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Drug Resistance of Normal Hematopoietic Stem Cells

Jan Moreb, M.D., and James R. Zucali, Ph.D.

We have previously shown that preincubation with interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFa) can protect normal hematopoietic progenitors but not leukemic cells from the toxicity of 4-hydroperoxy-cyclophosphamide (4-HC), an active derivative of cyclophosphamide. Diethylamino- benzaldehyde (DEAB), which inhibits aldehyde dehydrogenase class 1 (ALDH-1), the enzyme responsible for inactivation of 4-HC, abolishes this protection. Thus, the general goal of this proposal is to study the role of IL-1, TNFa, and ALDH in the protection of normal and tumor cells from 4-HC. Northern and Western analysis show induction of ALDH-1 in mRNA and protein in human bone marrow cells, with proportional two-fold increase in the ALDH-1 activity after incubation with IL-1 and TNFa. The full length of ALDH-1 cDNA was synthesized and subcloned in pLNCX retroviral vectors in the sense and antisense orientation. The expression of ALDH-1 in the sense or antisense orientations in appropriate cell lines will determine the relationship between ALDH-1 and resistance to 4-HC. Another specific aim in this proposal is to determine the effect of overexpression of ALDH-1 in human normal hematopoietic progenitors on their in vitro resistance to 4-HC using colony forming assay and long-term bone marrow cultures. These studies will impact molecular engineering in relation to drug resistance of normal and cancer cells, as well as gene therapy in general. Although our preliminary studies are being done with retroviral vectors, we are also exploring the use of AAV for hematopoietic gene delivery in collaboration with the Vector Core Laboratory.

This proposal will be the basis for future clinical trials where normal bone marrow cells will be targeted with such genes, e.g., aldehyde dehydrogenase and manganese superoxide dismutase (see the proposal of Dr. Zucali), with the aim to render these cells resistant to combination chemotherapy and radiotherapy and eliminate the need for support after high-dose therapy with stem cells. The HAL will be very critical in terms of providing the vectors for human use. The human studies will target diseases that can be treated with combination chemo/radiotherapy, such as hymphoma and multiple myeloma, or other hematopoietic malignancies, provided that purified normal stem cells can be obtained for the gene transduction. Clinical studies could start in 3-5 years. Both inpatient and outpatient GCRC facilities will be required to accommodate these patients.

Reference

  1. Zucali JR, Moreb J, Gibbons W, Alderman J, Suresh A, Zhang Y, Shelby B. Radioprotection of hematopoietic stem cells by interleukin-1. Exp Hematol 22:130-135, 1994.