In vivo antitumor activity of the NF-
B inhibitor dehydroxymethylepoxyquinomicin in a mouse model of adult T-cell leukemia
Takeo Ohsugi *,
Ryouichi Horie 1,
Toshio Kumasaka 2,
Akira Ishida 3,
Takaomi Ishida 4,
Kazunari Yamaguchi 5,
Toshiki Watanabe 4,
Kazuo Umezawa 6 and
Toru Urano
Division of Microbiology and Genetics, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan, 1 Department of Hematology, Faculty of Medicine, Kitasato University, 1-15-1 Sagamihara, Kanagawa 228-8555, Japan, 2 Department of Pathology (I), Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan, 3 Department of Computer and Media Science, Faculty of Engineering, Yamanashi University, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan, 4 Division of Pathology, Department of Cancer Research, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan, 5 Department of Safety Research on Blood and Biologics, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama-shi, Tokyo 208-0011, Japan and 6 Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-0061, Japan
* To whom correspondence should be addressed. Tel: +81 96 373 6549; Fax: +81 96 373 6552; Email: ohsugi{at}gpo.kumamoto-u.ac.jp
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Abstract
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Adult T-cell leukemia (ATL) is an aggressive neoplasm caused by human T-cell leukemia virus type I (HTLV-I). The nuclear transcription factor, NF-
B, is induced by HTLV-I and is central to the ensuing neoplasia. To examine the effect of a novel NF-
B inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on ATL in vivo, we developed an improved severe combined immunodeficiency (SCID) mouse model for ATL. Five-week-old SCID mice in which natural killer (NK) cell activity had been eliminated were inoculated intraperitoneally with the HTLV-I-infected cell lines, TL-Om1, MT-1, MT-2 and HUT-102. No engraftment of TL-Om1 cells and little tumorigenesis of MT-1 cells were detected 40 days after injection. In contrast, inoculation of mice with MT-2 and HUT-102 cells elicited high mortality, 100% frequency of gross tumor formation and tumor cell infiltration of various organs, all of which were reduced by coadministration of DHMEQ during the inoculation. Moreover, tumors from mice treated with DHMEQ had a high frequency of apoptosis. These results suggest that DHMEQ induces apoptosis in HTLV-I-transformed cells in vivo, resulting in inhibition of tumor formation and organ infiltration, thereby enhancing survival.
Abbreviations: ATL, adult T-cell leukemia; DHMEQ, dehydroxymethylepoxyquinomicin; DMSO, dimethylsulfoxide; HTLV-1, human T-cell leukemia virus type 1; NK, natural killer; SCID, severe combined immunodeficiency
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Introduction
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Adult T-cell leukemia (ATL) is an aggressive peripheral CD4-positive T-cell neoplasm caused by human T-cell leukemia virus type I (HTLV-I) infection (1,2). ATL is a lethal disease for which there is no effective treatment. It was recently reported that the nuclear transcription factor, NF-
B, plays a key role in oncogenesis induced by HTLV-I (3). NF-
B can be activated by HTLV-I-encoded tax and is constitutively activated in all ATL cells (3). Constitutively activated NF-
B appears to be the molecular basis for aberrant growth and cytokine gene expression observed in ATL cells. Therefore, NF-
B inhibitors may provide new therapeutic modalities for HTLV-I infections and ATL.
We have developed a novel NF-
B inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) (4), a 5-dehydroxymethyl derivative of epoxyquinomicin C, an antibiotic originally isolated from Amicolatopsis sp. (5). Most NF-
B inhibitors inhibit I
B
phosphorylation, whereas DHMEQ inhibits nuclear translocation of p65, a component of NF-
B (6). DHMEQ inhibits multiple myeloma cells in vitro (7) as well as in severe combined immunodeficiency (SCID) mice (7) and hormone-refractory prostate cancer in nude mice (8). DHMEQ has not shown toxicity in animals (4,6), suggesting the specificity of this compound for NF-
B.
ATL cells do not form tumors in nude mice, which are often used in cancer research. Leukemic cell lines from ATL patients or HTLV-I in vitro transformed cells have been engrafted successfully into SCID mice lacking functional T and B cells (911). However, in these models, a long period is required for tumor formation, and intraperitoneal inoculation of well-characterized HTLV-I transformed cell lines did not result in engraftment (12,13). Furthermore, although ATL patients suffer from short-term morbidity, ATL cells or HTLV-I transformed cell lines are rarely fatal in SCID mice. Using younger SCID mice than those used in previous studies, we recently established a small-animal model that is appropriate to assess therapeutic agents for ATL. This model is characterized by rapid tumor formation and death (14). In the present study, we evaluated the inhibitory effects of the novel NF-
B inhibitor, DHMEQ, on tumor formation and death in this improved animal model for ATL.
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Materials and methods
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Cell lines
Four HTLV-I infected T-cell lines, MT-1 (2), TL-Om1 (15), MT-2 (16) and HUT-102 (1), were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 µg/ml of streptomycin at 37°C in 5% CO2. MT-1 and TL-Om1 are leukemic T-cell lines derived from patients with ATL. MT-2 is an HTLV-I transformed T-cell line established by an in vitro coculture protocol. HUT-102 was established from a patient with cutaneous T-cell lymphoma, but later considered a lymphoma-type ATL. The clonal origin of this cell line is unclear, and can no longer be determined. HTLV-I negative T-cell lines, Jurkat and MOLT-4, were maintained in RPMI 1640 supplemented with 10% FBS and the above antibiotics. All cells lines were passaged twice a week.
Electrophoretic mobility shift analysis
The double-stranded oligonucleotide containing the mouse immunoglobulin kappa (Ig
) light-chain NF-
B consensus site (5'-AGTTGAGGGGACTTTCCCAGGC-3') (Promega, Madison, WI) was used as the DNA probe for NF-
B binding. Nuclear extracts were prepared essentially according to the method reported by Andrews and Faller (17). Briefly, cells were washed in cold phosphate-buffered saline (PBS) and suspended in buffer A [10 mM of HEPESKOH pH 7.9 at 4°C, 1.5 mM of MgCl2, 10 mM of KCl, 0.5 mM of dithiothreitol (DTT) and 0.2 mM of phenylmethyl sulfoxide (PMSF)]. Nuclei were pelleted by centrifugation, resuspended in cold buffer C [20 mM of HEPESKOH pH 7.9 at 4°C, 25% glycerol (v/v), 420 mM of NaCl, 1.5 mM of MgCl2, 0.2 mM of EDTA, 0.5 mM of DTT and 0.2 mM of PMSF], incubated on ice for 20 min and centrifuged. The supernatant was used as a nuclear extract. For binding assays, 5 µg samples of nuclear extract were incubated with poly-(deoxyinosinic-deoxycytidylic acid) (poly dI-dC) and
-32P end-labeled probe at room temperature for 30 min. DNAprotein complexes were resolved on 6% native polyacrylamide gels in 0.5x TBE buffer. The gels were then dried and analyzed by autoradiography.
Mice
SCID (C.B17-scid/scid) male mice, 4 weeks of age, were obtained from Charles River Japan (Tokyo, Japan). The mice were maintained under specific pathogen-free conditions in laminar-flow benches at 22 ± 2°C with a 12 h light/dark cycle. Mice were fed sterilized (
-irradiated) pellets and received hyperchlorinated water ad libitum. All procedures involving animals and their care were approved by the animal care committee of Kumamoto University in accordance with Institutional and Japanese government guidelines for animal experiments.
Inoculation of cells into SCID mice
Cells (0.510 x 107) were washed three times with PBS and injected intraperitoneally into 5-week-old SCID mice that had been pretreated 3 days earlier with monoclonal antibody (mAb) TM-ß1 (18) (1 mg/mouse) against mouse interleukin-2 receptor (IL2R) ß chain. This pretreatment eliminated natural killer (NK) cell activity. Surviving animals were killed 40 days after inoculation.
PCR
To detect HTLV-I provirus sequences, 0.5 µg of genomic DNA extracted from tumors or various organs was subjected to PCR analysis. Oligonucleotide primers for tax and human ß-globin genes were used and PCR was performed as described previously (19,20).
Histology and immunohistochemistry
Animals were killed on day 40. Organs were fixed in 10% neutral-buffered formalin immediately after removal, embedded in paraffin, cut into 4 µm sections and stained with hematoxylin and eosin (HE). For immunofluorescence assays, paraffin-embedded sections were incubated with anti-HTLV-I p19 mAb (Chemicon International Temecula, CA) or anti-HLA-DR mAb (DiaSorin, Stillwater, MN) and stained with FITC-conjugated anti-mouse IgG (Cappel, Durham, NC).
Growth inhibition assay
Racemic DHMEQ was synthesized as described (4), dissolved in dimethylsulfoxide (DMSO) and subsequently diluted in culture medium. The effect of DHMEQ on cell growth was assayed using a kit for the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) method (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega). In brief, 5 x 104 cells were incubated in a 96-well microculture plate with various concentrations of DHMEQ. Cells treated with the same concentrations of DMSO were used as controls. After incubation for 48 h, 20 µl of MTS solution was added and the cells were incubated for another 2 h. The absorbance of each well was determined at 490 nm by a microplate reader (Bio-Rad, Richmond, CA).
Nuclear staining
Cells (1 x 106) were cultured with 20 or 40 µg/ml of DHMEQ for 48 h. Cells treated with the same concentrations of DMSO served as controls (0 µg/ml of DHMEQ). The cells were then fixed with 1% glutaraldehyde for 30 min at room temperature and washed three times with PBS. Subsequently, they were stained with 5 µg/ml of Hoechst33342 (Calbiochem, San Diego, CA) for 5 min at room temperature and washed three times with PBS. The stained apoptotic and nonapoptotic nuclei were photographed under UV light with a digital CCD camera (DP70; Olympus, Tokyo, Japan), and a total of 1000 cells were counted.
Measurement of nuclear translocation of NF-
B p65
Cells (20 x 106) were cultured with 20 or 40 µg/ml of DHMEQ for 16 h. Cells treated with the same concentrations of DMSO served as controls (0 µg/ml of DHMEQ). The relative increase of NF-
B p65 translocation into the nucleus was measured using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (IMGENEX, San Diego, CA). In brief, the cells were centrifuged at 400 g for 1 min and washed with cold PBS. The cells were lyzed by 400 µl of hypotonic buffer and 30 µl of 10% NP-40 was added. The mixture was centrifuged at 18 000 g for 30 s. The supernatant was used as cytoplasmic extract. To the pellet was added 220 µl of nuclear extraction buffer and centrifuged at 18 000 g for 1 min. The supernatant was used as nuclear extract. The anti-p65 antibody coated plate captured nuclear or cytoplasmic free p65 of samples (0.51 mg/ml of protein) and the amount of bound p65 was detected by adding a secondary antibody followed by alkaline phosphatase-conjugated secondary antibody. The absorbance value for each well was determined at 405 nm by a microplate reader (Bio-Rad). The relative ratio of nuclear to cytoplasmic p65 was calculated from the absorbance value of nucleus divided by that of cytoplasm.
Administration of DHMEQ to SCID mice injected with MT-2 or HUT-102 cells
DHMEQ was dissolved in 0.5% carboxymethyl cellulose (CMC) to a final concentration of 1.2 mg/ml. SCID mice (5 weeks old) were pretreated with 1 mg/mouse of mAb TM-ß1, and 3 days later were administered 5 x 107 MT-2 or HUT-102 cells intraperitoneally. On day 0 of cell inoculation and three times a week thereafter (until day 30), 12 mg/kg of either DHMEQ or vehicle were administered intraperitoneally. Mice surviving for 40 days after MT-2 cell inoculation were autopsied and tumor formation was assessed. The mice injected with HUT-102 cells were observed until day 250, and the surviving mice were autopsied. We included a final group of SCID mice that did not receive a cell inoculation; these mice served as a tumor-free control and were observed until day 140.
Detection of apoptosis in vivo
Gross tumors were immediately fixed in 10% neutral-buffered formalin, appropriately cut and then embedded in paraffin. The tissues were sectioned to 4 µm thickness and stained with HE. Apoptosis in tissue sections was examined by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling) assay using the In Situ Cell Death Detection kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. DNA fragmentation was also assessed in gross tumors. Tumor cells were lyzed for 3 h at 55°C in lysis solution (10 mM of TrisHCl, pH 7.4, 10 mM of EDTA, 0.1% SDS and 150 mM of NaCl) containing 20 mg/ml of proteinase K. The cell lysates were then treated with RNase at 37°C for 30 min. DNA was recovered by phenolchloroform extraction and ethanol precipitation. Purified DNA was dissolved in 20 µl of TE buffer and separated by electrophoresis through a 2% agarose gel.
Statistical analysis
Statistical analysis was performed using Fisher's exact probability test and Student's t-test. Statistical significance was defined as P < 0.05.
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Results
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NF-
B activation of HTLV-I-infected cell lines
HTLV-I infected cell lines, MT-1, TL-Om1, MT-2 and HUT-102, were studied for activation of NF-
B using electrophoretic mobility shift analysis (EMSA). MT-1 and TL-Om1 are derived from leukemic cell clones, MT-2 is an in vitro HTLV-I transformed cell line and the clonal origin of HUT-102 is unclear and can no longer be determined. All these cell lines displayed significantly greater NF-
B/DNA binding activity than the HTLV-I uninfected T-cell line, Jurkat (Figure 1A), as reported previously (3).
Engraftment of HTLV-I infected cell lines in SCID mice
We assessed the death rate of SCID mice inoculated with HTLV-I infected cell lines. Intraperitoneal inoculation of 0.510 x 107 cells of the ATL-derived cell line MT-1 into 5-week-old NK-free SCID mice resulted in low death rates 40 days post-inoculation (Figure 1B). Death of tumor-bearing mice was not observed after inoculation with any dose of TL-Om1 cells. However, tumor-associated death was observed in 80 and 100% of mice inoculated with 5 x 107 and 10 x 107 MT-2 cells, respectively. Likewise, 40 and 67% tumor-associated death occurred in mice inoculated with 5 x 107 and 10 x 107 HUT-102 cells, respectively.
No engraftment or malignancy was detected when mice inoculated with TL-Om1 cells were killed 40 days after injection. Less than 50% of the mice inoculated with any number of MT-1 cells formed gross tumors, although it appears that no gross tumors were formed when mice were inoculated with 0.5 x 107 MT-1 cells (Figure 1C). In contrast, all mice injected with 1 x 107 or more HUT-102 cells formed gross tumors, and all mice inoculated with 5 x 107 or more MT-2 cells showed gross tumors. The tumors were <1 cm x 1 cm in the mice injected with MT-2 cells and >1 cm x 1 cm in mice inoculated with HUT-102.
To confirm that these tumors were derived from the inoculated cells, PCR was performed on tumor samples to detect HTLV-I tax and human ß-globin genes. All tumors formed in mice inoculated with MT-1, HUT-102 and MT-2 were positive for both genes. In contrast, the mice injected with TL-Om1 cells, which showed no gross tumors, were negative for both HTLV-I tax and human ß-globin genes in all organs tested by PCR (data not shown). All tumors reacted with anti-HTLV-I p19 [except those derived from mice inoculated with MT-1, which expresses low levels of HTLV-I (2)] and anti-HLA-DR monoclonal antibodies in immunofluorescence assays (data not shown). Consequently, we considered the SCID mice inoculated with either MT-2 or HUT-102 cells to be the most suitable animal model for evaluating new therapeutic agents to treat ATL.
DHMEQ induces apoptosis of MT-2 and HUT-102 cells
We chose to examine the effects of DHMEQ in the HTLV-I transformed cell lines MT-2 and HUT-102 because we found that these cells proliferated in SCID mice. These cells were cultured with various concentrations (040 µg/ml) of DHMEQ for 48 h. Cultivation with DHMEQ suppressed cell growth in a dose-dependent manner, as assessed by the MTS assay (Figure 2A). Treatment with >20 µg/ml DHMEQ was effective to suppress cell growth by 50% in both cell types. To examine whether the induction of apoptosis accounted for the cell growth inhibition, cells treated with 20 and 40 µg/ml of DHMEQ were stained with Hoechst 33342 and apoptotic nuclei were counted. There was significant apoptosis in both MT-2 and HUT-102 cell lines (Figure 2B).

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Fig. 2. DHMEQ reduces cell growth and induces apoptosis of HTLV-I transformed T-cell lines, MT-2 and HUT-102. Cells were cultured with or without DHMEQ for 48 h. (A) Cell growth was assessed by the MTS method and is expressed as a percentage of vehicle-treated control and represents the mean ± standard deviation. (B) Induction of apoptosis of MT-2 and HUT-102 cells by DHMEQ. The cells were fixed and stained with Hoechst33342. At least 1000 stained cells were counted and assessed as apoptotic or non-apoptotic nuclei.
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DHMEQ inhibits nuclear translocation of NF-
B p65
DHMEQ inhibits nuclear translocation of NF-
B in cells with TNF receptor-mediated activation of NF-
B (6). Thus, we measured the nuclear translocation of NF-
B p65 by ELISA in MT-2 and HUT-102 cells incubated with or without DHMEQ (Figure 3). After incubation for 16 h and before the initiation of cell death, NF-
B p65 translocation to the nucleus was inhibited in cells treated with DHMEQ (Figure 3A). The relative nuclear to cytoplasmic ratio of p65 in cells treated with DHMEQ was the same as in the HTLV-I uninfected T-cell line, MOLT-4 (Figure 3B). These results indicate that DHMEQ inhibits nuclear translocation of NF-
B p65 in HTLV-I transformed cell lines and restores the ratio of nuclear to cytoplasmic p65 in those cells to that of the HTLV-I uninfected T-cell line.
DHMEQ inhibition of tumor formation and death in mice inoculated with HTLV-I transformed cells
To evaluate the antitumor effect of DHMEQ against HTLV-I transformed cells, we inoculated 5-week-old SCID mice lacking NK activity with 5 x 107 HTLV-I transformed MT-2 or HUT-102 cells. A dose of 12 mg/kg of either DHMEQ or vehicle was administered intraperitoneally on day 0 of cell inoculation and three times a week thereafter until day 30. Whereas 10 of 12 DHMEQ-treated mice (83%) were alive 40 days after MT-2 cell injection, only 2 of 10 (20%) of the control (vehicle-treated) mice survived (P = 0.0048 by Fisher's exact test; Figure 4A). Likewise, only 2 of 11 (18%) of the control mice inoculated with HUT-102 cells survived to day 250, whereas 7 of 11 (64%) DHMEQ-treated mice survived (P = 0.0403 by Fisher's exact test; Figure 4B). In a tumor-free control, DHMEQ treatment at the dosage used was well tolerated, leading to no body weight loss in SCID mice when compared with the control (Figure 4C).

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Fig. 4. DHMEQ increases survival of SCID mice inoculated with HTLV-I transformed cell lines and does not affect the increase of body weight in SCID mice. Survival curves of SCID mice injected with MT-2 cells (A) or HUT-102 cells (B) in the absence or presence of DHMEQ (12 mg/kg body weight). The survival of MT-2 and HUT-102-inoculated mice treated with DHMEQ was statistically different from that of the control group. *P < 0.05; **P < 0.01. Increase of body weight in tumor-free SCID mice (C) with or without DHMEQ (12 mg/kg body weight).
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We next compared gross tumor formation and the distribution of inoculated cells to organs in control mice and mice treated with DHMEQ. In MT-2 inoculated mice, 2 of 12 animals (17%) treated with DHMEQ formed small gross tumors, whereas all mice (n = 10) that received vehicle alone had visible tumors (P = 0.0004 by Fisher's exact test; Figure 5A). To detect infiltration of inoculated cells into various organs, genomic DNA extracted from each organ was subjected to PCR using primers directed to the HTLV-I tax and human ß-globin genes. Inoculated cells were present in all organ types tested from both DHMEQ-treated and untreated mice. However, the incidence of organs positive for the DNA tumor markers in DHMEQ-treated mice was lower than that in untreated mice. In particular, the livers of 3 of 12 mice treated with DHMEQ were positive for MT-2 cells, compared with 10 of 10 livers from mice treated with vehicle alone (P = 0.0004 by Fisher's exact test; Figure 5A). For HUT-102 inoculated mice, 25% of mice treated with DHMEQ had visible tumors, whereas all mice treated with vehicle alone had gross tumors (P = 0.0035 by Fisher's exact test, n = 8 for each group; Figure 5B). Infiltration of HUT-102 cells in various organs was higher than that of MT-2 cells. Similarly, the incidence of infiltrated organs in DHMEQ-treated mice was lower than that of untreated controls (Figure 5B). In HUT-102 inoculated mice, lung infiltration was observed in 13% of mice treated with DHMEQ and in 75% of mice treated with vehicle alone (P = 0.0203 by Fisher's exact test, n = 8). Liver infiltration was found in 25% of mice treated with DHMEQ and in 100% of mice treated with vehicle alone (P = 0.0035 by Fisher's exact test, n = 8). The spleen was infiltrated in 25% of mice treated with DHMEQ and in 88% of mice treated with vehicle alone (P = 0.0203 by Fisher's exact test, n = 8). The kidney was infiltrated in 13% of mice treated with DHMEQ and in 75% of mice treated with vehicle alone (P = 0.0203 by Fisher's exact test, n = 8). Taken together, these data suggest that DHMEQ inhibits tumor formation and infiltration of HTLV-I transformed cells to various organs in this SCID mouse model of ATL.

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Fig. 5. DHMEQ inhibits tumor formation and infiltration of inoculated cells into various organs in mice inoculated with MT-2 or HUT-102 cells. PCR analysis to detect both HTLV-I tax and human ß-globin genes shows formation of tumors and infiltration of tumor cells into various organs in mice inoculated with MT-2 (A) or HUT-102 (B) cells treated with or without DHMEQ. MLN, mesenteric lymph node; PBMC, peripheral blood mononuclear cells. *P < 0.05; **P < 0.01; ***P < 0.001.
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DHMEQ-induced apoptosis in tumors
We examined the effect of DHMEQ on the induction of apoptosis in vivo in tumors of SCID mice inoculated with HTLV-I transformed cells. The tumor cells obtained from untreated mice proliferated well and possessed polygonal and bizarre nuclei with irregularly shaped nucleoli (Figure 6Aa). The TUNEL assay showed few apoptotic cells in tumors from untreated mice (Figure 6Ac, white arrow). Tumor cells from DHMEQ-treated mice were smaller than those from untreated mice and had polygonal or egg-shaped nuclei with pale chromatin and small nucleoli (Figure 6Ab). Tumors from DHMEQ-treated mice showed abundant apoptotic cells (TUNEL assay; Figure 6Ad) and higher DNA fragmentation compared with control mice (Figure 6B). These results suggest that DHMEQ strongly induces apoptosis in HTLV-I transformed cells in vivo.

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Fig. 6. DHMEQ induces apoptosis in tumors of SCID mice inoculated with HTLV-I transformed cell lines. (A) Histological examination of SCID mice inoculated with MT-2 cells. HE-stained sections of gross tumors showed vigorous proliferation of tumor cells from mice treated with a vehicle control (a), whereas those from mice treated with DHMEQ were small and had egg-shaped nuclei with pale chromatin and small nucleoli (b). TUNEL assays show apoptosis in gross tumors from mice treated with a vehicle control (c) or DHMEQ (d). Few apoptotic cells were observed in tumors from mice treated with a vehicle control (white arrow), whereas apoptotic cells were abundant (i.e. the majority of cells) in tumors from the DHMEQ-treated mice. Magnification, 100x. (B) DNA fragmentation in tumors from mice treated with DHMEQ. Genomic DNA was isolated from tumors of HUT-102 injected SCID mice treated with DHMEQ or vehicle controls and resolved by electrophoresis on a 2% agarose gel. The molecular marker lane contains DNA digested with Hind III.
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Discussion
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We recently reported that age is a critical factor influencing the efficiency of tumor formation and death in SCID mice injected with the HTLV-I transformed cell line MT-2 (14). In the present study, injection of 5 x 107 MT-2 cells into 5-week-old SCID mice lacking NK activity resulted in 80% mortality and 100% gross tumor formation. Likewise, inoculation with 5 x 107 HUT-102 cells caused death in 40% and gross tumor formation in 100% of SCID mice. The reason for the discrepancy in mortalities between mice inoculated with MT-2 cells and those inoculated with HUT-102 cells is not yet clear; however, it may be due to differences in cytokine production. Since both cell lines formed tumors in 100% of SCID mice, the difference of the cell growth in SCID mice could be eliminated. However, intraperitoneal inoculation of the ATL-derived cell line MT-1 into 5-week-old NK-free SCID mice resulted in low death rates and low tumor formation 40 days after inoculation. No engraftment or malignancy was detected in mice inoculated with any dose of TL-Om1 cells. All these cell lines displayed significantly greater NF-
B/DNA binding activity than the HTLV-I uninfected T-cell line. These results suggest that constitutively activated NF-
B is not sufficient to promote malignant cell proliferation in SCID mice lacking NK activity.
Infiltration of HTLV-I infected leukemic cells into various organs is well documented in ATL patients (21). We found that SCID mice inoculated with MT-2 or HUT-102 cells also showed infiltration into various organs. These results indicate that, in this mouse model, HTLV-I transformed cells infiltrate organs in a manner similar to leukemic cells in ATL patients. Thus, the effect of new therapeutic agents for ATL can be compared, both in vitro and in vivo, using this model and the same cell lines.
DHMEQ strongly inhibits constitutively activated NF-
B in both ATL-derived cell lines and in primary ATL cells from patients, inducing apoptosis of these cells at concentrations that do not affect the viability of peripheral blood mononuclear cells (M. Watanabe, T. Ohsugi, M. Shoda, T. Ishida, S. Aizawa, M. Maruyama-Nagai, A. Utsunomiya, S. Koga, Y. Yamada, S. Kamihira, A. Okayama, H. Kikuchi, K. Uozumi, K. Yamaguchi, M. Higashihara, K. Umezawa, T. Watanabe and R. Horie, submitted). In the present study, we also observed that DHMEQ induces apoptosis in HTLV-I infected cell lines, MT-2 and HUT-102 and inhibits nuclear translocation of the NF-
B subunit, p65. It is likely that DHMEQ itself would be active rather than a metabolite, given that we found that DHMEQ was stable at least for several hours in medium (data not shown). We then investigated whether DHMEQ has potential as a therapeutic agent in vivo in a SCID mouse model using the same cell lines. DHMEQ inhibited tumor formation, reduced mortality and induced apoptosis in tumor cells. Furthermore, DHMEQ inhibited infiltration of ATL cells into various organs. DHMEQ was well-tolerated at the dosage used (12 mg/kg), and no adverse effects were noted during the course of the study. The precise pharmacokinetics of DHMEQ in vivo have not been established. However, we are currently studying this problem.
The effect of the two NF-
B inhibitors, Bay-11-7082 and PS-341, against constitutive NF-
B activity in an in vivo model of ATL was examined recently (22,23). Bay-11-7082 inhibits I
B
phosphorylation and PS-341 is a proteasome inhibitor. After intraperitoneal injection of Bay-11-7082 into NOD-SCID/
cnull (NOG) mice inoculated subcutaneously with the ATL cell line, ED-40515(), there was no significant difference in tumor size between treated and control groups. However, Bay-11-7082 significantly inhibited the growth of established tumors when administered directly into the tumor site on a daily basis (23). Bay 11-7082 suppressed the infiltration of tumor cells into peripheral blood; however, data were not shown for other organs. Bay-11-7082 was reported to induce apoptosis of ATL cells in vitro (24), but its ability to induce apoptosis in vivo is not known. Tan and coworkers (22,25) reported that PS-341 in combination with the current clinically approved humanized anti-Tac significantly prolongs lifespan in a mouse model of ATL. Inhibition of NF-
B was not evident in cells treated by PS-341 alone. Furthermore, PS-341 alone did not prolong the survival of ATL tumor-bearing mice.
Our current results demonstrate that DHMEQ not only inhibits tumor formation and death in our mouse model but also inhibits infiltration of tumor cells and induces extensive apoptosis of tumor cells in vivo. This report is the first to detect apoptosis of ATL tumor cells following treatment with an NF-
B inhibitor in vivo.
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Acknowledgments
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This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science. Gratitude is expressed to Dr Masayuki Miyasaka of Osaka University for his generous donation of the anti-IL-2Rß mAb, TM-ß1. We wish to thank Noboru Sakio and Noriyuki Sakamoto for animal care, and Yoshiteru Tanaka for technical assistance.
Conflict of Interest Statement: DHMEQ is a patented compound. Dr. Kazuo Umezawa is a director of Signal Creation Inc. who deals with DHMEQ.
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Received December 28, 2004;
revised March 14, 2005;
accepted April 10, 2005.