HUMAN BREAST ADENOCARCINOMA MCF-7 0 CELLS ELECTROPORATED WITH CYTOSOLIC CLASS-3 ALDEHYDE DEHYDROGENASES OBTAINED FROM TUMOR-CELLS AND A NORMAL TISSUE EXHIBIT DIFFERENTIAL SENSITIVITY TO MAFOSFAMIDE/

The cytosolic class 3 aldehyde dehydrogenase (ALDH-3) present in human normal tissues/secretions is apparently much less able to catalyze th e oxidation of aldophosphamide to carboxyphosphamide than is the ALDH- 3 present in human tumor cells/tissues, suggesting that the former may be less able to protect cells from the cytotoxic action of cyclophosp hamide, mafosfamide, acid other oxazaphosphorines. To test this notion , relatively large and approximately equal amounts of human normal sto mach mucosa ALDH-3 and catechol-induced human breast adenocarcinoma MC F-7/0 ALDH-3 were first electroporated into cells ((MCF-7/0) that cons titutively express only very small amounts of the enzyme, The resultan t preparations were then tested for sensitivity to mafosfamide, ALDH-3 activities (NADP-dependent catalysis of benzaldehyde oxidation) were 1.7, 212, and 183 mIU/10(7) cells in shamelectroporated MCF-7/0 cells, and MCF-7/0 cells electroporated with stomach mucosa ALDH-3 and catec hol-induced MCF-7/0 ALDH-3, respectively. LC(50) values (concentration s of mafosfamide required to effect a 90% cell kill) were 62, 417, and > 1,000 mu M, respectively, The three preparations were equisensitive to phosphoramide mustard (LC(50) = similar to 850 mu M). Inclusion of benzaldehyde in the drug exposure medium fully restored the sensitivi ty of MCF-7/0 cells electroporated with either enzyme to mafosfamide, These observations support the notions that 1) cellular sensitivity to the oxazaphosphorines decreases as the cellular content of ALDH-3 inc reases, 2) the foregoing is the consequence of ALDH-3-catalyzed oxidat ion (thus detoxification) of aldophosphamide, and 3) the ALDH-3 presen t in at least some tumor cells/tissues is a slight variant of the ALDH -3 present in normal tissues/secretions. Furthermore, they illustrate the utility of electroporation used as a tool to determine whether a g iven enzyme, or even more generally, protein or other macromolecule, i s a determinant of cellular sensitivity to a given cytotoxic agent.