1 AMC Cancer Research Center, Denver, CO 80214, USA
2 Regulation of Cell Growth Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, PO Box B, Frederick, MD 21702, USA
3 N. N. Blokhin Cancer Research Center, Moscow, 115478, Russia
*Author for correspondence (e-mail: budunovai{at}amc.org)
Accepted October 2, 2001
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SUMMARY |
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Key words: NF-B, I
B
phosphorylation, IKK, Prostate cancer
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Introduction |
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Several lines of evidence suggest that aberrant NF-B regulation is associated with oncogenesis in mammalian systems. Amplification, overexpression or rearrangement of all genes coding for Rel/NF-
B factors with exception of RelB have been found in leukemias and lymphomas (Rayet and Gelinas, 1999). Constitutive activation of NF-
B is a common characteristic of many cell lines from hematopoietic and solid tumors (Rayet and Gelinas, 1999; Baldwin, 1996; Wang et al., 1999; Dejardin et al., 1999; Bours et al., 1994; Nakshatri et al., 1997; Sovak et al., 1997; Visconti et al., 1997, Palayoor et al., 1999; Duffey et al., 1999; Barkett and Gilmore 1999). The blockage of NF-
B activity in carcinoma cell lines by different approaches dramatically reduced their ability to form colonies in agar and reduced their growth in vitro and in vivo (Visconti et al., 1997; Duffey et al., 1999). It is important that NF-
B also plays a key role in cell protection against diverse apoptotic stimuli including chemotherapeutic drugs and
-irradiation through activation of the anti-apoptotic gene program in cells (Barkett and Gilmore, 1999).
In spite of the growing evidence of the important role of NF-B in tumorigenesis and resistance to chemotherapy, only a few attempts have been made to analyze the mechanisms of constitutive activation of NF-
B in transformed cells. It was found that mechanisms involved in NF-
B activation in tumor cell lines could be different, and include increased expression of NF-
B proteins, especially p50 and p52, mutations and deletions in I
B
gene and increased I
B
turnover (Devalaraja et al., 1999; Krappmann et al., 1999; Budunova et al., 1999; Rayet and Gelinas, 1999).
The aim of this study was to develop a comprehensive and detailed picture of changes in basal NF-B activity in a panel of prostate cells including primary prostate epithelial cells and six prostate carcinoma (PC) cell lines, and to elucidate the molecular mechanisms that could account for the NF-
B activation in PC cells, including the level of expression of Rel/NF-
B proteins, mutations in the I
B
gene, and I
B
turnover. Our results indicate that NF-
B is constitutively activated in human androgen-independent PC cells. We did not reveal any significant differences in the expression of various NF-
B and I
B proteins or I
B
mutations in any of the examined cell lines. Instead, in androgen-independent PC cells I
B
was heavily phosphorylated and displayed a shorter half-life. Our results indicate that aberrant IKK activation in androgen-independent PC cells leads to the constitutive activation of NF-
B survival signaling pathway, possibly contributing to their growth advantage.
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Materials and Methods |
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Preparation of cellular extracts and electrophoretic mobility shift and supershift assays (EMSA and EMSSA)
Nuclear and cytosolic proteins were isolated as described previously (Lyakh et al., 2000). The binding reaction for EMSA contained 10 mM Hepes (pH 7.5), 80 mM KCl, 1 mM EDTA, 1 mM EGTA, 6% glycerol, 0.5 µg of poly(dI-dC), 0.5 µg of sonicated salmon sperm DNA, [-32P]-labeled (2-3x105 cpm) double-stranded
B-consensus oligonucleotide (Promega Corp., Madison, WI), [
-32P]-labeled (2-3x105 cpm) double-stranded oligonucleotide representing Sp1-consensus binding site (Santa Cruz Biotechnology, Santa Cruz, CA), and 5-10 µg of the nuclear extract. DNA-binding reaction was performed at room temperature for 30-45 minutes in a final volume of 20 µl. For EMSSA antibodies against p65 (sc-109X), p50 (sc-114X), p52 (sc-298X), c-Rel (sc-71X) or RelB (sc-226X), were added 30 minutes after the beginning of reaction, and incubation was continued for an additional 30-45 minutes. All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). DNA-protein complexes were analyzed on 6% DNA retardation gels (Novex, Carlsbad, CA). Dried gels were subjected to radiography.
Western blot analysis
Proteins were resolved by electrophoresis on 10-12.5% SDS-PAAGs and transferred to Immobilon-P membrane (Millipore Corporation, Bedford, MA). Polyclonal anti-p50 (# 06-886), anti-p52 (# 06-413) anti-c-Rel (# 06-421) antibodies were from Upstate Biotechnology (Lake Placid, NY). Anti-p65 (sc-372), anti-RelB (sc-226) anti-IB
(sc-371), anti-I
Bß (sc-946), anti-I
B
(sc-7156) or anti-IKK
/ß (sc-7607) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Ser32 I
B
Ab was from Cell Signaling Technology Inc. (Beverly, MA). Anti-PARP Ab was from PharMingen (San Diego, CA). Membranes were blocked with 5% nonfat milk in TBST buffer and incubated with primary antibodies for 1.5 hours at room temperature. Anti-Phospho-Ser32 I
B
Ab required 6 hours incubation at 34°C. Peroxidase-conjugated anti-rabbit IgG (Sigma) was used as a secondary antibody. ECL Western blotting detection kit (Amersham Pharmacia Biotech, Sweden) was used for protein detection. The membranes were also stained with Ponceau Red to verify that equal amounts of proteins were loaded and transferred.
Pulse-chase analysis of IB
degradation
Metabolic labeling of cells was performed as described previously (Krappmann et al., 1999). Protein extracts were prepared at the indicated time points. Cells were washed twice with cold PBS and lysed in TNT buffer (20 mM Tris, pH 8.0, 200 mM NaCl, 1% Triton X-100) with protease inhibitors as described previously (Lyakh et al., 2000). Lysates were incubated on ice for 15 minutes and then centrifuged for 5 minutes at 13,000 g. Supernatant was used for immunoprecipitation. Immunoprecipitation of 400 µg of the total protein in 3 ml of TNT buffer was performed using IB
N (#1309) antiserum (a generous gift from N. Rice, NCI, Frederick, MD). Two hours later 20 µl of protein A-sepharose 4B (Sigma Chemical Co.) in TNT buffer were added to each sample and incubated with gentle rotation overnight. Then sepharose beads were washed 5 times with ice-cold TNT buffer and boiled for 5 minutes in SDS-loading buffer. The supernatant was resolved by SDS-PAAG followed by transfer to Immobilon-P membrane (Millipore Corporation).
In vitro IKK activity assay
Unstimulated prostate cells and LNCaP cells treated with TNF- (7.5 ng/ml) were lysed in TNT buffer with protease inhibitors. Immunoprecipitation of 450 µg of total protein was performed with 1 µl of rabbit IKK
(#1997) and IKKß (#4137) antisera (a kind gift of N. R. Rice, NCI, Frederick, MD), as described for pulse-chaise assay. Immunoprecipitate was washed three times in TNT buffer with protease inhibitors and twice with kinase buffer without protease inhibitors. Kinase reaction was performed in kinase buffer (20 mM Hepes, pH 7.4, 2 mM MgCl2, 2 mM MnCl2), containing 2 µCi of [
-32P]ATP and I
B
peptide (1-54) that has only Ser32 and Ser36 sites of phosphorylation (Boston Biologicals Inc., Boston, MA) as a substrate for 30 minutes at 30°C. Then 2x Tricine/SDS sample buffer (Novex, Carlsbad, CA) was added to each reaction, samples were boiled and subjected to PAAG on 10-20% gradient tricine PAAG (Novex). Gels were dried and exposed to film with an intensifying screen at 70°C.
IB
cDNA sequencing
IB
cDNA was obtained by RT-PCR from total RNA using previously described primers and conditions (Emmerich et al., 1999) except the modification in sense primer in the fourth pare of primers. We used the primer: 5'-GCTCAGGAGCCCTGTAATGGCC-GGACTG-3'. PCR products were resolved on 1.5% agarose gel, extracted by QIAquick gel extraction kit (Qiagen Inc., Valencia, CA) and subjected to direct sequencing.
Transfection of cell lines and luciferase activity
Prostate cells were plated on 35 mm dishes and at 50% confluence were co-transfected by Tfx-50 reagent (Promega Corp.) with the following constructs: B-luciferase reporter Fireflight luciferase (FL) under promoter with three copies of conventional
B site (Clontech Laboratories Inc., Palo Alto, CA); pRL-null construct Renilla luciferase (RL) under minimal promoter (Promega); MMTV.luciferase reporter Fireflight luciferase (FL) under control of MMTV promoter (Clontech); kinase-inactive mutants of either IKK
(IKK
K44M) or IKKß (IKKßK44M) which work in dominant-negative (d.n.) fashion; and I
B
d.n. mutant. Plasmids with IKK mutants were described earlier (Mercurio et al., 1997) and kindly provided by F. Mercurio (Signal Pharmaceutical Inc., San Diego, CA). Plasmid with the I
B
d.n. mutant lacking serine 32 and serine 36 (Van Antwerp et al., 1996) was a kind gift of I. Verma (Salk Institute, San Diego, CA). Tfx-50 reagent (2.25 µl/µg of plasmid DNA) and the plasmid DNAs (all at a dose of 2 µg/dish)
B.Luc, pRL-null, IKK
d.n., IKKß d.n., and I
B
d.n. were added to the dishes in antibiotic-free, serum-free medium. 24 hours after transfection, prostate cells were harvested in the lysis buffer and the luciferase activity was measured by dual luciferase assay (Promega) as recommended by the manufacturer. FL activity was normalized against RL activity to equalize for transfection efficacy.
Northern blot analysis
Total RNA from freshly harvested cells was isolated by TRI reagent (Molecular Research Center Inc., Cincinnati, OH) and subjected to northern blotting. 20 µg of total RNA was resolved in a 1% agarose/6% formaldehyde gel. The RNA was transferred to nylon membranes and probed for IB
and IL-6. The membranes were also hybridized with a 7S RNA probe to verify that equal amounts of RNA were loaded and transferred. The DNA probes were prepared by random-primed reactions using the complete coding sequence of human I
B
and IL-6 cDNAs (ATCC, Rockville, MD).
P65 immunostaining of prostate tumors
Prostate tissues were obtained from white male patients at the age 40-82 years during biopsy or surgery to remove prostate tumors. Paraffin sections of formalin-fixed prostate carcinoma samples with verified diagnosis and surrounding normal tissues were used for immunostaining. After microwave Ag retrieval and blocking with 5% nonfat milk in PBS, tissues were incubated with primary rabbit polyclonal p65 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) followed by secondary biotinylated anti-rabbit IgG. Immunostaining was visualized with streptavidin-alkaline phosphatase/histo mark red reagent (Kirkegaard & Perry, Gaithensburg, MD). Sections were counterstained in Mayers hematoxylin.
Adenovirus infection and apoptosis detection
Prostate cells were plated on 35 mm dishes and at 50% confluence were infected with type 5 recombinant Adenovirus (AdV) construct AdV-d.n.IB
encoding green fluorescent protein (GFP) and mutant human IkB
protein with substitution of serines 32 and 36 to alanines (32A36A) or adenovirus encoding only GFP (AdV-control). AdV-d.n.IkB
virus with deletions of E1 and E3 was generated using the AdEasy1 system. AdEasy1 system was a generous gift of T.-C. He (The Howard Hughes Medical Institute, Baltimore, MD) (He et al., 1998). Mutations of IkB
were constructed by site-directed mutagenesis with the Bio-Rad Muta-Gene Phagemid In Vitro Mutagenesis system (Bio-Rad Laboratories, Hercules, CA) as described (Whiteside et al., 1995). IkB
mutant has an N-terminal tag (ADRRIPGTAEENLQK) derived from the Equine Infectious Anemia Virus (EIAV) tat protein. Control E1/E3-deleted AdV 5 with GFP (AdV-control) was purchased from Quantum Biotechnologies (Montreal, QC, Canada). Adenoviruses were purified by CsCl gradient centrifugation. Cells were infected with adenoviruses (109 vp/dish) in 700 µl of medium with 0.5% serum overnight. 24 hours after infection cells were treated with 7.5 ng/ml TNF-
(R&D Systems, Minneapolis, MN) for 10 hours. Apoptosis was determined morphologically by counting the number of blebbing cells out of 200 fluorescent cells per slide. In addition, we used PARP proteolysis to determine apoptosis. Adherent cells and detached floaters were combined for whole-cell protein extract preparations. PARP cleavage was estimated by western blot analysis with PARP antibody (PharMingen, San Diego, CA).
Data in all figures are shown as results of the representative experiments. All experiments were repeated at least three times.
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Results |
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To evaluate NF-B DNA-binding activity, we performed an electrophoretic mobility shift assay (EMSA) using nuclear protein extracts. We found a strong increase of
B DNA-binding in androgen-independent DU145, PC3 and JCA1 cell lines compared with normal prostate epithelial cells and androgen-dependent LNCaP and MDA PCa 2b cells (Fig. 1A). It is important to note that
B DNA-binding was higher in androgen-independent CL2 cells derived from androgen-dependent LNCaP cells via an in vitro androgen deprivation (Fig. 1A). Significantly, the level of NF-
B binding in androgen-independent cells was similar to one in LNCaP cells treated with TNF-
(Fig. 1A, last lane). To rule out the general effects that some transcriptional regulators in androgen-independent PC cells have in
B-binding, we performed EMSA with Sp1 oligonucleotide. As shown in Fig. 1C, Sp1 binding activity did not correlate with androgen-dependence of growth. It was equally low in androgen-dependent LNCaP cells, their androgen-independent counterpart CL2, and androgen-independent DU145 cells. By contrast, Sp1 binding was much higher in androgen-dependent MDA PCa 2b cells and in androgen-independent JCA1 cells. Thus, NF-
B was specifically upregulated in androgen-independent PC cells.
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To study NF-B functional activity we performed transient transfection of primary prostate epithelial cells obtained from two different donors and several PC cell lines with exogenous
B-responsive gene,
B-luciferase reporter. The results of these experiments presented in Fig. 2A, in general correlated well with the EMSA results: the basal activity of
B reporter was much higher in androgen-independent PC3 and JCA1 cells than in primary prostate cells and LNCaP cells.
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To extend our observation of increased NF-kB activity in PC cells lines, we performed p65 immunostaining of ten samples of human PC obtained during biopsy and two samples of PC with apparently normal surrounding prostate tissues obtained during prostatectomy. The results clearly showed that p65 was overexpressed in the epithelial component of tumors in comparison with the surrounding tissues. Moreover, p65 was localized both in cytoplasm and in the nuclei of cells in PC: 23±8% of nuclei in PC were p65-positive compared to 10.5±0.7% of nuclei in normal tissues (Table 1). Translocation of p65 to the nucleus strongly suggests that NF-B is activated in prostate tumors. Unfortunately hormone-dependence of tumors could not be assessed because we used biopsies and surgically removed PC tissues from untreated patients.
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Even though we showed that IB
is not mutated in PC cells, we could not rule out that other I
B proteins are mutated or functionally impaired in those cells. Thus, in our next set of experiments we addressed the question whether NF-
B activation in androgen-independent PC cell lines is a result of altered interaction between NF-
B and I
B molecules using the universal inhibitor of all I
B degradation, MG132 (Sun and Carpenter, 1998). We expected that MG132, which blocks proteasome-dependent I
B proteolysis, will inhibit basal
B DNA binding if interaction between NF-
B and I
Bs in androgen-independent cells is normal. As shown in Fig. 4, MG132 indeed strongly inhibited basal
B DNA binding in PC3 and DU145 cells 30-60 minutes after treatment (Fig. 4). MG132 also decreased
B DNA binding in JCA1 cells 1 hour after treatment (Fig. 4). These results suggest that NF-
B is normally controlled by I
Bs in PC cells. Thus, the increased basal NF-
B activity in these cells is not a result of expression of mutated I
B or mutated NF-
B proteins constitutively present in the nucleus.
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Discussion |
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It is important to mention that amplification, overexpression and rearrangements of most genes coding for Rel/NF-B factors have been found in hematopoietic tumors and could underlie the constitutive NF-
B activation (Rayet and Gelinas, 1999). However, the most frequent finding in solid tumors and cell lines derived from solid tumors was the overexpression of p50 and p52 proteins (Rayet and Gelinas, 1999; Dejardin et al., 1995). p50 and p52 proteins have low transactivation activity, thus the biological role of p50 and p52 homodimers appears to be ambiguous (Budunova et al., 1999). The participation of RelA in solid tumors is the subject of many debates. RelA exhibits strong transactivation potential, however, alteration of RelA expression/function in solid tumors or cell lines derived from solid tumors has been only rarely reported (Rayet and Gelinas, 1999). Significantly, we found that the activation of p65/RelA-containing NF-
B complexes with the highest transactivation potential among other NF-
B dimers, was specific for PC cell lines and occurred without p65 or p50 overexpression in androgen-independent PC cells. In this regard it is important that nuclear p65 expression was increased in prostate carcinomas compared to surrounding apparently normal tissues.
The altered expression of IBs as well as mutations in I
B genes in tumor cells are implicated in the constitutive activation of NF-
B (Rayet and Gelinas, 1999; Emmerich et al., 1999; Cabannes et al., 1999). However, the results of our experiments strongly suggest that constitutive activation of NF-
B in PC cells is not a consequence of either altered expression or large rearrangements or mutations in NF-
B/I
B genes. Indeed, we did not find any changes in the level of expression of p65, p50 and three major I
B proteins (I
B
, I
Bß and I
B
) as well as deviations from expected sizes of those molecules in PC cells with activated NF-
B. Further, direct sequencing of I
B
cDNA has not predicted any mutations of the I
B
protein in cell lines with constitutive NF-
B activation. We cannot presently rule out the presence of mutations in I
Bß, I
B
, p50 or RelA genes in DU145, PC3 and JCA1 cells. However, our experiments with different NF-
B inhibitors and activators provided indirect evidence that NF-
B is normally controlled by I
Bs and fully functional in those PC cells. Indeed, the constitutive activity of NF-
B in DU145, PC-3 and JCA1 cells was inhibited by the IKK
d.n. mutant, IKKß d.n. mutant and by a proteasomal inhibitor MG132 that effectively blocks degradation of all I
B proteins (Sun and Carpenter, 1998). The analysis of the sensitivity of PC cells to the standard NF-
B inducers such as TNF-
, LPS and TPA, revealed that, in contrast to the Hodgkin lymphoma cells (Krappmann et al., 1999), and in spite of the high basal level of NF-
B activity, PC cells are highly sensitive to NF-
B activation (Gasparian et al., 2000).
Another recently described mechanism of NF-B activation in tumor cells implicates increased I
B
phosphorylation and turnover (Devalaraja et al., 1999; Krappmann et al., 1999). We found that in all studied androgen-independent PC cells, including CL2 cells derived from LNCaP cells, I
B
was heavily phosphorylated. Moreover, I
B
displayed a faster turnover in androgen-independent PC cells than in androgen-dependent PC cells. In addition, by in vitro kinase assay we demonstrated constitutive activation of IKK in androgen-independent cell lines. It is currently understood that the mechanisms of basal and induced NF-
B activation could be different. Activation of NF-
B through phosphorylation, ubiquitination and proteasome-dependent degradation of I
Bs is specific for cells treated with NF-
B inducers (Whiteside and Israel, 1997; Heissmeyer et al., 1999). The mechanisms responsible for the maintenance of the basal NF-
B activity are less clear and may not require I
B
phosphorylation at Ser32/36, ubiquitination or even proteasome-dependent degradation (Miyamoto et al., 1998; Krappmann et al., 1996). Our data strongly suggest that in androgen-independent PC cells, basal NF-
B activation employs a mechanism similar to that for NF-
B activation by inducers such as cytokines. It appears that constitutive NF-
B activity depends on the constitutive aberrant activation of IKKs and consequently, a faster I
B
turnover.
In this regard, it is important to mention that the androgen-independent PC cells produce numerous growth factors and cytokines, that are strong activators of IKK complex and consequently NF-B. Those cytokines and growth factors include TNF-
, different interleukins, FGF, EGF, NGF, HGF, PDGF and VEGF (Baldwin, 1996; Sun and Carpenter, 1998; Byrd et al., 1999; Gentry et al., 2000; Romashkova and Makarov, 1999). Knowing that the expression of genes encoding certain cytokines, for example IL-6, is regulated by NF-
B (Zhang et al., 1994), one could assume that activation of IKK in PC cells involves an established positive autocrine/paracrine loop.
Androgen-independent cell lines used in this study do not express androgen receptor (AR) (Tso et al., 2000; Mitchell et al., 2000). This allows to find an interesting parallel between NF-B activation in androgen-independent PC cells and estrogen receptor (ER)-deficient breast carcinoma cell lines (Nakshatri et al., 1997; Biswas et al., 2000) and to raise the question of the possible role of NF-
B in the development of growth autonomy and resistance to apoptosis in hormone-independent prostate and breast tumors. It is known that NF-
B is a key anti-apoptotic factor in most cells (Barkett and Gilmore, 1999). It has become clear recently that NF-
B could also play the pro-proliferative role in some cells through direct activation of genes involved in the cell cycle (Biswas et al., 2000; Hinz et al., 1999; Guttridge et al., 1999).
We found that NF-B blockage resulted in the increased apoptosis in LNCaP cells, and increased sensitivity to apoptosis induced by TNF-
in PC3 cells with high constitutive NF-
B activity. The latter result is in line with the previous finding on the essential role of NF-
B in resistance of PC cells to TNF-
(Sumitomo et al., 1999). The high resistance of PC3 cells with elevated constitutive level of NF-
B, to NF-
B blockage could be explained by the residual NF-
B activity in those cells (data not shown).
It is important to mention that despite some general similarities in the response of prostate cells to androgens and NF-B inducers, there is an evidence that NF-
B and AR mutually repress each other transcriptional activity. The repression involves either direct protein-protein interaction between AR and p65 or competition for intracellular transcriptional regulators (Palvimo et al., 1996; Valentine et al., 2000). Moreover, crosstalk between signaling mediated by AR and NF-
B also involves transcriptional repression of the AR gene by NF-
B (Supakar et al., 1995). This suggests that NF-
B blockage may result in restoration of AR function in PC cells.
In conclusion, the results presented here demonstrate that aberrant IKK activation leads to the constitutive activation of the NF-B survival signaling pathway in androgen-independent PC cells. Since NF-
B protects prostate cells from apoptosis, possibly stimulates proliferation of PC cells, and plays an important role in the selection for hormone-independence, NF-
B and IKK inhibition may prove useful both in the prevention of PC and in adjuvant therapy. Further studies are needed to identify the affected upstream signaling that results in IKK activation.
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ACKNOWLEDGMENTS |
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