Oxidative stress involvement in chemically induced differentiation of K562cells

The erythroid differentiation of K562 cells could be achieved by exposure t o several pharmacologic agents, including hemin, butyric acid (BA), and ant hracycline antitumor drugs such as aclarubicin (ACLA) and doxorubicin (DOX) . When used at subtoxic concentrations, these drugs induce the overexpressi on of erythroid genes, leading to hemoglobinization of cells. Because anthr acyclines are known to generate oxidative damage, we intended to demonstrat e the involvement of an oxidative stress in the chemically induced differen tiation process. The addition of antioxidants to anthracycline- and BA-indu ced cells decreased their growth and dramatically reduced the percentage of differentiated cells at day 3. Northern blot analysis showed that antioxid ants also decrease the expression of erythroid genes and related transcript ion factors in induced cells. Moreover, analyses of oxidative stress marker s showed that treatment with BA, ACLA, and DOX lead to a decrease in reduce d glutathione and antioxidant enzymes (glutathione peroxidase [GPx], glutat hione reductase [GRase], CuZn superoxide dismutase [SOD], and catalase [CAT ]). In addition, DOX increased thiobarbituric acid reactants (TBARs), and M nSOD activity was decreased by BA and DOX. Finally, the production of react ive oxygen species (ROS) by differentiating agents was demonstrated using t he dihydroethidium probe in a microspectrofluorometric assay. Altogether, t hese results strongly suggest the involvement of an oxidative stress genera ted by BA or anthracyclines as the first step in the irreversible different iation process. Additionally, these results underline the differences betwe en BA, ACLA, and DOX molecular mechanisms. (C) 2000 Elsevier Science Inc.