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Monoclonal Antibodies That Inhibit the Transport Function of the 190-kDa Multidrug Resistance Protein, MRP

Multidrug resistance in tumor cells is often accompanied by overexpression of multidrug resistance protein (MRP), a 190-kDa transmembrane protein that belongs to the ATP-binding cassette superfamily of transport proteins. MRP mediates ATP-dependent transport of a variety of conjugated organic anions and can also transport several unmodified xenobiotics in a glutathione-dependent manner. To facilitate structure-function studies of MRP, we have generated a panel of MRP-specific monoclonal antibodies (mAbs). Four of these mAbs, QCRL-2, -3, -4, and -6, bind intracellular conformation-dependent epitopes, and we have shown that they can inhibit the transport of several MRP substrates. Binding competition and immunoprecipitation assays indicated that mAbs QCRL-4 and -6 probably recognize the same detergent-sensitive epitope in MRP, whereas mAbs QCRL-2, -3, and -4 each bind distinct, non-overlapping epitopes. Fab fragments inhibit transport as effectively as the intact mAbs, suggesting that inhibition results from direct interactions of the mAbs with MRP. Immunodot blot and immunoprecipitation analyses revealed that the minimal regions of MRP sufficient for full reactivity of mAbs QCRL-2 and -3 are amino acids 617–858 and 617–932, respectively, which encompass the NH2-proximal nucleotide-binding domain (NBD). In contrast, the epitope bound by mAb QCRL-4 localized to amino acids 1294–1531, a region that contains the COOH-proximal NBD. However, none of the mAbs inhibited photolabeling of intact MRP with 8-azido-[α-32P]ATP. This suggests that rather than preventing nucleotide binding, the mAbs inhibit transport by interfering with substrate binding or by trapping MRP in a conformation that does not allow transport to occur. Our results also demonstrate for the first time that the NBDs of MRP can be expressed as soluble polypeptides that retain a native conformation.

The development of resistance to multiple natural product and semi-synthetic drugs commonly used in cancer chemotherapy, termed multidrug resistance, is a serious obstacle to successful treatment. This phenomenon has been extensively studied in vitro using tumor cell lines selected for resistance by chronic or intermittent exposure to drugs. The multidrug resistance observed in these cell lines is most commonly accompanied by overexpression of one or both of the integral membrane transporters, MRP 1 and P-glycoprotein (1)(2)(3). The 190-kDa MRP and 170-kDa P-glycoprotein are members of the ATP-binding cassette superfamily of transport proteins, which in eukaryotes are typically composed of one or two highly conserved nucleotide-binding domains (NBDs) and one to three membrane-spanning domains (MSDs) (4). The NBDs of ATPbinding cassette transporters are characterized by the presence of three short sequences, the so-called Walker A and B motifs and the transport family signature motif (3)(4)(5)(6). Outside of their two NBDs, however, MRP and P-glycoprotein show relatively little sequence homology, and it is now clear that there are significant structural and functional differences between these two proteins (7)(8)(9)(10)(11).
Transfection studies have confirmed that overexpression of either MRP or P-glycoprotein is sufficient to confer resistance to a similar, but not identical, spectrum of drugs (1,(12)(13)(14)(15). A substantial body of experimental evidence indicates that Pglycoprotein confers multidrug resistance by binding and transporting unmodified chemotherapeutic drugs and other compounds out of cells (1,2). In contrast, direct binding of drugs to which it confers resistance has not been demonstrated for MRP (3, 16 -19). However, MRP has been shown to be a primary active transporter of amphiphilic conjugated organic anions. Established substrates of MRP include a number of GSH-, glucuronide-, and sulfate-conjugated compounds, with the cysteinyl leukotriene LTC 4 and the GSH conjugate of the potent carcinogen aflatoxin B 1 being the highest affinity substrates identified to date (17,18,20,21). In addition to conjugated anions, MRP has been shown to mediate ATP-dependent transport of some unmodified compounds, such as aflatoxin B 1 and the anticancer drugs vincristine and daunorubicin, but only in a GSH-stimulatable manner (16,19,(21)(22)(23). Recently, we have shown that the GSH-stimulated transport of unmodified vincristine by MRP-enriched membrane vesicles involves cotransport of GSH (19). It may be that MRP confers resistance to at least some classes of xenobiotics in intact cells by such a cotransport mechanism.
We previously reported the isolation and initial characterization of a panel of five MRP-specific mAbs (QCRL-1, -2, -3, -4, and -6) (24). These mAbs were raised against crude membranes prepared from multidrug-resistant H69AR small cell lung cancer cells, which express very high levels of MRP. All five mAbs reacted only with permeabilized cells in immunocytochemical analyses, indicating that they recognize intracellular epitopes (24). The linear epitope bound by one of the mAbs, QCRL-1, was subsequently mapped to amino acids 918 -924 in the cytoplasmic region connecting the two halves of MRP (25). This knowledge has facilitated the use of mAb QCRL-1 for a number of purposes, including analysis of MRP expression in clinical tumor specimens (26 -31), structure-function studies (10,25,32,33), and immunoaffinity purification of MRP. 2 In contrast to mAb QCRL-1, the remaining four mAbs recognize conformation-dependent epitopes in the MRP molecule (24). mAbs QCRL-2, -3, and -4 also have the ability to inhibit the transport of several conjugated substrates (including LTC 4 , 17␤-estradiol 17-(␤-D-glucuronide), and aflatoxin B 1 -glutathionyl aflatoxin B 1 ) into inside-out MRP-enriched plasma membrane vesicles (16,21,23,34). Similarly, GSH-stimulatable transport of unmodified vincristine and aflatoxin B 1 is inhibited by these mAbs (19,21,23). These results suggest that the conformation-dependent epitopes recognized by mAbs QCRL-2, -3, and -4 are localized in or near functionally important regions of MRP. In this study, we show that mAbs QCRL-2, -3, and -4 recognize distinct, non-overlapping epitopes of MRP and that their ability to inhibit MRP-dependent transport results from direct interactions with the NBDs of the protein that do not prevent the binding of ATP.

MATERIALS AND METHODS
Cell Lines and Antibodies-The MRP-overexpressing multidrug-resistant H69AR small cell lung cancer cell line has been described previously (7,35). T5 cells are HeLa cells that have been transfected with a full-length MRP cDNA expression vector, pRc/CMV-MRP1, and C1 cells are HeLa cells transfected with empty vector (13,14). mAbs QCRL-1, -2, -3, -4, and -6 are murine mAbs raised against cell membranes from MRP-overexpressing H69AR cells (24). The mAbs were used in the form of crude or DEAE Blue-purified ascites fluid (26), were obtained as purified preparations from Centocor (Malvern, PA), or were purified using GammaBind Plus Sepharose resin (Amersham Pharmacia Biotech). Fab fragments of mAbs QCRL-1, -2, -3, and -4 were prepared using the ImmunoPure Fab kit (Pierce). MRP-1 and -2 are polyclonal antisera raised against homologous 15-amino acid peptides from the first and second NBDs of MRP (amino acids 765-779 and 1427-1441, respectively) (24,25,36). The rat mAb MRPr1 and the mouse mAbs MRPm5 and MRPm6 were kindly provided by Dr. R. J. Scheper (Free University Hospital, Amsterdam) (37,38). mAbs MRPr1 and MRPm6 recognize linear epitopes between MRP amino acids 238 -247 and 1511-1520, respectively (39), whereas the epitope for mAb MRPm5 is located between MRP amino acids 986 -1096. 3 Immunoprecipitations-For immunoprecipitation of MRP, membrane-enriched fractions were prepared from H69AR cells that had been metabolically labeled with [ 35 S]methionine (24). Membrane proteins were solubilized in PBS containing 1% CHAPS at detergent/ protein ratios ranging from 1:1 to 10:1 (w/w) for 3 h at 4°C. After centrifugation at 100,000 ϫ g, 1 l of crude ascites fluid was mixed with 22.5 g of solubilized membrane protein and incubated overnight at 4°C. For immunoprecipitation of polypeptides MRP-(617-932) and MRP-(1294 -1531) (prepared as described below), ϳ4 g of total cytosolic protein from infected Sf21 cells was diluted to 100 l with PBS, and 1 l of ascites (mAb QCRL-1, -2, -3, or -4) or 5 l of MRP-2 antiserum was added. After incubation for 2 h at 4°C, immune complexes were recovered by the addition of 30 l of 25% GammaBind Plus Protein G-Sepharose (Amersham Pharmacia Biotech) in PBS. After incubation for 1 h at room temperature, the beads were washed four times with PBS, and precipitated proteins were eluted with Laemmli buffer and analyzed by SDS-PAGE and fluorography. 125 I Labeling of mAbs-Sixty g each of DEAE Blue-purified mAbs QCRL-1, -2, -3, -4, and -6 were passed over an Econo-Pac 10DG column (Bio-Rad) and eluted in PBS. Protein-containing fractions were pooled, and the volume was brought up to 850 l with PBS. IODO-BEADS (Pierce) were washed with PBS and dried on filter paper. For each mAb, one IODO-BEAD was added to 150 l of PBS containing 400 Ci of 125 I (17 Ci/mg of iodine; ICN, Montreal, Quebec, Canada) and incubated for 5 min. The purified mAb was then added, and labeling was allowed to proceed for 15 min. To separate 125 I-labeled mAb from free 125 I, the reaction mixture was loaded onto an Econo-Pac 10DG column, the mAbs were eluted with PBS, and radioactive fractions were pooled.
Dot Blot Assay of Direct Binding Competition between Unlabeled and 125 I-Labeled mAbs-To quantitate the relative amount of mAb in DEAE blue-purified mAb samples and crude ascites fluid, the purified samples were separated by SDS-PAGE together with serial dilutions of the corresponding ascites fluid. The amount of IgG heavy chain protein in each was assessed by densitometric analysis of the Coomassie-stained gels on a Molecular Dynamics computing densitometer using Image-Quant software. Dot blots were prepared on Immobilon P membrane in a 96-well manifold by blotting 4 g of H69AR crude membranes in 50 l of TBS/well and draining slowly by gravity (see below). After washing with TBS containing Tween 20 (TBS-T), 100 l of blocking solution (10% calf serum and 1% bovine serum albumin in TBS-T) per well was added and allowed to drain slowly by gravity. Approximately 30 ng of each 125 I-labeled mAb was mixed with QCRL-1, -2, -3, -4, and -6 crude ascites or with mouse IgG (Pierce) in 200 l of blocking solution/well at ratios of unlabeled competing antibody to 125 I-labeled antibody ranging from 1:125 to 125:1. The labeled/unlabeled antibody mixtures were added to the blot and drained slowly by gravity. Finally, the wells were rinsed four times with 200 l of TBS-T, the blot was removed from the manifold and dried, and bound antibody was detected by exposure to Kodak X-Omat AR film at Ϫ70°C. were also prepared using similar methods. 4 All constructs were transferred into the recombinant donor plasmid pFASTBAC1 (Life Technologies, Inc., Burlington, Ontario, Canada), and recombinant bacmids and baculovirus were produced and used to infect Spodoptera frugiperda Sf21 cells as described previously (32).

Generation of Expression Constructs, Production of Recombinant
Cytosolic and Membrane Protein Isolation-H69AR, empty vectortransfected HeLa C1, MRP-transfected HeLa T5, or Sf21 cells were harvested, and crude membrane fractions were prepared (24,32). For some experiments, plasma membrane vesicles and cytosolic fractions were isolated. Briefly, cells were homogenized in buffer containing 50 mM Tris-HCl, 250 mM sucrose, 0.25 mM CaCl 2 , pH 7.5, and protease inhibitors (Complete TM , Roche Molecular Biochemicals). Cell pellets were frozen at Ϫ70°C for at least 1 h, thawed, and then disrupted by N 2 cavitation. EDTA was added to 1 mM, and after centrifugation at 500 ϫ g for 15 min, the supernatant was layered over 35% (w/w) sucrose in 50 mM Tris-HCl and 1 mM EDTA and centrifuged at 100,000 ϫ g for 1 h. The interface was collected and washed twice by centrifugation. The membrane pellet was resuspended in transport buffer (50 mM Tris-HCl and 250 mM sucrose, pH 7.5) and passed 20 times through a 27-gauge needle for vesicle formation. For Sf21 cells expressing polypeptides MRP-(617-932) and MRP-(1294 -1531), the layer above the 35% sucrose interface containing soluble cytosolic proteins was retained.
MRP-mediated LTC 4 Transport in Inside-out Membrane Vesicles-ATP-dependent [ 3 H]LTC 4 uptake into membrane vesicles was measured by a rapid filtration technique (16,40). Plasma membrane vesicles from MRP-transfected T5 cells (2 g of protein in a 60-l reaction volume) were preincubated alone or with mAb QCRL-1, -2, -3, or -4 (10 g/ml intact mAb or Fab fragment) for 1 h at room temperature. Uptake of [ 3 H]LTC 4 (50 nM, 40 nCi/reaction, 146 Ci/mmol; Life Science Products, Markham, Ontario) was then determined in the presence of 10 mM MgCl 2 and 4 mM ATP for 1 min at 23°C. Uptake was stopped by filtration through glass-fiber filters. Filters were washed with transport buffer, and associated radioactivity was quantitated.
Preparation of Dot Blots and Immunoblotting-Membrane proteins in TBS were blotted onto Immobilon P membrane by gravity using a 96-well vacuum manifold. After washing with TBS-T, the blots were removed from the manifold, cut into strips, and transferred to a 24-slot incubation tray. Blots were blocked for at least 1 h in 4% skim milk powder in TBS-T, incubated with primary antibody diluted in blocking solution for 1-18 h, and then processed as described previously (24). Primary antibody binding was visualized by enhanced chemiluminescence detection (Amersham Pharmacia Biotech) using horseradish peroxidase-conjugated F(abЈ) 2 fragments of goat anti-rat IgG (HϩL; for mAb MRPr1), goat anti-mouse IgG ϩ IgM (HϩL; for mAbs MRPm6, MRPm5, and QCRL-1, -2, -3, -4, and -6), or goat anti-rabbit IgG (HϩL; for antisera MRP-1 and MRP-2).
Labeling of MRP with 8-Azido-[ 32 P]ATP-Labeling of MRP-enriched T5 or control C1 membrane proteins was carried out as described previously (36,41). Briefly, 30 g of T5 or C1 membrane proteins was incubated with intact mAbs or their Fab fragments (40 g/ml) for 15 min at room temperature. 8-Azido-[␣-32 P]ATP (4 Ci, 20 Ci/mmol; ICN) was added, and incubation was continued on ice for 5 min. The samples were irradiated at 302 nm for 8 min, and proteins were then resolved by SDS-PAGE followed by autoradiography.

Immunoprecipitation of Full-length MRP-We demonstrated
previously that mAbs QCRL-1, -2, and -3 could immunoprecipitate MRP from 1% CHAPS-solubilized membranes, confirming that they are MRP-specific (24). In contrast, mAbs QCRL-4 and -6, which bound strongly and specifically to crude membranes from MRP-overexpressing cell lines in immunodot blots, did not immunoprecipitate MRP solubilized under similar conditions or with different detergents, including 0.1% SDS, 1% Nonidet P-40, or 0.5% deoxycholate (data not shown). Consequently, it could not be concluded with absolute certainty that they reacted exclusively with MRP. To test the possibility that mAbs QCRL-4 and -6 recognized conformation-dependent epitopes that were particularly sensitive to denaturation, milder conditions were used for extracting MRP from cell membranes prior to immunoprecipitation. At a detergent/protein ratio of 1:1, mAb QCRL-3 immunoprecipitated the 190-kDa MRP, as expected (Fig. 1). mAbs QCRL-4 and -6 also immunoprecipitated a protein of the same size under these conditions, indicating that they are indeed MRP-specific. Higher detergent/protein ratios (e.g. 10:1) had little effect on the interaction of mAb QCRL-3 with MRP. However, binding and precipitation of MRP by both mAbs QCRL-4 and -6 were substantially reduced, confirming the detergent sensitivity of the epitopes recognized by these mAbs.
Assay of Binding Competition between MRP-specific mAbs-Binding competition assays were carried out to determine whether mAbs QCRL-1, -2, -3, -4, and -6 recognize overlapping epitopes using H69AR membrane dot blots. A constant amount of 125 I-labeled mAb QCRL-1, -2, -3, -4, or -6 either alone or premixed with increasing amounts of unlabeled competing antibody (mouse IgG or mAb QCRL-1, -2, -3, -4, or -6) was added to individual wells. As expected, binding of each 125 I-labeled mAb was inhibited by the unlabeled form of the same mAb at an ϳ1:1 ratio or by an excess of unlabeled competitor (Fig. 2). This competition was specific because total mouse IgG did not compete for binding with any of the 125 I-labeled mAbs even when present at 125-fold excess. Of all the mAbs tested, only mAbs QCRL-4 and -6 competed reciprocally for binding to MRP. This finding, together with observations that they behave similarly in immunoprecipitations (Fig. 1) and other immunoassays (data not shown), suggested that mAbs QCRL-4 and -6 probably bind to the same epitope. Consequently, mAb QCRL-6 was not characterized further. No significant overlap between the epitopes of the other mAbs was apparent.
Inhibition of MRP-mediated LTC 4 Transport by Fab Fragments of mAbs QCRL-2, -3, and -4 -mAb QCRL-3 and, in some cases, mAbs QCRL-2 and -4 have been shown to inhibit MRPmediated transport of several conjugated substrates as well as GSH-stimulatable transport of several unmodified xenobiotics (16,19,21,23,34). However, intact (bivalent) mAbs were used in these studies, raising the possibility that inhibition resulted from steric effects caused by the Fc regions of these mAbs or by cross-linking of adjacent MRP molecules. To address this pos-FIG. 1. Immunoprecipitation of MRP with mAbs QCRL-4 and -6. Crude membranes prepared from H69AR cells that had been metabolically labeled with [ 35 S]methionine were solubilized in PBS containing 1% CHAPS at a detergent/protein ratio of 1:1 or 10:1 (w/w) as indicated. MRP was immunoprecipitated from 22.5 g of solubilized membrane protein using mAb QCRL-3, -4, or -6 crude ascites (1 l). Immune complexes were recovered using Protein G-Sepharose, and precipitated proteins were analyzed by SDS-PAGE and fluorography.
FIG. 2. Radioimmunoassay of binding competition between unlabeled and 125 I-labeled mAbs. Dot blots were prepared by blotting H69AR membrane protein (4 g/spot) onto Immobilon P membrane using a 96-well vacuum manifold. To each well was added a constant amount of 125 I-labeled mAb (QCRL-1, -2, -3, -4, or -6; indicated to the left of each blot) that had been preincubated with unlabeled competing antibodies (mAb QCRL-1, -2, -3, -4, or -6 crude ascites or mouse IgG; indicated to the right of each blot). The unlabeled/ 125 I-labeled mAb ratios are indicated at the top of the blots. After allowing the antibody mixtures to drain slowly by gravity, the wells were rinsed. The blots were dried, and 125 I-labeled mAb binding was detected by autoradiography.
sibility, Fab fragments of mAbs QCRL-2, -3, and -4 were prepared and tested to determine whether these monovalent fragments are also capable of inhibiting MRP-mediated transport activity. As expected, the Fab fragment of the non-inhibitory mAb QCRL-1 had little effect on MRP-mediated LTC 4 transport relative to the control sample with no mAb (Fig. 3). In contrast, Fab fragments of mAbs QCRL-2, -3, and -4 inhibited transport by Ͼ85% at 10 g/ml. Similar results were obtained with the intact mAbs, in agreement with previous observations. These results indicate that inhibition of MRP-mediated transport by mAbs QCRL-2, -3, and -4 is caused by their direct interaction with the protein.
Binding of mAbs to Sf21-expressed Modified MRP Molecules-To localize the conformation-dependent epitopes of mAbs QCRL-2, -3, and -4, the reactivity of these mAbs with a panel of truncated or otherwise altered MRP molecules expressed in Sf21 insect cells was examined in a series of immunodot blot analyses. In the first set of experiments, the mAbs were tested against MRP polypeptides with NH 2 -proximal truncations (MRP-(229 -1531) and MRP-(932-1531)) or COOHproximal truncations (MRP-(1-858) and MRP-(1-932)). In addition, a mutated protein (MRP ins708 ) that contains 13 additional amino acids (DIRTINVRFLREI) inserted at amino acid 708 between the Walker A and B motifs of NBD1 was examined. These amino acids are present in the comparable location of P-glycoprotein, but are absent from most MRP-related ATPbinding cassette transporters (3,7,11). A diagram of MRP showing the approximate locations of its three MSDs and two NBDs as well as the portions of MRP contained in the various polypeptides used in this analysis is shown in Fig. 4A.
As positive controls, the dot blots were probed in parallel with mAbs MRPr1, MRPm6, and QCRL-1, the epitopes of which have been defined previously (Fig. 4A) (25, 39). These blots provided standards against which the immunoreactivity of mAbs QCRL-2, -3, and -4 with each polypeptide could be compared (25,39). All three of the positive control mAbs showed patterns of immunoreactivity with the MRP fragments consistent with the locations of their epitopes (Fig. 4B). However, mAb MRPr1 reacted more strongly with the MRP-(229 -1531) polypeptide than the other two mAbs, and mAb QCRL-1 reacted more strongly with the MRP-(1-932) polypeptide than mAb MRPr1. In each case, the truncation in the MRP molecule is very near the site of the epitope of the more strongly reacting mAb (amino acids 238 -247 for mAb MRPr1 and amino acids 918 -924 for mAb QCRL-1) (25,39). This may increase accessibility of the mAbs to these epitopes and hence account for the stronger signal observed.
mAbs QCRL-2 and -3 showed similar patterns of immunore-activity with the MRP fragments (Fig. 4B). Both mAbs bound to the NH 2 -proximal half-molecule MRP-(1-932) and to the polypeptide lacking MSD1 (MRP-(229 -1531)), and neither bound to the COOH-proximal half-molecule MRP-(932-1531). In addition, the binding of both mAbs was markedly diminished by the insertion of 13 amino acids in the NH 2 -proximal NBD (MRP ins708 ). The only major difference between the two mAbs was that mAb QCRL-2 did not bind to MRP-(1-858), whereas mAb QCRL-3 reacted strongly with this polypeptide. These results suggest that the sequences bound by mAbs QCRL-2 and -3 are located somewhere in MSD2 and/or NBD1.
In addition, at least part of the mAb QCRL-2 epitope is between amino acids 859 and 932 in the region connecting NBD1 to MSD3. In contrast, mAb QCRL-4 reacted only with those polypeptides containing the COOH-proximal half of MRP (MRP-(932-1531) and MRP-(229 -1531)) and bound as well to MRP ins708 as the control mAbs. These observations suggest that the QCRL-4 epitope is in MSD3 and/or NBD2.
To further localize the QCRL-2 and -3 epitopes, a second series of immunodot blotting was carried out with these mAbs using MRP polypeptides containing MSD2 or MSD2 ϩ NBD1 (Fig. 5, A and B).  (Fig. 5B). mAbs QCRL-2 and -3 showed a pattern of reactivity similar to that of the MRP-1 antiserum, reacting only with polypeptides containing MRP amino acids 617-932 (Fig. 5B). This suggests that the conformation-dependent epitopes recognized by these mAbs are wholly or partially contained within this region corresponding to NBD1.
Immunoprecipitation of Soluble Polypeptides Corresponding to NBD1 (MRP-(617-932)) and NBD2 (MRP-(1295-1531))-To determine whether the epitopes recognized by the mAbs were entirely contained between amino acids 617 and 932 (QCRL-2 and -3) or amino acids 1296 and 1531 (QCRL-4), the ability of the mAbs to immunoprecipitate the MRP-(617-932) or MRP-(1294 -1531) polypeptides was examined. MRP-(617-932) migrated as a polypeptide of ϳ36 kDa in SDS-PAGE and could be immunoprecipitated from the cytosolic fraction of Sf21 cells with mAbs QCRL-2 and -3 as well as with mAb QCRL-1, as expected (Fig. 6A). Immunoprecipitation of MRP-(617-932) with mAbs QCRL-2 and -3 was specific because this polypeptide was not immunoprecipitated with mAb QCRL-4. MRP-(1294 -1531) was also present in the cytosolic fraction of Sf21 cells and migrated as a doublet between 25 and 30 kDa (Fig.  6B). The exact nature of these doublet MRP NBD-derived bands is not known. However, we speculate that there is a protruding "tail" at the NH 2 or COOH terminus of the folded NBD polypeptide that is susceptible to proteolysis. Cleavage of the tail would result in lower levels of the larger, but less stable full-length polypeptide and higher levels of the slightly smaller, but more stable protein. MRP-(1294 -1531) could be immunoprecipitated with mAb QCRL-4 as well as with the MRP-2 antiserum, as expected (Fig. 6B). Immunoprecipitation with these antibodies was specific because the only proteins detectable in the mAb QCRL-2 and -3 immunoprecipitates were the immunoglobulin light chains of the precipitating antibodies, which migrated at ϳ25 kDa.
Fab Fragments of mAbs QCRL-2, -3, and -4 Do Not Inhibit Azido-ATP Labeling of MRP-To determine whether mAbs QCRL-2, -3, and -4 interfere with the ability of MRP to bind nucleotide, membrane vesicle proteins from MRP-transfected T5 cells and control C1 cells were incubated with Fab fragments of the mAbs individually or together and then photolabeled with 8-azido-[ 32 P]ATP. As shown in Fig. 7, labeling of MRP in T5 membranes was unaffected by the Fab fragments, suggesting that the mAbs do not prevent nucleotide binding by the protein. DISCUSSION To provide molecular probes for studying both the structure and transport mechanism of MRP, we isolated a panel of mAbs that reacted preferentially with cell membranes from MRPoverexpressing cell lines (24). Four of these mAbs (QCRL-2, -3, -4, and -6) recognize intracellular conformation-dependent epitopes and are able to inhibit the transport activity of MRP. At the time of their initial characterization, immunodot blot analyses strongly suggested that mAbs QCRL-4 and -6 recognize MRP, but we were unable to confirm this directly by immunoprecipitation (24). By using very low detergent/protein ratios, we have now shown that mAbs QCRL-4 and -6 immunoprecipitate a single 190-kDa protein from solubilized membranes (Fig. 1). These observations establish the MRP specificity of mAbs QCRL-4 and -6 and indicate that both mAbs recognize detergent-sensitive epitopes. Binding competition assays revealed that mAbs QCRL-4 and -6 recognize identical or overlapping epitopes (Fig. 2). None of the remaining mAbs competed reciprocally for binding, demonstrating that mAbs QCRL-2, -3, and -4 recognize distinct, non-overlapping epitopes of MRP.
mAbs QCRL-2, -3, and -4 have been shown to be effective inhibitors of MRP-mediated ATP-dependent transport of several conjugated organic anion substrates, and mAb QCRL-3 has been shown to inhibit GSH-stimulatable transport of unmodified vincristine, daunorubicin, and aflatoxin B 1 (16,19,21,23,34). This inhibitory property is not shared by mAb QCRL-1, which may be explained by the fact that its linear heptapeptide epitope is within a region of MRP that is not required for LTC 4 transport activity in Sf21 cell membranes (25,32). The Fab fragments of mAbs QCRL-2, -3, and -4 inhibited MRP-mediated transport as effectively as the intact mAbs (Fig. 3). This finding indicates that inhibition is caused by direct interaction of the antigen-binding sites of these mAbs with MRP, rather than being the result of a steric effect caused by the Fc regions of the immunoglobulins or by cross-linking of adjacent MRP molecules.
As an initial step toward understanding the mechanism by which mAbs QCRL-2, -3, and -4 inhibit the transport activity of MRP, we have localized the conformation-dependent epitopes bound by these mAbs. With respect to mAbs QCRL-2 and -3, we have determined that the minimal regions of MRP sufficient for full reactivity are amino acids 617-932 and 617-858, respectively. These regions include NBD1 and different amounts of the segment that connects NBD1 to MSD3. Thus, a portion of the epitope recognized by mAb QCRL-2 is located between amino acids 859 and 932 in the connector region of the protein. Insertion of 13 amino acids at position 708 in NBD1 of MRP resulted in a markedly decreased reactivity of mAbs QCRL-2 and -3 and also reduced the transport activity of the protein. 4 Taken together, these observations indicate that the determinants of the epitopes bound by both of these mAbs are contained within NBD1 and that the relative positioning of these determinants is perturbed when the distance between the Walker A and B motifs of this domain is altered. In contrast to mAbs QCRL-2 and -3, the epitope bound by mAb QCRL-4 appears to reside wholly within the second NBD of MRP, be-tween amino acids 1294 and 1531. Since mAbs QCRL-2, -3, and -4 do not react with MRP in its denatured form, the ability of these mAbs to immunoprecipitate MRP-(617-932) (NBD1) and MRP-(1296 -1531) (NBD2) indicates that both of these soluble truncated polypeptides have folded correctly and assumed a configuration similar or identical to the NBDs when they are present in the context of the full-length protein in its native state. Consistent with this conclusion is our observation that the individual NBDs have the ability to bind and hydrolyze ATP. 4 This knowledge is important with respect to ongoing structural and functional studies of these MRP domains and their interactions with other regions of the protein as well as in comparisons with the NBDs of other ATP-binding cassette transporters such as P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (42)(43)(44)(45)(46). 4 The location of the QCRL-2, -3, and -4 epitopes in the NBDs of MRP raised the possibility that one or more of the mAbs might inhibit transport by interfering directly with ATP binding. However, the inability of both of the intact mAbs (data not shown) and their Fab fragments to inhibit labeling of MRP by 8-azido-ATP suggests that this is not the case (Fig. 7). We have shown previously that intact mAb QCRL-3 can inhibit photolabeling of MRP by [ 3 H]LTC 4 (16), indicating that this mAb may inhibit transport by directly limiting access of LTC 4 to its binding site. Further experiments examining the effects of the Fab fragments of mAbs QCRL-2, -3, and -4 on [ 3 H]LTC 4 photolabeling of MRP and MRP-derived polypeptides will be required to determine conclusively whether these mAbs inhibit transport of this substrate by preventing its binding to the protein. In turn, these studies may provide important information about the location of the LTC 4 and other substrate binding site(s) on MRP.
Structure-function studies of P-glycoprotein have been aided by several P-glycoprotein-specific mAbs that have been reported to interfere with substrate interactions and/or the transport activity of this protein. The first P-glycoprotein-reactive mAb to be characterized was mAb C219, which recognizes a linear cytoplasmic epitope located six amino acids downstream from the Walker B motif in both the NH 2 -and COOH-proximal NBDs of this protein (47,48). mAb C219 inhibited photolabeling of P-glycoprotein with the calcium channel blocker photoaffinity analog [ 3 H]azidopine (49), but unlike mAbs QCRL-2, -3, and -4 in the present study, also inhibited photolabeling with 8-azido-ATP (49,50). The inhibition of ATP binding by mAb C219 was associated with a decrease in ATPase activity present in membranes from P-glycoprotein-overexpressing cells (50), whereas we have noted no such effect of the MRP-specific mAbs on the ATPase activity of MRP. 2 Despite these effects on drug binding and ATPase activity, inhibition of P-glycoprotein transport activity by mAb C219 has not been reported. On the other hand, several other mAbs that bind extracellular epitopes of P-glycoprotein specifically reverse or reduce the drug resistance and/or drug accumulation deficit of viable, Pglycoprotein-overexpressing tumor cells (51)(52)(53)(54). The mechanism by which one of these mAbs, UIC2, inhibits P-glycoprotein function appears to involve trapping of the protein in an inactive conformation (55). Similarly, mAbs QCRL-2, -3, and -4 may stabilize or trap MRP in a given conformational state and, in doing so, prevent the conformational changes that are presumed to occur during the transport cycle of the protein. To explore this possibility, it will be necessary to determine if the mAbs are able to modulate MRP conformational transitions. mAbs QCRL-2, -3, and -4 cannot be used to detect MRP in intact, non-permeabilized cells because their epitopes are cytoplasmic. Nevertheless, it should be possible to test their reactivity with functional MRP in cell membranes, in the presence and absence of ATP and transport substrates, using filter binding assays. Such studies will provide important insight into the mechanism by which MRP transports its conjugated organic anion and drug substrates.


extfieldsofstudy: Medicine Biology

text:
Regulation of apoptosis in myeloid cells by interferon consensus sequence-binding protein.

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a disease with marked expansion of granulocytes and macrophages that frequently progresses to a fatal blast crisis, thus resembling human chronic myelogenous leukemia (CML). One important feature of CML is decreased responsiveness of myeloid cells to apoptotic stimuli. Here we show that myeloid cells from mice deficient in ICSBP exhibit reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. In contrast, apoptosis in thymocytes from ICSBP-deficient mice is unaffected. We also show that overexpression of ICSBP in the human U937 monocytic cell line enhances the rate of spontaneous apoptosis and the sensitivity to apoptosis induced by etoposide, lipopolysaccharide plus ATP, or rapamycin. Programmed cell death induced by etoposide was specifically blocked by peptides inhibitory for the caspase-1 or caspase-3 subfamilies of caspases. Studies of proapoptotic genes showed that cells overexpressing ICSBP have enhanced expression of caspase-3 precursor protein. In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-X(L). These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes.

ICSBP was originally identified as a transcription factor that, similar to IRF-2, acts as a repressor and inhibits IFNinducible promoter activities (8). Many attempts have been made to establish its contributions to IFN signaling, with recent studies revealing complex roles for this factor in immunity, cell cycle regulation, and hematopoiesis (9,10). Evidence that IRF family proteins play important roles in the growth of hematopoietic cells is seen in mice with null mutations of IRF-1 and IRF-2 (11), which are generally expressed, as well as IRF-4 (also called PIP or LSIRF) and ICSBP, which are almost exclusively expressed in hematopoietic cells (12). IRF-1 Ϫ / Ϫ mice have developmental defects in thymocytes and CD8 ϩ T cell differentiation, whereas IRF-2 Ϫ / Ϫ mice exhibit abnormalities of bone marrow hematopoiesis and B cell development (11). IRF-4 Ϫ / Ϫ mice exhibit profound alterations of the function and homeostasis of both mature B and T cells (12). ICSBP Ϫ / Ϫ mice are characterized by altered hematopoiesis that manifests as a syndrome similar to human chronic myelogenous leukemia (CML; reference 10). The most prominent early features of this disorder are marked expansions of the granulocytic, monocytic, and, to a lesser extent, lymphoid lin-eages. Older mice experience a transition from this chronic phase of disease to a clonal, malignant blast crisis (10). A striking clinical counterpart to myeloid malignancies of ICSBP Ϫ / Ϫ mice comes from the observation that ICSBP transcripts are greatly decreased in cells of patients with CML (13). Human CML is a complex disorder, with enhanced proliferation of granulocyte precursors and reduced sensitivity of myeloid cells to apoptosis suggested as contributing factors. A role for IRF family members in regulating cell death has precedent in the demonstration that DNA damageinduced apoptosis of peripheral T cells is dependent on IRF-1 (14). Here we show that myeloid cells of ICSBP Ϫ / Ϫ mice have increased resistance to apoptosis, and transfected cells overexpressing ICSBP have increased sensitivity.
For proliferative responses, single-cell preparations from spleen, lymph node, and bone marrow were cultured in 96-well plates at 2 ϫ 10 5 cells/ml for 24-72 h. Cells were pulsed with [ 3 H]thymidine for the last 18 h of culture and assayed for incorporation.
U937 ϩ and U937 Ϫ transfectants were washed and cultured for 24 h at a concentration of 3 ϫ 10 5 cells/ml in serum-free RPMI 1640 medium supplemented with 500 U/liter insulin (Eli Lilly and Co.) and 5 mg/liter transferrin (GIBCO BRL). Cells were treated with 20 g/ml etoposide or 30 ng/ml TNF-␣ plus 10 g/ml CHX, incubated for 18 h, and then harvested and assayed for apoptosis. In some experiments, transfectants were treated with 1 g/ml LPS (Sigma Chemical Co.) for 4 h and then with 2 mg/ml ATP (Sigma Chemical Co.) for 30 min, incubated for 6 h, and then harvested and assayed for apoptosis. For protease inhibition experiments, cysteine protease inhibitors ZVAD-fluoromethyl ketone (FMK), BD-FMK, and the control ZFA-FMK reagent (provided by Dr. P.A. Henkart, National Cancer Institute, Bethesda, MD) were added at a concentration of 50 M 1 h before induction of apoptosis.
TUNEL Assay. The presence of DNA nicking in apoptotic cells was assayed, with minor modifications, according to Gorczyca et al. (16). In brief, cell samples were collected and fixed in 1% formaldehyde in PBS on ice, washed once with PBS, resus-

Gene
Primers Probe pended in cold 70% ethanol in PBS, and stored at Ϫ20ЊC. After rehydration, cells were resuspended in cacodylate buffer, 25 mM CoCl 2 , and 0.5 nM biotin-dUTP in the presence or absence of 10 U TdT enzyme (Boehringer Mannheim Biochemicals) and incubated at 37ЊC. After washing in PBS, cells were resuspended in saline citrate buffer containing 4ϫ SSC, 2.5 g/ml FITC-avidin (Boehringer Mannheim Biochemicals), 0.1% Triton X-100 (Sigma Chemical Co.), and 5% nonfat dry milk. After a 30-min incubation in the dark, cells were washed in PBS with 0.1% Triton X-100 and 0.5% BSA and resuspended in Sorter medium (Quality Biological, Inc.). dUTP incorporation by individual cells was measured by green fluorescence on a FACScan™ flow cytometer using Cellquest™ data software (Becton Dickinson). Flow Cytometric Analysis and DNA Labeling. Cells were stained with FITC-labeled Abs, including those to CD4, CD8, B220, Mac-1, and 8C5 (GR-1; all from PharMingen). In brief, cells were resuspended in 100 l PBS containing 2% FCS and incubated with various mAbs (1 g/ml) for 20 min. After one wash with PBS, cells were stained for TUNEL assay. Analysis was performed on a FACScan™ flow cytometer.
Reverse Transcriptase PCR. mRNA was isolated using RNAzol (Tel-Test) followed by extraction with chloroform and isopropanol. 1 g of RNA was transcribed using MMLV-H(Ϫ) reverse transcriptase (RT; Promega Corp.) and then amplified by PCR using primers described in Table I under the following conditions: 1ϫ reaction buffer (Promega Corp.), 2.5 mM MgCl 2 , 2.5 mM dNTP, 5 M of each primer, and 1 U of Taq DNA polymerase (Promega Corp.) in a total volume of 40 l. Amplification was carried out through 30 cycles. PCR products were separated on 1.2% agarose gels and analyzed by Southern blot hybridization with FITC-labeled probes using an ECL-3Ј oligolabeling and detection system and Hyperfilm-ECL (Amersham International).
Western Blot Analysis. Whole cell extracts were prepared by lysing cells for 30 min at 4ЊC with lysis buffer (50 mM Tris, pH 7.5, 2 mM EDTA, 100 mM NaCl, 1 mM Na 3 VO 4 , 1% NP-40, 10 g/ml leupeptin, 5 g/ml aprotinin, and 10 g/ml PMSF) at a concentration of 10 6 cells/50 l. After centrifugation at 4ЊC for 15 min, aliquots of the extracts containing 100 g of protein were subjected to 10% SDS-PAGE. The proteins were then electrophoretically transferred to PVDF (polyvinylidine difluoride) membrane filters (Bio-Rad Labs.). Filters were probed with sc-515, a rabbit polyclonal Ab to human caspase-1; sc-625, a rabbit polyclonal Ab to caspase-2 of human origin; sc-1224, a goat polyclonal Ab to human caspase-3; sc-1041, a rabbit polyclonal Ab to human Bcl-X S/L ; or an Ab to p53 (all from Santa Cruz Biotechnology, Inc.) at a concentration of 1 g/ml in PBS with 5% dried milk and 0.2% Tween 20. Detection was carried out with secondary horseradish peroxidase-linked anti-rabbit or anti-goat Abs (Amersham International) and the Amersham ECL system. Transient Transfection and Luciferase Assay. Primers corresponding to the human Bcl-X L promoter at positions 77-101 (AAGT-CAGATTGCAGATCTGAGGCAG) and positions 745-769 (GAATTCACTTCATAGAACCTTGGAT) as previously reported (17; sequence available from EMBL/GenBank/DDBJ under accession number D30746) were used in a genomic PCR reaction to obtain the Bcl-X L promoter region. The PCR product was cloned into a basic pGL3 luciferase vector (Promega Corp.) cut with SacI and HindIII. The resulting plasmid was prepared by the CsCl gradient method as previously reported (18) and used for transient transfection assays. RAW cells, a mouse macrophage tumor line, were transfected by electroporation using the Cell-Porator (GIBCO BRL). In brief, up to 30 g of plasmid DNA was mixed with 1.0 ϫ 10 7 cells/ml and resuspended in 0.4 ml of fresh complete RPMI 1640 medium containing 10% FCS. The cell/DNA mixture was then incubated on ice for 10 min before electrical shock at 300 mV and 800 F. Cells were then transferred into 10 ml of fresh medium and left at room temperature for 10 min before incubation at 37ЊC. 24 h after transfection, luciferase assays were performed as reported (19).

Results
Apoptosis Is Decreased in Myeloid Cells from ICSBP Ϫ/Ϫ Mice. Although it was initially assumed that CML is caused by uncontrolled cell proliferation resulting in the clonal expansion evident in this disease, some studies have indicated that the rates of proliferation are not significantly increased (20,21). Other studies have shown that myeloid cells of CML patients have increased resistance to apoptosis, indicating that this disease may result from suppression of cell death rather than ungoverned proliferation (22,23). To investigate the possible relatedness of the disease of ICSBP Ϫ/Ϫ mice to CML, we first studied the levels of spontaneous proliferation and apoptosis of cells from nor- mal and mutant mice. As shown in Fig. 1 A, the proliferation of cultured spleen, lymph node, and bone marrow cells of mutant mice was increased over normal when assayed at 24-72 h. To examine whether reduced programmed cell death might contribute to the expansion of myeloid cells in ICSBP Ϫ/Ϫ mice, we examined spontaneous apoptosis of cultured cells from spleen, thymus, and bone marrow by TUNEL assay (Fig. 1 B). These studies revealed a significant reduction in the level of apoptotic death exhibited by spleen and bone marrow cells from ICSBP Ϫ/Ϫ mice. In contrast, thymocytes from ICSBP Ϫ/Ϫ and ICSBP ϩ/ϩ mice behaved similarly. To determine whether specific cell populations accounted for the reduced rate of apoptosis in spleen and bone marrow, cells were assayed simultaneously for apoptosis and expression of cell lineage markers. Mac-1 ϩ and, to a lesser extent, GR-1 ϩ cells from spleen and bone marrow of ICSBP Ϫ/Ϫ mice exhibited a significant reduction in the rate of apoptosis (Fig.  2, untreated cells). In contrast, the rates of apoptosis were comparable among CD4 ϩ /CD8 ϩ and B220 ϩ cells from mice of either genotype.
To further investigate the possible role of ICSBP in apoptosis of myeloid cells, we examined programmed cell death induced by etoposide, an agent that induces DNA breaks by inhibiting the religation activity of topoisomerase II. As shown in Fig. 2, apoptosis induced by etoposide was significantly reduced in GR-1 ϩ and Mac-1 ϩ cells from spleens and bone marrow of ICSBP Ϫ/Ϫ mice. In contrast, apoptosis of splenic B and T cells was not affected. Interestingly, bone marrow T cells from ICSBP Ϫ/Ϫ mice were significantly more sensitive to etoposide than were cells from heterozygotes. Finally, we tested cells from ICSBP Ϫ/Ϫ and ICSBP ϩ/ϩ mice for their sensitivity to apoptosis induced by TNF-␣ plus CHX. No differences in responsiveness were noted for cells of any phenotype for either genotype (Fig.  2). These findings indicate that ICSBP selectively affects distinct pathways leading to apoptosis of myeloid cells: induction by etoposide is clearly modulated by ICSBP, whereas the pathway activated by TNF-␣ plus CHX is not.
Apoptosis Is Enhanced in U937 Cells Overexpressing ICSBP. A full length ICSBP construct was stably transfected into U937 human monocytic cells (U937 ϩ ), resulting in levels of ICSBP proteins that were -03-01فfold higher than endogenous ICSBP (15). Transfectants containing the vector without insert (U937 Ϫ ) were used as controls. For these studies, three clones of each transfectant were selected and pooled. Using the TUNEL assay for detection of DNA strand breaks, we examined apoptosis of both cell populations cultured in serum-free medium supplemented with insulin and transferrin. During the first 18 h of culture, neither U937 ϩ nor U937 Ϫ cells showed clear evidence of apoptosis, but prominent differences were seen at 38 and 68 h (Fig. 3), indicating a proapoptotic role for ICSBP in myeloid cells.
Apoptosis is controlled by many distinct signals (24,25), but it appears that different signaling pathways ultimately converge to activate a common program (26). It has been shown that U937 cells undergo apoptosis in response to multiple agents (27,28). To further investigate the function of ICSBP in the regulation of distinct apoptotic pathways, we treated U937 ϩ and U937 Ϫ cells with different apoptosis-inducing agents. U937 ϩ cells exhibited greater susceptibility to DNA damage-induced death than did control cells. After 10 h of treatment with etoposide, almost 70% of U937 ϩ cells were TUNEL positive, in contrast to 30% of control cells (Fig. 4). Interestingly, apoptosis was induced by TNF-␣ plus CHX at the same levels in U937 Ϫ and U937 ϩ cells (data not shown), suggesting again that ICSBP is involved only in specific pathways of programmed cell death. A moderately enhanced sensitivity to apoptosis was also observed in U937 ϩ cells after treatment with LPS plus ATP. It was shown previously that ATP treatment leads to apoptosis via a caspase-1-independent pathway (29). We found that treatment with LPS for 4 h and then with ATP for 30 min induced apoptosis in 60% of U937 ϩ cells but only 45% of U937 Ϫ cells (Fig. 4). Moreover, U937 ϩ cells were significantly more sensitive to rapamycin, a potent immunosuppressive drug that has been shown to interfere with growth factor-induced cell proliferation and induce apoptosis in a murine B cell line (30) (data not shown). Although to date there is no direct evidence for the involvement of ICSBP in cell growth pathways, our results clearly show that ICSBP plays a role in the apoptotic response of U937 cells to specific deathinducing signals and confirm that ICSBP itself might contribute to spontaneous and DNA damage-induced apoptosis in this system.
Inhibition of Etoposide-induced Apoptosis by ZVAD-FMK and BD-FMK in U937 ϩ Cells. We next examined the role of caspases in programmed cell death of U937 ϩ cells. It is evident that caspases are important effector molecules of apoptosis (26,31). To determine whether specific caspase subfamilies contribute to apoptosis in U937 ϩ cells treated with etoposide, we tested the activity of two cell-permeable peptide FMK inhibitors of caspase-1 (IL-1␤ converting enzyme-family) proteases (32). ZVAD-FMK specifically blocks the caspase-1-like subfamily, and BD-FMK preferentially inhibits caspase-3-like proteases. ZFA-FMK, which lacks inhibitory activity for caspases, was used as a negative control. Both ZVAD-FMK and BD-FMK inhibited apoptotic death induced by etoposide in U937 ϩ (Fig.  5) and U937 Ϫ cells (data not shown). These results suggest that ICSBP enhances the sensitivity of U937 monocytic cells to apoptosis induced by etoposide and that the apoptotic process involves the activity of members of the caspase-1 and caspase-3 subfamilies.
Control of Caspase Gene Expression by ICSBP. To address the possibility that ICSBP may interfere with the activation of caspases, we examined the expression of caspase-1, -2, -3, and -7 (33)(34)(35)(36). Semiquantitative RT-PCR was performed on each cell population before and after treatment with etoposide (Fig. 6, left). Transcripts for caspase-2 and -3 were present at similar levels before and after treatment of U937 ϩ and control cells. In contrast, caspase-7 and, to a lesser extent, caspase-1 transcripts were increased in U937 ϩ before and after induction of apoptosis with etoposide. ␤2 microglobulin transcripts, run as a control, were comparable in both clones. These results suggest   that overexpression of ICSBP in U937 cells may affect apoptosis by enhancing expression of the proapoptotic genes of caspase-1 and -7. This study was extended by Western blot analysis. Whole cell lysates were prepared from U937 ϩ and U937 Ϫ cells before and after treatment with etoposide, and immunoblot analysis was performed. The 48-kD precursor protein of caspase-2 was detectable before treatment in both cell types (Fig. 6, right), whereas two smaller fragments were generated from the cleavage of the caspase-2 precursor after treatment with etoposide. Notably, the strength of the low-molecular-mass fragments correlated with the increased sensitivity of U937 ϩ cells to etoposide.
We next examined the expression of caspase-3 precursor protein. The most striking finding was markedly increased levels of the precursor in U937 ϩ cells. After treatment with etoposide, the precursor was processed to generate a lowermolecular-mass fragment, reflecting induction of apoptosis. Although our previous results showed that expression of caspase-3 mRNA was not altered in ICSBP clones, Western blot analysis revealed a markedly enhanced expression of caspase-3 protein. These data suggest that ICSBP might control caspase-3 activity through modulation of translation or protein stability rather than transcription. In light of enhanced caspase-1 mRNA expression in U937 ϩ cells, it was of interest to determine whether caspase-1 protein expression was also affected. We found the 45-kD precursor of caspase-1 was slightly overexpressed in U937 ϩ cells. Because U937 ϩ cells show enhanced sensitivity to apoptosis induced by ATP via a caspase-1-independent pathway (29), these new findings suggest that increased expression of caspase-1 is unlikely to be the basis for this phenotype. It thus seems that caspase-3 plays a major role in cell death signaling pathways controlled by ICSBP.
Bcl-X L Expression Is Reduced in U937 ϩ Cells. We next asked whether ISRE-binding activities could control ex-pression of antiapoptotic genes. Using semiquantitative RT-PCR analysis, we examined expression of members of the Bcl-2 family, including Bcl-X L , Bcl-2, and Mcl-1 (37), as well as the proapoptotic gene, Bax (38). Bcl-X L transcripts were decreased by %05ف in U937 ϩ cells (Fig. 7 A), whereas the levels of Bcl-2, Mcl-1, and Bax transcripts were comparable (data not shown). Treatment with etoposide resulted in downregulation of Bcl-X L expression in both cell types. Because U937 ϩ and U937 Ϫ cells were equally sensitive to TNF plus CHX and U937 ϩ cells were much more sensitive to etoposide, the importance of changes in Bcl-X L expression induced by ICSBP may contribute to changes in only some pathways of programmed cell death. The reduced levels of Bcl-X L transcripts observed in U937 ϩ cells suggested that IFN might have a previously unappreciated role in regulating expression of this gene. We therefore examined levels of Bcl-X L protein in U937 Ϫ cells stimulated with IFN-␣ or IFN-␥. Both cytokines induced significant increases in Bcl-X L expression that were maximal at 12 h of treatment (Fig. 7 B).
To further examine ICSBP regulation of Bcl-X L , we tested Bcl-X L promoter activity in RAW cells, a mouse  macrophage tumor line, using a transient transfection assay. As shown in Fig. 7 C, luciferase activity driven by a 645-bp Bcl-X L promoter was significantly reduced by ICSBP but was not significantly affected by cotransfection of IRF-1 or IRF-2. As a control, RSV (Rous sarcoma virus) promoter activity was also unaffected, indicating specificity of the effect and suggesting that ICSBP represses transcription of Bcl-X L .
Finally, we examined the expression of the antioncogenic transcription factor p53, which has been shown to be required to induce cell death in oncogene-expressing cells that have suffered DNA damage (39). p53 protein levels were significantly increased in U937 ϩ cells (Fig. 8), suggesting another mechanism by which ICSBP might be involved in the induction of spontaneous apoptosis and the DNA damage response pathway.

Discussion
It is widely recognized that myeloid cells die via apoptosis (40), but the mechanisms involved in determining apoptotic death in this lineage have only recently come to be partially understood (41,42). ICSBP knockout mice exhibit dysregulated hematopoiesis, defining a role for ICSBP in the proliferation and differentiation of hematopoietic cells. Here we report that myeloid cells from ICSBP-deficient mice exhibited reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. Moreover, overexpression of ICSBP in U937 cells was sufficient to enhance their sensitivity to spontaneous and induced apoptosis. Our results thus define ICSBP as a regulator of myeloid cell survival. We also point out that ICSBP is likely to normally function in regulation of the cell cycle in hematopoietic cells, because the spontaneous proliferation of bone marrow, spleen, and lymph node cells from mutant mice was significantly increased.
ICSBP has been identified as a negative transcription factor that binds to ISREs found in the promoters of type I IFNs and IFN-inducible genes (43,44). Unlike IRF-1, IRF-3, and ISGF3␥, ICSBP exhibits a tissue-restricted pattern of expression largely limited to cells of the immune system, particularly the monocytic and lymphoid lineages. Expression of ICSBP is constitutive and can be dramatically enhanced by IFN-␥. The intrinsically weak DNA binding affinity of ICSBP (45) is dramatically increased after heterodimerization with IRF-1 or IRF-2 (3,46). ICSBP/ IRF-2 binding to DNA is constitutive, whereas ICSBP/ IRF-1 DNA binding is only induced by IFN-␥. Several studies implicate IRF-1 as a critical tumor suppressor, regulating oncogene-induced cell transformation or apoptosis (39,47). It has been reported that IRF-1 regulates DNA damage-induced apoptosis in mitogen-activated peripheral T cells (14). IRF-1 is also required for the induction of apoptosis in fibroblasts carrying an activated c-Ha-ras gene after DNA damage or culture in low serum (39). Moreover, IRF-1 has been implicated in IFN-␥-induced apoptosis of hematopoietic progenitor cells (48). Our data provide evidence that ICSBP, another member of the IRF family, can regulate apoptosis of myeloid cells.
ICSBP Ϫ/Ϫ mice exhibit expanded populations of granulocytic, monocytic, lymphoid, and perhaps megakaryocytic lineages (10), suggesting that the decisive alteration occurs in an early common progenitor cell. Our studies revealed that the sensitivity to apoptosis of Mac-1 ϩ and GR-1 ϩ cells from spleen and bone marrow of ICSBP Ϫ/Ϫ mice was significantly reduced. Expression of these markers is different in myeloid subpopulations; granulocytes are GR-1 ϩ Mac-1 ϩ or GR-1 ϩ Mac-1 Ϫ , and macrophages are GR-1 Ϫ Mac-1 ϩ (49), indicating that both macrophages and granulocytes from ICSBP Ϫ/Ϫ mice exhibited impaired programmed cell death. In contrast, the rate of apoptosis was comparable in T and B cells from ϩ/ϩ or ϪրϪ mice, suggesting a specific role for ICSBP in apoptosis in the myeloid lineage. Analysis of apoptosis induced by etoposide confirmed the specific proapoptotic role of ICSBP in myeloid cells. GR-1 ϩ and Mac-1 ϩ cells from spleen and bone marrow of ICSBP Ϫ/Ϫ mice showed a significantly reduced sensitivity to apoptosis induced by this DNA-damaging agent. In contrast, etoposide-induced apoptosis of B and T cells from ICSBP Ϫ/Ϫ and wild-type mice was similar.
We also found that the proliferation index of bone marrow, spleen, and lymph node cells of ICSBP Ϫ/Ϫ mice was increased over normal. This suggests that the early accumulation of hematopoietic cells that characterizes these animals is mediated by enhanced proliferation as well as by reduced apoptosis. Studies of U937 cells overexpressing ICSBP provided strong support for the proposed role of ICSBP in regulating apoptosis in myeloid cells. ICSBP expressed at levels 10-30-fold higher than endogenous levels sensitized cells to spontaneous apoptosis and selective pathways of response to exogenous stimuli. U937 ϩ cells exhibited greater sensitivity to apoptosis induced by DNA damage, treatment with LPS plus ATP, and inhibition of TOR/FRAP kinase by rapamycin (reference 50; data not shown), but not to treatment with TNF-␣ plus CHX. This suggests that apoptotic pathways initiated by engagement of death receptors leading to rapid activation of caspase cascades are insensitive to effects mediated by ICSBP, whereas other pathways are sensitive. This pattern of sensitivity and resistance to the effects of ICSBP is similar to that suggested for Bcl-2 and Bcl-X L : little or no effect on apoptosis induced by death receptor engagement but prominent regulation of apoptosis induced by growth factor withdrawal or DNA damage (51)(52)(53). It is unclear whether these selective effects on apoptosis are mediated by ICSBP alone or in combination with other IRF family members. IRF-1 is an important requirement of DNA damage-induced apoptosis of peripheral T cells (14) but not myeloid cells, at least on its own, because it is expressed at normal levels in ICSBP Ϫ/Ϫ mice. Inhibition of TOR/FRAP by rapamycin (53) leads to G1 arrest in some systems (54) and acceleration of apoptosis in others (55). The results of TOR activation-either growth arrest or apoptosis-resemble the pattern of responses to p53 activation (56). Because p53 expression is enhanced in U937 ϩ cells, ICSBP may regulate apoptosis by acting as a rheostat to balance the activities of these bifunctional genes. Induction of apoptosis by p53 in some systems is thought to involve transcriptional induction of Bax (57). This activity is unlikely to be relevant to our system, because Bax transcripts were similar in control and U937 ϩ cells. The importance of TOR to apoptosis could reflect its regulation by Akt, which functions in the phosphatidylinositol-3 kinase pathway to inhibit apoptosis (58). Inhibition of TOR may induce apoptosis by inactivating downstream signaling pathways that involve p70 S6K kinase or 4E-BP-1 (59).
Further analysis of U937 ϩ cells revealed that ICSBP may affect expression of caspases involved in the central control and execution stages of cell death (60)(61)(62)(63). Our results revealed that transcripts for caspase-2 and -3 were expressed at the same level in U937 Ϫ and U937 ϩ cells before and after induction of apoptosis with etoposide. In contrast, caspase-7 and, to a lesser extent, caspase-1 transcripts were increased before and after induction of apoptosis of U937 ϩ cells with etoposide. These data show that expression of caspases belonging to distinct subfamilies is differentially regulated by ICSBP.
Our studies also revealed that caspase protein expression is specifically modulated in U937 ϩ cells. First, activation of caspase-2 was enhanced in U937 ϩ cells. Second, although caspase-3 transcripts were not increased in cells overexpressing ICSBP, levels of the caspase-3 precursor protein were significantly increased. Finally, caspase-1 precursor levels were higher in U937 ϩ than in control cells before and after induction of apoptosis with etoposide. These results clearly show that increased expression of caspase-1, -3, and -7 occurred in U937 ϩ cells in association with enhanced sensitivity to spontaneous and induced apoptosis. Although we have no direct evidence at present for the transcriptional or translational control of caspases by ICSBP, the enhanced levels of one or more could account for the priming of a cascade that leads to the effector phases of apoptosis.
Cell death is regulated by a number of genes that influence cell survival in either a positive or negative fashion. The Bcl-2 family consists of molecules that can either promote survival or augment programmed cell death (64,65). The capability of type I and type II IFNs to modulate the expression of genes belonging to the Bcl-2 family is a controversial issue (66,67). Recent reports revealed that IFN-␥ significantly inhibits eosinophil apoptosis but does not upregulate Bcl-2 expression (68). In addition, IFN-␣2 is reported to inhibit the growth of myeloma cells in a manner independent of Bcl-2 and Bcl-X L expression (69). On the other hand, a recent report has shown that leukemia inhibitory factor induces Bcl-X L mRNA via a STAT1-binding cis-element in cardiac myocytes, preventing a cytoprotective effect (70). We found that U937 ϩ cells exhibited a specific decrease in Bcl-X L transcripts. Moreover, our data show that Bcl-X L can be strongly induced by IFN-␣ and IFN-␥. Finally, our results clearly demonstrate that a consensus ISRE present in the promoter region of Bcl-X L is sufficient to confer efficient IFN-␣ inducibility. Surprisingly, Bcl-2 expression was not affected in U937 ϩ cells. These results argue for a Bcl-2-independent but Bcl-X Ldependent mechanism of apoptosis regulation by ICSBP. In our model, downregulation of Bcl-X L could promote caspase-1-dependent disruption of the mitochondrial inner transmembrane potential and the subsequent activation of caspase-3 and -7. Recently, posttranscriptional modifications of some Bcl-2 family members have been shown to regulate cell death (71,72). Because Bcl-X L by itself is able to form ion channels in membranes (73) and is present in both soluble and membrane-bound forms, it would be interesting to determine whether mRNA expression, posttranscriptional modifications, and/or subcellular localization of this antiapoptotic protein represent important steps in the pathway by which IRF family members regulate cell death. Furthermore, because the functional relationship between the Bcl-2 family of apoptotic modulators and the caspases remains unresolved, our model might be useful for elucidating specific interactions between these families of proteins.
Although the precise mechanism by which ICSBP modulates expression of genes involved in apoptosis remains to be elucidated, our data argue for a specific proapoptotic activity elicited in U937 cells overexpressing ICSBP. In addition, studies of ICSBP-deficient mice strongly support the involvement of this factor as a regulator in the apoptotic pathway of myeloid cells whose leukemic transformation might follow escape from normal apoptotic controls; however, the possible relations between the myeloid disease of ICSBP mice and human CML remain to be clearly defined. We also found that the proliferative rate of bone marrow cells from ICSBP Ϫ/Ϫ mice was increased, suggesting that enhanced proliferation as well as prolonged survival may contribute to this myeloid disorder.
We thank Dr. Luigi Aloj for assistance in primer preparation, Dr. Paola di Marzio for experimental advice, Dr. York Tomita for assistance in graphics, and Dr. Pierre A. Henkart for providing the caspase inhibitors ZVAD-, BD-, and ZFA-FMK and for stimulating discussion and critical review of the manuscript. We also thank Dr. Ted Torrey for helpful comments and Brenda Rae Marshall for superb editorial assistance in the preparation of the manuscript.


extfieldsofstudy: Biology Medicine

text:
Inhibition of Cyclooxygenase-2 Gene Expression by p53*

Oncogenes enhance the expression of cyclooxygenase (Cox)-2, but interactions between tumor suppressor genes and Cox-2 have not been studied. In the present work, we have compared the levels of Cox-2 and the production of prostaglandin E2 in mouse embryo fibroblasts that do not express any p53 ((10)1) versus the same cell line ((10.1)Val5) engineered to overexpress wild-type (wt) p53 at 32 °C or mutant p53 at 39 °C. Cells expressing wt p53 showed about a 10-fold decrease in synthesis of prostaglandin E2 compared with those expressing mutant p53. Levels of Cox-2 protein and mRNA were markedly suppressed by wt p53 but not by mutant p53. Nuclear run-offs revealed decreased rates of Cox-2 transcription in cells expressing wt p53. The activity of the Cox-2 promoter was reduced by 85% in cells expressing wt p53 but was reduced only by 30% in cells expressing mutant p53 compared with cells null for p53. The effect of p53 on the suppression of Cox-2 promoter activity was localized to the first 40 base pairs 5′ from the transcription start site. Electrophoretic mobility shift assay revealed that p53 competed with TATA-binding protein for binding to mouseCox-2 or human Cox-2 promoter extending from −50 to +52 base pairs. The results of this study suggest that interactions between p53 and Cox-2 could be important for understanding why levels of Cox-2 are undetectable in normal cells and increased in many tumors.

Two different isoforms of cyclooxygenase (Cox) 1 catalyze the synthesis of prostaglandins from arachidonic acid. Cox-1 is a constitutive enzyme, whereas Cox-2 is inducible (1,2). The Cox-2 gene is an early response gene that is rapidly induced by phorbol esters, cytokines, growth factors, and carcinogens (3)(4)(5)(6)(7). The different responses of the genes encoding Cox-1 and Cox-2 reflect, at least in part, differences in the regulatory elements in the 5Ј-flanking regions of the two genes (8).
Numerous studies support the idea that Cox-2 is important in carcinogenesis. It is known, for example, that Cox-2 is upregulated in transformed cells and in various forms of cancer (9 -16). A null mutation for Cox-2 markedly reduces the number and size of intestinal tumors in a murine model of familial adenomatous polyposis, i.e. APC ⌬716 knockout mice (17). Cox-2 deficiency also protects against the formation of extraintestinal tumors. Thus, Cox-2 knockout mice developed about 75% fewer chemically induced skin papillomas than control mice (18), and a selective inhibitor of Cox-2 caused a nearly complete suppression of azoxymethane-induced colon cancer (19). It is also known that the expression of Cox-2 is enhanced by oncogenes (9 -11), but the effects of tumor suppressor genes, e.g. p53, on Cox-2 are unstudied.
p53 is important in the suppression of cellular growth and transformation (20 -24). In addition, induction of wild-type (wt) p53 can cause cells to undergo apoptosis (25,26). By contrast, inactivation of p53 can lead to deregulation of the cell cycle and DNA replication, selective growth advantage, and tumor formation (27,28). Significantly, p53 can either increase or suppress the expression of a number of target genes (29 -32). Gene activation usually involves binding of p53 to specific consensus sequences. Recent studies indicate that repression of transcription by p53 may involve interaction with basal transcription machinery and may be important in mediating programmed cell death (33)(34)(35)(36).
In the present work, we have investigated the effects of p53 on Cox-2 gene expression. Our data show that wt p53 causes a marked decrease in the expression of Cox-2. In contrast, mutant p53 did not cause a significant reduction in levels of Cox-2. Interactions between p53 and Cox-2 could be important for understanding why levels of Cox-2 are essentially undetectable in normal epithelial cells and elevated in many cancers.  1 The abbreviations used are: Cox, cyclooxygenase; bp, base pair(s); f.p.u., foot print unit; GST, glutathione S-transferase; PGE 2 , prostaglandin E 2 ; TBP, TATA-binding protein; wt, wild-type; SSPE, salinesodium phosphate-EDTA buffer; a.u. arbitrary unit. p53, with the temperature-sensitive p53 allele encoding valine at amino acid 135 (37,38). The (10.1)Val5 line contains a temperature-sensitive p53 mutant that is wt at 32°C and mutant at 39°C (38). PGE 2 Production-(10)1 and (10.1)Val5 cell lines were plated in 6-well plates and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37°C until they were approximately 40% confluent. The cell lines were then shifted to 32°C or to 39°C for 18 h. The medium was then replaced with fresh medium containing 10 M sodium arachidonate. 30 min later, the medium was collected to measure the synthesis of PGE 2 . The levels of PGE 2 released by the cells were measured by enzyme immunoassay (9). Levels of PGE 2 production were normalized to protein concentrations.

Materials
Western Blotting-Cell lysates were prepared as described previously (39). SDS-polyacrylamide gel electrophoresis was performed under reducing conditions on 10% polyacrylamide gels as described by Laemmli (40). The resolved proteins were transferred onto nitrocellulose sheets as detailed by Towbin et al. (41). The nitrocellulose membrane was then incubated with a goat polyclonal anti-Cox-2 antiserum or a polyclonal anti-Cox-1 antiserum. A secondary antibody to IgG conjugated to horseradish peroxidase was used. The blots were probed with the ECL Western blot detection system according to the manufacturer's instructions.
Northern Blotting-Total cellular RNA was isolated from cell monolayers using a RNA isolation kit from QIAGEN Inc. For Northern blots, 10 g of total cellular RNA per lane were electrophoresed in formaldehyde-containing 1.2% agarose gels and transferred to nylon-supported membranes. After baking, membranes were prehybridized for 8 h and then hybridized in a solution containing 50% formamide, 5ϫ SSPE, 5ϫ Denhardt's solution, 0.1% SDS, and 100 g/ml single-stranded salmon sperm DNA. Hybridization was carried out for 16 h at 42°C with radiolabeled murine Cox-2 and 18 S rRNA probes. After hybridization, the membrane was washed for 20 min at room temperature in 2ϫ SSPE and 0.1% SDS, washed twice for 20 min in the same solution at 55°C, and washed twice for 20 min in 0.1ϫ SSPE and 0.1% SDS at 55°C. Washed membranes were then subjected to autoradiography. The density of the bands was quantified by densitometry.
Nuclear Run-off-For each cell line, 2.5 ϫ 10 5 cells were plated in four T150 dishes and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum until they were approximately 50% confluent at 37°C. The cell lines were then shifted to 32°C for 18 h. Nuclei were isolated and stored in liquid nitrogen. For the transcription assay, nuclei (1.0 ϫ 10 7 ) were thawed and incubated in reaction buffer (10 mM Tris, pH 8, 5 mM MgCl 2 , and 0.3 M KCl) containing 100 Ci of [␣-32 P]UTP and 1 mM unlabeled nucleotides. After 30 min, labeled nascent RNA transcripts were isolated. The Cox-2 (TIS10) and 18 S rRNA cDNAs were immobilized onto nitrocellulose and prehybridized overnight in hybridization buffer. Hybridization was carried out at 42°C for 24 h using at least 5 ϫ 10 5 cpm of labeled nascent RNA transcripts for each treatment group. The membranes were washed twice with 2ϫ SSC buffer for 1 h at 55°C and then treated with 10 mg/ml RNase A in 2ϫ SSC at 37°C for 30 min, dried, and autoradiographed.
Transient Transfection Assays-Cells were seeded at a density of 5 ϫ 10 4 cells/well in 6-well dishes and grown to 30 -40% confluence at 37°C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, unless stated otherwise. For each well, 2 g of plasmid DNA were transfected into cells using the calcium phosphate precipitation method according to the manufacturer's instructions. After 16 h, the medium was changed, and cells were then shifted to 32°C or 39°C. At the end of the treatment period, cells were harvested, and luciferase activity was measured. Each well was washed twice with phosphatebuffered saline. 100 l of 1ϫ lysis buffer (Analytical Luminescence Laboratories, San Diego, CA) were added to each well for 15 min. The lysate was centrifuged for 1 min at 4°C. The supernatant was used to assay luciferase activity with a Monolight 2010 Luminometer (Analytical Luminescence Laboratories) according to the manufacturer's instructions. Luciferase activity is expressed per microgram of protein in the cell extract.
Preparation of GST.p53-Purified p53 protein was prepared as a GST fusion protein according to methods described previously (34).
Gel Shift Assays-A fragment of the human COX-2 or mouse Cox-2 promoter extending from Ϫ50 to ϩ52 encompassing the in vivo transcription initiation site was made by polymerase chain reaction amplification. After purification from agarose gels, the fragment was dephosphorylated using calf intestinal phosphatase and end-labeled with [␥-32 P]ATP using T4 polynucleotide kinase. The labeled probe was separated from unincorporated nucleotides using purification columns from QIAGEN Inc.
Gel shift assays were performed by preincubating TBP (1 f.p.u./ reaction), GST.p53 (100 ng), or a combination of both proteins in 9 l of 20 mM Hepes (pH 7.5), 70 mM KCL, 12% glycerol, 0.05% Nonidet P-40, 100 M ZnSO 4 , 0.05 M dithiothreitol, 1 mg/ml bovine serum albumin, and 0.1 mg/ml poly(deoxyinosinic-deoxycytidylic acid) on ice. After 15 min, 100 pg of the labeled fragment were added, and the reaction was incubated for an additional 10 min at 25°C. Changes in mobility were assessed by electrophoresis through a 5% polyacrylamide gel that was run at 250 V for 2 h. After fixation, the gel was autoradiographed.
Statistics-Comparisons between groups were made by the Student's t test. A difference between groups of p Ͻ 0.05 was considered significant. Fig. 1 show that cells containing wt p53 synthesized about 90% less PGE 2 than cells that are null for p53. By contrast, mutant p53 did not inhibit production of PGE 2 . To evaluate whether the inhibition of prostaglandin synthesis could be related to differences in the levels of Cox, Western blotting of the cell lysate protein was carried out. Fig. 2 shows that wt p53 caused a nearly complete suppression of Cox-2 expression, whereas mutant p53 did not affect the levels of Cox-2. In other experiments, the suppressive effects of wt p53 on amounts of COX-2 protein were more modest. Cox-1 was not detectable by Western blotting in any of the cells tested. To further elucidate the mechanism responsible for the changes in the amount of Cox-2 protein, we determined the steady-state levels of Cox-2 mRNA by Northern blotting. Levels of Cox-2 mRNA were reduced in the presence of wt p53 but not mutant p53 (Fig. 3).

Wild-Type p53 Inhibits the Synthesis of Prostaglandins and Expression of Cox-2-The data in
Cox-2 Transcription Is Suppressed by Wild-Type p53-Because differences in the levels of mRNA could reflect altered rates of transcription or changes in the stability of mRNA, nuclear run-offs were performed to distinguish between these possibilities. As shown in Fig. 4, we found lower rates of synthesis of nascent Cox-2 mRNA in cells expressing wt p53.
To further investigate the importance of p53 in modulating p53 Inhibits Cyclooxygenase-2 Expression the expression of Cox-2, transient transfections were performed using a murine Cox-2 promoter luciferase construct. The activity of the Cox-2 promoter was reduced by about 85% in the cell line expressing wt p53 compared with the p53-null cell line (Fig. 5). In contrast, mutant p53 caused about a 30% decrease in Cox-2 promoter activity (Fig. 5). We also investigated whether p53 could inhibit v-src-mediated induction of Cox-2 promoter activity. As shown in Fig. 6, overexpressing v-src caused about a 6-fold increase in Cox-2 promoter activity in the p53-null cell line. The stimulatory effects of v-src were blocked by overexpressing wt p53 in the same cell system. In separate experiments, wt p53 also blocked phorbol ester-mediated induction of Cox-2 promoter activity (data not shown).
Localization of the Promoter Region Responsible for Repression of Transcription-We next attempted to define the region of the Cox-2 promoter that responded to p53. This was accomplished using a series of murine Cox-2 promoter deletion constructs. As shown in Fig. 7, Cox-2 promoter activity decreased as the length of the promoter decreased. However, with all promoter constructs including TIS10 -40, the activity of the Cox-2 promoter was higher in the p53-null cell line compared with the cell line expressing wt p53. The fact that these differences in promoter activity were maintained despite changes in promoter length indicates that the suppression of Cox-2 promoter activity by p53 was localized to the first 40 bp 5Ј of the transcription start site.
TBP is essential for the transcription of TATA-containing promoters such as the Cox-2 promoter. p53 has been reported to inhibit gene expression via interactions with TBP (33-35). To study possible interactions between p53 and TBP in the FIG. 6. Wild-type p53 suppresses v-src-mediated induction of Cox-2 promoter activity. (10)1 cells, which are null for p53, were grown under serum-free conditions. Cells were transfected with 1.0 g of Cox-2 luciferase construct TIS10L (963 bp) alone (Ϫp53, Ⅺ) or with 0.5 g of expression vector for wt p53 (ϩp53, Ⅺ). Cells were also co-transfected with 1.0 g of TIS10L plus 0.5 g of expression vector for v-src (Ϫp53, f) or 1.0 g of TIS10L plus 0.5 g of expression vectors for v-src and wt p53 (ϩp53, f). The total amount of DNA was kept constant at 2 g by using empty vector DNA. Twenty four h later, luciferase activity was measured. Six wells were used for each transfection condition. Columns, means; bars, S.D.
p53 Inhibits Cyclooxygenase-2 Expression regulation of Cox-2 promoter activity, gel retardation assays were performed. In these experiments, we used a series of labeled 100-bp fragments of murine and human Cox-2 promoter spanning from Ϫ400 to ϩ52 bp together with a GST.p53 fusion protein or purified TBP, or both. We did not observe binding of GST.p53 or TBP to regions of the Cox-2 promoter spanning from Ϫ400 to ϩ52 (data not shown). Binding was only observed with a DNA fragment (from Ϫ50 to ϩ52) that included the TATA box (Fig. 8). Incubation of TBP or GST.p53 with the labeled probe led to two different retarded bands. Moreover, the formation of the TBP-DNA complexes was inhibited by the addition of increasing amounts of GST.p53. We did not observe the formation of complexes with purified GST alone (data not shown).

DISCUSSION
The present data show that the expression of Cox-2 is markedly repressed by wt p53 but not by mutant p53. This observation is important for several reasons. For example, suppression of Cox-2 expression by wt p53 can explain why levels of Cox-2 protein are undetectable in normal epithelial cells and, by contrast, why mutations of p53 may contribute to the increased expression of Cox-2 that is observed in malignant tissues (12)(13)(14)(15)(16). Possibly, the induction of Cox-2 by oncogenes (9,11), growth factors (42), and cytokines (6) may be enhanced in cells expressing mutant p53. Moreover, increased expression of Cox-2 after the mutation of p53 could be an important link in the relationship between the function of p53 and cancer. Thus, the products of Cox-2 activity, i.e. prostaglandins, stimulate cell proliferation (43), inhibit immune surveillance (44,45), increase the invasiveness of malignant cells (46), and enhance the production of vascular endothelial growth factor, which promotes angiogenesis (47). It is already known that transduction of cancer cells with wt p53 decreases the synthesis of vascular endothelial growth factor and inhibits tumor cellinduced angiogenesis (48). Therefore, the present data provide a possible mechanistic explanation for the regulation of expres-sion of vascular endothelial growth factor and angiogenesis by p53. It thus becomes important to determine the correlation between the expression of mutant forms of p53 and amounts of Cox-2 in malignant tissue. It will also be important to determine whether inactivation or loss of other tumor suppressor genes alters the expression of Cox-2.
The repression of transcription by p53 is believed to be important for p53-mediated apoptosis, but the mechanism underlying this effect is not understood (36). On the other hand, overexpression of Cox-2 in intestinal epithelial cells inhibits apoptosis (49). Because Cox-2 interacts directly with nucleobindin, which is an apoptosis-associated protein, Cox-2 might inhibit apoptosis by sequestering nucleobindin (50). The effects of Cox-2 on apoptosis might also be indirect via the modulation of levels of intracellular arachidonic acid as suggested by Chan et al. (51). Levels of arachidonic acid, in turn, regulate the production of ceramide (52), which can induce apoptosis. Independent of the exact mechanism, the finding that wt p53 downregulates the transcription of Cox-2 could be important for understanding p53-mediated apoptosis.
p53 suppresses a variety of promoters that contain TATA elements (30,33,35). This suppression is thought to occur through direct interaction with components of the basal transcription machinery, such as TBP. For example, in prior studies, p53 inhibited the binding of TBP to several promoters, most probably through protein-protein interactions (33)(34)(35)53). Consequently, TBP is no longer able to assemble a functional transcription initiation complex. In the current study, p53 inhibited the formation of complexes between TBP and the murine and human Cox-2 promoters in a cell-free system. One possible explanation for this result is a direct competition between p53 and TBP for the TATA binding site. Interactions between TBP and p53 could decrease the rate of Cox-2 transcription, but this needs to be confirmed in intact cells.