The Response of Human Colonocytes to Folate Deficiency in Vitro: Functional and Proteomic Analyses Susan J. Duthie,*,† Yiannis Mavrommatis,† Gary Rucklidge,† Martin Reid,† Gary Duncan,† Mary P. Moyer,‡ Lynn P. Pirie,† and Charles S. Bestwick† Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, United Kingdom, and INCELL Corporation, San Antonio, Texas 78249 Received November 15, 2007
Low folate intake is associated with colon cancer. We combined a proteomics and biochemical approach to identify proteins and pathways affected by folate deficiency in human colonocytes. Folate differentially altered activity and expression of proteins involved in proliferation [e.g., PCNA], DNA repair [e.g., XRCC5, MSH2], apoptosis [e.g., BAG family chaperone protein, DIABLO and porin], cytoskeletal organization [e.g., actin, ezrin, elfin], and expression of proteins implicated in malignant transformation [COMT, Nit2]. Keywords: folic acid deficiency • human colonocytes • NCM460 • genomic stability • DNA repair • apoptosis • proteomics
Introduction Folate, one of the water-soluble B vitamins, is abundant in fruits and vegetables, particularly green leafy vegetables, and can lower cancer risk at several sites.1–3 Folate deficiency is widespread, affecting a substantial percentage of the population, notably adolescents, the institutionalized elderly and people in low-socioeconomic groups.4 The evidence linking low folate and malignant transformation is strongest for colon cancer. Prospective and case-control studies consistently describe an inverse relationship between colon adenoma and/or tumor incidence and folate status, measured as dietary intake or as blood plasma and erythrocyte vitamin concentrations.5,6 Folate deficiency perturbs biochemical and functional pathways supplied by 1-carbon metabolism with downstream consequences for genomic stability, malignant transformation and cancer risk. Folate is essential for the synthesis both of the pyrimidine thymidine and the purines adenosine and guanosine. Deoxyuridine monophosphate (dUMP) is converted to thymidine monophosphate (TMP) by thymidylate synthase using the folate coenzyme 5,10-methylenetetrahydrofolate as methyl donor, while 10-formylytetrahydrofolate is involved in the formation of both adenosine and guanosine. Continual production of these DNA precursors is essential for DNA synthesis, proliferation and repair. However, if folate is limiting, and the balance of these DNA precursors is altered, uracil is misincorporated into DNA in place of thymidine, resulting in mutagenesis, DNA strand breakage and chromosomal damage together with inhibition of normal DNA synthesis and repair.6–10 * To whom correspondence should be addressed. Dr Susan J. Duthie, Nutrition and Epigenetics Group, Division of Vascular Health, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, U.K. Tel., +44 1224 712751, Ext. 2324; fax, +44 1224 716629; e-mail,
[email protected]. † Rowett Research Institute. ‡ INCELL Corporation.
3254 Journal of Proteome Research 2008, 7, 3254–3266 Published on Web 07/03/2008
Perturbations in these systems increases the probability of DNA damage persisting in the cell and the risk of mutagenesis and carcinogenesis. It remains largely untested how folate depletion alters genes and proteins involved in genomic stability and malignant transformation. In this study, we used functional biomarkers combined with proteomics to investigate the mechanisms underlying DNA instability induced by folate deficiency in a human colon epithelial cell line. The influence of folate depletion on functional biomarkers of genomic stability such as DNA damage, DNA repair, cellular proliferation and apoptosis was determined. To better understand the molecular mechanisms underlying folate deficiency, a global proteomics approach was employed to identify molecular protein targets and metabolic processes significantly altered by folate status. These data provide information on the mechanisms through which folic acid maintains genomic stability in the colon in vitro.
Experimental Procedures Materials. Complete A52 medium (4 mg/L folic acid) and A52 medium without folic acid ( 8. *P < 0.001, where P values refer to differences between cells grown in the presence or absence of folic acid.
Figure 2. The effect of folate depletion on strand break DNA repair capacity. NCM460 cells were grown in the presence or absence of folic acid (4 mg/L) for 7 (A) or 14 days (B). DNA strand breakage was measured immediately (0 h), or 4, 8, 16, and 24 h after exposure to 10 µM hydrogen peroxide. Results are mean ( SEM for n ) 8. *P < 0.05, where P values refer to differences in repair between cells grown in the presence (squares) or absence (triangles) of folic acid.
population doublings in culture. The NCM460 cell line has been selected as a continuous cell line and is thus immortalized. These cells, which retain normal mucosal cell characteristics including expression of villin, cytokeratins and other colon epithelial cell antigens,11 have been used previously to examine how folate and other B vitamin are absorbed and metabolized in the normal human colon12,13 and how chemoprotective phytochemicals in the diet act differentially against normal and cancerous colon cells in vitro.14 However, the cell line appears to have partially progressed toward a colon cancer phenotype as it has a mutant p53 gene that likely contributed to the in vitro selection (www.incell.com). NCM460 cells were maintained in complete A52 medium supplemented with L-glutamine (2 mM), retinoic acid (100 nM), dexamethasone (1 nM), vitamin C (38 µg/mL), folic acid (4 mg/L) and bovine pituitary extract (120 µg/mL). Cells were incubated at 37 °C in an atmosphere of 95% air/5% CO2.
Figure 4. Effect of folic acid deficiency on colon cell proliferation. NCM460 cells were grown in the presence (triangles) or absence (squares) of folic acid (4 mg/L) for up to 14 days. Results are mean ( SEM for n > 8. *P < 0.005 or **P < 0.001, where P values refer to differences between cells grown in the presence or absence of folic acid.
Culture medium was changed every 3-4 days and the stocks were passaged into 75 cm2 flasks at a ratio of 1:3. For the folate deficiency studies, NCM460 cells were subcultured using trypsin/EDTA solution (0.25% trypsin in 0.02% EDTA). The cells were washed three times in PBS, plated at a density of 1 × 105 cells/25 cm2 and allowed to grow either in folatedeficient (F-) or folate-sufficient media (F+) for up to 14 days. Proteomic Analyses. Cells, grown for 14 days in either folatedepleted or folate-supplemented medium, were washed twice before scraping into 3 mL of Tris/sucrose (10 mM/250 mM) buffer, pH 7.4, and centrifuged at 13 000 rpm. The pellet was weighed and extraction buffer containing 7 M urea, 2 M Journal of Proteome Research • Vol. 7, No. 8, 2008 3255
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Figure 5. Effect of folic acid deficiency on caspase activity (A) and mitochondrial inner membrane potential (B). NCM460 cells were grown in the presence or absence of folic acid (4 mg/L) for 14 days. Results are mean ( SEM for n > 8. *P < 0.005 or **P < 0.001, where P values refer to differences between cells grown in the presence or absence of folic acid.
thiourea, 4% CHAPS and 2.0% Bio-Rad Biolite Ampholyte pH 3-10 added at a ratio of 3 µL buffer/mg of pellet. The sample was homogenized with an Eppendorf homogenizer on ice for 30 s and sonicated for 5 s.The homogenate was centrifuged at 16 000g for 5 min at 4 °C and the supernatant frozen at -80 °C. Protein concentration was determined using the Bio-Rad RC DC protein assay. A single 2-D gel was run per experimental flask (n ) 6 per group). Proteins were separated by isoelectric focusing in the first dimension [Bio-Rad immobilized pH gradient (IPG) strips (pI range 3-10)] and SDS-PAGE in the second dimension on 8-16% acrylamide gels (18 × 18 cm), as described previously.15 Gels were stained with Coomassie blue and imaged on a BioRad GS710 flat bed imager followed by image analysis using PD Quest Version 8.0.1. Quantitative analyses were performed using the Student’s t test between folate-deficient and control gels at a 95% confidence level. Spots with densities that significantly differed between treatments (p < 0.05) were excised from the SDS-PAGE gels using a robotic Bio-Rad spot cutter, trypsinized in a MassPrep station (Waters, MicroMass, Manchester, U.K.) and analyzed by LC-MS/MS using an Ultimate′ nano LC capillary chromatography system (LC Packings, Camberly, Surrey, U.K.) combined with an Applied Biosystems 4000 Q-Trap (Warrington, U.K.). Peptide fragment mass spectra yielded the sequence of separated peptides that were pasted into the fingerprinting Web resource program “Mascot” (Matrix Science Ltd., Boston, MA) for protein identification. Functional Biomarkers of NCM460 Cell Folate Status, Genomic Stability, and Apoptosis. After 7 and 14 days in culture, washed NCM460 monolayers were scraped into PBS and centrifuged at 2500 rpm for 5 min, the supernatant was removed, and the cell pellet snap frozen was in liquid nitrogen until folate determination by radioassay as described previously.16 3256
Journal of Proteome Research • Vol. 7, No. 8, 2008
Duthie et al. Cell growth over 14 days was determined in trypsin and versene recovered cells using a Neubauer Improved Hemocytometer.10 Endogenous DNA strand breakage and misincorporated uracil were measured in cells at Day 0, 7, 10 and 14 by alkaline single cell gel electrophoresis as described previously.10 Single strand break repair of oxidative DNA damage was determined in NCM460 cells grown in folate-replete or folatedeficient media for 7 and 14 days. Cells were recovered from the culture flask and exposed to H2O2 (10 µM) on ice for 5 min in microcentrifuge tubes and either immediately sampled or incubated in complete culture medium (F+ or F- as appropriate) at 37 °C in 95% air/5% CO2 for up to 24 h as described previously.10 Changes to mitochondrial membrane potential (∆Ψm) and polycaspase activity (caspases 1, 3, 4-9).were analyzed by flow cytometry (4 color FACS Calibur, Becton Dickinson, Oxford, U.K.) according to the manufacturer’s instructions using commercial assays (ApoAlert Mitochondrial Membrane Sensor and Vybrant FAM Polycaspase Assay kit, respectively, from Molecular probes, Invitrogen Detection Technologies, U.K.). Statistics and Pathway Modeling. For analyses of functional biomarkers, Student’s t test was carried out using Excel. For proteomic analysis, gel images were examined using PDQuest software (Bio-Rad). To compensate for nonexpression-related variations in spot intensity (due to experimental or sample inconsistencies), the gels were normalized by compensating for the total gel density. With this method, the raw quantity of each spot in a gel is divided by the total intensity of all the pixels in the image. After normalization and matching, spot densities were exported for statistical analysis using SIMCA-P (Umetrics UK Ltd., Winkfield, U.K.) and Exel. Spot densities were analyzed by Student’s t test at 5% significance to determine treatment effects. Data from all matched spots were analyzed by principal component analysis (PCA) and presented as 2D plots of the first and second component scores for individual cell treatments. Pathway analysis, data visualization and enrichment analysis were carried out using MetaCore software, a curated database of human protein-protein and protein-DNA interactions, transcription factors, signaling and metabolic pathways (www.genego.com, GeneGO, CA).
Results Functional Biomarkers of Folate Status and Impaired DNA Stability. Intracellular total folate decreased progressively in NCM460 cells cultured for up to 14 days in folate-deficient medium compared with cells grown in folate replete medium (Figure 1). Both folate deficient and control cells responded similarly to hydrogen peroxide-induced oxidative DNA damage after 14 days. However, NCM460 cells cultured for 7 and 14 days in folate-deficient medium were unable to effect complete repair of hydrogen peroxide-induced oxidative DNA strand breakage after 8, 16, or 24 h (Figure 2). Folate deficiency was associated with an approximately 5-fold elevated uracil misincorporation (Figure 3A), increased endogenous DNA strand breakage (Figure 3B) and impaired cell proliferation (Figure 4). Polycaspase activity (caspases 1,3,4-9) was increased by folate deficiency (Figure 5A) as was the proportion of cells with a decreased mitochondrial membrane potential, determined by flow cytometric analysis of JC-1 monomer and aggregate fluorescence (Figure 5B).
research articles
Response of Human Colonocytes to Folate Deficiency in Vitro
Table 1. Human Colonocyte Proteins Significantly Downregulated as a Consequence of Folate Deficiencya SSP
control
se
folate deficient
1819
221.5
43.9
100.8
5004 24
688.7 154.3
29.3 21.1
372 71.4
1
794.4
60.3
612
1706
434.4
46.0
236.7
6303 1102
290.2 1413.7
28.2 52.0
180.4 820.2
6004 1310 207 5902 5203 1004
377 215 852.2 703.5 391.5 2320.1
21.6 38.3 78.8 97.9 17.6 66.9
176.7 97.3 367.2 337.3 241.7 1677
6316
232
32.6
115.2
5202
1284
41.9
922.6
7601
189.7
28.9
106.7
6304
466.4
37.9
267.5
5510
430.5
36.8
299.5
1007 20 5007
390.6 199.1 866.6
43.3 12.2 63.7
161.5 92.1 358.1
7002
5240.1
596.1
7005
1709.9
65.1
1424.3
7408
315.1
21.3
203.5
4003
206.2
15.7
109
6201 6306 4405
1056.6 528.5 1896.1
117.3 76.2 204.2
567.4 215.9 1233.4
1204 5002 6410 4507 7314 3607
241.1 554.4 508.6 431.4 172.6 286.8
37.6 78.9 106.1 24.5 34.1 41.7
109.2 249.1 101.4 256.7 13.7 111.7
109
291.2
56.7
109.6
1812
1077.2
98.2
724.2
208 8601
1179.4 802
302.4 129.0
4108
319.9
15.8
16 2009
2121.5 486.6
101.7 73.0
5507
1341.5
204.6
3604
se
decrease (%)
protein
accession
Transcription, Protein Synthesis, and Catabolism 17.0 54.5 26S proteasome Q13200 non-ATPase regulatory subunit 2 90.7 46.0 40S ribosomal protein S12 P25398 17.2 53.7 60S acidic ribosomal protein P05387 P2 43.4 23.0 60S acidic ribosomal protein P05387 P2 18.5 45.5 Acylamino-acid-releasing P13798 enzyme 32.1 37.8 Aspartate aminotransferase P17174 119.7 42.0 Chloride intracellular O00299 channel protein 1 48.2 53.1 Cystatin B P04080 22.2 54.7 EEF1D protein Q9BW34 136.8 56.9 Elongation factor 1-b P24534 119.4 52.1 Elongation factor 2 P16369 44.6 38.3 Elongation factor Ts P43897 120.7 27.7 Eukaryotic translation P63241 initiation factor 5A 34.7 50.3 Fumarylacetoacetate Q96GK7 hydrolase domain-containing protein 2A 132.7 28.1 Heterogeneous nuclear P31942 ribonucleoprotein H3 16.6 43.8 Heterogeneous nuclear O60506 ribonucleoprotein Q 48.1 42.6 3-Hydroxyisobutyryl-coenzyme Q6NVY1 A hydrolase 42.7 30.4 Methylcrotonoyl-CoA Q9HCC0 carboxylase b chain 69.5 58.7 Myotrophin P58546 37.5 53.7 Nuclear transport factor 2 P61970 122.9 58.7 Peptidyl-prolyl cis-trans P62937 isomerase A 432.5 31.2 Peptidyl-prolyl cis-trans P62937 isomerase A 106.5 16.7 Phosphatidylethanolamine-bindingP30086 protein 1 43.3 35.4 Phosphoserine Q9Y617 aminotransferase 25.6 47.1 Low molecular weight P24666 phosphotyrosine protein phosphatase 97.3 46.3 Poly(rC)-binding protein 1 Q15365 58.3 59.1 Poly(rC)-binding protein 1 Q15365 153.1 35.0 ATP-dependent RNA P38919 helicase DDX48 29.0 54.7 Proteasome subunit a type 3 P25788 47.6 55.1 Proteasome subunit b type 2 P49721 83.0 80.1 Protein FAM50A Q14320 57.3 40.5 Ribonucleoprotein Q15367 7.4 92.1 RNA binding protein Raly Q9UKM9 48.8 61.1 Serine/threonine-protein Q13177 kinase PAK 2 25.2 62.4 Telomerase-binding protein Q15185 p23 60.2 32.8 Transitional endoplasmic P55072 reticulum ATPase
Cell Signaling, Cell Cycle, DNA Synthesis and Repair, Apoptosis 341.6 261.0 71.0 14-3-3 protein e 488.3 75.6 39.1 ATP-dependent DNA helicase Q1 239.1 20.6 25.3 BAG-family molecular chaperone regulator-2 1215.8 105.5 42.7 Calmodulin 105.5 41.2 78.3 Cellular retinoic acid binding protein 2 549.5 86.4 59.0 Glucose-6-phosphate 1-dehydrogenase
gene name
PSMD2
RPS12 RPLP2 RPLP2 APEH GOT1 CLIC1 CSTB EEF1D EEF1B2 EEF2 TSFM EIF5A FAHD2A
HNRPH3 SYNCRIP HIBCH MCCC2 MTPN NUTF2 PPIA PPIA PEBP1 PSAT1 ACP1
PCBP1 PCBP1 EIF4A3 PSMA3 PSMB2 FAM50A not available RALY PAK2 TEBP VCP
P62259 P46063
Ywhae RECQL
O95816
BAG2
P62158 P29373
CALM1/2/3 CRAPB2
P11413
G6PD
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Duthie et al.
Table 1. Continued SSP
control
se
folate deficient
se
decrease (%)
5508
740.8
73.8
501.9
72.0
32.2
2719 309
1488.4 789
156.5 256.3
612.9 114.5
145.8 113.2
58.8 85.5
5003
467.5
33.7
266.1
67.0
43.1
8010 1823 5410
754.4 959.3 480.2
173.5 140.4 44.1
258.2 425.2 288.4
63.5 91.1 72.4
65.8 55.7 39.9
2211 313
227.6 413.8
60.5 64.4
1.5 136.4
0.3 72.3
99.3 67.0
4001 4402
692.5 598.4
48.4 33.4
329.4 343.7
65.0 50.8
52.4 42.6
1108
548.2
21.4
183.9
38.5
66.5
404 6501 1203
279.9 684.5 199.4
24.8 44.0 10.6
135 409.5 99.9
23.5 94.6 28.2
51.8 40.2 49.9
7409 5414 5204 5411 6611 5405 6404 2204
338.6 186.9 506.1 224.3 927.1 1447.7 7016.9 2038.9
74.6 25.9 34.5 34.7 108.5 144.1 835.8 64.0
95.8 53 262.6 1.5 386 547.9 3117.8 1270.8
62.4 15.4 59.9 0.3 128.0 125.7 566.4 94.7
5207 5306 6101 6515
772.2 339 1402.2 318.7
48.9 30.2 54.1 81.8
483.7 177.5 954 1.5
5503 7525
2720.7 96.5
157.9 23.1
1670.3 1.5
3408 5408 5107 6103 6508
395.9 215.8 872.8 3892.8 574.2
40.7 39.8 47.1 89.7 64.8
191.6 66.4 455.4 2071.7 176.4
7316
722.8
126.3
1107
357.7
47.2
2102 4203
5522.1 465.3
688.1 60.0
4106 2005
2706.5 902.7
133.2 29.9
1002
5457.4
212.4
7312
1179.7
76.6
2305
227.5
20.6
2110
356.5
79.4
6601 3307 3411 22 27
1338.2 235.7 358.7 628.4 513.8
81.0 22.2 20.2 110.4 75.2
5205
1139.8
87.9
3258
protein
Glucose-6-phosphate 1-dehydrogenase Heat shock 70 kDa protein Hepatoma-derived growth factor Histidine triad nucleotide-binding protein 1 Histone H2B Caprin-1 Mitotic checkpoint protein BUB3 Prohibitin Proliferating cell nuclear antigen Protein DJ-1 Rab GDP dissociation inhibitor b Ran-specific GTPase-activating protein Reticulocalbin-1 Tyrosyl-tRNA synthetase Tumor protein D54
Intermediate Metabolism 71.7 Acetyl-CoA acetyltransferase 71.6 Adipophilin 48.1 Alcohol dehydrogenase 99.3 Alcohol dehydrogenase 58.4 Aldehyde dehydrogenase 62.2 Alpha-enolase 55.6 Alpha-enolase 37.7 L-lactate dehydrogenase B chain 90.7 37.4 Malate dehydrogenase 35.4 47.6 Malate dehydrogenase 164.5 32.0 Phosphoglycerate mutase 1 0.3 99.5 Pyruvate dehydrogenase E1 component 221.3 38.6 Retinal dehydrogenase 1 0.3 98.4 Succinate semialdehyde dehydrogenase 28.8 51.6 TALDO1 protein 19.2 69.2 TALDO1 protein 52.9 47.8 Triosephosphate isomerase 302.5 46.8 Triosephosphate isomerase 43.6 69.3 UDP-glucose 6-dehydrogenase
Xenobiotic Metabolism, Oxidative Stress, and Redox Regulation 70.8 25.5 90.2 Aldo-keto reductase family 1 member B10 1.5 0.3 99.6 Catechol O-methyltransferase 3642.6 357.9 34.0 Glutathione S-transferase Pi 183.3 52.8 60.6 Glutathione S-transferase omega-1 1850.4 162.2 31.6 Peroxiredoxin 6 552.7 91.2 38.8 Superoxide dismutase [Cu-Zn] 3711.6 448.7 32.0 Thioredoxin Structural Proteins, Cytoskeleton, and Cell Motility 795.5 130.5 32.6 Actin-related protein 2/3 complex subunit 2 134.9 25.4 40.7 F-actin capping protein a-2 subunit 1.5 0.3 99.6 F-actin capping protein b subunit 874.6 129.5 34.6 Lamin C 108 25.1 54.2 Macrophage capping protein 198.7 34.5 44.6 Macrophage capping protein 82.7 53.4 86.8 Myosin light polypeptide 6 203 47.1 60.5 Myosin regulatory light chain 2 459.3 95.6 59.7 PDZ and LIM domain protein 1(elfin)
Journal of Proteome Research • Vol. 7, No. 8, 2008
accession
gene name
P11413
G6PD
P08107 P51858
HSPA1A HDGF
P49773
HINT1
P57053 Q14444 O43684
H2BFS CAPRIN1 BUB3
P35232 P12004
PHB PCNA
Q99497 P50395
PARK7 GDI2
P43487
RANBP1
Q15293 P54577 O43399
RCN1 YARS TPD52L2
P24752 Q99541 P14550 P14550 P49419 P06733 P06733 P07195
ACAT1 ADFP AKR1A1 AKR1A1 ALDH7A1 ENO1 ENO1 LDHB
P40925 P40925 P18669 P08559
MDH1 MDH1 PGAM1 PDHA1
P00352 P51649
ALDH1A1 ALDH5A1
Q8WV32 Q8WV32 P60174 P60174 O60701
TALDO1 TALDO1 TPI1 TPI1 UGDH
O60218
AKR1B10
P21964
COMT
P09211 P78417
GSTP1 GSTO1
P30041 P00441
PRDX6 SOD1
P10599
TXN
O15144
ARPC2
P47755
CAPZA2
P47756
CAPZB
P02545 P40121 P40121 P60660 P19105
LMNA CAPG CAPG MYL6 MLCB
O00151
PDLIM1
research articles
Response of Human Colonocytes to Folate Deficiency in Vitro Table 1. Continued se
decrease (%)
196.9 904
36.3 151.4
46.4 47.3
169.3
2628.1
148.0
16.1
558.5
28.4
346.3
74.5
38.0
5610 5407
505.2 300.8
65.3 21.5
298.8 126
49.2 24.6
40.9 58
5608
1844.6
183.6
879.2
199.6
52.3
5307 4508
337.7 391
36.5 121.6
165.6 53.6
23.7 52.1
51.0 86.3
3601 6204
335.5 133.2
20.4 14.3
254.7 1.5
27.4 0.3
24.1 98.9
SSP
control
se
8009 104
367.3 1714.5
30.8 142.5
201
3133.4
5005
folate deficient
protein
gene name
accession
Profilin Tropomyosin a 3 chain Miscellaneous Complement component 1, Q subcomponent binding protein Cytochrome c oxidase polypeptide Vb FARSLB protein Hypothetical protein DKFZp564H1122 Hypothetical protein FLJ12506 Inorganic pyrophosphatase 2 Lamin C, ErbB3 binding protein EBP1, S-adenosylhomocysteine hydrolase chain A Leukotriene A-4 hydrolase Nitrilase family member 2
P07737 P06753
PFN1 TPM3
Q07021
C1QBP
P10606
COX5B
Q9NSD9 Q9H9W4
FARSLB
Q9H9W4 Q9H2U2 PPA2 545/Q9UQ80/P2:/INA/PA2G4/AHCY
P09960 Q8WUF0
LTA4H NIT2
a Spot number, mean spot density ( SEM for control gels (n ) 6) and folate-deficient gels (n ) 6). Change in abundance of protein as a consequence of folate deficiency is shown as percentage decrease in the normalized volume of the given protein spot compared with control. Proteins undetectable in folate-deficient gels or proteins downregulated more than 90% are shown as bold values. Accession number, gene, and protein name are shown for all identified proteins.
Proteomics. Comparative proteomics revealed approximately 800 spots in the total protein fraction that matched across all 12 gels. The 137 spots statistically significantly different between treatments were cut and identified. Of these, 103 spots were significantly downregulated (75%) in the deficient group (Table 1) and 34 spots (25%) were significantly upregulated (Table 2). Measures of confidence for protein identifications are presented in Tables 3 and 4. Duplicate identifications were made by LC/MS/MS for 13 proteins pairs that differed only by MW. Reanalyses of all 20 gels indicated that these were discrete spots. Eight identified proteins were either unique to all the gels from the control group or were absent from one single control gel [RNA binding protein Raly, prohibitin, alcohol dehydrogenase, pyruvate dehydrogenase E1 component, succinate semialdehyde dehydrogenase, aldo-keto reductase family 1 member B10, catechol O-methyltransferase and nitrilase family member 2]. It was not possible to make a definitive identification between 3 proteins for spot no. 4508 (Lamin C, ErB3 binding protein EBP1, adenosylhomocysteine hydrolase chain A). Principal component analysis (PCA) on spot density data derived from all spots revealed that the proteome of replicate gels separated according to folate treatment (Figure 6). 37% of variation in the data was explained by the first two components. A total of 65 of the identified proteins that differed significantly by folate treatment (P < 0.05 by Student’s t test) were in the first 100 top loadings by PCA analysis of all 780 spots. Proteins that differed by folate treatment were broadly categorized according to potential biochemical function (Tables 1 and 2). Major metabolic pathways related to protein biosynthesis, energy metabolism and on markers of cell proliferation [e.g., PCNA (downregulated 67%)], DNA repair [e.g., XRCC5 (upregulated 1.5-fold), MSH2 (upregulated 1.5-fold), ATPdependent DNA helicase Q1 (downregulated 39%)], and apoptosis [e.g., BAG family chaperone protein (downregulated 25%), DIABLO homologue (upregulated 2-fold) and voltagedependent anion channel protein 1 (porin; upregulated 1.5fold)] were affected by folate treatment.
Pathway analysis indicated those cellular processes (defined and annotated by GeneGo) most notably associated with proteins significantly affected by folate status included actin filament and cytoskeleton regulation, translation, and protein metabolism/catabolism and muscle metabolism (Table 5). Moreover, neoplastic metastasis, neoplastic processes, neoplasm by site and carcinoma were processes significantly affected by folate status when proteins were distributed by disease (Table 5). Biological networks, constructed using only the protein identities derived in this experiment indicated that cell motility, apoptosis and regulation of mitosis were significantly associated with folate treatment (Table 6).
Discussion Folate deficiency inhibited DNA synthesis and cell proliferation, increased DNA damage (seen as an elevation in both uracil misincorporation and DNA strand breakage), decreased DNA strand break repair capacity, and increased early markers of apoptosis (caspase activation and altered mitochondrial membrane potential) in NCM460 cells. Deregulated DNA repair occurred prior to DNA damage and apoptosis. Folate depletion induced changes in the total colonocyte proteome associated with alteration in the cell cycle and growth arrest, DNA repair and apoptosis. Several novel proteins implicated in malignant transformation were affected by folate status. This discussion focuses on proteins with potential implications for genomic stability and carcinogenesis. Genomic Stability (DNA Damage, DNA Repair, and Apoptosis). Folate deficiency significantly alters the cellular balance of DNA precursors, resulting in changed dinucleotide ratios and uracil misincorporation17,18 that increases DNA double strand break frequency and chromosomal damage.7,18,19 Folate-induced imbalances in DNA nucleotide precursor pools leads to error-prone DNA synthesis, cell replication and increased DNA mismatch repair (MMR) activity.17 Expression of MSH2, involved in MMR and ATP-dependent DNA helicase II (also known as XRCC5 or Ku80) involved in Journal of Proteome Research • Vol. 7, No. 8, 2008 3259
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Duthie et al. a
Table 2. Human Colonocyte Proteins Significantly Upregulated as a Consequence of Folate Deficiency SSP
control
se
folate deficient
se
fold increase
protein name
1512 1982.5 176.4 7104 117.9 12.8 802 8533.9 505.2 4302 293.1 30.2 803 297.4 68.6 3806 372.6 20.4 2912 122 18.1
3555.3 341.3 12734.8 430.9 686.2 499.3 259.5
429.2 31.8 1105.8 41.0 63.0 50.0 46.0
8102 229.6 56.2 1005 100.1 24.7 2003 4911.9 529.2 7004 3478.3 101.3 1006 126.9 27.9 3001 3147.4 341.3 2105 701.6 222.6 10 127.4 15.1 2708 501.4 26.5 1503 290.6 43.9 2808 125.3 6.5 2601 334.2 77.3 8105 599.8 124.4 8107 845.4 84.4
713.7 318.6 7330.8 4294 389.6 4622.2 2229.8 260.2 728.6 810.1 186.9 579.9 1213.3 1268.8
Cell Signaling, Cell Cycle, DNA Synthesis and Repair, Apoptosis 156.2 3.1 Annexin A1 36.6 3.2 S100 Calcium-binding protein A4 (metastatin) 501.3 1.5 S100 Calcium-binding protein A4 (metastatin) 139.3 1.2 S100 Calcium-binding protein A10 (calpactin) 83.2 3.1 S100 Calcium-binding protein A11 (calgizzarin) 529.3 1.5 S100 Calcium-binding protein A11 (calgizzarin) 161.9 3.2 Cathepsin D 36.5 2.0 Diablo homologue 57.2 1.5 ATP-dependent DNA helicase II, 80 kDa subunit 241.7 2.8 60 kDa Heat shock protein 24.4 1.5 Msh2 69.3 1.7 Lamin B2 138.7 2.0 Voltage-dependent anion-selective channel protein 1 (porin) 43.9 1.5 Voltage-dependent anion-selective channel protein 1 (porin)
4201 739.2 82.3 7701 300.5 36.5 7412 210 19.0 8302 1409.5 135.0 4904 107.7 15.1 6301 482.3 84.2 2402 1318.2 72.8
1100.2 461.1 345.9 2037.9 198.1 955.7 1775.2
113.7 60.2 56.0 231.8 26.4 139.4 159.7
1.5 1.5 1.6 1.4 1.8 2.0 1.3
2301 2100.7 359.8 3715 214.1 8.9 8106 580.5 32.6
3954.5 319.4 1204.2
597.7 33.5 97.3
Structural 1.9 1.5 2.1
7007 1620.7 101.3 7003 297.5 18.5 2807 289 42.0
2339 576.2 595.5
159.6 37.4 113.6
1.4 1.9 2.1
accession gene name
Transcription, Protein Synthesis, and Catabolism 1.8 FK506-binding protein 4 Q02790 2.9 Fumarylacetoacetate hydrolase domain containing protein 1 Q6P587 1.5 90 kDa Heat shock protein (endoplasmin) P14625 1.5 Heterogeneous nuclear ribonucleoprotein H P31943 2.3 Heterogeneous nuclear ribonucleoprotein U-like protein Q1KMD3 1.3 Neutral a-glucosidase AB Q14697 2.1 Transcription intermediary factor 1-b Q13263
FKBP4 FAHD1 HSP90B1 HNRPH1 HNRPUL2 GANAB TRIM28
P04083 P26447 P26447 P60903 P31949 P31949 P07339 Q9NR28 P13010 P10809 P43246 Q03252 P21796 P21796
ANXA1 S100A4 S100A4 S100A10 S100A11 S100A11 CTSD DIABLO XRCC5 HSPD1 MSH2 LMNB2 VDAC1 VDAC1
Intermediate Metabolism isomerase Aconitate hydratase Fumarate hydratase 3-Ketoacyl-CoA thiolase Oxoglutarate dehydrogenase (Lipoamide) Pyruvate dehydrogenase E1 Ubiquinol-cytochrome-c reductase complex core protein I
Q13011 Q99798 P07954 P42765 Q9UDX0 P08559 P31930
ECH1 ACO2 FH ACAA2 OGDH PDHA1 UQCRC1
Proteins, Cytoskeleton, and Cell Motility a-Actin Ezrin Galectin-3
P68133 P15311 P17931
ACTA1 VIL2 LGALS3
Miscellaneous Hemoglobin a chain mutant Hemoglobin e subunit 130 kDa leucine-rich protein
P69905 P02100 P42704
HBA1 HBE1 LRPPRC
D-3,5-D2,4-dienoyl-CoA
a Spot number, mean spot density ( SEM for control gels (n ) 6) and folate-deficient gels (n ) 6). Change in abundance of protein as a consequence of folate deficiency is shown as fold increase in the normalized volume of the given protein spot compared with control. Proteins upregulated more than 2-fold are shown as bold values. Accession number, gene, and protein name are shown for all identified proteins.
double strand break (DSB) repair, was increased in folatedeficient cells. MMR eliminates base-base mismatches and insertion/deletion loops before they become fixed postreplication. MSH2 is a lesion recognition protein in this system. DSB repair reduces chromosomal aberrations. XRCC5 acts to hold the broken ends of the DNA molecule together prior to rejoining. Downregulation of both these enzymes is associated with increased colon cancer risk. Mutations in MSH2 are associated with hereditary colon cancer (HNPCC) and sporadic colorectal tumors.20 XRCC5 is downregulated in colon adenomas.21 Upregulation of DNA repair protein expression in response to folate deficiency in this study reflects increased DNA damage. Despite significant upregulation of MMR and DSB DNA repair enzyme protein expression, folate deficiency functionally decreased DNA repair capacity (Table 2, Figure 2) in response to oxidative stress, suggesting that abnormal dinucleotide ratios retard complete repair despite upregulation of incision repair enzymes that 3260
Journal of Proteome Research • Vol. 7, No. 8, 2008
act early in the repair process. DNA strand breakage and imbalances in nucleotide precursors also stimulate apoptosis.20 Apoptosis allows removal of damaged cells before they can replicate. Folate deficiency increased the level of apoptosis (decreased mitochondrial membrane potential and increased polycaspase activity). Correspondingly, there was a differential effect on the expression of both proapoptotic proteins [e.g., heat shock protein 60, annexin 1, SMAC/Diablo, voltage-dependent anion selective channel protein 1 (porin) and cathepsin D] and antiapoptotic proteins (e.g., heat shock protein 70, tyrosyl-t RNA synthesis, galactin-3 and pyruvate dehydrogenase E1). Upregulation of Cathepsin D precedes release of cytochrome c and altered mitochondrial membrane potential in the mitochondrial apoptosis pathway, while annexin promotes apoptotic cell engulfment. Porin plays an important role in mitochondria-induced apoptosis. Folate deficiency increased cathepsin D, porin and annexin 1 expression 2-3-fold in this study. Folate deficiency also
research articles
Response of Human Colonocytes to Folate Deficiency in Vitro
Table 3. Measures of Confidence for Protein Identification of Human Colonocyte Proteins Significantly Downregulated as a Consequence of Folate Deficiencya SSP
Mr (exp) (kDa)
Mr (theor) (kDa)
pI
Mowse score -10 Log(P)
no. peptides matched
sequence coverage (%)
1819 5004 24 1 1706 6303 1102 6004 1310 207 5902 5203 1004 6316 5202 7601 6304 5510 1007 20 5007 7002 7005 7408 4003 6201 6306 4405 1204 5002 6410 4507 7314 3607 109 1812
96.7 13.5 15.3 15.4 84.1 41.1 31.2 12.5 35.6 30.5 101.5 32.4 16.2 32.7 35.9 67.5 38.9 60.3 11.5 11.9 15.5 15.3 20.4 41.2 16.9 38.3 38.8 47.3 29.2 24.5 37.2 48.9 36.7 60.7 20.4 96.8
100.2 14.4 11.7 11.7 81.2 46.1 26.8 11.1 60.9 24.6 95.2 35.4 16.7 34.6 36.9 69.6 42.9 61.3 12.8 14.5 17.9 17.9 20.9 40.4 17.9 37.5 37.5 46.9 28.3 22.8 40.2 40.6 32.5 58.0 18.7 89.2
5.7 7.2 4.7
