title: Complete replacement of membrane cholesterol with 4,4',14-trimethyl sterols in a human T cell line defective in lanosterol demethylation.
abstract: A3.01 is a hypoxanthine/aminopterin/thymidine-sensitive, human immunodeficiency virus-susceptible, human T cell line derived by Folks et al. (Folks, T., Benn, S., Rabson, A., Theodore, T., Hoggan, M. D., Martin, M., Lightfoote, M., and Sell, K. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4539-4543) following exposure of CEM cells to 8-azaguanine. In the present study, it is shown that A3.01 also contains a heretofore unrecognized mutation in cholesterol biosynthesis. A3.01 cells grown in the presence of 10% fetal bovine serum (FBS) contain primarily cholesterol in their membranes, but based on [14C]acetate labeling, synthesize only lanosterol and 24,25-dihydrolanosterol. Reduction in the amount of FBS provided resulted in decreased cellular levels of cholesterol with corresponding increases in the two 4,4',14-trimethyl sterols. In A3.01 cells cultured in 1% FBS medium, lanosterol and 24,25-dihydrolanosterol accounted for 7 and 45%, respectively, of total cellular sterols. Following dilution of the 1% FBS-grown cells into serum-free media, the level of membrane cholesterol gradually declined, such that after three passages it became virtually undetectable, whereas the proportions of lanosterol and 24,25-dihydrolanosterol rose to 25 and 75%, respectively. Even after eight passages in the serum-free media, A3.01 cells displayed a complete absence of cholesterol with no obvious effect on cell growth. Membranes isolated from A3.01 cells grown in the presence or absence of 10 micrograms/ml of cholesterol displayed similar phospholipid:sterol ratios, but membranes from the unsupplemented cells contained only approximately 5% as much cholesterol as the supplemented cell membranes. Finally, A3.01 cells grown in the absence of cholesterol were extremely resistant to the cytotoxic effects of amphotericin B, whereas cells cultured in the combined presence of 1% FCS and 10 micrograms/ml of cholesterol were sensitive to the drug. Collectively, these results demonstrate that 4,4',14-trimethyl sterols can effectively replace cholesterol in a human T cell lineage, indicating that not all mammalian cells have a requirement for cholesterol, per se. The A3.01 T cell lineage should prove useful in defining the role of cholesterol in membrane fusion and human immunodeficiency virus-mediated syncitia formation and cytopathic effects.
authors.affiliation: Department of Microbiology & Immunology, East Carolina University, School of Medicine, Greenville, North Carolina 27858-4354.
authors.forename: T M
authors.initials: TM
authors.lastname: Buttke
authors.forename: T M
authors.initials: TM
authors.lastname: Folks
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D001345:Autoradiography; D002460:Cell Line; D002784:Cholesterol / Q000096:biosynthesis / Q000378:metabolism*; D002849:Chromatography, Gas; D002855:Chromatography, Thin Layer; D016895:Culture Media, Serum-Free; D004352:Drug Resistance, Microbial; D006678:HIV; D006801:Humans; D007810:Lanosterol / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D008745:Methylation; D013261:Sterols / Q000096:biosynthesis / Q000378:metabolism*; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology
chemical_list: D016895:Culture Media, Serum-Free; D008563:Membrane Lipids; D013261:Sterols; D007810:Lanosterol; D000666:Amphotericin B; D002784:Cholesterol