Abstract
Chinese hamster ovary (CHO) cells incorporate 12-(1'-pyrene) dodecanoic acid (P12) into membrane lipids. Exposure of P12-labeled cells to long wavelength ultraviolet light causes cell killing, presumably because excitation of the pyrene moiety (a photosensitizer) leads to the generation of reactive oxygen species. Cytotoxicity is dependent upon the concentration of P12 used to label the cells, and time of UV exposure, and the presence of oxygen during irradiation. CHO mutant cells deficient in plasmalogen biosynthesis and peroxisome assembly (Zoeller, R.A., and Raetz, C.R.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5170-5174) are several orders of magnitude more sensitive to P12/UV treatment than wild-type cells, permitting direct selection of one wild-type cell in 1 X 10(4) mutant cells. A major factor responsible for the P12/UV hypersensitivity of these mutants appears to be the absence of plasmalogens. Supplementation of the mutants with 1-O-hexadecyl-sn-glycerol restores plasmalogen levels and nearly normal resistance to P12/UV treatment, whereas the biogenesis of peroxisomes is not restored. The P12/UV hypersensitivity of the plasmalogen-deficient mutants, together with the selective, P12/UV-induced decomposition of plasmalogens in wild-type cells, documented in the accompanying manuscript, suggest that the vinyl ether linkage of plasmalogens plays a direct role in protecting animal cell membranes against certain oxidative stresses.
Highlights
In the case of Chinese hamster ovary (CHO)-K1 cells (Fig. 31, labeling with 2 &I(or more) P12 for 20 h sensitized the cells to a 5-min irradiation with long wavelength UV light
We have discovered a phenotype associated with plasmalogen deficiency in CHO cells that suggests a special antioxidative function for plasmalogens, not previously appreciated
When mutant CHO cells lacking plasmalogens are labeled with the photosensitizer, P12, they are rendered much more susceptible to killing by long wavelength UV light than are P12-labeled, plasmalogen-containing cells
Summary
(Parallel vials containing untreated cells were used for protein determinations These cells were washed after P12 labeling with PBS lacking bovine serum albumin, and 0.8 ml of 1N NaOH was used to solubilize the cells prior to protein determination (30).) When larger amounts of cellular material were required for subfractionation of lipid species,cells were grownto mid-exponential in 100mm diameter tissue culture dishes, treated with P12, and harvested as above, with the exception that the cells were scraped from the dish in 5 ml of PBS with a rubber policeman, pelleted by centrifugation, resuspended in 0.8 mlof PBS, and added to ch1oroform:methanol (1:2) to form the single phase Bligh and Dyer mixture (28, 29). Fluorescence Microscopy":, X 10' cells were seeded in mlof medium in 60-mm tissue culture plastic dishes containing sterile glass coverslips.
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