ISSN 1007-7626 CN 11-3870 / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2011 11 27 11 1032 ~ 1038 DNMT 1 2 3 1 1 4 1 * 1 400016 2 400016 3 400016 4 400016 DNA DNA methyltransferase DNMT DNMT1 DNMT2 DNMT3A DNMT3B DNMT3L DNMT 60 5 DNMT DNMT2 DNMT3B DNMT3L DNMT3A DNMT1 DNMT DNMT1 DNMT2 DNMT3B Western GES-1 5 DNMT DNMT1 DNMT2 DNMT3A DNMT3B DNMT3L GES-1 DNMT 5- -2'- GES-1 DNMT DNMT DNA 5- -2'- Q34 Differential Expression of DNMTs Between Gastric Tissues with and without H Pylori Infection HE Miao 1 TANG Wei-Xue 2 JIANG Rong 3 ZHANG Neng 1 ZHA Lang 1 FAN Jing 4 WANG Zi-Wei 1 * 1 Department of General Surgery First Affiliated Hospital of Chongqing Medical University Chongqing 2 Laboratory Research Center First Affiliated Hospital of Chongqing Medical University Chongqing 3 Faculty of Basic Medical Sciences Chongqing Medical University Chongqing 4 Department of Emergency First Affiliated Hospital of Chongqing Medical University Chongqing 400016 China 400016 China 400016 China 400016 China Abstract DNA methyltransferase DNMT was the research focus of epigenetics Here we investigated protein expressions of DNMT1 DNMT2 DNMT3A DNMT3B DNMT3L in adult gastric mucosa and studied the effect of DNMT inhibitor 5-Aza-2'-dC on GES-1 gastric mucosa cell line Immunohistochemistry was implemented on 60 samples of adult gastric mucosa tissues We found that five kinds of DNMT proteins all existed in gastric tissues and the positive expression rates of DNMT2 DNMT3B were higher than those of other DNMT There were also differential expressions of DNMT1 DNMT2 and DNMT3B between gastric tissues with Helicobacter pylori infection and those without H pylori infection and the expression rates of infected group were higher than those of non-infected group P < 0 05 Immunofluorescence and Western blotting were used on GES-1 The results showed that DNMT proteins were expressed in gastric mucosa cell line And DNMT1 were distributed in nucleus and cytoplasm DNMT2 was distributed in nucleus DNMT3A DNMT3B DNMT3L were distributed in 2011-08-15 2011-10-18 * Tel 13098681910 E-mail wangziwei571@ sina com Received August 15 2011 Accepted October 18 2011 * Corresponding author Tel 13098681910 E-mail wangziwei571@ sina com
11 DNMT 1033 cytoplasm MTT assay was carried on for GES-1 with DNMT inhibitor 5-Aza-2'-deoxycytidine We found that 5-Aza-2 -deoxycytidine had anti-proliferative effect on GES-1 in a dose-and time-dependent manner These results suggested that DNMT proteins were expressed in adult gastric mucosa DNMT played a role as proliferation regulator more or less in gastric mucosa cell Abnormal expressions of DNMT1 DNMT2 and DNMT3B may be correlated with H pylori infection in gastric mucosa tissue Key words DNMT gastric mucosa 5-Aza-2'-deoxycytidine helicobacter pylori DNA FFP30 AA128 A0216 A0208 P0018 DNA MTT 5- -2'- 5-Aza-2'-deoxycytidine RNA Sigma M-2128 A3656 DNA 1 DNA 1 2 2 DNA 4% 24 h DNA DNA methyltransferase DNMT DNMT SP-9001 DNMT 4 1 50 ZLI-9017 5 min DNMT 3 1 3 DNMT GES-1 10% 4 RPMI-1640 5% CO 2 37 DNMT 3 d 1 DNMT DNMT1 DNMT2 1 4 DNMT3A DNMT3B DNMT3L GES-1 96 24 h DNMT GES-1 4% 20 min 0 5% Triton X-100 37 20 min 37 30 min 1 50 4 1 50 37 1 h 1 PBS 1 1 60 GES-1 RIPA 30 2011 2 6 min 4 12 000 / min 5 min P0010S 2011 6 100 10 min GES-1 P0012A 40 ~ 80 V 2 ~ 3 h DNMT1 DNMT2 DNMT3A DNMT3B DNMT3L SANTA CRUZ sc-20701 sc-20702 sc-20703 sc-20704 sc-20705 min TBST 5 min 3 1 3 000 DAB FITC IgG 37 1 5 h TBST 5 min 3 SP-9001 ZLI-9017 ZF-0311 RIPA PVDF Actin HRP HRP 250 μl / 5- -2'- ECL 1 μmol / L 5 μmol / L 10 μmol / L P0013E P0010S P0015 P0012A P0068 1 5 Western 250 ma 2 h 37 1 h 1 50 1 1 000 4 30 P0018 1 6 GES-1 96 10 4 / 24 48 72 96 120 h MTT 5 mg / ml 20 μl /
1034 27 4 h 150 μl / 2 3 DNMT 490 nm A DNMT = chi-square test A - A / A - A 5 1 7 SPSS17 0 5 DNMT DNMT3B P < 0 05 DNMT DNMT Fisher 5- -2'- GES-1 t P < 0 05 2 2 1 DNMT DNMT 5 DNMT DNMT2 1035 Fig 1A Fig 2 The positive expressions of DNMT in 60 gastric mucosa tissues DNMT proteins in 60 adult gastric mucosa 2 2 DNMT tissues were detected by Immunohistochemistry using anti- DNMT DNMT antibodies 1 50 Santa Cruz The positive Fig 1B-F Fig 2 DNMT1 expression rates of DNMT1 DNMT2 DNMT3A DNMT3B 63 3% 38 /60 DNMT2 93 3% 56 /60 DNMT3L were 63 3% 38 /60 93 3% 56 /60 71 7% DNMT3A 71 7% 43 /60 DNMT3B 43 /60 93 3% 56 /60 75% 45 /60 respectively 93 3% 56 /60 DNMT3L 75% 45 /60 5 Table 1 Helicobacter pylori H pylori DNMT1 DNMT2 DNMT P 0 05 These positive rates were different significantly P < 0 05 DNMT P < Row Column table chi-square test 0 05 DNMT3B DNMT2 DNMT3L DNMT3A DNMT1 2 4 GES-1 DNMT DNMT1 DNMT2 2 4 1 DNMT DNMT3A DNMT3B DNMT3L GES-1 GES-1 DNMT DNMT2 Fig 3 DNMT3B Table 1 Relationship between DNMT expressions and epidemiological features of gastric mucosa tissues n No of DNMT1 No of DNMT2 No of DNMT3A No of DNMT3B No of DNMT3L expression % P expression % P expression % P expression % P expression % P Sex Male 38 22 57 9 0 25 37 97 4 0 10 28 73 7 0 65 36 94 7 0 57 26 68 4 0 12 Female 22 16 72 7 19 86 4 15 68 2 20 90 9 19 86 4 Age years 50 41 28 68 3 0 24 40 97 6 0 05 30 73 2 0 70 39 95 1 0 42 32 78 0 0 42 < 50 19 10 52 6 16 84 2 13 68 4 17 89 5 13 68 4 Primary disease Benign 14 7 50 0 0 24 13 92 9 0 94 10 71 4 0 98 12 85 7 0 19 9 64 3 0 29 Malignant 46 31 67 4 43 93 5 33 71 7 44 95 7 36 78 3 H pylori infection Positive 52 36 69 2 0 02 * 51 98 1 0 00 * 37 71 2 0 82 52 100 0 00 * 40 76 9 0 38 Negative 8 2 25 0 5 62 5 6 75 0 4 50 0 5 62 5 There were significant correlations between H * P < 0 05 vs non-infection groups chi-square test pylori infection and DNMT expressions DNMT1 DNMT2 DNMT3B in gastric mucosa tissues
11 DNMT 1035 Fig 1 Expressions of DNMT in placenta tissues and gastric mucosa tissues DNMT proteins were detected by Immunohistochemistry using anti-dnmt antibodies 1 50 Santa Cruz A Expression of DNMT2 in human placenta tissue 400 B Expression of DNMT1 in adult gastric mucosa tissue 400 C Expression of DNMT2 in adult gastric mucosa tissue 400 D Expression of DNMT3A in adult gastric mucosa tissue 400 E Expression of DNMT3B in adult gastric mucosa tissue 400 F Expression of DNMT3L in adult gastric mucosa tissue 400 Fig 3 Fluorescence microscopy image of DNMT2 expression in GES-1 DNMT2 protein was detected by Immunofluorescence using anti-dnmt2 antibody 1 50 Santa Cruz and fluorescein-conjugated affinipure goat antirabbit IgG 1 100 ZSGB-BIO DNMT2 was distributed in nucleus 100
1036 27 DNMT3L DNMT1 DNMT3A DNMT GES-1 DNMT GES-1 2 5 5- -2'- GES-1 DNMT1 MTT assay DNMT DNMT2 DNMT3A DNMT3B DNMT3L GES-1 DNMT 2 4 2 Western Western GES-1 DNMT 5 DNMT Fig 4 5- -2'- 5-Aza-2'-deoxycytidine GES-1 Table 2 5- -2'- GES-1 P < 0 05 5- -2'-GES-1 Fig 4 Expressions of DNMT in GES-1 detected by Western blotting DNMT expressions were detected by Western blotting with anti-dnmt antibodies 1 100 Santa Cruz using proteins from GES-1 Actin was used as an internal control 1 1 500 Beyotime GES-1 expressed 5 kinds of DNMT proteins Table 2 Proliferation rates of GES-1 in different time after 5-Aza-2'-deoxycytidine treatment 24 h 48 h 72 h 96 h 120 h 0 μmol / L 1 1 1 1 1 1 μmol / L 0 983 ± 0 040 0 947 ± 0 034 * 0 913 ± 0 034 * 0 875 ± 0 031 * 0 832 ± 0 032 * 5 μmol / L 0 980 ± 0 023 0 892 ± 0 035 * 0 880 ± 0 030 * 0 838 ± 0 031 * 0 777 ± 0 042 * 10 μmol / L 0 933 ± 0 027 * 0 875 ± 0 031 * 0 860 ± 0 024 * 0 820 ± 0 050 * 0 767 ± 0 039 * The cell proliferation was evaluated by MTT assay The group was control GES-1 without 5-Aza-2'-deoxycytidine treatment 5-Aza-2'-deoxycytidine caused growth inhibition in GES-1 t-test * P < 0 05 vs control 3 DNMT DNMT1 DNMT2 DNMT3A DNA DNMT S- - DNMT3B DNMT3L DNMT1 DNA L- DNA 5 DNA 5- DNMT1 DNA DNA DNMT2 DNA trna DNMT2 DNA DNA 6 DNMT3A DNMT3B DNA 5 DNA 7 DNMT3L DNMT3A DNMT3B DNMT Kim 8 DNMT3L DNA DNMT 15
11 DNMT 1037 DNMT DNMT 5-Aza-2'-deoxycytidine 4 H DNMT pylori DNMT DNMT1 DNMT2 DNMT3B DNMT H pylori DNMT DNMT DNMT2 DNMT3B Western GES-1 DNMT DNMT2 DNMT DNMT DNA DNMT J Med Recapit 2 Esteller T H pylori 48-59 9 3 DNMT 10 H pylori DNMT DNMT1 DNMT2 DNMT3B H pylori DNMT J Cancer 2011 117 4 677-687 DNMT 5- -2'- DNMT DNMT 11 DNMT 7 12-14 5- -2'- J GES-1 DNMT 5- -2'- 1 5 DNMT DNMT2 DNMT3B DNMT3L DNMT3A DNMT1 DNMT 2 DNMT1 DNMT2 DNMT3A DNMT3B DNMT3L Ding 15 DNMT1 DNMT3A DNMT3B 3 DNMT 5-Aza-2'-deoxycytidine GES-1 DNMT References 1 DNA RNA J He Miao Wang Zi-Wei Current study of the correlation between DNA methylation and small RNA in tumor 2011 17 15 2296-2299 Detection and interpretation of altered methylation patterns in cancer cells J N Engl J Med 2008 358 11 DNA J Su Yu Wang Xi Zhu Wei-Guo DNA methyltransferases the role in regulation of gene expression and biological processes J Hereditas Beijing 2009 31 11 1087-1093 4 Daniel FI Cherubini K Yurgel LS et al The role of epigenetic transcription repression and DNA methyltransferases in cancer 5 Esteller M Cancer epigenomics DNA methylomes and histonemodification maps J Nat Rev Genet 2007 8 4 286-298 6 Goll MG Kirpekar F Maggert KA et al Methylation of trnaasp by the DNA methyltransferase homolog Dnmt2 J Science 2006 311 5759 395-398 J Liu Zhi-Jian Sun Ying-Li Epigenetics and cancer Target epigenetic alterations in tumor Chin J Biochem Mol Biol 2011 27 4 310-315 8 Kim H Park J Jung Y et al DNA methyltransferase 3-like affects promoter methylation of thymine DNA glycosylase independently of DNMT1 and DNMT3B in cancer cells J Int J Oncol 2010 36 6 1563-1572 9 Kague E Thomazini CM Pardini MI et al Methylation status of CDH1 gene in samples of gastric mucous from Brazilian patients with chronic gastritis infected by Helicobacter pylori J Arq Gastroenterol 2010 47 1 7-12 10 Kanai Y Ushijima S Kondo Y et al DNA methyltransferase expression and DNA methylation of CPG islands and pericentromeric satellite regions in human colorectal and stomach cancer J Int J Cancer 2001 91 2 205-212 11 Jung Y Park J Kim TY et al Potential advantages of DNA methyltransferase 1 DNMT1 -targeted inhibition for cancer therapy J J Mol Med 2007 85 10 1137-1148 12 Chiam K Centenera MM Butler LM et al GSTP1 DNA
1038 27 Methylation and Expression Status Is Indicative of 5-aza-2'- Deoxycytidine Efficacy in Human Prostate Cancer Cells J PLoS One 2011 6 9 e25634 13 Gu YM Ma YH Zhao WG et al Dickkopf3 overexpression inhibits pancreatic cancer cell growth in vitro J World J Gastroenterol 2011 17 33 3810-3817 14 Tsai KW Wu CW Hu LY et al Epigenetic regulation of mir- 34b and mir-129 expression in gastric cancer J Int J Cancer 2011 129 11 2600-2610 15 Ding WJ Fang JY Chen XY et al The expression and clinical significance of DNA methyltransferase proteins in human gastric cancer J Dig Dis Sci 2008 53 8 2083-2089 / WITHDRAWAL PMCA MCT DTT PrPsc 1 1 * 1 2 3 1 430030 2 430030 3 430023 The Inhibitory Effect of MCT in Different Concentration with DTT on Amplification of PrPscby PMCA in vitro BI Hao 1 LU Hua-Xin 1 * WANG Xian-Guang 1 LU Yan-Jun 2 LIU Zhi-Guo 3 1 Wuhan Blood Center Department of Clinical Laboratory Wuhan 430030 China 2 Department of Clinical Laboratory Tongji Hospital Tongji Medical University Huazhong University of Science and Technology Wuhan 430030 China 3 Department of Biotechnology and Chemical Engineering Wuhan Polytechnic University Wuhan 430023 China / Chinese Journal of Biochemistry and Molecular Biology 27 9 879-884 2011 / Attached letters to Journal Editorial Office from Hao BI and to Hao BI from Dr Wen-Quan ZOU 2008 2009 PMCA MCT DTT PrP SC 27 12 Alkylating Antitumor Drug Mechlorethamine Conceals A Structured PrP Domain and Inhibits in Vitro Prion Amplification 11 Journal of Toxicology and Environmental Health Part A 2011 11 1060