The modified IL-3 receptor agonist, daniplestim. binds to the interleukin-3 (IL-3) receptor-a subunit with 60-fold greater affinity and induces CPU formation 10 to 20-fold better than native IL-3. Myelopoietin-1 (MPO-1 ) binds both the IL-3 and G-CSF receptors. In CPU assays, MPO-1 demonstrated greater stimulation of CFU-GM than the co-addition of native IL-3 & G-CSF. While the in vivo effects of daniplestim and MPO-1 are well described, the mechanisms by which they stimulate growth are not well understood. We have investigated the effects of daniplestim and MPO-1 on the prevention of apoptosis and the induction of gene expression in human hematopoietic OCI-AML and AML193 cell lines. Daniplestim and MPO-1 prevented apoptosis 5 to 10-fold better than native recombinant IL-3 as determined by Annexin V/propidium iodide binding FACS assay and TUNEL assays. Daniplestim and MPO-1 also promoted the maintenance of the mitochondria! membrane potential at 10-fold lower concentrations than native IL-3 or G-CSF. MPO-1 promoted a lower redox potential as higher levels of free radicals were detected than after culture with native IL-3 which could imply increased respiration in MPO-1-treated cells. In addition, the expression of cytokine-regulated genes was examined by Clontech cDNA human cancer array analysis and the expression of the relevant genes confirmed by RT-PCR and western blot analysis. Changes in hybridization levels of 3-fold were considered significant. Increased levels of mRNA transcripts encoding many members of the MAPK pathway were detected after treatment of cells with daniplestim and MPO-1. In contrast, the expression of many Rho/Rac GTP/GDP exchange family members were decreased after treatment with these cytokines. Genes involved in the regulation of the redox potential such as Superoxide dismutase and glutathione synthetase were detected in elevated levels in cytokine treated cells. Many anti-apoptotic genes such as Bcl-XL, Oncostatin M, LIF, Pirn were detected at higher levels in cells treated with MPO-1 than cells treated with the coaddition of IL-3 & G-CSF. cDNAs were detected in 10-fold lower levels in cells cultured in the absence of cytokines. These results suggest that MPO-1 may be able to promote cell growth by inhibiting apoptosis and inducing expression of genes which induce cell proliferation.
|Issue number||11 PART II|
|Publication status||Published - 2000|
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