TCDD Treatment Eliminates the Long-Term Reconstitution Activity of Hematopoietic Stem Cells

Ruriko Sakai*,{dagger},{ddagger}, Teruyuki Kajiume*,{dagger},{ddagger}, Hiroko Inoue*,{ddagger}, Rieko Kanno*,{ddagger}, Masaki Miyazaki*,{ddagger}, Yuichi Ninomiya*,{ddagger} and Masamoto Kanno*,{ddagger},1

* Department of Immunology, Graduate School of Biomedical Science, Hiroshima University, Minami-ku, Hiroshima 734-8551, Japan; {dagger} Japanese Society for the Promotion of Science Research Fellowship for Young Scientists; and {ddagger} CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan

Received September 30, 2002; accepted November 11, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an endocrine disrupting chemical (EDC), can cause carcinogenesis, immunosuppression, and teratogenesis, through a ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). Despite remarkable recent advances in stem cell biology, the influence of TCDD on hematopoietic stem cells (HSCs), which possess the ability to reconstitute long-term multilineage hematopoiesis, has not been well investigated. In this study we examined the influence of TCDD on HSCs enriched for CD34-, c-kit*plus;, Sca-1+, lineage negative (CD34–KSL) cells. The number of the CD34–KSL cells was found to be increased about four-fold upon a single oral administration of TCDD (40 µg/kg body weight). Surprisingly, we found that these TCDD-treated cells almost lost long-term reconstitution activity. This defect was not present in AhR-/- mice. These findings suggest that modulation of AhR/ARNT system activity may have an effect on HSC function or survival.

Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; hematopoietic stem cells (HSCs); CD34-KSL (CD34-, c-kit+, Sca-1+, lineage negative) cells; long-term reconstitution activity; aryl hydrocarbon receptor; AhR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In recent years, many reports have focused on certain man-made toxins known as endocrine disrupting chemicals (EDCs) that persist in the environment and are capable of altering the endocrine homeostasis of animals, causing serious reproductive and developmental defects (Birnbaum, 1995Go; Sweeney, 2002Go). One such compound is the xenobiotic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), one of the most potent members of a family of EDCs, the polyhalogenated aromatic hydrocarbons. TCDD exposure can result in carcinogenesis, immunosuppression, and tissue and organ toxicity, as well as teratogenesis (Couture et al., 1990Go; Peters et al., 1999Go).

TCDD is believed to exert its effects primarily through a ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). AhR belongs to the basic helix–loop–helix (bHLH) superfamily of DNA binding proteins and heterodimerizes with the AhR nuclear translocator (ARNT) to form an AhR/ARNT transcription factor complex (Hoffman et al., 1991Go; Reyes et al., 1992Go). This complex binds to specific DNA sites in the regulatory domains of numerous target genes and mediates the biological effects of exogenous ligands (Okey et al., 1994Go). Indirubin and indigo were reported recently as potent natural ligands of AhR (Adachi et al., 2001Go). Studies using TCDD and its related congeners that activate AhR also suggest that the receptor has immunological functions. The recent generation of AhR knockout mice has provided evidence of a role for this protein in hepatic fibrosis development and in the immune system (Fernandez-Salguero et al., 1995Go; Mimura et al., 1997Go; Schmidt et al., 1996Go). Although it is also known that AhR is present in most cell and tissue types of the body, its functions in hematopoietic stem cells (HSCs) have not been examined in detail.

HSCs possess the ability to self-renew or differentiate into multiple distinct cell lineages. Substantial HSC self-renewal has been demonstrated in transplantation models (Iscove and Nawa, 1997Go; Pawliuk et al., 1996Go), in stroma-containing long-term marrow cultures supplemented with thrombopoietin (Yagi et al., 1999Go) and upon the ectopic expression of HOXB4 (Antonchuk et al., 2002Go; Sauvageau et al., 1995Go). Although there are some reports that in vitro HSC self-renewal has been achieved using various culture conditions (Audet et al., 2001Go; Ema et al., 2000Go; Holyoake et al., 1996Go; Miller and Eaves, 1997Go), the processes that govern HSC self-renewal remain poorly understood. Although the functional diversity of HSCs has been demonstrated unequivocally, it is less clear how such diversity is generated. The prevailing view is that HSC heterogeneity is regulated by both extrinsic and intrinsic events (Enver et al., 1998Go; Metcalf, 1998Go). Extrinsic (environmental) signals are derived predominantly from stromal cells and their products. Stromal cells are organized into niches that differ in their ability to maintain HSCs (Blazsek et al., 1995Go; Wineman et al., 1996Go). Thus, homing of HSCs to different types of stromal cell niches should contribute to HSC heterogeneity. In addition to extrinsic signals, intrinsic mechanisms control HSC decisions. Intrinsic mechanisms could induce HSC heterogeneity if HSCs make random decisions at the time of each HSC division. For example, each HSC has a choice of many fates, including self-renewal, differentiation, apoptosis, and migration. Evidence for stochastic processes comes from the analysis of the differentiation of myeloid and lymphoid precursors (Busslinger et al., 2000Go; Ogawa, 1999Go). For example, multipotent myeloid precursors generate more restricted precursors in an apparently random fashion (Ogawa, 1999Go). Recent studies of murine HSCs clearly demonstrated that expression of the surface antigens of stem cells is under the influence of the developmental stage and the kinetic state of the stem cells (Ogawa, 2002Go). It was demonstrated using the monocloncal anti-CD34 antibody RAM34 that only the CD34- fraction of lineage negative (Lin-), Sca-1+, c-kit+ bone marrow (BM) cells, known as CD34-KSL cells, of normal adult mice are capable of long-term hematopoietic reconstitution in lethally irradiated mice (Osawa et al., 1996Go).

Surprisingly, there have been only a few reports concerning the relationship between HSC and TCDD that describe an increase in the number of hematopoietic progenitor cells upon TCDD treatment (Frazier et al., 1994Go; Murante and Gasiewicz, 2000Go; Staples et al., 1998Go; Thurmond and Gasiewicz, 2000Go). Therefore, we decided to investigate more precisely whether TCDD influences the long-term reconstruction activity of CD34-KSL cells in BM.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (> 99% pure) was obtained from Cambridge Isotope Laboratories Inc. (Cambridge, MA). The compound was dissolved in corn oil as an administration vehicle (Katayama Chemicals, Inc., Chuo, Osaka, Japan).

Animals.
C57BL/6J (B6, CD45.2) mice were obtained from CREA JAPAN, INC. (Meguro, Tokyo, Japan) and B6 SJL Ptprca Pep3bBoyJ (B6 Ptprc, CD45.1) mice were obtained from Jackson Laboratory (Bar Harbor, ME) and kept in accordance with the Laboratory Animal Science guidelines of Hiroshima University. AhR-/- mice were kindly provided by Dr. Y. Fujii-Kuriyama (Tohoku University, Sendai, Japan). All mice were acclimated in-house for one week prior to treatment. Mice older than 10 weeks of age were used in the experiments.

TCDD treatment.
The experiments shown here were performed more than three times with at least five mice per group. Animals were administered either vehicle or TCDD at day zero and were euthanized at h 0, 3, and 12, and on days 1, 3, 7, 14, 28, 56, and 112. TCDD-treated animals were administered by gavage 40 µg/kg body weight (bw) TCDD and vehicle-treated animals were administered by gavage an equivalent dose of corn oil. The following TCDD doses were used: 0 (vehicle), 0.16, 0.63, 2.5, 10, 40, and 80 µg/kg bw. Animals were administered either vehicle or TCDD at day zero and were euthanized on day 7.

Antibodies.
The antibodies used in immunofluorescence staining included RAM34 (anti-CD34), D7 (anti-Sca-1), 2B8 (anti-c-kit), 104 (anti-CD45.2), and A20 (anti-CD45.1). Antibodies using for lineage markers included RA3-6B2 (anti-CD45R/B220), 145-2C (anti-CD3e), RM4-5 (anti-CD4), 53-6.7 (anti-CD8a), M1/70 (anti-Mac-1), RB6-8C5 (anti-Gr-1), and TER119. All the antibodies were purchased from Pharmingen (San Diego, CA).

Purification and analysis of CD34-KSL cells.
BM cells were flushed from femurs and tibiae of mice. Cell suspensions were then filtered through a sterile 100 µm Cell Strainer (No. 2360; Falcon, Lincoln Park, NJ) and stained with biotinylated antilineage makers. For analysis, cells were then stained with fluorescein isothiocyanate (FITC) anti-CD34, phycoerythrin (PE) anti-Sca-1, allophycocyanin (APC) anti-c-kit antibodies and streptavidin-PerCP (Pharmingen). Four-color analysis was performed on a FACS Calibur (Becton Dickinson, San Jose, CA) using CELLQuest software (Becton Dickinson). For cell sorting, bone marrow was depleted of lineage positive cells using MACS (Milteny Biotech GmbH, Bergisch Gladbach). Cells were stained with biotinylated antilineage markers and then were allowed to bind streptavidin-magnetic beads (Milteny Biotech). Ten µl of beads were used per 107 cells. Cells were then applied to a C-type MACS column and nonadherent (lineage negative; Lin-) cells were stained with the subsequent antibodies as described above (Randall and Weissman, 1998Go). CD34-KSL cells were sorted by FACS Vantage SE (Becton Dickinson). Dead cells were excluded by propodium iodide (PI) from analysis and sorting.

Competitive repopulation assay.
We applied and performed a competitive repopulation assay to which the CD45 (Ly5) system was adapted (Ema and Nakauchi, 2000Go); 100 sorted CD34-KSL cells from TCDD- or vehicle-treated B6 (CD45.2), AhR-/- (CD45.2), and AhR WT (CD45.2) mice were mixed with 1 x 105 BM competitor cells (B6 Ptprc, CD45.1) and were transplanted into B6 Ptprc mice irradiated at a dose of 10 Gy. After transplantation, peripheral blood cells of the recipients were stained with FITC anti-CD45.2 and PE anti-CD45.1. The cells were simultaneously stained with biotinylated anti-B220 or biotinylated anti-TER119, a mixture of biotinylated anti-Mac-1 and Gr-1, or a mixture of APC-conjugated anti-CD4 and -CD8. Biotinylated antibodies were developed with streptavidin-APC (Molecular Probes, Eugene, OR). The cells were analyzed on a FACS, and percentage chimerism was taken as the quadrant ratio of donor cells (CD45.2+ cells). When percent chimerism was more than 1% with all lineage reconstitution, recipient mice were considered to be multilineage reconstituted (positive mice). Dead cells were excluded by PI staining.

Two step single cell-methylcellulose colony assay.
Sorted CD34-KSL cells were deposited as single cells into 96-well microtiter plates by FACS Vantage SE and Clone Cyt (Becton Dickinson). The single cells were incubated in 100 µl of culture medium (MethocultTM GF, StemCell Technologies Inc., Vancouver, British Columbia, Canada) at 37°C in a humidified atmosphere 5% CO2 for seven days. Cultures were scored for colony-forming units using an inverted microscope. We evaluated and designated those containing more than 50 cells/colony as first colonies. Next, the first colonies were collected and sorted to deposit 100 cells/well and incubated for another seven days. Cultures were scored for colony-forming units in the same manner, and these colonies were designated second colonies.

Statistical analysis.
Means ± SD were compared with a Student’s t-test. Significance levels were set at p = 0.01–0.05 (*), p = 0.01–0.001 (**), and p < 0.001 (***).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Number of CD34-KSL Cells Was Increased by TCDD
To understand the effects of TCDD on the immune and/or hematopoietic systems, we focused on the study of HSCs in adult BM. Although recent studies reported that the number of hematopoietic progenitor cells was increased by TCDD administration (Murante and Gasiewicz, 2000Go), our first question was whether multilineage long-term HSCs are really affected by TCDD. Therefore, we investigated more precisely the effects of TCDD on long-term HSC populations, CD34-KSL cells, in BM. We treated 10-week-old C57BL/6 mice with 40 µg/kg bw TCDD or corn oil (vehicle) by single oral administration, and assessed HSC characteristics after seven days. When the lineage negative BM cells were further developed with Sca-1/c-kit, the percentage of Sca-1+/c-kit+ cells (gated in Fig. 1AGo) from TCDD-treated mice was found to be higher (7.73%) than that of cells isolated from vehicle-treated mice (2.32%). This observation is in good agreement with a recent report (Murante and Gasiewicz, 2000Go). We then assessed the percentages and cell number of the long-term HSC population as indicated by the CD34 marker. The percentage of CD34-negative cells was higher in TCDD-treated (12.21%) compared to vehicle-treated (4.44%) mice (Fig. 1AGo). Although there were no significant differences in the total number of BM cells between vehicle- and TCDD-treated mice, the number of CD34-KSL cells was four-fold higher in TCDD-treated mice (Fig. 1B Go,p = 0.0001, n = 20). It has been reported that TCDD administration increases the number of KSL progenitor cells in dose-dependent fashion (Murante and Gasiewicz, 2000Go). In corroboration with these prior results, we also assessed and found that the increase in CD34-KSL cells was TCDD dose-dependent at seven days after administration (Fig. 1CGo). The number of CD34-KSL cells is increased with doses as low as 2.5 µg/kg bw and we fixed the experimental dose at 40 µg/kg bw on which the significant difference was seen (Fig. 1C pGo, = 0.0083, n = 6).



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FIG. 1. Effects of TCDD on the number of BM cells and CD34-KSL cells at day 7 following treatment, and dose-response profile of CD34-KSL cells. (A) Flow cytometric analysis of CD34 expression profile on the KSL fraction of BM cells treated with vehicle (left panels) or TCDD (right panels). Representative flow cytometric counter plots of Sca-1/c-kit staining of lineage negative BM fractions (upper). Gated c-kit+Sca-1+Lin- (KSL) cells were examined for the expression of CD34. Fluorescence histogram (lower) shows CD34 staining profile of the gated, stem cell-enriched fraction. (B) The number of BM cells and CD34-KSL cells from C57BL/6 (B6) mice treated with vehicle (open bar) or TCDD (filled bar) after seven days. Although there was no difference in the number of BM cells, a significant difference (***p < 0.001, n = 20) was seen in the number of CD34-KSL cells. (C) Dose-response profile of the number of CD34-KSL cells. 0 µg/kg bw indicates vehicle-treated control; **p = 0.0083 (n = 6) and *p = 0.0482 (n = 5) for 40 and 80 µg/kg bw, respectively. These experiments were performed more than 3 times with at least 5 mice per group.

 
We next performed a time course analysis of the effects of a single oral administration of TCDD on CD34-KSL hematopoietic stem cell populations. As shown in Figure 2Go, TCDD exposure increased the number of CD34-KSL cells (Fig. 2BGo) from 12 h up to 56 days after treatment, although the number of total BM cells was not changed. Effects were observed as early as 12 h after treatment (p = 0.0058, n = 5), and peaked after about 1–2 weeks. Only after 112 days was this augmentation effect resolved. The increase in CD34-KSL cells could be due to changes in a number of processes, cell proliferation, cell death, differentiation, and homing. We could not, however, find any significant differences in cell proliferation or cell death by BrdU and TUNEL assays, respectively (data not shown).



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FIG. 2. Time course of BM and CD34-KSL cells. The numbers of BM (A) and CD34-KSL (B) cells were investigated at 0, 3, and 12 h, and at 1, 3, 7, 14, 28, 56, and 112 days following TCDD or vehicle treatment. No difference was seen in BM cell number at any timepoint between the vehicle- (open circles) or TCDD-treated (filled circles) groups. Significant differences were observed in CD34-KSL cell number between the vehicle- (open bars) and TCDD-treated (filled bars) groups from 12 h on. At 14 days after treatment, the number of CD34-KSL cells in the TCDD group was 3-fold that of the vehicle group. *p = 0.01 ~ 0.05; ** p = 0.001 ~ 0.001; *** p < 0.001. These experiments were performed more than 3 times with at least 5 mice per group.

 
TCDD Treatment Abolishes the Stem Cell Function of the CD34-KSL Cells
Next, we determined whether the TCDD-induced increased number of CD34-KSL cells maintain normal stem cell function. We performed a competitive repopulation assay with CD45.2 mice as donors and CD45.1 mice as recipients (Fig. 3AGo and Materials and Methods). The peripheral blood (PB) cells of the recipient mice were assessed for the long-term reconstitution ability of donor cells every four weeks after 100 CD34-KSL cells were transplanted. We monitored the reconstitution activity up to 28 weeks. In these experiments, "TCDD group" and "vehicle group" refer to recipient mice into which were transplanted CD34-KSL cells from TCDD- and vehicle-treated mice, respectively.



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FIG. 3. Function of CD34-KSL cells isolated from TCDD-treated mice. (A) BM transplantation experimental design. For details, please see Materials and Methods. (B) Representative flow cytometric dot plots of CD45.2 (donor cells)/CD45.1 (recipient and competitor cells) staining of PB at 16 weeks after BM transplantation. (C) Time course profile of chimerism. While mice transplanted with CD34-KSL cells isolated from vehicle-treated mice exhibited more than 5% chimerism, those transplanted with cells isolated from TCDD-treated mice exhibited less than 1% chimerism. Only 2 of 12 mice in the TCDD group had more than 1% chimerism, but they died at 20 weeks. (D) In vitro single cell methylcellulose colony assay. Both the first colony and the second colony were counted in the number of total colonies, regardless of the cell types. Total rates of first colony formation in one plate from single CD34-KSL cells isolated from vehicle- and TCDD-treated mice were 30 and 10%, respectively. Second colony formation rates of cells isolated from first colonies generated from vehicle- and TCDD-treated mice were 1 and 0.1%, respectively. These experiments were performed more than 3 times with at least 5 mice per group.

 
First, the cellularity of recipient peripheral blood was measured and percentage chimerism (ratio of the donor CD45.2+ cells) of nucleated PB cells was assayed with anti-CD45.2 and anti-CD45.1 monoclonal antibodies, as well as several lineage antibodies. Surprisingly, although the chimerism of the vehicle group was 20 to 80% (average 40%) after four months, we could not detect donor CD45.2+ cells in the TCDD group with the exception of two mice (Figs. 3B and 3CGo). These results strongly suggest that the CD34-KSL cells in TCDD-treated animals lose their hematopoietic long-term reconstitution activity.

To understand the mechanisms of this functional deterioration, we thought it could be a possibility that long-term reconstitution activity of HSC might shift from CD34-KSL fraction to CD34+KSL fraction, usually supporting short-term reconstitution activity, by stimulation of TCDD. One thousand CD34+KSL cells from both TCDD- and vehicle-treated adult mice were transplanted by the same method as described above. No long-term reconstitution activity was observed in either group (data not shown). Therefore, it is reasonable to conclude that the effect of TCDD on long-term reconstitution activity is targeted on the CD34-KSL cell itself rather than population shift.

Next we performed an in vitro two-step single-cell methylcellulose colony assay on CD34-KSL cells to analyze the effects of TCDD on self-renewal activity at the single cell level. Single CD34-KSL cells were sorted into individual wells filled with culture medium formulated to optimize the detection of murine hematopoieic progenitor cells, BFU-E, CFU-GM, CFU-G, CFU-M, and CFU-GEMM. Although we could not find a difference of cellularity of the colony between vehicle plates and TCDD plates (the plates where single CD34-KSL cells from vehicle- or TCDD-treated mice were sorted into each well), the first colony forming ability of CD34-KSL cells from the TCDD-treated mice was only one-third that of cells from the vehicle-treated mice (Fig. 3D pGo, = 0.0024, n = 6). Next we examined second colony forming activity. This process is an indicator of the self-renewal or progenitor-forming ability of HSCs. The second colony forming ability of CD34-KSL cells from the TCDD-treated mice was one-tenth of that of vehicle-treated mice, and the size of colonies generated from CD34-KSL cells from the TCDD-treated mice was 50% of that of vehicle-treated mice.

From these results, it is also conceivable that the long-term reconstitution activity of HSCs in CD34-KSL populations is damaged by TCDD, despite the increase in cell number.

Absence of Aryl Hydrocarbon Receptor Prevents the Elimination of Stem Cell Activity
As AhR is known to be a major dioxin receptor, we also examined the role of AhR in the TCDD-mediated elimination of stem cell activity. We performed the same experiments as described above using AhR-knockout (AhR-/-) mice. As shown in Figure 4AGo, we could not detect a significant TCDD-induced increase in the number of CD34-KSL cells in AhR-/- mice, although a three- to four-fold increase in cell number was observed in wild type (WT) mice. It is noteworthy that the total number of BM cells was not changed between AhR-/- and WT mice.



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FIG. 4. Participation of AhR in TCDD function. (A) Number of BM cells and CD34-KSL cells from AhR-/- mice and AhR WT mice (littermate). There were no differences between vehicle- and TCDD-treated conditions in the number of BM cells in either AhR-/- or WT mice. In WT mice, TCDD treatment increased the number of CD34-KSL cells compared to vehicle-treated control. However, no difference was seen in the number of CD34-KSL cells isolated from vehicle- or TCDD-treated AhR-/- mice. (B) Representative flow cytometric dot plots of CD45.2 (donor cells)/CD45.1 (recipient and competitor cells) staining of PB cells at 12 weeks after BM transplantation. Experimental procedure of BM transplantation was the same as described previously. These experiments were performed more than 3 times with at least 5 mice per group.

 
Next, we performed the competitive repopulation assay to assess the long-term reconstitution capability of CD34-KSL cells from AhR-/- mice treated with vehicle or TCDD. We used the same assay system described in Figure 3Go. We prepared three donor groups: CD34-KSL BM cells from the AhR-/- vehicle group, AhR-/- TCDD group, and WT(AhR+/+) TCDD group. Interestingly, we detected high percentages of chimerism (more than 10%) in both the AhR-/- vehicle group and the AhR-/- TCDD group (Fig. 4BGo), when assayed at 24 weeks. However, we could not detect significant chimerism (at least 1%) in the WT TCDD group.

These observations strongly suggest that both TCDD-induced increase in CD34-KSL cell number and suppression of CD34-KSL long-term reconstitution activity are AhR-dependent. Therefore, it is likely that TCDD affects long-term reconstitution activity through the AhR/ARNT pathway.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In spite of high levels of concern about both HSC and biological effects of TCDD, there were only a few reports that investigated the influence of TCDD on HSCs (Murante and Gasiewicz, 2000Go). Their data revealed increases in the number of bone marrow KSL cells, relative to control, over 24 h through 31 days following treatment of TCDD. They suggested that proliferation and/or differentiation processes of HSCs were affected by TCDD and that these effects contribute to a reduced capacity of bone marrow to generate pro-T lymphocytes.

The capacity for extensive self-renewal is commonly regarded as a property that is reserved for stem cells. Recently, a clonal BM-HSC transplantation system was used to study the function of HSCs. Here, we also used the BM-HSC transplantation system (competitive repopulation assay) to examine the effects of TCDD on long-term reconstitution activity. Our data demonstrate an unexpected elimination of long-term reconstitution activity in the population of CD34-KSL cells in BM. The abrogation seems to encompass all HSC behaviors, including self-renewal, cell death, homing, and lineage contribution (development). It strongly suggests that TCDD-induced alteration of all hematopoetic processes appeared from the beginning of hematopoiesis.

Therefore it is important to hypothesize that some regulation systems that are affected by TCDD might modulate hematopoiesis from the beginning. Recent studies have demonstrated the ability of intrinsic factors such as HOXB4, cyclin-dependent kinase (CDK) inhibitors, and Wnt signals to control HSC self-renewal and/or reconstitution activities. We hypothesized that misexpression of these intrinsic factors (genes) might abrogate HSC reconstitution activity after TCDD administration.

In the case of HOXB4, the quality of the HSCs induced by HOXB4 overexpression is not impaired, as demonstrated by their ability to fully repopulate all lineages (Antonchuk et al., 2002Go). Enhanced HSC regenerative ability in HOXB4-transduced bone marrow cells has also been demonstrated (Antonchuk et al., 2001Go; Sauvageau et al., 1995Go; Thorsteinsdottir et al., 1999Go). Studies in RAT-1 cells showed that HOXB4 overexpression activates the expression of AP-1 complex genes Fra-1 and Jun-B, with subsequent upregulation of cyclin D1 (Krosl and Sauvageau, 2000Go). It was also demonstrated that HOXB4 overexpression enhances ex vivo growth of total bone marrow cultures and that this effect is due to increased proliferation rather than reduced apoptosis (Antonchuk et al., 2001Go). Furthermore, the HOXB4-mediated growth advantage was found to be largely restricted to the most primitive fraction of hematopoietic cells (Antonchuk et al., 2002Go). The primitive cell-specific growth advantage suggests that HOXB4 overexpression either enhances HSC proliferation or self-renewal, or some combination of both. Indeed, we noticed that the upstream region of the HOXB4 gene possesses two xenobiotic response elements (TNGCGTG), which might interact with AhR/ARNT upon TCDD administration (M. Kanno, unpublished observation). Therefore, we speculate that AhR/ARNT might negatively control HOXB4 expression in HSCs.

Recently, the function of CDK inhibitor in HSCs has been studied quite extensively. In the absence of p21 (p21cip1/waf1), the G1 checkpoint-regulating CDK inhibitor, HSC proliferation and number are increased under normal homeostatic conditions. Furthermore, self-renewal of primitive cells is impaired in serially transplanted bone marrow from p21-/- mice, leading to hematopoietic failure (Cheng et al., 2000aGo,bGo; Enan et al., 1998Go; Kolluri et al., 1999Go).

In the case of the Wnt pathway, a central role of Wnt in the establishment and maintenance of cell fates has been demonstrated in vertebrates and invertebrates. To examine whether Wnt are involved in the regulation of hematopoietic stem/progenitor cell populations (HSCPs), several groups have investigated Wnt and frizzled gene expression in hematopoietic tissues and the response of HSCPs to WNTs (Austin et al., 1997Go; Van Den Berg et al., 1998Go).

There have been several reports describing two states of HSCs (resting and active). Seventy-five percent of HSCs are usually in a resting state, but external stimuli can induce cell cycle progression (Cheshier et al., 1999Go; Wright et al., 2001Go). HOXB4 and Wnt might be the candidates for this stimulus. It is conceivable that the signal pathways used by these stimuli are antagonized by the modulation AhR/ARNT system activity. This process of HSC activation has yet to be explored.


    ACKNOWLEDGMENTS
 
We thank S. Okamura and K. Yamashita for helpful suggestions and discussions. This study was supported by CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation, a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (12001243 for R.S.; 12215108 and 12490025 for M.K.), and the Uehara Life Science Foundation.


    NOTES
 
1 To whom correspondence should be sent at Department of Immunology, Graduate School of Biomedical Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Fax: +81-82-257-5179. E-mail: mkanno{at}hiroshima-u.ac.jp. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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