Within the bone marrow, growth factors (GF) and cytokines from stromal cells synergize to promote the proliferation and differentiation of stem cells/progenitors. Overall, this process is referred to as hematopoiesis. The arm of hematopoiesis which reflects development of the myelomonocytic lineages is myelopoiesis which is the process to be evaluated by this proposal. Drugs and toxicants which disrupt hematopoiesis can substantially affect health due to the lack of leukocytes as well as other cell types. Macrophage (M-phi) development and its dysregulation by doxorubicin (Dox) and p-benzoquinone (BQ), a metabolite of benzene, will be investigated. In addition, GSH and cysteine conjugates of BQ will be used as a potential negative control. Dox and BQ are known to be hematotoxic, but the mechanisms involved are unknown, except that both can oxidatively stress cells. Since cellular activation signals are initiated at the cell surface and the plasma membrane's (cell surface) structure/function is dependent on lipid-lipid, lipid-protein, and protein-protein domains, the ability of Dox and BQ to modify the plasma membrane (PM) via influences on cellular thiols will be examined. It is reasonable to hypothesize is that oxidative stress on hematopoietic progenitors can alter their redox state resulting in/from glutathione (GSH) depletion and lipid modulation which modifies PM structure/function including growth factor receptor expression or signal transduction. Analyses will focus on cellular thiols and lipids. Alterations in cellular GSH concentration/metabolism/distribution resulting from Dox or BQ treatment will be studied including adduct formation with toxicant (GS-BQ, GS-Dox) and endogenous factors such as LTA4. Evaluation of PM (or lipid) modifications will include quantitation of lipid constituents, membrane fluidity (fluorescence polarization and photobleaching time-recovery), arachidonate metabolism including quantitation of prostaglandin and leukotriene products, inositol metabolism (formation of PIP2 and conversion to IP3), and expression, topography, and lateral mobility of known surface antigens including GF receptors and markers of differentiation. Signal transduction will be assessed in terms of [Ca2+]j, PKC activity, phosphorylation patterns in general, post-translational modifications, and formation/production of transcription factors (which are known to be influenced by redox status). The effects of Dox and BQ on GF-induced gene expression also will be determined. In addition to comparing the effects of Dox and BQ on the stromal cells and progenitors plus or minus cytokines/GF, cell lines (and their drug resistant variants) will be used. The multiple drug resistant phenotype has been suggested to be GSH-dependent. This could be due to GSH-conjugates as suggested or GSH influences on PM structures such as a transporter. Our studies will assess the involvement of possible molecular mechanisms responsible for inhibition of hematopoiesis at the stromal cell and/or progenitor level.
| Colinas, R J; Hunt, D H; Walsh, A C et al. (1996) Hydroquinone resistance in a murine myeloblastic leukemia cell line. Involvement of quinone reductase and glutathione-dependent detoxification in nonclassical multidrug resistance. Biochem Pharmacol 52:945-56 |
