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Sp3 transcriptional activity in Ehrlich tumor cells
Cancer Letters 218 (2005) 91–98 www.elsevier.com/locate/canlet
Inhibition of glutaminase expression increases Sp1 phosphorylation and Sp1/Sp3 transcriptional activity in Ehrlich tumor cells Juan Antonio Seguraa,*, Ana Carolina Donadiob, Carolina Loboa, Jose´ Manuel Mate´sa, Javier Ma´rqueza, Francisco Jose´ Alonsoa a
Departamento de Biologı´a Molecular y Bioquı´mica. Facultad de Ciencias, Campus de Teatinos, Universidad de Ma´laga, 29071 Ma´laga, Spain b Departamento de Bioquı´mica Clı´nica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Pabello´n Argentina, ala I, subsuelo, Ciudad Universitaria, CP 5000 Co´rdoba, Argentina Received 2 June 2004; accepted 28 June 2004
Abstract Tumor cells expressing antisense glutaminase RNA show a drastic inhibition of glutaminase activity and they acquire a more differentiated phenotype. We have studied the expression of Sp1 and Sp3 transcription factors in both Ehrlich tumor cells and their derivative 0.28AS-2 antisense glutaminase expressing cells. The expression of phosphorylated Sp1 in 0.28AS-2 cells was 3-fold the expression in EATC. Full length Sp3 was also incremented in 0.28AS-2 cells. Sp1 and Sp3 binding to a consensus Sp1 probe was higher in 0.28AS-2 nuclear extracts, as determined by supershift assays. Sp1–DNA binding was inhibited by phosphatase treatment, demonstrating that phosphorylation of Sp1 is critical for its DNA binding capacity. The Sp1 and Sp3 DNA binding found in 0.28AS-2 cells was also correlated with an increased Sp1 activity, as shown in transient transfections assays carried out with a luciferase reporter plasmid. Incubation of Ehrlich tumor cells with the differentiation agent PMA could not totally reproduce the Sp1/Sp3 changes observed in 0.28AS-2 cells. However, it was demonstrated that the intracellular concentration of glutamine, but not glutamate or aspartate, is increased in 0.28AS-2 cells. In conclusion, the antisense inhibition of glutaminase leads to an increased expression of phosphorylated Sp1 and that correlates with an increase in Sp1 activity. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Glutaminase; Sp1; Sp3; Tumor; Antisense
1. Introduction * Corresponding author. Tel.: C34-9521-37601; fax: C34-952132000. E-mail address: [email protected] (J.A. Segura).
Glutamine is the most abundant free amino acid in plasma and a respiratory fuel for most neoplastic cells [1]. Glutamine plays an important role in a number of
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.06.054
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cellular processes such as gene expression regulation, apoptosis, cell swelling and proliferation signaling (see Ref. [2] for a review). Reduced availability of glutamine causes differentiation in human cancer cell lines, and its supplementation promotes a less differentiated phenotype [3,4]. Many experimental data provide evidence that an increased activity of phosphate activated glutaminase (EC 3.5.1.2) (GLS), the first step of glutamine catabolism, is positively correlated with proliferation and tumor malignancy [5–7]. Even though GLS is frequently up-regulated in tumors, neither energetic nor biosynthetic requirements can totally explain the very high glutaminolytic flux found in most cancer cells. Recently, using antisense technology, Lobo et al. [8] reported that inhibition of Ehrlich ascites tumor cell (EATC) GLS by antisense technology, diminished cellular proliferation and induced the acquisition of a more differentiated phenotype. Moreover, the antisense transfected cells, named 0.28AS-2, failed to grow in vivo as a consequence of their incapacity to evade the mouse immune response [9]. Sp1 is the first identified member of a family of transcription factors that binds DNA GC-rich boxes and regulates the expression of many different genes [10]. There is growing evidence that the Spfamily plays an important role in proliferation and differentiation and participates in the regulation of genes that are both ubiquitously expressed, as well as those expressed in a tissue specific manner [11,12]. Sp1 can be phosphorylated, a modification that affects its binding to the DNA [13–15] and Oglycosilated [16], which confers resistance to proteosome dependent degradation [17]. The GC-rich boxes bound by Sp1 are also recognized by the Sp3 transcription factor, competing for DNA binding. The regulation of Sp3 transcriptional activity is very complex and it has been described both as an activator or an inhibitor [10]. In the present work, Sp1 and Sp3 expression is studied in nuclear extracts prepared from EATC and their derivative 0.28AS-2 cells. The data presented here provide evidence that associates a differential regulation of Sp1 with the changes induced by antisense inhibition of GLS.
2. Materials and methods 2.1. Cell lines and culture conditions EATC, purchased from American Type Culture Collection (ATCC), and their derivative 0.28AS-2 cells [8] were grown in RPMI medium (Sigma) supplemented with 10% FCS and antibiotics. Cultures were incubated in a humidified atmosphere at 37 8C with 5% CO2/95% air; 0.28AS-2 cells were obtained from EATC after lipofection with the pcDNA3 plasmid (Invitrogene) containing an antisense 3 0 -cDNA segment (0.28 kb) from rat kidney GLS [8]. In some experiments, 100 nM PMA was added to the culture medium of exponentially growing cells, and after 48 h cells were harvested for processing. 2.2. Immunoblots Whole cell extracts were prepared as follows: cells were trypsinized, washed in PBS and lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 g/ml PMSF, 0.15–0.3 TIU/ml aprotinin and 1 mM sodium orthovanadate in PBS). The extracts were heat denatured in Laemmli sample buffer, resolved in a 7.5% acrylamide SDS gel and then electroblotted onto nitrocellulose membranes (Schleiger and Shuell). After blocking for 1 h with TTBS buffer (Tris–HCl 100 mM pH 7.5, 0.1% Tween-20, 0.9% NaCl) and 7% non fat dry milk, the blots were exposed to the Sp1 or Sp3 polyclonal antibodies (Santa Cruz Biotechnologies) for 1 h. As a secondary antibody, a goat anti-rabbit IgG-peroxidase (Sigma) was used. Finally, the resulting protein– antibody complexes were detected by chemiluminescence with the ECL kit (Amersham Pharmacia Biotech). Bands were quantified by using a GS-800 calibrated densitometer (Bio-Rad) and the Quantityone software. The immunoprecipitation of phosphorylated Sp1 was carried out using an anti-phospho-serine agaroseconjugated antibody (Sigma). Immunoprecipitated complexes were dissolved in SDS sample buffer, electrophoresed and immunoblotted. Finally, Sp1 was detected using Sp1 polyclonal antibodies.
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2.3. Preparation of nuclear extracts Nuclear extracts were obtained with a modification of the method described by Kovarik et al. [18]. Briefly, approx. 5!107 cells were trypsinised, collected and washed once with cold phosphate buffered saline. Cells were then lysed for 30 min at 4 8C with 2 ml of lysis buffer (0.6% Nonidet P-40, 0.15 M NaCl, 10 mM Tris–HCl, pH 7.9 and 1 mM EDTA). Nuclei were pelleted at 2000g for 5 min and washed once with lysis buffer without detergent. Nuclear proteins were extracted from the final pellet with one volume (packed volume of cell pellet) of high salt extraction buffer (10 mM HEPES, pH 7.9, 0.42 M NaCl, 0.1 mM EGTA, 0.1 mM EDTA, 1.5 mM MgCl2, 0.5 mM dithiothreitol, and 25% glycerol) for 30 min at 4 8C with occasional shaking. Nuclear debris was removed by centrifugation (twice at 10000g for 10 min at 4 8C). The protein concentrations were estimated with the Bradford reagent (Bio-Rad). All buffers contained the following protease inhibitors: 0.5 mM PMSF, 0.1 mM benzimidine and 1 g/ml leupeptin. The nuclear proteins were kept at K70 8C and used without dialysis in band shift assays. 2.4. Oligonucleotide band shifts Consensus double stranded oligonucleotides for Sp1 and mutant control were obtained from Santa Cruz Biotechnologies (consensus: 5 0 -ATTCGATCGGGGCGGGGCGAGC-3 0 ; mutant: 5 0 -ATTCGATCGGTTCGGGGCGAGC-3 0 ). Oligonucleotides were labeled at the 5 0 ends with 125 mCi of [gamma-32P]ATP (Amersham Corp., 3000 Ci/mmol) in a T4 polynucleotide kinase reaction. A 20 ml binding mixture contained: 50000 cpm of end-labeled oligonucleotide probe, 4% glycerol, 10 mM Tris–HCl, pH 7.6, 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1mg of poly(dI-dC) (Roche), and 5mg of nuclear extract. The binding reaction was performed for 30 min at 4 8C. In some experiments, nuclear extracts were preincubated with 5U of alkaline phosphatase (Roche) for 20 min at 30 8C, prior to the addition of the probe. For Sp1 and Sp3 DNAbinding detection, specific goat polyclonal antibodies (Santa Cruz Biotechnologies) were used. Antisera were incubated with the binding mixture for 10 min at 4 8C
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prior to the addition of the probe. The retarded complexes were electrophoresed on 4% native polyacrylamide gels at 75 V at room temperature. After that, the gels were dried and exposed to films. 2.5. Transient transfection and luciferase assay The Sp1-dependent luciferase reporter plasmid pGAGC6 was a kind gift from Dr Jeffrey Kudlow (School of Medicine. University of Alabama). The pGAGC6 plasmid contains six Sp1 consensusbinding sites cloned into a luciferase reporter plasmid upstream of the adenovirus major late initiator TATA box. The plasmid pGAM, containing only the adenovirus TATA box, was used as a control. The plasmid pCMV-bgal (containing the human cytomegalovirus promoter upstream the bgalactosidase gene) was used to normalize luciferase activity. Cotransfections of EATC and 0.28AS-2 cells with reporters and the pCMV-bgal plasmids were carried out using Lipofectin Reagent (GibcoBRL) according to the manufacturer’s instructions. The combined detection of luciferase and b-galactosidase was performed with the Dual-Light System (Applied Biosystems). Expression of luciferase enzymatic activity was normalized to b-galactosidase activity. Each transfection was repeated at least three times, and statistical differences were determined by using the U-Mann–Whitney test. 2.6. Measurement of glutaminase activity and determination of intracellular amino acid concentrations Glutaminase activity was determined in EATC and 0.28AS-2 cells as it is described by Lobo et al. [8]. In order to determine the intracellular concentration of amino acids, 75!106 cells were harvested, washed with PBS and deproteinized with 1.5 ml of 10% (v/v) perchloric acid. After centrifugation 3 min at 3000g, the supernatant fluid was neutralized with 20% KOH (w/v) using Universal indicator. The concentration of glutamine and glutamate was determined as described by Lund [19], and aspartate as described by Mo¨llering [20].
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Fig. 1. Sp1 and Sp3 protein content and Sp1 phosphorylation in EATC and 0.28AS-2 cells. Nuclear extracts were prepared from exponentially growing cells, and then analyzed by immunoblotting for Sp1 (A) and Sp3 (C) protein content. Immunoprecipitated Sp1 protein using an anti-phosphoserine antibody is shown in panel (B). The blots shown are representative of results obtained with at least three independently prepared nuclear extracts and they were carried out as described under ‘Section 2’.
3. Results In order to study the effects of GLS inhibition on the expression level of Sp1 and Sp3 transcription
factors, we carried out Western blots experiments using nuclear extracts made up from EATC and their derivative, the antisense GLS expressing 0.28AS-2 cells. Two proteins of 105 and 95 kDa, corresponding to the phosphorylated and non-phosphorylated Sp1 forms, respectively, were detected. While the nonphosphorylated form did not change, the intensity of the phosphorylated form increased 3-fold in 0.28AS-2 cells as compared to EATC (Fig. 1A). The phosphorylated form could be immunoprecipitated by using an anti-phospho-serine agarose-conjugated antibody (Fig. 1B). The Sp3 isoforms expression pattern obtained (Fig. 1C) was similar to that reported by other authors [21,22]: a primary band of 110 kDa representing the full length protein and two bands corresponding to truncated isoforms of about 60– 70 kDa [10]. Immunoblot experiments revealed that Sp3 full length protein expression is increased 8-fold in 0.28AS-2 cells. In order to determine the Sp1 and Sp3 DNA binding capacity of EATC and 0.28AS-2 nuclear extracts, an electrophoretic mobility shift assay using a consensus Sp1 oligonucleotide was developed. Two retarded complexes were detected in the mobility shift assay of both EATC and 0.28AS-2 nuclear extracts (Fig. 2A). Significantly, the band intensities obtained with 0.28AS-2 nuclear extracts were greater than with EATC. When a specific Sp1 antibody was used, only the lower band could be observed. On the contrary, only the upper band remained when a Sp3 specific antibody was used. This seems to indicate that the upper band corresponds to Sp1–DNA complexes and the lower band to Sp3–DNA complexes.
Fig. 2. EATC and 0.28AS-2 band shift assays. (A) Nuclear extracts were prepared from exponentially growing cells and incubated with a radiolabelled Sp1 consensus oligonucleotide. Anti-Sp1 or anti-Sp3 antibodies were used to identify Sp complexes. As a control for specificity 100!Sp1 unlabelled consensus (100!Cons.) or mutated (100!Mut.) oligonucleotides were used. (B) Nuclear extracts from 0.28AS-2 cells were treated with alkaline phosphatase and subjected to band shift assay. Consensus and mutated probes sequencies are described under ‘Section 2’. The autorradiograms shown are representatives of the results obtained with three independently prepared nuclear extracts.
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Fig. 3. Sp1 activity in EATC and 0.28AS-2 cells using a luciferase reporter plasmid. EATC and 0.28AS-2 cells were transiently cotransfected with the pGAGC6 Sp1 reporter plasmid and a b-Galactosidase plasmid (pCMV-bgal). pGAM plasmid, lacking the Sp1 consensus motives, was used as a control. Luciferase activity was quantified 48 h later. The results are reported as mean valuesGstandard deviations from three different experiments. Each experiment was measured in triplicate. *P!0.01 as tested with the Mann–Whitney U test.
The increased Sp1–DNA binding obtained with 0.28AS-2 nuclear extracts could be explained by its increased expression of phosphorylated Sp1 protein. In order to test this possibility, 0.28AS-2 nuclear extracts were treated with alkaline phosphatase prior to carry out the mobility shift assay experiment. As shown in Fig. 2B most of the upper band, assigned to Sp1, was absent after alkaline phosphatase treatment. Because Sp1–DNA binding is not always positively correlated with Sp1 activity, we carried out measurements of Sp1 activity in EATC and 0.28AS-2 cells transfected with the Sp1 reporter plasmid pGAGC6. This plasmid contains six Sp1 consensus sites and an adenovirus TATA box upstream of a luciferase reporter gene. A plasmid vector without
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the Sp1 sites (pGAM) was used as a control and the PCMV-bgal plasmid containing the b-galactosidase gene was used to normalize luciferase activity. As shown in Fig. 3, luciferase activity in 0.28AS-2 cells was 3.5-fold the value obtained with the parental EATC. Since members of the Sp-family of transcription factors are implicated in differentiation and proliferation processes, we wanted to test whether the induction of differentiation in EATC could also reproduce the Sp1/Sp3 pattern observed in 0.28AS-2 cells. To achieve this, we used the differentiation agent PMA. Culture medium from exponentially growing EATC was supplemented with 100 nM PMA. The cells were further incubated for 2 days and then nuclear extracts were prepared and analyzed by immunoblot and band shift assays. In Western blot analyses (Fig. 4A), nuclear extracts from control EATC showed a pattern of Sp 1 protein very similar to the one obtained above (Fig. 1A). PMA treatment caused an enhanced expression of both 105 and 95 kDa forms of Sp1, but did not affect the Sp3 protein (Fig. 4B). As observed in band shift assays (Fig. 4C), PMA treatment did not caused any significant change in the binding activity of Sp1 to the consensus Sp1 oligo. Next we decided to study if the inhibition of GLS activity could influence the concentration of intracellular glutamine and the products of glutamine metabolism glutamate and aspartate. The antisense inhibition of GLS decreased almost 5 fold the GLS activity in 0.28AS-2 cells, and increased to the same extent the intracellular glutamine concentration. Just the opposite situation can be observed in EATC
Fig. 4. Effects of glutamine deprivation and PMA treatment on Sp1 and Sp3 expression in EATC. Culture medium from exponentially growing EATC was replaced either with glutamine free medium or medium supplemented with 100 nM PMA. Fresh cell culture medium was used in control plates. Cells were further incubated for 2 days. Nuclear extracts were then prepared as described under ‘Section 2’. Total Sp1 and Sp3 protein content were analyzed by immunoblotting (A and B) and the DNA binding activity of proteins from the nuclear extracts were studied by band shift assay (C) as detailed under ‘Section 2’. Results shown are a representative of three independently prepared extracts.
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Table 1 Determination of GLS activity and quantification of intracellular concentration of amino acids
EATC 0.28AS-2
GLS activity (mU/106 cells)
Glutamine (mM)
Glutamate (mM)
Aspartate (mM)
23.0G2.8* 5.0G0.9*
1.3G0.5* 5.2G1.7*
6.7G1.0 6.9G1.1
3.8G0.2 4.6G0.3
Each value is the meanGSD for three independent cell preparations. *P!0.05 as determined by the U-Mann–Whitney statistical test.
(Table 1). However, the concentration of glutamate and aspartate did not significantly change.
4. Discussion The influence of GLS inhibition on Sp1 and Sp3 expression is studied by using wild type EATC and in their derivative the 0.28AS-2 antisense GLS expressing cells. It is remarkable that the expression of phosphorylated Sp1 protein is incremented in 0.28AS2 cells. Immunoprecipitation experiments revealed that Sp1 is mostly phosphorylated on a serine residue (Fig. 1). Furthermore, the Sp1 phosphorylation also induces an increase in Sp1-DNA binding, as it is manifested in band shift assays (Fig. 2). The fact that phosphatase treatment inhibits DNA Sp1 binding highlights the, importance of phosphorylation in the DNA binding capacity of Sp1 in 0.28AS-2 cells. The increase in Sp1 DNA binding is also correlated with a similar increase in Sp1 activity in 0.28AS-2 cells, as it is shown in transient transfection experiments carried out with luciferase reporter plasmids (Fig. 3). These results clearly correlate Sp1-phosphorylation with an increased Sp1 activity in 0.28AS-2 cells in agreement with previous reports showing that an increased Sp1 phosphorylation enhances DNA binding and/or transactivation [23,24]. An important mechanism that can modulate Sp1 activity is exerted by Sp3 protein. We have shown by immunoblotting that the 110 kDa form of Sp3 protein was 8-fold incremented in 0.28AS-2 cells (Fig. 1C), and this is also accompanied by an increase in Sp3 DNA binding (Fig. 2A), however, the increase in Sp3 DNA binding does not lead to an inhibition of Sp1 activity, as it is shown in the transfections with luciferase reporter plasmid. Reports on the transcriptional properties of Sp3 appear, at first sight, contradictory. Sp3 has been shown to act as a transcriptional positive regulator
similar to Sp1 [25,26]; in other experiments, Sp3 remained inactive or acted only as a very weak activator [27,28]. The structure and arrangement of the recognition sites appears to determine whether Sp3 is transcriptionally inactive and can repress Sp1 mediated activation or it acts as a strong activator. Promoters containing single binding sites are activated, whereas promoters containing multiple binding sites often are not activated or they respond weakly to Sp3 [29,30]. In 0.28AS-2 cells, Sp3 seems to remain inactive or acts as a weak activator as judge by the Sp1 activity measured in the luciferase experiments carried out with the pGAGC6 plasmid. It has been previously reported that changes in the phosphorylated forms of Sp1 occur during liver differentiation [31] and in cell cycle regulation [32]. That suggests a possible role of Sp1 in the maintenance of the more differentiated phenotype of 0.28AS-2 cells. When EATC were treated with PMA, phosphorylated and non-phosphorylated forms of Sp1 were increased, whereas Sp3 protein content and binding to DNA remained unchanged. This suggests that the mechanisms underlying PMA driven differentiation and GLS antisense inhibition are not coincident. The inhibition of GLS in 0.28AS-2 cells must influence the glutamine consumption and metabolization. It should be affected both the level of free intracellular glutamine and the intermediate metabolites of the glutamine oxidation pathway. In fact, we have demonstrated that the intracellular concentration of glutamine, but not glutamate or aspartate, is increased in 0.28AS-2 cells. The higher GLS activity of EATC keep the intracellular glutamine at low concentrations; this is in accordance with previous data showing that in vivo growing EATC have undetectable levels of free intracellular glutamine [33]. Glutamate and aspartate are maintained at similar levels both in FATC and 0.28AS-2 cells, however, the higher GLS activity in EATC should
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lead to a higher glutamate consumption and metabolization. The differences in free intracellular glutamine is interesting per se, and could influence a number of cellular processes, since there is a growing body of evidence pointing to glutamine as a signal molecule involved in apoptosis, proliferation and gene expression regulation [34–36]. In conclusion, the results obtained show that antisense glutaminase inhibition enhances Sp1 phosphorylation, leading to an increased Sp1 DNA binding and activity. This could be implicated in the reversion of the malignant phenotype observed in 0.28AS-2 antisense expressing cells. Further studies are needed to clarify the mechanisms underlying the molecular links between glutaminase inhibition and Sp1 activation.
Acknowledgements This work was supported by the grant SAF20011894 from the Comisio´n Interministerial de Ciencia y Tecnologı´a and the Fundacio´n Ramo´n Areces. This paper is dedicated to Professor Ignacio Nu´n˜ez de Castro in the year of his retirement.
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Inhibition of glutaminase expression increases Sp1 phosphorylation and Sp1/Sp3 transcriptional activity in Ehrlich tumor cells Juan Antonio Seguraa,*, Ana Carolina Donadiob, Carolina Loboa, Jose´ Manuel Mate´sa, Javier Ma´rqueza, Francisco Jose´ Alonsoa a
Departamento de Biologı´a Molecular y Bioquı´mica. Facultad de Ciencias, Campus de Teatinos, Universidad de Ma´laga, 29071 Ma´laga, Spain b Departamento de Bioquı´mica Clı´nica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Pabello´n Argentina, ala I, subsuelo, Ciudad Universitaria, CP 5000 Co´rdoba, Argentina Received 2 June 2004; accepted 28 June 2004
Abstract Tumor cells expressing antisense glutaminase RNA show a drastic inhibition of glutaminase activity and they acquire a more differentiated phenotype. We have studied the expression of Sp1 and Sp3 transcription factors in both Ehrlich tumor cells and their derivative 0.28AS-2 antisense glutaminase expressing cells. The expression of phosphorylated Sp1 in 0.28AS-2 cells was 3-fold the expression in EATC. Full length Sp3 was also incremented in 0.28AS-2 cells. Sp1 and Sp3 binding to a consensus Sp1 probe was higher in 0.28AS-2 nuclear extracts, as determined by supershift assays. Sp1–DNA binding was inhibited by phosphatase treatment, demonstrating that phosphorylation of Sp1 is critical for its DNA binding capacity. The Sp1 and Sp3 DNA binding found in 0.28AS-2 cells was also correlated with an increased Sp1 activity, as shown in transient transfections assays carried out with a luciferase reporter plasmid. Incubation of Ehrlich tumor cells with the differentiation agent PMA could not totally reproduce the Sp1/Sp3 changes observed in 0.28AS-2 cells. However, it was demonstrated that the intracellular concentration of glutamine, but not glutamate or aspartate, is increased in 0.28AS-2 cells. In conclusion, the antisense inhibition of glutaminase leads to an increased expression of phosphorylated Sp1 and that correlates with an increase in Sp1 activity. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Glutaminase; Sp1; Sp3; Tumor; Antisense
1. Introduction * Corresponding author. Tel.: C34-9521-37601; fax: C34-952132000. E-mail address: [email protected] (J.A. Segura).
Glutamine is the most abundant free amino acid in plasma and a respiratory fuel for most neoplastic cells [1]. Glutamine plays an important role in a number of
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.06.054
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cellular processes such as gene expression regulation, apoptosis, cell swelling and proliferation signaling (see Ref. [2] for a review). Reduced availability of glutamine causes differentiation in human cancer cell lines, and its supplementation promotes a less differentiated phenotype [3,4]. Many experimental data provide evidence that an increased activity of phosphate activated glutaminase (EC 3.5.1.2) (GLS), the first step of glutamine catabolism, is positively correlated with proliferation and tumor malignancy [5–7]. Even though GLS is frequently up-regulated in tumors, neither energetic nor biosynthetic requirements can totally explain the very high glutaminolytic flux found in most cancer cells. Recently, using antisense technology, Lobo et al. [8] reported that inhibition of Ehrlich ascites tumor cell (EATC) GLS by antisense technology, diminished cellular proliferation and induced the acquisition of a more differentiated phenotype. Moreover, the antisense transfected cells, named 0.28AS-2, failed to grow in vivo as a consequence of their incapacity to evade the mouse immune response [9]. Sp1 is the first identified member of a family of transcription factors that binds DNA GC-rich boxes and regulates the expression of many different genes [10]. There is growing evidence that the Spfamily plays an important role in proliferation and differentiation and participates in the regulation of genes that are both ubiquitously expressed, as well as those expressed in a tissue specific manner [11,12]. Sp1 can be phosphorylated, a modification that affects its binding to the DNA [13–15] and Oglycosilated [16], which confers resistance to proteosome dependent degradation [17]. The GC-rich boxes bound by Sp1 are also recognized by the Sp3 transcription factor, competing for DNA binding. The regulation of Sp3 transcriptional activity is very complex and it has been described both as an activator or an inhibitor [10]. In the present work, Sp1 and Sp3 expression is studied in nuclear extracts prepared from EATC and their derivative 0.28AS-2 cells. The data presented here provide evidence that associates a differential regulation of Sp1 with the changes induced by antisense inhibition of GLS.
2. Materials and methods 2.1. Cell lines and culture conditions EATC, purchased from American Type Culture Collection (ATCC), and their derivative 0.28AS-2 cells [8] were grown in RPMI medium (Sigma) supplemented with 10% FCS and antibiotics. Cultures were incubated in a humidified atmosphere at 37 8C with 5% CO2/95% air; 0.28AS-2 cells were obtained from EATC after lipofection with the pcDNA3 plasmid (Invitrogene) containing an antisense 3 0 -cDNA segment (0.28 kb) from rat kidney GLS [8]. In some experiments, 100 nM PMA was added to the culture medium of exponentially growing cells, and after 48 h cells were harvested for processing. 2.2. Immunoblots Whole cell extracts were prepared as follows: cells were trypsinized, washed in PBS and lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 g/ml PMSF, 0.15–0.3 TIU/ml aprotinin and 1 mM sodium orthovanadate in PBS). The extracts were heat denatured in Laemmli sample buffer, resolved in a 7.5% acrylamide SDS gel and then electroblotted onto nitrocellulose membranes (Schleiger and Shuell). After blocking for 1 h with TTBS buffer (Tris–HCl 100 mM pH 7.5, 0.1% Tween-20, 0.9% NaCl) and 7% non fat dry milk, the blots were exposed to the Sp1 or Sp3 polyclonal antibodies (Santa Cruz Biotechnologies) for 1 h. As a secondary antibody, a goat anti-rabbit IgG-peroxidase (Sigma) was used. Finally, the resulting protein– antibody complexes were detected by chemiluminescence with the ECL kit (Amersham Pharmacia Biotech). Bands were quantified by using a GS-800 calibrated densitometer (Bio-Rad) and the Quantityone software. The immunoprecipitation of phosphorylated Sp1 was carried out using an anti-phospho-serine agaroseconjugated antibody (Sigma). Immunoprecipitated complexes were dissolved in SDS sample buffer, electrophoresed and immunoblotted. Finally, Sp1 was detected using Sp1 polyclonal antibodies.
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2.3. Preparation of nuclear extracts Nuclear extracts were obtained with a modification of the method described by Kovarik et al. [18]. Briefly, approx. 5!107 cells were trypsinised, collected and washed once with cold phosphate buffered saline. Cells were then lysed for 30 min at 4 8C with 2 ml of lysis buffer (0.6% Nonidet P-40, 0.15 M NaCl, 10 mM Tris–HCl, pH 7.9 and 1 mM EDTA). Nuclei were pelleted at 2000g for 5 min and washed once with lysis buffer without detergent. Nuclear proteins were extracted from the final pellet with one volume (packed volume of cell pellet) of high salt extraction buffer (10 mM HEPES, pH 7.9, 0.42 M NaCl, 0.1 mM EGTA, 0.1 mM EDTA, 1.5 mM MgCl2, 0.5 mM dithiothreitol, and 25% glycerol) for 30 min at 4 8C with occasional shaking. Nuclear debris was removed by centrifugation (twice at 10000g for 10 min at 4 8C). The protein concentrations were estimated with the Bradford reagent (Bio-Rad). All buffers contained the following protease inhibitors: 0.5 mM PMSF, 0.1 mM benzimidine and 1 g/ml leupeptin. The nuclear proteins were kept at K70 8C and used without dialysis in band shift assays. 2.4. Oligonucleotide band shifts Consensus double stranded oligonucleotides for Sp1 and mutant control were obtained from Santa Cruz Biotechnologies (consensus: 5 0 -ATTCGATCGGGGCGGGGCGAGC-3 0 ; mutant: 5 0 -ATTCGATCGGTTCGGGGCGAGC-3 0 ). Oligonucleotides were labeled at the 5 0 ends with 125 mCi of [gamma-32P]ATP (Amersham Corp., 3000 Ci/mmol) in a T4 polynucleotide kinase reaction. A 20 ml binding mixture contained: 50000 cpm of end-labeled oligonucleotide probe, 4% glycerol, 10 mM Tris–HCl, pH 7.6, 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1mg of poly(dI-dC) (Roche), and 5mg of nuclear extract. The binding reaction was performed for 30 min at 4 8C. In some experiments, nuclear extracts were preincubated with 5U of alkaline phosphatase (Roche) for 20 min at 30 8C, prior to the addition of the probe. For Sp1 and Sp3 DNAbinding detection, specific goat polyclonal antibodies (Santa Cruz Biotechnologies) were used. Antisera were incubated with the binding mixture for 10 min at 4 8C
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prior to the addition of the probe. The retarded complexes were electrophoresed on 4% native polyacrylamide gels at 75 V at room temperature. After that, the gels were dried and exposed to films. 2.5. Transient transfection and luciferase assay The Sp1-dependent luciferase reporter plasmid pGAGC6 was a kind gift from Dr Jeffrey Kudlow (School of Medicine. University of Alabama). The pGAGC6 plasmid contains six Sp1 consensusbinding sites cloned into a luciferase reporter plasmid upstream of the adenovirus major late initiator TATA box. The plasmid pGAM, containing only the adenovirus TATA box, was used as a control. The plasmid pCMV-bgal (containing the human cytomegalovirus promoter upstream the bgalactosidase gene) was used to normalize luciferase activity. Cotransfections of EATC and 0.28AS-2 cells with reporters and the pCMV-bgal plasmids were carried out using Lipofectin Reagent (GibcoBRL) according to the manufacturer’s instructions. The combined detection of luciferase and b-galactosidase was performed with the Dual-Light System (Applied Biosystems). Expression of luciferase enzymatic activity was normalized to b-galactosidase activity. Each transfection was repeated at least three times, and statistical differences were determined by using the U-Mann–Whitney test. 2.6. Measurement of glutaminase activity and determination of intracellular amino acid concentrations Glutaminase activity was determined in EATC and 0.28AS-2 cells as it is described by Lobo et al. [8]. In order to determine the intracellular concentration of amino acids, 75!106 cells were harvested, washed with PBS and deproteinized with 1.5 ml of 10% (v/v) perchloric acid. After centrifugation 3 min at 3000g, the supernatant fluid was neutralized with 20% KOH (w/v) using Universal indicator. The concentration of glutamine and glutamate was determined as described by Lund [19], and aspartate as described by Mo¨llering [20].
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Fig. 1. Sp1 and Sp3 protein content and Sp1 phosphorylation in EATC and 0.28AS-2 cells. Nuclear extracts were prepared from exponentially growing cells, and then analyzed by immunoblotting for Sp1 (A) and Sp3 (C) protein content. Immunoprecipitated Sp1 protein using an anti-phosphoserine antibody is shown in panel (B). The blots shown are representative of results obtained with at least three independently prepared nuclear extracts and they were carried out as described under ‘Section 2’.
3. Results In order to study the effects of GLS inhibition on the expression level of Sp1 and Sp3 transcription
factors, we carried out Western blots experiments using nuclear extracts made up from EATC and their derivative, the antisense GLS expressing 0.28AS-2 cells. Two proteins of 105 and 95 kDa, corresponding to the phosphorylated and non-phosphorylated Sp1 forms, respectively, were detected. While the nonphosphorylated form did not change, the intensity of the phosphorylated form increased 3-fold in 0.28AS-2 cells as compared to EATC (Fig. 1A). The phosphorylated form could be immunoprecipitated by using an anti-phospho-serine agarose-conjugated antibody (Fig. 1B). The Sp3 isoforms expression pattern obtained (Fig. 1C) was similar to that reported by other authors [21,22]: a primary band of 110 kDa representing the full length protein and two bands corresponding to truncated isoforms of about 60– 70 kDa [10]. Immunoblot experiments revealed that Sp3 full length protein expression is increased 8-fold in 0.28AS-2 cells. In order to determine the Sp1 and Sp3 DNA binding capacity of EATC and 0.28AS-2 nuclear extracts, an electrophoretic mobility shift assay using a consensus Sp1 oligonucleotide was developed. Two retarded complexes were detected in the mobility shift assay of both EATC and 0.28AS-2 nuclear extracts (Fig. 2A). Significantly, the band intensities obtained with 0.28AS-2 nuclear extracts were greater than with EATC. When a specific Sp1 antibody was used, only the lower band could be observed. On the contrary, only the upper band remained when a Sp3 specific antibody was used. This seems to indicate that the upper band corresponds to Sp1–DNA complexes and the lower band to Sp3–DNA complexes.
Fig. 2. EATC and 0.28AS-2 band shift assays. (A) Nuclear extracts were prepared from exponentially growing cells and incubated with a radiolabelled Sp1 consensus oligonucleotide. Anti-Sp1 or anti-Sp3 antibodies were used to identify Sp complexes. As a control for specificity 100!Sp1 unlabelled consensus (100!Cons.) or mutated (100!Mut.) oligonucleotides were used. (B) Nuclear extracts from 0.28AS-2 cells were treated with alkaline phosphatase and subjected to band shift assay. Consensus and mutated probes sequencies are described under ‘Section 2’. The autorradiograms shown are representatives of the results obtained with three independently prepared nuclear extracts.
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Fig. 3. Sp1 activity in EATC and 0.28AS-2 cells using a luciferase reporter plasmid. EATC and 0.28AS-2 cells were transiently cotransfected with the pGAGC6 Sp1 reporter plasmid and a b-Galactosidase plasmid (pCMV-bgal). pGAM plasmid, lacking the Sp1 consensus motives, was used as a control. Luciferase activity was quantified 48 h later. The results are reported as mean valuesGstandard deviations from three different experiments. Each experiment was measured in triplicate. *P!0.01 as tested with the Mann–Whitney U test.
The increased Sp1–DNA binding obtained with 0.28AS-2 nuclear extracts could be explained by its increased expression of phosphorylated Sp1 protein. In order to test this possibility, 0.28AS-2 nuclear extracts were treated with alkaline phosphatase prior to carry out the mobility shift assay experiment. As shown in Fig. 2B most of the upper band, assigned to Sp1, was absent after alkaline phosphatase treatment. Because Sp1–DNA binding is not always positively correlated with Sp1 activity, we carried out measurements of Sp1 activity in EATC and 0.28AS-2 cells transfected with the Sp1 reporter plasmid pGAGC6. This plasmid contains six Sp1 consensus sites and an adenovirus TATA box upstream of a luciferase reporter gene. A plasmid vector without
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the Sp1 sites (pGAM) was used as a control and the PCMV-bgal plasmid containing the b-galactosidase gene was used to normalize luciferase activity. As shown in Fig. 3, luciferase activity in 0.28AS-2 cells was 3.5-fold the value obtained with the parental EATC. Since members of the Sp-family of transcription factors are implicated in differentiation and proliferation processes, we wanted to test whether the induction of differentiation in EATC could also reproduce the Sp1/Sp3 pattern observed in 0.28AS-2 cells. To achieve this, we used the differentiation agent PMA. Culture medium from exponentially growing EATC was supplemented with 100 nM PMA. The cells were further incubated for 2 days and then nuclear extracts were prepared and analyzed by immunoblot and band shift assays. In Western blot analyses (Fig. 4A), nuclear extracts from control EATC showed a pattern of Sp 1 protein very similar to the one obtained above (Fig. 1A). PMA treatment caused an enhanced expression of both 105 and 95 kDa forms of Sp1, but did not affect the Sp3 protein (Fig. 4B). As observed in band shift assays (Fig. 4C), PMA treatment did not caused any significant change in the binding activity of Sp1 to the consensus Sp1 oligo. Next we decided to study if the inhibition of GLS activity could influence the concentration of intracellular glutamine and the products of glutamine metabolism glutamate and aspartate. The antisense inhibition of GLS decreased almost 5 fold the GLS activity in 0.28AS-2 cells, and increased to the same extent the intracellular glutamine concentration. Just the opposite situation can be observed in EATC
Fig. 4. Effects of glutamine deprivation and PMA treatment on Sp1 and Sp3 expression in EATC. Culture medium from exponentially growing EATC was replaced either with glutamine free medium or medium supplemented with 100 nM PMA. Fresh cell culture medium was used in control plates. Cells were further incubated for 2 days. Nuclear extracts were then prepared as described under ‘Section 2’. Total Sp1 and Sp3 protein content were analyzed by immunoblotting (A and B) and the DNA binding activity of proteins from the nuclear extracts were studied by band shift assay (C) as detailed under ‘Section 2’. Results shown are a representative of three independently prepared extracts.
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Table 1 Determination of GLS activity and quantification of intracellular concentration of amino acids
EATC 0.28AS-2
GLS activity (mU/106 cells)
Glutamine (mM)
Glutamate (mM)
Aspartate (mM)
23.0G2.8* 5.0G0.9*
1.3G0.5* 5.2G1.7*
6.7G1.0 6.9G1.1
3.8G0.2 4.6G0.3
Each value is the meanGSD for three independent cell preparations. *P!0.05 as determined by the U-Mann–Whitney statistical test.
(Table 1). However, the concentration of glutamate and aspartate did not significantly change.
4. Discussion The influence of GLS inhibition on Sp1 and Sp3 expression is studied by using wild type EATC and in their derivative the 0.28AS-2 antisense GLS expressing cells. It is remarkable that the expression of phosphorylated Sp1 protein is incremented in 0.28AS2 cells. Immunoprecipitation experiments revealed that Sp1 is mostly phosphorylated on a serine residue (Fig. 1). Furthermore, the Sp1 phosphorylation also induces an increase in Sp1-DNA binding, as it is manifested in band shift assays (Fig. 2). The fact that phosphatase treatment inhibits DNA Sp1 binding highlights the, importance of phosphorylation in the DNA binding capacity of Sp1 in 0.28AS-2 cells. The increase in Sp1 DNA binding is also correlated with a similar increase in Sp1 activity in 0.28AS-2 cells, as it is shown in transient transfection experiments carried out with luciferase reporter plasmids (Fig. 3). These results clearly correlate Sp1-phosphorylation with an increased Sp1 activity in 0.28AS-2 cells in agreement with previous reports showing that an increased Sp1 phosphorylation enhances DNA binding and/or transactivation [23,24]. An important mechanism that can modulate Sp1 activity is exerted by Sp3 protein. We have shown by immunoblotting that the 110 kDa form of Sp3 protein was 8-fold incremented in 0.28AS-2 cells (Fig. 1C), and this is also accompanied by an increase in Sp3 DNA binding (Fig. 2A), however, the increase in Sp3 DNA binding does not lead to an inhibition of Sp1 activity, as it is shown in the transfections with luciferase reporter plasmid. Reports on the transcriptional properties of Sp3 appear, at first sight, contradictory. Sp3 has been shown to act as a transcriptional positive regulator
similar to Sp1 [25,26]; in other experiments, Sp3 remained inactive or acted only as a very weak activator [27,28]. The structure and arrangement of the recognition sites appears to determine whether Sp3 is transcriptionally inactive and can repress Sp1 mediated activation or it acts as a strong activator. Promoters containing single binding sites are activated, whereas promoters containing multiple binding sites often are not activated or they respond weakly to Sp3 [29,30]. In 0.28AS-2 cells, Sp3 seems to remain inactive or acts as a weak activator as judge by the Sp1 activity measured in the luciferase experiments carried out with the pGAGC6 plasmid. It has been previously reported that changes in the phosphorylated forms of Sp1 occur during liver differentiation [31] and in cell cycle regulation [32]. That suggests a possible role of Sp1 in the maintenance of the more differentiated phenotype of 0.28AS-2 cells. When EATC were treated with PMA, phosphorylated and non-phosphorylated forms of Sp1 were increased, whereas Sp3 protein content and binding to DNA remained unchanged. This suggests that the mechanisms underlying PMA driven differentiation and GLS antisense inhibition are not coincident. The inhibition of GLS in 0.28AS-2 cells must influence the glutamine consumption and metabolization. It should be affected both the level of free intracellular glutamine and the intermediate metabolites of the glutamine oxidation pathway. In fact, we have demonstrated that the intracellular concentration of glutamine, but not glutamate or aspartate, is increased in 0.28AS-2 cells. The higher GLS activity of EATC keep the intracellular glutamine at low concentrations; this is in accordance with previous data showing that in vivo growing EATC have undetectable levels of free intracellular glutamine [33]. Glutamate and aspartate are maintained at similar levels both in FATC and 0.28AS-2 cells, however, the higher GLS activity in EATC should
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lead to a higher glutamate consumption and metabolization. The differences in free intracellular glutamine is interesting per se, and could influence a number of cellular processes, since there is a growing body of evidence pointing to glutamine as a signal molecule involved in apoptosis, proliferation and gene expression regulation [34–36]. In conclusion, the results obtained show that antisense glutaminase inhibition enhances Sp1 phosphorylation, leading to an increased Sp1 DNA binding and activity. This could be implicated in the reversion of the malignant phenotype observed in 0.28AS-2 antisense expressing cells. Further studies are needed to clarify the mechanisms underlying the molecular links between glutaminase inhibition and Sp1 activation.
Acknowledgements This work was supported by the grant SAF20011894 from the Comisio´n Interministerial de Ciencia y Tecnologı´a and the Fundacio´n Ramo´n Areces. This paper is dedicated to Professor Ignacio Nu´n˜ez de Castro in the year of his retirement.
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