The differentiation-inducing agents, N-methylformamide and retinoic acid, are currently in clinical study for cancer therapy. The proposed experiments with HL-60 human promyelocytic leukemia cells may provide useful information for designing rational combinations of differentiation-inducing agents and nucleoside analogs for use in chemotherapy. Preliminary studies have shown that nucleoside transport in HL-60 cells is reduced more than 25 fold during N,N-dimethylformamide (DMF)-induce differentiation. The kinetic parameters of adenosine and deoxyadenosine transport are affected differently. Thus transport studies can be used to elucidate the actions of different inducing agents, and the inducers provide a probe for studying the mechanism(s) of nucleoside transport. The modulation of nucleoside transport will be studied with the following agents: N-methylformamide, dimethylsulfoxide, hypoxanthine, 3-deazauridine, retinoic acid, 1 Alpha, 25-dihydroxyvitamin D3, and 12-0-tetradecanoylphorbol-13-acetate. The effects of these compounds on nucleoside transport will be compared to those of DMF with respect to magnitude, timing, reversibility, and commitment and related temporally to other differentiation-associated changes. Influx will be measured by rapid transport assays with radiolabeled permeants and by adenosine deaminase titration assays with deoxycoformycin and coformycin. Turnover of nucleoside transport protein(s) will be determined by pulse-chase labeling with [35S]methionine, followed by photoaffinity labeling with [3H]p-nitrobenzyl-6-mercaptopurine ribonucleoside (NBMPR), a specific nucleoside transport inhibitor. Physicochemical studies of the transporter(s) will include sensitivity to sulfhydryl group reactive compounds, SDS polyacrylamide gel electrophoresis, and chromatofocusing. The affinity of natural and analog nucleosides for the nucleoside transporter(s) will be determined kinetically; analog nucleotide formation will be measured by radioisotope or HPLC assays. Also, the HL-60 cells will be cultured in the presence of NBMPR and dipyridamole to determine whether these high-affinity hydrophobic transport inhibitors affect growth, responsiveness to differentiation inducers, or the turnover of the HL-60 nucleoside transport proteins. Since NBMPR is in clinical trials as a host-protective adjunct for nucleoside chemotherapy, this information would help predict its usefulness when added to a combination of nucleoside analog plus differentiation inducer.
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| Stoeckler, J D; Li, S Y (1987) Influx of 5'-deoxy-5'-methylthioadenosine into HL-60 human leukemia cells and erythrocytes. J Biol Chem 262:9542-6 |
| Chen, S F; Cleaveland, J S; Hollmann, A B et al. (1986) Changes in nucleoside transport of HL-60 human promyelocytic cells during N,N-dimethylformamide induced differentiation. Cancer Res 46:3449-55 |
