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 test this hypothesis cDNA encoding bcl-2 was transfected into cells of the continuous lines OCI/AML-2 and OCI/AML-5. Four transfectant lines were isolated; three contained transfected bcl-2 in the sense orientation (AML5-BCL2sa, AML5-BCL2sb and 2-bcl2) and one with anti-sense bcl-2(AML5-bcl2as). The presence of the transfected gene was demonstrated by Northern blot; translation of the sense transfected genes into protein was demonstrated by Western blotting. Lines with sense-oriented transfected 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 statistical significance. The effect of ATRA on bcl-2 expression was compared in sense-transfected cells and their parents; by Northern blotting it was shown that the endogenous but not the transfected genes were down-regulated after ATRA exposure. The capacity of cells with transfected genes to respond to ATRA was tested by obtaining Ara-C survival curves for ATRA-treated cells. Compared to controls not exposed to ATRA, the transfected cells showed little or statistically insignificant changes 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 association with regulatory elements.
Hu
Z B
ZB
Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Canada.
Minden
M D
MD
McCulloch
E A
EA
D000208:Acute Disease; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002470:Cell Survival; D003561:Cytarabine / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007951:Leukemia, Myeloid / Q000188:drug therapy*; D009857:Oncogenes / Q000187:drug effects / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
D000970:Antineoplastic Agents; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D003561:Cytarabine; D014212:Tretinoin; D006861:Hydrogen Peroxide