Laboratoire d'Hématologie, Hôpital Haut Lévêque
Absolute quantitation of Apo1/Fas was performed, using a quantitative flow cytometry kit (APOCYT FAS (cell), Biocytex, Marseille, F) on Jurkat, U937, HL60 and K562 leukemic cell lines and on several variants exhibiting different levels of mdr-1 expression. Fas expression was found to be 25,000, 11,600, 3,700 and <500 sites/cell for Jurkat, U937, HL60 and K562 respectively. Three independent determinations were performed on 11 different cell lines with expression levels ranging from 1,600 to 25,000 sites/cell. Pending on the cell line, the coefficient of variation varied from 1.4 to 20 % of the expression value. The ability to complete apoptosis induced by anti-Fas IgM was found related to the number of Fas sites/cell (r=0.94). Four variants of Jurkat with different mdr-1 expression levels were developed and assayed for Fas expression. A negative relationship between mdr-1 expression (as measured using anti P-gp antibody and FCM) and Fas expression was found (r=-0.70). A very low level of mdr-1 expression (only detected by RT-PCR) was sufficient to induce a decrease in Fas expression of respectively 40 and 56 % in Jurkat and HL60 mdr-1 variants. Treatment of parental HL60 cells with low concentrations of Daunorubicine induced a dose dependent overexpression of Fas in 18 h, suggesting that induction of Fas expression could be one of the cell responses to antitumoral drug treatment. These results confirm that acquired multidrug resistance is a multifactorial mechanism and that regulation of Fas expression could participate from such a phenotype.