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[Cancer Research 62, 932-940, February 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Autocrine Interleukin-6 Production in Renal Cell Carcinoma

Evidence for the Involvement of p53

Laura S. Angelo, Moshe Talpaz and Razelle Kurzrock1

Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-6 is an autocrine growth factor for renal cell carcinoma (RCC). We sought to determine whether p53 regulates constitutive IL-6 production. RCC cell lines containing mutant (mut) p53 produced higher levels of IL-6 than those containing wild-type (wt) p53 (P < 0.05). Transfection of wt p53 into RCC cell lines bearing mut p53 (UOK 121LN) or wt p53 (A498 and ACHN) resulted in repression of IL-6 promoter chloramphenicol acetyltransferase activity (P < 0.05). Mutant p53 was either less effective at repressing IL-6 promoter activity (ACHN cells) or enhanced IL-6 promoter activity (A498 cells). A498 cells stably transfected with mut p53 produced higher levels of IL-6 than A498 cells transfected with an empty expression vector (P < 0.05). Electrophoretic mobility shift assays showed decreased binding of CAAT enhancer binding protein, cyclic AMP responsive element binding protein, ± nuclear factor-{kappa}B transcription factors to the IL-6 promoter in various RCC cell lines transfected with wt p53 (P < 0.05) but not in those transfected with mut p53. These data suggest that: (a) mutation of p53 contributes to the overexpression of IL-6 in RCC; and (b) wt p53 represses IL-6 expression, at least in part, by interfering with specific transcription factor binding to the IL-6 promoter.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-6 is a pleiotropic cytokine that plays a major role in the host’s response to injury or infection (1 , 2) . IL-6 has also been implicated in the pathogenesis and/or prognosis of several different tumors including multiple myeloma (3) , lymphoma (4, 5, 6) , ovarian cancer (7) , prostate cancer (8) , and RCC (9, 10, 11) .

In RCC, IL-6 serves as an autocrine growth factor (12, 13, 14, 15) . IL-6 is produced by a wide variety of normal cells upon stimulation and constitutively by various tumor cell lines (15, 16, 17, 18, 19, 20) . Normal kidney cells express low levels of IL-6 (16) . RCC cell lines and fresh tumor tissue have been shown to express much higher levels (15, 16, 17 , 20 , 21) . These findings suggest that the transcriptional pathways for IL-6 expression are constitutively activated in renal epithelial cells but are maintained at low basal levels by mechanisms that are disrupted in tumor tissue.

The molecular mechanism(s) allowing enhanced IL-6 expression in renal cell tumors has not been elucidated. However, it is known that IL-6 mRNA levels are increased by exposure of cells to cycloheximide, suggesting that a labile repressor protein maintains basal levels (22 , 23) . Of interest in this regard, Santhanum et al. (24) have demonstrated that p53 can repress transcription from the IL-6 promoter in an inducible system (HeLa cervical carcinoma cells, which are null for p53; Refs. 24, 25, 26 ). Furthermore, p53 mutations have been detected in 20–30% of primary kidney tumors and in 70–80% of metastatic tumors (27, 28, 29, 30, 31) , suggesting that mutations are associated with progression of RCC (32 , 33) .

On the basis of the above results, we sought to determine whether p53 mutations play a role in the deregulation of IL-6 expression in RCC cell lines. Our studies demonstrate that: (a) IL-6 levels are significantly higher in RCC cell lines expressing a mut p53 than those expressing wt p53; (b) wt p53 suppresses IL-6 promoter activity, whereas mutant p53 has a less suppressive or an enhancing effect; (c) stable transfection of various p53 mutants into RCC cell lines results in a substantial increase in IL-6 secretion; and (d) suppression of IL-6 promoter activity by p53 appears to be mediated through interference with several transcription factors important in IL-6 gene regulation (C/EBP, CREB, and NF-{kappa}B).


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Culture Conditions.
The A498 and ACHN cell lines were purchased from the American Type Culture Collection (Rockville, MD). A498 cells were grown in Eagle’s minimal essential medium (BioWhittaker, Walkerville, MD), nonessential amino acids, sodium pyruvate, and L-glutamine. ACHN cells were grown in Eagle’s minimal essential medium. The UOK cell lines are a series of renal tumor cell lines generously provided by Dr. W. Marston Linehan (National Cancer Institute, Bethesda, MD). UOK 121LN is a lymph node metastases of a primary RCC. The UOK cell lines were maintained in DMEM with high glucose plus L-glutamine. RPTEC cells are normal renal proximal tubule epithelial cells purchased from Clonetics (San Diego, CA). All cell lines were supplemented with 10% heat-inactivated FCS and maintained at 37°C and 5% CO2 in a humidified chamber.

Antibodies.
All p53-specific antibodies were purchased from Calbiochem (Cambridge, MA). Clone PAb 421 (Ab-1) is a pantropic antibody that recognizes both wild-type and mut p53 (34, 35, 36) . Clone PAb 1620 (Ab-5) recognizes a conformational epitope of wt p53 and therefore does not recognize mut p53 (37) . Clone PAb 240 (Ab-3) detects amino acid residues 212–217 of human p53. It is specific for mutant p53 in immunoprecipitation experiments (36 , 38) . All p53-specific antibodies are murine. The isotypic control for murine IgG2a antibodies was anti-Aspergillus niger glucose oxidase, an enzyme that is neither present nor inducible in mammalian tissues (DAKO Corp., Carpinteria, CA). A murine anti-thyroglobulin antibody was used as an isotypic control for IgG1 (DAKO).

Anti-p65 NF-{kappa}B, anti-p50 NF-{kappa}B, anti-C/EBPß, anti-C/EBP{delta}, and anti-jun antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). These antibodies are derived from rabbit polyclonal sera. Normal rabbit serum was used as a control.

ELISA.
To determine the amount of IL-6 protein and other inflammatory cytokines being produced by the cell lines, ELISAs were performed. Cells were plated at 1 x 106 per 100-cm2 dish. At confluency, cell culture supernatants were harvested, centrifuged, and placed at -20°C. A human-specific IL-6 ELISA (R&D Systems, Minneapolis, MN) was used to determine the amount of IL-6 present in the supernatant. All samples were run in duplicate and repeated three times. The lower limit of detection of the ELISA is 0.7 pg/ml. TNF-ß (lymphotoxin) levels were measured using an ELISA purchased from R&D systems as well (lower limit of detection, <7 pg/ml). IL-1{alpha}, IL-1ß, and TNF-{alpha} were measured by ELISAs purchased from Endogen Corp (Cambridge, MA; lower limit of detection <5 pg/ml, <2 pg/ml, and <1 pg/ml, respectively).

RT-PCR/DNA Sequencing.
RT-PCR followed by DNA sequencing was used to determine p53 status in the A498 and ACHN cell lines. Oligonucleotide primers used to amplify two overlapping regions of the p53 gene were used. RNA was extracted from the cells using the RNAzol reagent (Cinna Scientific, Friendswood, TX). One µg of RNA was reverse transcribed into cDNA using 1 µM of the reverse strand primer (GeneAmp RNA PCR kit; Perkin-Perkin-Elmer Corp., Norwalk, CT). PCR reactions were then carried out. PCR products were run on a 1% agarose gel, and bands of the appropriate size were excised from the gel and subcloned by ligation into the TA cloning vector (Invitrogen, Carlsbad, CA). Ligation reactions were used to transform One Shot bacterial cells (Life Technologies, Inc., Bethesda, MD), and ampicillin-resistant colonies were analyzed for the presence of PCR insert. Positive colonies were sent for sequencing at the core sequencing facility at University of Texas M. D. Anderson Cancer Center. Sp6 and T7 primers were used to sequence both strands of the DNA.

Expression Vectors and Reporter Constructs.
The human IL-6 promoter CAT construct extends 1.2 kb upstream of the transcription start site of the IL-6 gene (a generous gift of Dr. Towia Libermann, Harvard Medical School, Boston, MA; Ref. 39 ). Dr. Guillermina Lozano, University of Texas M.D. Anderson Cancer Center, Houston, TX, graciously provided the p53 expression vectors used in this work. PC53-SN contains the sequence for human wt p53 cDNA under the control of the human cytomegalovirus promoter/enhancer (40, 41, 42) . PC53-CX3 contains a temperature-sensitive mut human p53 (codon 143 GTG->GCG, Val to Ala). An empty p53 expression vector was used as a control for all transfection experiments. PG13 CAT and MG15 CAT served as positive and negative controls for p53 transactivation activity, respectively (generously donated by Dr. Bert Vogelstein, Johns Hopkins, Baltimore, MD; Ref. 43 ).

Transient Transfection.
Transient transfection of p53 into A498, ACHN (wt p53-expressing), or UOK 121LN (mut p53-expressing) was performed. Cells were grown on 100-mm dishes to 60% confluence. Cell lines were transfected using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate liposomal transfection reagent (Boehringer Mannheim, Indianapolis, IN) as described in the manufacturer’s instructions. IL-6 promoter construct (2.5 µg) was used per dish. In the figures shown, P53 plasmids were used at a concentration of 5.0 µg per 100-mm dish, as published previously (24 , 25) .

CAT Assays.
We transfected with multiple concentrations of p53 plasmid (2.5, 5.0, and 10.0 µg per 100-mm dish), and each CAT assay was performed three to five times with consistent results (44) . CAT assays were performed 48 h after transient transfection. The amount of cell lysate added to each reaction tube was normalized to total protein (44) . Protein in the cell lysates was quantified using the Bradford Protein assay (Bio-Rad, Hercules, CA). Typically, 100 µg were added to each reaction tube. Samples were loaded onto TLC plates and then exposed to X-ray film (Kodak). TLC plates were quantified using a PhosphorImager (Molecular Dynamics).

Immunoprecipitation and Western Blot Analysis.
Immunoprecipitation followed by Western blot analysis was used to determine the levels of p53 protein in the various cell lines and clones. For p53 protein expression, 5 x 106 cells were lysed in ice-cold 1x PBS-TDS (1x PBS, 1% TritonR X-100, 0.5% sodium deoxycholate, and 0.1% SDS). Immediately prior to use, 1 mM EDTA, 1 µg/ml pepstatin, and 0.2 mM phenylmethylsulfonyl fluoride were added. Samples were rotated for 10 min at 4°C and centrifuged to remove cell debris. Samples were immunoprecipitated with either a nonspecific isotypic control antibody or a p53-specific antibody for 2 h at 4°C. After immunoprecipitation, samples were loaded onto a 12% polyacrylamide gel. Recombinant human mut and wt p53 baculovirus lysates were purchased from PharMingen (San Diego, CA). Proteins were transferred to nitrocellulose membranes (Nitropure; Micron Separations, Inc.) using electroblotting. Blots were incubated with primary antibody at a concentration of 1.0 µg/ml. For p53 detection, blots were incubated with sheep anti-p53 (Ab-7; Calbiochem) for 1 h at room temperature. Blots were washed in Tris-buffered saline with 0.1% Tween 20, then probed with a rabbit antisheep biotinylated antibody, followed by streptavidin-horseradish peroxidase. p53 protein was visualized using the Enhanced Chemiluminescence (ECL) detection system (Amersham, Buckinghamshire, United Kingdom) and autoradiography.

To determine the effect of transfected wt p53 on c-jun levels in the cells, direct Western blot analysis was performed. Cells were lysed in 1.0% NP40R, 0.5% sodium deoxycholate, and 0.1% SDS. One hundred µg/ml phenylmethylsulfonyl fluoride, 63 µg/ml aprotinin, and 1 mM sodium orthovanadate were added as protease inhibitors. Total protein was measured using the DC protein assay (Bio-Rad). Twenty-five µg of total protein per lane were loaded onto 12% polyacrylamide gels. Gels were electroblotted onto nitrocellulose membranes and probed with 1.0 µg/ml antibody in TBS-T. ECL detection was performed.

EMSA.
To determine the effect of transfection of wt or mut p53 on the binding of various transcription factors to the IL-6 promoter, EMSA was performed. Nuclear extracts were prepared as described (45) . The Bradford protein assay (Bio-Rad) was used to determine the amount of protein in the extracts. All oligonucleotides were purchased from Genosys Biotechnologies (The Woodlands, TX). Single-stranded oligonucleotides were as follows: NF-{kappa}B anti-sense, 5'-CGGTATCATGGGAAAATCCCACA-3'; NF-IL6 sense, 5'-CGGTACATTGCACAATCT-3'; NF-IL6 antisense, 5'-CGGTAGATTGTGCAATGT-3'; MRE I long sense, 5'-CGGTATGCTAAAGGACGTCACATTGCA-3'; and MRE I long antisense, 5'-CGGTTGCAATGTGACGTCCTTTAGCAT-3'. Oligonucleotides were labeled with dCTP labeling mix (Amersham Pharmacia Biotech, Piscataway, NJ), 2 units of Klenow (Promega Corp., Madison, WI), and 50 µCi of [{alpha}-32P]dCTP (New England Nuclear, Cambridge, MA). Probes were then run through a G-50 Sephadex column (5'->3', Inc., Boulder, CO) to eliminate free nucleotides. Binding reactions consisted of 6 µg of nuclear extract, 10x binding buffer [20 mM HEPES (pH 7.9), 50 mM KCl, 1.0 mM EDTA, 1.0 mM DTT, and 5.0% glycerol], and 1 µg of poly(deoxyinosinic-deoxycytidylic acid). All supershift antibodies were purchased from Santa Cruz Biotechnology, Inc.

TUNEL Assay.
TUNEL assays were used to determine whether cells were dying of apoptosis after transient transfection of wt p53. Cells (1 x 105) cells were plated into flaskettes containing glass slides (Nunc, Naperville, IL). Cells were transfected with either empty p53 expression vector or wt p53, as described previously. Forty-eight h after transfection, slides were washed once with PBS, air-dried, and then fixed for 30 min at room temperature in 4% paraformaldehyde in PBS (pH 7.4). TUNEL reaction mixture (Boehringer Mannheim, Indianapolis, IN) was incubated with the slides for 60 min at 37°C in the dark. Slides were photographed using a fluorescent microscope.

Generation of Stable Transfectants.
A498 cells were grown in 100-mm dishes to 60% confluence prior to transfection. Ten µg of either empty p53 expression vector or mut p53 (pC53-CX3) plus Geneporter transfection reagent (San Diego, CA) were mixed and incubated together for 30–60 min at room temperature prior to placing on the cells. Forty-eight h after transfection, cells were placed in medium containing 200–300 µg/ml Geneticin (Sigma Chemical Co., St. Louis, MO). Medium was replaced every 3–4 days thereafter, and individual colonies were selected after ~2 weeks using cloning cylinders and vacuum grease. Clones were analyzed for expression of p53 and IL-6. Conditioned medium was harvested at 72 h after plating for IL-6 secretion. (The cells had a very steady doubling time, and 72 h consistently represented the time of confluency).

Statistical Analysis.
The statistical significance of changes in IL-6 production in RCC cell lines expressing either wt or mut p53 was determined by the Mann-Whitney test. The statistical significance of repression of the IL-6 promoter by wt p53 and of increased IL-6 production in A498 clones was also determined by the Mann-Whitney test. The statistical significance of changes in binding of transcription factors to the IL-6 promoter after transfection of p53 was determined using either a one-sided or two-sided t test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RCC Cell Lines Expressing a mut p53 Secrete More IL-6 Than Those Expressing a wt p53.
To determine the influence of p53 status on the level of IL-6 expression in RCC cell lines, we obtained several RCC cell lines containing either a wt or mut p53. The RPTEC cell line represents normal renal proximal tubule epithelial cells. Caki-1, A704, A498, and ACHN express wt p53, as reported previously (46) and/or RT-PCR analysis in our laboratory. The UOK series of cell lines all contain p53 mutations, as reported previously (30) . Table 1Citation shows the level of IL-6 protein in the conditioned medium of the various RCC cell lines at confluency. Conditioned medium from RPTEC cells contains a basal level of 218 pg/ml. Conditioned medium from the RCC cell lines expressing wt p53 contained a median level of 416 pg/ml, whereas the medium from the UOK cell lines (mut p53) contained a significantly higher median IL-6 level of 4,663 pg/ml (P < 0.05, Mann-Whitney test).


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  		Table 1 IL-6 levels in conditioned medium of RCC cell lines

 
RCC Cell Lines Do Not Secrete TNF or IL-1.
Several cytokines including IL-1{alpha}, IL-1ß, TNF-{alpha}, and TNF-ß can induce IL-6 (47) . We therefore analyzed several of our RCC cell lines (A498, ACHN, and UOK 121LN) for expression of these cytokines. These cell lines were also the ones selected for mechanistic studies of the effects of p53 on IL-6 expression (CAT assays and EMSAs). ELISAs performed on conditioned medium indicated that IL-1{alpha}, IL-1ß, TNF-{alpha}, and TNF-ß protein levels were below the lower limit of sensitivity of the assays. Therefore, secretion of these cytokines is unlikely to account for the high level of constitutive IL-6 expression by these cells.

wt p53 Inhibits IL-6 Promoter CAT Activity in RCC Cell Lines as Compared with Mutant p53.
We transiently cotransfected a full-length human IL-6 promoter CAT construct (39) and p53 expression vectors into our RCC cell lines to determine the effect of p53 on the constitutive activation of the IL-6 promoter. Transfection of wt p53 into wt p53-expressing A498 and ACHN cells resulted in a 50% decrease in IL-6 promoter CAT activity when compared to transfection with empty p53 expression vector (P < 0.05; Fig. 1, A and BCitation ). Transfection of wt p53 into the mut p53-expressing UOK 121LN cell line results in over 75% decrease in IL-6 promoter CAT activity when compared to transfection with empty vector (P < 0.05; Fig. 1CCitation ). Transfection of mut p53 (codon 143, Val to Arg) either had less of an inhibitory effect (ACHN cells) or actually enhanced IL-6 promoter activity (A498 cells; Fig. 1ACitation and Fig. 2Citation ). The data from these experiments are depicted graphically in Fig. 2Citation .



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  		Fig. 1. CAT assay showing the effect of transfection of p53 on the constitutive activity of the human IL-6 promoter in A498, ACHN, and UOK 121LN cells. Cells were transiently transfected with the full-length human IL-6 promoter construct either alone or in combination with empty expression vector, wt p53, or mut p53 (A498 cells only). A, A498 cells; B, ACHN cells; C, UOK 121LN cells. CAT enzyme refers to recombinant CAT enzyme and is used as a positive control for the assay. The results show that wt p53 significantly decreased IL-6 promoter activity in all cell lines tested (P < 0.05). mut p53 was a less effective repressor of IL-6 promoter activity in ACHN cells and enhanced IL-6 promoter activity in A498 cells.

 


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  		Fig. 2. Graphic representation of CAT assays. All TLC plates from CAT assays were quantified using a phosphorimager. The means of three to five experiments are shown; bars, SD. CAT activity was normalized to the empty vector control, which was assigned a value of 100%. The results show that wt p53 significantly (P < 0.05) decreased IL-6 promoter CAT activity in all three RCC cell lines tested.

 
We also cotransfected the cells with reporter constructs that are positive and negative controls for the function of wt p53 in the cells. As expected, wt p53 transactivated the PG13CAT reporter construct, which contains 13 p53 consensus binding sites (43) . MG15CAT, which contains 15 repeats of a mutated p53 consensus binding site, was activated weakly or not at all (Ref. 43 ; Fig. 3Citation ). These control experiments confirm that transfection of wt p53 resulted in appropriate transactivation in our cell lines, and that under these conditions wt p53 significantly suppressed IL-6 promoter activity, whereas mutant p53 had a less suppressive or an enhancing effect.



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  		Fig. 3. CAT assay showing transactivation of the PG13CAT vector by wt p53 after transient transfection into A498 cells (A) and UOK 121LN cells (B). The MG15CAT plasmid, which contains 15 mutated p53 binding sites, is not transactivated by wt p53 in A498 cells (A) and only weakly in UOK 121LN (B). These results show that p53 is present in the cells and capable of transactivating promoters containing p53 binding sites.

 
wt p53 Inhibits the Binding of C/EBP, CREB, and NF-{kappa}B Transcription Factors to the IL-6 Promoter in wt p53-expressing A498 Cells.
To determine whether transcription factors critical to IL-6 gene regulation were being affected by p53, EMSA was used. In Fig. 4Citation , wt p53-expressing A498 cells were transiently transfected with empty vector, wt, or mut p53 (143 Val). The MRE I, NF-IL6, and NF-{kappa}B binding sites of the human IL-6 promoter were examined in our study. [These sites are known to be important in the regulation of IL-6 gene expression (48) ]. Fig. 4ACitation shows transcription factor binding to the MRE I region of the IL-6 promoter in A498 cells. The MRE I contains both the C/EBP (NF-IL6) and the CREB protein binding sites (1) . A498 cells that have been transfected with wt p53 show decreased binding of CREBs and C/EBPs to the MRE I site when compared with those cells transfected with empty vector (Fig. 4ACitation , Lanes 3 and 4). EMSA were quantified using densitometry (Fig. 4E)Citation . Binding of CREB family members is decreased by 42% in A498 cells transfected with wt p53.



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  		Fig. 4. EMSA of various regions of the IL-6 promoter in A498 cells after transfection of either wt or mut p53. Cells were transiently transfected with empty expression vector, wt, or mut p53, and nuclear extracts were harvested 48 h later. Equal amounts of extract were added to binding reactions and loaded onto 6% native polyacrylamide gels in Tris glycine buffer. A, MRE. B, NF-IL6 site. C, NF-{kappa}B site. D, MRE and NF-{kappa}B site after transfection with mut p53. N.E., nuclear extract; + 50x cold, 50x cold competitor oligonucleotide; N.S., nonspecific. These results show that transfection of wt p53 into the A498 cell line results in decreased binding of transcription factors to various sites of the IL-6 promoter, whereas transfection of mut p53 does not result in a decrease in binding. E, densitometry of EMSAs from wt p53-expressing A498 cells transfected with empty expression vector, wt p53, or mut p53. Binding of each family of transcription factors was measured individually, with NF-{kappa}B divided into p50 homodimers and p50/p65 heterodimers. Binding of transcription factors upon transfection with empty expression vector was assigned a value of 100%. All EMSAs were performed two to six times, and mean values are shown; bars, SD. These results show that wt and mut p53 have differential effects on the binding of transcription factors to the IL-6 promoter in A498 cells.

 
Fig. 4BCitation shows C/EBP proteins bound to the NF-IL6 binding site located within the MRE. Three specific complexes are formed, as demonstrated by cold competition with unlabeled oligonucleotide (Fig. 4BCitation , Lane 7). Specific antibodies to C/EBP family members and a preimmune serum (negative control) were used to determine which of the C/EBP family members were binding to the NF-IL6 site in our cell line. The two upper complexes in Lanes 1 and 11 undergo a further shift in mobility after incubation with anti-C/EBP ß antibody (anti-NF-IL6; indicated by arrow 2). Thus, C/EBP ß (NF-IL6) is present in the top two complexes. There is no supershift after addition of anti-C/EBP {alpha} antibody, indicating that C/EBP {alpha} is not present in any of the complexes (Fig. 4BCitation , Lanes 3 and 9). However, incubation with anti-C/EBP {delta} antibody results in a supershift of all three complexes, indicating that C/EBP {delta} is present in all three complexes (arrow 1). Most importantly, binding of C/EBP ß and {delta} to the NF-IL6 site is decreased in A498 cells transfected with wt p53 when compared with cells transfected with empty vector (Fig. 4BCitation , Lanes 5 and 6). Densitometry revealed a 32% decrease in binding of C/EBP family members to the IL-6 promoter in A498 cells (Fig. 4ECitation ; P = 0.005 by one-sided t test).

NF-{kappa}B is also an important factor in the regulation of IL-6 gene expression (39 , 49) . It can bind C/EBP ß and synergistically up-regulate the IL-6 promoter (50) . To determine whether wt p53 had any effect on the binding of NF-{kappa}B to the IL-6 promoter in our cells, supershift analysis with antibody directed against the p50 and p65 subunits of NF-{kappa}B was performed. Both subunits of NF-{kappa}B are constitutively bound to the IL-6 promoter in our cells (Fig. 4CCitation , Lanes 1, 2, 7, and 8). Competition with unlabeled oligonucleotide demonstrates the specificity of the complexes (Fig. 4CCitation , Lane 5). Furthermore, binding of both NF-{kappa}B p50 and p65 is decreased in A498 cells after transfection with wt p53 (Fig. 4CCitation , Lanes 3 and 4). There is a 60% decrease in binding of p50p50 homodimers and a 49% decrease in p50/p65 complexes of NF-{kappa}B in A498 cells transfected with wt p53.

Fig. 4DCitation compares the effect of transfection of wt and mut p53 on the binding of transcription factors to the MRE I and NF-{kappa}B binding sites of the IL-6 promoter in A498 cells. The left panel is an EMSA using the MRE I as a probe. Lane 1 contains cold competitor oligonucleotide. Lanes 2, 3, and 4 show binding of C/EBP and CREB transcription factors to the MRE in cells transfected with empty vector, wt, or mut p53, respectively. Again, there is a substantial decrease in the binding of C/EBP (32% decrease; P = 0.005) and CREB (42% decrease; P < 0.05) transcription factors to the MRE upon transfection of wt p53. Transfection of mut p53 (Val 143) resulted in enhanced binding as determined by densitometry (see Fig. 4ECitation ). The right panel shows binding of NF-{kappa}B p50 and p65 after transfection with empty vector, wt p53, or mut p53. There is a 62% decrease in binding of p50/p50 and a 49% decrease in binding of p50/p65 complexes of NF-{kappa}B in A498 cells transfected with wt p53, whereas there are only minimal decreases in binding in cells transfected with Val 143 mut p53. These data demonstrate that wt and mut p53 have differential effects on the binding of transcription factors to the IL-6 promoter in A498 cells.

wt p53 Inhibits the Binding of C/EBP and CREB but not NF-{kappa}B to the IL-6 Promoter in mut p53-expressing UOK 121LN Cells.
We examined the effect of wt p53 transfection on the binding of transcription factors to the IL-6 promoter in mut p53-expressing UOK 121LN cells. When cells were transfected with wt p53, binding of the C/EBP family members was significantly decreased (P = 0.001 by one-sided t test). Binding of CREB proteins was also significantly decreased (P = 0.009 by one-sided t test; Fig. 5ACitation , Lanes 1 and 2, Lanes 4 and 5; and Fig. 5BCitation ). In contrast, binding of the p50 homodimer and p50/p65 heterodimer of NF-{kappa}B remained unaffected (Fig. 5B)Citation .



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  		Fig. 5. EMSA of the MRE and NF-{kappa}B region of the IL-6 promoter in mut p53-expressing UOK 121LN cells after transfection of empty vector or wt p53. A, binding of transcription factors to the MRE region of the IL-6 promoter. Two separate experiments are shown here (Lanes 1 and 2, and Lanes 4 and 5). Nuclear extracts from the Jurkat T cell line treated with phorbol myristate acetate were added as a positive control (Lane 3). Unlabeled oligonucleotide was added as competition in Lane 6. B, densitometry of EMSAs from mut p53-expressing UOK 121LN cells transfected with empty vector or wt p53. EMSA was performed two to six times, and mean values are shown. Films were quantified using densitometry. Binding of transcription factors in cells transfected with empty vector was assigned a value of 100%. Bars, SD. The results demonstrate inhibition of binding of C/EBP and CREB transcription factors but not NF-{kappa}B in UOK 121LN cells transfected with wt p53.

 
RCC Cell Lines Transiently Transfected with wt p53 Are Not Undergoing Apoptosis.
Both wt and mut p53-expressing RCC cell lines in our study showed a decrease in binding of various transcription factors to the IL-6 promoter after transfection with wt p53. To verify that this effect was not attributable to apoptosis of the cells, TUNEL assays were performed. HL-60 cells treated with UV radiation for 20 min and harvested after 4 h of incubation at 37°C were used as a positive control for the assay. These assays demonstrated that, at the time the cells were harvested for EMSAs (48 h), neither A498 nor UOK 121LN cells had undergone apoptosis (Fig. 6)Citation .



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  		Fig. 6. TUNEL assay of wt p53-expressing A498 cells and mut p53-expressing UOK 121LN cells after transfection with either empty expression vector or wt p53. Cells were plated onto glass slides, transfected, and then treated as described in "Materials and Methods." A, HL60 cells treated with UV light (positive control). B, untreated HL60 (negative control). C, A498 after transfection with empty vector. D, A498 after transfection with wt p53. E, UOK 121LN cells after transfection with empty vector. F, UOK 121LN cells after transfection with wt p53. The results show that neither RCC cell line undergoes apoptosis after transfection with wt p53.

 
c-jun Protein Levels Are Unaffected in UOK 121LN Cells Transfected with wt p53.
Because wt p53 did not affect binding of NF-{kappa}B family members to the IL-6 promoter in UOK 121LN cells, we sought to determine whether transfection of wt p53 was affecting some other aspect of IL-6 promoter regulation. Another site of the IL-6 promoter that may be affected by p53 is the AP-1 site (51 , 52) . wt p53 has been shown to down-regulate c-jun at the level of transcription (53) . Therefore, we examined UOK 121LN cells for changes in c-jun protein expression after transfection with wt p53. As demonstrated in Fig. 7Citation , Western blotting did not show significant changes in c-jun protein levels after transfection with either empty vector or wt p53.



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  		Fig. 7. Western blot showing the levels of c-jun protein in UOK 121LN cells transfected with either empty expression vector or wt p53. Cells were harvested 48 h after transfection and lysed as described in "Materials and Methods." Equal amounts of protein were loaded onto 12% SDS-PAGE gels, followed by blotting onto nitrocellulose membranes. Blots were probed with a human specific anti-c-jun antibody. Lane 1, A431 cell lysates served as a positive control; Lane 2, UOK 121LN cells transfected with empty vector; Lane 3, UOK 121LN cells transfected with wt p53. Densitometry results show that c-jun protein levels remain unchanged in UOK 121LN cells after transfection with wt p53.

 
Secretion of IL-6 Is Increased in A498 Cells Stably Transfected with mut p53.
On the basis of the observation that p53 can regulate the IL-6 promoter in RCC cell lines, stable transfectants of the wt p53-expressing A498 cell line were generated to determine whether overexpression of mut p53 contributes to the up-regulation of IL-6 production in this cell line. Fig. 8ACitation shows the results of IL-6 ELISAs performed on the conditioned medium of various clones stably transfected with mut p53 (143 Val). A clone stably transfected with empty p53 expression vector was generated as a control for the effect of transfection of the vector into the cells (Fig. 8Citation , A+E.1). A143.8, A143.6, A143.5, and A143.7 are all clones of the A498 cell line stably transfected with the 143 Val mut p53. Each clone produces significantly more IL-6 than the A+E.1 clone (P = 0.05). However, there does not appear to be a direct correlation between the amount of IL-6 secreted and the level of p53 protein (clone 421 p53 antibody) expressed in the clones (Fig. 8B)Citation .



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  		Fig. 8. A, levels of IL-6 produced by various A498 clones stably transfected with mut p53. Cells were transfected with mut p53 (143 Val), and resistant colonies were selected using G418. Clones were grown to confluency, and conditioned medium was collected. Human IL-6 ELISA was performed as described. The X axis shows analysis of the clones; the Y axis shows pg/ml of IL-6 produced. A+E.1, A498 stably transfected with empty expression vector. Columns, means of two to six repeats of the experiment; bars, SD (where three or more repeats were performed). B, Western blot analysis showing levels of p53 protein expression in A498 clones. Cells (5 x 106) were lysed and immunoprecipitated with either an anti-p53-specific antibody or an isotypic control antibody as labeled. Samples were run on a 12% SDS-PAGE gel, blotted onto nitrocellulose membranes, and probed with another p53-specific antibody. rp53, recombinant p53. All clones stably expressing mut p53 produced significantly more IL-6 than the clone transfected with empty expression vector (P = 0.05).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Renal epithelial cells produce low levels of IL-6 (16 , 17) , suggesting that the IL-6 transcriptional machinery is constitutively activated in these cells. However, much higher levels of IL-6 are produced by RCC cells (16, 17, 18, 19, 20, 21) , and IL-6 can serve as an autocrine growth factor for these cells (12, 13, 14, 15) . The latter results suggest that IL-6 levels are kept at basal levels in normal renal cells, but that this regulatory mechanism is disrupted in renal cancer.

In the present study, we demonstrate that p53, when mutated, can contribute to increased levels of IL-6 production by RCC cell lines. Previous studies demonstrated that p53 can also regulate IL-6 gene expression in an inducible system (24 , 25) . It appears that wt p53 and mut p53 have differential effects on the IL-6 promoter. wt p53 inhibits the promoter, whereas mut p53 either enhances IL-6 promoter activity or represses it to a much lesser degree than wt p53 (Ref. 24 and Figs. 1Citation and 2Citation ). Thus, it is conceivable that IL-6 levels remain basal in RCC cell lines until p53 is mutated. This hypothesis is supported by our observation that IL-6 production is increased in RCC cell lines with mut p53 (Table 1)Citation and in wt p53-bearing RCC A498 cells after stable transfection with mut p53 (Fig. 8)Citation . Our results are consistent with published observations indicating that several p53 mutants are capable of enhancing transcription and act as dominant negative mutants (54 , 55) . There have also been reports suggesting an association between p53 mutation and elevated IL-6 production in other tumors including fallopian tube carcinoma (56) , oral squamous cell carcinoma (57) , and ovarian cancer (51) . Interestingly, Han et al. (58) also found two p53 mutants in the joints of rheumatoid arthritis patients. These particular p53 mutants increased IL-6 gene expression when transfected into dermal fibroblasts (58) .

Although mutant p53 may contribute to IL-6 production in RCC and other tumors, according to our experiments, wt p53 has a strong repressive effect on the IL-6 promoter (Fig. 2)Citation . Overexpression of wt p53 in RCC cell lines resulted in significant repression of the full-length human IL-6 promoter (Figs. 1Citation and 2Citation ). EMSA analysis using an anti-p53 antibody (clone 421) demonstrated no supershift of the complexes at either the MRE I or the NF-IL6 binding site of the IL-6 promoter in A498 cells (data not shown). These results are consistent with published data indicating that the IL-6 promoter does not have a p53 binding site (24) . In general, when p53 acts to enhance transcription, the target gene contains a p53 consensus element (59) , whereas when p53 acts as a repressor, the target gene lacks a p53 binding site (60, 61, 62) . It has been postulated previously that p53 acts as a repressor by protein/protein interaction with C/EBP family members or with TATA binding protein (24, 25, 26 , 61 , 62) .

Our experiments show that, in the A498 cell line (wt p53-expressing), transfection of wt p53 resulted in a decrease in binding of both C/EBP-ß and C/EBP-{delta}. CREB and NF-{kappa}B transcription factor binding was also inhibited in this cell line. NF-IL6 and NF-{kappa}B interact with one another and synergistically up-regulate the IL-6 promoter (50) , as can NF-IL6 and C/EBP-{delta} (63) . Thus, the transcription factors important in IL-6 promoter regulation can form multiple protein complexes (50 , 63 , 64) . Previous reports have also suggested that wt p53 repressed the IL-6 promoter through a possible protein/protein interaction with the C/EBP family of transcription factors (24, 25, 26) . Therefore, it is possible that, in A498 cells, wt p53-associated attenuation of transcription factor binding is due to interference with the ability of NF-IL6 to form transcription factor complexes with NF-{kappa}B, CREB, and C/EBP {delta}.

In contrast to our results, binding of C/EBP-ß or C/EBP-{alpha} to the albumin promoter in hepatocellular carcinoma by either wt or mut p53 was not inhibited (65) . However, transfection of wt p53 reduced binding of C/EBP proteins to the insulin promoter (66) . Thus, it appears that wt p53 can repress different promoters in different tissues using different mechanisms.

wt p53 may have differential effects on the IL-6 promoter, depending on background gene expression in the cell lines. One difference between our cell lines is that A498 contains wt p53, whereas UOK 121LN contains mut p53. wt p53 had a greater inhibitory effect on the binding of C/EBP, CREB, and NF-{kappa}B to the IL-6 promoter by EMSA in A498 as compared with UOK 121LN. If protein/protein interactions are responsible for the effects of p53 on IL-6 transcription factors (24, 25, 26) , then it is conceivable that mut p53 has a higher affinity for C/EBPs, NF-IL6, and NF-{kappa}B than wt p53. mut p53 in this scenario would function to enhance binding of at least some of these transcription factor complexes, as seen in the case of the MRE in our experiments (Fig. 4D)Citation . A higher affinity would make it more difficult for transfected wt p53 to displace the endogenous interaction of mut p53 with the transcription factors.

The AP-1 site is also a potential target for regulation by p53. c-jun binds to the AP-1 site and is down-regulated by wt p53 at the level of transcription (53 , 67) . Even so, we were unable to detect any change in c-jun protein levels after transfection of wt p53 into UOK 121LN cells (Fig. 7)Citation . Other mechanisms may, however, also be operative. For instance, wt and mut p53 can influence the composition of transcription factor complexes at the AP-1 site of the IL-6 promoter (51) . wt p53 also down-regulates TATA-mediated transcription (68, 69, 70) by binding TATA-binding protein and other proteins in the basal transcription factor complex, TFIID (68) . Analysis of the effects of p53 on these promoter sites merits exploration.

Because p53 is crucial to certain forms of apoptosis, loss or mutation of this tumor suppressor gene product would presumably result in decreased cell death (71, 72, 73, 74) . IL-6 expression may mediate, at least in part, enhanced survival. IL-6 can prevent p53-mediated cell death in M1 myeloid leukemia cells (75) . IL-6 can also prevent apoptosis in a variety of lymphoid cells and cell lines, as well as intestinal epithelium, through activation of the bcl-2 proto-oncogene (76) . Thus, p53 and IL-6 appear to be opposing signals. wt p53 may keep IL-6 levels low; IL-6 inhibits p53-mediated apoptosis and also acts as a proliferative stimulus.

The data presented here suggest that mutation of p53 is one mechanism whereby IL-6 becomes overexpressed in RCC. A similar model for p53 involvement in the development of a more aggressive phenotype has been proposed for follicular lymphoma (73) and human adult T-cell leukemia (74) . When p53 is mutated or inactivated in adult T-cell leukemia, several cytokines including IL-6, granulocyte-macrophage colony-stimulating factor, IL-1{alpha}, and the IL-2 receptor {alpha} chain become constitutively activated (74) . In RCC, wt p53 has a suppressive effect on binding of specific transcription factors to the IL-6 promoter as compared with mut p53, and this effect may mediate the ability of wt p53 to maintain IL-6 at low basal levels. In the presence of mut p53, the suppressive effect of wt p53 appears to be attenuated in RCC lines, resulting in overexpression of IL-6, which then acts as an autocrine growth factor (12, 13, 14, 15 , 72) . (Even so, because not all mutant p53-bearing cell lines have elevated IL-6 expression, other factors must also be operative.)


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Bioimmunotherapy, Box 422, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 794-1226; Fax: (713) 745-2374; E-mail: rkurzroc{at}mdanderson.org Back

2 The abbreviations used are: IL, interleukin; RCC, renal cell carcinoma; mut, mutant; wt, wild type; C/EBP, CAAT enhancer binding protein; CREB, cyclic AMP responsive element binding; NF-{kappa}B, nuclear factor-{kappa}B; AP-1, activator protein-1; CAT, chloramphenicol acetyltransferase; EMEM, Eagle’s minimal essential media; EMSA electrophoretic mobility shift assay; TNF, tumor necrosis factor; MRE, multiresponse element I; RT-PCR reverse transcription-PCR; TUNEL, terminal deoxynucleotidyl transferase-mediated UTP nick end labeling. Back

Received 7/24/01. Accepted 11/30/01.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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