The broad long-term objective of this research is to understand the mechanism of drug resistance in normal hematopoietic stem cells in order to develop new therapies aimed at increasing their resistance and thus the therapeutic index of chemotherapeutic agents used to treat cancer patients. The overall goal of the present proposal is to investigate the role of cytosolic aldehyde dehydrogenase (ALDH1) in the protection of normal hematopoietic progenitors from 4-hydroperoxycyclophosphamide (4- HC), an active derivative of cyclophosphamide. ALDH1 is the enzyme responsible for the inactivation of 4-HC. Diethylaminobenzaldehyde, an inhibitor of ALDH1, can prevent the cytoprotection induced by IL-1 and TNFalpha. The first specific aim will investigate the effect of IL-1 and TNFalpha on ALDH1 mRNA and protein in normal as well as malignant cells. Northern analysis, mapping the ALDH1 mRNA extracted from purified cells with radiolabeled RNA probes, and transcriptional run on assay will be performed. In addition, Western blotting and ALDH1 activity assay will evaluate the ALDH1 protein. Indeed, in preliminary experiments, I have shown that the ALDH1 mRNA and protein are increased in bone marrow cells after incubation with IL-1 and TNFalpha for 20hrs. The second specific aim is to study the isolated effects of ALDH1 in certain cell lines. Full-length ALDH1 cDNA will be synthesized by reverse transcriptase PCR, incorporated in an appropriate retroviral vector and used to infect candidate cell lines to study its effect on their resistance to 4-HC. The third specific aim is to determine the effect of overexpression of ALDH1 in normal hematopoietic progenitors in terms of their in vitro resistance to 4-HC. In the second as well as the third specific aim, the cloning of ALDH1 cDNA into the retroviral vector will be in the orientation to act as a sense or antisense to ALDH1 which will result in the increase or decrease of the ALDH1 protein, respectively, in the transduced cells. These studies will allow me to verify the exact role of ALDH1 in the protection against 4-HC toxicity in normal and malignant cells. It will also give insight into the mechanisms of drug resistance in normal hematopoietic progenitors and whether its amplification may give a therapeutic advantage for the normal progenitors over some malignant cells. The result of these experiments may lead to future studies that will evaluate the ability of human stem cells engineered to overexpressed ALDH1 to increase the therapeutic index of cyclophosphamide in vivo.
