COMPARTMENTAL ISOLATION OF CHOLESTEROL PARTICIPATING IN THE CYTOPLASMIC CHOLESTERYL ESTER CYCLE IN CHINESE-HAMSTER OVARY 25-RA CELLS

Using the Chinese hamster ovary cell line, 25-RA, we have demonstrated that lipoprotein-derived cholesterol and endogenously synthesized cho lesterol are selectively differentiated with respect to their cellular locations, These cells lack sterol-mediated regulation, spontaneously storing large amounts of esterified cholesterol, which turns over wit h a half-time of 7.5 h. When [H-3]cholesterol was provided to the cell s in serum to trace cellular cholesterol, the specific activities of c ellular free and esterified cholesterol (6238 +/- 273 and 5128 +/- 277 cpm/mu g, respectively) failed to equilibrate, indicating that bulk c ellular free cholesterol is isolated from that participating in the ch olesteryl ester cycle. Using [H-3]acetate to trace the fate of endogen ously synthesized cholesterol, a failure of equilibration was also obs erved (specific activities of free and esterified cholesterol = 280 +/ - 37 and 458 +/- 8 cpm/mu g, respectively), The lower specific activit y of the precursor indicates that endogenously synthesized cholesterol is preferentially esterified, When cells radiolabeled with [H-3]aceta te were post-incubated in the absence of radiolabel, the specific acti vity of the esterified cholesterol pool remained significantly higher than that of the free cholesterol, suggesting that cholesterol derived from hydrolysis of esterified cholesterol is preferentially re-esteri fied.