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N-(4-Hydroxyphenyl)retinamide Elevates Ceramide in Neuroblastoma Cell Lines by Coordinate Activation of Serine Palmitoyltransferase and Ceramide Synthase¹

John Wayne Cancer Institute at Saint John’s Health Center, Santa Monica, California 90404 [H. W., M. C. C.]; Division of Hematology-Oncology, Children’s Hospital, Los Angeles, California 90027 [B. J. M., C. P. R.]; and Departments of Pediatrics [B. J. M., C. P. R.], Cell and Neurobiology [B. J. M.], and Pathology [C. P. R.], University of Southern California Keck School of Medicine, Los Angeles, California 90033

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR; fenretinide) is cytotoxic to a variety of cancer cell lines, and we previously showed an association between ceramide generation and 4-HPR cytotoxicity for neuroblastoma cell lines (B. J. Maurer et al., J. Natl. Cancer Inst. (Bethesda), 91: 1138–1146, 1999). Here we determine whether the increased ceramide mediated by 4-HPR in the CHLA-90 human neuroblastoma cell line results from de novo ceramide synthesis. Treatment of CHLA-90 with 4-HPR for 2 h, in the presence of [³H]palmitic acid, caused sequential formation of [³H]sphinganine (220% over control) and [³H]ceramide (160% over control), with sphinganine returning to baseline at 4 h, and ceramide continuing to increase (215% over control). 4-HPR treatment did not accelerate cellular decay of sphingomyelin. Preincubation of cells with either L-cycloserine, an inhibitor of serine palmitoyltransferase (SPT), or fumonisin B₁, an inhibitor of ceramide synthase, retarded ceramide formation in response to 4-HPR treatment, although sphinganine was still generated when 4-HPR and FB₁ were present. Data from in vitro enzyme assays using microsomes showed that preexposure of intact cells to 4-HPR resulted in a time (175% over control; 6 h)- and dose-dependent increase (173% over control; 10 µM) in SPT activity as well as a time (265% over control)- and dose-dependent increase (215% above control; 10 µM) in ceramide synthase activity. Our results show that 4-HPR-mediated ceramide generation is derived from the de novo synthetic pathway by coordinate activation of SPT and ceramide synthase. Knowledge of these biochemical events is of utility when downstream modulators of ceramide metabolism are used to heighten the cytotoxic response to chemotherapy.

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