Methylprednisolone Influences Zap-70 in B-CLL In Vitro.

Zap-70 is accepted as a surrogate marker for mutational status of immunoglobulin heavy-chain variable region genes in B-CLL. Whether Zap-70 is a functional target for treatment of the more aggressive CLL cells that express unmutated IgV_H is still under investigation.

The use of methylprednisolone (Mp) is widespread in the treatment of B-CLL related concomitant auto-immune disorders. We have recently demonstrated that Zap-70+ B-CLL cells are more resistant to chemotherapy but more susceptible to apoptotic cell death by steroids than Zap-70 negative cells in vitro. We investigated the possible influence of Mp on the expression of Zap-70 in B-CLL cells.

Mononuclear cells of 15 patients with B-CLL were isolated by Ficoll centrifugation and incubated with different concentrations of Mp (range 0.1–500 μM). After 24h and 48h, Zap-70 expression, phospho-ZAP-70 (Tyr 319 and Tyr 493) and apoptosis were evaluated by flow cytometry (indirect staining ZAP-70 and phospho-ZAP-70 and Annexin V-FITC/propidium iodide double staining for apoptosis) and Western blotting when appropriate. Student’s t-test was used for statistical analysis. Mp induces time and dose dependent apoptosis in B-CLL cells (LD₅₀ 48h=16.8 μM). After 24h and 48h of incubation at doses of 50 μM, Zap-70 expression decreased significantly (50 μM: p<0.001) and more prominently in B-CLL than in T cells. We also observed a time but not concentration dependent decrease of phospho-ZAP-70 expression, on flow cytometry and western blotting. The observed down-regulation of phospho-ZAP-70 after Mp treatment is probably more important than the down-regulation of ZAP-70, since the phosphorylated form of ZAP-70 is responsible for signal transduction. The effect on P-ZAP-70 can theoretically be explained by an increase in phosphatase activity, a reduction in kinases or an increased susceptibility of the unphosphorylated form to proteasomal degradation. The effect of Mp on P-Zap-70 was inhibitable with the pan-tyrosine-phosphatase inhibitor sodium orthovanadate, but not with the proteasome inhibitor MG0132. Inhibition of the glucocorticoid receptor with mifepristone inhibited induction of apoptosis but not ZAP-70-decrease. Fludarabine treatment of the cells caused comparable levels of apoptosis but negligible decrease of ZAP-70 expression suggesting the decrease in Zap-70 phosphorylation is not due to the apoptosis by itself. On the other hand since mifepristone can inhibit the apoptotic effect of Mp but not the decrease of Zap-70 expression, the potential inhibiting Zap-70 phosphorylation as a therapeutic target should be questioned, and apoptosis induced by methylprednisolone may not depend on it.