From by guest on October 20, 2011. For personal use only.

1996 87: 657-665

Anticoagulant effects of retinoic acids on leukemia cells
T Saito, T Koyama, K Nagata, R Kamiyama and S Hirosawa

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Japan). Japan. We have further investigated the effects of ATRA on leukemic cells freshly isolated from patients at diagnosis. HL60. Initial clinical trial of 9 4 s RA for treatment of APL is ongoing! In this study. were provided by Corvas International Inc (Greenwich.00/0 657 .’ ATRA has high binding affinity to RARs but very low-affinity to RXRs. 0 1996 by The American Society of Hematology.6 Monoclonal mouse antihuman TF IgGs. Tokyo Medical and Dental University. leads to disseminated intravascular coagulation syndrome (DIC) with considerably high frequency. Tokyo Medical and Dental University. We have recently found that ATRA upregulates thrombomodulin (TM) and downregulates tissue factor (TF) expression in APL (NB4) and monoblastic leukemia (U937) cell lines. are applied not only t o established cell lines of specific subtypes (M3 and M51 but also t o more universal AML (most cases of M3 and M5 and a part of the other types of AML) cells freshly isolated from patients. 1995. upregulation of TM expression and downregulation of TF expression. For personal use only. KA-3 and KA-4. Since we have suggested a potential efficiency of ATRA as a preventive and therapeutic agent for DIC in AML M3 and acute monoblastic leukemia. AML MS. We have also studied the effects of 9 4 s RA and 1 3 4 s RA on the expressions of TM and TF in NB4 and U937 cells. Human protein C (inactivated and activated) and human antithrombin I11 were generously provided by the ChemSero-Therapeutic Research Institute (Kumamoto.01 t o 1 pmol/L) compared with ATRA was optimal for prolongation of normal plasma-based f e recalcification time (reduction o c lsurface TF activity). 1996: pp 657-665 A From School of Allied Health Sciences and the First Department of Internal Medicine. Vol 87. 2011.From bloodjournal. The publication costs of this article were defrayed in part by page charge payment. The chromogenic substrate S2266 was from Kabi Vitrum (Stockholm.’ Furthermore. in vitro3 and does not induce its own catabolism. Ryuichi Kamiyama. Polyclonal antihuman TM antibody was from Teijin Ltd (Tokyo. 1 3 4 s RA. acute myelogenous leukemia (AML) M3 according to the French-American-British classification. we have examined the regulation of TM and TF expressions in patients’ leukemic cells and have compared the effects of 9cis RA with those of ATRA and 13-cis RA on leukemic cell lines. Tokyo. These features may distinctly be advantageous for 9-cis RA for treatment of patients with AML M3 and other diseases. (n = 5). B/ood84:3001. All other chemicals were reagent-grade products. 0006-4971/96/8702-0040$3. which is a stereoisomer of RA. Science.hematologylibrary. Israel. Submitted June 5.000 NIHU/mg protein) were from Sigma (St Louis. distinct expressions of TM and TF in human leukemic cells and their regulation by ATRA were shown. MO). Chemicals were purchased from the following suppliers. section 1734 solely to indicate this fact. No 2 (January 15). is a high-affinity ligand for the RA receptors (RARs) and the retinoid X receptors. A relatively wide range of 9cis RA concentrations (0. MATERIALS AND METHODS Reagents. 6B4 and 5G9. 9-cis RA was kindly provided by F. and all M3 and M5 patients. and M6 (n = 1). Decrease of TF antigen was observed in 4 M2. Address reprints requests to Takatoshi Koyama. ie. Japan).1 M4. MD. Western blot analysis under nonreducing conditions showed that both ATRA and 9-cis RA markedly induced the prominent band at 75 kD of TM and reduced the band at 45 kD of TF. solely bind t o RARs. Anticoagulant Effects of Retinoic Acids on Leukemia Cells By Takako Saito. which possesses the entire extracellular domain. no TM and TF antigens were detected in all chronic lymphocytic leukemia cells (n = 3) with or without ATRA treatment. ATRA induces not only terminal differentiation of malignant cells but also rapid amelioration of DIC associated with marked hyperfibrinolysis. NB4 and U937. Increase of TM antigen O M2 was documented in all AML cells: M (n = l). 13-cis RA. Sports and Culture and by a grant for intractable diseases from the Ministry of Health and Welfare of Japan. was a gift from Asahi Chemical Industry CO (Fuji. KA-3 binds to N-terminal part of the epidermal growth factor-homology domain and KA-4 recognizes the “serine/ threonine-rich’’ polypeptide core near the primary thrombin binding site of TM molecule. The biologic effects of ATRA are mediated by its binding to the retinoic acid receptors (RARs). M4 (n = 3). 9 4 s RA. Takatoshi Koyama. 9 4 s RA may be more effective than ATRA as an inducer of differentiation of AML M3 cells and as an anticoagulant agent for patients with certain types of AML as well. Japan) and Green Cross (Osaka. thrombin (4. This article must therefore be hereby marked “advertisement” in accordance with 18 U. 1-5-45. Sweden). all-trans retinoic acid (ATRA) has been introduced for treatment of APL and has become practically and consistently effective in inducing complete remission. accepted August 30. Musashino Red Cross Hospital. ATRA. Kaoru Nagata. Jerusalem. CT) by guest on October 20. Changes of TM and TF antigen levels were associated with those of TM and TF cofactor levels on thecell surface. Northern blot analysis has shown similar changes of mRNA levels. Presented in part in a Symposium at the XVthCongress of International Society on Thrombosis and Haemostasis. Monoclonal mouse antihuman TM IgGs.Bunkyo-ku. Anticoagulant effects of ATRA. Switzerland). Recently. June 1995. Japan). were obtained by established hybridomas techniques as previously reported? KA-3 and KA-4 recognize different epitopes of TM. Supported in part by a research grantfrom the Ministry of Education. porcine mucosal heparin. decrease of TF antigen. Hoffmann-La Roche Ltd (Basel. Germany). we were urged to study ATRA effects on expressions of TM and TF in primary leukemic cells freshly isolated from patients with AML. although ATRA and another isomer. 113 Japan. Human placenta-derived “F (Thromborel S ) was from Behringwerke AG (Marburg. and increase of TM antigen on the surface and in the lysates of NB4 and U937 cells. The First Department of Internal Medicine. Tokyo. Recombinant human soluble TM. 9-cis RA is slightly more potent inducer of differentiation of a AML M2 cell line. 0 1996 by The American Society of Hematology. respectively. and Shinsaku Hirosawa We have recently found that all-trans retinoic acid (ATRA) upregulates thrombomodulin (TM) and downregulates tissue factor (TF) expression in acute myelogenous leukemia (AML) M3 cells (NB41and acute monoblastic leukemia cells (U9371 (Koyama et al. A stereoisomer of RA. M5 (n = 31. Yushima. and the Department of Hematology. has been found to be a high-affinity ligand for RARs and retinoid X receptors (RXRs).1994). However.C. DIC is often exacerbated by treatment with cytotoxic drugs. which indicates that RAs regulate TM and TF expression in leukemic cells at transcriptional levels. 9-cis RA. 1995. M3 (n = 3). CUTE PROMYELOCYTIC leukemia (APL). andhuman aBlood.

The cell numbers were counted and adjusted. Leukemiccellsisolated from patients were incubated with ATRA (1 pmol/L) for 24 hours. and -3). M5b-1and -2. Total TM antigen in cell lysates was measured by enzyme-linked immunosorbent assay (ELISA) using an antihuman TM monoclonal antibody U .~ patient from whom NB4 was established suffered from DIC at diagnosi~. -2. Paris. -2. 2 patients with acute lymphoblastic leukemia (ALL. Leukemic cells were incubated with agents for the times indicated and washed with PBS three times. Measurement of TM and TF antigens.- ATRA (-l (+l (-1 (+l Fig 1. Cell lysates were stored at -80°C until the assay. Japan). Japan) supplemented with 10% fetal calf serum. Peripheral blood samples were obtained at diagnosis before the start of chemotherapy and the mononuclear fraction was isolated by sedimentation on Ficoll-Hypaque density gradients. 9-cis R A . Mononuclear cells were then washed three times in phosphate-buffered saline (PBS) and suspended in RPM1 1640 medium (Nissui Pharmaceutical CO. NB4 (APL. For personal use only."' Total TF antigen in cell lysates was also measured by ELISA using antihuman TF monoclonal antibodies 6B4 and 5G9 as already reported. M2-1. ATRA. and -3. and M6). Effects of ATRA on total TM and TF antigens in leukemic cells freshly isolatedfrom patients.1 %. The control leukemia cells (those incubated without RAs) were exposed to an appropriate amount of ethanol in the culture medium.1. Total TM (A) and TF (B) antigen levds were shown on ordinates.~ U937 wasprovided by the Japanese Cancer Research Bank (Tokyo. -3. Cells were then extracted for 30 minutes with by guest on October 20. 'DIC was documented or suspected. and reagents were purchased from Wako Pure Chemicals (Osaka. ALL-l and -2) and 3 patients with chronic lymphocytic leukemia (CLL. AML M3) was kindly provided by Dr M. -2. Cell culture. Cell debris was removed by centrifugation at 12. no significant differences of TM and TF expressions were generally observed. unless otherwise indicated. Blood samples were selected for study only when they were shown to contain more than 85% blasts. M5a.000g for 20 minutes. All the procedures involving RAs were performed in subdued lights. and 13cis RA (MS) were dissolved in absolute ethanol to a concentration of 8. and -5.7. M4-1. Lanotte (HBpital Saint Louis. The following human leukemic cell lines were used in this study. Whether primary leukemia cells were analyzed immediately after isolation or after 24 hours of culture." Cell surface TM cofactor activity.' Surface TM cofactor activity was determined by the hydrolysis of chromogenic substrate . M3-1.32 m m o K and further diluted into growth medium at the desired concentrations so that the final ethanol concentration in the culture media was less than 0. Tokyo. CLL. and -3. France). 658 SAlTO ET AL " 3 M5b-1 M2-l M3-1 M6 M2-4 M3-2 M2-3 M2-5 M2-2 M O CLL-l 2.3 and a polyclonal antihuman TM antibody as reported before.3 ALL-IIP ATRA ATRA- .5% Triton X-100 in PBS at 4'C. -2. -4. Leukemic cells freshly isolated from patients. 17) withthe rearrangements of RARa and promyelocytic leukemia (PML) genes as shown in the most cases with APL and the presence of PMLA RARa fusion pr0tein. TM cofactor activity on cell surface was measured as described previously.hematologylibrary. Peripheral blood was drawn from 16 patients with AML (MO. NB4 is characterized by the presence of chromosomal translocation t( 15. 2011.From bloodjournal. Japan).

Cell surface TM (AI and TF IBI activities were I determinedfor 10' suspended ATRA cells as described in the Materials and Methods. Cells ( I X IO6) were added to 0. F 3 " T l ATRA ATRA - CLL-1 ATRA (-1 (+l (-1 (+l . Japan).TM (-1 (+) placenta M4-3 M5b-2 M3-1 M5b-1 M5a TF Fig 2. Cell surface TF cofactor activity: anai. MA). *S 0 c B 200- MSb-2 0 E M4-2 MS1 . After transfer t o lmmobilon (Millipore Corp.' TF cofactor activity was quantitated by reference to standard curves (log-log plot) constructed with human placenta TF and TF activity. Cell surface TM and TF 100activitiesof patients' leukemic cells modulated by ATRA. lane 11). soluble recombinant TM was used (A. OF ANTICOAGULANT EFFECTS A kD B kD 662009766451 ATRA(-) 45- 31 22 - 2 (+) 3 4 5 6 7 (-) 8 (+) 9 1 0 1 1 2 3 1 (-1 (+l (-1 (+l (-1 (+l rec. As a control.From bloodjournal. that yielded a 50-second recalcification time was defined as 1 UlmL. and standard indirect immunofluorescence A 300 M4-3 1 . cvtometric anal?f sis. The prominent 1 band at approximately75 kD represents TM (A). Analysis o surface TM and TF antigens byflow. and 1 3 4 s RA. placenta TF was used (B.E .Jminitue or as a percentage of the initial velocity of activated protein C formation (100% for the formation with the cell surface TM under basal conditions). A monoclonal anti-TF antibody 5G9 identified thenonreduced form of TF as a band at approximately45 kD (B).org by guest on October 20.. Leukemic cell suspensions were adjusted to 1 X 107/mL in PBS. Leukemic cell samples freshly obtained from several patients were incubated with 1 pmol/L ATRA for 24 hours. For personal use only. Bedford.. As a control. Each set of leukemic cells was treated with p n o l / L ATRA for 24 hours. Because our previous report has already shown that the procoagulant activities on the surface of NB4 and U937 cells determined as described above are deduced from TF expression. The suspensions were incubated with an inhibitory monoclonal anti-TF antibody 6B4 or an anti-TM antibody KA-4 at a concentration of 10 &mL for 30 minutes at 4°C. prolongation of recalcification time ismainly due to downregulation of TF expression by RAs. After incubation at 37°C for 3 minutes. Kobe.W c 0 (D Fig 3.1 mL ofpooled human normal plasma. The results were expressed as Am0D4.1 mL of 25 mmol/L calcium chloride was added and plasma recalcification time was determined with a semiautomatic coagulometer CA1 0 0 (Sysmex. 0. NB4 cells and U937 cells were washed with PBS and IO" cells were resuspended in I mL PBS. lane 31. Western blot analysis of patients' leukemic cell preparationswith anti-TM andanti-TF antibodies. Leukemic cells (10'1 were lyredin detergent and the preparations were subjected t o sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE1under nonreducing conditions. immunoblotting wasaccomplished with a monoclonal anti-TM antibody KA-3 (A) and a monoclonal anti-TF antibody 5G9 (B). After incubation for 24 hours with or without I pmol/L ATRA.hematologylibrary. S-2266. 2011.vsis of procoagulant acrivity in clotting assay. Controls of the cells in the absence of thrombin and protein C were treated similarly and no activation of protein C was observed. 9 4 s RA.

A and 13-cis R A ) for 24 hours.OOO1 to B pmollL) for 24 hours. For personal use only. o I us37 10-4 10-3 IO-* 10-1 I 5 @M) (-1 all-bm. Total cytoplasmic RNAwas prepared from NB4 and U937 cells after treatment with ATRA. and M5b-1 and -2. Both TM and TF expression in all CLL cells was not detected before and after ATRA treatment. techniques were used for the analysis of surface TM and TF by guest on October 20. 6. or 13cis RA for 5 hours by the acid guanidium thiocyanate-phenol-chloroform method. Furthermore. lane 1). Daiichi Pharmaceutical CO. NB4 cells were incubated with various concentrations 9of cis RA (O. Isolated RNA was analyzed in Northern blotting using Digoxigenin Luminescent Detection Kit (Boehringer Mannheim. Wakita. respectively (Molecular Biology Research Laboratory.24 ng TF protein per lo7 cells.From bloodjournal. whereas TF expression was documented in 1 patient. The monoclonal antibody KA-3 identified the nonreduced form of TM as a prominent band at approximately 75 kD. Concentration dependency of S-cis RA on the TM (A) and TF (B) antigenexprsssion in NB4 cell lysates and cell surfaceTF activity of NB4 cells (C) were determined. and 13A TF cis RA on cell surface activity were compared (Dl. t U . Cell surface TM and TF activities o patients' leukemic f cells modulated by ATRA. -2. Horseradish peroxidaseconjugated anti-TF monoclonal antibody 5G9 was usedfor the detection of TF. and all M5 [M5a and M5b-1 and -21 cells) examined (Fig 2A. Effects of RAs on total TM and TF antigens in NB4 cell lysates and cell surface TF activity of NB4 and US37 cells. Northernblotting. TM markedly increased in all AML cells (M3-1. The cases in which DIC was documented or suspected are marked with an asterisk in Fig 1B.'* RESULTS Effects of ATRA on TM and TF antigens in leukemic cells When untreated. Nawa and Dr K. M4-3." Immunoblorting analysis.The monoclonal antibody against human TF 5G9 identified the nonreduced form of TF in M3-l cells as a band at approximately 45 kD (Fig 2B. After treatment with ATRA. 9 4 s RA. M3-1. The presence of TM and TF molecules in the patients' leukemic cells was determined by immunoblotting with the antibodies specific for human TM and TF. After incubation with ATRA. The TF cDNA probe was prepared by a polymerase chain reaction (PCR) method from a cDNA library of tumor necrosis factor a-stimulated human umbilical vein endothelial cells. 4. S-cis R . Fig 1B). TM activities were increased on the . lane 2). 660 SAITO ET AL 150 - A cn c B 100- 3 10- l - l& l& 9 4 s RA (PM) l& lo" C IOW - D z 3 E 3 ATRA 3 E E oooOciaRA T 8 F 2 t E 500- j RA JNB4 U . A T t h l l l I-Nhe I fragment in the coding region of the human TM gene and a TF cDNA probe were kind gifts from Dr K. and were used as hybridization probes. M4-3. -4. S-cis R . Western blot analysis for TM and TF in leukemic cells from patients. The same amount of lysates from lo6 cells was applied to each lane. and -5. In 2 patients with ALL. Japan). -2. Western blot analyses of TM and TF in cell lysates were performed essentially as described previously. lanes 2. 9-ci8 RA(+) RA(+) 134s RA(+) Fig 4.' KA-3 and horseradish peroxidase-conjugated rabbit antimouse IgG antibody were used for the detection of TM. and -3. the effects of ATRA. After treatment of the leukemic cells with ATRA. TF of the leukemic cells was markedly decreased after treatment with ATRA (Fig 2B. TM expression in the leukemic cells was not detected before and after treatment. ATRA decreased TF antigen levels in I1 of 16 AML cells (M2-l. and 10). TM and freshly isolated frompatients. Germany). total TM antigen was increased in all 16 AML cells (Fig 1A).hematologylibrary. Effects of ATRA on Th4 and TF activities on the surface of leukemic cell samples freshly obtained from several patients are shown in Fig 3. M5a. In contrast. 2011. Tokyo. NB4 and US37 cells were incubated with 1 pmollL RAs ( A M . TF antigen levels in freshly isolated leukemic cells were varied in the range of 0 to 865 ng TM protein and 0 to 9. Mannheim. 8.

9-cis RA. We found 40. which is an easily attainable level in patients treated with 9-cis RA. and 13-cisRA. 2011. the concentration at which ATRA decreases expression of TF most efficiently. The TM antigen levels in U937 cells were increased by treatment with ATRA. 9-cis RA. As shown in Fig 4C. and 13-cis RA on T M antigen in NB4 and U937 cells. or 13-cis RA for 24 hours. induced an efficient decrease of TF antigen and an increase of TM antigen (Fig 4A and B). on total TM and TF antigens f in NB4 andU937 c e l l s . M4-3. 9 4 s (+) 4 134s (+) . "1 Fig 5. ATRA. ATRA. on the expression of TM and TF in NB4 cells were examined. and 134s RA (lane 4) for 24 houn. 9-cis R A .8% of the NB4 cells positive for the anti-TM antibody KA-4 by FCM analysis (Fig 6A). ie.1%. 0.4% of the NB4 cells to . respectively (Fig 6B. F C M analysis of T M and TF antigens on the cell sugace of NB4 and U937 modulated by RAs. 9-cis RA increased TM antigen level while decreasing the TF antigen level dose-dependently. the TF activities were reduced by all RAs (Fig 4D). 4 1 3 4 s (+) . and 94.8%.01 to 1 pmol/L. ANTICOAGULANT EFFECTS OF RETlNOlCACIDS 661 A B NB4 c t so} U937 1 l - 2 3 1 4 4 *B LL I 3 1 T 1. and 13-cis RA (1 pmoVL) reduced TF antigen levels in NB4 and U937 cells (Fig 5C and D). 8 . NB4 T U937 1. control 2 all-rrans(+) . Cell su$ace TF activity of NB4 cells modulated by 9-cis RA and ATRA. A relatively wide range of 9-cis RA concentrations.1% by treatmentwith ATRA.From bloodjournal. In both NB4 and U937 cells.01 to 1 pmol/L) was optimal for reduction of TF activity when compared with ATRA. and 13-cis RA on TF antigen in NB4 and U937 cells. NB4 (A and C) andU937 (B and D) cells were incubated with l WmollL ATRA (Iana 2). Effects of 9-cis RA and ATRA on TF activity on the surface of NB4 cells were examined. 92. and 13-cis RA. The percentage of positive cells for KA-4 was increased to 94. a relatively wide range of 9 4 s RA concentrations (0. Effects of 9-cis RA on total T M and TF antigens in NB4 cells lysates (dose-dependency). In NB4 cells. 3. Effects of ATRA. The effects of a stereoisomer. 3 %cis (+) . and M5b-2) were significantly reduced. and 13-cis RA at a concentration of 1 pmoVL increased TM antigen to similar levels (Fig 5A). Effects o RA. control 2 all-rrens(+) . 9-cis RA was slightly more potent than ATRA at 1 pmol/L. When NB4 and U937 cells were pretreated with 1 pmol/L of ATRA. 9-cis RA. We found by guest on October 20. For personal use only.hematologylibrary. 9-cis RA was most effective for increase of the TM antigen level (Fig 5B). whereas those of another 2 patients with CLL-1 and M4-2 were not significantly changed (Fig 3B). and D). 9-cis R A . Cell surface TF activity o NB4 and U937 cells modulated f by R A s . 9-cia RA (lana 31. C. 9-cis RA. Lane lrepfwents the control.g m P 5 e 0 h1l 2L 1 2 3 + m O 1 1 2 3 4 surface of all AML cells studied (Fig 3A). 9-cis RA. TF activities on the surface of 3 patients' cells (M3-1. TotalTM (A and B) and TF (C and D) antigen levels are shown on the ordinates. Effects of ATRA. 9-cis R A .9-cis RA is slightly more effective than ATRA and 13-cis RA.

and 13-cis RA. changes of TM and TF mRNAs by ATRA were similar to those of NB4 and U937 cells (data not shown). Expression of specific mRNA for TF found as a single band at 2. The abscissa shows fluorescence intensities and the ordinate shows the number cells. In leukemic cells freshly isolated from patients. Although overall procoag- . G. 2011. whereas the percentage the positive cells decreased to 3. and 13-cis RA. The flow cytometric analyses of both untreated and treated cells incubated with KA-4 (anti-TM: A. and H). Although a rough correlation between relatively high TF antigen expression and the development of DIC seems to be present. E.0%. 3. development of DIC can be estimated by evaluating overall procoagulant and anticoagulant activities. and 98.5%. and 3. and 1 3 4 s RA (D and H). and 4). lanes 2. respectively (Fig 6F. whereas the percentage of positive cells decreased to 9.9-cis RA.From bloodjournal. a prominent band of TM at 75 kD was markedly increased to the level as high as when the cells were treated withATRA (data not shown). and H) monoclonal antibodyfollowed by goat FITC-antimouse IgGwere shown. 9. C. and 4).1 % of the untreated U937 cells to be positive for KA-4. Of the untreated U937 cells. 3. After treatment of NB4 and U937 cells with 9-cis RA and 13-cis RA. B.$ace TM activity in NB4 and U937 cells by 9-cis RA. F.108 2 0. When treated with ATRA.O03/min/5 X lo6 NB4 cells (34 2 2 ng activated protein C/106 cells) and 0.004/min/5 X lo6U937 cells (87. Increased f expression of specific mRNA for TM was detected as a single band at 3.1%. and D) or 684 (antiTF. 9-cis RA. Flow cvtometricanalysis of surface M and T TF antigensin NB4 and U937 cells modulatedby RAs.7% when treated with ATRA.036 -C O.1%. by guest on October 20. 18. Northern blot for TM and TF: effects o RAs. 1. we have shown that anticoagulant effects of RAs are inducible not only to specific subtypes (M3 and M5) of the established cell lines but also to certain AML subtypes (most cases of M3 and M5 and a part of the other types of AML) of the leukemic cells freshly isolated from patients. Western blot for TM: effects of RAs. We also found 79.4% when treated with ATRA. anticoagulant glycoprotein TM is also expressed variedly in leukemic cells. 9-cis RA increased the rate of protein C activation to about threefold in NB4 cells and to about 2. respectively. lanes 2. 99.3 kb was markedly decreased when NB4 cells were treated with RAs (Fig 8B. For personal use only. 9-cis RA. Enhancement of sur.hematologylibrary. NB4 and U937cells were untreated (A and E) or treated with l pmollL ATRA (B and F1.7 kb when NB4 and U937 cells were treated with RAs (Fig 8A. of be positive for the anti-TF antibody 6B4 (Fig 6E). Constitutive expression levels of TM and TF were vaned in untreated leukemic cells that were freshly isolated. and 7.2%. Surface TM activity induced by 9-cis RA was determined by the acceleration of thrombin-catalyzed protein C activation.5%. Basal AOD40s.0% were positive for 6B4. and 13-cis RA.6 5 3 ng activated protein C/106cells). respectively.9-cis RA (C and G).5-fold in U937 cells (Fig 7A and B). as shown in Fig 1. 99. Thus.7% of U937 cells were positive for KA-4. DISCUSSION In the present study. 662 SAITO ET AL A B C D E F G H control all-trans Qcis 134s A B C D E F TF control all-trans Qcis 134s Fig 6.m/min levels were 0.

as cytotoxic drugs do. Cell surface TF activities measured by recalcification time were reduced in 3 of 4 AML cells. For personal use only. 5.o- l . Surface TM activity was determined for 5 x lo6 suspended cells as described in the Materials and Methods. Twenty microgramsof total RNA was extracted from cultured NB4 and U937 cells after exposure to 1 pmol/L ATRA (lane 2). ATRA therapy exerts rapid improvement in coagulopathy and hyperfibrinolysis. whereas downregulation of its expression by ATRA was not shown. (2) Rapid amelioration of DIC is induced ATRA by through downregulation of TF and cancer procoagulant expressions in APL cells and upregulation of TM expression in APL andendothelial cells. and 13-cis RA (lane 4) for 5 hoursandelectrophoresedon a 1% agarose/ by guest on October 20. The presence of a unique procoagulant with cysteine proteinase activity distinct from TF in leukemic cells including NB4 cells has been reported.'' (3) Rapid reductionof elevated fibrinolytic and proteolytic activities in APL cells is exerted byATRA. in ALL and CLL cells. with a rapid correction of the bleeding tendency.". These observations suggest that differentiation into mature lymphocytic cells implies the loss ofTMand TF inducibility but that premature lymphoblastic cells may express TF." Upregulation ofTM antigen and activity expression by ATRA was shown in all primary leukemic cells from AML patients studied.9- . In this investigation.'" which leads to severe hemorrhagic complications.'~Upregulation ofTMmay also reduce hyperfibrinolysis induced by U-PA in patients with APL. TF expression was not documented either. as shown in NB4 and U937 cells.hematologylibrary. expressions of the other procoagulant and anticoagulant proteins should be considered to be involved in DIC. 9 4 s RA(+) ulant activity in leukemic cells may be controlled mainly by levels of TF and TM expressed. ( I ) The differentiation process induced by ATRA does not exacerbate the release of procoagulant and fibrinolytic substances from the leukemic cell. The blot was probed with a digoxigenin-labeled cDNA fragment specifically encoding TM (A) or TF (B). In 1 of 2 patients withALL. 9-cis RA (lane 3). These data suggest that upregulation of TM and downregulation of TF. l . although the levels of induction were different in each leukemic cells. 1. Lane l 1 2 3 4 1 represents untreated control. thrombin-antithrombin 111 complexes. and plasmin-a2-plasmin inhibitor c ~ m p l e x . control 2 . RNAmolecular weight markers are given along the left margin. 11 1 2 2 1. Northern blot analysis: effects of RAs.13 Differentiation therapy with RA induces complete remission in APL patients. In this context.32. 2011. ATRA decreased the level of TF antigen expression in 1 1 of 16 AML cells. we have also shown upregulation of A kb B kb +TM I.8Fig 8. Changes in TM cofactor activity for protein C activation on NB4 and U937 cell surfaces after exposure to 9-cis RA (1 pmollL1. However. the past and thefuture clinical trials of RAs for AML and other malignant cells to induce differentiation should be evaluated from the coagulation andfibrinolysis points of view. Results were expressed as the percentage of initial velocity of activated protein C (lane 2)compared with the control (lane 1) that was not treated with the reagent. TM may possess not only natural anticoagulant properties but also antifibrinolytic properties by acceleration of thrombin inactivation of single-chain urokinase-type plasminogen activator (U-PA). In CLL cells.9- 1.'.From bloodjournal.". TF expression was documented. Antifibrinolytic activity of TM should be further evaluated. no TM expression was documented in the absence and presence of ATRA.o- 2 3 4 1 2 3 4 NB4 U937 NB4 . are major factors in the improvement of DIC. cross-linked fibrin degradation product (D-dimer fragment). with normalization of plasma fibrinogedfibrin degradation product (fragment E). Several possible explanations for '~ marked reduction of hemorrhagic complications byATRA are proposed. ANTICOAGULANT EFFECTS OF RETlNOlC ACIDS 663 B U937 :TM Fig 7.

Marchetti M. Parkinson JF.01 to 1 pmol/L) than was ATRA. Nakajima H. Blood 83: 1883. Am J Hematol 46:184. Horiuchi T: Up-regulation of tissue factor mRNA in humanumbilical vein endothelial cells by calmodulin inhibitor W7. Am J Hematol 40:252. including ATRA and 9-cis RA. Miller WH Jr. Bang NU. 1992 10. 9 4 s RA has high binding affinity to both RARs and RXRs. Clerici M. 1994 . Sakamaki H. Hirokawa K. For personal use only. Kizaki M. Such concentrations are easily attainable level in patients treated with 9 4 s RA. Rigas JR. are intriguing and indispensable subjects for further investigation. Watanabe K. Najiman S. Evans RM. on proliferation. a recent report” has shown that TM induction in AML M2 cell line HL60 cells cultured with ATRA reflecting TM biosynthesis levels is independent of HL60 differentiation into neutrophilic cells. 1994 2.” More detailed analysis of cell differentiation and TM/TF expressions may be necesS T . Matsuzawa K. UT-7. Nervi by guest on October 20. High-affinity binding of RARs to RAREs takes place only if a separate retinoid receptor family. 1991 8. This may be one of the reasons why 9-cis RA is more effective than ATRA and 1 3 4 s RA in the regulation ofTM and TF in the leukemic cells. Formelli F. Morikawa M. Tapiovaara H. and embryonal morphogenesis. Mangelsdorf DJ. Sakashita A. Lanotte M. recombinant mutants of human thrombomodulin. but ATRA and 13-cis RA bind to RXRs with very low affinity. 1993 4. Aoki N: Frequent rearrangements of retinoic acid receptor alpha gene and My1 gene. cell proliferation. 1994 13. 1995 5. Falanga A. TM inducibility may not depend on cell differentiation by ATRA. Thromb Res 73:177. Gordon SG. The TM antigen level induced by ATRA in NB4 cells reaches a maximum level within a few days. 1l-cis. Kizaki M. The excellent skillful technical assistance of Misako Shibakura is gratefully acknowledged. Aoki N:Monoclonal antibodies to human thrombomodulin whose binding is calcium dependent. Rambaldi A. Dermime S. Pelicci PG. Miller VA. ATRA binds to RARs with high affinity. REFERENCES Retinoids derived from retinol (vitamin A) are critical regulators of vision. whereas cyclic AMP increases TM mRNA stability (our unpublished observations). Yokoyama K. 2011.Aoki N: Determination of plasma tissue factor antigen and its clinical significance. Hirosawa S. 9-cis RA was especially more effective in a relatively wide range of concentrations (0. KomatsuN. Tokyo. is present. Marchesi E. Ohdama S. Kawai Y. RXRs. Ulm E. Japan) for helpful discussions and supplying us some patient sam- I . Blood 82:1573. and rare mutations of RAS and F M S genes in acute promyelocytic leukemia. Kawamata N. Hurme M. On the other hand. Kimura S. Consonni R. Koyama T. Martin-Thouvenin V. Aoki N: Alltrans retinoic acid (ATRA) upregulates thrombomodulin and downregulates tissue factor expression in acute promyelocytic leukemia cells: Distinct expression of thrombomodulin and tissue factor in human leukemic cells Blood 84:300l. 1992 3. Kuriyama R. Stein RB. Obashi K. The regulation of RA-target genes is caused by the mediation of the nuclear receptors that are capable of binding to RA-responsive elements (RAREs) of a direct repetition of two 5’-PuGG(G/T)TCA-3’. Murata M. NB4 cells. Aoki N: The biosynthesis of thrombomodulin and its enhancement by dibutyryl CAMP in a human megakaryoblastic cell line. Blood 85:3021. In the experiment of RAs for the effects on total TM and TF antigens in the cell lysates and procoagulant activities on the surface of NB4 cells. Br J Haematol 87:343. Barbui T: Cancer procoagulant inthe human promyelocytic cell line NB4 and its modulation by all-trans retinoic acid. Preissner KT: Relationship between post-translational glycosylation and anticoagulant function of secretable. 1994 14. Moriki T. differentiation. Shirley M. GambacortiPasserini C: Occurrence of resistance to retinoic acid in the acute promyelocytic leukemia cell line NB4 is associated with altered expression of the pml/RARa protein. 1991 7. Panniani G. Muller-Berghaus G. Berger R: NB4. Cell 68:397. Nagoya T. Yamamoto K. 1994 15. Tokuhira M. Dyck JA. according to a recent report using RA-resistant NB4 cells. Tong WP. 1993 9. Koeffler HP: Effects of novel retinoic acid compound. Lanotte M. 9-cis RA may be slightly more effective than ATRA not only as an inducer of differentiation of AML M3 cells but also as an anticoagulant agent for treatment of patients with certain types of AML. Tohda S. Blood 82:3592. and 13-cis RAs. a maturation inducible cell line with t(15. J Biochem 105:478. Sawada M. Koyama T. Furthermore. Because ATRA upregulates TM expression in all primary AML cells. Tanosaki R. Eichele G. Int J Hematol54:341. Ikeda Y: Rapid improvement of coagulopathy by all-trans retinoic acid in acute promyelocytic leukemia. 1991 I 1. Wakita K. ples. 664 SAITO ET AL TM expression and downregulation of TF expression in NB4 andU937 cell lines by stereoisomer RAs. Thaller C: 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. ACKNOWLEDGMENT The authors are gratefully indebted to emeritus Prof Nobuo Aoki and Dr Shuji Tohda (Tokyo Medical and Dental University. Kamata T. Hasegawa R. 1994 12. the concentration at which ATRA has its most optimal effect. Murakami N. Br J Haematol 78:515. NB4 cells’ sensitivity to maturation by ATRA may be crucial to the loss of their procoagulant activity. but it is not yetknown whether RAs change TF mRNA stability. Matikainen S. Heyman RA. Vaheri A: Induction of differentiation of promyelocytic NB4 cells by retinoic acid is associated with rapid increase in urokinase activity subsequently downregulated by production of inhibitors. In human umbilical vein endothelial cells. Intracellular isomerases further convert some intermediates to 9-cis. Hirosawa S. Mielicki WP.hematologylibrary. and expression of retinoic acid receptor-a and retinoid X receptor-a RNA by HL60 cells. Ikeda Y. Handa M. Uchida H. Grignani F. Talamo GP. Nakajima H. Koyama T. Benedetti F. Gill GM. differentiation. Hirosawa S. Blood 77:1080. Truglia JA. Marumoto Y. Aoki N. However. 9-cis RA was slightly more effective than other types of RAs at 1 pmoV L. ATRA increases transcription of TM gene without changing TM mRNA stability. 1989 6. Nishida K.From bloodjournal. 13-cis RA and 9 4 s RA are naturally occumng ligands of the nuclear RARs. including TF and cancer procoagulant activities. Valensi F. Balerini P. Jakubowski A. Identification of specific pathways responsible for the anticoagulant effects of RAs. RAs thus regulate TM expression in leukemia cells at transcriptional level. Warrell RP Jr: 9-cis retinoic acid induces complete remission but does not reverse clinically acquired retinoid resistance in acute promyelocytic leukemia. Sozzi G. and U937 cells. 9-cis-retinoic acid. 17) marker isolated from a human acute promyelocytic leukemia (M3). Leukemia 8:156.

Mannucci PM: Thrombomodulin is a cofactor for thrombin degradation of recombinant single-chain urokinase plasminogen acti- vator “in vitro” andin a perfused rabbit heart model. Dawson AA: The bleeding disorder in acute promyelocytic leukaemia: Fibrinolysis due to U-PA rather than defibrination. ANTICOAGULANT EFFECTS OF RETlNOlC ACIDS 665 16. Thromb Haemost 73:1280. GambacortiPasserini C. 1989 20. Marchetti M. Orsini G. Daniel M-T. Br J Haematol 71:511. Thromb Haemost 72573. 2011. Castaigne S. Croll A. Dombret H. Consonni R. Vaghi F. Bennett B. Ishii H. Molinari A.From bloodjournal. Fenaux P. Rijken D: Acceleration of the thrombin inactivation of single-chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin. Leukemia 9: 19. Groeneveld E. Scrobohaci M-L.hematologylibrary. Chomienne C. Duncan A. Booth NA. Lanotte M. Degos L: In vivo thrombin and plasmin activities in patients with acute promyelocytic leukemia (APL): Effect of all-trans retinoic acid (ATRA) therapy. 1994 21. Lansen J. J Clin Invest 88:1680. Kizaki K. 1992 19. Giorgetti C.Oldani E. Barbui T Effect of all-trans-retinoic acid on the procoagulant activity of retinoic acid-“resistant” human promyelocytic cell lines. For personal use only. DeMunk G. Falanga A. MiclCa J-M. 1991 18. Kazama M: Thrombomodulin induction by all-trans retinoic acid is independent of HL-60 cells differentiation to neutrophilic cells. Faioni EM. 1995 (abstr) . Hone S .org by guest on October 20. Thromb Haemost 67:226. 1995 17.

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