Tungsten accumulates in the intervertebral disc and vertebrae stimulating disc degeneration and upregulating markers of inflammation and pain.

Tungsten is incorporated in many industrial goods, military applications and medical devices due to its ability to impart flexibility, strength and conductance to materials. Emerging evidence has questioned the safety of tungsten exposure as studies have demonstrated it can promote tumour formation, induce pulmonary disease and alter immune function. Although tungsten is excreted from the body it can accumulate in certain organs such as the brain, colon, liver, kidneys, spleen and bones, where most of the bioaccumulation occurs. Whether prolonged tungsten exposure leads to accumulation in other tissues is unknown. The present study demonstrated that mice exposed to 15 ppm sodium tungstate for 4 weeks in their drinking water showed comparable accumulation in both the bony vertebrae and intervertebral discs (IVDs). Lumbar IVD height was significantly reduced in tungsten-exposed mice and accompanied by decreased proteoglycan content and increased fibrosis. In addition to catabolic enzymes, tungsten also increased the expression of the inflammatory cytokines IL-1ß and tumour necrosis factor (TNF)-α as well as the neurotrophic factors nerve growth factor (NGF) and brain-derived nerve factor (BDNF) in IVD cells. Tungsten significantly increased the presence of nociceptive neurons at the endplates of IVDs as observed by the expression of calcitonin gene-related peptide (CGRP) and anti-protein gene product 9.5 (PGP9.5) in endplate vessels. The present study provided evidence that tungsten may enhance disc degeneration and fibrosis as well as increase the expression of markers for pain. Therefore, tungsten toxicity may play a role in disc degeneration disease.

Trolox enhances the anti-lymphoma effects of arsenic trioxide, while protecting against liver toxicity.

Arsenic trioxide (As2O3) is an effective therapy in acute promyelocytic leukemia (APL), but its use in other malignancies is limited by the higher concentrations required to induce apoptosis. We have reported that trolox, an analogue of alpha-tocopherol, increases As2O3-mediated apoptosis in a variety of APL, myeloma and breast cancer cell lines, while non-malignant cells may be protected. In the present study, we extended previous results to show that trolox increases As2O3-mediated apoptosis in the P388 lymphoma cell line in vitro, as evidenced by decrease of mitochondrial membrane potential and release of cytochrome c. We then sought to determine whether this combination can enhance antitumor effects while protecting normal cells in vivo. In BDF1 mice, trolox treatment decreased As2O3-induced hepatomegaly, markers of oxidative stress and hepatocellular damage. In P388 tumor-bearing mice, As2O3 treatment prolonged survival, and the addition of trolox provided a further significant increase in lifespan. In addition, the combination of As2O3 and trolox inhibited metastatic spread, and protected the tumor-bearing mice from As2O3 liver toxicity. Our results suggest, for the first time, that trolox might prevent some of the clinical manifestations of As2O3-related toxicity while increasing its pro-apoptotic capacity and clinical efficacy in hematological malignancies.

Differential effects of the widely expressed dMax splice variant of Max on E-box vs initiator element-mediated regulation by c-Myc.

dMax, a naturally occurring splice variant of the Myc binding protein Max, lacks the DNA binding basic region and helix 1 of the Helix-Loop-Helix domain; dMax interacts with c-Myc in vitro and in vivo, and inhibits E-box Myc site driven transcription in transient transfection assays. Here we have investigated the expression, function and interactions of dMax. RT/PCR analyses detected dmax mRNA in multiple tissues of the developing, newborn and adult mouse. Functionally, dMax reduced the ability of c-Myc to cooperate with the progression factor A-Myb to promote S phase entry of quiescent smooth muscle cells. In contrast, dMax failed to ablate inhibition of initiator element (Inr)-mediated transcription by c-Myc in Jurkat T cells. In in vitro protein:protein association assays, dMax interacted with c-Myc, N-Myc, L-Myc, Mad1, Mxi1, Mad3 and Mad4, but not with itself or wild-type Max. These interactions required an intact leucine zipper. Inhibition of E-box-mediated transactivation by induction of dMax overexpression resulted in apoptosis of WEHI 231 B cells. Thus, dMax is a widely expressed, naturally occurring protein, with the capacity to bind most members of the Myc/Max superfamily; dMax has little effect on Inr-mediated repression by c-Myc, but can significantly decrease E-box-mediated events promoting proliferation and cell survival.

Phenotype-associated lectin-binding profiles of normal and transformed blood cells: a comparative analysis of mannose- and galactose-binding lectins from plants and human serum/placenta.

Surface glycoconjugates of normal and transformed blood cells are commonly characterized by plant lectins. To infer physiological significance of protein-carbohydrate interactions, mammalian lectins are obviously preferable as research tools. So far, human serum lectins have not been used to assess their binding to immunophenotyped human normal or transformed blood cells. Thus, our study combines two groups of lectins with different specificity from plant and human sources. Besides concanavalin A (ConA) we have isolated the mannose-binding protein and serum amyloid P component from human serum. Especially the mannose-binding protein is believed to play a role in host defence against bacteria and yeast cells with unknown impact on normal and tumor cells. These three lectins establish the first group. In addition to the immunomodulatory mistletoe lectin, whose binding can elicit enhanced cytokine secretion from mononuclear blood cells, we included the beta-galactoside-binding lectin (14 kDa) from human placenta in the second group. The initial series of measurements was undertaken using two-color flow cytometry to determine the phenotype-associated binding (based on cluster designation; CD) of the lectins to blood and bone marrow cells from normal donors and the cell line CEM (T-lymphoblastoid), KG1-A (primitive myeloid leukemia) and Croco II (B-lymphoblastoid). Heterogeneity was apparent for each lectin in the CD-defined cell populations. Significant differences in binding were noted between Viscum album agglutinin (VAA) and other lectins for CD4+ cells from blood and between mannose-binding protein (MBP) and VAA versus 14 kDa, ConA and serum amyloid P component (SAP) for CD19+ cells from bone marrow.(ABSTRACT TRUNCATED AT 250 WORDS)

The aryl hydrocarbon receptor, a basic helix-loop-helix transcription factor of the PAS gene family, is required for normal ovarian germ cell dynamics in the mouse.

The aryl hydrocarbon receptor (AhR), so-designated based on the ability of the protein to bind with and be activated by polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons, is part of an emerging family of ligand-activated transcriptional regulators that are distinct from the steroid-thyroid hormone receptor superfamily. Once bound by ligand, the AhR interacts with the AhR nuclear translocator (ARNT) protein to form the aryl hydrocarbon receptor complex (AHRC). Both subunits of the AHRC contain sequences corresponding to basic helix-loop-helix domains, a motif that is shared by a number of other dimeric transcription factors. Although the natural ligand(s) for the AhR remains to be elucidated, to date over fifteen genes, including enzymes, growth factors and other transcription factors, have been identified as potential targets for transcriptional regulation by the chemically-activated AHRC. In the ovary, PAH exposure is known to cause destruction of oocytes within immature follicles, implying that one function of the AhR is to mediate cell death signaling in the female germ line. To assess this possibility, we explored AhR expression patterns in the murine ovary, and then determined the impact of AhR-deficiency (gene knockout) on female germ cell dynamics. Immunohistochemical analysis of ovaries of wild-type female mice indicated that AhR protein was abundantly and exclusively expressed in oocytes and granulosa cells of follicles at all stages of development. Histomorphometric analysis of serial ovarian sections revealed a two-fold higher number of primordial follicles in Ahr-null versus wild-type females at day 4 postpartum. This phenotype likely results from a cell-intrinsic death defect in the developing germ line since AhR-deficiency attenuated the magnitude of oocyte apoptosis in fetal ovaries cultured without hormonal support for 72 h. We propose that the AhR, activated by an as yet unknown endogenous ligand(s), serves to regulate the size of the oocyte reserve endowed at birth by affecting germ cell death during female gametogenesis.

Identification of NF-kappaB in the marine fish Stenotomus chrysops and examination of its activation by aryl hydrocarbon receptor agonists.

Members of the Rel family of proteins have been identified in Drosophila, an echinoderm, Xenopus, birds and mammals. Dimers of Rel proteins form the transcription factor nuclear factor kappaB (NF-kappaB) that rapidly activates genes encoding cytokines, cell surface receptors, cell adhesion molecules and acute phase proteins. Evidence suggests that xenobiotic compounds also may alter the activation of NF-kappaB. This study had a dual objective of identifying members of the Rel family and examining their activation by xenobiotic compounds in a marine fish model, scup (Stenotomus chrysops). A DNA-protein crosslinking technique demonstrated that liver, kidney and heart each had at least three nuclear proteins that showed specific binding to an NF-kappaB consensus sequence, with molecular weights suggesting that the proteins potentially corresponded to mouse p50, p65 (RelA) and c-rel. In addition, an approximately 35kD NF-kappaB binding protein was evident in liver and kidney. The 50 kD protein was immunoprecipitated by mammalian p50-specific antibodies. The presence of Rel members in fish implied by those results was confirmed by RT-PCR cloning of a Rel homology domain (an apparent c-rel) from scup liver. NF-kappaB activation occurred in vehicle-treated fish, but this appeared to decrease over time. In fish treated with 0.01 or 1 mg 3,3',4,4', 5-pentachlorobiphenyl per kg, NF-kappaB activation in liver did not decrease, and there was a 6-8-fold increase in activation 16-18 days following treatment. Treatment with 10 mg benzo[a]pyrene/kg had no effect on NF-kappaB-DNA binding, either at 3 or 6 days following treatment. The data show that the Rel family of proteins is present in fish, represented at least by a p50/105 homologue, and support a hypothesis that some aryl hydrocarbon receptor agonists can activate NF-kappaB in vivo.

The eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NF-κB and is aberrantly regulated in acute myeloid leukemia.

The eukaryotic translation initiation factor eIF4E is a potent oncogene elevated in many cancers, including the M4 and M5 subtypes of acute myeloid leukemia (AML). Although eIF4E RNA levels are elevated 3- to 10-fold in M4/M5 AML, the molecular underpinnings of this dysregulation were unknown. Here, we demonstrate that EIF4E is a direct transcriptional target of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) that is dysregulated preferentially in M4 and M5 AML. In primary hematopoietic cells and in cell lines, eIF4E levels are induced by NF-κB activating stimuli. Pharmacological or genetic inhibition of NF-κB represses this activation. The endogenous human EIF4E promoter recruits p65 and cRel to evolutionarily conserved κB sites in vitro and in vivo following NF-κB activation. Transcriptional activation is demonstrated by recruitment of p300 to the κB sites and phosphorylated Pol II to the coding region. In primary AML specimens, generally we observe that substantially more NF-κB complexes associate with eIF4E promoter elements in M4 and M5 AML specimens examined than in other subtypes or unstimulated normal primary hematopoietic cells. Consistently, genetic inhibition of NF-κB abrogates eIF4E RNA levels in this same population. These findings provide novel insights into the transcriptional control of eIF4E and a novel molecular basis for its dysregulation in at least a subset of M4/M5 AML specimens.

The novel arsenical Darinaparsin circumvents BRG1-dependent, HO-1-mediated cytoprotection in leukemic cells.

Darinaparsin (Dar) is a more potent cytotoxic arsenical than arsenic trioxide (ATO). We hypothesized that the increased cytotoxicity of Dar may be because of a decreased cytoprotective response. We observed that, unlike ATO, Dar does not induce heme oxygenase-1 (HO-1), even though it induces expression of other nuclear factor (erythroid-derived 2)-like 2 (NRF2)-dependent detoxifying enzymes to a greater extent than ATO, in both cancer cell lines and patient-derived leukemic cells. This strengthens the emerging evidence, showing that response to reactive oxygen species (ROS) is stimuli specific. Dar treatment prevents recruitment of the transcriptional coregulator Brahma-related gene 1 (BRG1) to the HMOX1 promoter, which is required for HMOX1 expression. The inability of Dar to induce HO-1 correlates with arrest in G2/M cell cycle phase and BRG1 phosphorylation. Inhibition of HO-1 increases the toxicity of ATO, but has no effect on Dar-induced apoptosis. Accordingly, the lack of HO-1 induction is involved in Dar's enhanced antileukemic properties. Our data highlight cytoprotective responses mediated by HO-1 and BRG1 as a novel target for enhancing the therapeutic range of arsenicals.

A novel arsenical has antitumor activity toward As2O3-resistant and MRP1/ABCC1-overexpressing cell lines.

Inorganic arsenic trioxide (As(2)O(3)) is a highly effective treatment for acute promyelocytic leukemia (APL). However, other cancers do not respond well to this form of arsenic at clinically achievable doses. We tested a novel arsenical, S-dimethylarsino-glutathione (darinaparsin) for efficacy in various malignancies in vitro. Darinaparsin is significantly more potent than As(2)O(3) at mediating apoptosis in various malignant cell lines and is highly active against APL cells derived for As(2)O(3) resistance. We provide evidence that darinaparsin triggers apoptosis by inducing signaling pathways that do not completely overlap with As(2)O(3). We show that darinaparsin induces apoptosis and oxidative stress to a greater extent than As(2)O(3), although like As(2)O(3), darinaparsin-induced toxicity is c-Jun NH(2)-terminal kinase-dependent. However, darinaparsin does not induce promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha) degradation or rearrange PML nuclear bodies in APL cells, nor is its toxicity increased by glutathione depletion. Darinaparsin treatment results in higher intracellular arsenic accumulation when compared to As(2)O(3) treatment. This may be explained by our finding that As(2)O(3), but not darinaparsin, is efficiently exported by ABCC1, suggesting increased therapeutic efficacy of darinaparsin in ABCC1-overexpressing tumors. Our studies indicate that darinaparsin efficiently kills tumor cells with increased antioxidant capacity and drug exporters and suggest that darinaparsin may have a broader therapeutic spectrum than As(2)O(3).

The biology of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a disease associated with fusion oncoproteins invariably involving the retinoic acid receptor (Raralpha). Retinoic acid induces differentiation in APL cells and is successfully used in conjunction with chemotherapy to treat and cure a significant percentage of patients with APL. APL is also a model for disruption of normal retinoid-mediated transcription resulting in blocked differentiation. The study of the molecular mechanisms of APL oncogenesis has revealed novel interactions between fusion oncoproteins and transcriptional coregulators, already leading to new treatment strategies.

The role of NF-kappaB as a survival factor in environmental chemical-induced pre-B cell apoptosis.

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental chemicals that suppress the immune system at multiple levels, including at the level of B cell development in the bone marrow microenvironment. Specifically, PAH induce preB cell apoptosis in primary bone marrow cultures and in cocultures of an early preB cell line (BU-11) and a bone marrow stromal cell line (BMS2). Previous studies focused on the molecular mechanisms through which PAH induce stromal cells to deliver an apoptosis signal to adjacent preB cells. Apoptosis signaling within the preB cell itself was not investigated. Here, the role of NF-kappaB, a lymphocyte survival factor, in PAH-induced preB cell apoptosis was assessed. Analysis of DNA-binding proteins extracted from the nuclei of untreated BU-11 cells indicated DNA-binding complexes comprising NF-kappaB subunits p50, c-Rel, and/or Rel A. NF-kappaB down-regulation with previously described inhibitors induced BU-11 cell apoptosis, demonstrating that the default apoptosis pathway blocked by NF-kappaB is functional at this early stage in B cell development. Similarly, exposure of BU-11/BMS2 cocultures to 7,12-dimethylbenz[a]anthracene (DMBA), a prototypic PAH, down-regulated nuclear Rel A and c-Rel before overt apoptosis. Finally, ectopic expression of Rel A or c-Rel rescued BU-11 cells from DMBA-induced apoptosis. These results extend previous observations by demonstrating that 1) NF-kappaB is a survival factor at an earlier stage of B cell development than previously appreciated and 2) NF-kappaB down-regulation is likely to be part of the molecular mechanism resulting in PAH-induced preB cell apoptosis. These results suggest nonclonally restricted, PAH-mediated suppression of B lymphopoiesis.

The bioflavonoid galangin blocks aryl hydrocarbon receptor activation and polycyclic aromatic hydrocarbon-induced pre-B cell apoptosis.

Bioflavonoids are plant compounds touted for their potential to treat or prevent several diseases including cancers induced by common environmental chemicals. Much of the biologic activity of one such class of pollutants, polycyclic aromatic hydrocarbons (PAH), is mediated by the aryl hydrocarbon receptor/transcription factor (AhR). For example, the AhR regulates PAH immunotoxicity that manifests as pre-B cell apoptosis in models of B cell development. Because bioflavonoids block PAH-induced cell transformation and are structurally similar to AhR ligands, it was postulated that some of them would suppress PAH-induced, AhR-dependent immunotoxicity, possibly through a direct AhR blockade. This hypothesis was tested using a model of B cell development in which pre-B cells are cultured with and are dependent on bone marrow stromal or hepatic parenchymal cell monolayers. Of seven bioflavonoids screened, galangin (3,5,7-trihydroxyflavone) blocked PAH-induced but not C(2)-ceramide- or H(2)O(2)-induced pre-B cell apoptosis. Because galangin blocked AhR-dependent reporter gene expression, AhR complex-DNA binding, and AhR nuclear translocation, inhibition of a relatively early step in AhR signaling was implicated. This hypothesis was supported by the ability of galangin to bind the AhR and stabilize AhR-90-kDa heat shock protein complexes in the presence of AhR agonists. These studies demonstrate the utility of pre-B cell culture systems in identifying compounds capable of blocking PAH immunotoxicity, define at least one mechanism of galangin activity (i.e., repression of AhR activation), and motivate the use of this and similar dietary bioflavonoids as relatively nontoxic inhibitors of AhR agonist activity and as pharmacologic agents with which to dissect AhR signaling pathways.

Regulation of preB cell apoptosis by aryl hydrocarbon receptor/transcription factor-expressing stromal/adherent cells.

Polycyclic aromatic hydrocarbons (PAH) are environmental chemicals that mediate immunosuppression. In long-term bone marrow B-cell lymphopoiesis models, PAH induce apoptosis in immature (preB) lymphocytes. Since the biologic function of PAH is often mediated by the aryl hydrocarbon receptor/transcription factor (AhR), the role of the AhR or AhR-regulated genes was assessed in preB cell apoptosis. Specifically, a bone marrow-derived preB cell line (BU-11) was cultured on monolayers of the AhR + bone marrow-derived stromal cell line BMS2, hepatoma sublines that express various levels of AhR activity (Hepa-1c1c7 and variants), AhR+ thymic epithelial cells, and primary bone marrow stromal cells from wildtype or AhR-/- mice. Cultures were treated with one of two prototypic PAH, 7,12-dimethylbenz[a] anthracene (DMBA) or benz[a]pyrene (B[a]P), and the percentage of cells undergoing apoptosis measured. The data demonstrated that: 1) bone marrow- and hepatic-derived stromal/adherent cells support preB cell growth and regulate apoptosis induced by DMBA or B[a]P; 2) B[a]P is more effective than DMBA when preB cells are maintained on Hepa-1c1c7 monolayers than when maintained on BMS2 monolayers; 3) DMBA is more effective than B[a]P when preB cells are cultured on BMS2 monolayers; 4) alpha-naphthoflavone, an AhR antagonist and cytochrome P-450 inhibitor, blocks preB cell apoptosis in both BU-11/Hepa-1c1c7 and BU-11/BMS2 cultures; 5) although preB cells grow well in Hepa-1c1c7 or BMS2 supernatants, addition of PAH in the absence of hepatic- or bone marrow-derived adherent cells does not result in preB cell apoptosis; 6) preB cell apoptosis is dependent on AhR activity in adherent hepatic- or bone marrow-derived stromal cells; and 7) apoptosis is induced by DMBA when preB cells are maintained on primary bone marrow stromal cell monolayers from wildtype but not from AhR-/- mice. Collectively, the data indicated that AhR-regulated activities in the hematopoietic microenvironment influence the susceptibility of immature lymphocytes to low-dose PAH exposure.

Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture.

Epidemiological evidence suggests tea (Camellia sinensis L.) has chemopreventive effects against various tumors. Green tea contains many polyphenols, including epigallocatechin-3 gallate (EGCG), which possess anti-oxidant qualities. Reduction of chemically induced mammary gland carcinogenesis by green tea in a carcinogen-induced rat model has been suggested previously, but the results reported were not statistically significant. Here we have tested the effects of green tea on mammary tumorigenesis using the 7,12-dimethylbenz(a)anthracene (DMBA) Sprague-Dawley (S-D) rat model. We report that green tea significantly increased mean latency to first tumor, and reduced tumor burden and number of invasive tumors per tumor-bearing animal; although, it did not affect tumor number in the female rats. Furthermore, we show that proliferation and/or viability of cultured Hs578T and MDA-MB-231 estrogen receptor-negative breast cancer cell lines was reduced by EGCG treatment. Similar negative effects on proliferation were observed with the DMBA-transformed D3-1 cell line. Growth inhibition of Hs578T cells correlated with induction of p27(Kip1) cyclin-dependent kinase inhibitor (CKI) expression. Hs578T cells expressing elevated levels of p27(Kip1) protein due to stable ectopic expression displayed increased G1 arrest. Thus, green tea had significant chemopreventive effects on carcinogen-induced mammary tumorigenesis in female S-D rats. In culture, inhibition of human breast cancer cell proliferation by EGCG was mediated in part via induction of the p27(Kip1) CKI.

The role of polycyclic aromatic hydrocarbon metabolism in dimethylbenz[a]anthracene-induced pre-B lymphocyte apoptosis.

Previous studies indicated that two prototypic PAH, benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA), suppress the developing immune system by inducing apoptosis in bone marrow pre-B lymphocytes. In bone marrow cultures consisting of pre-B cells growing on bone marrow stromal cell monolayers, pre-B cell apoptosis was shown to be dependent on the aryl hydrocarbon receptor/transcription factor (AhR) expressed in stromal cells. However, it was not determined if AhR activation alone is sufficient or if DMBA metabolism is required for induction of a stromal cell-derived apoptosis signal. To address these issues we assessed: 1) the ability of poorly metabolized AhR ligands to induce pre-B cell apoptosis and 2) the capacity for and the mechanism through which an early DMBA metabolite induces pre-B cell apoptosis. Three poorly metabolized AhR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3,3',4,4',5-pentachlorobiphenyl, and 3,3',4,4'-tetrachlorobiphenyl failed to induce pre-B cell apoptosis in bone marrow cultures, indicating that AhR activation alone is not sufficient to induce apoptosis and suggesting a role for PAH metabolism in induction of an apoptosis signal. Consistent with this hypothesis, DMBA-3, 4-dihydrodiol, an early DMBA metabolite, induced significant pre-B cell apoptosis. The ability of DMBA-3,4-dihydrodiol to activate the AhR, inhibition of DMBA-3,4-dihydrodiol-induced apoptosis by alpha-naphthoflavone, and the significantly lower levels of DMBA-3, 4-dihydrodiol-induced apoposis in pre-B cell populations maintained on AhR(-) stromal cells strongly support a role for the AhR in DMBA-3,4-dihydrodiol-induced apoptosis. Of two DMBA-metabolizing enzymes evaluated, CYP1A1 and CYP1B1, the latter appeared to be the more likely to play a role in DMBA-induced apoptosis. These data confirm a role for the AhR in PAH-induced pre-B cell apoptosis, indicate a role for DMBA metabolism, and suggest a feedback loop in which at least one product of DMBA metabolism augments AhR signaling, leading to induction of an apoptosis stimulus.

Induction of PreB cell apoptosis by 7,12-dimethylbenz[a]anthracene in long-term primary murine bone marrow cultures.

Numerous studies demonstrate that polycyclic aromatic hydrocarbons (PAH) suppress immunity by modifying the function of both B and T cells. Relatively few studies have assessed the effects of these common environmental chemicals on immature lymphocytes. In the present study, long-term primary bone marrow cultures were employed to investigate the effects of a prototypic PAH and aryl hydrocarbon receptor (AhR) agonist, 7,12-dimethylbenz[a]anthracene (DMBA), on immature B lymphocytes. In this system, immature preB cells are maintained in a supportive microenvironment provided by bone marrow stromal cells. Results presented here demonstrate that (1) exposure of primary bone marrow cultures to DMBA results in preB cell death by apoptosis; (2) notably low doses of DMBA (> or = 10(-8) M) induce preB cell apoptosis; (3) in long-term cultures, bone marrow stromal cells, but not preB cells, express AhR mRNA and protein as determined by in situ hybridization, RT-PCR, and immunoblotting; (4) freshly isolated unfractionated bone marrow cells, but not purified bone marrow B cells, express AhR protein as assessed by immunohistochemistry; (5) alpha-naphthoflavone, a competitive AhR inhibitor and cytochrome P450 antagonist, completely blocks DMBA-induced preB cell apoptosis in primary bone marrow cultures; and (6) DMBA or benzo[a]pyrene injection in vivo results in bone marrow cell apoptosis consistent with the death of hematopoietic cells clustered around stromal elements. The results implicate programmed cell death as a mechanism underlying DMBA-mediated immunosuppression and suggest that preB cell death is influenced by local interactions with AhR+ bone marrow stromal cells.