RESULTS and DISCUSSION

Differential 2-D maps were generated for primary tumor and liver metastasis samples. Figure 2 shows an example of the 2D-PAGE maps thus obtained. Five replicates maps were obtained for each sample.

(a) (b) Figure 2. 2-D maps representing primary tumor (a) and liver metastasis (b) samples.

Figure 3 shows the master map of the Neuroblastoma cells: about 270 spots could be counted with Sypro Ruby staining and 85 spots were found to be differently expressed of which 30 were up-regulated, 15 down-regulated, 28 were highly expressed (more than 10 times) and 12 highly silenced (less than 10 times). All these spots were eluted and treated as described in section 4.2.3. The 44 identified spots, by means of LC-MS/MS analysis, are circled in the standard map and are indicated with their SSP numbers.

Figure 3. Master map of Neuroblastoma cells. The 44 identified spots are circled and numbered (see Table 1 for their classsification). Pie chart: summary of all proteins up- or dow-regulated and/or switched on/off.

35%

18%

33%

14% Increased

Decreased ON

OFF

Table 1. Identified proteins

SSP ID

Fold of

variaotion Protein name NCBI accesion no.

HUGO

gene name Reported function

601 ↑ 2,4 Y box-binding protein 1 gi|112410 YB1 signaling DNA damage and cell proliferation 3303 ↑ 3,5 cytoskeleton associated protein gi|736704 CKAP1 protein folding, microtubule-based process

4310 ↑ 2,7 eukaryotic translation initiation

factor 4E gi|3219774 eIF-4E translational initiation, protein biosynthesis, regulation of translation

5006 ↑ 3,9 Histidine triad

nucleotide-binding protein 1 gi|1633186 HINT1 signal transduction 5011 ↑ 3,9 Histidine triad

nucleotide-binding protein 1 gi|1633186 HINT1 signal transduction 5307 ↑ 2,5 DNAJC14 protein gi|16877384 DNAJC14 protein folding 3-hydroxyanthranilate

3,4-dioxygenase gi|20810280 HAAO synthesis of the excitotoxin quinolinic acid (QUIN) from 3-hydroxyanthranilic acid

5613 ↑ 2,5

Chain A, Human Mitochondrial Aldehyde Dehydrogenase Complexed With Nad+ And Mn2+

gi|6137677 ALDH2 carbohydrate metabolism, metabolism, alcohol metabolism

6203 ↑ 2,4 thioredoxin peroxidase gi|5453549 PRDX2 oxygen and reactive oxygen species metabolism, response to oxidative stress, anti-apoptosis 6405 ↑ 3,9 arginase gi|178995 ARG arginine catabolism, response to wounding, urea

cycle, arginine metabolism

7204 ↑ 3,4 calreticulin precursor gi|4757900 CALR regulation of transcription, DNA-dependent, protein folding

7208 ↑ 2,2 peroxiredoxin 1 gi|4505591 PRX1 skeletal development, cell proliferation, response to oxidative stress

7311 ↑ 2,8 mitochondrial short-chain enoyl-coenzyme A hydratase 1 precursor

gi|12707570 ECHS1 fatty acid metabolism, energy pathways, metabolism, fatty acid beta-oxidation 7503 ↑ 3,1 acetyl-CoA C-acetyltransferase

1 precursor, mitochondrial gi|86728 ACAT1

mitochondrially localized enzyme that catalyzes the reversible formation of acetoacetyl-CoA from two molecules of acetyl-CoA.

7506 ↑ 3,6 proteasome endopeptidase

complex p42 chain gi|7435741 PSMC6 ATP-dependent degradation of ubiquitinated proteins

7507 ↑ 5,2 Phosphoglycerate kinase 1 gi|6679291 PGK1 glycolysis

7603 ↑ 2,6 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (mitochondrial)

gi|5031751 HMGCS2 ketone body biosynthesis, acetyl-CoA metabolism, cholesterol biosynthesis

8508 ↑ 2,7 acetyl-CoA C-acetyltransferase

1 precursor, mitochondrial gi|86728 ACAT1

mitochondrially localized enzyme that catalyzes the reversible formation of acetoacetyl-CoA from two molecules of acetyl-CoA.

2106 > 10 DNAJC14 protein gi|16877384 DNAJC14 protein folding 3-hydroxyanthranilate

3,4-dioxygenase gi|20810280 HAAO synthesis of quinolinic acid 5510 > 10 26S proteasome regulatory

chain, p40 gi|1085272 PSMD7 protein biosynthesis, ribosome biogenesis

6509 > 10 fumarylacetoacetate hydrolase,

FAH gi|253320 FAH

tyrosine catabolism, regulation of transcription, DNA-dependent, metabolism, aromatic amino acid family metabolism, L-phenylalanine catabolism 6510 > 10 poly(rC) binding protein 1 gi|5453854 single-stranded nucleic acid binding protein

7704 > 10

Delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrial precursor

gi|2506350 ALDH4A1 aldehyde metabolism, retinoic acid metabolism, metabolism

7705 > 10

Delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrial precursor

gi|2506350 ALDH4A1 aldehyde metabolism, retinoic acid metabolism, metabolism

7609 > 10 catalase gi|179950 CAT response to oxidative stress, electron transport 7610 > 10 catalase gi|179950 CAT response to oxidative stress, electron transport 7611 > 10 ATP synthase, H+ transporting,

mitochondrial F1 complex gi|4757810 ATP5A1 ATP synthesis coupled proton transport, proton transport, ATP biosynthesis

8509 > 10 Phosphoglycerate kinase 1 gi|129902 PGK1 glycolysis

1302 ↓ 2,0 GTP binding protein gi|4092054 RAN

intracellular protein transport, protein-nucleus import, DNA metabolism, RNA-nucleus export, cell growth and/or maintenance, spermatid

development, small GTPase mediated signal transduction, signal transduction, regulation of cell cycle

1702 ↓ 2,2 calreticulin precursor gi|4757900 CALR regulation of transcription, DNA-dependent, protein folding

2710 ↓ 3,0 heat shock 90kDa protein 1,

beta gi|20149594 HSPCB

response to heat, response to unfolded protein, positive regulation of nitric oxide biosynthesis, protein folding

3306 ↓ 2,2 ubiquitin thiolesterase gi|88254 protein metabolism

4101 ↓ 2,0 stathmin 1 gi|5031851 OP18

cell growth and/or maintenance, mitotic spindle assembly, intracellular signaling cascade, microtubule depolymerization, microtubule-based process, axonogenesis

4108 ↓ 2,0 stathmin 1 gi|5031851 OP18 see spot no. 4101

4201 ↓ 2,1 glutathione transferase gi|87564 GST

Conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles.

ubiquitin thiolesterase gi|88254 OTUB

hydrolase that can remove conjugated ubiquitin from proteins in vitro and may therefore play an important regulatory role at the level of protein turnover by preventing degradation.

5004 ↓ 2,7 S100 calcium binding protein

A11 (calgizzarin) gi|5032057 S100A11 negative regulation of cell proliferation, negative regulation of DNA replication

BolA-like protein 2 (My016

protein) gi|12001972 BOLA2 transcription regulator activity 6501 ↓ 2,2 actin-related protein gi|381964 ACTR1A vesicle-mediated transport pancreatic tumor-related

protein gi|189597 anchoring to other cellular components 7202 ↓ 2,6 raf kinase inhibitor protein gi|913159 RKIP interacts with Raf-1 and seems to inhibit it 3204 < 10 40s ribosomal protein S7 gi|337518 RPS7 protein biosynthesis, ribosome biogenesis

ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform a

gi|5453559 ATP5J energy pathways, ATP synthesis coupled proton transport, proton transport

thiol-specific antioxidant protein gi|438069 PRX2 oxygen and reactive oxygen species metabolism, response to oxidative stress, anti-apoptosis 3401 < 10 Fructose-1,6-bisphosphatase gi|119740 FBP1 gluconeogenesis, carbohydrate metabolism,

fructose metabolism 4001 < 10 Retinoic acid-binding protein I,

cellular gi|266904 CRABP1 development, signal transduction, transport 5505 < 10 Human Diff6,H5,CDC10

homologue gi|1040689 NEDD5 cell cycle, cytokinesis 6001 < 10 Histidine triad

nucleotide-binding protein 1 gi|1633186 HINT1 see spots no. 5006, 5011

6301 < 10 Chain A, Triosephosphate

Isomerase gi|999892 TPI1

metabolism, fatty acid biosynthesis, glycolysis, pentose-phosphate shunt, lipid biosynthesis, gluconeogenesis

Chain A, Horf6 A Novel Human

Peroxidase Enzyme gi|3318841 8507 < 10 Phosphoglycerate kinase 1 gi|129902 PGK1 glycolysis

Figure 4 shows a small gel area with a few spots up- and down-regulated in liver metastasis (LM) vs primary tumor (PT).

Figure 4. Examples of identified proteins: YB1, eIF-4E, S100A11, RKIP. For each protein is shown the position in the gel and the expression trend in the comparison.

PT

LM

YB1

(SSP 601)

increased 2,4 fold

PT

LM

increased 2,7 fold