טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKatzir Hagar
SubjectMultidrug Resistance in a Cell-Line Derived from a
Ph+-ALL Patient; Influx of Rhodamines into
Liposomes and Cells
DepartmentDepartment of Biology
Supervisor Professor Gera Eytan


Abstract

We examined the effect of Gleevec, an inhibitor of BCR-ABL kinase activity used for treatment of CML, on doxorubicin resistance exhibited by a human (Ph)-positive ALL cell line (TMD5).

TMD5 cells exhibited an ATP-dependent resistance to doxorubicin. This resistance is not mediated by either P-glycoprotein (P-gp) or MRP1. Gleevec did not modulate doxorubicin uptake into TMD5 cells. No major changes in expression of proteins potentially capable of mediating multidrug resistance (MDR), occurred upon treatment with Gleevec.

Protection from apoptosis has been postulated to be one of the crucial biological consequences of BCR-ABL kinase activity. Inhibition of this activity by Gleevec promoted apoptosis in TMD5 cells. Exposure of cells to a combination of Gleevec with cytotoxic drugs induced apoptosis in a fraction of the cells similar to the sum of effects of exposure to each agent separately.

MDR is the result of competition between passive uptake and efflux by the MDR pumps. In this study we used rhodamines as indicators for the following stages of passive drug transport: Rhodamines affinity to lipid membranes was measured using dialysis, rate of rhodamines flip-flop across membranes was measured using fluorescence resonance energy transfer (FRET) with NBD-labeled liposomes, and rate of rhodamines influx into liposomes was measured using trypan-blue containing liposomes.

Uptake rates by living cells were measured using FRET between calcein trapped in the cells` cytoplasm and the rhodamines. All rhodamines exhibited similar influx rates, and the exact rates depended on the rhodamine tested as well as the type of cells.