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Originally published In Press as doi:10.1074/jbc.M507443200 on September 21, 2005

J. Biol. Chem., Vol. 280, Issue 47, 39582-39593, November 25, 2005
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5-Aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro and in Vivo via AMP-activated Protein Kinase*

Ramandeep Rattan{ddagger}, Shailendra Giri{ddagger}, Avtar K. Singh§, and Inderjit Singh{ddagger}1

From the {ddagger}Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425 and the §Department of Pathology and Laboratory Medicine, Ralph Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425

Received for publication, July 8, 2005 , and in revised form, September 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
5-Aminoimidazole-4-carboxamide-1-{beta}-4-ribofuranoside (AICAR) is widely used as an AMP-kinase activator, which regulates energy homeostasis and response to metabolic stress. Here, we investigated the effect of AICAR, an AMPK activator, on proliferation of various cancer cells and observed that proliferation of all the examined cell lines was significantly inhibited by AICAR treatment due to arrest in S-phase accompanied with increased expression of p21, p27, and p53 proteins and inhibition of PI3K-Akt pathway. Inhibition in in vitro growth of cancer cells was mirrored in vivo with increased expression of p21, p27, and p53 and attenuation of Akt phosphorylation. Anti-proliferative effect of AICAR is mediated through activated AMP-activated protein kinase (AMPK) as iodotubericidin and dominant-negative AMPK expression vector reversed the AICAR-mediated growth arrest. Moreover, constitutive active AMPK arrested the cells in S-phase by inducing the expression of p21, p27, and p53 proteins and inhibiting Akt phosphorylation, suggesting the involvement of AMPK. AICAR inhibited proliferation in both LKB and LKB knock-out mouse embryo fibroblasts to similar extent and arrested cells at S-phase when transfected with dominant negative expression vector of LKB. Altogether, these results indicate that AICAR can be utilized as a therapeutic drug to inhibit cancer, and AMPK can be a potential target for treatment of various cancers independent of the functional tumor suppressor gene, LKB.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AMP-activated protein kinase (AMPK)2 is a highly conserved serine/threonine protein kinase. It is a heterotrimer containing a catalytic ({alpha}) and two regulatory subunits ({beta} and {gamma}), each of which have at least two isoforms (1). AMPK is called the "fuel gauge" of the biological system, because it is activated under conditions that deplete cellular ATP and elevate AMP levels, such as glucose deprivation, heat shock, hypoxia, and ischemia (2, 3), and also by hormones like leptin (4), adiponectin (5), catecholamine (6), and interleukin-6 (7). Upon activation, AMPK phosphorylates and inactivates a number of metabolic enzymes involved in ATP-consuming pathways like fatty acid, cholesterol synthesis, and protein synthesis that include enzymes like acetyl-Co enzyme A carboxylase (ACC), fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and mammalian target of rapamycin (mTOR) and activates ATP-generating process like fatty acid oxidation and glucose uptake (8). The mechanisms of activating AMPK include direct allosteric binding of AMP to the {gamma} subunits and phosphorylation, catalyzed by an upstream AMP kinase (AMPKK), recently identified to be LKB1 (STK11) (9-11). Recent studies have demonstrated that AMPK can also be activated by other stimuli that do not cause a detectable change in the AMP/ATP ratio, like hyperosmotic stress and pharmacological agents like thiazolidinediones, metformin, and 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR) (11-14).

Activation of AMPK has been related with protection from injury and apoptosis caused by myocardial ischemia (15, 16) and apoptosis due to metabolic stress (17-19). In these scenarios, AMPK has been proposed as an anti-apoptotic molecule. However, recent reports have indicated anti-proliferative and pro-apoptotic action of activated AMPK using pharmacological agents or AMPK overexpression. AMPK activation has been shown to induce apoptosis in human gastric cancer cells (20), lung cancer cells (21), prostate cancer (22), pancreatic cells (23), and hepatic carcinoma cells (24) and enhance oxidative stress induced apoptosis in mouse neuroblastoma cells (25), by various mechanisms that includes inhibition of fatty acid synthase pathway and induction of stress kinases and caspase 3.

AMPK is an anti-growth molecule because of its relationship with two tumor suppressor genes: LKB and TSC2 (tuberous sclerosis complex 2). LKB functions as an upstream kinase (AMPKK) that activates AMPK (26). LKB mutations result in Peutz-Jeghers syndrome, which results in predisposition to cancers of the colon, pancreas, breast, and other sites (27-29). Mutations of LKB1 typically occur in the catalytic domain, leading to loss of its kinase activity and presumably a failure to phosphorylate and activate AMPK (30). TSC2 forms a complex with TSC1 and inhibits mTOR, leading to inhibition in protein synthesis and negative regulation of cell size and growth (31). Mutations of TSC1·TSC2 causes tuberous sclerosis, which is associated with hamartomatous polyps in multiple tissues and an increased risk of cancers (32).

In the present study we have investigated the effect of AICAR on cell proliferation in vivo and in vitro in various cancer cell lines. AICAR is converted to its triphosphorylated form ZMP, inside the cell, by an adenosine kinase (14), which acts as an AMP analogue and activates AMPK and its upstream kinase LKB without affecting the ATP:AMP ratio in the cell (14). AICAR-mediated AMPK activation was found to be a proficient inhibitor of cell proliferation and the mechanism of its anti-proliferative effect may be mediated via inhibition of PI3K-Akt pathway and increased expression of cell cycle inhibitory proteins p21, p27, and p53, thereby exhibiting potential as an anti-cancer drug.



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  		FIGURE 1.
AICAR inhibits proliferation of cancer cells. Specified numbers of cells (C6, MCF-7, PC3, CEM, and K562) were plated, treated with AICAR (0.25-1 mM), followed by exposure to [3H]thymidine for 6 h and subsequent counts. The data is representation of three separate experiments done in triplicates. ***, p < 0.001 compared with control; **, p < 0.01 compared with control, NS, non-significant compared with control.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Cell Culture—DMEM/F-12, DMEM/4.5 g of glucose medium, fetal bovine serum (FBS), and Hanks' balanced salt solution were obtained from Invitrogen as was RPMI 1640. AICAR was purchased from Toronto Research Chemicals (Ontario, Canada). Iodotubericidin was obtained from Calbiochem. [3H]Thymidine ribotide ([3H]TdR) was purchased from PerkinElmer Life Sciences. Propium iodide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and transfection reagent, FuGENE, were purchased from Roche Applied Science. The enhanced chemiluminescence (ECL) detecting reagent was from Amersham Biosciences, and the luciferase assay system was from Promega (Madison, WI). C6 glioma, T98G astrocytoma, U87MG astrocytoma, MCF-7 breast cancer, and PC-3 prostrate carcinoma cell lines were obtained from ATCC (Rockville, MD), hematological cancer cell lines (CEM T-lymphoblast cells, K-562 chronic myelogenous leukemia cells) were a kind gift from Dr. J. Barredo (Medical University of South Carolina). Primary antibodies, p21, p27, and p53 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phosphospecific as well as pan-Akt, mTOR, and AMPK were from Cell Signaling (Beverly, MA). AMPK{alpha}2-dominant negative expression vector (D157A) was a kind gift from Dr. David Carling (MRC Clinical Sciences Centre, London, UK), and AMPK{alpha}1 and {alpha}2 constitutive expression vectors were kind gifts from Dr. Jin-Zhong Zhang (Case Western Reserve University, Cleveland, OH) and Dr. Benoit Viollet (René Descartes University, Paris, France), respectively. LKB wild type, LKB dominant negative (kinase dead), STRAD{alpha} and MO25{alpha} expression vectors were kind gifts from Dr. Dario R. Alessi (Wellcome Trust Biocenter, University of Dundee, Dundee, UK).

Cell Culture—C6 glioma cells, T98G, U87MG, and PC-3 were maintained in DMEM/F-12 medium supplemented with 10% FBS and antibiotics. MCF-7 cells were maintained in DMEM/4.5 g of glucose with 10% FBS. CEM and K-562 were maintained in RPMI 1640 supplemented with 10% FBS. LKB knock-out and wild-type mouse embryo fibroblasts (MEFs) were a kind gift from Dr. Tomi P. Makela (Institute of Biomedicine and Helsinki University Central Hospital, Biomedicum Helsinki, University of Helsinki, Finland) and were maintained in DMEM/4.5 g of glucose with 10% FBS, essential amino acids, and antibiotics. All treatments were done in the presence of serum.

Thymidine Incorporation—Proliferation of cells determined by was [3H]thymidine ribotide ([3H]TdR) incorporation into DNA. 1 x 106 cells per well of adherent cell lines (C6, MCF-7, and PC3) and 0.25 x 106 cells/well of suspension cell lines (NALM-6, CEM, CEMP, and K562) were plated in respective medias. Cells were incubated for 18-24 h with or without the presence of AICAR at the indicated concentrations. Each group was exposed to 37 kBq/ml [methyl-3H]thymidine in the same medium for 6 h. The adherent cells were fixed by 5% trichloroacetic acid and lysed in SDS/NaOH lysis buffer overnight. Radioactivity was measured by Beckman LS3801 liquid scintillation counter (Canada). Suspension cell culture was harvested by cell harvester (Packard instrument Co., Meriden, CT), and radioactivity was measured by 1450 microbeta liquid scintillation counter (PerkinElmer Life Sciences).

Clonogenic Assay—Cells were treated with AICAR for 18-24 h, trypsinized, counted, and 300 cells/100-mm plate were plated. The cells were allowed to form colonies, and media was changed every third day for 2-3 weeks. The colonies were stained with MTT and enumerated (33).

Flow Cytometry Assessment of Cell Cycle—Cellular DNA content was assessed by flow cytometry. Cells were cultured in 6-well plates and treated with AICAR or transfections were performed. Cells attached to the plate were collected with trypsin, washed, and resuspended in 100 µl of PBS, and 5 ml of 70% ethanol was added slowly while continuous vortexing of cells and were fixed overnight. Next day, cells were spun, washed, and suspended in 400 µl of PBS with addition of 10 mg/liter RNase A and 75 µM propidium iodide. Cells were acquired by flow cytometry (BD Biosciences FACSCalibur flow cytometer) using Modfit LT software.

Immunoblot—After a stipulated time of incubation in the presence or absence of AICAR, cells were scraped, washed with Hanks' buffer, and sonicated in 50 mM Tris-HCl (pH 7.4) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml antipain, 5 µg/ml pepstatin A, and 5 µg/ml leupeptin). Proteins (50 µg/lane) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h in 5% nonfat dry milk in TTBS (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20, pH 7.5) and incubated overnight in primary antibody (p21, p27, p53, Akt, {beta}-actin, mTOR, PCNA at 1:2000 dilution) containing 5% nonfat dry milk for non-phospho antibodies and containing 5% albumin for phospho-antibodies (Akt-p, mTOR-p at 1:1000 dilution). The blots were washed four times with TTBS (5 min/wash) and incubated for 45 min at room temperature with respective horseradish peroxidase-conjugated secondary antibody (1:5000). The blots were washed three times in TTBS and once in 0.1 M PBS (pH 7.4) at room temperature; protein expression was detected with ECL.

Animals—Adult male Wistar rats weighing 200-250 g were purchased from Charles-River Laboratories. Animals were maintained, and all protocols were approved by the animal use committees of the Medical University of South Carolina in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).

Tumor Implantation—C6 glioma cells were prepared fresh from culture to ensure optimal viability of cells during tumor inoculation. The cells were trypsinized, and 106 tumor cells prepared in 100 µl of PBS were injected subcutaneously in the lateral side of the right hind leg of the rats, after shave and sterile preparation. On the 5th day of implantation 100 mg/kg body weight/day of AICAR was given intra-peritoneal until the 14th day, when the animals were sacrificed and the tumor was excised, weighed, and fixed in formalin (34).

Immunohistochemistry—Tumor sections were processed as previously described (35). In brief, deparaffinized and rehydrated sections were microwaved for 10 min in antigen unmasking fluid (Vector Laboratories, Burlingame, CA), treated with 3% hydrogen peroxide in methanol to eliminate endogenous peroxidase activity and blocked to reduce nonspecific staining. Sections were incubated overnight with primary antibody (1:100) followed by tyramide signal enhancement technique (Renaissance TSA for Immunocytochemistry, PerkinElmer Life Sciences) per manufacturer's instructions. After washing, slides were air-dried and mounted with aqueous mounting media (Vectashield, Vector Laboratories). The sections were examined under a fluorescence microscope (Olympus BX-60) with an Olympus digital camera (Optronics, Goleta, CA) using a dual band pass filter. Images were captured and processed using Adobe Photoshop 7.0.



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  		FIGURE 2.
AICAR inhibits clonogenic potential of transformed cells. C6 glioma and PC3 prostate cells were treated with AICAR for 18 h, trypsinized and 300 cells/100-mm plate were plated to form colonies. The data are a representation of three separate experiments done in triplicates. ***, p < 0.001 compared with control; **, p < 0.01 compared with control.

 
Transfection Studies—Plasmids were purified using the endotoxin-free plasmid midi prep kit (Qiagen). For transient transfections, C6 glioma cells were seeded in 6-well plates and grown to 60-80% confluence in DMEM/F-12 plus 5% FBS without antibiotics and transfected using FuGENE reagent. 1-3 µg of AMPK DN or AMPK{alpha}1CA or AMPK{alpha}2 CA expression vector along with 1 µg of eGFP expression vector or insertless expression vector (pcDNA3.1) were used for transfecting. Cells were treated with AICAR for 24 h and processed for GFP-gated DNA analysis by flow cytometry. Similarly, LKB1 wild type (1 µg) and dominant negative (1 µg) along with STRAD{alpha} (0.5 µg) and MO25{alpha} (0.5 µg) expression vectors were used for transfection studies.

Antisense Experiments—To decrease the levels of endogenous AMPK, C6 glioma cells were transfected for 48 h with 25 µM phosphothiorated antisense (AS) oligonucleotide (5'-CGCCCGTCGTCGTGCTTCTGC-3') directly against both the {alpha}1- and {alpha}2-subunits of AMPK (36, 37) and a missense (MS) oligonucleotide (5'-CTCCCGGCTTGCTGCCGT-3') along with eGFP expression vector (36). Oligonucleotides were transfected with FuGENE reagent per the manufacturer's instructions. The cells were then treated with AICAR for 24 h and analyzed for cell cycle analysis by flow cytometry.

PI3K Activity—After 2 h of AICAR (1 mM) treatment, cells were lysed with ice-cold lysis buffer containing 1% v/v Nonidet P-40, 100 mM NaCl, 20 mM Tris (pH 7.4), 10 mM iodoacetamide, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitors (Sigma-Aldrich). Lysates were incubated at 4 °C for 15 min, followed by centrifugation at 13,000 x g for 15 min. The supernatant was precleared with protein A/G-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C, followed by the addition of 1 µg/ml p85 mAb. After 2-h incubation at 4 °C, protein G-Sepharose beads were added, and the resulting mixture was further incubated for 1 h at 4 °C. The immunoprecipitates were washed twice with lysis buffer, once with PBS, once with 0.5 M LiCl and 100 mM Tris (pH 7.6), once in water, and once in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, and 0.25 mM EDTA). PI3K activity was determined using a lipid mixture of 100 µl of 0.1 mg/ml phosphatidylinositol and 0.1 mg/ml phosphatidylserine dispersed by sonication in 20 mM HEPES (pH 7.0) and 1 mM EDTA. The reaction was initiated by the addition of 20 µCi of [{gamma}-32P]ATP (3000 Ci/mmol, PerkinElmer Life Sciences) and 100 µM ATP, and terminated after 15 min by the addition of 80 µl of 1 N HCl and 200 µl of chloroform:methanol (1:1). Phospholipids were separated by TLC and visualized by exposure to iodine vapor and autoradiography (38).



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  		FIGURE 3.
AICAR causes cell cycle arrest in S-phase. C6 glioma and U87MG astrocytoma cells were treated with AICAR at indicated concentrations. After overnight fixation cells were suspended in PBS with RNase A and propidium iodide and acquired for DNA content by flow cytometry using Modfit LT software. The first peak represents the cells in G0/G1 phase, the second peak with slashed bars represents the cells accumulated in S-phase, and the third peak represents cells in the M-phase. The data are also graphically represented as percentage of cells in S-phase and M-phase. The data are a representative of three separate experiments. ***, p < 0.001 compared with control; **, p < 0.01 compared with control.

 



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  		FIGURE 4.
AICAR inhibits cell proliferation in vivo. A, weight of excised tumor implanted in lateral side of the right flank of rats (n = 8) from vehicle (saline) and AICAR (100 mg/kg body weight/day) treated. AICAR significantly reduced the tumor mass as compared with vehicle treated animals. *, p < 0.05 compared with vehicle (Bi). Immunofluorescent microscopy images showing decreased PCNA expression in AICAR-treated tumor sections as compared with vehicle-treated rats, stained as described under "Materials and Methods." The number of enumerated cells is depicted graphically (Bii). Data are mean ± S.D. of 10 fields from three different experiments. ***, p < 0.001 compared with control. C, Western blot depicting decreased expression of PCNA in the AICAR-treated tumor tissue from 2 different sets of animals as compared with vehicle-treated data. Each set had n = 6.

 
Statistical Analysis—The data were statistically analyzed by performing the Student-Newman-Keuls Test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AICAR Inhibits Proliferation of Cancer Cells—To investigate the effect of AICAR on the growth of various cancer cell lines, namely PC-3 (human prostate cancer cell), MCF-7 (human breast cancer cell line), C6 glioma (rat transformed brain glial cells), U87MG (human astrocytoma cell line), K-562 (human chronic myelogenous leukemia cells), and CEM (human T-lymphoblast cells), cells were plated in their respective medium for growth and treated with different concentrations of AICAR (0.25-1 mM) for 24 h, and cell proliferation was examined by [3H]thymidine uptake. AICAR inhibited the proliferation of all cell lines tested significantly in a dose-dependent manner (Fig. 1). All tested cell lines underwent significant proliferation inhibition, indicating that this phenomenon is widespread and not limited to a specific cell type/line. To further confirm this observation, a clonogenic assay was performed, where cells were treated with AICAR for 24 h, trypsinized and plated at a density of 300 cells/100-mm plate without AICAR. After 3 weeks, formed colonies were counted by staining the live cells with MTT. AICAR treatment significantly reduced the number of colonies being formed as compared with the untreated cells (Fig. 2), suggesting that a single treatment of AICAR treatment is sufficient to cause a sustained inhibition of proliferation in different cancer cell lines.



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  		FIGURE 5.
AICAR induces AMPK and ACC phosphorylation in vitro and in vivo. A, C6 glioma and PC3 prostate cells were treated with AICAR at indicated concentrations and harvested at specified time points, cell lysates were processed for the detection of phospho-AMPK (p-Thr-172) and phospho-ACC by immunoblot as discussed under "Materials and Methods." The blots are representatives of three individual experiments done. B, cell lysates were prepared from the vehicle- and AICAR-treated tumor tissues from two different set of animals (each set had n = 6) and processed for the detection of phospho-AMPK (p-Thr-172) and phospho-ACC as above.

 
AICAR Causes Cell Cycle Arrest in S-phase—Inhibition in proliferation would indicate an anomaly in the cell cycle. To examine this, cells were treated with AICAR (0.5-1 mM), and phases of cell cycle were analyzed by flow cytometry. Treatment of cells with AICAR resulted in accumulation of cells in S-phase (peak with slashed bars), with almost no cells detected in M-phase (third peak) suggesting that inhibition in proliferation by AICAR is due to the arrest of cell cycle at S-phase (Fig. 3).

AICAR Inhibits Proliferation in Vivo—To investigate whether the anti-proliferative effects of AICAR extends to the in vivo system, we utilized the rat flank tumor model (34). Wherein, C6 glioma cells (1 x 106) were implanted aseptically in the right flank of the rat, and after 5 days of tumor formation, animals were treated with 100 mg/kg body weight of AICAR intraperitoneally. On day 14, animals were sacrificed, and the tumors were excised, weighed, and fixed. Weight of the tumors was taken as an index of tumor development and progression. AICAR treatment was able to reduce the growth of tumors in animals significantly (>50%) when compared with untreated animals (Fig. 4A). To examine the status of proliferating cells in vivo, immunohistochemistry was performed on the sections of excised tumor tissues for PCNA (proliferating cell nuclear antigen), a marker for proliferating cells. AICAR significantly reduced the expression and number of cells exhibiting PCNA expression, indicating that the number of proliferating cells is reduced by AICAR treatment in vivo as demonstrated by immunohistochemistry and its expression by Western blot (Fig. 4, B and C). Thus, the anti-proliferative effect of AICAR is effective in vivo as well and can be exploited for applications in attenuating cancer cell growth.

AICAR Mediates Its Anti-proliferative Action via AMP-activated Protein Kinase—AICAR, is the earliest known AMPK activator, and most of its effects have been shown to be because of AMPK activation, although few reports of its AMPK-independent effects exist (22). To investigate if AMPK activation is responsible for the anti-proliferative effects observed by AICAR treatment, the phosphorylation of AMPK and its downstream target, ACC, an enzyme in the fatty acid synthesis pathway, was taken as an indicator of AMPK activation. AICAR induced the phosphorylation of AMPK and ACC in a dose- and time-dependent manner as demonstrated in C6 glioma and PC3 prostate cell lines (Fig. 5, A and B). Similar phosphorylation of ACC and AMPK was observed in vivo, in the AICAR-treated excised tumor tissue (Fig. 5C). Iodotubericidin is an inhibitor of adenosine kinase and inhibits the conversion of AICAR to its activated form ZMP inside the cell and thus inhibits activation of AMPK by AICAR. Cells were pretreated with iodotubericidin 30 min before the addition of AICAR (0.5-1 mM), and proliferation was measured after 16 h by [3H]thymidine uptake. Iodotubericidin treatment inhibited the proliferation arrest caused by AICAR thus indicating the involvement of AMPK (Fig. 6A). To further confirm the role of AMPK, C6 glioma cells were transiently transfected with dominant negative (DN) and constitutive active (CA) forms of AMPK along with eGFP expression vector. The cells were treated with AICAR for 18 h, and GFP-positive cells were analyzed by flow cytometry for DNA content to determine the cells in S-phase. C6 glioma cells transfected with AMPK dominant negative were not able to undergo S-phase arrest when treated with AICAR (Fig. 6B). Inversely, C6 glioma cells transfected with expression vector of constitutive active AMPK{alpha}1 were found to be arrested at S-phase similar to AICAR-treated cells (Fig. 6C). Further, we used the antisense (AS) approach to knock out the expression of AMPK. C6 glioma cells were transfected with AMPK antisense (AS) and missense (MS) oligonucleotides, and levels of AMPK were observed 48 h post transfection. The level of AMPK protein was attenuated by AS, whereas MS had no effect (Fig. 6D, inset). Moreover, transfection with antisense of AMPK along with GFP expression vector in C6 glioma cells significantly reduced the AICAR-mediated S-phase arrest; however, MS oligonucleotide did not affect the potential of AICAR to arrest cells in S-phase (Fig. 6D). Taken together, these evidences point strongly toward a role for AMPK as an effective anti-proliferative system.



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  		FIGURE 6.
AICAR mediated its anti-proliferative action via AMPK. A, cells (CEM, K-526, and PC-3) were pretreated with iodotubericidin (0.1 µM) before addition of AICAR and assayed for [3H]thymidine incorporation. Iodotubericidin reversed the AICAR-induced proliferation block. The data are representative of three separate experiments performed in triplicates. ***, p < 0.001 compared with control; ###, p < 0.001 compared with AICAR (B and C) C6 glioma cells were transiently co-transfected with 2 µg of AMPK dominant negative (DN) (B) or AMPK constitutive active (CA) (C) and 1 µg of eGFP expression vector. The DNA content was normalized by pcDNA3. AICAR was added where indicated, and after 18 h cells were fixed overnight and analyzed for arrest in S-phase as detailed under "Materials and Methods." AICAR was not able to arrest the cells in the presence of AMPK DN (B), whereas AMPK CA expression was sufficient to arrest the cells in S-phase and showed additive effect with AICAR (C). The data are representative of three separate experiments. ***, p < 0.001 compared with control; ###, p < 0.001 compared with AICAR; ##, p < 0.01 compared with AICAR; #, p < 0.05 compared with AICAR; NS, non-significant compared with control. D, cells were transfected with AMPK antisense (AS) and missense (MS) oligonucleotides along with eGFP expression vector and treated with AICAR. The level of AMPK protein was reduced by the transfection of AS, whereas MS had no effect (inset) at 72 h. The cells were fixed and processed for GFP-gated S-phase arrest. Antisense of AMPK abolished the AICAR-mediated S-phase arrest, whereas MS oligonucleotide had no effect. These data are representative of 3 separate experiments. ***, p < 0.001 compared with control; ###, p < 0.001 compared with AICAR; NS, non-significant compared with AICAR.

 



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  		FIGURE 7.
AICAR inhibits PI3K-Akt Pathway. A, C6 cells were treated with AICAR (1 mM) for 2 h and processed for PI3K activity as described under "Materials and Methods." AICAR significantly reduced the PI3K activity as assessed by inositol 1,4,5-bisphosphate levels. The inositol 1,4,5-bisphosphate levels were measured by densitometry analysis. The blot is representative of three separate experiments. **, p < 0.01 compared with control (B) C6 cells were treated with AICAR for increasing time points as indicated, cells lysates were prepared and analyzed for Akt (Ser-473) and mTOR (Ser-1448) phosphorylation by Western blot as detailed under "Materials and Methods." C, cell lysates were prepared from the treated tumor tissue from two different set of animals and processed for the detection of phospho-Akt, which was reduced by AICAR treatment.

 



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  		FIGURE 8.
AICAR regulates the expression of cdk inhibitors via AMPK. Protein expression of cell cycle inhibitors p21, p27, and p53 was increased by AICAR treatment as analyzed by immunoblot in C6 cells treated with AICAR (A) and tumor tissue (B). C, immunofluorescent microscopy images of tumor sections from vehicle and treated rats, stained with p21, p27, and p53 antibodies as described under "Materials and Methods." D, C6 glioma cells were transiently transfected with 2 µg of AMPK dominant negative (DN) or AMPK constitutive active (CA) with DNA normalization done with pcDNA and treated with AICAR where indicated. Cell lysates were prepared and assessed by immunoblot for p21, p27, and phospho-Akt expressions. AICAR induced p21 and p27 expression and down-regulated phospho-Akt as before (lane 2), which was reversed by AMPK DN expression (lane 3) and had no effect with AICAR treatment (lane 4). AMPK CA {alpha}1 and {alpha}2 overexpression was able to induce the p21 and p27 expression and attenuate phospho-Akt by itself (lanes 5 and 6). The blots are representatives of three individual experiments done.

 
AICAR/AMPK Inhibits the PI3K-Akt Pathway—Because AICAR inhibits cell proliferation and PI3K-Akt is one of the most important pathways regulating proliferation, we examined the effect of AICAR on the PI3K-Akt pathway. C6 glioma cells were treated with AICAR (1 mM) for 2 h, and PI3K activity was assessed using phosphoinositol as a substrate, and we observed that AICAR treatment significantly reduced the PI3K activity (Fig. 7A). One of the downstream effectors of PI3K, Akt is the main mediator regulating proliferation (39). AICAR also reduced the phosphorylation of Akt in vitro and in vivo (Fig. 7, Bi and C). It also inhibited the phosphorylation (Ser-1448) of mTOR (Fig. 7Bii), which is a downstream target of Akt and regulates protein synthesis and cell growth (40). Thus, attenuation of the PI3K-Akt pathway may be one of the mechanisms by which AMPK negatively regulates growth.

AICAR Regulates the Expression of Cyclin-dependent Kinase Inhibitors via AMPK—Because AICAR inhibits the cell proliferation by arresting cells at S-phase in vitro as well as in vivo, we examined the expression of cyclin-dependent kinase (cdk) inhibitors, which bind to cyclin-cdk complexes and inhibit the progression of cell cycle. AICAR induced the expression of p21 and p27, the cip/kip protein cdk inhibitors in a time-dependent manner (Fig. 8A). It also induced the expression of p53, which is known to regulate the cell cycle as well as p21 expression (41). The expression of p21, p27, and p53 proteins, were also increased in vivo, as assessed by immunohistochemistry of tissue sections and by Western blot analysis of protein isolated from excised tumor tissue (Fig. 8, B and C). The effect of AICAR on the expression of growth regulators is mediated via activation of AMPK, because transfected AMPK DN abolished the AICAR-mediated induction of p21, whereas the CA form of AMPK{alpha}1 and {alpha}2 induced the expression by itself (Fig. 8D). In case of p27, AMPK DN reduced the AICAR-induced expression, but AMPK{alpha}1 and {alpha}2 CA forms were only able to induce p27 protein marginally compared with AICAR. In case of Akt, DN AMPK{alpha}2-transfected cells did not respond to the AICAR-mediated inhibition in Akt phosphorylation, whereas, in CA-transfected cells, there was significant inhibition (Fig. 8E), indicating that AMPK activation is responsible for increase in cdk inhibitor protein expressions and inhibition of Akt phosphorylation.

LKB (AMPKK) Status Does Not Affect AICAR-mediated Growth Arrest—LKB is a recently discovered upstream target of AMPK (AMPK kinase, AMPKK), which phosphorylates AMPK at Thr-172 for its full activation. LKB itself is a tumor suppressor gene and inactivation of LKB results in predisposition to various cancers (27-29). It is being hypothesized that the anti-tumor effects of LKB are due to AMPK activity. To examine the possible involvement of LKB in AICAR/AMPK-induced growth arrest, we utilized LKB knock-out (LKB-/-) and LKB WT (LKB+/+) MEF cell lines. AICAR was able to induce the phosphorylation of AMPK and ACC to a similar extent in both MEFs (Fig. 9A). AICAR was able to inhibit proliferation in both knock-out and wild-type MEFs to a similar extent (Fig. 9B). This was further supported by similar S-phase arrest observed when C6 cells were transiently transfected with LKB dominant negative and wild-type expression vector along with expression vectors of its cofactors, STRAD{alpha} and MO25{alpha}, and treated with AICAR (Fig. 9C). These data indicate that AMPK activation by AICAR is sufficient to cause growth arrest and does not require activation by LKB.



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  		FIGURE 9.
LKB (AMPKK) status does not affect AICAR mediated growth arrest. A, LKB knock-out (LKB-/-) and wild-type (LKB+/+) mouse embryo fibroblast were treated with AICAR and analyzed by immunoblot for AMPK and ACC phosphorylation. The blots are representatives of three individual experiments done. B, LKB knock-out (LKB-/-) and wild-type (LKB+/+) mouse embryo fibroblast were treated with varying concentrations of AICAR and assayed for [3H]thymidine incorporation. The data are representative of three separate experiments. ***, p < 0.001 compared with control. C, C6 glioma cells were transiently transfected with 1 µg of LKB dominant negative (DN) or LKB wild type (WT) with STRAD{alpha} (0.5 µg) and MO25{alpha} (0.5 µg) expression vectors, eGFP, and pcDNA and treated with AICAR where indicated. After 18 h cells were fixed overnight and analyzed for arrest in S-phase as detailed under "Materials and Methods."

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have demonstrated that AMPK activation by AICAR results in growth arrest at S-phase due to inhibition of PI3K-Akt pathway and up-regulation of cdk inhibitors, independent of its upstream kinase LKB. This inference is based on the following observations: 1) Treatment of various cancer cell lines by AICAR attenuated the proliferation both in vitro and in vivo studies. 2) The attenuation of cell proliferation was due to the activation of AMPK as evident from adenosine kinase inhibitor studies (iodotubericidin), the use of expression vectors (dominant negative and constitutive active) and AMPK antisense experiments. 3) The growth arrest is mediated by inhibition of PI3K activity and Akt phosphorylation and up-regulation of cell-cycle inhibitor proteins p21, p27, and p53. 4) Activation of AMPK in the absence of LKB also results in growth arrest. We show here the direct relation between AMPK activation and growth inhibition in vitro and in vivo. In addition, these observations strongly indicate AICAR, an AMPK activator to be an efficient anti-proliferative agent in vitro and in vivo.



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  		FIGURE 10.
A schematic representation of effect of AICAR on proliferation of cancer cells. AICAR, upon entering the cell, is converted to ZMP, which activates AMPK. Upon activation AMPK increases the expression of p21, p27, and p53 proteins, which may be responsible for the S-phase arrest being observed. On the other hand it inhibits the PI3K activity and Akt phosphorylation, which results in inhibition of mTOR and speculative regulation of other targets like MDM2, Bad, and caspase 9, resulting in a proliferation and cell growth block. The overall signaling taking place results in a sustained proliferation arrest, which can ultimately lead to loss of viability due to onset of senescence or apoptotic pathways.

 
Being a pharmacological activator of AMPK, AICAR has been used extensively to study its role in physiology (13, 14). It has recently been shown to have anti-inflammatory properties that were reported to be mediated by AMPK activation (36, 42), although its AMPK-independent effects have also been reported (22). In our study, AICAR mediates its effect via activation of AMPK, which is supported by both pharmacological (iodotubericidin) as well as molecular approaches (DN, CA, and AS AMPK transfections). Treatment of iodotubericidin, inhibitor of conversion of AICAR to ZMP, abrogated the S-phase arrest of cells (Fig. 6A). Moreover, inhibition of AMPK either by its DN expression vector or AS oligonucleotide also resulted in the reversal of AICAR-mediated growth arrest, whereas AMPK CA expression was able to accumulate cells in S-phase (Fig. 6, B-D). AMPK DN also blunted the elevated expression of p21 and p27, whereas AMPK CA itself was able to induce their expression (Fig. 8). The induction of p27 by AMPK CA {alpha}1 and {alpha}2 seems to be marginal as compared with p21. The observation represented here is consistent and reproducible. Right now we do not have an explanation for this disparity. One of the possible explanations could be that p27 regulation is related to the differential localization of the {alpha}1 (cytosol) and {alpha}2 (nuclear) isoforms of the catalytic subunits of AMPK, which is yet to be established in terms of p27 regulation. AICAR seems to affect the PI3K-Akt proliferation pathway, because AICAR and AMPK CA inhibited PI3K activity and Akt phosphorylation (Fig. 7). These evidences point strongly toward AMPK activation being the major cause of growth arrest. Thus, AMPK can be considered as a negative regulator of proliferation and can modulate protein expression to this effect, classifying it as a tumor suppressor system that can be exploited for attenuation of cancers.

Activation of AMPK by AICAR, metformin, or thiazolidinediones or expression of constitutively active mutants has been shown to cause death or attenuate the growth of cancer cells. AICAR- and rosiglitazone-mediated AMPK activation caused proliferation block and cell death by inhibiting fatty acid and protein synthesis pathways and increasing p21 expression in prostate cells (22). Adenosine-induced AMPK was shown to cause apoptosis in gastric cancer cells (20), and activation of AMPK by AICAR and its CA form was shown to cause apoptosis in pancreatic cells by inducing JNK pathway (23). Similarly, AMPK induced JNK and caspase 3 activity resulting in apoptosis in liver cells (24). AMPK activation was also demonstrated to enhance H2O2-mediated apoptosis in neuroblastoma cells by inducing NF-{kappa}B and p38-JNK pathways (25). These studies, along with the present study, suggests AMPK as an efficient growth inhibitor and apoptosis inducer. On the other hand, it also has been shown to have a protective effect on stress-injured cells in heart ischemia and reperfusion injury model (15, 18). AMPK activation protects primary astrocytes from fatty acid-induced death by inhibiting de novo ceramide synthesis (17) and protects human umbilical vein endothelial cells from hyperglycemia by inhibition of caspase 3 and Akt activation (18) and by similar mechanism in thymocytes (19). In pancreatic cancer cells, AMPK was shown to bestow tolerance toward nutrient deprivation (43). These studies presented AMPK as a protective agent. The reason for these apparently opposing effects of AMPK activation in cell survival and cell death is not known, but it can be speculated that in actively dividing cancer cells, the inhibition of ATP-consuming processes by AMPK may be less compatible with their survival, whereas in non-dividing cells, where the protective effects of AMPK have been observed under acute stress, the shutdown of ATP-consuming pathways may not alter the balance for survival.

AMPK as a tumor suppressor system has gained more scientific interest because of its link with two tumor suppressors. One of these is the tuberous sclerosis complex 2 (TSC2), mutations of which cause tuberous sclerosis, an autosomal dominant disorder, which in humans is associated with hamartomatous polyps in multiple tissues and an increased risk of cancers. TCS2 forms an inhibitory complex with TSC1 and inhibits mTOR, leading to negative regulation of cell size and growth (31, 32). TSC2 is phosphorylated and inhibited by Akt. The observation that AMPK phosphorylates and activates TSC2 suggests yet another mechanism for the inhibitory effect of AMPK on growth (44, 45). In this regard our observation that AMPK inhibits PI3K activity and Akt phosphorylation (Fig. 7), points toward another route for regulating not only the TSC2-mTOR pathway but also other downstream players of Akt that control proliferation. The tumor suppressor, LKB1 (Stk11) functions as an upstream kinase, which phosphorylates and activates AMPK in physiological settings. Mutations of LKB1 have been found in Peutz-Jeghers syndrome, an autosomal dominant disorder characterized by multiple hamartomatous polyps (benign overgrowth of differentiated tissues) of the colon and a predisposition to cancers of the colon, pancreas, and other locations in the gastrointestinal tract (27, 29). Mutations of LKB1 typically occur in the catalytic domain, leading to loss of its kinase activity and presumably a failure to phosphorylate AMPK. According to our observation in LKB-null MEF cells, AICAR was able to activate AMPK and inhibit proliferation in both cells to a similar extent (Fig. 9), indicating that AMPK can act as a tumor suppressor even in absence of functional LKB. Thus, activation of AMPK by any other means or agents can also be exploited for the therapeutics for Peutz-Jeghers syndrome, where LKB is mutated.

Taken together, we conclude that activation of AMPK, by AICAR in this case results in activation of p21, p27, and p53, which results in cell cycle arrest. The elevation in the expression of p21 may be a direct effect of AMPK or may be occurring via p53, which remains to be explored. On the other hand it not only inhibits TSC2 complex as well documented by others (44, 45) but also attenuates the PI3K and Akt activity, which would further add to the negative regulation of TSC2-mTOR pathway (Fig. 10). Inhibition of Akt would also result in negative regulation of MDM2 that would help in up-regulating p53 activity, and forkhead, BAD, and caspase 9 (46), which would all assist in manifesting the anti-proliferative effect of AMPK activation. The exact mechanisms need to be worked out, because the regulation of proliferation and cell growth by AMPK appears to be quite complex. Sustained inhibition of cell cycle and/or proliferation can be hypothesized to lead to cell death by senescence or apoptosis. Overall, AMPK activation by AICAR or any other pharmacological agent is an attractive target for cancer therapy.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants NS-22576, NS-34741, NS-40810, NS-37766, and NS-40144. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Children Research Institute, Medical University of South Carolina, 173 Ashley Ave., 5th Floor, Charleston, SC 29425. Tel.: 843-792-7542; Fax: 843-792-7130; E-mail: singhi{at}musc.edu.

2 The abbreviations used are: AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; mTOR, mammalian target of rapamycin; AMPKK, AMPK kinase; AICAR, 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside; TSC2, tuberous sclerosis complex 2; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; TdR, thymidine ribotide; PBS, phosphate-buffered saline; PCNA, proliferating nuclear antigen; GFP, green fluorescent protein; eGFP, enhanced GFP; AS, antisense; MS, missense; PI3K, phosphatidylinositol 3-kinase; DN, dominant negarive; CA, constitutively active; MEF, mouse embryo fibroblasts; JNK, c-Jun NH2-terminal kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ZMP, AICA riboside monophosphate metabolite. Back


   ACKNOWLEDGMENTS
 
We thank Dr. J. Barredo (Medical University of South Carolina, SC) for his kind gift of CEM T-lymphoblast and K-562 chronic myelogenous leukemia cell lines. We thank Dr. David Carling (MRC Clinical Sciences Centre, London, UK) for his kind gift of AMPK{alpha}2-dominant negative expression vector (D157A). We thank Dr. Jin-Zhong Zhang (Case Western Reserve University, Cleveland, OH) and Dr. Benoit Viollet (René Descartes University, Paris, France) for their kind gift of AMPK{alpha}1 and {alpha}2 constitutive active constructs, respectively. We thank Dr. Dario R. Alessi (Wellcome Trust Biocentre, University of Dundee, and Dundee, UK) for the kind gift of LKB wild type, LKB dominant negative (kinase-dead), STRAD{alpha}, and MO25{alpha} expression vectors. We thank Dr. Tomi P. Makela (Institute of Biomedicine and Helsinki University Central Hospital, Biomedicum Helsinki, University of Helsinki, Finland) for his kind gift of LKB knock-out and wild-type mouse embryo fibroblasts. We thank Joyce Bryan, Carrie Barnes, and Hope Terry for laboratory assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hardie, D. G., and Carling, D. (1997) Eur. J. Biochem. 246, 259-273[Medline] [Order article via Infotrieve]
  2. Davies, S. P., Carling, D., and Hardie, D. G. (1989) Eur. J. Biochem. 186, 123-128[Medline] [Order article via Infotrieve]
  3. Kemp, B. E., Stapleton, D., Campbell, D. J., Chen, Z. P., Murthy, S., Walter, M., Gupta, A., Adams, J. J., Katsis, F., van Denderen, B., Jennings, I. G., Iseli, T., Michell, B. J., and Witters, L. A. (2003) Biochem. Soc. Trans. 31, 162-168[Medline] [Order article via Infotrieve]
  4. Minokoshi, Y., Kim, Y. B., Peroni, O. D., Fryer, L. G., Muller, C., Carling, D., and Kahn, B. B. (2002) Nature 415, 339-343[CrossRef][Medline] [Order article via Infotrieve]
  5. Yamauchi, T., Kamon, J., Minokoshi, Y., Ito, Y., Waki, H., Uchida, S., Yamashita, S., Noda, M., Kita, S., Ueki, K., Eto, K., Akanuma, Y., Froguel, P., Foufelle, F., Ferre, P., Carling, D., Kimura, S., Nagai, R., Kahn, B. B., and Kadowaki, T. (2002) Nat. Med. 8, 1288-1295[CrossRef][Medline] [Order article via Infotrieve]
  6. Ruderman, N. B., Park, H., Kaushik, V. K., Dean, D., Constant, S., Prentki, M., and Saha, A. K. (2003) Acta Physiol. Scand. 178, 435-442[CrossRef][Medline] [Order article via Infotrieve]
  7. Kelly, M., Keller, C., Avilucea, P. R., Keller, P., Luo, Z., Xiang, X., Giralt, M., Hidalgo, J., Saha, A. K., Pedersen, B. K., and Ruderman, N. B. (2004) Biochem. Biophys. Res. Commun. 320, 449-454[CrossRef][Medline] [Order article via Infotrieve]
  8. Luo, Z., Saha, A. K., Xiang, X., and Ruderman, N. B. (2005) Trends Pharmacol. Sci. 26, 69-76[CrossRef][Medline] [Order article via Infotrieve]
  9. Hong, S. P., Leiper, F. C., Woods, A., Carling, D., and Carlson, M. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 8839-8843[Abstract/Free Full Text]
  10. Woods, A., Johnstone, S. R., Dickerson, K., Leiper, F. C., Fryer, L. G., Neumann, D., Schlattner, U., Wallimann, T., Carlson, M., and Carling, D. (2003) Curr. Biol. 13, 2004-2008[CrossRef][Medline] [Order article via Infotrieve]
  11. Hawley, S. A., Boudeau, J., Reid, J. L., Mustard, K. J., Udd, L., Makela, T. P., Alessi, D. R., and Hardie, D. G. (2003) J. Biol. 2, 28[CrossRef][Medline] [Order article via Infotrieve]
  12. Fryer, L. G., Parbu-Patel, A., and Carling, D. (2002) J. Biol. Chem. 277, 25226-25232[Abstract/Free Full Text]
  13. Sullivan, J. E., Brocklehurst, K. J., Marley, A. E., Carey, F., Carling, D., and Beri, R. K. (1994) FEBS Lett. 353, 33-36[CrossRef][Medline] [Order article via Infotrieve]
  14. Corton, J. M., Gillespie, J. G., Hawley, S. A., and Hardie, D. G. (1995) Eur. J. Biochem. 229, 558-565[Medline] [Order article via Infotrieve]
  15. Russell, R. R., 3rd, Li, J., Coven, D. L., Pypaert, M., Zechner, C., Palmeri, M., Giordano, F. J., Mu, J., Birnbaum, M. J., and Young, L. H. (2004) J. Clin. Invest. 114, 495-503[CrossRef][Medline] [Order article via Infotrieve]
  16. Nishino, Y., Miura, T., Miki, T., Sakamoto, J., Nakamura, Y., Ikeda, Y., Kobayashi, H., and Shimamoto, K. (2004) Cardiovasc. Res. 61, 610-619[Abstract/Free Full Text]
  17. Blazquez, C., Geelen, M. J., Velasco, G., and Guzman, M. (2001) FEBS Lett. 489, 149-153[CrossRef][Medline] [Order article via Infotrieve]
  18. Ido, Y., Carling, D., and Ruderman, N. (2002) Diabetes 51, 159-167[Abstract/Free Full Text]
  19. Stefanelli, C., Stanic, I., Bonavita, F., Flamigni, F., Pignatti, C., Guarnieri, C., and Caldarera, C. M. (1998) Biochem. Biophys. Res. Commun. 243, 821-826[CrossRef][Medline] [Order article via Infotrieve]
  20. Saitoh, M., Nagai, K., Nakagawa, K., Yamamura, T., Yamamoto, S., and Nishizaki, T. (2004) Biochem. Pharmacol. 67, 2005-2011[CrossRef][Medline] [Order article via Infotrieve]
  21. Li, J., Jiang, P., Robinson, M., Lawrence, T. S., and Sun, Y. (2003) Carcinogenesis 24, 827-834[Abstract/Free Full Text]
  22. Xiang, X., Saha, A. K., Wen, R., Ruderman, N. B., and Luo, Z. (2004) Biochem. Biophys. Res. Commun. 321, 161-167[CrossRef][Medline] [Order article via Infotrieve]
  23. Kefas, B. A., Cai, Y., Ling, Z., Heimberg, H., Hue, L., Pipeleers, D., and Van de Casteele, M. (2003) J. Mol. Endocrinol. 30, 151-161[Abstract]
  24. Meisse, D., Van de Casteele, M., Beauloye, C., Hainault, I., Kefas, B. A., Rider, M. H., Foufelle, F., and Hue, L. (2002) FEBS Lett. 526, 38-42[CrossRef][Medline] [Order article via Infotrieve]
  25. Jung, J. E., Lee, J., Ha, J., Kim, S. S., Cho, Y. H., Baik, H. H., and Kang, I. (2004) Neurosci. Lett. 354, 197-200[CrossRef][Medline] [Order article via Infotrieve]
  26. Hardie, D. G., Scott, J. W., Pan, D. A., and Hudson, E. R. (2003) FEBS Lett. 546, 113-120[CrossRef][Medline] [Order article via Infotrieve]
  27. Hemminki, A., Markie, D., Tomlinson, I., Avizienyte, E., Roth, S., Loukola, A., Bignell, G., Warren, W., Aminoff, M., Hoglund, P., Jarvinen, H., Kristo, P., Pelin, K., Ridanpaa, M., Salovaara, R., Toro, T., Bodmer, W., Olschwang, S., Olsen, A. S., Stratton, M. R., de la Chapelle, A., and Aaltonen, L. A. (1998) Nature 391, 184-187[CrossRef][Medline] [Order article via Infotrieve]
  28. Jenne, D. E., Reimann, H., Nezu, J., Friedel, W., Loff, S., Jeschke, R., Muller, O., Back, W., and Zimmer, M. (1998) Nat. Genet. 18, 38-43[CrossRef][Medline] [Order article via Infotrieve]
  29. Nakanishi, C., Yamaguchi, T., Iijima, T., Saji, S., Toi, M., Mori, T., and Miyaki, M. (2004) Oncology 67, 476-479[Medline] [Order article via Infotrieve]
  30. Forcet, C., Etienne-Manneville, S., Gaude, H., Fournier, L., Debilly, S., Salmi, M., Baas, A., Olschwang, S., Clevers, H., and Billaud, M. (2005) Hum. Mol. Genet. 14, 1283-1292[Abstract/Free Full Text]
  31. Inoki, K., Zhu, T., and Guan, K. L. (2003) Cell 115, 577-590[CrossRef][Medline] [Order article via Infotrieve]
  32. Li, Y., Corradetti, M. N., Inoki, K., and Guan, K. L. (2004) Trends Biochem. Sci. 29, 32-38[CrossRef][Medline] [Order article via Infotrieve]
  33. Giocanti, N., Hennequin, C., Rouillard, D., Defrance, R., and Favaudon, V. (2004) Br. J. Cancer 91, 2026-2033[CrossRef][Medline] [Order article via Infotrieve]
  34. Parsa, A. T., Chakrabarti, I., Hurley, P. T., Chi, J. H., Hall, J. S., Kaiser, M. G., and Bruce, J. N. (2000) Neurosurgery 47, 993-999; discussion 999-1000[Medline] [Order article via Infotrieve]
  35. Nath, N., Giri, S., Prasad, R., Singh, A. K., and Singh, I. (2004) J. Immunol. 172, 1273-1286[Abstract/Free Full Text]
  36. Giri, S., Nath, N., Smith, B., Viollet, B., Singh, A. K., and Singh, I. (2004) J. Neurosci. 24, 479-487[Abstract/Free Full Text]
  37. Culmsee, C., Monnig, J., Kemp, B. E., and Mattson, M. P. (2001) J. Mol. Neurosci. 17, 45-58[CrossRef][Medline] [Order article via Infotrieve]
  38. Giri, S., Rattan, R., Singh, A. K., and Singh, I. (2004) J. Immunol. 173, 5196-5208[Abstract/Free Full Text]
  39. Brazil, D. P., Yang, Z. Z., and Hemmings, B. A. (2004) Trends Biochem. Sci. 29, 233-242[CrossRef][Medline] [Order article via Infotrieve]
  40. Harrington, L. S., Findlay, G. M., and Lamb, R. F. (2005) Trends Biochem. Sci. 30, 35-42[CrossRef][Medline] [Order article via Infotrieve]
  41. el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) Cell 75, 817-825[CrossRef][Medline] [Order article via Infotrieve]
  42. Nath, N., Giri, S., Prasad, R., Salem, M. L., Singh, A. K., and Singh, I. (2005) J. Immunol. 175, 566-574[Abstract/Free Full Text]
  43. Kato, K., Ogura, T., Kishimoto, A., Minegishi, Y., Nakajima, N., Miyazaki, M., and Esumi, H. (2002) Oncogene 21, 6082-6090[CrossRef][Medline] [Order article via Infotrieve]
  44. Kimura, N., Tokunaga, C., Dalal, S., Richardson, C., Yoshino, K., Hara, K., Kemp, B. E., Witters, L. A., Mimura, O., and Yonezawa, K. (2003) Genes Cells 8, 65-79[Abstract]
  45. Bolster, D. R., Crozier, S. J., Kimball, S. R., and Jefferson, L. S. (2002) J. Biol. Chem. 277, 23977-23980[Abstract/Free Full Text]
  46. Song, G., Ouyang, G., and Bao, S. (2005) J. Cell Mol. Med. 9, 59-71[Medline] [Order article via Infotrieve]

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