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Lysosomal Sequestration of Polyamine Analogues in Chinese Hamster Ovary Cells Resistant to the S-Adenosylmethionine Decarboxylase Inhibitor, CGP-48664¹

Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263 [D. L. K., J. D. B., C. W. P.]; Research Laboratories, Novartis Pharmaceuticals AG., CH-4002, Basel, Switzerland [H. M.]; and Department of Medicinal Chemistry, University of Florida, J. Hillis Miller Health Center, Gainesville, Florida 32610 [R. J. B.]

CGP-48664, an inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), is presently undergoing Phase 1 clinical trials as an experimental anticancer agent. We have shown previously (D. L. Kramer et al., J. Biol. Chem., 270: 2124–2132, 1995) that Chinese hamster ovary (CHO) cells that are made resistant to the growth inhibitory effects of the drug overexpress AdoMetDC because of a stable gene amplification. Unexpectedly, these same cells (CHO/644) were found to be insensitive to the growth inhibitory effects of N¹,N¹¹-diethylnorspermine (DENSPM)—a polyamine analogue also undergoing Phase 1 clinical trials—despite accumulating 5 times more analogue than parental cells. We now report that treatment of CHO/664 cells with DENSPM results in the formation of numerous large cytoplasmic vacuoles, which on the basis of electron microscopy and cytochemical staining seem to be lysosomal in origin. A series of newly established CHO cell lines made differentially resistant to 1, 3, 10, 30, and 100 µM CGP-48664 by chronic exposure were used to demonstrate that vacuole formation correlated with the accumulation of extremely high levels of DENSPM without increasing growth inhibition. These same cells were used to show that AdoMetDC gene overexpression as indicated by mRNA levels was unrelated to vacuole formation; cells resistant to 100 µM CGP-48664 displayed a 170-fold increase in AdoMetDC mRNA levels and formed vacuoles in response to DENSPM, whereas those resistant to 10 µM CGP-48664 displayed a 120-fold increase in AdoMetDC mRNA levels and failed to form vacuoles. Despite accumulating to high intracellular levels, DENSPM was much less effective than spermine at down-regulating ornithine decarboxylase and polyamine transport activities in highly resistant cells. Similarly, DENSPM was less able to induce spermidine/spermine N¹-acetyltransferase activity in cells that formed vacuoles than in those that did not. Overall, natural polyamines failed to induce vacuoles and various analogues of DENSPM were used to probe the structural specificity of the effect. The data are consistent with the probability that DENSPM is sequestered to high concentrations in lysosomal vacuoles of CGP-48664-resistant cells and is, therefore, not available to interact with polyamine regulatory sites or to cytotoxically affect cell growth. In addition to implicating the lysosome as a potential new site of CGP-48664 drug action that could be involved in antitumor activity and/or host toxicities, the findings also suggest a potential mechanism of cell resistance to analogues such as DENSPM.

¹ This work was supported in part by National Cancer Institute Grant RO1 CA22153 (to C. W. P.) and Roswell Park Cancer Institute Core Grant CA 16056 (to J.B.).

² To whom requests for reprints should be addressed, at Grace Cancer Drug Center, Roswell Park Cancer Institute Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-3002; Fax: (716) 845-8857; E-mail: Porter@sc3101.med.buffalo.edu.

Received 4/ 1/98. Accepted 7/ 2/98.

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