The benzene metabolite hydroquinone (HQ) and the antitumor agent doxorubicin (DX) are chemically-related compounds that cause hematotoxicity through a mechanism that is likely to involve oxidative stress. Tumor Necrosis Factor-a (TNF) is capable of directly protecting human hematopoietic stem/progenitor cells (HPC) from numerous oxidative stress-inducing hematotoxic agents, including HQ and DX. TNF also affects normal HPC proliferation and differentiation and may stimulate myelodysplastic and leukemic HPC to proliferate. However, the mechanism by which TNF affects the biological activities of HPC remains largely unknown. It is hypothesized that the TNF-induced genes that have been identified by differential display reverse transcription PCR and RNase protection analyses encode products that are important in the protective (or other biological) effects of TNF on human HPC.
The specific aims, research design and methods are: 1) to determine whether the TNF-induced genes identified are preferentially expressed in primitive or committed HPC by RNase protection on double purified HPC. Full-length cDNAs of these TNF-induced genes will be isolated by 5' rapid amplification of cDNA ends. The promoter regions will also be isolated by subcloning these sequences from human genomic bacterial artificial chromosomes. Sequencing and DNA database searches with the coding and promoter regions of the induced genes will be done to gain insight into the functions of the products of the unknown genes and to identify putative transcriptional regulatory sequences. 2) to assess the involvement of the TNF-induced genes in protection of HPC from HQ or DX using antisense methods to selectively inhibit gene expression in HPC or by transiently transfecting a leukemic cell line, not normally protected by TNF, or purified HPC with vectors expressing candidate protective genes to determine whether resistance to HQ or DX can be conferred. 3) to create profiles of the expression levels of known genes in HPC treated with TNF and/or HQ or DX, using DNA array technology. The long-term objectives of this proposal are to determine what TNF-activated genes and biochemical pathways are involved in protection of human HPC from hematotoxic agents.
| 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 |
