DIRECT EVIDENCE FOR THE PARTICIPATION OF BCL-2 IN THE REGULATION BY RETINOIC ACID OF THE ARA-C SENSITIVITY OF LEUKEMIC STEM-CELLS

All-trans retinoic acid (ATRA) increases the sensitivity of AML blast cells to cytosine arabinoside (Ara-C) or daunorubicin (DNR) when ATRA is given after drug. We have proposed that down-regulation of bcl-2 is part of the mechanism by which ATRA regulates drug sensitivity. To te st this hypothesis cDNA encoding bcl-2 was transfected into cells of t he continuous lines OCl/AML-2 and OCl/AML-5. Four transfectant lines w ere isolated; three contained transfected bcl-2 in the sense orientati on (AML5-BCL2sa, AML5-BCL2sb and 2-bcl2) and one with anti-sense bcl-2 (AML5-bcl2as). The presence of the transfected gene was demonstrated b y Northern blot; translation of the sense transfected genes into prote in was demonstrated by Western blotting. Lines with sense-oriented tra nsfected bcl-2 were significantly less sensitive to Ara-C or H2O2 than the parental lines; the cells with anti-sense transfected genes were more sensitive than their parent but the difference did not reach stat istical significance. The effect of ATRA on bcl-2 expression was compa red in sense-transfected cells and their parents; by Northern blotting it was shown that the endogenous but not the transfected genes were d own-regulated after ATRA exposure. The capacity of cells with transfec ted genes to respond to ATRA was tested by obtaining Ara-C survival cu rves for ATRA-treated cells. Compared to controls not exposed to ATRA, the transfected cells showed little or statistically insignificant ch anges in Ara-C sensitivity after ATRA treatment. We conclude that data from the transfectants provides evidence that expression of bcl-2 is a determinant of sensitivity to Ara-C and H2O2; and that the effect of ATRA on sensitivity requires the presence of bcl-2 genes in associati on with regulatory elements.