Matrix and serine protease expression during leukemic cell differentiation induced by aclacinomycin and all-trans-retinoic acid

In myeloid leukemia, immature leukemic cells are able to egress into peripheral blood to infiltrate extra-medullary organs. We therefore analyzed the migrating and invasive potential of human HL-60 and NB4 cell lines, representative of acute myelogenous leukemia, their ability to express matrix metalloproteases (MMPs), tissue inhibitors of metalloproteases (TIMPs) and urokinase plasminogen activator (uPA) in response to differentiating agents. Granulocytic differentiation by all-trans-retinoic acid (ATRA) and aclacinomycin (ACLA) strongly increased HL-60 and NB4 cell migration and invasion. At mRNA and protein levels, these cell lines produced significant amounts of MMP-9 (HL-60 < NB4). Granulocytic differentiation by ACLA increased both pro and active forms of MMP-9 whereas ATRA decreased them and stimulated uPA mRNAs. TIMP-1, the physiological MMP inhibitor, increased during granulocytic differentiation whereas TIMP-2 did not significantly vary. Use of Batimastat and aprotinin suggests that ATRA was active by modulating the uPA system while ACLA interfered with MMP expression. In conclusion, our data demonstrate that HL-60 and NB4 cells express MMPs and uPA which are differentially regulated by the differentiating agents ATRA and ACLA and suggest the clinical usefulness of MMPs and serine protease inhibitors in the prophylaxis and treatment of the ATRA syndrome. (C) 2002 Elsevier Science Inc. All rights reserved

Time-course of butyric acid-induced differentiation in human K562 leukemic cell line: rapid increase in g-globin, porphobilinogen deaminase and NF-E2 mRNA levels

International audienceButyric acid (BA) was shown to induce hemoglobinization of dence of globin gene expression on the NF-E2 transcription K562 cells in a dose-and time-dependent manner. The maximal factor has been demonstrated in murine erythroleukemia differentiation (54% of hemoglobinized cells) was obtained with cells. 15 The GATA-1 transcription factor has been shown to the 0.5 mM concentration, which induced a 60% inhibition of play a central role in the control of several erythroid genes cell growth at day 3 without cytotoxicity. Parallel to the kinetics including g-globin, PBGD, erythropoietin receptor (EPOR) of hemoglobinization, a rapid increase in g-globin and porpho-bilinogen deaminase (PBGD) mRNAs was observed in BA-and GATA-1 itself. 16 treated cells. This increase was time-dependent and higher for In view of the above, we propose in this report to study the g-globin than for PBGD (six-and twofold at day 3, time-course of BA-induced hemoglobinization and erythroid respectively). In contrast, erythropoietin receptor mRNAs were mRNA overexpression in human K562 cells. By so doing, we not affected by BA treatment. Analysis of erythroid transcrip-intend to bring new insights into the involvement of GATA-1 tion factor mRNA levels during the time course of BA treatment and, especially, NF-E2 transcription factors in the BA-triggered showed, for the first time, an early and marked (up to threefold) increase in p45 NF-E2 mRNA, contrasting with that of GATA-1 differentiation process. mRNA (,1.5-fold). Taken together, these results showed the rapid differentiating effect of BA and suggest the involvement of the NF-E2 transcription factor. Materials and method

Time-course of butyric acid-induced differentiation in human K562 leukemic cell line: rapid increase in g-globin, porphobilinogen deaminase and NF-E2 mRNA levels

International audienceButyric acid (BA) was shown to induce hemoglobinization of dence of globin gene expression on the NF-E2 transcription K562 cells in a dose-and time-dependent manner. The maximal factor has been demonstrated in murine erythroleukemia differentiation (54% of hemoglobinized cells) was obtained with cells. 15 The GATA-1 transcription factor has been shown to the 0.5 mM concentration, which induced a 60% inhibition of play a central role in the control of several erythroid genes cell growth at day 3 without cytotoxicity. Parallel to the kinetics including g-globin, PBGD, erythropoietin receptor (EPOR) of hemoglobinization, a rapid increase in g-globin and porpho-bilinogen deaminase (PBGD) mRNAs was observed in BA-and GATA-1 itself. 16 treated cells. This increase was time-dependent and higher for In view of the above, we propose in this report to study the g-globin than for PBGD (six-and twofold at day 3, time-course of BA-induced hemoglobinization and erythroid respectively). In contrast, erythropoietin receptor mRNAs were mRNA overexpression in human K562 cells. By so doing, we not affected by BA treatment. Analysis of erythroid transcrip-intend to bring new insights into the involvement of GATA-1 tion factor mRNA levels during the time course of BA treatment and, especially, NF-E2 transcription factors in the BA-triggered showed, for the first time, an early and marked (up to threefold) increase in p45 NF-E2 mRNA, contrasting with that of GATA-1 differentiation process. mRNA (,1.5-fold). Taken together, these results showed the rapid differentiating effect of BA and suggest the involvement of the NF-E2 transcription factor. Materials and method

Oxidative stress involvement in chemically induced differentiation of K562 cells.

International audienceThe erythroid differentiation of K562 cells could be achieved by exposure to several pharmacologic agents, including hemin, butyric acid (BA), and anthracycline antitumor drugs such as aclarubicin (ACLA) and doxorubicin (DOX). When used at subtoxic concentrations, these drugs induce the overexpression of erythroid genes, leading to hemoglobinization of cells. Because anthracyclines are known to generate oxidative damage, we intended to demonstrate the involvement of an oxidative stress in the chemically induced differentiation process. The addition of antioxidants to anthracycline- and BA-induced cells decreased their growth and dramatically reduced the percentage of differentiated cells at day 3. Northern blot analysis showed that antioxidants also decrease the expression of erythroid genes and related transcription factors in induced cells. Moreover, analyses of oxidative stress markers showed that treatment with BA, ACLA, and DOX lead to a decrease in reduced glutathione and antioxidant enzymes (glutathione peroxidase [GPx], glutathione reductase [GRase], CuZn superoxide dismutase [SOD], and catalase [CAT]). In addition, DOX increased thiobarbituric acid reactants (TBARs), and MnSOD activity was decreased by BA and DOX. Finally, the production of reactive oxygen species (ROS) by differentiating agents was demonstrated using the dihydroethidium probe in a microspectrofluorometric assay. Altogether, these results strongly suggest the involvement of an oxidative stress generated by BA or anthracyclines as the first step in the irreversible differentiation process. Additionally, these results underline the differences between BA, ACLA, and DOX molecular mechanisms