Aryl Hydrocarbon Receptor Regulation of Ceramide-induced Apoptosis in Murine Hepatoma 1c1c7 Cells
A FUNCTION INDEPENDENT OF ARYL HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR*

John J. Reiners Jr.Dagger and Russell E. Clift

From the Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 48201

    ABSTRACT
Top
Abstract
Introduction
References

The relationship between aryl hydrocarbon receptor (AHR) content and susceptibility to apoptosis was examined in the murine hepatoma 1c1c7 cell line and a series of variants having different levels of AHR expression. Exposure of 1c1c7 cultures to N-acetylsphingosine (C2-ceramide) caused a concentration-dependent inhibition of cell proliferation, loss of viability, and induction of apoptosis as monitored by analyses of DNA fragmentation and caspase activation. A variant cell line (Tao) having ~10% of the AHR content of 1c1c7 cells also arrested following exposure to C2-ceramide, but did not undergo apoptosis. Modulation of 1c1c7 and Tao AHR contents by transfection of Ahr antisense and sense constructs, respectively, confirmed the relationship between AHR content and susceptibility to C2-ceramide-induced apoptosis. C2-ceramide also induced the apoptosis of an AHR-containing cell line lacking the aryl hydrocarbon receptor nuclear translocator protein. AHR ligands (i.e. 2,3,7,8-tetrachlorodibenzo-p-dioxin and alpha -naphthoflavone) neither induced apoptosis nor modulated the development of apoptosis in C2-ceramide-treated 1c1c7 cultures. AHR content did not affect staurosporine- or doxorubicin-induced apoptosis. These results suggest the AHR modulates aspects of ceramide signaling associated with the induction of apoptosis but not cell cycle arrest, and does so by a mechanism that is independent of its interaction with aryl hydrocarbon receptor nuclear translocator and exogenous AHR ligands.

    INTRODUCTION
Top
Abstract
Introduction
References

The aryl hydrocarbon receptor (AHR)1 is a ligand-activated transcription factor (1-3). Numerous xenobiotics, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ligands of the AHR. In many tissues and cell lines, the non-liganded AHR appears to be a cytosolic protein (4, 5). Upon binding TCDD, the AHR translocates to the nucleus where it forms a dimer with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. AHR/ARNT heterodimers interact with specific enhancer sequences in target genes designated dioxin-responsive elements, and stimulate the transcriptional activation of such genes (1-3). Many of the biological processes initiated by TCDD can be attributed to this transcriptional activation of target genes (1).

In general, the cellular functions of the AHR have been defined or inferred from comparisons of the effects of ligands of the AHR in wild type and AHR-deficient or null cell lines or mice (1-3,6-11). Such approaches have delineated the need for the AHR in mediating the teratogenic, immunomodulating, cytostatic, and apoptotic activities of TCDD, and the role of the AHR in TCDD-initiated, transcriptional activation of several phase I and II genes involved in biotransformation (1-3, 6-11). However, studies employing exogenous ligands do not address the issue of whether the AHR, in the absence of exogenous ligand, has other cellular functions. Recent studies, performed in the absence of exogenous ligand, have implicated a role for the AHR in cell cycle progression. These studies used murine hepatoma 1c1c7 and Tao cells, the latter being an AHR-deficient variant derived from the 1c1c7 line (12). The doubling time of Tao cells is considerably longer than parental 1c1c7 cells due to a prolongation of G1, but can be shortened by the introduction of an Ahr expression construct (12). Conversely, introduction of an Ahr antisense expression construct into 1c1c7 cells increases cell doubling times due to a lengthening of G1. This inverse relationship between AHR content and cell doubling times has also been observed with rat hepatoma 5L and BP8 cells, the latter being an AHR-deficient variant derived from 5L cells (11). An unresolved issue in these studies is whether the observed AHR-dependent effects were ARNT-dependent and mediated by an endogenous AHR ligand.

Apoptosis is a physiological process that entails the programmed death of a cell. It plays a critical role during development and in the maintenance of tissue and organ homeostasis (13, 14). Indeed, disregulation of processes controlling apoptosis often contribute to the development of neoplasia. Apoptosis is also the process by which many genotoxic and chemotherapeutic drugs exert their cytotoxic effects. Several studies implicate the AHR as having a role in influencing or mediating apoptotic processes. For example, immunohistochemical analyses of embryonic tissues show that AHR expression is developmentally regulated and occurs independently of ARNT expression in regions undergoing remodeling, a process that involves apoptosis (15, 16). Similarly, resting T cells express very low levels of the AHR (17, 18). Upon mitogenic stimulation they enter the cell cycle, divide, and subsequently undergo apoptosis (19, 20). Expression of the AHR is markedly increased following such stimulation, and the kinetics of expression parallel the onset of apoptosis (17, 18). Although the role of the AHR in the above examples is speculative, there are at least two examples in which the AHR protein has been clearly associated with an apoptotic process. The first of these entails the thymic atrophy seen in AHR+/+ mice, but not AHR null mice, following exposure to TCDD (9, 21). The second example relates to the fibrosis and reduced liver size seen in AHR null mice (6, 7). Recent studies suggest that these phenotypic traits reflect the autocrine production by AHR null hepatocytes of a cytokine (e.g. transforming growth factor-beta ) that causes hepatocyte apoptosis (22). Presumably, absence of the AHR facilitates conditions that lead to the activation of latent transforming growth factor-beta (22).

Ceramide is an endogenous lipid generated by either de novo biosynthesis or sphingomyelinase degradation of sphingomyelin (23, 24). Intracellular levels of ceramide are elevated by a variety of stimuli/agents that induce apoptosis, including Fas ligand engagement of CD95, ionizing radiation, ultraviolet radiation, chemotherapeutic and genotoxic chemicals, and several cytokines (23-26). Numerous physiological processes are affected by ceramide. It has been reported to be a modulator of immune cell differentiation, mitochondrial respiration, inflammation, cell cycle progression, apoptosis, and the stress response (23, 24, 27-29). Many of these processes are also affected by TCDD and other ligands of the AHR. Furthermore, ceramide has several structural features which suggest that it might be an AHR ligand. Specifically, structure-activity analyses suggest that the AHR preferentially binds aromatic, hydrophobic planar molecules, and has a binding site with dimensions of ~6.8 × 13.7 Å (30, 31). Although not aromatic, ceramide is a hydrophobic molecule. The amide group, which serves as the link between ceramide's two acyl chains, imparts a planar conformation on the molecule in which the axes of the two acyl chains lie parallel to one another (32). Furthermore, the overall dimensions of some ceramide molecules do not dramatically differ from those of some AHR ligands (e.g. 4 × 20 Å for palmitoyl ceramide (C16); Ref. 33).

The current studies evolved from a preliminary characterization of ceramide as a putative AHR ligand. As an approach we examined the transcriptional activation of Cyp1a1 in AHR-containing (1c1c7) and AHR-deficient (Tao) cells treated with N-acetylsphingosine, a cell-permeable analog of ceramide. Although initial studies did not support the hypothesis being tested, we noted a pronounced difference in the viability of the two cell lines following ceramide exposure.2 In the current investigation, we determined whether manipulation of the AHR contents of cells of the 1c1c7 lineage could modulate susceptible to ceramide-induced apoptosis, and two additional inducers of apoptosis: staurosporine and doxorubicin (23, 34). Susceptibility of the various 1c1c7 variant lines to ceramide-induced apoptosis correlated directly with cellular AHR contents, but was independent of the presence of a functional ARNT protein. In contrast, AHR content did not influence susceptibility to staurosporine- or doxorubicin-mediated apoptosis. These findings implicate a novel role for AHR in ceramide-mediated apoptosis that is independent of its functioning as an ARNT-associated transcription factor.

    EXPERIMENTAL PROCEDURES

Materials-- alpha -Naphthoflavone, doxorubicin, and staurosporine were purchased from Sigma. TCDD was the gift of Dr. S. Safe (Texas A & M University, College Station, TX). N-Acetyl-D-erythro-sphingosine (C2-ceramide) and dihydro-N-acetyl-D-erythro-sphingosine (DHC2-ceramide) were purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). Ac-DEVD-AMC and 7-amino-4-methyl-coumarin (AMC) were obtained from PharMingen (San Diego, CA) and Aldrich, respectively. Geneticin, alpha -minimal essential medium, fetal bovine serum, and penicillin-streptomycin were purchased from Life Technologies, Inc.

Cell Culture-- The murine hepatoma 1c1c7, BPrc1, Tao, WCMV, WARV, TCMV, and TAHR cell lines were obtained from Dr. J. Whitlock, Jr. (Stanford University, Palo Alto, CA). The origins and characterizations of these cell lines have been described in detail (12, 35, 36). All cell lines were cultured at 32 °C in alpha -minimal essential medium containing 5% fetal bovine serum and 100 units/ml penicillin and 100 µg/ml streptomycin. The TCMV, TAHR, WCMV, and WARV lines were maintained in medium containing 500 ng/ml Geneticin.

Analyses of a chemical's cytostatic/cytotoxic effects were performed with cultures initially seeded at a density of 5-20 × 104 cells/60-mm dish in 3 ml of medium. The following day cultures were treated with either solvent (Me2SO or ethanol), ceramides (dissolved in Me2SO or ethanol), or TCDD, alpha -naphthoflavone, doxorubicin, or staurosporine (dissolved in Me2SO). At various times after treatment, culture medium was lightly sprayed over the culture surface to dislodge loosely attached cells, which were transferred to a centrifuge tube. Attached cells were removed with a 0.25% trypsin-EDTA mixture and added to the centrifuge tube. In some instances the pooled cell suspensions were pelleted by centrifugation and resuspended in phosphate-buffered saline. Cells were counted with a hemacytometer. Viability was assessed by trypan blue exclusion. Each sample was counted a minimum of three times.

Caspase Assay-- Cells were plated at a density of ~1 × 106/100-mm culture dish in 10 ml of medium. Two days after plating, cultures were treated with either solvent or chemicals of interest. At various times after treatment, cells were dislodged into the culture medium with a cell scraper, pelleted by centrifugation, resuspended in 0.5 ml of lysis buffer (10 mM Tris, pH 7.5, 130 mM NaCl, 1% Triton X-100, 10 mM NaF, 10 mM NaPi, and 10 mM NaPPi), quick frozen in liquid nitrogen, and stored at -70 °C. On the day of assay, lysates were thawed on ice, homogenized for 20 s (PT 1200 Polytron, Kinematica, Switzerland), and centrifuged at 18,000 × g for 12 min at 4 °C. Supernatant fluids were kept on ice until assayed by a modified version of a protocol supplied by the provider of Ac-DEVD-AMC (PharMingen). Assays were performed at room temperature and initiated by the addition of 100 µl of supernatant fluid to 1.9 ml of protease assay buffer (final concentrations: 20 mM Hepes, pH 7.4, 10% glycerol, 2 mM dithiothreitol, and 20 µM Ac-DEVD-AMC). AMC release was monitored with a Perkin-Elmer LS-5 fluorescence spectrophotometer using an excitation wavelength of 380 nm and an emission wavelength of 460 nm. A standard curve created with AMC was used to convert fluorescence intensity into picomoles of product produced in the presence of supernatant fluids. Protein concentrations were determined with Bio-Rad protein assay reagent using bovine serum albumin as a standard. Caspase activities are reported as nanomoles of AMC released/min/mg of protein of supernatant fluid. Initial characterization studies demonstrated that substrate cleavage was linear with time and directly proportional to the amount of protein in the assay.2

DNA Fragmentation Assay-- Cells were plated at ~1.5 to 2 × 106/100-mm dish in 10 ml of medium. Cultures were treated 1 day later with solvent or chemicals. At various times after treatment, the culture medium was removed and the cultures were washed with phosphate-buffered saline, aspirated dry, and stored at -70 °C. Non-adherent cells in the culture medium were recovered by centrifugation and also stored at -70 °C.

Adherent cells were lysed in the plates by addition of 5 ml of 4 M guanidine isothiocyanate, 25 mM sodium citrate, pH 7.0, 100 mM beta -mercaptoethanol, and 0.5% sarkosyl. Lysates were transferred to the tubes containing pellets of non-adherent cells. Lysates were extracted with 5 ml of Tris-equilibrated phenol (pH >7.6) plus 1 ml of chloroform:isoamyl alcohol (49:1). After centrifugation for 1 h at 6500 × g at 4 °C, the aqueous phase was transferred to new tubes. Nucleic acids were precipitated by addition of an equal volume of isopropanol and subsequently pelleted by centrifugation. Pellets were resuspended in 0.5 ml of 10 mM Tris, 1 mM EDTA, pH 8.0 (TE), 0.1% SDS, and 40 µg/ml RNase A and incubated at 37 °C, for 1 h. After successive extractions with Tris-equilibrated phenol (pH >7.6):chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1), DNA was precipitated by the addition of 1/50 volume of 5 M NaCl and 2 volumes of 95% EtOH. DNA was pelleted by centrifugation, resuspended in TE (~10 µl per 1 × 106 cells), and quantified by spectrophotometry. DNAs were mixed with ethidium bromide (80 ng/ml) and separated on 2% agarose, 1× TAE gels. DNA was visualized under UV light and recorded with a IS-1000 Digital Imaging System (Alpha Innotech Corp., San Leandro, CA). Nucleosomal ladders were visualized with exposures of ~2 s. Briefer exposures (<= 1/15 s) were used to visualize high molecular weight DNA.

RNA Preparation and Northern Blot Analyses-- Total cellular RNA was isolated according to the acidic phenol extraction method of Chomczynski and Sacchi (37). RNA was resolved on 1.2% agarose/formaldehyde gels and transferred to nylon membranes as described previously (5). The probes used for the detection of CYP1A1 and 7 S RNAs, and the conditions used for hybridization have been described in detail (38).

Immunoblotting and Quantification of AHR-- The conditions detailed by Reiners et al. (5) were used for the preparation of cellular extracts, separation of proteins on SDS-polyacrylamide gels, transfer of proteins to nitrocellulose, and immunodetection of the AHR with an affinity-purified polyclonal rabbit antibody to the murine AHR protein (provided by Dr. A. Poland while at the University of Wisconsin, Madison, WI). Antigen-immunoglobulin conjugates were detected with an ECL detection kit (Amersham Pharmacia Biotech) and recorded on x-ray film.

    RESULTS

Ceramide Induction of Apoptosis in 1c1c7 and AHR-deficient Variant Cell Lines-- A variety of cell types commit to, and undergo apoptosis upon exposure to N-acetylsphingosine (C2-ceramide), a cell-permeable analog of ceramide (23, 39). Exposure of 1c1c7 cultures to varying concentrations of C2-ceramide resulted in a concentration-dependent suppression of cell proliferation (Fig. 1A). Concentrations >= 30 µM completely suppressed cell division (Fig. 1A), enhanced cell permeability to trypan blue (Fig. 1C), and caused dramatic morphological changes. Specifically, within 4-6 h of exposure to >= 30 µM C2-ceramide, the cells began to vacuolate, and by 12-16 h had shrunken, as had the nuclei.2 DNA fragmentation and the development of nucleosomal ladders, a characteristic of late stage apoptotic cells, were detected within 16 h of C2-ceramide treatment (Fig. 1E) and preceded alterations in membrane permeability to trypan blue (Fig. 1, compare C and E).


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Fig. 1.   Ceramide-induced apoptosis in 1c1c7 and Tao cells. Suspensions of 1c1c7 (first column) and Tao (second column) were plated 1-2 days prior to onset of treatment. At time zero cultures were treated with Me2SO (bullet ) or varying concentrations of C2-ceramide (down-triangle, 1 µM; , 10 µM; open circle , 30 µM; triangle , 60 µM;). Cultures were harvested at various times after treatment for analyses of cell numbers (panels A and B,), cell survival (panels C and D), and isolation of DNA. To facilitate comparison, the 60 µM concentration from panel D is indicated in panel C by a dashed line. Purified DNA (24 µg) from the cultures treated with 30 µM C2-ceramide for indicated times was mixed with ethidium bromide and separated on 2% agarose gels. High molecular weight DNA was detected with UV and photographed using a 1/30th-s exposure (panels G and H). Fragmented DNA on the same gel was photographed using a 2-s exposure (panels E and F). C-48 denotes Me2SO-treated cultures harvested at the same time as the 48-h ceramide treatment group (panels E-H). Data in panels A-D represent means ± S.E. of 3-6 plates.

Tao cells were derived from 1c1c7 cells and have been reported to contain normal amounts of ARNT protein, but only 10% of the AHR protein content of 1c1c7 cells (12, 36). The data present in the top panel of Fig. 2 confirm the markedly different AHR contents of the 1c1c7 and Tao cell lines. These differences were also reflected in how the two cell lines responded to TCDD. Although steady state CYP1A1 mRNA levels were elevated above basal levels in both cell lines following TCDD exposure, the induced CYP1A1 mRNA contents of 1c1c7 cells were severalfold greater than those observed in its AHR-deficient counterpart (bottom panel of Fig. 2).


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Fig. 2.   AHR content and TCDD responsiveness of 1c1c7 cells and variant lines. Exponentially growing cultures were harvested and processed for AHR protein analyses without any treatment (top panel) or were exposed to Me2SO or 2 nM TCDD for 6 h prior to being harvested for isolation of RNA and analyses of CYP1A1 and 7 S RNAs (bottom panel). Analyses were made on 30 µg of protein (top panel) or 10 µg of total RNA (bottom panel).

Tao cells grew considerably slower than 1c1c7 cells (Fig. 1, compare A and B). Proliferation of Tao cells, like the parental 1c1c7 line, was completely suppressed by exposure to concentrations of C2-ceramide >= 30 µM (Fig. 1B). However, the killing effects of anti-proliferative concentrations of C2-ceramide were muted in Tao cells (Fig. 1D). Specifically, the kinetics of killing were slower and the absolute level of killing was much less than that seen in parental 1c1c7 cells (Fig. 1, compare C and D). Indeed, ~30% of C2-ceramide-treated Tao cells were viable after even 4 days of treatment (n = 3 experiments).2 Furthermore, at comparable loadings of DNA, nucleosomal ladders were not detected in Tao cells following 48 h (Fig. 1, compare E and F), or even 72 h of exposure to C2-ceramide.2

Ma and Whitlock (12) recently reported the characterization of a series of cell lines derived by transfection of 1c1c7 and Tao cells with Ahr antisense and sense expression constructs, respectively. Transfection of 1c1c7 cells with an Ahr antisense construct gave rise to a cell line designated WARV having AHR contents similar to those measured in Tao cells (top panel of Fig. 2). Conversely, transfection of Tao cells (also called AhR-D; Ref. 12) with an Ahr sense expression vector gave rise to a cell line designated TAHR having AHR contents similar to that seen in 1c1c7 cells (Fig. 2). The AHR contents of 1c1c7 and Tao cells stably transfected with just the vector (resulting lines are designated WCMV and TCMV, respectively) were very similar to the contents of the corresponding parental cells (Fig. 2). The responsiveness of the four engineered cell lines to TCDD, as monitored by measuring steady state CYP1A1 mRNA contents 6 h after treatment, also correlated with cellular AHR contents (bottom panel of Fig. 2). We used these lines to further examine the relationship between AHR content and susceptibility to ceramide-induced apoptosis (Fig. 3). The sensitivity of WCMV cells to C2-ceramide was virtually identical to the parental line (compare Fig. 1, A and C, with Fig. 3, A and E). However, WARV cells were markedly less sensitive to C2-dependent killing, as judged by trypan blue staining (Fig. 3, compare E and F) and DNA laddering (Fig. 3, I and J). Conversely, TAHR cells were more susceptible to C2-ceramide-induced killing and DNA fragmentation than the TCMV cell line (Fig. 3, compare G with H, and K with L).


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Fig. 3.   Ceramide-induced apoptosis in transfected 1c1c7 and Tao cells. Suspensions of WCMV (first column), WARV (second column), TCMV (third column), and TAHR (fourth column) were plated 1-2 days prior to onset of treatment. At time zero, cultures were treated with ethanol (bullet ) or varying concentrations of C2-ceramide (down-triangle, 1 µM; open circle , 30 µM). Cultures were harvested at various times after treatment for analyses of cell numbers (panels A-D), cell survival (panels E-H), and isolation of DNA. To facilitate comparison, the 30 µM concentrations from panels F and G are indicated by dashed lines in panels E and H, respectively. Analyses of fragmented DNA (panels I-L) and high molecular weight DNA (panels M-P) are as described in the legend of Fig. 1. Data in panels A-H represent the means ± S.E. of 3-6 plates.

The data presented in Fig. 3 merit two additional comments. First, manipulation of AHR contents modulated cell doubling times. AHR-deficient variant lines (WARV and TCMV) grew much slower than the engineered cell lines (WCMV and TAHR) expressing wild type AHR levels (Fig. 3, compare A, B, C, and D). These results are similar to those reported by Ma and Whitlock (12). Second, irrespective of AHR content, proliferation was arrested in all four engineered cell lines following exposure to 30 µM C2-ceramide (Fig. 3, A-D).

The studies reported in Figs. 1 and 3 were performed with cultures grown at 32 °C in the presence of 5% fetal bovine serum (our standard conditions; Ref. 38). Similar studies were performed with cultures grown in variable amounts of FBS, or at 37 °C.2 The concentration-response curves were influenced by the amount of serum present in the culture medium. Decreasing media serum concentrations decreased the amount of ceramide required to achieve cell killing. Increasing the culturing temperature to 37 °C slightly accelerated the kinetics of cell killing. However, the differential responsiveness of the parental and variant cell lines to C2-ceramide was unaffected by either temperature or serum content.2 Furthermore, the differential responsiveness of the 1c1c7 and Tao lines was also observed when cultures were treated with N-hexanoylsphingosine (C6-ceramide), another cell-permeable analog of ceramide having a longer acyl chain.2

Detection of Caspase Activity-- Endonuclease activation and fragmentation of DNA into nucleosome multimers occurs in the later stages of the apoptotic process. Activation of a series of cysteine proteases, referred to as caspases, occurs earlier. Caspases 3 and 7 are activated by a variety of apoptotic inducers, and are routinely assayed by monitoring cleavage of the synthetic substrate N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC; Refs. 40 and 41). Activated caspases 3 and 7 cleave Ac-DEVD-AMC between Asp and AMC, resulting in the release of a fluorescent product.

Caspase specific activities in 1c1c7 (Fig. 4A) or TAHR cells (Fig. 4B) increased ~10-20-fold over 72 h following exposure to the solvent. These increases may reflect the time-dependent development of focally packed areas and the induction of apoptosis in these areas. Independent studies demonstrated that caspase activities were basically indistinguishable in non-treated and ethanol-treated cultures, and directly related to the confluence of the cultures.2 In contrast, treatment of 1c1c7 (Fig. 4A) or TAHR cells (Fig. 4B) with a concentration of C2-ceramide (30 µM) that induced DNA fragmentation increased caspase specific activities ~400-500-fold. The kinetics of caspase activation and DNA fragmentation paralleled one another in C2-ceramide-treated 1c1c7 and TAHR cultures (compare Fig. 1E with Fig. 4A and Fig. 3L with Fig. 4B).


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Fig. 4.   Kinetics of caspase activation in 1c1c7 and 1c1c7 variant cell lines. Suspensions of 1c1c7, Tao, TAHR, and TCMV cells were plated 2 days prior to treatment with ethanol or 30 µM C2-ceramide. Cultures were harvested at various times after treatment for analyses of caspase activities. Data represent the means ± S.E. of analyses performed on 3 plates. A: , 1c1c7-C2; diamond , Tao-C2; open circle , 1c1c7-EtOH; triangle , Tao-EtOH. B: , TAHR-C2; diamond , TCMV-C2; open circle , TAHR-EtOH; triangle , TCMV-EtOH.

Caspase specific activities in the AHR-deficient Tao and TCMV cells were also elevated following exposure to C2-ceramide. However, the level of caspase activation in the two AHR-deficient lines was less than that measured in the 1c1c7 and TAHR lines (Fig. 4, A and B). Indeed, caspase specific activities in C2-ceramide-treated TCMV cells were, at most, only 3-fold higher than the values measured in solvent-treated cultures. Hence, the absence of DNA fragmentation in C2-ceramide-treated AHR-deficient lines correlated with the diminished activation of caspases (compare Fig. 1F with Fig. 4A and Fig. 3K with Fig. 4B).

Analyses of caspase activation in TAHR and TCMV cells at varied C2-ceramide concentrations are presented in Fig. 5. No effects were seen in either cell line at concentrations of <= 10 µM. In TAHR cells a small activation was observed at 20 µM, and near-maximal activation occurred at 30 µM. In marked contrast, no activation occurred in the AHR-deficient TCMV line at concentrations up to 60 µM C2-ceramide (Fig. 5). Similar results were obtained in analyses of the 1c1c7 and Tao lines.2 Specifically, caspases were not activated in Tao cells treated with concentrations of C2-ceramide as high as 60 µM, whereas maximal activation occurred at 30 µM in 1c1c7 cells. Thus, the differential responsiveness of AHR-containing and AHR-deficient cell lines to C2-ceramide did not reflect a shift in the concentration-response curves.


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Fig. 5.   Caspase activation in TCMV and TAHR cells as a function of C2-ceramide concentration. Suspensions of TAHR (open circle ) and TCMV cells (bullet ) were plated 2 days prior to treatment with varied concentrations of C2-ceramide. Cultures were harvested 26 h after treatment for analyses of caspase activities. Data represent the means ± S.E. of analyses performed on 3 culture dishes.

Ceramide Induction of Apoptosis in a 1c1c7 Variant Cell Line Lacking ARNT-- Ligand-activated functions of the AHR are thought to require its heterodimerization with ARNT (1-3). Although ceramide has not been identified as a ligand of the AHR, we investigated whether the effects of ceramide require a functional ARNT protein. BPrc1 cells were derived from the 1c1c7 line and contain comparable amounts of the AHR, but no functional ARNT (35). Exposure of BPrc1 cells to 60 µM C2-ceramide (30 µM concentration was not tested) suppressed cell proliferation (Fig. 6A), and enhanced permeability to trypan blue (Fig. 6B). Like the parental 1c1c7 line, BPrc1 cells were more sensitive than Tao cells to the killing actions of C2-ceramide (Fig. 6B). Exposure of BPrc1 cultures to C2-ceramide resulted in caspase activation (Fig. 6, C and D), and the development of nucleosomal DNA ladders (Fig. 6E), and loss of high molecular weight DNA (Fig. 6F). Indeed, 1c1c7 and BPrc1 cells were quite similar to one another with respect to the concentrations of C2-ceramide required for caspase activation (compare Figs. 5 and 6C) and the magnitude of caspase activation following C2-ceramide exposure (compare Figs. 4A and 6D).


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Fig. 6.   Ceramide-induced apoptosis in BPrc1 cells. Suspensions of BPrc1 were plated 1-2 days prior to onset of treatment. At time zero, cultures were treated with solvent or varying concentrations of C2-ceramide (see symbols in figure). Cultures were harvested at various times after treatment for analyses of cell numbers (panel A), survival (panel B), caspase activation (panels C and D), and DNA. Analyses of fragmented DNA (panel E) and high molecular weight DNA (panel F) are as described in the legend of Fig. 1. C-48 denotes Me2SO-treated cultures harvested at the same time as the 48-h ceramide treatment group. Data in panels A-D represent means ± S.E. of 3-6 plates. To facilitate comparison to an AHR-deficient cell line, the 60 µM C2-ceramide survival data for the Tao cell line from Fig. 1D is indicated in panel B by a dashed line.

Effects of Ceramide Analogs and AHR Ligands on Ceramide induced Apoptosis-- Dihydro-N-acetyl-D-erythro-sphingosine (DHC2-ceramide) differs from C2-ceramide in only one respect; it lacks the double bond between carbons 4 and 5 of the sphingoid backbone. This single alteration drastically alters its biological activities. Specifically, it does not induce apoptosis in cells that normally respond to C2-ceramide (39). Concentrations of DHC2-ceramide <= 60 µM neither affected 1c1c7 proliferation nor viability (Fig. 7, A and B, respectively). These data suggest that the apoptosis seen in 1c1c7 cells following C2 exposure represents a ceramide-specific process, as opposed to a nonspecific lipid effect.


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Fig. 7.   Dihydro-N-acetyl-D-erythro-sphingosine-induced apoptosis. Suspensions of 1c1c7 cells were plated 1 day prior to treatment with either solvent or DHC2-ceramide. Cultures were harvested for analyses of cell numbers (panel A) and survival (panel B) at various times after treatment. Data represent the means ± S.E. of analyses performed on 3 culture dishes. , ethanol; triangle , 30 µM DHC2; open circle , 60 µM DHC2.

C2-ceramide has dimensions and structural features which approximate some of the modeled characteristic of AHR ligands (30-33). TCDD is a well characterized agonist of the AHR (1). Exposure of 1c1c7 cultures to a concentration of TCDD that transcriptionally activates Cyp1a1 (38) had no effects on cell proliferation or viability (Fig. 8, A and B). Furthermore, preincubation of 1c1c7 cultures with TCDD did not affect the kinetics of cell killing caused by subsequent exposure to C2-ceramide (Fig. 8B).


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Fig. 8.   Effects of AHR ligands on ceramide-induced apoptosis. Suspensions of 1c1c7 cells were plated 1 day prior to treatment with either solvent or C2-ceramide and/or TCDD (left column) or C2-ceramide and/or alpha -NF (right column). Cultures were treated with AHR ligands 2 h prior to the addition of C2-ceramide. Solvent controls and cultures treated with only C2-ceramide were also pretreated with solvent in order to mimic the double solvent exposure occurring in cultures treated with C2-ceramide + AHR ligand. Cultures were harvested at various times after ceramide treatment for analyses of cell numbers (panels A and C) and survival (panels B and D). Data represent the means ± S.E. of analyses performed on 3 culture dishes. A and B: , Me2SO; diamond , 30 µM C2; open circle , 60 µM C2; triangle , 5 nM TCDD; box-plus , 30 µM C2 + 5 nM TCDD; black-diamond , 60 µM C2 + 5 nM TCDD. C and D: , Me2SO; diamond , 1 µM alpha -NF; open circle , 30 µM C2; triangle , 1 µM alpha -NF + 30 µM C2.

alpha -Naphthoflavone (alpha -NF) is also a ligand of the AHR. However, it functions as an antagonist in many cell types at low micromolar concentrations, and as an agonist at concentrations of >= 10 µM (42-44). An antagonist concentration of alpha -NF was weakly cytostatic to 1c1c7 cultures (Fig. 8C), but had no effect on cell viability (Fig. 8D). Incubation of 1c1c7 cultures with alpha -NF prior to treatment with C2-ceramide affected neither the kinetics nor magnitude of cell killing caused by C2-ceramide (Fig. 8D).

Doxorubicin- and Staurosporine-induced Apoptosis in Variant and Parental 1c1c7 Cells-- Numerous chemicals are capable of inducing apoptosis. Staurosporine and doxorubicin, like ceramide, induce caspase activation through a Bcl-2 inhibitable process that probably involves the Apaf-1 complex (34). Exposure of 1c1c7 and AHR-deficient Tao cultures to concentrations of doxorubicin >= 0.1 µM strongly suppressed the proliferation of both cell lines (Fig. 9, A and B). Based upon analyses of trypan blue permeability at various concentrations of doxorubicin, Tao and 1c1c7 cultures appeared to be fairly similar in their sensitivities to the killing actions of the drug (Fig. 9, E and F). Indeed, the kinetics of caspase activation and levels of activation, at each concentration of doxorubicin tested, were very similar in the two cell lines (Fig. 9, compare I and J).


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Fig. 9.   Staurosporine- and doxorubicin-induced apoptosis in 1c1c7 and Tao cells. Suspensions of 1c1c7 and Tao cells were plated 1 or 2 days prior to treatment with Me2SO, or varied concentrations of doxorubicin or staurosporine. Cultures were harvested at various times after treatment for analyses of cell numbers (panels A-D), survival (panels E-H), and caspase activities (panels I-L). Data represent the means ± S.E. of analyses performed on three culture dishes. A, B, E, F, I, and J: bullet , Me2SO; down-triangle, 0.01 µM; open circle , 0.1 µM; triangle , 1 µM; , 10 µM. C, D, G, H, K, and L: bullet , Me2SO; down-triangle, 5 nM; open circle , 20 nM; triangle , 100 NM.

Exposure of 1c1c7 and Tao cultures to concentrations of staurosporine >= 5 nM totally suppressed the proliferation of both cell lines (Fig. 9, C and D). Although analyses of trypan blue exclusion suggested that Tao cultures were slightly less susceptible than 1c1c7 cultures to the killing actions of low concentrations of staurosporine, this differential response was lost at a concentration of 100 nM staurosporine (Fig. 9, compare G and H). Furthermore, analyses of caspase activation suggested no differences in the responsiveness of 1c1c7 and Tao cultures to staurosporine. The kinetics and magnitude of caspase activation, at each concentration of staurosporine tested, were very similar in the two cell lines (Fig. 9, compare K and L).

    DISCUSSION

The murine hepatoma 1c1c7 cell line and its AHR/ARNT variants have been used extensively as tools in the study of AHR function. Use of these lines led to the characterization of the AHR as a ligand-activated transcription factor that functions in concert with ARNT to activate a series of genes having dioxin-responsive elements in their flanking sequences (1, 35, 36, 45). A recent investigation employing 1c1c7 cells and AHR variants demonstrated that AHR content, in the absence of exogenous AHR ligands, affects cell shape, the duration of the G1 phase of the cell cycle, and the expression of a hepatic-specific, differentiation-related gene (12). The current investigation defines a new function for the AHR in cells of the 1c1c7 lineage. Specifically, within the series of cell lines surveyed, those having the lowest levels of AHR expression were the least susceptible to ceramide-induced apoptosis. Thus, susceptibility to ceramide-induced apoptosis was directly related to the AHR contents of these cell lines.

One plausible explanation for the differential sensitivities of AHR-containing and AHR-deficient cell lines to ceramide-induced apoptosis is that ceramide is an AHR ligand. We reasoned that if C2-ceramide were acting as an AHR ligand, its effects might be duplicated by other known AHR ligands. However, this was not the case. Neither TCDD nor alpha -NF triggered the induction of apoptosis in 1c1c7 cells or modulated the induction of apoptosis by ceramide (see Fig. 8). Reciprocally, we found that treatment of 1c1c7 cultures with concentrations of C2- or C6-ceramide of <= 60 µM neither reproducibly elevated CYP1A1 mRNA contents (a 2.5-fold elevation was noted at 4 h after treatment in only one of five experiments), nor affected the transcriptional activation of Cyp1a1 by TCDD.2 These latter studies demonstrate that ceramide is not an AHR antagonist. Although our studies do not rule out the possibility that ceramide affects the AHR by some indirect mechanism, they suggest that ceramide is not an AHR ligand. In addition, ceramide-induced apoptosis occurred in a variant of 1c1c7 cells (e.g. BPrc1 cell line) having parental AHR contents, but no functional ARNT. This is an exciting finding, as it demonstrates that the mechanism by which the AHR modulates ceramide-induced apoptosis can not involve ligand-activated AHR/ARNT heterodimer. To the best of our knowledge, this is the first demonstration of an AHR-modulated process that does not require ARNT.

Although the AHR content of cells of the 1c1c7 lineage influenced caspase activation by ceramide, it had no affect on the kinetics or magnitude of caspase activation following exposure to doxorubicin or staurosporine. Thus, the differential sensitivities of wild type and AHR-deficient cell lines to ceramide-induced apoptosis reflect a specific action of ceramide, as opposed to a generalized resistance of the AHR-deficient lines to apoptotic inducers. These data raise the issue of whether ceramide signaling, in general, is regulated by AHR content. Ceramide induces cell cycle arrest in many cell types (for review, see Ref. 24). This activity reflects a G1 block and is characterized by the accumulation of hypophosphorylated retinoblastoma protein (24, 46). We were unable to define a cytostatic, non-apoptotic concentration of C2-ceramide in 1c1c7 cells. In contrast, concentrations of C2-ceramide were identified that arrested both 1c1c7 and TCMV proliferation, which did not induce apoptosis of the AHR-deficient TCMV cell line (as assessed by DNA laddering and caspase activation). Hence, AHR content appears to differentially influence the cytostatic and apoptotic activities of ceramide. It should be noted that an uncoupling of the cytostatic and apoptotic activities of C2-ceramide has also been observed in Raji cells (47), and can be operationally induced in ceramide-treated Molt-4 and U937 cells by cotreatment with a cell-permeable diacylglycerol analog or phorbol 12-myristate 13-acetate (29).

Intracellular ceramide levels are often elevated following receptor-triggered (e.g. CD95/Apo1 or tumor necrosis factor-alpha ) and non-receptor-triggered (e.g. ionizing radiation, staurosporine, daunorubicin, etc.) apoptosis (24, 25, 48, 49). These elevations can reflect either de novo biosynthesis (49) or sphingomyelinase activation and degradation of sphingomyelin (25). Because exogenously added, cell-permeable ceramides induce apoptosis in several cell types, it has been hypothesized that ceramide produced in situ may be the initiator of the execution phase of apoptosis. This hypothesis is supported by two additional lines of experimentation. First, lymphoblasts and fibroblasts derived from humans (e.g. individuals suffering from Neimann-Pick disease) or mice defective in acidic sphingomyelinase are resistant to ionizing radiation or doxorubicin induced-apoptosis, but apoptose if treated with an exogenous source of ceramide (48, 50). Second, some multiple drug-resistant cell lines express a glucosylceramide synthase that inactivates ceramide via conjugation (51, 52). Treatment of such lines with an inhibitor of glucosylceramide synthase renders them sensitive to the apoptotic actions of cell-permeable ceramide analogs and a variety of chemotherapeutic agents (52). Nevertheless, the observed sensitivity of the AHR-deficient Tao cells to exogenously added doxorubicin or staurosporine, but not C2-ceramide, strongly suggests that the first two agents activate caspases by a ceramide-independent process. The 1c1c7 and AHR variant lines may be useful tools for the characterization of this process, and assessment of the contributions of ceramide to the initiation of apoptosis by apoptotic inducers. Meaningful interpretation of comparisons of the sensitivities of wild type and AHR "knockout" mice to the actions of apoptotic agents, or physiological processes that entail an apoptotic process (e.g. regulation of T cell proliferation following stimulation) will be dependent upon such information.

Ceramide affects a variety of signaling molecules and pathways (24). It is a direct activator of a PPA2-like phosphatase termed ceramide-activated protein phosphatase (CAPP; Refs. 24 and 53) and a membrane-bound, Ser/Thr protein kinase designated ceramide-activated protein kinase (CAP kinase; Refs. 54 and 55). This latter enzyme was recently shown to be identical to kinase suppressor of Ras (56), and is capable of activating raf-1 kinase. Ceramide is also an activator of the zeta  isozyme of protein kinase C (57, 58) and members of the stress-activated protein kinase (SAPK) cascade (27, 59). It is also a potent inhibitor of complex III of the mitochondrial respiratory chain (60). Of these various effects, ceramide activation of the SAPK cascade and inhibition of complex III activity have been linked to the induction of apoptosis (27, 28, 59, 61).

Treatment of cells and isolated mitochondria with ceramide increases mitochondrial production of reactive oxygen species (ROS; 28, 61). The ROS arise as a consequence of ceramide inhibition of complex III, blockage of respiration, and the subsequent diversion of electrons from ubisemiquinone to molecular oxygen (61, 62). The ROS, in turn, lead to the development of oxidative stress, mitochondrial damage, and disruption of mitochondrial membrane potential and permeability (61, 63). Such a scenario could lead to the release of cytochrome c, which is required for caspase activation by the mitochondrial-associated Apaf-1 complex (64). In preliminary studies we have found that C2-ceramide exposure of suspended 1c1c7 cells results in a concentration-dependent suppression of respiration.3 In contrast, the effects of ceramide on respiration appear to be muted in Tao cells.3 These observations may provide a basis for the observed lack of caspase activation in the AHR-deficient cells. We are currently investigating this line of research.

Nucleosomal DNA fragmentation is a late stage marker of apoptotic cells, and is catalyzed by a 40-kDa DNase designated DNA fragmentation factor (DFF40; Refs. 65-67). DFF40 normally exists in the cytosol in an inactive form as a consequence of its association with DFF45 (66, 66). In vitro studies in which DFF40 and DFF45 are mixed with caspase-3 (66, 67) and investigations employing caspase-3-deficient cells (68) and caspase inhibitors (69-71) collectively demonstrate that caspase-3 activates DDF40 by cleaving DFF45, which facilitates dissociation of the heterodimeric complex. Given the role of caspase-3 in activating DFF40, and the inability of C2-ceramide to activate caspase-3 in our AHR-deficient cell lines, it is easy to rationalize the absence of DNA laddering in these cells following C2-ceramide exposure.

Although C2-ceramide-treated AHR-deficient 1c1c7 variant cell lines did not exhibit characteristics commonly associated with apoptotic cells (morphology, caspase-3 activation, DNA laddering), they were killed by ceramide, as determined by measurements of trypan blue exclusion. The sensitivities of the parental and AHR-deficient variant cell lines to the cytotoxic effects of C2-ceramide, however, were markedly different. At comparable concentrations of C2-ceramide, a lower percentage of AHR-deficient cells died, and the kinetics of cell killing were considerably slower than those observed in their AHR-containing counterparts. Hence, the mechanisms by which C2-ceramide killed 1c1c7 cells and its AHR-deficient variants are probably different. Additional studies are required to determine if the AHR-deficient variants die by a necrotic pathway following C2-exposure. It should be emphasized that other examples exist documenting cell killing by apoptotic agents via a pathway that does not lead to the development of features characteristic of apoptotic cells. Specifically, MCF-7 cells do not express functional caspase-3 as a consequence of a 47-base pair deletion within exon 3 of the CASP-3 gene (68). Nevertheless, numerous apoptotic agents kill MCF-7 cells by a process that neither involves caspase-3 or DFF40 activation nor results in the morphological characteristics of apoptotic cells (see Ref. 68, and references therein). In this respect, the effects of apoptotic agents on MCF-7 cells are very similar to the effects of C2-ceramide on AHR-deficient 1c1c7 variant lines.

In summary, our studies define a new role for the AHR, which is related to its regulation of ceramide-initiated apoptosis. The extent to which the AHR regulates the numerous signaling pathways affected by ceramide is not known. However, there appears to be some specificity since ceramide was cytostatic, but not apoptotic to AHR-deficient cells of the 1c1c7 lineage. Furthermore, our studies are the first to define a biological function for the AHR that is independent of its interaction with ARNT, and its functioning as a ligand-activated, ARNT-associated transcription factor.

    ACKNOWLEDGEMENT

We thank Dr. Cornelis Elferink for introducing the idea that ceramide may be an AHR ligand.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant CA34469, by Pilot Project Grant MCT26 awarded by P30 ES06639, and by assistance from the services of the Cell Culture Facility Core and Cell Imaging and Cytometry Facility Core, which are supported by National Institutes of Health NIEHS Grant P30 ES06639.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Inst. of Chemical Toxicology, Wayne State University, 2727 Second Ave., Rm. 4000, Detroit, MI 48201. Fax: 313-577-0082; E-mail: john.reiners.jr{at}wayne.edu.

The abbreviations used are: AHR, aryl hydrocarbon receptor; alpha -NF, alpha -naphthoflavone; Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; AMC, 7-amino-4-methyl-coumarin; ARNT, aryl hydrocarbon receptor nuclear translocator; C2-ceramide, N-acetyl-D-erythro-sphingosine; DFF, DNA fragmentation factor; DHC2-ceramide, dihydro-N-acetyl-D-erythro-sphingosine; ROS, reactive oxygen species; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2 R. E. Clift and J. J. Reiners, Jr., unpublished observations.

3 B. Taffe and J. J. Reiners, Jr., unpublished observations.

    REFERENCES
Top
Abstract
Introduction
References

  1. Hankinson, O. (1995) Annu. Rev. Pharmacol. Toxicol. 35, 307-340[CrossRef][Medline] [Order article via Infotrieve]
  2. Schmidt, J. V., and Bradfield, C. A. (1996) Annu. Rev. Cell Dev. 12, 55-89[CrossRef][Medline] [Order article via Infotrieve]
  3. Nebert, D. W. (1994) Biochem. Pharmacol. 47, 25-37[CrossRef][Medline] [Order article via Infotrieve]
  4. Pollenz, R. S., Sattler, C. A., and Poland, A. (1995) Mol. Pharmacol. 45, 428-438[Abstract]
  5. Reiners, J. J., Jr., Jones, C. L., Hong, N., Clift, R., and Elferink, C. (1997) Mol. Carcinog. 19, 91-100[CrossRef][Medline] [Order article via Infotrieve]
  6. Fernandez-Salguero, P., Pineau, T., Hilbert, D. M., McPhail, T., Lee, S. S. T., Kimura, S., Nebert, D. W., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1995) Science 268, 722-726[Medline] [Order article via Infotrieve]
  7. Schmidt, J. V., Su, G. H.-T., Reddy, J. K., Simon, M. C., and Bradfield, C. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6731-6736[Abstract/Free Full Text]
  8. Mimura, J., Yamashita, K., Nakamura, K., Norita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., and Fujii-Kuriyama, Y. (1997) Genes Cells 2, 645-654[Abstract/Free Full Text]
  9. Fernandez-Salguero, P. M., Hilbert, D. M., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1996) Toxicol Appl. Pharmacol. 140, 173-179[CrossRef][Medline] [Order article via Infotrieve]
  10. Ge, N.-L., and Elferink, C. J. (1998) J. Biol. Chem. 273, 22708-22713[Abstract/Free Full Text]
  11. Weib, C., Kolluri, S. K., Kiefer, F., and Göttlicher, M. (1996) Exp. Cell Res. 226, 154-163[CrossRef][Medline] [Order article via Infotrieve]
  12. Ma, Q., and Whitlock, J. P., Jr. (1996) J. Mol. Cell. Biol. 16, 2144-2150
  13. Jacobson, M. D., Weil, M., and Raff, M. C. (1997) Cell 88, 347-354[Medline] [Order article via Infotrieve]
  14. Scott, D. W., Grdina, T., and Shi, Y. (1996) J. Immunol. 156, 2352-2356[Abstract]
  15. Abbott, B. D., Birnbaum, L. S., and Perdew, G. H. (1995) Dev. Dyn. 204, 133-143[Medline] [Order article via Infotrieve]
  16. Abbott, B. D., and Probst, M. R. (1995) Dev. Dyn. 204, 144-155[Medline] [Order article via Infotrieve]
  17. Lawrence, B. P., Leid, M., and Kerkvliet, N. I. (1996) Toxicol. Appl. Pharmacol. 138, 275-284[CrossRef][Medline] [Order article via Infotrieve]
  18. Crawford, R. B., Holsapple, M. P., and Kaminski, N. E. (1998) Mol. Pharmacol. 52, 921-927[Abstract/Free Full Text]
  19. Chrest, F. J., Buchholz, M. A., Kim, Y. H., Kwon, T.-K., and Nordin, A. A. (1993) Cytometry 14, 883-890[Medline] [Order article via Infotrieve]
  20. Su, X., Zhou, T., Yang, P., Wang, Z., and Mountz, J. D. (1996) J. Immunol. 156, 4198-4208[Abstract]
  21. Kamath, A. B., Nagarkatti, P. S., and Nagarkatti, M. (1997) Toxicol. Appl. Pharmacol. 142, 367-377[CrossRef][Medline] [Order article via Infotrieve]
  22. Zaher, H., Fernandez-Salguero, P. M., Letterio, J., Sheikh, M. S., Fornace, A. J., Jr., Roberts, A. B., and Gonzalez, F. J. (1998) Mol. Pharmacol. 54, 313-321[Abstract/Free Full Text]
  23. Hannun, Y. A., and Obeid, L. M. (1995) Trends Biochem. Sci. 20, 73-77[CrossRef][Medline] [Order article via Infotrieve]
  24. Hannun, Y. A. (1996) Science 274, 1855-1859[Abstract/Free Full Text]
  25. Jaffrezou, J.-P., Levade, T., Bettaieb, A., Andrieu, N., Bezombes, C., Maestre, N., Vermeersch, S., Rousee, A., and Laurent, G. (1996) EMBO. J. 15, 2417-2424[Abstract]
  26. Haimovitz-Friedman, A., Kan, C.-C., Ehleiter, D., Persaud, R. S., McLaughlin, M., Fuks, Z., and Kolesnick, R. N. (1994) J. Exp. Med. 180, 525-535[Abstract]
  27. Jarvis, W. D., Fornari, F. A., Jr., Auer, K. L., Freemerman, A. J., Szabo, E., Birrer, M. J., Johnson, C. R., Barbour, S. E., Dent, P., and Grant, S. (1997) Mol. Pharmacol. 52, 935-947[Abstract/Free Full Text]
  28. García-Ruiz, C., Colell, A., Marí, M., Morales, A., and Fernández-Checa, J. C. (1997) J. Biol. Chem. 272, 11369-11377[Abstract/Free Full Text]
  29. Jayadev, S., Liu, B., Bielawska, A. E., Lee, J. Y., Nazaire, F., Pushkareva, M. Y., Obeid, L. M., and Hannun, Y. A. (1995) J. Biol. Chem. 270, 2047-2052[Abstract/Free Full Text]
  30. Gillner, M., Bergman, J., Cambillau, C., Alexandersson, M., Fernström, B., and Gustafsson, J.-A. (1994) Mol. Pharmacol. 44, 336-345[Abstract]
  31. Kafafi, S. A., Said, H. K., Mahmoud, M. I., and Afeefy, H. Y. (1992) Carcinogenesis 13, 1599-1605[Abstract]
  32. Pascher, I. (1976) Biochim. Biophys. Acta 455, 433-451[Medline] [Order article via Infotrieve]
  33. Shah, J., Atienza, J. M., Duclos, R. I., Jr., Rawlings, A. V., Dong, Z., and Shipley, G. G. (1995) J. Lipid Res. 36, 1936-1944[Abstract]
  34. Yang, E., and Korsmeyer, S. J. (1996) Blood 88, 386-401[Free Full Text]
  35. Miller, A. G., Israel, D., and Whitlock, J. P., Jr. (1983) J. Biol. Chem. 258, 3523-3527[Abstract/Free Full Text]
  36. Whitlock, J. P., Jr., and Galeazzi, D. R. (1984) J. Biol. Chem. 259, 980-985[Abstract/Free Full Text]
  37. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
  38. Schöller, A., Hong, N. J., Bischer, P., and Reiners, J. J., Jr. (1994) Mol. Pharmacol. 45, 944-954[Abstract]
  39. Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Science 259, 1769-1771[Medline] [Order article via Infotrieve]
  40. Cohen, G. M. (1997) Biochem. J. 326, 1-16[Medline] [Order article via Infotrieve]
  41. Thornberry, N. A., Rano, T. A., Peterson, E. P., Rasper, D. M., Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., Chapman, K. T., and Nicholson, D. W. (1997) J. Biol. Chem. 272, 17907-17911[Abstract/Free Full Text]
  42. Santostefano, M., Merchant, M., Arellano, L., Morrison, V., Denison, M. S., and Safe, S. (1993) Mol. Pharmacol. 43, 200-206[Abstract]
  43. Wilhelmsson, A., Whitelaw, M. L., Gustafsson, J.-A., and Poellinger, L. (1994) J. Biol. Chem. 269, 19028-19033[Abstract/Free Full Text]
  44. Lu, Y.-F., Santostefano, M., Cunningham, B. D. M., Threadgill, M. D., and Safe, S. (1996) Biochem. Pharmacol. 51, 1077-1087[CrossRef][Medline] [Order article via Infotrieve]
  45. Reyes, H., Reisz-Porszasz, S., and Hankinson, O. (1992) Science 256, 1193-1195[Medline] [Order article via Infotrieve]
  46. Dbaibo, G. S., Pushkareva, M. Y., Jayadev, S., Schwarz, J. K., Horowitz, J. M., Obeid, L. M., and Hannun, Y. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1347-1351[Abstract]
  47. Kuroki, J., Hirokawa, M., Kitabayashi, A., Lee, M., Horiuchi, T., Kawabata, Y., and Miura, A. B. (1996) Leukemia 10, 1950-1958[Medline] [Order article via Infotrieve]
  48. Herr, I., Wilhelm, D., Bohler, T., Angel, P., and Debatin, K. M. (1997) EMBO. J. 16, 6200-6208[Abstract/Free Full Text]
  49. Bose, R., Verheij, M., Haimovitz-Friedman, A., Scotto, K., Fuks, Z., and Kolesnick, R. (1995) Cell 82, 405-414[Medline] [Order article via Infotrieve]
  50. Santana, P., Pena, L. A., Haimovitz-Friedman, A., Martin, S., Green, D., McLoughlin, M., Cordon-Cardo, C., Schuchman, E. H., Fuks, Z., and Kolesnick, R. (1996) Cell 86, 189-199[Medline] [Order article via Infotrieve]
  51. Lavie, Y., Cao, H., Bursten, S. L., Giuliano, A. E., and Cabot, M. C. (1996) J. Biol. Chem. 271, 19530-19536[Abstract/Free Full Text]
  52. Lavie, Y., Cao, H., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682-1687[Abstract/Free Full Text]
  53. Wolff, R. A., Dobrowsky, R. T., Bielawska, A., Obeid, L. M., and Hannun, Y. A. (1994) J. Biol. Chem. 269, 19605-19609[Abstract/Free Full Text]
  54. Liu, J., Mathias, S., Yang, Z., and Kolesnick, R. N. (1994) J. Biol. Chem. 269, 3047-3052[Abstract/Free Full Text]
  55. Yao, B., Zhang, Y., Delikat, S., Mathias, S., Basu, S., and Kolesnick, R. (1995) Nature 378, 307-310[CrossRef][Medline] [Order article via Infotrieve]
  56. Zhang, Y., Yao, B., Delikat, S., Bayoumy, S., Lin, X.-H., Basu, S., McGinley, M., Chan-Hui, P.-Y., Lichenstein, H., and Kolesnick, R. (1997) Cell 89, 63-72[Medline] [Order article via Infotrieve]
  57. Lozano, J., Berra, E., Municio, M. M., Diaz-Meco, M. T., Dominguez, I., Sanz, L., and Moscat, J. (1994) J. Biol. Chem. 269, 19200-19202[Abstract/Free Full Text]
  58. Muller, G., Ayoub, M., Storz, P., Rennecke, J., Fabbro, D., and Pfizenmaier, K. (1995) EMBO J. 14, 1961-1969[Abstract]
  59. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., and Kolesnick, R. N. (1996) Nature 380, 75-79[CrossRef][Medline] [Order article via Infotrieve]
  60. Gudz, T. I., Tserng, K.-Y., and Hoppel, C. L. (1997) J. Biol. Chem. 272, 24154-24158[Abstract/Free Full Text]
  61. Pastorino, J. G., Simbula, G., Yamamoto, K., Glascott, P. A., Jr., Rothman, R. J., and Farber, J. L. (1996) J. Biol. Chem. 271, 29792-29798[Abstract/Free Full Text]
  62. Turrens, J. F., Alexandre, A., and Lehninger, A. L. (1985) Arch. Biochem. Biophys. 237, 408-414[Medline] [Order article via Infotrieve]
  63. Decaudin, D., Marzo, I., Brenner, C., and Kroemer, G. (1998) Int. J. Oncol. 12, 141-152[Medline] [Order article via Infotrieve]
  64. Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413[Medline] [Order article via Infotrieve]
  65. Liu, X., Li, P., Widlak, P., Zou, H., Luo, X., Garrard, W. T., and Wang, X. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8461-8466[Abstract/Free Full Text]
  66. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S. (1998) Nature 391, 43-50[CrossRef][Medline] [Order article via Infotrieve]
  67. Mitamura, S., Ikawa, H., Mizuno, N, Kaziro, Y., and Itoh, H. (1998) Biochem. Biophys. Res. Commun. 243, 480-484[CrossRef][Medline] [Order article via Infotrieve]
  68. Janicke, R. U., Sprengart, M. L., Wati, M. R., and Porter, A. G. (1998) J. Biol. Chem. 273, 9357-9360[Abstract/Free Full Text]
  69. Jaeschke, H., Fisher, M. A, Lawson, J. A., Simmons, C. A., Farhood, A., and Jones, D. A. (1998) J. Immunol. 160, 3480-3486[Abstract/Free Full Text]
  70. Yue, T. L., Wang, C., Romanic, A. M., Kikly, K., Keller, P., DeWolf, W. E., Jr., Hart, T. K., Thomas, H. C., Storer, B., Gu, J. L., Wang, X., and Feuerstein, G. Z. (1998) J. Mol. Cell Cardiol. 30, 495-507[CrossRef][Medline] [Order article via Infotrieve]
  71. Kimura, C., Zhao, Q. L., Kondo, T., Amatsu, M., and Fujiwara, Y. (1998) Exp. Cell Res. 239, 411-422[CrossRef][Medline] [Order article via Infotrieve]


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