TRANSDUCTION OF MDR1 INTO HUMAN AND MOUSE HEMATOPOIETIC PROGENITOR CELLS - USE OF RHODAMINE (RH123) TO DETERMINE TRANSDUCTION FREQUENCY ANDIN-VIVO SELECTION

The MDR1 gene product P-glycoprotein (P-gp) extrudes several anticance r drugs including taxol and fluorescent dyes such as rhodamine (Rh123) , Modulation of the level of P-gp expression has the potential of over coming multidrug resistance. One possible approach is the retroviral t ransfer of the human MDR1 gene into murine and human bone marrow (BM) progenitor cells, The rationale for this approach is increased chemopr otection, which allows chemotherapy of a greater level of intensity to be delivered. In this study, flow cytometric measurement of Rh123 ext rusion was used to test P-gp function in human and mouse haemopoietic progenitor cells, which had been transduced with a virus containing th e human MDR1 transcription unit, Human CD34(+) selected cells were ana lysed immediately following transduction. In two successive experiment s MDR1 cDNA transduction resulted in a 7% and 11% increase of P-gp exp ressing Rh123 dull cells. To monitor transduction efficiency over time as well as the possibility of in vivo selection of drug-resistant BM cells in mice treated with increasing numbers of taxol cycles, the ass ay was also successfully applied to peripheral blood lymphocytes of mi ce transplanted with MDR1 transduced BM cells, demonstrating increased Rh123 efflux in transduced cells. Analysis of another fluorescence as say using fluorescein di-beta galactopyranoside as a substrate for bet a-galactosidase in cells transduced with a MDR1:beta-gal double transc ription unit was hindered by background staining due to endogenous bet a-gal activity, We conclude that the Rh123 efflux assay is a sensitive method to monitor P-gp function in MDR1 cDNA transduced cells, and ma y be used to enrich transduced cells via now cytometric cell sorting f or Rh123 dull cells.