Reduction of Rhodamine-123 Efflux by MDR modulators and detection of MDR1 gene expression in acute leukemia: prognostic implications.

Expression of the multidrug resistance gene MDR1 is reported to be determinant for response to therapy and survival in some tumors and in leukemia. Several studies have been performed measuring either expression of MDR1 mRNA or its product, the P-glycoprotein. In our study we investigated the MDR expression by a functional flow cytometric assay based on the fluorescent dye Rhodamine-123 (Rhd123) efflux/retention in presence or absence of MDR modulators. The CEM parental cell line and the derived Vinblastine-100 resistant cells (CEM/VLB100) were used as negative and positive controls. Rhd efflux was not seen in the parental cell line whereas in the CEM/VBL resistant it was markedly present (> 1 log reduction of fluorescence intensity) and efficiently blocked by verapamil (10 ug/mL), cyclosporin A (5 ug/mL) and its analog PSC 833 (SANDOZ) (5 uM), respectively by 45 %, 48.9%, and 54.2%. Rt-PCR detection of mRNA for MDR1 was used to confirme the MDR expression in the CEM/VBL cell line. We then studied 92 patients with acute leukemia, 66 of them were at diagnosis and subsequently treated with at least one MDR-related chemoterapeutic agent, the mean blast percentage was > 80%, 60 cases were AML and 32 ALL. Among the AML patients 33% failed to achieve complete remission and a significant (p=0.005) difference in the mean Rhd efflux percentage was observed in presence of MDR modulator compared with CR patients (30.1% vs 11.3%). Among the group of non responding cases 78.5% of them showed a significant higher value of Rhd efflux compared to 36% of responding patients. When Rt-PCR was performed the expression of MDR1 was found in 62.5% of the cases and a correlation with the functional test was found in 87.5% of resistant cases. In patients with ALL we did not found difference in terms of efflux of fluorescent dye and MDR1 gene expression between responding and non responding cases, rather in these patients could be important to establish correlation between MDR expression and response duration.