemia, and gene transfer of the IFN-alpha gene into hematopoietic progenitor cells may have great potential for the treatment of chronic myelogenous leukemia (CML). Therefore, we examined the ability of adenovirus (Ad)-IFN-alpha gene construct to transfect normal bone marrow hematopoietic CD34+ stem cells and the production of IFN-alpha protein by these cells. Ad-cytomegalovirus (CMV) promoter-driven IFN-alpha at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture. Optimal transduction of CD34+ cells with the AdCMV-IFN-alpha construct was achieved using 120 plaque forming units (pfu). Flow cytometric determinations revealed that there was no significant difference in CD34+ cell viability for the 8 or 12-h transfection periods. Immunoassay of IFN-alpha produced by CD34+ cells shows that IFN-alpha levels increased several fold in transfected cells and this was not seen in CD34+ cells transfected with the heme oxygenase gene (HO-1). These in vitro data suggest that adenovirus-mediated gene transfer of IFN-alpha into hematopoietic stem cells can be achieved and that the IFN-alpha protein is produced by viable CD34 progenitor cells.