MECHANISM OF CYTOSINE-ARABINOSIDE TOXICITY TO THE BLAST CELLS OF ACUTE MYELOBLASTIC-LEUKEMIA - INVOLVEMENT OF FREE-RADICALS

Retinoic acid and hydrocortisone (HC) have been shown to regulate the drug sensitivity of the blast cells of acute myeloblastic leukemia (AM L). We asked if the proto-oncogene bcl-2 played a role in this regulat ion. As target cells we used the continuous lines, OCI/AML-1, OCI/AML- 2 or OCI/AML-5; expression of bcl-2 can be detected by Northern analys is of RNA from OCI/AML-2 or OCI/AML-5 cells; bcl-2 expression can be f ound in OCI/AML-1 cells only by using RT-PCR. Exposure of OCI/AML-5 or OCI/AML-5 cells to retinoic acid (all-trans retinoic acid, ATRA) led to a down-regulation of bcl-2 expression that was first seen after 2 h of exposure and was complete after a day. The down-regulation could b e prevented by exposing the cells to ara-C either before or after ATRA ; decrease in bcl-2 protein was moderate and only obvious after 36 h o f ATRA treatment. Nuclear run-on experiments provided evidence that bc l-2 down-regulation was occurring at transcriptional and post-translat ional levels. Since bcl-2 is considered to have antioxidant activity, we tested the sensitivity of the three cell lines to H2O2; we found th at OCI/AML-1, the line with very low bcl-2 expression, was a inn-fold more H2O2-sensitive than OCI/AML-2 or OCI/AML-5, where bcl-2 expressio n can be detected readily. We then asked if H2O2 sensitivity could be regulated. We found that exposure of cells to HC before H2O2 was prote ctive while ATRA after peroxide treatment increased killing; this is t he same pattern of regulation observed when AML blasts are exposed to HC before, or ATRA after ara C. Finally, we asked whether N-acetylcyst eine (NAG), a known radical scavenger would protect cells against ara- C killing. Significant protection was observed when NAC was given befo re drug, but not if given after drug. NAC protection against ara-C kil ling was seen for OCI/AML-1 and 2 cells, but not for OCI/AML-5 cells. We interpret the results as follows: ara-C kills cells in two ways: fi rst, directly, by incorporation into DNA and chain termination; second , indirectly, by inducing the production of toxic radicals. Bcl-2 redu ces the oxidant activity of such radicals, and is protective. ATRA reg ulates ara-C toxicity by its action on bcl-2. Left unexplained are the action of HC, which does not affect bcl-2 expression and the mechanis m by which ara-C prevents down-regulation of bcl-2 by ATRA.