TY - JOUR
T1 - Transduction of MDR1 into human and mouse haemopoietic progenitor cells
T2 - use of rhodamine (Rhl23) to determine transduction frequency and in vivo selection
AU - HEGEWISCH‐BECKER, SDSANNA
AU - HANANIA, ELTE G.
AU - FU, SIQUING
AU - KORBLING, MARTIN
AU - DEISSEROTII, ALBERT B.
AU - ANDREEFF, MICHAEL
PY - 1995/8
Y1 - 1995/8
N2 - Summary. The MDR1 gene product P‐glycoprotein (P‐gp) extrudes several anticancer drugs including taxol and fluorescent dyes such as rhodamine (Rhl23). Modulation of the level of P‐gp expression has the potential of overcoming multidrug resistance. One possible approach is the retroviral transfer of the human MDR1 gene into murine and human bone marrow (BM) progenitor cells. The rationale for this approach is increased chemoprotection, which allows chemotherapy of a greater level of intensity to be delivered. In this study, flow cytometric measurement of Rhl23 extrusion was used to test P‐gp function in human and mouse haemopoietic progenitor cells, which had been transduced with a virus containing the human MDR1 transcription unit. Human CD34+ selected cells were analysed immediately following transduction. In two successive experiments MDR1 cDNA transduction resulted in a 7% and 11% increase of P‐gp expressing Rhl23 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 assay was also successfully applied to peripheral blood lymphocytes of mice transplanted with MDR1 transduced BM cells, demonstrating increased Rhl23 efflux in transduced cells. Analysis of another fluorescence assay using fluorescein di‐beta galactopyranoside as a substrate for β‐galactosidase in cells transduced with a MDRl:d‐gal double transcription unit was hindered by background staining due to endogenous β‐gal activity. We conclude that the Rhl23 efflux assay is a sensitive method to monitor P‐gp function in MDR1 cDNA transduced cells, and may be used to enrich transduced cells via flow cytometric cell sorting for Rhl23 dull cells.
AB - Summary. The MDR1 gene product P‐glycoprotein (P‐gp) extrudes several anticancer drugs including taxol and fluorescent dyes such as rhodamine (Rhl23). Modulation of the level of P‐gp expression has the potential of overcoming multidrug resistance. One possible approach is the retroviral transfer of the human MDR1 gene into murine and human bone marrow (BM) progenitor cells. The rationale for this approach is increased chemoprotection, which allows chemotherapy of a greater level of intensity to be delivered. In this study, flow cytometric measurement of Rhl23 extrusion was used to test P‐gp function in human and mouse haemopoietic progenitor cells, which had been transduced with a virus containing the human MDR1 transcription unit. Human CD34+ selected cells were analysed immediately following transduction. In two successive experiments MDR1 cDNA transduction resulted in a 7% and 11% increase of P‐gp expressing Rhl23 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 assay was also successfully applied to peripheral blood lymphocytes of mice transplanted with MDR1 transduced BM cells, demonstrating increased Rhl23 efflux in transduced cells. Analysis of another fluorescence assay using fluorescein di‐beta galactopyranoside as a substrate for β‐galactosidase in cells transduced with a MDRl:d‐gal double transcription unit was hindered by background staining due to endogenous β‐gal activity. We conclude that the Rhl23 efflux assay is a sensitive method to monitor P‐gp function in MDR1 cDNA transduced cells, and may be used to enrich transduced cells via flow cytometric cell sorting for Rhl23 dull cells.
KW - membrane transport
KW - multidrug resistance
KW - p‐glycoprotein. gene transfer
KW - rhodamine 123
UR - http://www.scopus.com/inward/record.url?scp=0029146909&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029146909&partnerID=8YFLogxK
U2 - 10.1111/j.1365-2141.1995.tb05209.x
DO - 10.1111/j.1365-2141.1995.tb05209.x
M3 - Article
C2 - 7669666
AN - SCOPUS:0029146909
SN - 0007-1048
VL - 90
SP - 876
EP - 883
JO - British Journal of Haematology
JF - British Journal of Haematology
IS - 4
ER -