Arsenic trioxide (As2O3) gradually downregulates tissue factor expression without affecting thrombomodulin expression in acute promyelocytic leukemia cells

Abstract

Arsenic trioxide (As 2 O 3 ) was shown to induce complete remission in a high proportion of patients with relapsed and all-trans retinoic acid (ATRA)-resistant acute promyelocytic leukemia (APL). 1 The clinical response to As 2 O 3 is associated with the induction of nonterminal cytodifferentiation and the activation of cysteine proteases (caspases) that are characteristic of apoptosis. We have recently found that vitamin A derivatives, retinoids such as ATRA, exert anticoagulant effects by upregulating thrombomodulin (TM) and downregulating tissue factor (TF) expression in APL and acute monoblastic leukemia cells. 2,3 The effect of ATRA explains the rapid improvement of disseminated intravascular coagulation syndrome (DIC), which is a complication, observed in almost all APL patients. Since the mechanism of action of As 2 O 3 does not involve its binding to retinoic acid receptors (RARs), in contrast to that of retinoic acids (RAs), 4 we have examined the regulation of TM and TF expression in APL cell line, NB4 cells treated with As 2 O 3 alone or in combination with ATRA. Our data generally support a very recent report by Zhu et al, 5 which has appeared in this journal.After we began treating NB4 cells with As 2 O 3 , we replaced half of NB4 cell-suspended medium by fresh medium containing 10% fetal calf serum and 1 M As 2 O 3 every 3 days. Such a culture condition avoids serum starvation, makes cells more resistant to apoptosis induced by As 2 O 3 and keeps cell concentration rather more constant than that previously reported in studies in vitro. Treatment of NB4 cells with 1 M As 2 O 3 for 14 days induced morphological changes, such as condensed chromatin, smaller nuclei, a decreased nuclei/cytoplasm ratio, and the appearance of granules in the cytoplasm. About 30% of the cells were found to resemble myelocyte morphologically, and the maturation stage of the remainder of the cells still corresponded to the promyelocyte stage. Less than 10% of total cells appeared apoptotic showing nuclear fragmentation. More than 90% of cells still kept alive by Trypan blue exclusion method. Viable cell numbers were counted and adjusted.We measured the changes in the total levels of TF and TM antigens in NB4 cells incubated with 1 M As 2 O 3 (from Sigma, St Louis, MO, USA). The TF antigen levels in cell lysates treated with As 2 O 3 decreased more gradually and modestly (1.37 ± 0.04 at 7 days, 0.81 ± 0.04 at 14 days vs control 2.20 ± 0.12 ng/10 7 cells) compared to those in cell lysates treated with 0.1 M ATRA. ATRA induced a marked decrease in TF expression at 1 day (0.40 ± 0.02 ng/10 7 cells) as reported before. 2,3 On the other hand, As 2 O 3 did not induce a change in TM antigen levels even after treatment for 14 days (2.46 ± 0.16 vs control 2.69 ± 0.10 ng/10 7 cells). ATRA markedly increased the TM antigen level (32.4 ± 4.9 ng/10 7 cells) at 1 day as reported. 2,3 NB4 cells were treated with As 2 O 3 and the cell surface TF and TM activities were measured. Treatment of NB4 cells with As 2 O 3 for 3 days resulted in a de...

Arsenic trioxide, a therapeutic agent for APL

“…In APL patients receiving As 2 O 3 treatment, the improvement of disseminated intravascular coagulation (DIC) or hyper®brinolysis paralleled the amelioration of bleeding symptoms, the correction of plasma ®brinogen level, and the decrease in membrane procoagulant activity (PCA) as well as TF contents of APL blasts Agis et al, 1999). Consistent with the in vivo ®ndings, the membrane PCA and TF at both protein and mRNA levels in APL cell line NB4 were rapidly down-regulated by 1 mM As 2 O 3 Ohsawa et al, 2000).…”

Characteristics of fibrinolytic disorders in acute promyelocytic leukemia

“…As a differentiation therapy, ATO induces APL cell differentiation by degrading the PML-RARα fusion protein. Studies have shown that TF and procoagulant activity were significantly decreased in ATO-treated APL cells [15,16]. There are also reports that observed a significant reduction in Pn generation on ATO-treated APL cells [12].…”

Orchestration of multiple arrays of signal cross-talk and combinatorial interactions for maturation and cell death: another vision of t(15;17) preleukemic blast and APL-cell maturation

“…Since arsenic trioxide was shown to induce maturation of ATRA-resistant APL cells (Chen et al, 1996) and was eectively used to treat ATRA-resistant APL relapses Shen et al, 1997;Bergstrom et al, 1998), numerous investigations have been designed to determine its mechanisms of action (MuÈ ller et al, 1998;Shao et al, 1998;Dai et al, 1999;Koken et al, 1999;Sternsdorf et al, 1999;Ohsawa et al, 2000;Kitamura et al, 2000a). This aspect is exhaustively reviewed by others in this issue.…”