Increased nuclear factor-{kappa}B activation is related to the tumor development of renal cell carcinoma

Mototsugu Oya1,3, Atsushi Takayanagi2, Akio Horiguchi1, Ryuichi Mizuno1, Masafumi Ohtsubo2, Ken Marumo1, Nobuyoshi Shimizu2 and Masaru Murai1

1 Department of Urology and
2 Department of Molecular Biology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan


    Abstract
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 Materials and methods
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Although an aggressive phenotype of renal cell carcinoma (RCC) is known to frequently be associated with inflammatory paraneoplastic syndrome including serum C-reactive protein (CRP) elevation, the molecular mechanism underlying this clinical phenomenon as well as what yields the malignant phenotype leading to the progression of RCC has yet to be elucidated. Based on the increased level of inflammatory cytokines such as interleukin-6 in advanced cases of RCC, a cytokine-inducible transcription factor, namely, nuclear factor-{kappa}B (NF-{kappa}B), may thus play a role in the progression of RCC. An electrophoretic mobility shift assay (EMSA) was carried out to determine the activity of NF-{kappa}B. Out of 45 cases of RCC, 15 cases (33%) showed a >200% increase in the NF-{kappa}B activity in comparison with that seen in normal renal tissue. In locally advanced cases (>=pT3), 64% (9/14) showed an increased activity whereas it was only observed in 19% (6/31) of localized cases (<=pT2). All three cases with metastases showed an increased NF-{kappa}B activity. The NF-{kappa}B activity determined by EMSA was further confirmed by an immunohistochemical analysis using an antibody recognizing the nuclear localization signal (NLS) in p65 subunit of NF-{kappa}B. The serum CRP elevation correlated with the increased NF-{kappa}B activation, and therefore NF-{kappa}B may be a causative transcription factor of inflammatory paraneoplastic syndrome. A high NF-{kappa}B activity was associated with an increased expression of both the p65 and p50 subunits of NF-{kappa}B and a concomitant decreased expression of I{kappa}B{alpha}. No functional mutations of the I{kappa}B{alpha} gene were detected. The NF-{kappa}B activity may therefore be a late event in carcinogenesis related to tumor development, thereby representing a possible molecular target in the treatment of RCC.

Abbreviations: CRP, C-reactive protein; EMSA, electrophoretic mobility shift assay; IL, interleukin; NF-{kappa}B, nuclear factor-{kappa}B; RCC, renal cell carcinoma.


    Introduction
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 Abstract
 Introduction
 Materials and methods
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With the advent of such imaging modalities as ultrasonography and computed tomography, renal cell carcinomas (RCC) can now be frequently detected asymptomatically. These carcinomas are usually low grade and low stage and tend to be successfully treated by a surgical resection. However, there are also aggressive RCC phenotypes, which are normally identified as far advanced cases with metastases. These patients have an extremely poor prognosis and the therapeutic options for such patients are few because chemotherapy and irradiation are ineffective (1). Interestingly, these carcinomas are occasionally associated with inflammatory paraneoplastic syndrome, which can be detected by an increased amount of serum C-reactive protein (CRP) (24). As a result, an aberrant expression of inflammatory cytokines by cancer cells may thus play a role in cancer development and also accelerate cancer cell growth in an autocrine or paracrine manner (5,6). Interleukin (IL)-6 has been thought to be a major causative cytokine because 50% or more of patients with metastatic RCC have an increased level of IL-6, which correlates with an increased amount of CRP (2,7). The predominant mechanism by which these cytokines are newly synthesized involves an inducible transcriptional initiation of their respective genes. This phenomenon is governed by transcription factors, which bind to the regulatory regions of the genes. The transcription factor nuclear factor (NF)-{kappa}B has been recognized to be a major cytokine-inducible transcription factor. Cytokines and growth factors such as IL-6, tumor necrosis factor (TNF)-{alpha} and granulocyte-colony stimulating factor are considered to be target genes of NF-{kappa}B (8). NF-{kappa}B might contribute to the deregulated cytokine expression in RCC. Therefore, in patients with RCC associated with inflammatory paraneoplastic syndrome, NF-{kappa}B may be aberrantly activated and thereby govern the induction of the causative cytokines.

The active NF-{kappa}B complex is a compound of two subunits designated p65 (RelA) and p50 (9). The genes encoding p65 and p50 have been cloned and the N-terminal of both proteins show considerable homology to the product of the oncogene rel. NF-{kappa}B is present in most cells, where it remains in an inactive form in the cytoplasm bound with an inhibitory protein I{kappa}B which masks the NLS. Active NF-{kappa}B is released and then is translocated to the nucleus with the rapid degradation of I{kappa}B, after stimulation with a number of agents including cytokines such as TNF-{alpha} and IL-1, phorbor ester, viral infections (8).

The oncogenic role of NF-{kappa}B in solid cancers has been described previously in the breast, colon, ovarian, thyroid and pancreas cancer (10). We have recently described that some RCC cell lines have an increased NF-{kappa}B activity (11). We herein studied the role of NF-{kappa}B in RCC carcinogenesis and/or tumor development of RCC using RCC tissue samples. We describe the increased activity of NF-{kappa}B in RCC, which is related to both tumor invasiveness and metastases. Our findings suggest that NF-{kappa}B may thus be a potentially useful molecular target to treat patients with advanced RCC.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient characteristics and tissue samples
A total of 45 RCC were surgically removed in the Department of Urology, Keio University School of Medicine. Tumor tissue specimens as well as normal kidney tissue specimens were taken and immediately cast into liquid nitrogen. The samples were stored in a deep freezer at –80°C. The patient data and tumor characteristics are shown in Table IGo. Tumor grading and staging were done according to the TNM classification (12). Patients #10, #17 and #45 had metastatic disease in the lung, the bone and the pancreas, respectively. The materials included 42 clear cell carcinomas and three papillary cell carcinomas. Preoperative blood CRP was analyzed in all patients. We defined 0.15 as the cut off level based on a 95 percentile value of the 776 healthy volunteers. Eighteen patients demonstrated increased serum CRP levels. The differences in NF-{kappa}B activation between the subgroups of RCC were tested for significance using Fisher’s exact probability test. A level of P < 0.05 was considered to indicate significance.


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Table I. NF-{kappa}B activation in RCC

 
Extraction of proteins
Stored samples were crushed in liquid nitrogen and homogenized using a homogenizer in cell lysis buffer [20 mM HEPES (pH 7.9), 0.2% NP-40, 10% glycerol, 400 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM sodium vanadate, 0.5 mM PMSF (all Sigma, St Louis, MO)] and a protease inhibitor mix (complete protease inhibitor cocktail set, Boehringer Mannheim, Indianapolis, IN) and then were incubated on ice for 15 min. The supernatants were taken and stored at –80°C.

Electrophoretic mobility shift assay (EMSA), competition assay and supershift assay
Twenty micrograms of the supernatants were incubated with poly dIdC (3 µg; Amersham Pharmacia Biotech, Buckinghamshire, UK) in a binding buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 mM DTT and 4 vol% glycerol] with double-stranded oligonucleotides for the consensus binding sites of NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3') labeled with [{gamma}-32P]dATP by T4 polynucleotide kinase (New England Biolabs Inc., Beverly, MA) for 20 min at room temperature. For the competition assay, proteins were pre-incubated with double-stranded unlabeled NF-{kappa}B binding oligonucleotide or mutant oligonucleotide (5'-AGTTGAGGCGACTTTCCCAGGC-3') at a 100-fold molar excess. For the supershift assay 2 µg of antibody for p65 and p50 (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the samples and incubated for 30 min. As a control for EMSA, Sp-1 activation was investigated using double-stranded oligonucleotides for the consensus binding sites of Sp1 (5'-ATTCGATCGGGGCGGGGCGAGC-3'). For the competition assay, mutant oligonucleotide (5'-ATTCGATCGGTTCGGGGCGAGC-3') was used. The samples were separated on non-denaturing 4% polyacrylamide gel in 0.5x TBE. The gels were then dried and exposed on an imaging plate overnight and then were analyzed using FLA2000 (Fuji Film Company, Tokyo, Japan). The signals were quantified using a densitometer. The values (tumor tissue/normal tissue) are shown in Table IGo. Using densitometric analyses, we defined aberrant activation as a ratio (T/N) of more than two.

Immunoblotting
The extracted protein (10 µg for p65 and p50; 20 µg for I{kappa}B{alpha}) with sample buffer containing 2-mercaptoethanol were separated on SDS–PAGE. The gels were blotted onto a PVDF membrane using the semi-dry method. The membrane was incubated with 5% skim milk in TBS overnight. The primary antibody used was either rabbit anti-p65 (CHEMICON International, Inc., Temecula, CA) or anti-p50 polyclonal antibody (Santa Cruz Biotechnology) or anti-I{kappa}B{alpha} polyclonal antibody (Upstate Biotechnology, Lake Placid, NY). ß-Actin was used as an internal control (Sigma). Immunodetection was performed using an enhanced detection system with avidin–biotin complex (Vectastain ABC kit, Vector Lab., Burlingame, CA).

Immunohistochemistry
Five-micron sections from formalin-fixed paraffin-embedded tissue specimens were deparaffinized in xylene and dehydrated in graded ethanol, followed by PBS. Antigen was retrieved by heating at 121°C for 10 min in 10 mM sodium citrate (pH 6.0), and then was incubated with 0.3% H2O2 to suppress the endogenous peroxidase activity. The slides were blocked in 10% goat serum and incubated with mouse monoclonal anti-NF-{kappa}B p65 antibody (1:50; Boehringer Mannheim) for 24 h at 4°C. To confirm the specific reaction of the antibody, a control reaction was performed in the absence of the primary antibody. After washing, the slides were incubated with Simple stain max po (Nichirei, Tokyo, Japan) for 30 min and 3,3'-diaminobenzidine, and thereafter were counter stained by hematoxylin.

Mutation analysis of I{kappa}B{alpha} gene
The genomic DNA samples were purified by standard procedures using proteinase K and SDS followed by extraction with phenol–chloroform. The genomic DNA fragments contained I{kappa}B{alpha} exons were amplified in 20 µl volume containing 100 ng genomic DNA template, 1 µM primer and 1 M betain using the Expand High-Fidelity PCR system (Roche Diagnostics, Tokyo). For exon 1, PCR cycling condition was 94°C for 2 min followed by 40 cycles of 94°C for 20 s, 50°C for 10 s, 68°C for 30 s, supplemented 5% DMSO with primers, I{kappa}B1b'(5'-CTGGCTTGGAAATTCCCCGAGCCTGAC-3') and I{kappa}B1r(5'-CGCGCGCGTCCCGCCCTCCCGACGA-3'); for exon 2, 94°C for 2 min followed by 40 cycles of 94°C for 20 s, 55°C for 10 s, 68°C for 40 s with primers, I{kappa}B2a(5'-CGAAGTCCCCGGTTGCATAAGG-3') and I{kappa}B2c(5'-GGATCTGGGGTGACTCTGCTAC-3'); for exon 3, 94°C for 2 min followed by 40 cycles of 94°C for 20 s, 60°C for 10 s, 68°C for 40 s with primers, I{kappa}B3a'(5'-TCTAGGAGGAGCAGCACCCAACG-3') and I{kappa}B3d (5'-TAGGAGTTTAAGCTCTTGCCTGGA-3'); for exon 4 to 5, 94°C for 2 min followed by 40 cycles of 94°C for 20 s, 61°C for 10 s, 68°C for 40 s with primers, I{kappa}B4b(5'-AAAGAATAGGTGAAAGGAGTGAGG-3') and I{kappa}B5d'(5'-CAGCTCTAGGGGCCTGGGAGGGT-3'); for exon 6, 94°C for 2 min followed by 40 cycles of 94°C for 20 s, 60°C for 10 s, 68°C for 30 s with primers, I{kappa}B6a(5'-GAGTTATTTCCAGTAGTGGCCTC-3') and I{kappa}B6d'(5'-GGGGTCAGTCACTCGAAGCACA-3'). The amplified products were purified from agarose gel using a gel extraction kit (Qiagen, Tokyo) and then were sequenced directly using a BigDye terminator cycle sequencing kit and the same primers for PCR while performing an analysis on a 377 automated sequencer (Applied Biosytems Japan, Tokyo).


    Results
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 Materials and methods
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Increased NF-{kappa}B activation correlates with the invasiveness of RCC
According to the classification of UICC (12), pT1 and pT2 are localized tumors whereas pT3 is an invasive disease into the perinephric tissue (pT3a) or renal veins (pT3b). A representative example of EMSA is shown in Figure 1AGo. In the pT1 cases [localized and 7.0 cm or less; pT1 is subdivided to pT1a (<=4.0 cm) or pT1b (> 4.0 cm)] 16% (4/24) had a >200% enhanced activity (defined as an increased activity) of NF-{kappa}B in comparison with normal tissue (Table IGo). In the pT2 cases (localized and >7.0 cm) this rate of increased NF-{kappa}B activation was 29%(2/7). In contrast, in the pT3 cases, 64% (9/14) showed an increased activity of NF-{kappa}B (Table IIGo). This rate of increased activity is significantly higher than that of the localized cases. This suggests that the increased NF-{kappa}B activation may be closely related with the invasiveness of RCC. A competition assay was performed to identify the band, which represents the NF-{kappa}B binding activity. Competition was done using a 100-fold molar excess of wild-type oligonucleotides or mutant oligonucleotides. The NF-{kappa}B bands shown in Figure 1BGo were totally diminished by competition with wild-type oligonucleotides, but not with mutant oligonucleotides. A supershift assay was performed using p65 and p50 antibodies to further confirm the bands of EMSA, which demonstrated specific binding of NF-{kappa}B to consensus oligonucleotides (Figure 1CGo). Furthermore, we also performed EMSA for a transcription factor that should not be modified in carcinogenesis as a control. The activation status of a ubiquitously activated nuclear transcription factor Sp1 in RCC was similar to that in normal renal tissues (Figure 1DGo). Pathologically, our series included 42 clear cell carcinomas and three papillary carcinomas. Two papillary cases showed a modest increase in the NF-{kappa}B activity. Regarding the tumor grade, 71% (5/7) of grade three tumors showed an increased NF-{kappa}B activation, and this rate was higher than that for grade 1 or 2 tumors (26%; 10/38). RCC rarely involves lymph nodes. In our series one patient out of three with lymph node involvement had an increased NF-{kappa}B activity. Three patients with metastases showed an increased NF-{kappa}B activation.






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Fig. 1. Binding to the NF-{kappa}B consensus sequence of proteins in normal (N) and tumor (T) tissue specimens in a series of consecutive patients (#1–5). The ratio (T/N) is described in Table 1Go. An increased activity (T/N >2) is observed in #1, 2 and 3. A competition assay was carried out using a 100-fold molar excess of wild-type oligonucleotides or mutant oligonucleotides. Note that the specific NF-{kappa}B bands are completely abolished by competition with the wild-type oligonucleotides, but not with mutant oligonucleotides. Supershift bands were observed by adding NF-{kappa}B p65 and p50 antibody to confirm that the bands were specific to NF-{kappa}B. Binding to the Sp1 consensus sequence of proteins in normal and tumor tissue specimens. A competition assay was carried out using a 100-fold molar excess of wild-type oligonucleotides or mutant oligonucleotides. The Sp1 activities of the normal and the tumor tissue specimens demonstrated almost the same level.

 

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Table II. Correlation between NF-{kappa}B activation and the pathological findings
 
Immunohistochemical detection of translocated NF-{kappa}B in RCC tissues
Immunohistochemistry was carried out to confirm that the NF-{kappa}B activation detected by EMSA reflects the activation of RCC cells in tissues. The monoclonal antibody used for immunohistochemistry recognizes an epitope of the NLS of p65. As a result, the activated and translocated p65 is stained in the nucleus. We analyzed 14 specimens (#16–29) and representative pictures are shown in Figure 2Go. In a specimen from patient #28 which had an increased NF-{kappa}B activity as determined by EMSA, almost all the cancer cells (>90%) were positive for the nuclear staining with p65 antibody (Figure 2AGo). The specificity of nuclear staining for the RCC tissue specimens with p65 antibody was ascertained by abolishing the staining in the absence of the primary antibody (Figure 2BGo). In a specimen from patient #19 which showed a moderate increase (T/N = 1.65), fewer positively stained RCC cells were observed (Figure 2CGo). In contrast, in a specimen from patient #21, which showed a decreased NF-{kappa}B activity, positively stained cancer cells were scarcely observed (Figure 2DGo). In normal kidney tissues, weak nuclear staining was observed in renal tubular epithelial cells (Figure 2EGo). A correlation between p65 staining and the level of {kappa}B binding activity was observed in the other specimens investigated. In summary, an immunohistochemical analysis confirmed the NF-{kappa}B activation in RCC tissues.



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Fig. 2. RCC stained with the antibody detecting the NLS of p65. (A) Almost all cancer cells (>90%) show positive staining in the nucleus (Patient #28). (B) In control immunostaining of the same patient as (A) in the absence of the primary antibody, no positively stained cells are observed (C). Fewer cancer cells are positively stained compared with (A) (Patient #19). (D) No positively stained cells are observed (Patient #21). (E) Normal kidney tissue. Weak positive cells are observed in renal tubular epithelial cells. Scale bar: 50 µm.

 
Serum CRP elevation correlates with the increased NF-{kappa}B activation in RCC tissues
Among the circulating markers of the acute inflammatory responses, CRP is the most commonly used marker in clinical practice (13). CRP is the prototype of an acute phase reactant, which is virtually absent in normal conditions and appears in large quantities in inflammation (14). Therefore, the elevation of the serum CRP level represents inflammatory paraneoplastic syndrome of RCC. In our study, an elevation of CRP correlated with an increased NF-{kappa}B activation (Table IIGo). This observation supports the idea that inflammatory cytokines are produced in RCC, which are induced by NF-{kappa}B activation. The produced cytokines circulate in the body, thus resulting in an elevation in the serum CRP level.

Increased NF-{kappa}B activation associated with an augmented p65 and p50 expression with a concomitant decreased I{kappa}B{alpha} expression
To investigate the contribution to the increased NF-{kappa}B activation by the expression of the components of NF-{kappa}B, the expression of p65, p50 and I{kappa}B{alpha} was analyzed by immunoblotting. Figure 3Go shows the immunoblotting findings of the same patients shown in Figure 1Go (#1–5). An augmented expression of both p65 and p50 was observed in patients #1, 2, 3 and 5 but not in 4. In #4, the expression of p65 was up-regulated but not of p50. In patients with increased NF-{kappa}B activation (#1–3), the I{kappa}B{alpha} expression decreased, whereas, cases without any increased NF-{kappa}B activation (#4 and #5), the I{kappa}B{alpha} expression was not altered. Therefore, the increased NF-{kappa}B activation associated with the augmented expression of both p65 and p50 with a concomitant decreased I{kappa}B{alpha} expression. For an increased NF-{kappa}B activation, an augmented expression of both p65 and p50 is thought to be needed because the binding activity needs both subunits, which conjugate with each other. An augmented expression of either p65 or p50 was not sufficient to obtain an increased NF-{kappa}B activation.



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Fig. 3. Immunoblotting of NF-{kappa}B p65, p50 and I{kappa}B{alpha} in identical patients as Figure 1Go. An augmented expression of both p65 and p50 was observed in patients #1, 2, 3 and 5 but not 4. In #4, the expression of p65 was up-regulated but not of p50, in which no enhanced NF-{kappa}B activation was observed. Note that in cases with an increased NF-{kappa}B activity (#1–3), the I{kappa}B{alpha} expression decreased, whereas, in cases without any increased activation (#4 and #5), the I{kappa}B{alpha} expression did not change. ß-Actin was used as an internal control. Any relative increases quantified using a densitometer (tumor tissue/normal tissue) are shown as graphs.

 
No functional mutations were found in the I{kappa}B{alpha} gene
The increased NF-{kappa}B activation may be due to the presence of defective I{kappa}B{alpha} caused by a mutation of the gene. Therefore, a mutation analysis of I{kappa}B{alpha} was performed in 10 patients (#1, 2, 3, 6, 12, 13, 17, 25, 28 and 29). All exons of the I{kappa}B{alpha} gene in the tumors and normal tissue specimens were sequenced, however, no obvious mutation causing a loss of function was found. Only silent mutations were found in exons 1 and 4 of both normal tissues and tumors. Patients #2, 3, 6 and 12 were heterozygotes of 27Asp (GAC and GAT) in exon 1. Patients #17, 25 and 28 were heterozygotes of 183Gly (GGC and GGG) in exon 4. Other base-exchanges were found in the introns but not in the consensus sequence between the exon and intron.


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Positive inflammatory reactions in an aggressive phenotype are typical features of RCC. Although a high blood level of inflammatory cytokines such as IL-6 has been observed in these patients, the mechanism underlying this clinical phenomenon has not yet been clarified. We therefore studied the activation of NF-{kappa}B, which is an important transcriptional regulator of cytokine expression.

We herein described the increased NF-{kappa}B activation in RCC, which correlated with invasiveness and metastases. The increased activation of NF-{kappa}B may be responsible for the clonal selection of RCC through the production of inflammatory cytokines, which provide autocrine growth and selective survival. In breast cancer, the activation of NF-{kappa}B appears to be an early event that occurs prior to the malignant transformation of human mammary epitherial cells in vitro and in rats treated with carcinogenesis in vivo (15). In contrast, in RCC, we demonstrated that the activation of NF-{kappa}B correlates with its progression to invasion and metastases. In clinical situations, these advanced RCC have so far been shown to be associated with paraneoplastic syndrome. As a result, NF-{kappa}B might be the major responsible molecule that yields malignant phenotype, thus governing both tumor invasiveness and metastases.

Furthermore, recent studies have shown that NF-{kappa}B induces the expression of several apoptosis inhibitors as well as inflammatory cytokines. These inhibitors include c-IAP1, c-IAP2, TRAF1, TRAF2, A20, IEX-1L, Bfl-1/A1, Bcl-x (10). We have also recently shown that the inhibition of the constitutive activation of NF-{kappa}B in RCC cell lines leads to the induction of apoptosis (11). Therefore, NF-{kappa}B is considered to play a role in allowing the RCC cells to survive and also progress. However, it remains to be elucidated as to which molecules help RCC cells survive, thereby enhancing the progression of RCC as downstream targeting molecules of NF-{kappa}B.

NF-{kappa}B induces a variety of genes, which are supposed to play a role in the invasion and metastases of cancer cells. These include angiogenic factor such as VEGF (16), proteolytic enzymes such as urokinase plasminogen activator (17) and cell adhesion molecules such as ICAM-1 (18). Accordingly, RCC is characterized by the prominent neovasculization and an overexpression of VEGF (19,20). These characteristics can be explained by the observed increase in NF-{kappa}B activation. Furthermore, the expression of extracellular proteases associated with tumor invasive phenotype is also controlled by NF-{kappa}B (21). Excessive NF-{kappa}B activity can up-regulate the extracellular proteases leading to a degradation of both the extracellular matrix and the basal membrane, thus leading to cancer invasion.

Another characteristic of RCC is its resistance to chemotherapy. Although the specific mechanisms of drug resistance have been described (22), no unifying concept has emerged to explain the general resistance of RCC to a wide range of chemotherapeutic agents. Wang et al. reported the NF-{kappa}B activity and sensitivity to chemotherapy to be inversely correlated (23). Therefore, resistance to chemotherapy in RCC may in part be due to the increased NF-{kappa}B activation. The underlying mechanism of resistance to chemotherapy has been partly explained by the de novo induction of multi-drug resistant gene product. Interestingly, NF-{kappa}B has been implicated to induce this gene expression (24).

Regarding the mechanism of enhanced NF-{kappa}B activation, we have shown a high NF-{kappa}B activity was associated with an augmented expression of the subunits of NF-{kappa}B, p65 and p50. Visconti et al. showed that p65 expression but not p50 expression was augmented in human thyroid carcinoma cell lines (25). Specific antisense oligonucleotides of p65 successfully reduced the growth of the cell lines whereas antisense oligonucleotides of p50 did not. Conversely, in non-small cell lung carcinoma, an overexpression of p50 was observed (26).

The other determinant of NF-{kappa}B activation is I{kappa}B{alpha}. Recent reports have shown a constitutive activation of NF-{kappa}B to be associated with a low I{kappa}B{alpha} expression (27). However, conflicting reports have also described an enhanced expression of I{kappa}B{alpha} in the cell lines with high NF-{kappa}B activity because I{kappa}B{alpha} is an inducible gene of NF-{kappa}B (28,29). In pancreatic cancers, the level of I{kappa}B{alpha} proteins was reported to increase at least 10-fold (30). We demonstrated that RCC with an increased NF-{kappa}B activity had a decreased amount of I{kappa}B{alpha} expression. It is conceivable that this is due to extracellular stimuli by cytokines, which lead to the degradation of I{kappa}B{alpha}.

Another conceivable mechanism of the increased NF-{kappa}B activity is a functional defect of I{kappa}B{alpha} caused by a mutation of the I{kappa}B{alpha} gene. In Hodgkin’s disease, mutations or deletions of I{kappa}B{alpha} gene have been reported which result in a constitutive NF-{kappa}B activation (3133). The mutation of I{kappa}B{alpha} gene was analyzed in RCC tissues; however, no functional mutations of I{kappa}B{alpha} gene were found as far as we could investigate.

Taken together, NF-{kappa}B may thus be a major molecule, which characterizes the invasiveness and metastasis of RCC as well as the inflammatory paraneoplastic syndrome associated with a serum CRP elevation. NF-{kappa}B may also be a potentially useful molecular target to treat patients with RCC. As we recently described, the inhibition of active NF-{kappa}B by the adenoviral transduction of a stable form of I{kappa}B{alpha} gene may therfore be a potentially novel gene therapy for RCC, for which there is otherwise no effective therapeutic modality at present (11).


    Notes
 
3 To whom correspondence should be addressed Email: moto-oya{at}sc.itc.keio.ac.jp Back


    Acknowledgments
 
This study was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 21, 2002; revised October 2, 2002; accepted October 8, 2002.