Role of Methionine Adenosyltransferase 2A and S-adenosylmethionine in Mitogen-Induced Growth of Human Colon Cancer Cells
2007; Elsevier BV; Volume: 133; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2007.03.114
ISSN1528-0012
AutoresHui Chen, Meng Xia, Mark T. Lin, Heping Yang, J Kuhlenkamp, Tony Li, Nicole M. Sodir, Yongheng Chen, Heinz Josef–Lenz, Peter W. Laird, Steven Clarke, José M. Mato, Shelly C. Lu,
Tópico(s)Folate and B Vitamins Research
ResumoBackground & Aims: Two genes (MAT1A and MAT2A) encode for methionine adenosyltransferase, an essential enzyme responsible for S-adenosylmethionine (SAMe) biosynthesis. MAT1A is expressed in liver, whereas MAT2A is widely distributed. In liver, increased MAT2A expression is associated with growth, while SAMe inhibits MAT2A expression and growth. The role of MAT2A in colon cancer in unknown. The aims of this study were to examine whether MAT2A expression and SAMe and its metabolite methylthioadenosine (MTA) can modulate growth of colon cancer cells. Methods: Studies were conducted using resected colon cancer specimens, polyps from Min mice, and human colon cancer cell lines RKO and HT-29. MAT2A expression was measured by real-time polymerase chain reaction and cell growth by the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: In 12 of 13 patients and all 9 polyps from Min mice, the MAT2A messenger RNA levels were 200%–340% of levels in adjacent normal tissues, respectively. Epidermal growth factor, insulin-like growth factor 1, and leptin increased growth and up-regulated MAT2A expression and MAT2A promoter activity in RKO and HT-29 cells. SAMe and MTA lowered the baseline expression of MAT2A and blocked the growth factor–mediated increase in MAT2A expression and growth in colon cancer cell lines. Importantly, the mitogenic effect of these growth factors was inhibited if MAT2A induction was prevented by RNA interference. SAMe and MTA supplementation in drinking water increased intestinal SAMe levels and lowered MAT2A expression. Conclusions: Similar to the liver, up-regulation of MAT2A also provides a growth advantage and SAMe and MTA can block mitogenic signaling in colon cancer cells. Background & Aims: Two genes (MAT1A and MAT2A) encode for methionine adenosyltransferase, an essential enzyme responsible for S-adenosylmethionine (SAMe) biosynthesis. MAT1A is expressed in liver, whereas MAT2A is widely distributed. In liver, increased MAT2A expression is associated with growth, while SAMe inhibits MAT2A expression and growth. The role of MAT2A in colon cancer in unknown. The aims of this study were to examine whether MAT2A expression and SAMe and its metabolite methylthioadenosine (MTA) can modulate growth of colon cancer cells. Methods: Studies were conducted using resected colon cancer specimens, polyps from Min mice, and human colon cancer cell lines RKO and HT-29. MAT2A expression was measured by real-time polymerase chain reaction and cell growth by the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: In 12 of 13 patients and all 9 polyps from Min mice, the MAT2A messenger RNA levels were 200%–340% of levels in adjacent normal tissues, respectively. Epidermal growth factor, insulin-like growth factor 1, and leptin increased growth and up-regulated MAT2A expression and MAT2A promoter activity in RKO and HT-29 cells. SAMe and MTA lowered the baseline expression of MAT2A and blocked the growth factor–mediated increase in MAT2A expression and growth in colon cancer cell lines. Importantly, the mitogenic effect of these growth factors was inhibited if MAT2A induction was prevented by RNA interference. SAMe and MTA supplementation in drinking water increased intestinal SAMe levels and lowered MAT2A expression. Conclusions: Similar to the liver, up-regulation of MAT2A also provides a growth advantage and SAMe and MTA can block mitogenic signaling in colon cancer cells. Methionine adenosyltransferase (MAT) is an essential cellular enzyme that catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor and the ultimate source of the propylamine moiety used in polyamine biosynthesis.1Lu S.C. Mato J.M. Role of methionine adenosyltransferase and SAMe in alcohol-associated liver cancer.Alcohol. 2005; 35: 227-234Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar In mammals, 2 different genes, MAT1A and MAT2A, encode for 2 homologous MAT catalytic subunits, α1 (forms either a dimer MAT III or tetramer MAT I) and α2 (forms MAT II), while a third gene, MAT2β, encodes for a regulatory subunit β that regulates MAT II.1Lu S.C. Mato J.M. Role of methionine adenosyltransferase and SAMe in alcohol-associated liver cancer.Alcohol. 2005; 35: 227-234Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 2Kotb M. Mudd S.H. Mato J.M. Geller A.M. Kredich N.M. Chou J.Y. Cantoni G.L. Consensus nomenclature for the mammalian methionine adenosyltransferase genes and gene products.Trends Genet. 1997; 13: 51-52Abstract Full Text PDF PubMed Scopus (189) Google ScholarMAT1A is expressed mostly in liver and is a marker for normal differentiated liver.1Lu S.C. Mato J.M. Role of methionine adenosyltransferase and SAMe in alcohol-associated liver cancer.Alcohol. 2005; 35: 227-234Abstract Full Text Full Text PDF PubMed Scopus (131) Google ScholarMAT2A is widely distributed. MAT2A also predominates in the fetal liver and is progressively replaced by MAT1A during liver development.1Lu S.C. Mato J.M. Role of methionine adenosyltransferase and SAMe in alcohol-associated liver cancer.Alcohol. 2005; 35: 227-234Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar In hepatocytes, increased MAT2A and MAT2β expression is associated with increased growth and malignant degeneration.1Lu S.C. Mato J.M. Role of methionine adenosyltransferase and SAMe in alcohol-associated liver cancer.Alcohol. 2005; 35: 227-234Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 3Cai J. Sun W.M. Hwang J. Stain S. Lu S.C. Changes in S-adenosylmethionine synthetase in human liver cancer: molecular characterization and significance.Hepatology. 1996; 24: 1090-1097Crossref PubMed Google Scholar, 4Huang Z. Mao Z. Cai J. Lu S.C. Changes in methionine adenosyltransferase during liver regeneration in the rat.Am J Physiol. 1998; 38: G14-G21Google Scholar, 5Cai J. Mao Z. Hwang J. Lu S.C. Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.Cancer Res. 1998; 58: 1444-1450PubMed Google Scholar Even though the MAT isoenzymes catalyze the same reaction, they are regulated differently by the product SAMe. In liver cells, SAMe maintains MAT1A expression and suppresses MAT2A expression.6Garcia-Trevijano E.R. Latasa M.U. Carretero M.V. Berasain C. Mato J.M. Avila M.A. S-Adenosylmethionine regulates MAT1A and MAT2A gene expression in cultured rat hepatocytes: a new role for S-adenosylmethionine in the maintenance of the differentiated status of the liver.FASEB J. 2000; 14: 2511-2518Crossref PubMed Scopus (96) Google Scholar, 7Yang H.P. Huang Z.Z. Zeng Z.H. Chen C.J. Selby R.R. Lu S.C. Role of promoter methylation in increased methionine adenosyltransferase 2A expression in human liver cancer.Am J Physiol. 2001; 280: G184-G190Google Scholar In addition, SAMe and its metabolite methylthioadenosine (MTA) can inhibit liver cell growth and induce apoptosis in liver cancer cells while protecting normal hepatocytes against apoptosis.5Cai J. Mao Z. Hwang J. Lu S.C. Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.Cancer Res. 1998; 58: 1444-1450PubMed Google Scholar, 8Ansorena E. García-Trevijano E.R. Martínez-Chantar M.L. Huang Z.Z. Chen L.X. Mato J.M. Iraburu M. Lu S.C. Avila M.A. S-adenosylmethionine and methylthioadenosine are anti-apoptotic in cultured rat hepatocytes but pro-apoptotic in human hepatoma cells.Hepatology. 2002; 35: 274-280Crossref PubMed Scopus (120) Google Scholar, 9Yang H.P. Sadda M.R. Li M. Zeng Y. Chen L.X. Bae W.J. Ou X.P. Runnegar M.T. Mato J.M. Lu S.C. S-Adenosylmethionine and its metabolite induce apoptosis in HepG2 cells: role of protein phosphatase 1 and Bcl-xS.Hepatology. 2004; 40: 221-231Crossref PubMed Scopus (73) Google Scholar Outside of the liver, the only cell type studied is the lymphocyte, where MAT2A expression and SAMe levels have been shown to increase during T-lymphocyte activation.10Tobeña R. Horikawa S. Calvo V. Alemany S. Interleukin-2 induces γ-S-adenosyl-L-methionine synthetase gene expression during T-lymphocyte activation.Biochem J. 1996; 319: 929-933Crossref PubMed Scopus (22) Google Scholar The increase in SAMe level was believed to be necessary to support polyamine synthesis during rapid growth.10Tobeña R. Horikawa S. Calvo V. Alemany S. Interleukin-2 induces γ-S-adenosyl-L-methionine synthetase gene expression during T-lymphocyte activation.Biochem J. 1996; 319: 929-933Crossref PubMed Scopus (22) Google Scholar The influence of MAT2A and SAMe levels on cell growth in other cell types has not been studied. One small study examined MAT protein expression and activity in colorectal carcinoma and normal colon. The ratio of MAT activity in tumor tissue versus normal tissue correlated well with the stage of the colorectal tumor. The higher activity correlated with higher immunohistochemical staining for MAT II.11Ito K. Ikeda S. Kojima N. Miura M. Shimizu-Saito K. Yamaguchi I. Katsuyama I. Sanada K. Iwai T. Senoo H. Horikawa S. Correlation between the expression of methionine adenosyltransferase and the stages of human colorectal carcinoma.Surg Today. 2000; 30: 706-710Crossref PubMed Scopus (15) Google Scholar However, the mechanism of MAT II up-regulation in colon cancer was not investigated, and whether this may play a role in colon cancer pathogenesis is also unknown. Epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and leptin are 3 well-known growth factors that have been implicated in colon cancer growth and invasion. Higher levels of both IGF-1 and IGF-1 receptor (IGF-1R) have been shown in colon cancer,12Reinmuth N. Fan F. Liu W. Parikh A.A. Stoeltzing O. Jung Y.D. Bucana C.D. Radinsky R. Gallick G.E. Ellis L.M. Impact of insulin-like growth factor receptor-1 function on angiogenesis, growth, and metastasis of colon cancer.Lab Invest. 2002; 82: 1377-1389Crossref PubMed Scopus (162) Google Scholar, 13LeRoith D. Roberts Jr, C.T. The insulin-like growth factor system and cancer.Cancer Lett. 2003; 195: 127-137Abstract Full Text Full Text PDF PubMed Scopus (948) Google Scholar and IGF-1R signaling plays an important role in tumor growth, angiogenesis, and metastasis.12Reinmuth N. Fan F. Liu W. Parikh A.A. Stoeltzing O. Jung Y.D. Bucana C.D. Radinsky R. Gallick G.E. Ellis L.M. Impact of insulin-like growth factor receptor-1 function on angiogenesis, growth, and metastasis of colon cancer.Lab Invest. 2002; 82: 1377-1389Crossref PubMed Scopus (162) Google Scholar Increased EGF receptor signaling has also been shown to provide growth advantage and correlate with colon cancer progression and metastatic potential.14Roberts R.B. Min L. Washington M.K. Olsen S.J. Settle S.H. Coffey R.J. Threadgill D.W. Importance of epidermal growth factor receptor signaling in establishment of adenomas and maintenance of carcinomas during intestinal tumorigenesis.Proc Natl Acad Sci U S A. 2002; 99: 1521-1526Crossref PubMed Scopus (241) Google Scholar, 15Yokoi J. Thaker P.H. Yazici S. Rebhun R.R. Nam D.H. He J. Kim S.J. Abbruzzese J.L. Hamilton S.R. Fidler I.J. Dual inhibition of epidermal growth factor receptor and vascular endothelial growth factor receptor phosphorylation by AEE788 reduced growth and metastasis of human colon carcinoma in an orthotopic nude mouse model.Cancer Res. 2005; 65: 3716-3725Crossref PubMed Scopus (121) Google Scholar Likewise, elevated leptin levels were found to be a risk factor for colon cancer in men,16Stattin P. Lukanova A. Biessy C. Söderberg S. Palmqvist R. Kaaks R. Olsson T. Jellum E. Obesity and colon cancer: does leptin provide a link?.Int J Cancer. 2004; 109: 149-152Crossref PubMed Scopus (260) Google Scholar and leptin has been shown to promote invasiveness of colon cancer cells.17Attoub S. Noe V. Pirola L. Bruyneel E. Chastre E. Mareel M. Wymann M.P. Gespach C. Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase, Rho- and Rac-dependent signaling pathways.FASEB J. 2000; 14: 2329-2338Crossref PubMed Scopus (219) Google Scholar Leptin levels correlate with body mass index, and obesity is a well-recognized risk factor for colon cancer.16Stattin P. Lukanova A. Biessy C. Söderberg S. Palmqvist R. Kaaks R. Olsson T. Jellum E. Obesity and colon cancer: does leptin provide a link?.Int J Cancer. 2004; 109: 149-152Crossref PubMed Scopus (260) Google Scholar The aims of the current study were to examine (1) expression of MAT2A in resected colon cancer specimens, (2) the effects of mitogens on expression of MAT2A genes and growth in colon cancer cell lines RKO and HT-29, and (3) the influence of SAMe and MTA on MAT2A expression and effects of mitogens. We found that similar to the liver, MAT2A expression is important for cell growth and is required for the mitogens to induce growth in colon cancer cells. Furthermore, SAMe and MTA also lower MAT2A expression and modulate the growth response of colon cancer cells to mitogens. These findings may have important implications in the pathogenesis and treatment of colon cancer. Cell culture media, fetal bovine serum, and primers were obtained from Gibco BRL Life Technologies (Grand Island, NY). The Luciferase Assay System was obtained from Promega (Madison, WI). All restriction endonucleases were obtained from either Promega or Gibco. RNA interference (RNAi) against MAT2A was obtained from Invitrogen (Carlsbad, CA). SAMe in the form of disulfate p-toluenesulfonate spray dried powder (97.18% purity) was generously provided by Gnosis SRL (Cairate, Italy). MTA was purchased from Sigma (St Louis, MO). All other reagents were of analytical grade and were obtained from commercial sources. We had available to us colon cancer and paired normal colon specimens from 13 patients, which were from a repository based on availability of both normal and cancerous colon specimens from the same patient. Each patient signed an informed consent for analysis of resected tissue for molecular markers. These tissues were immediately frozen in liquid nitrogen for subsequent analysis of messenger RNA (mRNA), Western blot, and SAMe levels as described in the following text. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Keck School of Medicine University of Southern California's human research review committee. Nine adenomatous polyps were obtained from three 3-month-old male Min mice (4 from different parts of the small intestine from one mouse and 5 from different parts of the colon from 2 other mice). Normal intestinal tissues adjacent to the polyps were included for comparison. These tissues were immediately snap frozen for subsequent analysis of mRNA and SAMe levels as described in the following text. Three-month-old male C57/B6 mice were fed ad libitum a standard diet (Harland Teklad irradiated mouse diet 7912; Madison, WI), were caged individually, and had free access to water supplemented with SAMe (75 or 150 mg · kg−1 · day−1), MTA (75 mg · kg−1 · day−1), phosphate-buffered saline (vehicle for SAMe), or dimethyl sulfoxide (0.2% vehicle for MTA) for 6 days. SAMe and MTA were made fresh daily. The amount of water intake, body weight, and animal behavior were closely monitored. After 6 days, animals were killed, intestine was cut open along the longitudinal axis, and mucosa was stripped from the intestine as we described.18Sodir N.M. Chen X. Park R. Nickel A.E. Conti P.S. Moats R. Bading J.R. Shibata D. Laird P.W. Smad3 deficiency promotes tumorigenesis in the distal colon of ApcMin/+ mice.Cancer Res. 2006; 66: 8430-8438Crossref PubMed Scopus (81) Google Scholar Mucosa was weighed and a portion processed for SAMe measurement as described in the following text, and the rest was snap frozen for subsequent RNA extraction. Animals were treated humanely, and all procedures were in compliance with our institutions' guidelines for the use of laboratory animals. HT-29 and RKO cells were obtained from the Cell Culture Core of the University of Southern California Liver Disease Research Center and grown according to instructions provided by the American Type Culture Collection (Rockville, MD). Before treatment with growth factors SAMe or MTA, medium was changed to 0.1% fetal bovine serum overnight. Medium was then changed to withhold serum and cells were treated with leptin, IGF-1, or EGF (all at 100 ng/mL), SAMe (0.5–5 mmol/L), MTA (0.5–1 mmol/L), or respective vehicle controls for 1–24 hours for various assays as described in the following text. These growth factors were shown to exert mitogenic effects in colon cancer cell lines at the dose chosen.12Reinmuth N. Fan F. Liu W. Parikh A.A. Stoeltzing O. Jung Y.D. Bucana C.D. Radinsky R. Gallick G.E. Ellis L.M. Impact of insulin-like growth factor receptor-1 function on angiogenesis, growth, and metastasis of colon cancer.Lab Invest. 2002; 82: 1377-1389Crossref PubMed Scopus (162) Google Scholar, 17Attoub S. Noe V. Pirola L. Bruyneel E. Chastre E. Mareel M. Wymann M.P. Gespach C. Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase, Rho- and Rac-dependent signaling pathways.FASEB J. 2000; 14: 2329-2338Crossref PubMed Scopus (219) Google Scholar, 19Hardwick J.C.H. Van Den Brink G.R. Offerhaus G.J. Van Deventer S.J.H. Peppelenbosch M.P. Leptin is a growth factor for colonic epithelial cells.Gastroenterology. 2001; 121: 79-90Abstract Full Text PDF PubMed Scopus (347) Google Scholar RNAi experiments were performed using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's instructions. Small interfering RNA (siRNA) oligonucleotides for MAT2A and scrambled siRNA were synthesized by the University of Southern California Norris Comprehensive Cancer Center Microchemical Core Laboratory and annealed to form duplexes. Stealth RNAi for MAT2A and stealth RNAi negative control duplexes were synthesized by Invitrogen. The following siRNA sequences were used: si-MAT2A #1, 5′-ACACAUUGGAUAUGAUGAUTT-3′ (sense) and 5′-AUCAUCAUAUCCAAUGUGUTT-3′ (antisense); si-control with scrambled sequence (negative control siRNA having no perfect matches to known human genes), 5′-UUCUCCGAACGUGUCACAUdTdT-3′ (sense) and 5′-AUGUGACACGUUCGGAGAAdTdT-3′ (antisense); stealth RNAi-MAT2A #5, 5′- CCACCUACAGCCAAGUGGCAGAUUU-3′ (sense) and 5′-AAAUCUGCCACUUGGCUGUAGGUGG-3′ (antisense). Transfection was allowed to proceed 72 hours before collection for different assays. Total RNA was isolated by the EZgeno Total RNA Isolation Kit (Genemega, San Diego, CA) and subjected to reverse transcription by using Moloney murine leukemia virus reverse transcriptase (Invitrogen). A total of 2 μL of reverse transcription product was subjected to quantitative real-time polymerase chain reaction (PCR) analysis. The primers and TaqMan probes for MAT2A, MAT2β, and Universal PCR Master Mix were purchased from ABI (Foster City, CA). Hypoxanthine phosphoribosyl-transferase 1 and ubiquitin C were used as housekeeping genes as described.20Vandesomeple J. De Preter K. Pattyn F. Poppe B. Van Roy N. De Paepe A. Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol. 2002; 3: 34.1-34.11Google Scholar The thermal profile consisted of 1 cycle at 95°C for 15 minutes followed by 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute. The expression of MAT2A and MAT2β was checked by normalizing the Ct of MAT2A and MAT2β to that of the control housekeeping gene (hypoxanthine phosphoribosyl-transferase 1 or ubiquitin C).21Giulietti A. Overbergh L. Valckx D. Decallonne B. Bouillon R. Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression.Methods. 2001; 25: 386-401Crossref PubMed Scopus (1124) Google Scholar The ΔCt obtained was used to find the relative expression of MAT genes according to the following formula: Relative Expression = 2-ΔΔCt, where ΔΔCt represents ΔCt of MAT genes in colon cancer or treated cells minus ΔCt of MAT genes in normal colon or control cells. The human MAT2A promoter construct –571/+60-LUC and glutathione synthetase (GSS) promoter construct –1686/-46-LUC were previously described7Yang H.P. Huang Z.Z. Zeng Z.H. Chen C.J. Selby R.R. Lu S.C. Role of promoter methylation in increased methionine adenosyltransferase 2A expression in human liver cancer.Am J Physiol. 2001; 280: G184-G190Google Scholar, 22Yang H.P. Sadda M.R. Yu V. Zeng Y. Lee T.D. Ou X.P. Chen L.X. Lu S.C. Induction of human methionine adenosyltransferase 2A expression by tumor necrosis factor alpha: Role of NF-μB and AP-1.J Biol Chem. 2003; 278: 50887-50896Crossref PubMed Scopus (54) Google Scholar, 23Lee T.D. Yang H.P. Whang J. Lu S.C. Cloning and characterization of the human glutathione synthetase 5'-flanking region.Biochem J. 2005; 390: 521-528Crossref PubMed Scopus (60) Google Scholar and subcloned in the sense orientation upstream of the luciferase coding sequence of the pGL-3 enhancer vector (Promega). Both promoter constructs contain maximal promoter activity. To study the effect of mitogens on human MAT2A promoter activity in HT-29 cells, HT-29 cells (5 × 105 cells in 2 mL serum-free medium) were transiently transfected with 2 μg MAT2A promoter firefly luciferase gene construct or promoterless pGL3-enhancer vector (as negative control) using the Superfect Transfection Reagent (Qiagen, Valencia, CA) as we described.22Yang H.P. Sadda M.R. Yu V. Zeng Y. Lee T.D. Ou X.P. Chen L.X. Lu S.C. Induction of human methionine adenosyltransferase 2A expression by tumor necrosis factor alpha: Role of NF-μB and AP-1.J Biol Chem. 2003; 278: 50887-50896Crossref PubMed Scopus (54) Google Scholar to control for transfection efficiency, cells were cotransfected with the Renilla phRL-TK vector (Promega). Cells were treated with mitogens (100 ng/mL), SAMe (5 mmol/L), MTA (1 mmol/L), or vehicle control during the last 3 (IGF-1 and EGF) to 6 hours (leptin, SAMe, and MTA) of the transfection (18 hours total). The luciferase activity driven by the MAT2A promoter construct was normalized to Renilla luciferase activity. Each experiment was performed with triplicate samples. Specificity of SAMe and MTA on promoter activity was examined by transfecting HT-29 cells with the GSS promoter construct and treating cells with SAMe or MTA as previously described. Cell growth was measured by the cell growth determination kit 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from Sigma. The MTT assay measures the cell proliferation rate and reduction in cell viability. HT-29 or RKO cells (1 × 104/well) were plated in 96-well plates and treated with growth factors (all 100 ng/mL) SAMe (0.5–5 mmol/L) and MTA (0.5–1 mmol/L), alone or in combination for 16 hours in serum-free medium. To determine the effect of MAT2A RNAi on mitogen-induced changes in MTT, cells were first treated with RNAi for 48 hours, followed by mitogen treatment for another 24 hours. Apoptosis was assessed by Hoechst staining as we described.22Yang H.P. Sadda M.R. Yu V. Zeng Y. Lee T.D. Ou X.P. Chen L.X. Lu S.C. Induction of human methionine adenosyltransferase 2A expression by tumor necrosis factor alpha: Role of NF-μB and AP-1.J Biol Chem. 2003; 278: 50887-50896Crossref PubMed Scopus (54) Google Scholar Briefly, RKO cells were grown on coverslips and treated with RNAi#1 for 24 hours, and cells were fixed with paraformaldehyde and stained with 8 μg/mL Hoechst 33258 dye for 30 minutes. Cells with bright, fragmented, condensed nuclei were identified as apoptotic cells using the Nikon Eclipse TE300 fluorescent microscope (Melville, NY). At least 5 random fields (at 300×) were counted. Cellular SAMe and MTA levels were measured as we described.24Farrar C. Clarke S. Altered levels of S-adenosylmethionine and S-adenosylhomocysteine in the brains of L-isoaspartyl (D-aspartyl) O-methyltransferase-deficient mice.J Biol Chem. 2002; 277: 27856-27863Crossref PubMed Scopus (33) Google Scholar Polyamine levels were determined according to the method described.25Brown N.D. Strickler M.P. Femtomolar ion-pair high-performance liquid chromatographic method for determining Dns-polyamine derivatives of red blood cell extracts utilizing an automated polyamine analyzer.J Chromatogr. 1982; 245: 101-108Crossref PubMed Scopus (48) Google Scholar Western blot analysis for MAT II in colon cancer and HT-29 cells treated with IGF-1 (100 ng/mL) for 8 hours was performed as we described9Yang H.P. Sadda M.R. Li M. Zeng Y. Chen L.X. Bae W.J. Ou X.P. Runnegar M.T. Mato J.M. Lu S.C. S-Adenosylmethionine and its metabolite induce apoptosis in HepG2 cells: role of protein phosphatase 1 and Bcl-xS.Hepatology. 2004; 40: 221-231Crossref PubMed Scopus (73) Google Scholar using anti–MAT II antibodies (GenWay Biotech, Inc, San Diego, CA). Data are given as mean ± SEM. Statistical analysis was performed using Student t test for comparison of paired samples and analysis of variance followed by Fisher test for multiple comparisons. Significance was defined by P < .05. We first examined MAT expression in colon cancer and adjacent normal tissue control by real-time PCR. We had available to us colon cancer and paired normal colon specimens from 13 patients, which were randomly selected from a repository based on availability of both normal and cancerous colon specimens from the same patient. Table 1 summarizes the clinical and molecular data. Note that MAT2A is induced in 12 of 13 colon cancer specimens. By comparison, the gene that encodes the regulatory subunit MAT2β is not significantly changed as compared with normal colon. MAT1A is not expressed in normal or cancerous colon. Increased MAT II (α2 and α2′) level was confirmed using Western blot analysis (Figure 1). The smaller α2′ subunit is believed to be derived from α2 by posttranslational modification.26Halim A.B. Legros L. Geller A. Kotb M. Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells.J Biol Chem. 1999; 274: 29720-29725Crossref PubMed Scopus (85) Google Scholar Consistent with this, SAMe levels were higher in all colon cancer specimens (normal colon, 0.26 ± 0.03 nmol/mg protein; colon cancer, 0.73 ± 0.11 nmol/mg protein; results are mean ± SE from 6 specimens each; P < .01 by paired Student t test). Tissue MTA level was below detection limit.Table 1MAT Expression in Normal Colon and Colon Cancer SpecimensPatient no.SexAge (y)Cancer siteStageHistologyMAT2AaPercentage relative expression, tumor/normal colon. MAT expression determined by real-time PCR.MAT2βaPercentage relative expression, tumor/normal colon. MAT expression determined by real-time PCR.1M65RectosigmoidT3N1M0Moderate222312F47RectumTXN2M0Unknown171903M53RectosigmoidT3N1M0Poor1521894M66RectumT3N1M1Moderate1742205M59SigmoidT3N2M1Moderate531146F61Left colonT3N2M1Moderate2031227F77Right colonT2N2M1Well differentiated245888M67Right colonT3N2M1Unknown1381749M62RectumT3N2M0Moderate17110310F56Right colonUnknownModerate1876011M52RectumT3N2M0Poor29515312M73RectumTXNXM1Moderate2668913F68Right colonT4N1M1Poor284234a Percentage relative expression, tumor/normal colon. MAT expression determined by real-time PCR. Open table in a new tab We also compared MAT2A mRNA levels in 9 adenomatous polyps with adjacent normal tissues from 3 Min mice, and all of the polyps had higher MAT2A mRNA levels (338% ± 93%; range, 132%–909%; expressed as percentage of matched normal tissue mRNA level; P < .05). SAMe levels in 4 polyps (average MAT2A mRNA levels were 200% ± 50% of normal) were also higher than adjacent normal tissues (normal, 0.28 ± 0.38 nmol/mg protein; polyps, 0.38 ± 0.22 nmol/mg protein; P < .05). MTA levels were unchanged (normal, 0.06 ± 0.03 nmol/mg protein; polyps, 0.08 ± 0.04 nmol/mg protein). We next determined the effect of leptin on these 2 MAT genes in RKO and HT-29 cells and found that leptin treatment resulted in a time-dependent increase in MAT2A but not MAT2β mRNA levels, with near doubling of the MAT2A mRNA levels 5 hours after leptin treatment in both cell types (Figure 2A and C). IGF-1 and EGF treatment of both cell types also doubled the MAT2A mRNA levels (Figure 2B and 3B). Similar to leptin, these growth factors also did not affect MAT2β mRNA level in the 2 colon cancer cell lines (not shown). IGF-1 treatment also increased MAT II level in HT-29 cells (Figure 1).Figure 3SAMe and MTA (A) lower MAT2A mRNA levels and (B) prevent a growth factor–induced increase in MAT2A expression. HT-29 cells were treated with SAMe (5 mmol/L) or MTA (1 mmol/L) or vehicle control for 3–6 hours, and MAT2A mRNA levels were determined by real-time PCR (A). To examine whether SAMe and MTA can modulate the effect of the growth factors on MAT2A expression, HT-29 cells were treated with growth factors SAMe or MTA alone or in combination. Cotreatment of SAMe or MTA with leptin was for 6 hours, while cotreatment for IGF-1 or EGF was for 3 hours. All results represent mean ± SEM from 3–4 independent experiments performed in duplicate. *P < .05 vs control, †P < .05 vs respective growth factors.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We have previously shown that SAMe treatment lowers MAT2A expression in hepatocytes.6Garcia-Trevijano E.R. Latasa M.U. Carretero M.V. Berasain C. Mato J.M. Avila M.A. S-Adenosylmethionine regulates MAT1A and MAT2A gene expression in cultured rat hepatocytes: a new role for S-adenosylmethionine in the maintenance of the differentiated status of the liver.FASEB J. 2000;
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