RESISTANCE TO ERUCIC-ACID AS A SELECTABLE MARKER FOR PEROXISOMAL ACTIVITY - ISOLATION OF REVERTANTS OF AN INFANTILE REFSUM-DISEASE CELL-LINE

A system based on the ability of cells to oxidize very long-chain fatt y acids (VLCFA) was developed to select in vitro normal human fibrobla sts from fibroblasts of patients suffering from peroxisomal disorders with multienzymatic deficiencies: Zellweger syndrome, neonatal adrenol eukodystrophy, infantile Refsum disease (IRD). Cells treated with vari ous concentrations of erucic acid (C-22:1 n-9 ) revealed an enhanced t oxicity of this fatty acid for the fibroblasts of patients compared wi th normal cells. This differential toxicity is correlated with variabl e accumulations of C-22:1 n-9 and the absence of beta-oxidation produc ts 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 absenc e of selective pressure, the variants exhibited a resistance level to C-22:1 n-9 identical to that of normal cells. Furthermore, beta-oxidat ion of VLCFA is re-established in these selected cells as well as dihy droxyacetone phosphate acyltransferase activity. Immunoblot experiment s also demonstrated a restored pattern of acyl-CoA oxidase molecular f orms. Last, immunofluorescence studies revealed the presence of cytopl asmic structures that were absent in the original IRD cells. Thus, bot h the deficiencies in metabolic pathways and paucity of the organelle are at least partially corrected in the selected clones.