Resistance to erucic acid as a selectable marker for peroxisomal activity: isolation of revertants of an infantile Refsum disease cell line.

Abstract

A system based on the ability of cells to oxidize very long-chain fatty acids (VLCFA) was developed to select in vitro normal human fibroblasts from fibroblasts of patients suffering from peroxisomal disorders with multienzymatic deficiencies: Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease (IRD). Cells treated with various concentrations of erucic acid (C22:1 n-9) revealed an enhanced toxicity of this fatty acid for the fibroblasts of patients compared with normal cells. This differential toxicity is correlated with variable accumulations of C22:1 n-9 and the absence of beta-oxidation products in the mutants. Revertants from clonal IRD cell lines were isolated in the selective medium at frequencies ranging from 3 x 10(-7) to 4 x 10(-6) depending on the line. After six weeks of growth in the absence of selective pressure, the variants exhibited a resistance level to C22:1 n-9 identical to that of normal cells. Furthermore, beta-oxidation of VLCFA is re-established in these selected cells as well as dihydroxyacetone phosphate acyltransferase activity. Immunoblot experiments also demonstrated a restored pattern of acyl-CoA oxidase molecular forms. Last, immunofluorescence studies revealed the presence of cytoplasmic structures that were absent in the original IRD cells. Thus, both the deficiencies in metabolic pathways and paucity of the organelle are at least partially corrected in the selected clones.