Arsenic Trioxide (As 2 O 3) Gradually Downregulates Tissue Factor

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

TO THE EDITOR

Arsenic trioxide (As2O3) 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 As2O3 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 vit- amin 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 As2O3 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 As2O3 alone or in combination with ATRA. Our data generally Figure 1 Changes in TF and TM cofactor activity on the surface 5 support a very recent report by Zhu et al, which has appeared in of NB4 cells after exposure to As2O3. (a) NB4 cells were exposed to µ µ this journal. 1 M As2O3 for 1, 3 or 7 days, or exposed to 0.1 M ATRA for 1 day. 6 After we began treating NB4 cells with As2O3, we replaced half of Cell surface TF activity (assay of 10 cells) was determined on the NB4 cell-suspended medium by fresh medium containing 10% fetal basis of normal plasma-based one-stage recalcification clotting time µ calf serum and 1 M As2O3 every 3 days. Such a culture condition and quantitatively measured by reference to standard curves con- avoids serum starvation, makes cells more resistant to apoptosis structed using human placenta TF as described previously.3 (b) Cell induced by As2O3 and keeps cell concentration rather more constant surface TM cofactor activity in cell suspensions was determined as than that previously reported in studies in vitro. Treatment of NB4 described previously.2,3 Basal ∆OD405 nm/min levels were 0.033 ± µ 6 6 cells with 1 M As2O3 for 14 days induced morphological changes, 0.002/min/5 × 10 cells (0.72 ± 0.04 APC nM/min/10 cells). These such as condensed chromatin, smaller nuclei, a decreased assays were repeated independently five times and the results are nuclei/cytoplasm ratio, and the appearance of granules in the cyto- expressed as the mean ± s.d. The difference between the TF cofactor plasm. About 30% of the cells were found to resemble myelocyte activity on the surface of NB4 cells treated with As2O3 for 7 days and morphologically, and the maturation stage of the remainder of the that of untreated cells is statistically significant (P = 0.0005). 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. (Figure 1b). As O treatment of THP-1 monoblastic leukemia cells for 7 We measured the changes in the total levels of TF and TM antigens 2 3 µ and 14 days did not decrease TF activity on the cell surface. in NB4 cells incubated with 1 M As2O3 (from Sigma, St Louis, MO, The downregulation of TF induced by ATRA was partially abolished USA). The TF antigen levels in cell lysates treated with As2O3 decreased ± ± by pretreatment of NB4 cells with As2O3 for a 1-day period more gradually and modestly (1.37 0.04 at 7 days, 0.81 0.04 at 14 (Figure 2a). Such treatment inhibited the upregulation of TM to an ± 7 days vs control 2.20 0.12 ng/10 cells) compared to those in cell lys- extent about one-third of that induced by ATRA (Figure 2b). ates treated with 0.1 µM ATRA. ATRA induced a marked decrease in TF expression at 1 day (0.40 ± 0.02 ng/107 cells) as reported before.2,3

On the other hand, As2O3 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/107 cells). ATRA markedly increased the TM antigen level (32.4 ± 4.9 ng/107 cells) at 1 day as reported.2,3

NB4 cells were treated with As2O3 and the cell surface TF and TM activities were measured. Treatment of NB4 cells with As2O3 for 3 days resulted in a decrease in cell surface TF activity, which occurred more gradually, and not to the extent induced by 0.1 µM ATRA treatment for 1 day. ATRA promptly induced downregulation of TF activity as previously reported.3 Treatment for a 7-day period was required to decrease the TF activity to about one-third of the pretreatment levels of the NB4 cells (Figure 1a).2,3 The percent decrease in cell surface TF activity after 7 days was two-fold greater than that in TF antigen levels in total cell lysates (Figure 1a). Cell surface TM activity was measured as the degree of accel- eration of thrombin-catalyzed protein C activation. As2O3 stimulation did not change the rate of protein C activation on the surface of NB4 cells Figure 2 Effect of As2O3 and ATRA in combination on the TF (a) and TM (b) activities on the surface of NB4 cells. NB4 cells were µ pretreated with 1 M As2O3 for 1 day, and subsequently exposed to µ + As2O3 and 0.1 M ATRA for 1 more day (As ATRA). The effects of Correspondence: T Koyama, School of Allied Health Sciences, Faculty As2O3 treatment for a 2-day period and ATRA treatment for a 1-day of Medicine, Tokyo Medical and Dental University, 1–5–45 Yushima, period of NB4 cells are also shown. These assays were repeated inde-

Bunkyo-ku, Tokyo 113–8519, Japan; Fax: 81–3–5803–5882 pendently four times. The inhibitory effect of pretreatment with As2O3 Received 21 October 1999; accepted 10 January 2000 on TM upregulation by ATRA was significant (P = 0.02). Correspondence 942 tive of coagulopathy and hyperfibrinolysis fibrinogen/fibrin degradation products (FDP) and D-dimers gradually decreased and thus clinical improvement of DIC was also observed.1,5 Whereas APL patients treated with ATRA show rapid improvement of coagulopathy within a

few days, DIC in patients treated with As2O3 improves in 1 week or more (personal communication by Dr Zhou Jin, First College of Harbin Medical University, Harbin, People’s Republic of China). The effect of

As2O3 in inducing modest TF downregulation without TM upregulation, as shown in this study, seems to be compatible with the gradual and

slow improvement of DIC in patients treated with As2O3. Since As2O3 may neither interact nor activate a RA-responsive element in the TM gene, it may not be able to upregulate TM

expression. The effects of As2O3 could be due to the rapid modulation α and degradation of PML/RAR fusion protein induced by As2O3.As a result of the disappearance of the PML/RARα fusion protein, which Figure 3 RT-PCR analysis of TF and TM mRNAs in NB4 cells might have some stimulatory effect on TF gene expression in the APL treated with As2O3. Total RNAs were extracted from cultured NB4 cell line NB4, the level of TF expression may gradually decrease. µ cells after exposure to 1 M As2O3 for 7 or 14 days. RT-PCR analysis Whereas ATRA has a positive- or negative-acting effect by inducing 3 of TF (a) or TM (b) mRNA was performed as previously described. the formation of complexes with its receptors, As2O3 does not form Base pair markers are shown to the left. Glyceraldehyde-3-phosphate such complexes to directly regulate TM and TF gene expression. The

dehydrogenase (GAPDH) mRNA was used as a control for assessing repression of TF by As2O3, therefore, progresses more slowly than that the quantity of mRNA. in the case of ATRA. For the effect on cell differentiation induced by ATRA, the presence of the PML/RARα fusion protein is rather necessary. Also, it has been

reported that As2O3 inhibits ATRA-induced, rapid differentiation of NB4 cells when applied in vitro in combination with ATRA.6 In our

To examine the changes in TF and TM mRNA levels, NB4 cells study, pretreatment with As2O3 partially abolished the rapid TM upre- were treated with As2O3 for 7 or 14 days. In cells treated with As2O3, gulation and TF downregulation effects of ATRA. The rapid disappear- the level of expression of TF mRNA decreased more gradually than ance of the PML/RARα fusion protein might interfere with the action 3 that observed in cells treated with ATRA (Figure 3a), and As2O3 did of ATRA on TM and TF regulation. These phenomena indicate that

not induce any change in the level of expression of TM mRNA As2O3 inﬂuences the RA-induced pathway for regulation of these (Figure 3b). genes. Whereas the modulation of TF by As2O3 appears to occur in Treatment of NB4 cells with 0.1 µM ATRA for 1 day markedly parallel with cell differentiation or apoptosis, further investigations are reduced the level of expression of TF mRNA (Figure 4a, lane 2), necessary to elucidate the precise mechanism involved in these µ whereas treatment with 1 M As2O3 for 2 days only slightly reduced functions. the TF mRNA level (Figure 4a, lane 3). The downregulation of TF mRNA expression induced by ATRA was partially inhibited by pre-

treatment of the cells with As2O3 (Figure 4a, lane 4). Expression of TM mRNA was markedly increased in NB4 cells treated with ATRA for 1 day (Figure 4b, lane 2), whereas the level of expression of TM Acknowledgements

mRNA did not change in cells treated with As2O3 for 2 days (Figure 4b, lane 3). The upregulation of TM mRNA expression induced This work was supported in part by research grants from the Atsuko by ATRA was apparently inhibited by pretreatment with As2O3 Ouchi Memorial Fund, Tokyo Medical and Dental University, from (Figure 4b, lane 4). According to Western blotting, treatment with the Research Foundation for Clinical Pharmacology. 0.1 µM ATRA appreciably reduced the level of promyelocytic leukemia (PML)/RARα fusion protein, but the level of PML/RARα decreased µ 11 more markedly after treatment with 1 M As2O3. The downregulation M Ohsawa School of Allied Health Sciences, and α 12 of PML/RAR expression induced by As2O3 was partially inhibited by T Koyama First Department of Internal Medicine, cotreatment with ATRA. M Shibakura1 Tokyo Medical and Dental University, 1 During the period of As2O3 therapy, the levels of the markers indica- S Kamei Tokyo, Japan S Hirosawa2

References

1 Shen ZX, Chen GQ, Ni JH, Li XS, Xiong SM, Qui QY, Zhu J, Tang W, Sun GL, Yang KQ, Chen Y, Zhou L, Fang ZW, Wang YT, Ma J, Zhang P, Zhang TD, Chen SJ, Chen Z, Wang ZY. Use of arsenic

trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efﬁcacy and pharmacokinetics in relapsed patients. Blood 1997; 89: 3354–3360. 2 Koyama T, Hirosawa S, Kawamata N, Tohda S Aoki N. All-trans retinoic acid (ATRA) upregulates thrombomodulin and downregulates tissue factor expression in acute promyelocytic leukemia cells. Blood 1994; 84: 3001–3009. 3 Shibakura M, Koyama T, Saito T, Miyasaka N, Kamiyama R, Hiro- sawa S. Anticoagulant effects of synthetic retinoids mediated via different receptors on human leukemia and umbilical vein endothelial cells. Blood 1997; 90: 1545–1551. 4 Wang Z-G, Rivi R, Delva L, Ko¨nig A, Scheinberg DA, Gambacorti- Figure 4 RT-PCR analysis of TF (a) and TM (b) mRNAs in NB4 Passerini C, Gabrilove J, Warrell RP, Pandolﬁ PP. Arsenic trioxide

cells treated with As2O3 and/or ATRA. Total RNAs were extracted and melarsoprol induce programmed cell death in myeloid leuke- from cultured NB4 cells after exposure to 0.1 µM ATRA for 1 day (lane mia cell lines and function in a PML and PML-RARα independent µ 2), 1 M As2O3 for 2 days (lane 3), or ATRA and As2O3 for 1 day after manner. Blood 1998; 92: 1497–1504. 1-day preincubation with As2O3 (lane 4). 5 Zhu J, Guo WM, Yao YY, Zhao WL, Pan L, Cai X, Ju B, Sun GL,

Leukemia Correspondence 943 Wang HL, Chen SJ, Chen GQ, Caen J, Chen Z, Wang ZY. Tissue K, Lamph WW, Waxman S, Pelicci PG, LoCoco F, Avvisati G, factors on acute promyelocytic leukemia and endothelial cells are Testa U, Peschle C, Gambacorti-Passerini C, Nervi C, Miller WH differently regulated by retinoic acid, arsenic trioxide and chemo- Jr. Arsenic trioxide as an inducer of apoptosis and loss of therapeutic agents. Leukemia 1999; 13: 1062–1070. PML/RARα protein in acute promyelocytic leukemia cells. J Natl 6 Shao W, Fanelli M, Ferrara FF, Riccioni R, Rosenauer A, Davison Cancer Inst 1998; 90: 124–133.

Cellular cytotoxic drug sensitivity in children with acute leukemia and Down’s syndrome: an explanation to differences in clinical outcome?

TO THE EDITOR

During the last decade several groups have reported that acute myeloid leukemia (AML) in children with Down’s syndrome (DS) has a significantly better outcome than AML in other children.1,2 In acute lymphoblastic leukemia (ALL), on the other hand, the clinical outcome tends to be worse for DS than for non-DS children.3,4 The reason for this difference between AML and ALL is not known. In an ongoing study we prepared leukemic cells at diagnosis from 62 children with AML and 222 children with ALL for test of in vitro sensitivity to cytotoxic drugs. Five children with AML (8%) and five children with ALL (2.3%) also had DS, figures similar to those previously reported in population-based studies, and this enabled us to compare drug sensitivity in tumor cells from DS and non-DS children. Leukemic cells from fresh bone marrow or blood samples were assessed for their in vitro drug sensitivity by the fluorometric microcul- ture cytotoxicity assay (FMCA) against a panel of drugs used in the current Nordic protocols for AML2 and ALL,5 respectively. The FMCA is a non-clonogenic assay, very similar to the more widely used MTT assay,6 and is based on the measurement of fluorescence generated from hydrolysis of fluorescein diacetate to fluorescein by cells with intact plasma membranes.7 A survival index (SI) was calculated as the fluorescence signal of cells after 72 h of drug incubation as a percentage of that of unexposed controls. Leukemic cells from DS children with AML were significantly more sensitive (P = 0.03–0.002) than blast cells from non-DS children to cytosine arabinoside (Ara-C), doxorubicin, dexamethasone (see Figure 1a) and amsacrine (not shown). A trend in the same direction was found for etoposide (VP16) and 6-thioguanine. DS patients with AML were significantly younger (mean 2.1 years) than non-DS patients (7.9 years), but a statistically significant difference was found also when comparing the DS samples with those of age-matched controls, and there was no clear correlation between age and in vitro sensitivity among non-DS patients with AML. Three Down patients had FAB subtype M1 and two had M7 AML. In contrast, leukemic cells from DS children with ALL were significantly less sensitive (P = 0.02–0.001) than cells from non-DS patients to dexamethasone and asparaginase, with a similar trend for vincristine, doxorubicin and Ara-C (Figure 1b). The mean age for DS children with ALL was 10.8 years and for non-DS patients 6.5 years (NS). Two DS patients fulfilled standard risk and three intermediate risk criteria according to the Nordic ALL protocol. Figure 1 In vitro sensitivity of leukemic blast cells from children Findings similar to ours have previously been described in DS chil- with Down’s syndrome and acute myeloid leukemia (AML; n = 5), dren with AML,8,9 while in ALL a single study on six DS patients upper panel, or acute lymphoblastic leukemia (ALL; n = 5), lower showed no significant differences for most drugs tested (ALL cells of panel, compared to non-Down children with leukemia (n = 57 and DS patients were slightly more sensitive to anthracyclines).9 217, respectively). SI denotes surviving cells, in percentage of The inferior clinical outcome in ALL has been attributed to excess- untreated control, after 72 h incubation with cytosine arabinoside ive therapy-related toxicity in DS children, but this would not explain 0.5 µg/ml (Ara-C), doxorubicin 0.5 µg/ml (Dox), etoposide 5 µg/ml the difference between AML and ALL. Our data, which have to be (VP16), 6-thioguanine 10 µg/ml (6-TG), dexamethasone 7.1 µg/ml confirmed in larger series of patients, indicate that at least part of the (Dexa), vincristine 0.5 µg/ml (Vcr), asparaginase 10 U/ml (Asp). The explanation for the differences in clinical outcome between DS and results are presented as mean values plus s.e.m. The statistical signifi- non-DS children should be sought among mechanisms for tumor cell cance levels (Student’s t-test) are indicated; NS, non-significant. drug resistance. The fact that the difference in drug sensitivity between DS and other children was diametrically opposed for AML and ALL, suggests cell lineage-specific rather than general cellular alterations associated with the presence of three copies of chromosome 21 as suggested by Taub et al.8 Presently, we do not understand the Correspondence: G Lo¨nnerholm, Akademiska Barnsjukhuset, SE- mechanism(s) behind the differences in drug sensitivity between DS 75185 Uppsala, Sweden; Fax: 46 18 665853 and non-DS children, but we think our data point at an area for further Received 20 October 1999; accepted 22 December 1999 fruitful research.

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