C-JUN-NH2-Terminal Kinase Promotes Apoptosis, Suppresses P-Glycoprotein Mediated Multidrug Resistance but Enhances MRP Mediated Efflux in AML Cells.

Abstract

JNK has been implicated in distinct cellular events, as proliferation, cellular transformation and apoptosis. JNK has also been recently reported to reverse MDR1 mediated drug resistance and increase sensitivity to chemotherapeutic agents in non-hematopoietic cancer cells. As acquired drug resistance represents a major obstacle in successful therapy of Acute Myeloid Leukemia (AML), the significance of JNK activation in relation to apoptosis induction and drug resistance in AML was sought. JNK is active in U937 leukemia cells and undergoes further activation upon treatment with therapeutically relevant concentrations of daunomycin. Daunomycin induces massive apoptosis in these cells, which is significantly reduced if U937 cells are preincubated with JNK inhibitor 420116. MDR1 and MRP expression levels are low in U937 cells. URD cells, a U937 deritive cell line characterized by resistance to adriamycin, was tested. Interestingly, no basal JNK activity was detected in URD cells and daunomycin treatment did not activate JNK nor induced apoptosis, albeit abundant JNK protein expression. JNK has been proposed to be prerequisite for mediating drug resistance in HL60 cells, by up regulating GSTP1 and MRP efflux pump. GSTP1 was strongly expressed in URD cells in the absence of JNK activation, but MRP expression was insignificant. Moreover, GSTP1 contributed to JNK inactivation in these cells, as immunoprecipitation experiments revealed a complex of GSTP1 with JNK. MDR1 activity in URD cells was 90%, as detected by the calcein AM efflux assay. Transfection of URD cells with SEKED plasmid expressing JNK decreased MDR1 activity by 35% while MRP activity exhibited >3 fold increase, from 5% to 18,4%. 29% of URD cells underwent apoptosis after 24hr of treatment with 1μM daunomycin compared to 5% of cells transfected with empty vector, as measured by annexin V/ PI and DAPI staining. To validate our results to primary AML cells, 25 AML bone marrow samples were tested for JNK activation, apoptosis induction after daunomycin treatment and GSTP1, MDR1 and MRP expresion levels. Basal JNK activity was detected in 58% of patients and did not correlate with apoptosis susceptibility or MDR1 and MRP expression levels. JNK activation within 1 hr of treatment occurred in 9 samples (36%) and strongly correlated with statistically significant apoptosis (p=0,0044< 0,05) after 12hr of continuous drug exposure. In 16 samples (64%), JNK activation was not observed upon daunomycin treatment and apoptosis did not reach a statistically significant level (p=0,3>0,05). Chemotherapy induced JNK activation was irrelevant to basal JNK activity status. GSTP1 was strongly expressed in all samples examined. Our experimental data indicate that JNK activation is implicated in distinct cellular processes in response to chemotherapy. Activation of JNK correlated with apoptosis induction in U937 cells and in primary leukemia samples. Moreover, JNK activity was lost in resistant URD cells, perhaps due to GSTP1 inhibition, and when expressed induced MDR1 downregulation and apoptosis. The proapoptotic function of JNK in URD cells can not be attributed only to MDR1 downregulation, since it is also observed in U937 cells, characterized by low MDR1 activity. Interestingly, JNK expression in URD cells also upregulated the MRP efflux pump. Further studies are needed to elucidate whether JNK represents a possible drug target to overcome drug resistance in AML.