Nutritional Limitation Sensitizes Mammalian Cells to GSK-3β Inhibitors and Leads to Growth Impairment
2011; Elsevier BV; Volume: 178; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2010.12.047
ISSN1525-2191
AutoresPaola de Candia, Giuseppina Minopoli, Viola Verga, Anna Gargiulo, Marco Vanoni, Lilia Alberghina,
Tópico(s)Nutrition, Genetics, and Disease
ResumoThe serine/threonine kinase GSK-3β was initially described as a key enzyme involved in glucose metabolism, but it is now known to regulate a wide range of biological processes, including proliferation and apoptosis. We previously reported a transformation-dependent cell death induced by glucose limitation in K-ras–transformed NIH3T3. To address the mechanism of this phenomenon, we analyzed GSK-3β regulation in these cells in conditions of high versus low glucose availability. We found that glucose depletion caused a marked inhibition of GSK-3β through posttranslational mechanisms and that this inhibition was much less pronounced in normal cells. Further inhibition of GSK-3β with lithium chloride, combined with glucose shortage, caused specific activation of AMP-activated protein kinase and significant suppression of proliferation in transformed but not normal cells. The cooperative effect of lithium and low glucose availability on cell growth did not seem to depend exclusively on ras pathway activation because two human cell lines, A549 and MDA-MB-231, both harboring an activated ras gene, showed very different sensitivity to lithium. These findings thus provide a rationale to further analyze the biochemical bases for combined glucose deprivation and GSK-3β inhibition as a new approach to control transformed cell growth. The serine/threonine kinase GSK-3β was initially described as a key enzyme involved in glucose metabolism, but it is now known to regulate a wide range of biological processes, including proliferation and apoptosis. We previously reported a transformation-dependent cell death induced by glucose limitation in K-ras–transformed NIH3T3. To address the mechanism of this phenomenon, we analyzed GSK-3β regulation in these cells in conditions of high versus low glucose availability. We found that glucose depletion caused a marked inhibition of GSK-3β through posttranslational mechanisms and that this inhibition was much less pronounced in normal cells. Further inhibition of GSK-3β with lithium chloride, combined with glucose shortage, caused specific activation of AMP-activated protein kinase and significant suppression of proliferation in transformed but not normal cells. The cooperative effect of lithium and low glucose availability on cell growth did not seem to depend exclusively on ras pathway activation because two human cell lines, A549 and MDA-MB-231, both harboring an activated ras gene, showed very different sensitivity to lithium. These findings thus provide a rationale to further analyze the biochemical bases for combined glucose deprivation and GSK-3β inhibition as a new approach to control transformed cell growth. GSK-3 is a multifunctional serine/threonine kinase that plays a major role in Wnt and Hedgehog signaling pathways, the regulation of cell division cycle, cell fate and survival, stem cell renewal, apoptosis, neuronal cell growth and differentiation, and circadian rhythm.1Kockeritz L. Doble B. Patel S. Woodgett J.R. Glycogen synthase kinase-3: an overview of an over-achieving protein kinase.Curr Drug Targets. 2006; 11: 1377-1388Crossref Scopus (230) Google Scholar In mammals, two isoforms are encoded by distinct genes, GSK-3α and GSK-3β, that, despite a high degree of similarity, are not functionally redundant.2Rayasam G.V. Tulasi V.K. Sodhi R. Davis J.A. Ray A. Glycogen synthase kinase 3: more than a namesake.Br J Pharmacol. 2009; 156: 885-898Crossref PubMed Scopus (389) Google Scholar The protein function of GSK-3β is much better investigated. Consistent with the observation that GSK-3β is involved in many cellular pathways, its dysregulation has been implicated in various human diseases, such as bipolar mood disorder, neurodegenerative pathologic conditions, and diabetes.3Martinez A. Preclinical efficacy on GSK-3 inhibitors: towards a future generation of powerful drugs.Med Res Rev. 2008; 28: 773-796Crossref PubMed Scopus (91) Google Scholar Regarding the role of GSK-3β in cancer, conflicting results are found in the literature. Oncogenic transcription factors (eg, c-Jun and c-Myc) and proto-oncoproteins (ie, β-catenin) are putative GSK-3β substrates for phosphorylation-dependent inactivation4Manoukian A.S. Woodgett J.R. Role of glycogen synthase kinase-3 in cancer: regulation by Wnts and other signaling pathways.Adv Cancer Res. 2002; 84: 203-229Crossref PubMed Scopus (130) Google Scholar; therefore, GSK-3β could interfere with tumor development.5Polakis P. The oncogenic activation of beta-catenin.Curr Opin Genet Dev. 1999; 9: 15-21Crossref PubMed Scopus (605) Google Scholar However, although pharmacologic inhibition of GSK-3 would be expected to promote cancer, no direct in vivo evidence has indicated that such a phenomenon occurs on administration of GSK-3 antagonists. In fact, the risk of cancer development among psychiatric patients treated with lithium salts, long known as an inhibitor of GSK-3β,6Stambolic V. Ruel L. Woodgett J.R. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells.Curr Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar is significantly lower than that in the general population, suggesting that inhibition of GSK-3β may have a generally protective effect toward carcinogenesis.7Cohen Y. Chetrit A. Cohen Y. Sirota P. Modan B. Cancer morbidity in psychiatric patients: influence of lithium carbonate treatment.Med Oncol. 1998; 15: 32-36Crossref PubMed Scopus (95) Google Scholar Accordingly, recent studies show that GSK-3β inhibitors lead to significant reduction in cell growth and proliferation of prostate,8Mazor M. Kawano Y. Zhu H. Waxman J. Kypta R.M. Inhibition of glycogen synthase kinase-3 represses androgen receptor activity and prostate cancer cell growth.Oncogene. 2004; 23: 7882-7892Crossref PubMed Scopus (106) Google Scholar pancreatic,9Ougolkov A.V. Fernandez-Zapico M.E. Savoy D.N. Urrutia R.A. Billadeau D.D. Glycogen synthase kinase-3β participates in nuclear factor κB-mediated gene transcription and cell survival in pancreatic cancer cells.Cancer Res. 2005; 65: 2076-2081Crossref PubMed Scopus (289) Google Scholar colorectal,10Shakoori A. Ougolkov A. Yu Z.W. Zhang B. Modarressi M.H. Billadeau D.D. Mai M. Takahashi Y. Minamoto T. Deregulated GSK3β activity in colorectal cancer: its association with tumor cell survival and proliferation.Biochem Biophys Res Commun. 2005; 334: 1365-1373Crossref PubMed Scopus (235) Google Scholar ovarian,11Cao Q. Lu X. Feng Y.J. Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells.Cell Res. 2006; 16: 671-677Crossref PubMed Scopus (160) Google Scholar medullary thyroid,12Kunnimalaiyaan M. Vaccaro A.M. Ndiaye M.A. Chen H. Inactivation of glycogen synthase kinase-3β, a downstream target of the raf-1 pathway, is associated with growth suppression in medullary thyroid cancer cells.Mol Cancer Ther. 2007; 6: 1151-1158Crossref PubMed Scopus (94) Google Scholar and pheochromocytoma13Kappes A. Vaccaro A. Kunnimalaiyaan M. Chen H. Lithium ions: a novel treatment for pheochromocytomas and paragangliomas.Surgery. 2007; 141: 161-165Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar cancer cell lines and that GSK-3β activity is essential for maintenance of a subset of leukemias.14Wang Z. Smith K.S. Murphy M. Piloto O. Somervaille T.C. Cleary M.L. Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy.Nature. 2008; 455: 1205-1209Crossref PubMed Scopus (233) Google Scholar The first role identified for GSK-3β almost 30 years ago recognized it as a crucial factor in glucose metabolism, being the kinase that phosphorylates and inactivates glycogen synthase, the first enzyme in glycogen biosynthesis.15Rylatt D.B. Aitken A. Bilham T. Condon G.D. Embi N. Cohen P. Glycogen synthase from rabbit skeletal muscle: amino acid sequence at the sites phosphorylated by glycogen synthase kinase-3, and extension of the N-terminal sequence containing the site phosphorylated by phosphorylase kinase.Eur J Biochem. 1980; 107: 529-537Crossref PubMed Scopus (180) Google Scholar It is well-known that even in the presence of oxygen, tumors consistently rely on glycolysis to generate a substantial fraction of total cellular ATP production. Furthermore, tumor cells maintain ATP production by increasing glucose influx to fuel the energy requirements of unrestricted proliferation.16Gatenby R.A. Gawlinski E.T. The glycolytic phenotype in carcinogenesis and tumor invasion: insights through mathematical models.Cancer Res. 2003; 63: 3847-3854PubMed Google Scholar The role of GSK-3β in this phenotype is still unclear. Nonetheless, it is conceivable, although still not completely demonstrated, that GSK-3β could be involved in glucose metabolism of tumor cells through its effect on glycogen metabolism. To explore whether altered carbon metabolism of transformed cells involves GSK-3β dysregulation, we used murine NIH3T3 cells, a largely studied immortalized cell line established as a model parental cell line for the study of cell transformation.17Kahn S. Yamamoto F. Almoguera C. Winter E. Forrester K. Jordano J. Perucho M. The c-K-ras gene and human cancer.Anticancer Res. 1987; 7 (review): 639-652PubMed Google Scholar Similar to most cancer cells, NIH3T3 cells transformed by an activated form of the K-ras oncogene exhibit a higher rate of glucose consumption associated with mitochondrial dysfunction.18Chiaradonna F. Gaglio D. Vanoni M. Alberghina L. Expression of transforming K-Ras oncogene affects mitochondrial function and morphology in mouse fibroblasts.Biochem Biophys Acta. 2006; 1757: 1338-1356PubMed Google Scholar So, whereas normal NIH3T3 cells cope with glucose shortage by relying on oxidative metabolism and decelerating cell proliferation, K-ras–transformed cells are remarkably sensitive to glucose deprivation, losing their growth advantage and high survival rate.19Chiaradonna F. Sacco E. Manzoni R. Giorgio M. Vanoni M. Alberghina L. Ras-dependent carbon metabolism and transformation in mouse fibroblasts.Oncogene. 2006; 25: 5391-5404Crossref PubMed Scopus (92) Google Scholar Analysis of GSK-3β regulation in K-ras–transformed fibroblasts and their parental counterparts in conditions of high versus low glucose availability showed posttranslational inhibition of GSK-3β in the latter condition. This pattern was uncoupled from regulation of glycogen synthase and β-catenin. After this observation, we exposed normal and transformed cells to glucose deprivation and GSK-3β chemical inhibition and found that nutrient limitation sensitizes transformed cells, but not parental NIH3T3, to growth impairment by two different GSK-3β inhibitors. Glucose limitation also rendered more effective the growth inhibition properties of lithium chloride (LiCl) in the human neoplastic MDA-MB-231 cells. Normal murine fibroblasts (obtained from the American Type Culture Collection, Manassas, VA) and a K-ras–transformed derived cell line (226.4.1),20Pulciani S. Santos E. Long L.K. Sorrentino V. Barbacid M. Ras gene amplification and malignant transformation.Mol Cell Biol. 1985; 5: 2836-2841Crossref PubMed Scopus (162) Google Scholar A549 lung carcinoma, HeLa cells, and MDA-MB-231 breast cancer cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) containing 10% newborn calf serum (or 10% fetal bovine serum for human cell lines), 2 mmol/L glutamine, 100 U/mL of penicillin, and 100 mg/mL of streptomycin (normal growth medium) at 37°C in a humidified atmosphere of 5% CO2. To verify the cell response to glucose depletion, the cells were grown in medium without glucose and sodium pyruvate (Invitrogen) supplemented with the appropriate concentration of glucose (25 and 1 mmol/L). For cell growth assays, cells were plated in six-well plates (NIH3T3 and K-ras–transformed cells at 28,800 cells per well, A549 at 133,000 cells per well, HeLa at 77,000 cells per well, and MDA-MB-231 at 106,000 cells per well) and were allowed to attach overnight. The medium was changed with different concentrations of glucose in the presence of either carrier (or NaCl) or GSK-3 inhibitors (three wells were used for each condition). Cells were collected using trypsin-EDTA (Invitrogen) at different time points and were counted using a Bürker counting chamber. Cell death was determined by trypan blue (Sigma-Aldrich, St. Louis, MO) dye exclusion assay. For synchronization in G2-M phase, cells were incubated for 16 hours with nocodazole, 100 ng/mL; then, synchronized cells were recovered by gentle shaking, released by the drug-induced block in fresh medium, and harvested after 4, 6, and 8 hours. For protein kinase B (PKB) inhibition, K-ras–transformed cells were plated in six-well plates at 28,800 cells per well, and the medium was changed as described. After 72 hours of growth, either wortmannin, 10 μmol/L, or dimethyl sulfoxide alone was added to the medium for 30 minutes, and then cells were harvested for protein evaluation. For AMP-activated protein kinase (AMPK) activation, cell growth assay was performed, as described previously herein, in the presence of either carrier or 500 μmol/L aminoimidazole carboxamide ribonucleotide (Sigma-Aldrich). For K-ras overexpression, 1 × 106 HeLa cells were transfected with 10 μg of a porcine cytomegalovirus–transforming–K-ras expression vector as previously reported21Baker S.J. Markowitz S. Fearon E.R. Willson J.K. Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53.Science. 1990; 249: 912-915Crossref PubMed Scopus (1605) Google Scholar or with 10 μg of a porcine cytomegalovirus empty vector using FuGENE (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer's instructions. Twelve hours after transfection, cells were plated in six-well plates (77,000 cells per well) and were grown as described previously herein. Total RNA was extracted from NIH3T3 and K-ras–transformed cells using the SV Total RNA Isolation Kit (Promega, Madison, WI). First-strand cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The first-strand cDNA was then used as a template for quantitative RT-PCR with the TaqMan Gene Expression Master Mix and the specific TaqMan Gene Expression Assays for the GSK3-β, c-src, and β-actin genes (Applied Biosystems). The cycling conditions were as follows: initial denaturation at 94°C for 2 minutes, followed by 40 cycles of denaturation at 94°C for 15 seconds and annealing/extension at 60°C for 1 minute. β-actin was used as normalizing internal control for gene expression analyses. Differences in gene expression were evaluated using a t-test. Cells were lysed in radioimmunoprecipitation assay buffer plus phosphatase and protease inhibitors. After incubation for 15 minutes on ice, the extracts were centrifuged at 13,200 rpm for 10 minutes. The protein concentration of supernatant was measured using the Bradford procedure (Bio-Rad Laboratories, Richmond, CA), using bovine serum albumin as a standard. Total and fractionated cellular extracts were subjected to electrophoresis in SDS–polyacrylamide gel and were transferred to a nitrocellulose membrane (Amersham, Otelfingen, Switzerland). Membranes were preincubated in Tris-buffered saline [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl]/−5% defatted milk powder for 1 hour at room temperature and then were incubated in Tris-buffered saline/–0.01% Tween 20/5% defatted milk powder containing the appropriate antibodies overnight at 4°C. The antibodies used were against GSK-3β, phospho–GSK-3β (Ser9), phospho–GSK-3β (Tyr216), glycogen synthase, phospho-glycogen synthase (Ser641), phospho-AKT substrate, phospho-AMPK (Thr172), and AMPKα (Cell Signaling Technology, Beverly, MA); β-catenin (BD Transduction Laboratories, Heidelberg, Germany); N-terminal anti–β-catenin S/T-nonphosphorylated and pan ras (Upstate Biotechnology, Lake Placid, NY); and vinculin and actin (Sigma-Aldrich). After three washings (5 minutes each) in Tris-buffered saline/−0.05% Tween 20, the membranes were incubated with a peroxidase-coupled secondary antibody (Amersham) for 30 minutes at room temperature. After incubation, the membranes were washed three times in Tris-buffered saline/−0.05% Tween 20. The reaction was visualized using ECL (Amersham), followed by exposure to an X-ray film. Images were scanned at a minimum resolution of 300 dpi. Protein levels were quantified by densitometry of JPEG images using the NIH image-based software ImageJ. For the 5-bromo-2′-deoxyuridine (BrdU) incorporation assay, K-ras–transformed cells were grown in duplicate on polylysine-precoated glass coverslips in six-well plates and were treated as described previously herein. The cells were then incubated with 10 μmol/L 5-bromo-2′-deoxyuridine (Roche Molecular Biochemicals) for 5 hours. Cells on coverslips were fixed with paraformaldehyde (4% in PBS, pH 7.4), permeabilized with 0.5% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany), and incubated in 0.5-N HCl for 30 minutes. Coverslips were washed three times with PBS in a 10-minute period and were incubated with a primary mouse anti-BrdU antibody (5-Bromo-2′-deoxyuridine Labeling and Detection Kit; Roche Molecular Biochemicals) following the instructions of the supplier. Then, the cells were stained with Alexa Fluor 546–conjugated secondary antibody (Molecular Probe, Invitrogen), with nuclear counterstaining with DAPI (Sigma-Aldrich), 1:1000. The coverslips were mounted in Mowiol (Calbiochem, San Diego, CA) onto a glass microscope slide, and fluorescence was examined using an Axiophot microscope (Zeiss Jena GmbH, Jena, Germany). The number of total and BrdU-positive cells in each condition were counted in 10 nonoverlapping fields per coverslip, and the quantification of BrdU-positive cells per total number of DAPI–positive nuclei was determined as a mean ± SD percentage. The distribution of cells at specific cell cycle phases was evaluated by flow cytometry as previously described.22Gaglio D. Soldati C. Vanoni M. Alberghina L. Chiaradonna F. Glutamine deprivation induces abortive s-phase rescued by deoxyribonucleotides in k-ras transformed fibroblasts.PLoS One. 2009; 4: e4715Crossref PubMed Scopus (110) Google Scholar Briefly, cells were trypsinized, washed with PBS, and fixed in 75% ethanol at 4°C. Subsequently, samples were stained with propidium iodide (Sigma-Aldrich) and were analyzed using a fluorescence-activated cell sorter (FACScan; BD Biosciences, Franklin Lakes, NJ), using Cell Quest software (BD Biosciences). Data analysis was performed using WinMDI software. Parental and K-ras–transformed fibroblasts were plated at 3000 cells/cm2 density. After 16 hours of growth, the medium was changed with a glucose concentration maintained at 25 mmol/L. Twenty-four hours after medium change, in the condition of exponential growth, we harvested parental and transformed cells and prepared total RNA to evaluate the mRNA levels of GSK-3β using real-time PCR. Transformed cells showed a lower mRNA level for GSK-3β, and the protein level was also sensibly lower in transformed versus parental cells as assessed by Western blotting (Figure 1A). Normal and transformed cells were then plated as described previously herein, and after 16 hours, the medium was changed (cells were also harvested at this time, referred to as time 0). Media were supplemented with either high (25 mmol/L) or low (1 mmol/L) glucose concentration. Cells were then harvested after 24, 48, and 72 hours of growth. All the experiments reported in this and the following paragraphs refer to the previously mentioned experimental setup, already used for several studies.18Chiaradonna F. Gaglio D. Vanoni M. Alberghina L. Expression of transforming K-Ras oncogene affects mitochondrial function and morphology in mouse fibroblasts.Biochem Biophys Acta. 2006; 1757: 1338-1356PubMed Google Scholar, 19Chiaradonna F. Sacco E. Manzoni R. Giorgio M. Vanoni M. Alberghina L. Ras-dependent carbon metabolism and transformation in mouse fibroblasts.Oncogene. 2006; 25: 5391-5404Crossref PubMed Scopus (92) Google Scholar To evaluate the effect of glucose shortage on GSK-3β regulation, we prepared RNA from normal and K-ras–transformed NIH3T3 cells grown in either high or low glucose availability. GSK-3β mRNA levels were measured using real-time PCR. In normal and transformed cells, the level of mRNA for GSK-3β was constant until 48 hours of growth and increased at 72 hours in low glucose, more substantially in transformed cells (Figure 1B, top). This time-dependent expression pattern was specific for GSK-3β mRNA and was not a general response to glucose limitation because expression of an unrelated gene, c-src, remained constant over the timeframe of the experiment (Figure 1B, bottom). The GSK-3β protein level remained fairly stable over time in normal and transformed cells, with no increase at late time points in low glucose (Figure 2A), suggesting a compensatory mechanism for the observed transcriptional activation. In K-ras–transformed cells, the level of GSK-3β inhibitory phosphorylation at Ser9 was consistently higher in low compared with high glucose, with the largest difference at 72 hours. On the other hand, activating Tyr216 phosphorylation was slightly decreased in low versus high glucose at the same time point (Figure 2A). Through quantification of Western blotting by ImageJ, we showed an increase in the ratio between Ser9 phosphorylated and total protein over time specifically in low glucose and a 12-fold difference of this ratio in low versus high glucose at 72 hours (Figure 2B, top). In normal cells, on the other hand, Ser9 phosphorylation did not increase significantly or even decreased over time also in low glucose, and the difference between high and low glucose at 72 hours was less pronounced than in transformed cells (Figure 2B, bottom). Given that GSK-3 is known to inhibit cell proliferation and glycogen accumulation mainly through phosphorylation of β-catenin and glycogen synthase, respectively, we analyzed the regulation of these targets in cells grown in either high or low glucose. Activation of β-catenin was evaluated by using an antibody specific for the active form of the protein, dephosphorylated on Ser37 or Thr41. As shown in Figure 2A, neither the phosphorylation pattern nor total protein accumulation seemed to be affected by glucose deprivation in normal and transformed cells. Quantification analysis performed on triplicates at 24 and 72 hours confirmed that the ratio between the activated and total forms of the protein was not modulated in the two cell lines (Figure 2B). We also evaluated β-catenin nuclear localization in transformed cells: at 24 and 72 hours, the total and active forms of β-catenin were mostly in the nuclear/insoluble fraction, and this pattern was likewise not affected by glucose availability (data not shown). In contrast to the observed GSK-3β phosphorylation pattern, glycogen synthase seemed to be phosphorylated at a higher level after 72 hours of growth in low compared with high glucose in normal and transformed cells (Figure 2A). The quantitative analysis of the ratio between the phosphorylated and total forms of the protein confirmed that glycogen synthase phosphorylation was increased in low glucose– versus high glucose–grown cells, with the difference being larger in normal cells than in transformed cells (Figure 2B). The observation that GSK-3β posttranslational inhibition increased in conditions of nutrient limitation prompted us to evaluate the effect of GSK-3β inhibition on proliferation of normal and transformed NIH3T3 cells. To this end, we treated both cell lines grown in either high or low initial glucose with the GSK-3β inhibitor LiCl, using NaCl as an isotonic control. During the experiment, K-ras–transformed cells grew significantly less in media supplemented with low as opposed to high initial glucose concentrations (Figure 3A, left), as previously reported.19Chiaradonna F. Sacco E. Manzoni R. Giorgio M. Vanoni M. Alberghina L. Ras-dependent carbon metabolism and transformation in mouse fibroblasts.Oncogene. 2006; 25: 5391-5404Crossref PubMed Scopus (92) Google Scholar Cell growth was significantly further inhibited by the addition of LiCl (Figure 3A, left; P < 0.05). Sb-216763, a cell-permeable maleimide compound that selectively inhibits GSK-3,23Coghlan M.P. Culbert A.A. Cross D.A. Corcoran S.L. Yates J.W. Pearce N.J. Rausch O.L. Murphy G.J. Carter P.S. Roxbee Cox L. Mills D. Brown M.J. Haigh D. Ward R.W. Smith D.G. Murray K.J. Reith A.D. Holder J.C. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription.Chem Biol. 2000; 7: 793-803Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar failed to inhibit proliferation of K-ras–transformed cells grown in media supplemented with a high initial glucose concentration (Figure 3A, right) when used at a concentration of 25 μmol/L. On the other hand, this concentration was sufficient to effectively inhibit growth of transformed cells grown with low glucose availability (Figure 3A, right; P < 0.05). A higher concentration (50 μmol/L) of the drug was necessary to observe an effect similar to that observed with lithium (data not shown). These data thus suggest that K-ras–transformed NIH3T3 fibroblasts are exquisitely sensitive to GSK-3β inhibition when grown under conditions of glucose limitation. Normal NIH3T3 cells grew similarly in media supplemented with different glucose concentrations (Figure 3B), and LiCl had little, if any, inhibitory effect (Figure 3B, left). Similar to LiCl, Sb-216763 did not affect the growth of parental cells (Figure 3B, right). K-ras–transformed cells could not sustain their growth through the citric acid cycle because cells did not restore a normal growth rate when medium was supplemented with sodium pyruvate (Figure 4A). This result is in keeping with previous studies showing that K-ras–transformed cells have defective mitochondria19Chiaradonna F. Sacco E. Manzoni R. Giorgio M. Vanoni M. Alberghina L. Ras-dependent carbon metabolism and transformation in mouse fibroblasts.Oncogene. 2006; 25: 5391-5404Crossref PubMed Scopus (92) Google Scholar and, more specifically, that the reduction in respiration observed is due to a decrease in complex I activity.24Baracca A. Chiaradonna F. Sgarbi G. Solaini G. Alberghina L. Lenaz G. Mitochondrial Complex I decrease is responsible for bioenergetic dysfunction in K-ras transformed cells.Biochim Biophys Acta. 2010; 1797: 314-323Crossref PubMed Scopus (114) Google Scholar To ascertain whether the PI3K/PKB pathway was responsible of the increased Ser9 phosphorylation of GSK-3β in the condition of glucose shortage, transformed cells were grown in either high or low glucose, and 72 hours after medium change, cells were treated with wortmannin, a specific inhibitor of PI3K. The subsequent inhibited activity of PKB on wortmannin exposure was evaluated by Western blotting using an antibody that recognizes the phospho-(Ser/Thr) PKB substrate motif (RXXS/T). This control showed similar suppression of the phosphorylation level for different PKB substrates (Figure 4B). On the other hand, whereas wortmannin treatment dramatically inhibited GSK-3β phosphorylation in high glucose, it only marginally decreased it in low glucose (Figure 4B), suggesting that in the condition of nutrient depletion, kinases other than PKB are responsible for the observed Ser9 phosphorylation. Normal cells are known to have a lower rate of nutrient consumption than transformed cells and not to consume the glucose completely during the exponential phase of growth in conditions of glucose shortage.19Chiaradonna F. Sacco E. Manzoni R. Giorgio M. Vanoni M. Alberghina L. Ras-dependent carbon metabolism and transformation in mouse fibroblasts.Oncogene. 2006; 25: 5391-5404Crossref PubMed Scopus (92) Google Scholar Consequently, we observed that normal cells did not activate AMPK after 72 hours of growth in 1 mmol/L glucose, as assessed by the lack of activating phosphorylation on AMPK Thr172 (Figure 4D). On the other hand, transformed cells do consume glucose very rapidly.19Chiaradonna F. Sacco E. Manzoni R. Giorgio M. Vanoni M. Alberghina L. Ras-dependent carbon metabolism and transformation in mouse fibroblasts.Oncogene. 2006; 25: 5391-5404Crossref PubMed Scopus (92) Google Scholar We confirmed this by observing complete consumption of glucose already at 48 hours of growth in 1 mmol/L glucose (data not shown). Unexpectedly, though, glucose depletion was unable to activate AMPK, not even at 72 hours, and only when glucose shortage was combined with GSK-3β inhibition, phospho-AMPK was clearly detected by Western blotting (Figure 4D). In transformed cells, AMPK activation was a strong signal of cell growth inhibition; in fact, treatment with a specific AMPK-activating drug, aminoimidazole carboxamide ribonucleotide, caused complete suppression of proliferation, independent of glucose availability (Figure 4C). Reduced cell growth of K-ras–transformed cells in low glucose has been shown to be partly due to increased apoptosis.19Chiaradonna F. Sacco E. Manzoni R. Giorgio M. Vanoni M. Alberghina L. Ras-dependent carbon metabolism and transformation in mouse fibroblasts.Oncogene. 2006; 25: 5391-5404Crossref PubMed Scopus (92) Google Scholar To ascertain whether the reduced growth in the presence of GSK-3 inhibitors was due to an effect on cell death and/or cell proliferation, the overall cell viability of K-ras–transformed cells and their ability to enter S phase were monitored. To evaluate the level of cell death, we harvested adherent and floating cells and ran a trypan blue exclusion assay. The percentage of trypan blue positive over total cell number was comparable for different treatments at 24 and 48 hours (Fig
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