Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues and its potential post-translational modification

Abstract

Gastric cancer is one of the most common cancers worldwide. The purpose of this study was to find out potential markers for gastric cancer. Tumor and normal tissues from 152 gastric cancer cases were analyzed by two-dimensional gel electrophoresis (2-DE). The images of silver stained gels were analyzed and statistical analysis of spot intensities revealed that spot 4262 showed higher expression (5.7-fold increase) in cancer tissues than in normal tissues (P< 0.001). It was identified by peptide mass fingerprinting as nicotinamide N-methyltransferase (NNMT). A monoclonal antibody with a detection limit down to 10 ng was produced against NNMT in mouse. Using the prepared monoclonal antibody, western blot analysis of NNMT was performed for gastric tissues from 15 gastric cancer patients and two gastric ulcer patients. The results corroborated those of 2-DE experiments. A single spot was detected in gastric ulcer tissues while four to five spots were detected in gastric cancer tissues. In cancer tissues, two additional spots of acidic and basic form were mainly detected on 2-DE gels. This suggests that NNMT receives a post-translational modification in cancer- specific manner.

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 38, No. 5, 455-465, October 2006

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues and its potential post-translational modification

Bo-Hyun Lim1, Bok-Im Cho1,Yu Na Kim1, Jae Won Kim1,2, Soon-Tae Park3 and Chang-Won Lee1,2,4

1Department of Microbiology College of Natural Science

2Research Institute of Life Science

3Department of Surgery College of Medicine Gyeongsang National University Jinju 600-701, Korea

4Corresponding author: Tel, 82-55-751-5941;

Fax, 82-55-759-0187; E-mail, [email protected]

Accepted 7 June 2006

Abbreviations: 2-DE, 2-dimensional electrophoresis; NNMT, nicotinamide N-methyltransferase

Abstract

Gastric cancer is one of the most common cancers worldwide. The purpose of this study was to find out potential markers for gastric cancer. Tumor and normal tissues from 152 gastric cancer cases were analyzed by two-dimensional gel electrophoresis (2-DE). The images of silver stained gels were analyzed and statistical analysis of spot intensities revealed that spot 4262 showed higher expression (5.7-fold increase) in cancer tissues than in normal tissues (P 0.001). It was identified by peptide mass fingerprinting as nicotinamide N-methyltransferase (NNMT). A monoclonal antibody with a detection limit down to 10 ng was produced against NNMT in mouse. Using the prepared monoclonal antibody, western blot analysis of NNMT was performed for gastric tissues from 15 gastric cancer patients and two gastric ulcer patients. The results corroborated those of 2-DE experiments. A single spot was detected in gastric ulcer tissues while four to five spots were detected in gastric cancer tissues. In cancer tissues, two additional spots of acidic and basic form were mainly detected on 2-DE gels. This suggests that NNMT receives a post-translational modification in cancer- specific manner.

Keywords: electrophoresis, gel, two-dimensional; nicotinamide N-methyltransferase; protein processing, post- translational; proteomics; stomach neoplasms

Introduction

Gastric cancer is the most common cancer in Korea and other Asian countries. Although incidence rates in western world are much lower than in Asia, gastric carcinoma is still a significant worldwide health burden, second only to lung tumors as a leading cause of cancer deaths (Lyman, 1992; Ha et al., 2002; Kim et al., 2002). To reduce the mortality and improve the effectiveness of therapy, many studies have tried to find key biomarkers. Biomarkers are important molecular signposts of the biologic state of a cell at a specific condition (Tahara et al., 1996; Loging et al., 2000). Two-dimensional gel electrophoresis (2-DE) is widely used for in investigation of protein complements between diseased and healthy tissue with the purpose of developing diagnostic markers and detecting novel drug targets (Wilkins et al., 1996; Gygi et al., 2000). Proteomic technologies are providing the tools needed to discover and identify biomarkers associated with diverse diseases and biological processes (Blackstock et al., 1999; Hanash, 2003; Choi et al., 2004; Eun et al., 2004). There have been many studies including analysis of prognostic factors, development of chemotherapeutic agents, and the search for the early detecting molecules, but there are currently very few molecular markers that are clinically in use. Proteomics is one of the technologies that rapidly change our approach to cancer research and can lead to the molecular characterization of cellular events associated with cancer progression, signaling, and developmental stages. 152 cases of gastric cancer and normal tissues were analyzed by two-dimensional electrophoresis, and western blotting. As a result of this study, we suggest that nicotinamide N-methyltransferase will be an efficient tumor marker in gastric cancer diagnosis.

Materials and Methods

Materials

IPG strips of pH 3-10, 4-7 were purchased from

456 Exp. Mol. Med. Vol. 38(5), 455-465, 2006

Amersham Pharmacia Biotech (APS, Immobiline Dry-Strip, 0.5 3 180 mm, Uppsala, Sweden) and Bio-Rad (ReadyStrip; 0.5 3 170 mm, Hercules, CA). Bio-Lyte (pH 3-10) was from Bio-Rad. SDS, acrylamide, methylenebisacrylamide, TEMED, ammonium persulfate, DTT, urea, Tris, glycine, glycerol, and CHAPS were purchased from Bio-Rad or USB (Cleveland, OH). Silver nitrate, coomassie Brilliant Blue G-250, TCA, iodoacetamide, and -cyano-4-hydroxycinnamic acid were from Sigma (St. Louis, MO). Methanol, ethanol, phosphoric acid, acetic acid and formaldehyde were purchased from Merck (Darmstadt, Germany). Sequencing grade-modified trypsin was obtained from Promega (Madison, WI). Other reagents were obtained from Sigma or Merck.

Stomach tissue samples

Human stomach tissue samples were prepared from resection materials of gastric cancer patients in Gyeongsang National University Hospital. Resections were examined by a pathologist and normal tissue samples were prepared from noncancerous regions.

Preparation of stomach tissue protein samples

Frozen stomach tissue samples (100-200 mg) were homogenized in 2 ml homogenization buffer (50 mM Tris-HCl, pH 7.2) containing a protease inhibitor cocktail [1 mM 4-(2-aminoethyl)benzenesulfonyl fluo-ride hydrochloride (AEBSF), 0.8 M aprotinin, 21 M leupeptin, 36 M bestatin, 15 M pepstatin A, 14 M E-64] using an utraturrax homogenizer (type T8, IKA Labortechnik, Staufen, Germany) at 25,000 rpm. The mixture was centrifuged at 1,000 g for 5 min to remove tissue and cell debris. The supernatant was used as total homogenate. The total homogenate was centrifuged in a Beckman TL-100 table top ultra-centrifuge at 100,000 rpm (approx. 430,000 g) in a TLA-100.2 rotor for 10 min at 4oC. The supernatant was taken as soluble fraction and the pellet was used for membrane protein preparation. TCA (50% w/v) was added to the supernatant to a final concentration of 10% w/v and the solution was allowed to stand on ice for 30 min. Protein precipitate was collected by spin in a microcentrifuge at 15,000 rpm for 10 min at 4oC, and washed three times in 10% TCA. The precipitate was washed once in diethyl ether and dried under air stream. The dry pellet was dissolved with sonication in the lysis solution [8 M urea, 4% (w/v) CHAPS, 40 mM Tris, 100 mM DTT, 2% w/v Bio-Lyte pH 3-10] and allowed to stand for 1 h at room temperature. After centrifugation at 15,000 g for 10 min at 15oC, the super-natant was used as the 2-DE sample for the soluble fraction. The protein samples were stored in aliquots

at -70oC until use. For the preparation of the membrane fraction, the ultracentrifugal pellet was washed once in PBS, dissolved in the lysis solution with sonication, centrifuged at 15,000 g for 10 min at 15oC, and the supernatant was used as membrane fraction sample. Protein concentration of 2-DE samples was estimated according to using a commercial Bradford reagent (Bio-Rad). BSA was used as standard.

Two-dimensional electrophoresis

IEF was carried out using commercially available, dedicated apparatuses: IPGphor (Amersham Pharmacia Biotech) or Protean IEF Cell (Bio-Rad). IPG strips were used according to the manufacturers instructions. Samples containing up to 200 g protein for analytical gels or up to 1 mg for micro-preparative gels, were diluted to 300-350 l with

rehydration solution (8 M urea, 2% CHAPS, 100 mM DTT, 0.5% v/v pH 3-10 IPG buffer, trace bromophenol blue), and applied to strips by overnight rehydration at 50 V. Proteins were focused succeedingly for 1 h at 200 V, 1 h at 500 V, 1 h at 1,000 V, then a gradient was applied from 1,000 to 8,000 V in 30 min, and focusing was continued at 8,000 V for 8.5 h to give a total of 70 kVh on an IPGphor. With Protean IEF Cell, focusing was done initially at 250 V for 15 min, then the voltage was increased to 10,000 V within 3 h, and maintained at 10,000 V for 7 h for a total of 70 kVh. All IEF steps were carried out at 20oC. After the first-dimensional IEF, IPG gel strips were placed in an equilibration solution (6 M urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl, pH 8.8) containing 1% DTT for 10 min with shaking at 50 rpm on an orbital shaker. The gels were then transferred to the equilibration solution containing 2.5% iodoacetamide and shaken for a further 10 min before placing them on a 7.5-17.5% gradient polyacrylamide gel slab. Separation in the second dimension was carried out using Protean II xi electrophoresis equipment and Tris-glycine buffer (25 mM Tris, 192 mM glycine) containing 0.1% SDS at a current setting of 5 mA/gel for the initial 1 h and 10 mA/gel thereafter. The second-dimensional SDS-PAGE was developed until the bromophenol blue dye marker had reached the bottom of the gel.

Protein visualization and image analysis

For silver staining, following second-dimensional SDS-PAGE, analytical gels were immersed in methanol/acetic acid/water (50:12:38) for 1.5 h, followed by washing twice in 50% ethanol for 20 min. Gels were pretreated for 1 min in a solution of 0.02% Na2S2O3. This was followed by three 1 min washes in deionized water. Proteins were stained in a

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues457

solution containing 0.2% AgNO3 and 0.075% (v/v) formalin (37% formaldehyde in water) for 20 min, and washed twice in deionized water for 1 min.Subsequently, gels were developed in a solution of 0.06% (v/v) formalin, 2% Na2CO3, and 0.0004%

Na2S2O3. When the desired intensity was attained, the developer was discarded and stopped by 1% acetic acid. For Coomassie blue staining of micro-preparative gels, gels were fixed three times in 30% ethanol containing 2% phosphoric acid for 20 min and rinsed three times in 2% phosphoric acid. Gels were then equilibrated in a solution containing 18% ethanol, 2% phosphoric acid, and 15% ammonium sulfate for 30 min and Coomassie brilliant blueG-250 was added to a final concentration of 1%.Staining was carried out overnight. Protein patterns in the gels were recorded as digitalized images using a high-resolution scanner (GS-710 Calibrated Imaging Densitometer, Bio-Rad). Gel image matching was done with PDQuest software (Bio-Rad).

In-gel digestion

In-gel digestion of protein spots on Coomassie- or silver-stained gels was performed essentially as described by Jensen et al. (1999). After the completion of staining, the gel slab was washed twice with water for 10 min. The spots of interest were excised with a scalpel, cut into pieces, and put into 1.5 ml microtubes. The particles were washed twice with water for 15 min, and then twice with water/acetonitrile 1:1 (v/v) for 15 min. The solvent volumes were about twice the gel volume. Liquid was removed, acetonitrile was added to the gel particles and the mixture was left for 5 min. Liquid was removed and the particles were rehydrated in 0.1 M NH4HCO3 for 5 min. Acetonitrile was added to give a 1:1 v/v mixture of 0.1 M NH4HCO3/acetonitrile and the mixture was incubated for 15 min. All liquid was removed and gel particles were dried in a vacuum centrifuge (Heto- Holten, Allerd, Denmark), reswelled in 10 mM DTT/0.1 M NH4HCO3, and incubated for 45 min at 56oC to reduce the peptides.After chilling tubes to room temperature and removing the liquid, 55 mM iodoacetamide in 0.1 MNH4HCO3 was added, the tubes were incubated for 30 min at room temperature in the dark to S-alkylate the peptides. Iodoacetamide solution was removed, the gel particles were washed with 0.1 M NH4HCO3 and acetonitrile, dried in a vacuum centrifuge, rehydrated on ice in digestion buffer containing 50 mM NH4HCO3, 5 mM CaCl2, and 12.5 ng/l of trypsin, and incubated for 45 min on ice. Excess liquid was removed and about 20 l of digestion buffer without trypsin was added. After overnight digestion at 37oC, 25 mM NH4HCO3 was added, and

the tube was incubated for 15 min. Acetonitrile was added and the tube was incubated for a further 15 min. The supernatant was recovered, and the extraction was repeated twice with 5% formic acid/acetonitrile (1:1, v/v). The three extracts were pooled and dried in a vacuum centrifuge.

MALDI-TOF-MS and data base search

Tryptic peptides were redissolved in a solution containing water, acetonitrile, and trifluoroacetic acid (93:5:2, v/v), and the solution was treated for 5 min in a bath sonicator. Target preparation was carried out by solution-phase nitrocellulose method. Saturated solution of -cyano-4-hydroxycinnamic acid (about 40 mg/ml) and nitrocellulose solution (20 mg/ml) were prepared separately in acetone. A mixture of the -cyano-4-hydroxycinnamic acid solution, nitrocellulose solution, and 2-propanol was prepared at a ratio of 2:1:1. Peptide calibrants (50-200 fmole of each), des-Arg-bradykinin (monoisotopic mass, 904.4681), and neurotensin (1672.9715) were added and the mixture solution was then spotted on the target and dried. Dried samples were

pH 4 pH 7

A

Molecular weight

B

Molecular weight

Figure 1. 2-DE protein pattern of normal and cancer stomach tissues. Stomach tissue proteins (50 g) from the normal (A) and cancer tissues (B) were separated on pH 4-7 IPG strip, in the first dimension and 7.5-17.5% gradient SDS-PAGE gel in the second dimension. Spots were visualized by silver staining. On the right, image crops of the gel region containing the spot 4,262 are shown. The position of the spot indicated by arrows.

458 Exp. Mol. Med. Vol. 38(5), 455-465, 2006

washed with 5 l of 5% formic acid for 10 s, followed by 5 l of Milli-Q water for 10 s, and then dried spots were analyzed in a Voyager-DE STR MALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, NA). The spectrometer was run in positive ion mode and in reflector mode with the settings: accelerating voltage, 20 kV; grid voltage, 76%; guide wire voltage, 0.01%; and a delay of 150 ns. The low mass gate was set at 500 m/z. Proteins were identified by peptide mass fingerprinting with the search programs MS-FIT (http://prospector.ucsf.edu/ ucsfhtml3.4/msfit.htm) and ProFound (http://129.85. 19.192/profound_ bin/WebProFound.exe). The following search parameters were applied: SWISS-PROT and NCBI were used as the protein sequence databases; a mass tolerance of 50 ppm and one incomplete cleavage were allowed; acetylation of the N-terminus, alkylation of cysteine by carbamidomethylation, oxidation of methionine, and pyroGlu formation of N-terminal Gln were considered as possible modifications.

Generation of monoclonal antibodies

His-tagged fusion protein was expressed in E. coli and purified as affinity chromatography method (Ni-NTA agarose, QIAGEN). They were used for immunization of mice and screening of hybridomas secreting Ag-specific monoclonal antibodies, respectively. Each of the 6-week-old female Balb/c mice was given primary injection (intraperitoneal) with 100 g of the protein emulsified in complete Freunds adjuvant (Sigma-Aldrich Inc.). Two weeks later, each mouse was given one secondary booster injection using the same amount of antigen mixed with incomplete Freunds adjuvant (Sigma-Aldrich Inc.). Booster injections were administered to each mouse at 2-week intervals three more times. Ag-specific antibodies in the serum of immunized mice were tested by Western blot assays prior to hybridoma fusion. The spleen was excised, and hybridoma fusion was performed by standard techniques (Harlow, 1988). mAbs in supernatants of hybridoma cultures were screened in ELISA. Ascites fluid was produced by injecting the hybridoma cells into a pristane-primed Balb/c.

Normal Cancer

No.26

No.64

No.80

Figure 2. Differentially expressed NNMT in normal and cancer tissues of gastric cancer patients. The positon of NNMT was indicated by squares.

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues459

Table 1. Expression level of spot 4262 in normal and cancer tissue of gastric cancer tissues.

Sample No. N C C/N

Sample No. N C C/N

1 347 1064 3.06

45 108 882 8.17

2 246 1630 6.62

46 182 522 2.87

3 257 3156 12.28

47 511 1972 3.86

4 96 515 5.36

48 138 706 5.12

5 0 0 0a

49 202 0 b

6 295 829 2.81

50 159 1544 9.71

7 373 1840 4.93

51 866 2686 3.1

8 215 507 2.36

52 0 0 0a

9 0 688 +c

53 58 1655 28.5

10 0 0 0a

54 0 0 0a

11 0 0 0a

55 234 228 0.97

12 0 0 0a

56 235 0 b

13 0 0 0a

57 0 0 0a

14 0 0 0a

58 0 0 0a

15 0 0 0a

59 218 0 b

16 0 0 0a

60 0 0 0a

17 0 0 0a

61 242 2934 12.1

18 138 873 6.33

62 0 0 0a

19 0 0 0a

63 1044 165 0.16

20 134 0 b

64 320 419 1.31

21 0 0 0a

65 0 0 0a

22 0 0 0a

66 94 548 5.83

23 0 0 0a

67 160 2989 18.7

24 0 0 0a

68 108 4298 39.8

25 0 4019 +c

69 265 215 0.81

26 92 922 10

70 102 1098 10.8

27 0 0 0a

71 178 709 3.98

28 0 0 0a

72 474 616 1.3

29 0 0 0a

73 132 1089 8.25

30 0 0 0a

74 359 602 1.68

31 115 1364 11.9

75 0 1181 +c

32 231 3260 14.1

76 178 709 3.98

33 0 0 0a

77 621 1622 2.61

34 773 2063 2.67

78 633 1307 2.06

35 209 268 1.28

79 153 1075 7.03

36 195 506 2.59

80 232 750 3.23

37 17 1160 68.2

81 308 4480 14.5

38 116 1694 14.6

82 438 828 1.89

39 518 1438 2.78

83 436 390 0.89

40 405 999 2.47

84 84 2204 26.2

41 160 660 4.13

85 238 2467 10.4

42 214 2664 12.4

86 198 0 b

43 760 1056 1.39

87 706 1150 1.63

44 606 538 0.89

88 411 363 0.88

460 Exp. Mol. Med. Vol. 38(5), 455-465, 2006

Table 1. Continued.

Sample No. N C C/N

Sample No. N C C/N

89 200 1230 6.15

122 84 401 4.77

90 451 1147 2.54

123 323 1992 6.17

91 402 0 b

124 105 1030 9.81

92 177 735 4.15

125 211 2067 9.8

93 73 2650 36.3

126 400 1859 4.65

94 0 3190 +c

127 918 1899 2.07

95 514 274 0.53

128 187 1138 6.09

96 337 1465 4.35

129 175 2225 12.7

97 0 0 0a

130 598 552 0.92

98 63 1218 19.3

131 272 1666 6.13

99 425 1236 2.91

132 23 1077 46.8

100 0 0 0a

133 446 1492 3.35

101 168 956 5.69

134 253 3332 13.2

102 0 3235 +c

135 403 1591 3.95

103 160 903 5.64

136 482 3196 6.63

104 1092 1424 1.3

137 323 1992 6.17

105 378 1601 4.24

138 718 1684 2.35

106 44 179 2.07

139 272 1942 7.14

107 435 563 1.29

140 551 919 1.67

108 248 1812 7.31

141 105 463 4.41

109 197 419 2.13

142 911 3530 3.87

110 349 2835 8.12

143 232 4045 17.4

111 0 2069 +c

144 391 1567 4.01

112 0 389 +c

145 643 875 1.36

113 511 0 b

146 620 1259 2.03

114 1202 1297 1.08

147 604 392 0.65

115 0 466 +c

148 568 2358 4.15

116 318 3385 10.6

149 670 2149 3.21

117 166 980 5.9

150 683 506 0.74

118 560 485 0.87

151 691 1760 2.55

119 232 1452 6.26

152 1238 1502 1.21

120 374 531 1.42

A.V.E 269 1120 5.7

121 255 1555 6.1

S.D 21 86

aNo spot detection. bExpressed only in normal tissues. cExpressed only in cancer tissues.

Table 2. The results of t-test of spot 4262 expression in gastric

cancer tissues.

Average spot volume Parametric method

Normal Cancer Cancer-Normal P value

Test statistic ( 0.001)

269 1120 851 10.18 7.1310-17

Western blot analysis

The initial sample for immunoblotting was prepared in the same way as for silver staining. After a run on the 7.5-17.5% gradient SDS-PAGE, the separated proteins were transferred to nitrocellulose membranes. Blocking was performed with a TBST buffer (10 mM Tris-HCl, pH 8.8, 150 mM NaCl, 0.5% Tween-20) containing 5% skim milk (DIFCO). The membranes were then incubated with a mouse monoclonal anti-NNMT antibody (dilution to 1:10,000) for overnight at room temperature. The immunoblots

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues461

Figure 3. Search results of peptides mass fingerprinting. The spot was identified as nicotinamide N-methyltransferase by MALDI-TOF MS, with 8 matching peptides which corresponded to a sequence coverage of 26%.

Table 3. Summary of ELM search results. Total 21 modification sites were reported.

Modification site Total 21 Cell compartment

CK1 phosphorylation site 6 Nucleus, cytoplasmCK2 phosphorylation site 2 Nucleus, cytoplasmGlycosaminoglycan attachment site 4 Extracellular golgi appartusGSK phosphorylation recognition site 5 Nucleus, cytoplasmPKA (cell signalling) 1 Cytoplasm, cAMP dependent protein kinase complex PKA phosphorylation 1 CytoplasmMAPK phosphorylation site 1 Nucleus, cytoplasmSUMO-1 recognition motif 1 Nucleus, PML body

were washed for 30 min, changing the TBST buffer every 10 min, then incubated with a alkaline phosphatase conjugated secondary antibody (dilution to 1:50,000) for 4 h at room temperature. The immunoblots were washed every 10 min for 1 h with changing TBST buffer, and developed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP, USB) and p-nitro

blue tetrazolium chloride (NBT, USB) in alkaline phosphate buffer (100 mM Tris-HCl, pH 9.5, 10 mM NaCl, 5 mM MgCl2).

In vitro phosphorylation

Poly His-tag removed NNMT was mixed with a

462 Exp. Mol. Med. Vol. 38(5), 455-465, 2006

kinase reaction buffer comprised of 20 mM Tris HCl (pH 7.5), 120 mM KCl, 10 mM MgCl2, 100 M ATP and CK2 (1 unit transfers 1 mol of phosphate from ATP to casein per min at pH 8.5 and 37oC) in a final volume of 50 l and incubated for overnight at 30oC.

Reactions were easily stopped by treatment with heat, trypsin digestion, or urea. All experiments were repeated at least twice and confirmed by western blotting.

152 cases of gastric cancer and normal tissues were analyzed by two-dimensional electrophoresis. The images of silver stained gels were analyzed with the software PDQustTM (Figure 1, 2). The statistical analysis revealed that the levels of the spot 4,262 in tumor tissues were significantly higher than in normal tissues (Table 1, 2). The spot was identified as nicotinamide N-methyltransferase by MALDI-TOF MS, with 8 matching peptides which corresponded to a sequence coverage of 26% (Figure 3). Nicotinamide N-methyltransferase (NNMT, E.C.2.1.1.1) is an S-adenosyl-L-methionine dependent cytosolic enzyme which catalyses the N-methylation of nicotinamide, its predominant route of metabolism.

Results

Proteome analysis of gastric cancer tissues reveals overexpression of NNMT

M P1 P2 P3 P4 P5 P6

Kd

113

92

52

35

29

21

M 1000 100 10 1 ng

Figure 4. 12% SDS-PAGE of overexpression and purification of NNMT. Cells were induced with 1 mM IPTG and overexpressed protein was purified with Ni-NTA agarose. M, low MW marker; P1, non-induced cells; P2, cells induced with IPTG; P3, cleared lysate; P4, flow-through; P5, wash; P6, eluate.

Figure 5. Sensitivity of monoclonal antibody against NNMT. Poly-His tag removed recombinant NNMT was applied to 12% SDS-PAGE and transferred on PVDF membrane. Monoclonal antibody (LP 16-1) against NNMT from ascites fluid was used as a primary antibody (dilution to 1:10,000).

A

pH 4 pH 7 pH 4 pH 7

B

No.38

No.68

No.81

Patient 1

Patient 2

No.74

No.83

pH 4 pH 7

Normal Cancer

Figure 6. Modification of NNMT in gastric cancer tissues. (A) 100 g of proteins from normal and cancer tissues were analyzed by 2-DE. LP 16-1 monoclonal antibody was used as a primary antibody. (B) 100 g of proteins from gastric ulcer tissues were analyzed by 2-DE. LP 16-1 monoclonal antibody was used a primary antibody (dilution to 1:10,000).

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues463

Generation of anti-NNMT monoclonal antibody

We produced monoclonal antibody against NNMT to perform further study. Balb/c mice were immunized with purified NNMT, and the spleen cells were fused with myeloma cells. The hybridoma cells producing monoclonal antibody against the NNMT were screened by indirect ELISA, and the monoclonal antibody was obtained from cloning and ascites fluid producing process. The antibody reacted specifically with NNMT protein. The detection limit of the prepared monoclonal antibody was down to about 10 ng (Figure 5).

Western blot analysis of NNMT in gastric cancer tissues and gastric ulcer tissues

Gastric tissues from 15 gastric cancer patients were analyzed by Western blot and the results revealed that NNMT was expressed more highly in cancer tissues than in normal tissues (Figure 6). This result

of Western blotting coincided with those of 2-DE experiments. Western blotting revealed that NNMT exists in multiple spots in gastric tissues. In normal gastric tissues, mainly a single spot was predominant in most cases. However, four to five spots were observed in most cancer tissues. We also analyzed gastric tissues from 2 gastric ulcer patients and found only one spot in both cases. The results indicate that the presence of multiple spots of NNMT is highly specific to tumor tissues of stomach. The presence of multiple NNMT spots suggested its post-translarational modification in gastric cancer tissues.

Post-translational modification of NNMT

The pattern of multiple spots with nearly identical MW and distinct pI values suggested that the modification involved a relatively small group with charges, possibly a phosphorylation. By eukaryotic

Figure 7. Summary of features reported by ELM search after globular domain filtering and context filtering.

464 Exp. Mol. Med. Vol. 38(5), 455-465, 2006

pH 4

pH 7

A

B

C

Figure 8. Western blot analysis of NNMT. (A) Recombinant NNMT treated with thrombin. (B) NNMT from gastric ulcer tissues and thrombin-treated recombinant NNMT. (C) NNMT reacted with CK2. n additional spot was detected on the left of the original spot, as indicated by the square.

linear motif (ELM) search (http://elm.eu.org), NNMT amino acid sequence was found to contain several consensus sequences for modification (Figure 7). Interestingly, casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser/Thr protein kinase and phosphorylates more than 100 substrates and is up-regulated in rapidly dividing cells including most human tumors. So the possibility of NNMT phosphorylation by CK2 was examined. Overexpressed and affinity-purified NNMT was reacted with CK2 in vitro and then was analyzed by Western blotting. NNMT treated with CK2 was found in two spots on 2-DE gel, while non-treated control showed a single spot. The additional spot had same molecular weight as the original spot but had a lower pI value (Figure 8). Thus CK2 was capable of phosphorylation of NNMT in vitro.

Discussion

Proteomic technologies have been used to identify cancer-specific proteins that are useful for cancer diagnosis, progression, and therapeutic targets (Wilkins et al., 1996; Loging et al., 2000; Pandey and Mann, 2000). The objective of present study was to identify cancer-associated proteins using a proteomic approach by analyzing protein expression in healthy and cancerous stomach tissues. We found one protein that was expressed at markedly higher levels in cancerous tissue compared with normal stomach tissue. It was identified as nicotinamide N-methyltransferase by peptide mass fingerprinting.

In a small scale proteomics study of human gastric cancer tissues,a higher expression of NNMT was reported in 8 out of 18 cases examined (Jang et al., 2004). However, no statistical analysis of the results was presented and statistical significance of its differential expression between normal and tumor tissues could not be ascertained. We analyzed 152 cases of gastric cancer and normal tissues and statistical analysis showed extremely high significance of the difference (P 0.001). Furthermore, these 2-DE results were verified by Western blotting. Interestingly, Western blotting revealed that NNMT exists in multiple spots in gastric tissues and the presence of multiple NNMT spots is highly specific to tumor tissues of stomach.

Nicotinamide N-methyltransferase is an S-adenosyl-L-methionine dependent cytosolic enzyme which catalyses the N-methylation of nicotinamide, its predominant route of metabolism (Aksoy et al., 1995). Methylation is also an important conjugation reaction in the biotransformation of many drugs and xenobiotics including pyridine and other structurally related compounds (Weinshilboum., 1989). NNMT was first identified by cDNA cloning from the liver and the protein is predicted to be present in the cytosol (Aksoy et al., 1995). Several diseases are associated with abnormal nicotinamide metabolism resulting in the production of elevated levels of N-methylnicotinamide; these include Parkinsons disease and hepatic cirrhosis (Cuomo et al., 1994; Parsons et al., 2002). Recently, NNMT was reported as novel serum markers of human colorectal cancer as potential candidates for noninvasive detection of early colorectal neoplasm (Roessler et al., 2005).

To our knowledge, NNMT modification has not been reported previously in the human gastric cancer. We performed eukaryotic linear motif search for NNMT to find potential modification sites. NNMT amino acid sequence was found to possess the motifs recognized by CK1 and CK2 for Ser/Thr phosphorylation, glycosaminoglycan attachment sites, GSK phosphorylation sites, PKA recognition sites, a MAPK phosphorylation site and a sumoylation site. Among the various possibilities, we showed that recombinant NNMT was phosphorylated by CK2 in vitro.

In the present study, we observed NNMT could be a sensitive and specific tumor marker of gastric tissues. We reported for the first time that NNMT was modified in gastric cancer tissue by Western blot analysis. The mechanism by which NNMT is modified is still unknown and further studies are needed to understand the relationship between gastric cancer and post-translational modification.

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues465

Acknowledgement

This study was supported partially by Clinical Research Fund of Gyeongsang National University Hospital for the year 2004.

References

Aksoy S, Brigitte FB, Aviva W, Peter L, Richard MW. Human nicotinamide N-methyltransferase gene: Molecular cloning, structural characterization and chromosomal localization. GENOMICS 1995;29:555-61

Berndt P, Hobohm U, Langen H. Reliable automatic protein identification from matrix-assisted laser desorption/ionization mass spectrometric peptide fingerprints. Electrophoresis 1999;20:3521-6

Blackstock WP, Weir MP. Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol 1999; 17:121-7

Choi BH, Kim JS. Age-related decline in expression of calnexin. Exp Mol Med 2004;36:499-503

Cuomo R, Dattilo M, Pumpo R, Capuano G, Boselli L, Budillon G. Nicotinamide methylation in patients with cirrhosis. J

Hepatol 1994;20:138-42

Eun JP, Choi HY, Kwak YG. Proteomic analysis of human cerebral cortex in epileptic patients. Exp Mol Med 2004;36:185-91

Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R. Evaluation of two-dimensional gel electrophoresis based proteome analysis technology. Proc Natl Acad Sci USA 2000; 97:9390-5

Ha GH, Lee SU, Kang DK, Ha NY, Kim SH, Kim JN, Bae JM, Kim JW, Lee CW. Proteome analysis of human stomach tissue: Separation of soluble proteins by two-dimensional polyacrylamide gel electrophoresis and identification by mass spectrometry. Electrophoresis 2002;23:2513-24

Hanash S. Disease proteomics. Nature 2003;422:226-32

Harlow E, Lane D. Antibodies: a laboratory manual, 1988, Cold Spring Harbor Laboratory, USA

Humphery-Smith I, Williams KL, Hochstrasser DF. From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Biotechnology (NY) 1996;14:61-5

Jang JS, Cho HY, Lee YJ, Ha WS, Kim HW. The differential proteome profile of stomach cancer: identification of the bio-marker candidates. Oncol Res 2004;14:491-9

Jensen ON, Wilm M, Shevchenko A, Mann M. Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels. Methods Mol Biol 1999;112:513-30

Kim HJ, Chang WK, Kim MK, Lee SS, Choi BY. Dietary factors and gastric cancer in Korea: a case-control study. Int J Cancer 2002;97:531-5

Loging WT, Lal A, Siu IM, Loney TL, Wikstrand CJ, Marra MA,

Prange C, Bigner DD, Strausberg RL, Riggins GJ. Identifying potential tumor markers and antigens by database mining and rapid expression screening. Genome Res 2000;10:1393-402

Lyman GH. Risk factors for cancer. Prim Care 1992;19:465-79 Smith ML, Burnett D, Philip B, Rosemary HW, Helen MB, Adrian CW, David BR. A direct correlation between nicotinamide N-methyltransferase activity and protein levels in human liver cytosol. Biochimica et Biophysica Acta 1998; 238-44

OFarrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975;250:4007-21

Pandey A, Mann M. Proteomics to study genes and genomes. Nature 2000;405:837-46

Parsons RB, Smith ML, Williams AC, Waring RH, Ramsden DB. Expression of nicotinamide N-methyltransferase (E. C. 2.1.1.1) in the Parkinsonian brain. J Neuropathol Exp Neurol 2002;61:111-24

Parsons RB, Smith SW, Waring RH, Williams AC, Ramsden DB. High expression of nicotinamide N-methyltransferase in patients with idiopathic Parkinsons disease. Neurosci Lett 2003;342:13-6

Roessler M, Rollinger W, Palme S, Hagmann ML, Berndt P, Engel AM, Schneidinger B, Pfeffer M, Andres H, Karl J, Bodenmuller H, Ruschoff J, Henkel T, Rohr G, Rossol S, Rosch W, Langen H, Zolg W, Tacke M. Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for color-ectal cancer. Clin Cancer Res 2005;11:6550-7

Ryu JW, Kim HJ, Lee YS, Myong NH, Hwang CH, Lee GS, Yom HC. The Proteomics approach to find biomarkers in gastric cancer. J Korean Med Sci 2003;18:505-9

Saime A, Carol LS, Richard M. Liver nicotinamide N-methyltransferase. J Biol Chem 1994;269:14635-840

Smith ML, Burnett D, Bennett P, Waring RH, Brown HM, Williams AC, Ramsden DB. A direct correlation between nicotinamide N-methyltransferase activity and protein levels in human liver cytosol. Biochim Biophys Acta 1998;1442:238-44

Steel LF, Haab BB, Hanash SM. Methods of comparative proteomic profiling for disease diagnostics. J Chromatogr B Analyt Technol Biomed Life Sci 2005;815:275-84

Tahara E, Semba S, Tahara H. Molecular biological observations in gastric cancer. Semin Oncol 1996;23:307-15

Weinshilboum R. Methyltransferase pharmacogenetics. Pharmacol Ther 1989;43:77-90

Werner M, Becker KF, Keller G, Hofler H. Gastric adenocarcinoma: pathomorphology and molecular pathology. J Cancer Res Clin Oncol 2001;127:207-16

Wilkins MR, Pasquali C, Appel RD, Ou K, Golaz O, Sanchez JC, Yan JX, Gooley AA, Hughes G, Wilkins MR, Sanchez JC, Gooley AA, Appel RD, Humphery-Smith I, Hochstrasser DF, Williams KL. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev 1996;13:19-50