Introduction

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The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor. In the absence of ligand it resides in the cytoplasm complexed with two heat shock protein 90 molecules and an immunophilin-like molecule (Schmidt and Bradfield, 1996; Carver et al., 1998). Upon ligand binding the AHR translocates to the nucleus where it complexes with the aryl hydrocarbon nuclear translocator (ARNT) protein. The resulting heterodimer recognizes and binds to dioxin responsive elements in target genes, and in conjunction with a series of coactivating proteins, stimulates target gene transcription (Nebert, 1994; Hankinson, 1995; Schmidt and Bradfield, 1996). To date, a variety of coplanar aromatic molecules have been identified as AHR ligands. Among them are several flavonoids, polycyclic aromatic hydrocarbons, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Genes of the "Ah battery" encode proteins involved in the phase I and II metabolism of xenobiotics and are transcriptionally activated by TCDD through an AHR-dependent process. For example, TCDD treatment elevates CYP1A1 (nomenclature used for CYP genes, mRNAs, and proteins is as recommended by Nelson et al., 1996), CYP1A2, and CYP1B1 expression in a variety of tissues (Nebert, 1994). These P450s have been implicated in the bioactivation of numerous procarcinogens. Conversely, TCDD also stimulates the transcription of GSTA1, AHD4, NQO1, NQO2, and UGT1A6 (Nebert, 1994). The proteins corresponding to these latter genes are involved in the detoxification of many carcinogens and cytotoxic agents used in chemotherapy.

In addition to its aforementioned activities, the AHR may also be a cell cycle regulatory protein. For example, the cytostatic activity of TCDD in the rat hepatoma 5L cell line is dependent upon the presence of the AHR (Weiss et al., 1996; Elferink et al., 2001). Similarly, recent studies suggest that a portion of the cytostatic activities of several flavonoids relates to their functioning as AHR ligands (Reiners et al., 1999). The AHR may also influence cell cycle progression via a ligand-independent mechanism. At least three groups have shown that the time it takes to traverse the cell cycle, in the absence of exogenous AHR ligand, is inversely related to the amount of AHR protein expressed (Ma and Whitlock, 1996; Weiss et al., 1996; Reiners and Clift, 1999).

Although several studies suggest that the AHR can function as a modulator of cell cycle progression, very little is known about the regulation of AHR function and expression as a function of the cell cycle. It has been reported that contact arrested murine NMuLi c1 mouse liver cells (Becker and Bartholomew, 1979), and serum/growth factor arrested murine 3T3 fibroblasts fail to respond to AHR ligands (Vaziri et al., 1996). In the case of the fibroblasts this response was shown to reflect AHR turnover and a suppression of AHR expression in the arrested G_o population (Vaziri et al., 1996). We previously reported that the transcriptional activation of Cyp1a1 by AHR ligands was suppressed in 1c1c7 hepatoma cells arrested in G₂/M as a consequence of exposure to the microtubule disrupter nocodazole (Schöller et al., 1994). These findings raise the issues of whether the transcriptional activation of Cyp1a1 and AHR function may be cell cycle-dependent. In the current study we investigated these problems by analyzing the expression of two TCDD-inducible genes in 1c1c7 cultures arrested in G₂/M by treatment with a series of microtubule disrupters (Colcemid, vinblastine, and estramustine) and a microtubule stabilizer (Taxol). As a complement to these studies we also analyzed the TCDD-dependent induction of CYP1A1 in elutriated subpopulations of myelomonocytic leukemia U937 cells representing distinct phases of the cell cycle. These latter studies circumvented any complications associated with chemical-induced cell cycle blockage. They also provided information on all phases of the cell cycle in cycling cells. Our studies demonstrate that AHR-dependent activation of CYP1A1 is cell cycle phase-dependent and is markedly suppressed in G₂/M cells.

	Materials and Methods

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Chemicals. TCDD was purchased from Chemsyn (Lenexa, KS). Colcemid, vinblastine sulfate, and protein A-Sepharose were obtained from Sigma (St. Louis, MO). Taxol was obtained as a gift from the Bristol-Myers Squibb Pharmaceutical Research Institute (Wallingford, CT). Estramustine was obtained from Kabi Pharmacia (Lund, Sweden). Cyclin B antibody was purchased from Invitrogen (Carlsbad, CA).

Cell Culture and Treatment. Wild-type murine Hepa 1c1c7 cells (obtained from Dr. J. Whitlock, Stanford University, Palo Alto, CA) were cultured in -minimal essential medium supplemented with 5% fetal bovine serum and 100 U/ml penicillin and 100 µg/ml streptomycin, and grown at 32°C in a humidified atmosphere containing 5% CO₂. Cultures were passaged by exposure to trypsin/EDTA.

Centrifugal Elutriation of U937 Cells. A Beckman Coulter JE-6B elutriation system (Beckman Coulter, Inc., Fullerton, CA) and rotor were used to enrich for populations of U937 cells in different phases of the cell cycle. In general, 1 × 10⁸ cells (1 × 10⁷ cells/ml suspended in RPMI 1640 + 1% fetal bovine serum) were loaded into the separation chamber (sterilized with 6% H₂O₂ and maintained at ambient temperature at a rotor speed of 2500 rpm) by using an entry flow rate of 15% of the maximum flow rate capacity. After loading the flow rate was increased by 5% increments until 70% of the maximum flow rate capacity was reached. Flow rates of 25, 32, 38, 44, 51, 57, 63.6, 70, 76.5, and 83 ml/min corresponded to maximum flow rate capacities of 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65%, respectively. Approximately 100 ml of flow through was collected at each step. For simplicity, cells recovered at a specific percentage of maximum flow rate capacity are referred to as that elutriation fraction (i.e., cells eluted at 25% of the maximum flow rate capacity constitute elutriation fraction 25). Debris and dead cells were removed in elutriation fractions 20 and 25. Elutriation fractions were pelleted by centrifugation and subsequently washed twice with PBS. Samples of washed cells were removed for isolation of RNA, analyses of DNA contents by FACs, and cell counting. Samples removed for RNA extraction were frozen in liquid nitrogen and stored at 80°C until the time of RNA isolation.

Flow Cytometry and Chromosome Staining. 1c1c7 cultures were harvested and processed for FACs analyses of DNA content as described by Reiners et al. (1999). Suspensions of U937 cells were pelleted by centrifugation, washed twice with PBS before being fixed, and subsequently processed like 1c1c7 cells. DNA analyses were made with a BD Biosciences FACScalibur instrument (BD Biosciences, San Jose, CA). Percentages of cells in the G₁, S, and G₂/M stages of the cell cycle were determined with a DNA histogram-fitting program (MODFIT; Verity Software, Topsham, ME). A minimum of 10⁴ events/sample was collected for subsequent analyses. Colcemid-treated cells were fixed and stained with aceto-orcein to detect chromosomes as described by Lopes et al. (1993).

RNA Preparation and CYP1A1 mRNA Detection by Northern Analyses. Total cellular RNA was isolated using either commercially available TRIzol reagent, or the acidic phenol extraction method of Chomczynski and Sacchi (1987). RNA was resolved on 1.2% agarose/formaldehyde gels and transferred to nitrocellulose membranes as described by Reiners et al. (1997). The probes used for the detection of 7S and murine and human CYP1A1 RNAs and the conditions used for hybridization have been described in detail (Schöller et al., 1994; Reiners et al., 1997).

Quantitative RT-PCR of CYP1A1 Heterogeneous Nuclear RNA. Total cellular RNA was isolated from 1c1c7 cultures by the method of Chirgwin et al. (1979). The RT-PCR primer sets and protocol used for the quantitative RT-PCR amplification and detection of murine CYP1A1 hnRNAs have been described in detail by Elferink and Reiners (1996). In brief, the protocol uses a CYP1A1 recombinant RNA (rcRNA) internal standard identical to the target hnRNA except for the addition of an engineered unique NcoI restriction site. This rcRNA standard contains Cyp1a1 genomic sequences from nucleotides 47 to +913 and includes all of Cyp1a1 exon 1 and 826 bp of intron 1. The same primers sets are used for RT and PCR amplification of cellular CYP1A1 hnRNA and the CYP1A1 rcRNA internal standard. Digestion of the internal standard PCR product with NcoI cleaves it into two fragments (318 and 350 bp) that can be resolved from the 658-bp cellular CYP1A1 hnRNA PCR product on nondenaturing 5% polyacrylamide gels.

Preparation of rcRNA Internal Standards for CYP1A1 and NMO1 RT-PCR. Total cellular RNA was isolated from 1c1c7 cells cultured in the presence of 1 nM TCDD for 1 h by the method of Chirgwin et al. (1979). Reverse transcription reactions were primed using gene-specific CYP1A1 and NMO1 RT primers (Table 1). The resulting cDNAs were subsequently used in PCR reactions to create Cyp1a1 and Nmo1 cDNA deletion constructs having internal deletions of 134 bp (nucleotides 1661-1794 of CYP1A1 mRNA) and 169 bp (nucleotides 469-637 of NMO1 mRNA), respectively. Specifically, forward PCR/reverse deletion PCR primers and RT/forward deletion primers were used in PCR reactions to create the 5' and 3' arms, respectively, of the deletion cDNA constructs (Table 1). The resulting 5' and 3' arms were annealed and cloned first into a PCRII vector (Invitrogen). The PCR product was removed using BamHI and EcoRV and then directionally cloned into the BamHI and SmaI sites of pKST₇/A₇₀, a plasmid containing a T7 RNA polymerase start site and a polyadenylated tail (Elferink and Reiners, 1996). After selection for ampicillin resistance, plasmids were subsequently sequenced. Linearized plasmids were used in in vitro transcription assays as described by Elferink and Reiners (1996) to produce rcRNAs. In vitro-generated rcRNAs were purified by oligo(dT)-Sepharose chromatography, quantitated spectrophotometrically, diluted, and stored at 80°C as single-use aliquots.

Competitive RT-PCR Assay. Total cellular RNA was isolated from treated 1c1c7 cultures by the method of Chirgwin et al. (1979). Reverse transcription assays contained 2 µg of total cellular RNA, and varying numbers of molecules of rcRNA, and were primed with a gene-specific RT primer (Table 1). After reverse transcription, reactions were diluted 1/10.5 in water, and used for PCR. Amplification with the forward and reverse PCR primers listed in Table 1 yielded a 799-bp product for wild-type CYP1A1 mRNA (nucleotides 1325-2129), a 671-bp PCR product for the internal standard CYP1A1 rcRNA, a 799-bp product for wild-type NMO1 mRNA (nucleotides 115-913), and a 630-bp PCR product for the internal standard NMO1 rcRNA. For PCR amplification cDNAs were heated to 95°C for 2 min and cycled 17 to 25 times (for Cyp1a1 cDNAs) or 27 to 35 times (for Nmo1 cDNAs) at 95°C for 30 s, 61°C for 1 min, and 72°C for 2 min. After the final cycle an extension at 72°C for 10 min was performed. PCR products were labeled by the inclusion of [-³²P]dCTP in the synthesis reactions, separated on 3.5% acrylamide gels, and visualized/quantitated with a Bio-Rad GS-525 molecular imager (Bio-Rad, Hercules, CA) and Molecular Dynamics (Sunnyvale, CA) software.

Orotic Acid Incorporation. Two days after plating 1c1c7 cultures were treated with 0.2 µM Colcemid. Approximately 21 h later nontreated and Colcemid-treated cultures were washed two times with PBS and refed with -minimal essential medium containing 5% fetal bovine serum and 1 µCi/ml [¹⁴C]orotic acid. After a 90-min incubation at 32°C the cultures were processed for estimation of the amount of orotic acid incorporated into RNA. The procedure used for culture fixation and extraction has been described in detail by Schöller et al. (1994). A second set of dishes was treated with trypsin/EDTA to estimate cell numbers. [¹⁴C]Orotic acid incorporation is expressed as dpm/10³ cells.

Immunofluorescent Detection of AHR and ARNT. The procedures used for immunofluorescent detection of AHR and ARNT in 1c1c7 cultures have been described in detail (Jones and Reiners, 1997). The antibodies used in these studies were rabbit polyclonal, affinity-purified antibodies made to amino acids 61 to 419 of the murine AHR, and amino acids 318 to 773 of the human ARNT (both antibodies were the generous gift of Dr. A. Poland, NIOSH, Morgantown, WV). Fluorescence was observed using a Nikon Optiphot microscope (Nikon, Tokyo, Japan) equipped with an oil emersion lens and recorded on Kodak TMAX 100 film (Eastman Kodak, Rochester, NY).

Western Blot Analyses. Cytosolic extracts were prepared as described by Pollenz et al. (1994) with the exception that the 2× lysis buffer also contained 2 µg/ml leupeptin and 2 µg/ml aprotinin. Cytosolic and nuclear proteins were subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrophoretically transferred from the gel to nitrocellulose. The procedure described by Schöller et al. (1994), with the substitution of a 2-h incubation with primary antibody, was used for the immunological detection of AHR and ARNT. The primary antibodies used in these studies are described above. Antigen-immunoglobulin conjugates were detected with an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ) and recorded on X-ray film.

p34^cdc2(Cyclin B/cdc2) Kinase Assay. 1c1c7 cells were washed with cold PBS before the addition of lysis buffer [10 mM Tris, pH 7.5, 130 mM NaCl, 1% Triton X-100, 10 mM NaF, 10 mM NaP, 10 mM NaPP_i, 1 mM NaVO₄, and 1/10 (v/v) of a 10× solution of protease inhibitor cocktail (product P2714; Sigma)]. After 5 min on ice the lysate was collected and centrifuged for 15 min at 11,000g. A volume of supernatant fluid corresponding to 30 µg of protein was incubated on ice with 3 µg of a cyclin B antibody in a total volume of 30 µl for 90 min. The volume was adjusted to 50 µl with lysis buffer to which was added 20 µl of protein A-Sepharose. After a 30-min incubation on ice the protein A-Sepharose was washed three times with lysis buffer and pelleted by centrifugation. Histone kinase activity in the pellets was assayed as described by Kung et al. (1990). Phosphorylated histone H1 was detected by autoradiography after separation of proteins in the kinase assay supernatants on 12% SDS polyacrylamide gels.

Statistical Analyses. Data were analyzed by the Tukey's Honestly Significant Difference test. The Statistica 5.0 software package (StatSoft, Tulsa, OK) was used to perform these calculations. Differences were consider statistically significant if P < 0.05.

	Results

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Induction of G₂/M Arrest by Microtubule Disrupters and Stabilizers. 1c1c7 cultures were treated with varying concentrations of microtubule disrupters/stabilizers to identify conditions sufficient to cause a G₂/M arrest in the absence of cytotoxicity. The panels in the top two rows of Fig. 1 demonstrate that concentrations of Colcemid, vinblastine, and Taxol existed that were capable of suppressing 1c1c7 proliferation in the absence of overt cytotoxicity. The doubling time of 1c1c7 cells grown at 32°C is ~23 h. Because the cultures were harvested 20 to 23 h after the addition of mitotic blocker the 50% value for control cell content noted in the first row of panels represents a total suppression of proliferation.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Effects of microtubule disrupters/stabilizers on 1c1c7 proliferation, viability, and cell cycle progression. Cultures were treated with DMSO or microtubule disrupters/stabilizers for 20 to 23 h prior to harvesting for analyses of cell number, morphology, viability, and DNA content. Cell number and viability data represent the means ± S.D. of four to eight plates (two experiments). Cell cycle data represent the means ± S.D. of three plates. , total cells; , cells with round morphology; and , cells with flat morphology.

Suppression of Cyp1a1 and Nmo1 Induction in G₂/M Arrested Cultures. Treatment of 1c1c7 cultures with mitotic blockers for ~20 h before the addition of TCDD resulted in concentration-dependent reductions in induced, steady-state CYP1A1 mRNA contents (Fig. 2). In general, reductions were seen at concentrations sufficient to induce G₂/M arrest (compare Figs. 2 and 1, bottom row). It should be emphasized that the effects of Colcemid and Taxol on CYP1A1 mRNA contents were only seen when the cells were allowed sufficient time to arrest in G₂/M before the addition of TCDD. Specifically, analyses of CYP1A1 mRNA contents in cultures treated with TCDD at various times after the addition of 0.2 µM Colcemid demonstrated an inverse relationship between the percentage of cells in G₂/M and induced, steady-state CYP1A1 mRNA content (J. J. Reiners, unpublished data).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   TCDD-induced steady-state CYP1A1 mRNA contents in G2/M arrested 1c1c7 cultures. Cultures were pretreated with DMSO or varied concentrations of the microtubule disrupters/stabilizers for 20 to 21 h prior to the addition of TCDD (1 nM). Total RNA was isolated 5 h after the addition of inducer or solvent from cells having normal, flat (F), or rounded (R) morphology for analyses of CYP1A1 or 7S RNAs.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Quantitative RT-PCR analyses of CYP1A1 and NMO1 mRNAs in Colcemid-arrested TCDD-treated 1c1c7 cultures. Cultures were pretreated with 0.2 µM Colcemid for 22 h prior to the addition of TCDD (1 nM). Total RNA was isolated 5 h later from nonfractionated cultures and Colcemid-treated cells having the flat or rounded morphology. Parallel control cultures were treated with DMSO. A, representative of the type of data obtained in the quantitative RT-PCR assay. The triangles represent increasing ratios of the standard (rcRNA) to target mRNA molecules used in the RT reaction. The actual number of competing rcRNA molecules differed between treatment groups and RNA species. B and C, relative amounts of cellular CYP1A1 and NMO1 mRNAs, respectively, determined by quantitative RT-PCR analyses in three independent experiments. The data represent means ± S.D. Treatment groups were no treatment control (I), TCDD-treated (II), Colcemid + TCDD-treated nonfractionated cells (III), Colcemid + TCDD-treated flat cells (IV), and Colcemid + TCDD-treated round cells (V). *, significantly less than TCDD-treated controls, P < 0.05.

Analyses of CYP1A1 Transcription in Colcemid-Arrested Cultures. Nuclear run-on assays are generally used to examine the transcriptional status of a gene. However, it is impossible to isolate nuclei from prophase and pre-/metaphase cells because they lack a nuclear envelope. We recently developed and validated a quantitative RT-PCR assay as a surrogate for nuclear run-on assays that uses whole cell RNA preparations (Elferink and Reiners, 1996). This assay amplifies CYP1A1 hnRNA corresponding to the first exon and intron of the Cyp1a1 gene and includes in the RT and PCR portions of the assay an rcRNA internal standard. This internal standard is identical to the target hnRNA except for the addition of an engineered unique NcoI restriction site. Digestion of the PCR products with NcoI cleaves the internal standard PCR product into two fragments (318 and 350 bp) that can be resolved from the cellular CYP1A1 hnRNA PCR product (658 bp).

View larger version (57K): [in this window] [in a new window]   Fig. 4.   Quantitative RT-PCR analyses of CYP1A1 hnRNAs in Colcemid-arrested TCDD-treated 1c1c7 cultures. Cultures were pretreated with 0.2 µM Colcemid for 22 h prior to the addition of DMSO or TCDD (1 nM). Total RNA was isolated 1 h later from nonfractionated cultures and Colcemid-treated cells having the flat (F) or rounded (R) morphology. Parallel control cultures were treated with DMSO. Bottom, representative of the type of data obtained in the assay. Top, analysis of the depicted gel in which signals have been normalized to the internal standard rcDNA-derived PCR product.

Effects of Colcemid on Ligand-Induced AHR Translocation. Transcriptional activation of Cyp1a1 or Nmo1 by TCDD involves its binding to the AHR and the subsequent translocation of the AHR to the nucleus. These two steps can be qualitatively assessed by indirect fluorescent monitoring of AHR translocation after exposure to TCDD (Pollenz et al., 1994; Reiners et al., 1997). The panels in the top and bottom rows of Fig. 5 demonstrate that both ARNT and AHR, respectively, are expressed in asynchronous and Colcemid-arrested 1c1c7 cultures. ARNT was nuclear in both asynchronous and Colcemid-arrested cultures (Fig. 5, A and C, respectively), and its location was not affected by TCDD treatment (Fig. 5, B and D). In contrast, the AHR was predominately cytoplasmic in asynchronous and Colcemid-arrested cultures (Fig. 5, E and G, respectively) but translocated to the nucleus after the addition of TCDD (Fig. 5, F and H). It should be noted that our interpretations are restricted to those Colcemid-treated cells having the flat phenotype (and primarily in G₂). The rounded cells (examples are highlighted by arrows) represent M phase cells and lack a nuclear envelope.

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Cellular distribution of AHR and ARNT in normal and Colcemid-arrested 1c1c7 cells. Cells grown on glass coverslips were treated for 20 h with DMSO or Colcemid (0.2 µM) prior to exposure to DMSO or 2 nM TCDD for 90 min. After fixation the cultures were first incubated with rabbit anti-mouse ARNT (A-D) or AHR (E-H) antibody and then goat anti-rabbit tetramethylrhodamine B isothiocyanate antibody. The immune complexes were visualized by indirect fluorescence microscopy. Treatments and antibodies used are DMSO, ARNT (A); TCDD, ARNT (B); Colcemid + DMSO, ARNT (C); Colcemid + TCDD, ARNT (D); DMSO, AHR (E); TCDD, AHR (F); Colcemid + DMSO, AHR (G); and Colcemid + TCDD, AHR (G). The arrowheads in D and G indicate cells having the round morphology. Scale bar, 20 µm.

Effects of Colcemid on Orotic Acid Incorporation. It is conceivable that the reduced CYP1A1 and NMO1 mRNA contents seen in TCDD-treated Colcemid-arrested 1c1c7 cultures reflect a general suppression of transcription in G₂/M phase cells. To test this possibility, control and Colcemid-arrested cultures were pulsed with the RNA precursor [¹⁴C]orotic acid and monitored for incorporation into acid-insoluble material. Figure 6 demonstrates that Colcemid-treated cultures incorporated ~50% of the amount measured in solvent-treated control cultures. However, Colcemid-derived flat and round cells differed markedly in their abilities to incorporate [¹⁴C]orotic acid. Specifically, the round cells showed very poor [¹⁴C]orotic acid incorporation, whereas, the flat G₂ phase cells incorporated more [¹⁴C]orotic acid than asynchronous control cultures.

View larger version (44K): [in this window] [in a new window]   Fig. 6.   Effects of Colcemid on orotic acid incorporation. Cultures of 1c1c7 cells were treated with DMSO or 0.2 µM Colcemid for ~21 h prior to addition of [14C]orotic acid. After a 90-min incubation at 32°C the cultures were processed to determine orotic acid incorporation into RNA in cells having the flat (F) and round (R) morphology. Parallel cultures were processed for determination of cell numbers. Data represent means ± S.D. of analyses performed on three to six plates. The F + R "reconstituted" group represents an estimate made by adding the specific activities of the flat and round populations after correcting for the relative proportions of each cell type in the Colcemid-treated cultures. *, significantly less than DMSO and Colcemid (F) groups, P < 0.01. **, significantly greater than all other groups, P < 0.01.

Responsiveness to TCDD after Release from G₂/M Arrest. The replating of asynchronous control cultures did not affect the relative distribution of cells in the various phases of the cell cycle (Fig. 7A, left). Colcemid-arrested 1c1c7 cultures resumed progression through the cell cycle within 12 h of replating (Fig. 7A, right). This reentry was accompanied by the loss of cyclin B-dependent kinase activity, a marker for G₂/M phase cells (monitored by assessing histone H1 phosphorylation in an in situ kinase assay; Fig. 7B). The replating of Colcemid-arrested cultures did not result in tightly synchronized cultures. However, relative to replated asynchronous cultures, Colcemid-derived cultures were enriched in S phase cells 24 h after replating (compare panels in Fig. 7A).

View larger version (43K): [in this window] [in a new window]   Fig. 7.   Responsiveness to TCDD after release from G2/M arrest. Cultures of 1c1c7 cells were treated with DMSO or 0.2 µM Colcemid for 20 h before being exposed to 1 nM TCDD, or replated in fresh medium lacking Colcemid. Zero time in the figure corresponds to when the cultures were pulled for replating. Cultures were analyzed with respect to DNA contents of replated DMSO-treated (left) and replated Colcemid-arrested (right) cultures (A), histone H1 kinase activity and AHR and ARNT protein content (B), and CYP1A1 mRNA content after a 5-h exposure to 1 nM TCDD (C).

TCDD Induction of CYP1A1 in U937 Cells. As a complement to the chemical synchronization studies, we attempted to use centrifugal elutriation to enrich for phase-specific populations of 1c1c7 cells. However, we were unable to reproducibly obtain fractions highly enriched in distinct phases of the cell cycle. For that reason we turned our attention to U937 cells, a monocytoid cell line that expresses the AHR (Hayashi et al., 1995), which can be separated into phase-specific populations by centrifugal elutriation (McCabe et al., 2000). CYP1A1 mRNA was not detected in nontreated or DMSO-treated asynchronous U937 cultures (Fig. 8, first three lanes and last lane). In contrast, CYP1A1 mRNA was detected by Northern blot analysis within 3 h of treatment of cultures with TCDD (Fig. 8). Thereafter, steady-state CYP1A1 mRNA contents continued to increase for minimally an additional 15 h. The lowest and highest concentrations of TCDD tested (2 and 10 nM) were very similar in their abilities to stimulate the accumulation of CYP1A1 mRNA at each of the time points investigated. The 2 nM concentration of TCDD was used in subsequent studies.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   TCDD-mediated CYP1A1 induction in U937 cells. Log-phase cultures of U937 cells were treated for 3, 6, or 18 h with 2, 5, or 10 nM TCDD before being harvested for isolation of RNA and subsequent analyses of CYP1A1 and 7S RNAs. Parallel cultures were treated with DMSO and harvested 3, 6, and 18 h after treatment. Analyses were performed on 10 µg of RNA.

Centrifugal Elutriation of U937 Cells. TCDD is cytostatic to some cell lines (Vogel and Abel, 1995; Weiss et al., 1996; Wang et al., 1997). Exposure to 2 nM TCDD did not affect the proliferation of U937 cells (Fig. 9A). Figure 9B demonstrates that centrifugal elutriation can be used to prepare enriched populations of U937 cells in all phases of the cell cycle after short-term exposure to TCDD. Specifically, highly enriched preparations of G₁ phase (elutriation fractions 30 and 35), S phase (elutriation fraction 40), and G₂/M phase cells (elutriation fractions 50 and 55 + 60) could be reproducibly isolated. In general, the sum of the cells in the various elutriation fractions equaled 95 ± 3% of the initial, preseparated population (R. P. Santini and J. J. Reiners, unpublished data). Furthermore, the sum of the cells in phase-specific fractions, when normalized to the total number of cells recovered after elutriation, yielded cell cycle distributions identical to that measured in the starting population. For example, elutriation fractions 30 and 35 collectively represented ~55% of the elutriated population and were almost exclusively G₁ cells (Fig. 9, B and C). The pre-elutriation population was ~50% G₁ cells. Similarly, elutriation fractions 50 + 55 + 60 represented ~9% of the total elutriated population and were ~70% G₂/M cells. The starting pre-elutriation population was ~10% G₂/M cells.

View larger version (28K): [in this window] [in a new window]   Fig. 9.   Effects of TCDD on U937 cell cycle progression. Asynchronous cultures of U937 cells were treated with 2 nM TCDD or DMSO. A, cultures were harvested at various times after the addition of TCDD () or DMSO () for estimation of cell numbers. Data represent means ± S.D. of three culture dishes. B, cultures were harvested ~3 h after DMSO or TCDD treatment for analyses of cell cycle stage by flow cytometry (first two columns). The remaining columns in B represent flow cytometry analyses of elutriated fractions of cells harvested ~3 h after TCDD treatment. C, percentage of the total elutriated population in individual elutriation fractions. The data in B and C represent means ± S.D. of three experiments.

CYP1A1 mRNA Contents in TCDD-Treated and Elutriated U937 Cells. We reasoned that analyses of elutriated cells harvested within 3 h of TCDD treatment could provide a relative estimate of the cell cycle responsiveness of CYP1A1 to TCDD. CYP1A1 mRNAs were detected in all elutriation fractions prepared from cultures harvested 3 h after TCDD treatment (Fig. 10A). However, there were marked differences among the fractions in their CYP1A1 mRNA contents. When normalized to 7S RNA contents, the highest CYP1A1 cellular content was noted in elutriation fraction 35. The lowest contents were noted in elutriation fraction 50 (Fig. 10B, top, and three additional experiments). The maximum difference between elutriation fractions in Fig. 10 was ~3-fold. In a single experiment we obtained sufficient cells in elutriation fractions 55 + 60 to isolate amounts of mRNA sufficient for Northern blot analyses. The CYP1A1 mRNA content in this pooled fraction was ~52% of the CYP1A1 mRNA content of nonseparated TCDD-treated cultures (R. P. Santini and J. J. Reiners, unpublished data).

View larger version (41K): [in this window] [in a new window]   Fig. 10.   CYP1A1 mRNA content of TCDD-treated U937 cells separated by elutriation. A, U937 cells were treated with 2 nM TCDD or DMSO for 3 h before being harvested for subsequent isolation of RNAs for analyses of CYP1A1 and 7S RNAs. RNA was also isolated from the elutriated fractions of TCDD-treated cultures. Northern blots in A represent signals obtained on 10 µg of RNA. B, intensities of CYP1A1 and 7S mRNA signals depicted in A, and three additional independent experiments were measured to establish signal strength ratios. The ratio for nonseparated, TCDD-treated cells was set as 1. Data represent means ± S.D. of four experiments. *, significantly greater than all other groups, P < 0.01; **, significantly different from all other groups except elutriation fraction 45, P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The current investigation was designed to determine whether the TCDD-mediated induction of CYP1A1 is a cell cycle-regulated process, and specifically suppressed in G₂/M. As an approach we used four chemicals to arrest 1c1c7 cultures in G₂/M by two different mechanisms (i.e., microtubule disruption or stabilization). As a complement to these studies we also used a model system that did not use chemical synchronization. Specifically, centrifugal elutriation was used to separate TCDD-treated asynchronous cultures of U937 cells into highly enriched populations of G₁, S, and G₂/M phase cells. G₂/M cells prepared by this later procedure contained 52 ± 6% of the CYP1A1 mRNA content of the TCDD-treated asynchronous starting population. This value was similar to that measured in 1c1c7 cultures arrested with the minimum concentration of Taxol required to achieve optimal G₂/M arrest. All of the microtubule disrupters also suppressed the TCDD-dependent activation of CYP1A1 at concentrations that caused G₂/M arrest. However, CYP1A1 mRNA contents continued to decrease with increasing concentrations of Colcemid, estramustine, and vinblastine. This continued decrease should not have occurred if the effects of the three microtubule disrupters only reflected their abilities to cause G₂/M arrest. Furthermore, a concentration of vinblastine was identified that suppressed the TCDD-induced accumulation of CYP1A1 mRNA in non-G₂/M phase cells. These studies emphasize both the caveats associated with chemical synchronization and the need for complementary procedures in the study of cell cycle-regulated processes. Nevertheless, both approaches demonstrated that G₂/M phase cells have lower steady-state CYP1A1 mRNA contents after TCDD treatment than similarly treated asynchronous cultures.

Treatment of 1c1c7 cultures with microtubule disrupters and stabilizers lead to a tetraploid population having either a flat or rounded morphology. Staining with aceto-orcein revealed that the flat and rounded cells represented predominantly G₂ and mitotic cells, respectively. Analyses of the separated cell types demonstrated that the TCDD-dependent induction of Cyp1a1, as well as Nmo1, was suppressed in both cell types. The Nmo1 data are significant because this gene is also a member of the Ah battery, and transcriptionally activated by TCDD via an AHR-dependent process (Nebert, 1994). To date, we know of only one other example in which the activities of a ligand-activated transcription factor are down-regulated in G₂ phase cells. Specifically, several glucocorticoid-responsive genes cannot be induced in the G₂ phase of the cell cycle (Martin et al., 1969; Fanger et al., 1986; Hsu et al., 1992). This suppression has been shown to correlate with an inhibition of glucocorticoid receptor nuclear accumulation, and site-specific alterations in glucocorticoid receptor phosphorylation (Hsu et al., 1992; Bodwell et al., 1998).

It has been known for some 40 years that transcription is suppressed in mitotic cells (Taylor, 1960; Prescott and Bender, 1962; Hartl et al., 1993). Analyses of orotic acid incorporation into RNA demonstrated that overall RNA synthesis was strongly suppressed in mitotic 1c1c7 cultures generated by Colcemid exposure. Hence, it is conceivable that the suppressed responses we detected in M phase cultures may reflect a general suppression of transcription and are AHR-independent. However, the same explanation may not account for the suppression seen in the Colcemid-derived G₂ population. Orotic acid incorporation in these latter cells was comparable with, if not greater than that measured in asynchronous 1c1c7 cultures. Although the identities of the RNAs synthesized in G₂ phase cells are not known, we assume that cyclin B and p21Cip1 mRNAs are in this population because their basal transcription is cell cycle-dependent and occurs in G₂ (Li et al., 1994; Dulic et al., 1998).

The transcriptional activation of Cyp1a1 and Nmo1 by TCDD is mediated by the AHR, in conjunction with ARNT. The suppressed inductions of both genes in G₂/M 1c1c7 cultures did not correlate with reductions in either AHR or ARNT, as monitored by Western blot and indirect immunofluorescence analyses. It is also unlikely that ligand binding or AHR transformation is affected in G₂ cells. This conclusion is based upon the observed translocation of the AHR to the nucleus after TCDD exposure. AHR translocation requires both ligand binding and receptor transformation. In a previous study we demonstrated by EMSA that AHR/ARNT binding to a radiolabeled oligonucleotide containing a dioxin-responsive element was markedly suppressed in nuclear extracts obtained from nocodazole-derived G₂/M cultures (Schöller et al., 1994). However, recent studies suggest that the procedure used for the isolation of nuclei in these studies also enriched for particles that looked like nuclei, contained DNA, but lacked a true nuclear envelope and had reduced levels of ARNT (J. J. Reiners, unpublished data). It is likely that the EMSA assay used in these studies underestimated AHR/DNA binding. Hence, it is conceivable that the G₂ effects seen in the current study could reflect suppressions of AHR/ARNT dimerization, DNA binding, or the transactivating activity of DNA bound AHR/ARNT complex.

Analyses of elutriated U937 cells and replated Colcemid-arrested 1c1c7 cultures demonstrate that TCDD is capable of inducing CYP1A1 throughout the cell cycle. However, the elutriation studies also show that cells in different phases of the cell cycle do not respond identically to TCDD. Specifically, relative to the CYP1A1/7S mRNA ratio determined for TCDD-treated asynchronous cultures (set as 1), ratios of 1.41 ± 0.09 (range 1.31-1.68) and 0.52 ± 0.06 (range 0.46-0.63) occurred in elutriation fractions 35 and 50, respectively. If one accepts that the elutriation fractions depicted in Fig. 9B represent from left to right the progression of cells from G₁ to M, our data demonstrate that late G₁ cells accumulate ~3-fold more CYP1A1 mRNA than do G₂/M cells. It is intriguing that the extremes in the CYP1A1/7S ratios correspond approximately to the sites of the two major checkpoints of the cell cycle. It is conceivable that agents and conditions that lead to G₁ and G₂ arrest may influence the biological activities of TCDD and other AHR ligands.

The fluctuating responsiveness of CYP1A1 to TCDD accompanying cell cycle progression may reflect pRb coactivation of the AHR. Recent studies with rat hepatoma 5L/BP8 cells demonstrate that the AHR interacts directly with pRb, but only the hypophosphorylated form (Ge and Elferink, 1998; Elferink et al., 2001). This interaction appears necessary for maximal AHR-mediated CYP1A1 expression, consistent with pRb acting as an AHR coactivator (Elferink et al., 2001). Because hypophosphorylated pRb is restricted to the G_o and G₁ phases, AHR-mediated transactivation of the CYP1A1 gene would be predicted to be cell cycle-dependent, with maximal induction detected in G_o and G₁ phase cells. Our current data are entirely consistent with this prediction.

TCDD is cytostatic to a variety of cell types. It suppresses the proliferation of several human breast cell lines, including MCF-7, T47D, and MDA-MD-486 (Fernandez and Safe, 1992; Vogel and Abel, 1995; Wang et al., 1997, 1998). It also inhibits the proliferation of rat hepatoma 5L cells (Weiss et al., 1996; Elferink et al., 2001) and sparsely seeded primary rat hepatocytes in response to epidermal growth factor (Schrenk et al., 1994). In the former case the effects of TCDD have been clearly shown to be mediated by its interaction with the AHR (Weiss et al., 1996). In contrast, we previously reported that TCDD is not cytostatic to 1c1c7 cells (Reiners and Clift, 1999; Reiners et al., 1999), and in the current study demonstrate that it has no effects on the proliferation of U937 cells. In addition, we have observed that TCDD is not cytostatic to the human breast epithelial cell line MCF10A at concentrations sufficient to transcriptionally activate members of the Ah battery (J. J. Reiners, unpublished data). Hence, TCDD-dependent transformation of the AHR does not ensure the induction of a cytostatic process. The basis for the differential responses of varied cell types to the cytostatic effects of TCDD is unknown. However, it may be related to the loss of components involved in checkpoint control in the nonresponsive lines. For example, U937 cells are p53 null (Dou and Lui, 1995; Dou et al., 1995). The MCF10A line is null for p15 and p16 (Brenner and Aldaz, 1995). Lastly, preliminary studies indicate that the 1c1c7 line does not express pRb (J. J. Reiners and A. Joiakim, unpublished data).

In summary, these are the first studies that we know of to demonstrate that a microtubule stabilizer and a series of microtubule disrupters suppress the activation of members of the Ah battery by an AHR ligand. These are also the first studies that we know of to suggest that responsiveness to TCDD may differ at the sites of the two major checkpoints of the cell cycle.