Pierre Alain

Approach, by flow cytometry, to the mechanism of action of S9788, a new multidrug resistance modifier

TIc otdy surface starker preseudy associated with hunan hematopoictic stem cells is the CD 34 antigon. P-glycoprolein (P-gp) is fomd on bone n~rrow progcuitor cell (BMPC) and some Icul~mic blasls, with different teclmologi~ (hlmmnologieal slaining or molecular biology for Acute Non Lynkohobl~lic Letda:mia (ANLL) Rhodamine 123 cfflux in BMPC[II). Tim energy dependent waramembran elflux pump is respomiblc for MuhiDrug Resislance (MDR) in Ion)or cells. The aim of fl~ work is to detcnuine if CD 34 pesitivc ptcnotype is associated with MDR phononlonom in nonual or leukemic cells. CD 34+ cells of nonual bone m:~rrow from 5 autologom Iraesplanlation domlor5 wen= collecled and frozen in liquid uitrogen. CD 34+ cells we~ selected by panning (AIS ,TECHGEN) or by inmnmo-lrmgnetic beads (DYNAL-BIOSYS). Leul~mic CD 34+ ceils were collected before treaunent (5 ANLL md 4 ALl.). Pgp expression is detcmtined by Rhc~danfine (Rho) 123 cfflux at 40C, 370C and in tbe presence of P-gp itdtibito~ (Ve~pamil). To determine if fluorescent dye cfflux is associaled with CD 34 phonotype, ceils were staiz~ed with Rho 123 md after cffiux at 37C, d¢y were labeled by iudirect inmnulofluorescon~ with motlocloual antibody HPCA2 (BECTON-DICKINSON) wlvnt recognizes CD34 molecules. Phyceerydu-in (PE) labeled geel anti-momc Ig-G were obtained from IMMUNOTECll. Flow aualysis was couducted with a CYiORON ABSOLUTE (ORTIIO). Rho123, PE, were excited with a 488 nm light from an argon laser. Selection of emission light was aclfieved by baud pa~s fdtet,a (515-530 nm for Rho ; 565-592 tun for PE) hi normal CD 34+ cells Rho 122, elflux is correlated with the purity of irnmonoselection.Eighty-three percenl ~_11%) of CD 34+ cells are MDR~ wiflt ftmetiorm,~d Rhol 9-3 lest in dmd l~rametcrs assays. Resuhs in CD 34+ Ion~mic cells are heterogenom. MDR+ is present in 47%, 79%, 43%, 43%, 0% of cells in dtml par.asnelc~ assays, hi 3 CD 34+ ALL ~suhs are m follows : 0%, 0%, 95% M DR+ cells in dual color ~says. These resul~ eotffimt fie expression of functiomml P-gp in normal bone nmrrow CD 34+ cells (83%). Ntis P-gp cxpressinn is Ictcrogcnons in CD 34+ leui~mic cells. The clinical role or fmlctioonal MDR phenolnenom expression d~refore will require further study.