Detection Of Expression And Function Of P-glycoprotein In The Patients With Acute Leukemia

Multidrug resistance ( MDR) will eventually lead to failure of chemotherapy in acute leukemia. It was caused by complicated reasons , among which the expression of the membrane transport - associated drug resistant protein, P - glycoprotein (P - gp) is the most common and important one.P - gp is a transmembrane glycoprotein, which can lead to drug resistance by transporting intracellular lipophilic drugs outside, e. g. anthracyclins and vinca ?alkuloids etc. Suppression of P - gp function may recover the sensitivity of drug resistant tumor cells to anticancer drugs due to overexpression of P - gp. Detection of P - gp is prerequisite for judging the relation between P - gp and drug resistance and for applying inhibitory effect of P - gp. To elucidate the relation between acute leukemia and P ?gp and to evaluate the sensitivifty of different methods to detect P - gp, we used immunohistochemistry and flow cy-tometry to detect the expression and function of P - gp. The results showed that the positive expressing rate of P - gp was high in CD34+ ≥50years old and relapsed/refractory groups, and functional assay of P ?gp was more accurate and clinically significant comparing with oth-er detecting assays.Methods1. Cell cultureSensitive cell line k562/S and drug resistant one K562/D of leu-kemia K562 cell were cultured routinely in our laboratory. Exponen-tially growing cells were used for all experiments. The two cell lines were used as negative and positive controls respectively to detect ex-pression and function of P ?gp.2. Patient samples.- 50 acute leukemia (AL) cases, 22 were male,28 were female;average age was 42. 6, all cases are confirmed according to FAB diagnostic standard. Among them there are 14 acute lymphoblastic leukemia ( ALL) cases at diagnosis, one case of re-plased/refractory ALL,30 acute myeloid leukemia cases at diagnosis, 13 of which are acute promyelocytic (APL) ,5 of relapsed/refractory AML,one of which is relapsed/refractory APL. The following APL is not included in AML.3. Isolation of mononuclear cells:3 ?5 ml heparinized peripheral blood or bone marrow fluid (blasts >70% ) were drawed mononuclear cells were isolated with Fi-coll density gradient centrifugation.4. Detection of P ?gp expressionP - gp expression was monitored by immunohistochemistry and was judged positive if positive cell > 20%. Examining P - gp expres-sion by flow cytometry if positive cell > 10% , then P - gp expression was judged positive.5. Functional assay of P - gp-We use flow cytometry to detect the effect of CsA on absorption and release of RH123. If accumulative rate >30% or efflusive rate > 15% , P - gp function was judged positive.6. Detection of CD34 expressionPrimary antibody is CD34 antibody, other steps were the same as immunohistochemistry of P - gp mentioned above.Results1. Expression of P - gpPositive cases of P - gp expression detected by flow cytometry was few, so comparison between groups could not be conducted. The following P - gp expression was equal to its expression acquired with immunohistochemistry. There were no positive cases of P - gp expression in de novo APL group, which was obviously lower than de novo AML one; there were no differences of expression rate between de novo ALL group and AML one, the same as de novo and replased/refractory ones.2. Functional assay of P - gpNo P - gp functionally positive cases was found in de novo APL group, which was apparently lower than de novo AML group; de novo ALL group had no differences in positive rate compared with de novo AML group; replased/refractory group had higher positive rate than de novo group.The expression of P - gp had direct relation with its function.3. Epression of CD34CD34+ rates were the same in de novo ALL group and de novo AML group. Except APL, there were no differences between de novogroup and replased/refractory group.The expression of CD34 had direct relation with P - gp expression and its function.4. Excep...