Effect of all-trans retinoic acid on chemotherapy induced apoptosis and down-regulation of Bc1-2 in human myeloid leukaemia CD34 positive cells

Abstract 

Acute myeloid leukaemia (AML) is a heterogeneous malignant disease in which disease progression at the level of CD34 positive cells has a major impact in drug resistance and relapse. The multi-drug resistance (MDR1) gene product, P-glycoprotein is expressed mainly in CD34 positive AML cells and Bc1-2 is expressed simultaneously with several putative drug resistance parameters in these cells. Bc1-2 over-expression is associated with CD34 positivity, poor response to chemotherapy and reduced overall survival in AML patients. Recently, all-trans retinoic acid (RA) has been reported to enhance cytarabine-induced apoptosis and downregulate Bc1-2 in several human myeloid leukaemia CD34 negative cells. The two CD34 positive human myeloid leukaemia cell lines: KG1 and KGla have the unique feature of expressing significant functional P-glycoprotein. Thus, the efficacy of RA in enhancing cytrabine- and fludarabine-induced apoptosis and overcoming the resistance was examined in both KG1 (CD34+CD7−) and KGla (CD34+CD7+) human myeloid leukaemia cells in the present study. Both cytarabine and fludarabine induced a dose–dependent increase in the number of apoptotic cells in both CD34 positive cell types. Interestingly, the cytarabine-induced apoptosis was significantly more than fludarabine-induced apoptosis in both cell types. All-trans RA alone failed to induce apoptosis or inhibit proliferation of either of the two human CD34 positive leukaemia cell types. However, RA enhanced cytarabine- or fludarabine-induced apoptosis and inhibition of proliferation in KG1 CD34+CD7− but not in KGla CD34+CD7+ myeloid leukaemia cells. As single agents, RA, cytarabine and fludarabine reduced Bc1-2 expression in a dose–dependent manner in both cell types. Using a quantitative ELISA assay, the Bc1-2 protein concentration was reduced by 86 or 100% after 72 h of treatment with 10 μM cytarabine or fludarabine, respectively, in both CD34 positive leukaemia cell types. The addition of RA to cytarabine enhanced its induced reduction of Bc1-2 in KG1 CD34+CD7− but not in KGla CD34+CD7+ human myeloid leukaemia cells. Meanwhile, RA failed to augment fludarabine-induced reduction of Bc1-2 in both cell types. In conclusion, the present results suggest a potential role for the combination of RA and cytarabine in the treatment of refractory and/or relapsed AML patients with CD34+CD7− but not CD34+CD7+ blast cells.

Keywords: Apoptosis, Bc1-2, CD34, Fludarabine, Leukaemia, Retinoic acid

Abbreviations: AML, acute myeloid leukaemia, MDR, multi-drug resistance, RA, all-trans retinoic acid