The human p53 expression vectors were obtained from Dr. Bert Vogelstein (19), while the murine p53 expression vectors were obtained from Dr. Moshe Oren ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1993by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 268, No. 20, Issue of July 15, pp. 1509&15100,1993 Printed in U.S.A.
Modulation of the Human Interleukin-6 Promoter (IL-6) and Transcription FactorCIEBPP (NF-IL6) Activity by p53 Species* (Received for publication, February 8, 1993, and in revised form, April 6, 1993)
Lola MarguliesS andPravin B. SehgalSQ From the Department of $Microbiology and Immunology and §Medicine, New York Medical College, Valhalla, New York 10595
Constitutiveup-regulation of interleukin-6 (IL-6) The contribution of mutations in p53, a frequent occurrence gene expression is observed in many neoplastic cell in neoplastic cells (reviewed in Refs. 9 and lo), toalterations lines. The contribution of mutations inp53 to the up- in the synthesis of cytokines by tumor cells or in the response regulation of the IL-6 promoter was evaluated in tran- of tumor cells to cytokines remains largely unexplored. sient transfection experiments. InHeLa cells, wildTranscriptional modulation of target genes by p53 is under type (wt) humanormurine p53 preferentially re- intensive investigation in many laboratories (11-27). On the pressed the IL-6 promoter. The p53 mutants Val-135 one hand, wild-type ( w t ) p53 can activate transcription from and Phe-132 up-regulated IL-6 promoter activity in a class of promoters that contain p53-binding sites either in these cells at both 32.5 and 37 “C. The temperaturesensitive Val-135 mutant was not only not inhibitory the native promoter (e.g. the muscle-specific creatine kinase or “wt-like”at the lower temperature, but had gained promoter) or as engineered binding sites (11, 14-16, 18, 19, a transcriptional activator phenotype whichwas tem- 22, 23, 26, 27). On the other hand, wt p53 can repress transcription from promoters such as interleukin-6, c-fos, c-myc, perature-independentinHeLa cells. Thefunctional DNA target for transcriptional modulation of the IL-6 @-actin, amajor histocompatibility gene, the retinoblastoma promoter by p53 species included the multiple cyto- gene, proliferating cell nuclear antigen, the multidrug resistkine- and second messenger-response element (- 173 ance gene, and numerous viral promoters (13, 17, 24, 25, 28). The ability of wt p53 to bind to andblock the function of the to -146); point mutations in the transcription factor C/EBPB-bindingsite within the second messenger-re- TATA-binding protein hasbeen suggested to be a mechanism sponse element largely blocked the ability ofp53 mu- contributing to repression by p53 of several different protants Val-135 and Phe-132 to up-regulate this pro- moters (29). Transcriptional modulation by p53 is, in turn, moter. The up-regulation of IL-6 promoter constructs regulated by interactions between p53 and other proteinssuch by co-transfection intoHeLa cells of a C/EBP@consti- as the simian virus 40 (SV40) large T antigen, the papillotutive expression vector was blocked in a dominant mavirus E6 oncoprotein, or the murine double minute-2 pronegative manner by wt p53. In contrast, the p53 mu- tein (MDM-2) (12, 20, 21, 30). tants Val-135 and Phe-132 further enhancedC/EBPj3Mutations in tumor-derived p53 species have been found to mediated up-regulation of IL-6 promoter constructs. lead to a reduction or loss of function in assays for transcripThemodulationofC/EBPj3 function by p53 species tional activation or repression (11-27). As an example, the provides a basis for the involvement of p53 not only in murine p53 Val-135 mutant, which is temperature-sensitive the regulation of cytokine synthesis but also in the (ts) for its effects oncell growth (inhibitory or wt-like at altered responsivenessof tumor cells to cytokines. 32.5 “C and stimulatoryor mutant-like at 37 “C) (31-33), was observed to be ts for enhancing transcription from the MCK promoter andfrom p50-2, a reporter constructthat contained Tumor cells often display aberrations in cytokine synthesis two copies of the 50-nucleotide p53-binding element from (1-4). As an example, “constitutive” production of interleu- within the MCK promoter (27). p53 Val-135 enhanced trankin-6 (IL-6)’ has been observed in various neoplastic cell lines scription at 32.5 “C (thew t phenotype), butwas less active at derived from multiple myeloma cells, other B cell dyscrasias, 39 “C (the mutant phenotype). Serum-induced c-fos mRNA renal and ovarian carcinomas, and Kaposi’s sarcoma (1-6). expression in a rat fibroblast cell line stably transfected with In many of these instances, the tumor cells also display an a Val-135 expression vector was inhibited at 32.5 “C but not alteration in the responsiveness to cytokines. For example, at 37 “C, suggesting that this mutant may also have a ts oncostatin M and IL-6 contributeto enhanced cell prolifera- phenotype with respect to transcriptional repression (17). In a previous study, we reported the transcriptional represtion in Kaposi’s sarcoma cell lines (6). In contrast, proliferation of breast carcinoma cell lines is inhibited by IL-6 (7, 8). sion of the IL-6 promoter by wt p53 and the relief of this repression by transforming mutations (SCX3 and c5) (24). * This work was supported by National Institutes of Health Grant We now describe the up-regulation of the IL-6 promoter by AI-16262 and a contract from the National Foundation for Cancer the p53 mutants Val-135 and Phe-132. An intact C/EBP@Research. The costs of publication of this article were defrayed in binding site in the IL-6 promoter (34-40) was a requirement part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18U.S.C. Section for up-regulation of the IL-6 promoter by these p53 mutants suggesting that the transcription factor C/EBPfi might be a 1734 solelyto indicate this fact. The abbreviations used are: IL, interleukin; Pgal, P-galactosidase; target for p53 modulation. In functional experiments, wt ~ 5 3 CAT, chloramphenicol acetyltransferase; C/EBP, CAAT-enhancer blocked transcriptional activation of the IL-6 promoter by C/ binding protein; CMV, cytomegalovirus; LTR, long-terminal repeat; ESP& In contrast, the p53 mutant species Val-135 and PheMCK, muscle-specific creatine kinase; MRE, multiple cytokine- and second messenger-response element; NF-IL6, nuclear factor-interleu- 132 enhanced C/EBP@-mediated gene activation. These data kin-6; RSV, Rous sarcoma virus; TK, herpes virus thymidine kinase; identify a novel mechanism by which the p53 species could affect cytokine synthesis and function. ts, temperature-sensitive; wt, wild-type.
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IL-6 Modulation byp53 MATERIALS ANDMETHODS
Cell Culture and Transient DNA Transfection Assays-HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented as described (35). Procedures for DNA transfection into cells using the calcium-phosphate method were essentially as described (24, 35, 36, 38). The plasmid pRSV(3gal (5 bg) was included as a marker for overall effectiveness of transfection and to control for the effects of p53 on basal transcription factors. In all experiments, transfected cultures were kept in serum-containingmedium (5%,v/v, fetal bovine serum; GIBCO) for 24 h, then depleted of serum for 20 h, followed by a “serum induction” (20% v/v) of the IL-6 promoter constructs for 20-22 h. For temperature shiftexperiments, all cells were initially transfected at 37 “C for 24 h and then shifted to 32.5 “C. Cells were harvested for preparation of extracts approximately 66-70 h afterthe beginning of transfection. All cell extracts were first assayed for protein content using a microassay (Bio-Rad) and for P-galactosidase (opal) activity as described previously (24, 35, 36, 38). Two sets of chloramphenicol acetyltransferase (CAT) assays were performed for each experiment: in one set,the totalprotein in extracts was used as the basis for normalization between different experimentalgroups, in the other, &galactosidase activity was used as thebasis for normalization between extracts. Following thin layer chromatography and autoradiography, spots corresponding to acetylated and nonacetylated [14C]chloramphenicolwere cut out, and the radioactivity was measured by liquid scintillation spectroscopy. Fold activationor repression was calculated as described earlier (24, 35,36, 38). All transfection experiments were repeated at least three times, and the data presented represent consistently replicable observations. DNA Phmids-The various CAT reporter constructs and control plasmids used in this study are listed in Table I. The various IL-6 constructs have been described earlier (24,35,36, 38) and were prepared by Dr. Anuradha Ray. pTKC-los and pTKC-, constructs were those described by McKnight and colleagues (41,42). p50-2 was obtained from Dr. Arnold Levine (27). The various p53 expression vectors, the C/EBP@expression vector (pNF-ILG), and various control plasmids used are listed in Table 11. The human p53 expression vectors were obtained from Dr. Bert Vogelstein (19), while the murine p53 expression vectors were obtained from Dr. Moshe Oren (31, 32). pNF-IL6 was obtained from Dr. S. Akira (34, 43). RESULTS
Activation of IL-6 PromoterConstructs by Mutant p53 Specks in HeLa Cells Fig. 1 summarizes the effect of overexpression of wt p53 species and of various mutant p53 species on IL-6 promoter activity. The wt p53 (human and murine) strongly repressed the IL-6 promoter in pIC225, mutants SCX3 andc5 no longer repressed this promoter, and mutants Val-135 and Phe-132 consistently enhanced IL-6 promoter activity. It is noteworthy that, in HeLa cells, the “ts” mutant Val-135 did not display a TABLEI IL-6/CAT and other reporter constructs used in the present study Plasmid
pIC225 pmRCE-1 pmRCE-2 pmNF-KB pInrC pAR12TKC PTKC-los pTKC-W p50-2 pCHllO pRSVBgal
Comment
-225 to +13 IL-6/CAT pIC225 with CG to GTmutation in MRE-1 at -161, -162 pIC225 with TG toCA mutation in MRE-2 a t -153, -154 in C/EBP site pIC225 with GGG to AAT mutation in NFKBsite at -71, -72, -73 -60 to +13 IL-6 TATA and Inr site/CAT IL-6 MRE (-173 to -145) in PTKC-~, -105 herpes TK with CAAT box (C/EBPbinding site)/CAT -80 herpes TK with CAAT box-deleted/CAT 2 X 50-bp p53-binding element from MCK/ CAT SV40 early enhancer-promoter/pgal (used occasionally) RSV LTR/Bgal (used routinely)
15097
TABLEI1 p53 and other expression vectors used (unless stated otherwise driven by cytomegalovirus (CMV) early enhuncer/promoter) Plasmid Comment Human p53, wt Human p53, transforming mutant (Val-143 to Ala) Murine p53, wt Murine p53, transforming mutant (Glu-168 to Gly; Met-234 to Ile) p53Va1135 Murine p53, transforming mutant with “ts” phenotype, Harvey sarcoma virus LTR promoter p53Phe132 Murine p53, transforming mutant, “non-ts”phenotype, Harvey sarcoma virus LTR promoter Human NF-IL6 (alias CIEBPP) invector pCDM8 pNF-IL6 Vector control derived from p53-SN3 by removal of pCMV p53 cDNA insert; used as a control to adjust total transfected DNA amounts CMV promoter vector with expression cloning site inpCDM8 tact pSVneo Additional control plasmid used to adjust total transfected DNA amounts in experiments with p53 Val135 or ~ 5 Phe-132 3 p53-SN3 p53-scx3 pCMVNc9 pCMVc5
“wt-like” or inhibitory phenotype at 32.5 “C (compare panels A and B in Fig. 1). We have previously identified the segment in the IL-6 promoter from -145 to -173 as acomplex “multiple cytokineand second messenger-responsive enhancer” (MRE) that is responsive to a wide variety of inducers (e.g. serum, interleukin-1, tumor necrosis factor, phorbol ester, or forskolin) (3538). The IL-6 MREcontainspartially overlapping motifs MRE-1(-173 to -151) and MRE-2 (-158 to -145) that each mediate response to all of these stimuli (38). Significantly, MRE-2 corresponds to thetranscription factor CIEBPP (alias NF-IL6)-binding site (34, 38). The IL-6 MRE confers cytokine responsiveness on the herpes virus thymidine kinase promoter (TK-1~5)(36-38). This construct, pARlBTKC, was used in assays designed to investigate the effect of p53 species on the isolated IL-6 MRE (Fig. 2). Humanand murine wt p53 species strongly repressed expression from the reporter construct pARlSTKC, but the mutants SCX3 and c5 did not. Fig. 2, B and C, shows that whether CAT assays were normalized to total protein (panel B ) or to P-galactosidase activity (panel C ) in the cell extracts, up-regulation of pAR12TKC by both p53 Val-135 and Phe132 was observed at both temperatures.
Effect of p53 Val-135 on Reporter Construct p50-2 in HeLa Cells Because a ts phenotype of p53 Val-135 wasnot observed by us in HeLa cells using IL-6 reporter constructs,we tested the effect of Val-135 on reporter construct p50-2 in these cells. This reporter construct contains two copies of a 50-base pair p53-binding DNA element from within the muscle-specific creatine kinase promoter (MCK) (27). Zambetti et al. (27) have previously shown that p50-2 transfected into rat fibroblasts was up-regulated by w t p53 and by Val-135 at 32.5 “C, but less so at 39 “C. As expected (Fig. 3), wt p53 enhanced expression from p50-2 at both 32.5 and 37 “C. Strikingly, in HeLa cells, Val-135 resulted in a higher level of activation of p50-2 than did wt p53 at both temperatures. Although Val135 was not ts in HeLa cells, additional experiments using both the p50-2 and IL-6 reporter constructs verified that p53 Val-135 was clearly ts in CV1 cells in our hands (data not shown). Mechanism of Modulation of the IL-6 Promoter by p53 Species The DNATarget-In preliminary experiments, we used the DNA-binding immunoprecipitation assay (44) employing 32P-
IL-6 Modulation by p53
15098
B
A mwt.. .._. - mc5.-
hSCX3 hwt
~ o " r w " + + + + +
Fold repression
+ + + + + +
+ + +
Serum -
Temp 32 37 32 37 32
Val135 Phe132 p53
.
pIC225
37 32 37 32 37 32 37 5.3 8.6 1.6 1.3 10.513.1 1.0 1.3
32 37
32 37 32 37
1.8 3.2 1.5 2.4 Fold activation
FIG.1. Modulation of the IL-6 promoter (-226 to +13) in construct pIC226 in HeLa cells co-transfected by various p53
expression vectors at different temperatures. HeLa cell cultures in 100-mm plastic Petri dishes were transfected with pIC225 (10pg)
and pRSVBgal(5 pg) together with each of various p53 expression vectors (5 pg each) or with 5 pg of the pCMV (panel A ) or pSVneo (panel B ) control DNA. Cultures were incubated in the absence (-) or presence of serum (+) a t 32.5 or 37 "C using the regimen described under "Materials and Methods." CAT assays were performed by normalizing between groups using &galactosidase activity. Fold repression and fold activation were calculated with respect to theserum-treated controls at thecorresponding temperature.
c PAR I 2TKC
B pAR12TKC
A PAR I2TKC
Phe I32 b
..,
. I ,
1
'
0"OO
3231323732
Temp
6 1.2 25.3 1.3
Fold change
37
5.9 5
3.1
2
3132
32 3237
3
2.1
JI
2.1
1.3
FIG. 2. Modulation of the function of the IL-6 MRE construct pAR12TKC in HeLa cells by various pS3 species at different temperatures. HeLa cell cultures were transfected with pAR12TKC (2.5 pg) and pRSV8gal (5 pg) together with 5 pg each of various p53
expression vectors or control plasmids (pCMV in panel A, pSVneo in panels B and C ) . Following transfection, the cells were incubated a t either 37 "C(panelA and as indicated in panels B and C ) or a t 32.5 "C. Panel A illustrates a CAT assay that was 8-galactosidase-normalized; panels B and C are, respectively, protein (200 pg/assay) or &galactosidase-based CAT assays camed outin the same experiment.
P53
mwt
Val135
Val135
32 37 3732 32 37 Temp 37 32 Fold activation 3.2 3.9 2.7 5.9 5.1 13.5 FIG.3. Modulation of the function of the pS3-binding MCK DNA element in construct p60-2 in HeLa cells by p53 species. HeLa cell cultures were transfected with p50-2 (10 pg) and pRSVagal (5 pg) together with various p53 expression vectors or the pCMV control DNA (5 pg each). Transfected cells were incubated a t either 32.5 or 37 "C as indicated. CAT assays were performed using volumes of cell extracts corresponding to 100 pg of protein/assay.
labeled segments of IL-6 promoter DNA, extracts from HeLa cells transfected with p53 expression vectors, and various anti-p53 monoclonal antibodies (PAb421, P1801, and PAb420), but were unable to detect specific binding of IL-6 promoter DNA fragments to p53-containing complexes. Therefore, we focussed our effort toward a series of functional
assays in order to gain insight into the mechanism(s) by whichp53species affect the function of the human IL-6 promoter. An evaluation of the effects of p53 species on a series of point-mutation constructs engineered within the context of the intactIL-6 promoter in pIC225 (-225 to +13) was informative. Whereas mutations in the NF-KBsite (pmNF-KB) and the MRE-1 element (pmRCE-1) reduced overall CAT signal strength derived from the IL-6 promoter in HeLa cells, they did not affect the ability of the reporter to be modulated by p53 species (repression by wt p53, up-regulation by Val-135) (data not shown). In contrast,a point mutation in MRE-2 in the C/EBPB (alias NF-IL6)-binding site (pmRCE-2)blocked modulation by p53 (Fig. 4). Additionally, the core IL-6 Inr/ CAT construct pInrC was neither repressed nor up-regulated by p53 species (data not shown). Taken together, the data pointed to the requirement for an intact C/EBPB site for modulation by p53 species. Modulation of Transcription Factor CIEBPB Alias NF-IL6 Function by p53 Species-As a direct extension of the DNA target studies, we evaluated the ability of p53 speciesto affect CIEBPB function. Fig. 5 shows that pNF-IL6 up-regulated the IL-6 promoter in pIC225. However, wt human p53 but not its mutantSCX3, inhibited C/EBPB-mediatedactivation of pIC225 in a dominant manner. Thus, transcription factor
IL-6Modulation by p53 C/EBPB was a functional target for repression by wt p53 in HeLa cells. Table I11 shows that C/EBPB function is further up-regulated by Val-135 and Phe-132 provided that the reporter constructs have an intact C/EBP-binding site. Table 111, experiment A, shows that pIC225, but not the C/EBPBsite mutant pmRCE-2, was up-regulated by C/EBPB in conjunction with Val-135 and Phe-132. The inability of Val-135 or Phe-132 to affect pmRCE-2, even in the presence of exogenously expressed CIEBPB, indicates that this transcription factor and itscognate binding site in the IL-6 promoter DNA are part of a major mechanism accounting for up-regulation of the IL-6 promoter by p53Val-135 and Phe-132. This inference was confirmedby the data in Table 111, experiment B, which demonstrate dramatic transcriptional activation effects of C/EBPB in association with p53 Val-135or Phe-132 on the herpes virus TK promoter, provided that the reporter construct included the C/EBP-binding site (PTKC-,~~) but not when this site was deleted (pTKC-so). DISCUSSION
Although wt p53repressed the IL-6 promoter, specific tumor-derived mutations in p53 (e.g. Val-135 and Phe-132) up-regulated this promoter. The p53 ts mutant Val-135 upregulated the IL-6 promoter at both 32.5 and 37 “C in HeLa cells. This phenotype defines a “gain in function” mutation in p53 becauseunder no experimental conditions did Val-135 or Phe-132 display the repression phenotype that is characteristic of wt p53 withrespect to the the human IL-6 promoter in these cells. Modulation of the function of transcription factor C/EBPB (alias NF-IL6) by wt p53 (inhibition) andby Val-135 (enhancement) was identified as a mechanism forthe effects of p53species on IL-6 gene expression. The data support the possibility that mutations in p53 may contribute
P53 pmRCE-2
Fold change 1.0 1.2 1 . 1 1.3 1.3 1.2 1.4 FIG. 4. Lack of modulation of the MRE-2 point mutant pmRCE-2 by p53 species and C/EBPP. HeLa cell cultures were transfected with pmRCE-2 (10 pg) and pRSV@gal (5 pg) together with p53 (5 pg) and/or C/EBP@(10 pg) expression vectors or pCMV control DNA (10 pg). CAT assays were performed by normalizing between groups based on @-galactosidaseactivity. FIG. 5. Inhibition of C/EBPB-mediated activation of the IL-6 promoter in pIC225 by wt but not mutant p63 species. HeLa cell cultures were transfected with pIC225 (10 pg) and pRSVBgal (5 pg) with or without additional p53 expression vector(huDNA man wt and/or its mutant SCXB) (each at 5 pg). In addition, varying amounts of pNF-IL6, an expression plasmid for C/ EBPB, or pCMV control DNA were also included in the transfections (DNA amounts indicated in micrograms). CAT assays werenormalized based on 8-palactosidase activity.
P53 pNF-IL6 2.5 5
15099
to dysregulated cytokine gene expression in tumor cells and alterations in the responsiveness of tumor cells to different cytokines. p53 Val-135 was originally reported to be ts for its effects on cell growth; in contrast, p53 Phe-132 was not ts in these assays (31,321. In rat fibroblast lines stably transfected with a constitutive p53Val-135 expression vector, proliferation was inhibited at 32.5 ‘C but not at 37 “C (31). As an extension of these observations, Ginsberget al. (17) reported that seruminduced c-fos mRNA expression in the p53 Val-135-transfected rat cell line was inhibited at 32.5 “C, but not at 37 “C. These investigators inferred from this ohservation that p53 Val-135 had a ts phenotype with respect to transcriptional repression of the c-fos promoter independent of the effects of Val-135 on cellproliferation (31, 32). Thus, in this system, it was inferred that p53 Val-135 hadthe “wt” or transcriptional repression phenotype at 32.5‘C withrespect to the c-fos promoter. The datapresented in this article reveal that transcriptional modulation by p53 Val-135 or Phe-132 is not temperaturedependent in HeLa cells. The IL-6 promoter and the p50-2 reporter construct were both up-regulated at both 32.5 and 37 “C. This is in contrast to the effects of authentic wt p53 which repressed the IL-6 promoter but activated the MCK promoter-derivative p50-2. Thus, in HeLa cells, the phenotype of p53 Val-135 at 32.5 “C was wt-like only with respect to p50-2. The mutants p53 Val-135 and Phe-132 had gained a transcriptional activator phenotype with respect to the IL6 promoter. The biochemical basis for why wt p53 activates the p50-2 promoter construct butrepresses the IL-6 construct is unclear. A possibility to consideris that wt p53may transcriptionally activate those promoters that contain a direct p53-DNA-binding site (as in the p50-2 promoter), but repress those that do not have a p53-DNA-binding site by indirectly interfering with the activity of transcription factors such as TATA-binding protein and CPBPB. The functional target for modulation by p53 in the IL-6 promoter DNA included the CIEBPB-binding site in MRE-2. Although, in preliminary experiments, we have been unable to detect specific direct binding of p53or p53-containing protein complexes to fragments of IL-6 promoter DNA using the DNA-binding immunoprecipitation assay, the question of direct binding of p53species to the IL-6 promoter DNA remains open. The ability of mutant p53 speciesto up-regulate IL-6 promoter activity suggests a novel mechanism forthe “constitutive” up-regulation of cytokine synthesis seen in many neoplastic cell lines. Furthermore, that p53 species functionally modulate the transcription factor C/EBPB ties p53 into a large body of work that relates to the response of cells to cytokines and to cell differentiation in general. C/EBPB is the major transcription factor involvedin activating gene I
human wt
+I
human s C x 3 + ;
1 _ 0_ , 0 2.5 5 10; 0 2.5 5
IO
“m -”
PIC225
*”
Fold activation 4.1 2.8 2.7 Fold repression 7.3 7.8 7.6
0 0.6 2.7 3.6 5.9
5.5
I
5
7.5 10
pCMV 15 carrier
15100
IL-6 Modulation by p53
TABLE111 DNA sequence requirementsfor the effects of mutant p53 species and of C/EBPP on the IL-6 and TK promoters Fold increase in CAT activitf Reporter construct
CmBpB
p53 Phe-132
+CIEBPi3
p53 Val-135
+C/EBPB
Experiment A" pIC225 2.8 1.5 4.4 1.6 9.9 pmRCE-2 0.9 0.9 0.9 1.0 0.9 Experiment B 3.8 PTK-IOSC 13.1 77.2 3.5 68.9 pTK-WC 1.3 1.1 2.0 1.1 1.7 a Values for percent conversion of radiolabeled chloramphenicol to the acetylated forms in control cultures were 1.83,0.64,1.0, and 0.55% in cultures transfected with pIC225, pmRCE-2, pTK-,,,&, and pTK-WC, respectively. * In Experiment A, HeLa cells were transfected with pIC225 (10 pg) or the C/EBPP-binding site mutant pmRCE-2 (2.5 pg). In experiment B, HeLa cells received pTKC-,, (2.5 pg) or the C/EBPBbinding site deletion mutant pTKC-80 (2.5 pg). In addition, each culture received pRSVPgal(5 pg) together with various p53 expression vectors (5 pg each) and the additional inclusion oftheC/EBP@ expression vector pNF-IL6 in the transfection (10 pg). Control DNA plasmids used included pCMV (10 pg) to match the CMV-driven plasmid pNF-IL6 and pSVneo (5 pg) to match the p53 vectors; thus, all the cultures received the same amount of DNA in each transfection.
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