Open Access

Dietary exposure to soy or whey proteins alters colonic global gene expression profiles during rat colon tumorigenesis

Molecular Cancer20054:1

DOI: 10.1186/1476-4598-4-1

Received: 08 September 2004

Accepted: 11 January 2005

Published: 11 January 2005

Abstract

Background

We previously reported that lifetime consumption of soy proteins or whey proteins reduced the incidence of azoxymethane (AOM)-induced colon tumors in rats. To obtain insights into these effects, global gene expression profiles of colons from rats with lifetime ingestion of casein (CAS, control diet), soy protein isolate (SPI), and whey protein hydrolysate (WPH) diets were determined.

Results

Male Sprague Dawley rats, fed one of the three purified diets, were studied at 40 weeks after AOM injection and when tumors had developed in some animals of each group. Total RNA, purified from non-tumor tissue within the proximal half of each colon, was used to prepare biotinylated probes, which were hybridized to Affymetrix RG_U34A rat microarrays containing probes sets for 8799 rat genes. Microarray data were analyzed using DMT (Affymetrix), SAM (Stanford) and pair-wise comparisons. Differentially expressed genes (SPI and/or WPH vs. CAS) were found. We identified 31 induced and 49 repressed genes in the proximal colons of the SPI-fed group and 44 induced and 119 repressed genes in the proximal colons of the WPH-fed group, relative to CAS. Hierarchical clustering identified the co-induction or co-repression of multiple genes by SPI and WPH. The differential expression of I-FABP (2.92-, 3.97-fold down-regulated in SPI and WPH fed rats; P = 0.023, P = 0.01, respectively), cyclin D1 (1.61-, 2.42-fold down-regulated in SPI and WPH fed rats; P = 0.033, P = 0.001, respectively), and the c-neu proto-oncogene (2.46-, 4.10-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively) mRNAs were confirmed by real-time quantitative RT-PCR. SPI and WPH affected colonic neuro-endocrine gene expression: peptide YY (PYY) and glucagon mRNAs were down-regulated in WPH fed rats, whereas somatostatin mRNA and corresponding circulating protein levels, were enhanced by SPI and WPH.

Conclusions

The identification of transcripts co- or differentially-regulated by SPI and WPH diets suggests common as well as unique anti-tumorigenesis mechanisms of action which may involve growth factor, neuroendocrine and immune system genes. SPI and WPH induction of somatostatin, a known anti-proliferative agent for colon cancer cells, would inhibit tumorigenesis.

Keywords

colon cancer soy whey gene expression profiling neuro-endocrine microarray rat

Background

Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer-related mortality in the U.S. [1, 2]. Estimated new cases of colon cancer were 79,650 for men and 73,530 for women in 2004 [1]; approximately $6.3 billion is spent in the United States each year on treatment of CRC [2]. Accumulating evidence suggests that diet is an important environmental factor in the etiology of CRC. High consumption of red meats, animal fats, chocolate, alcohol and refined cereals are linked to higher incidence of these cancers in Western societies [35], whereas protective effects of fruits, vegetables and whole grains have been suggested [5].

Soy foods and soybean constituents have received considerable attention for their potential role in reducing cancer risk [6, 7]. Our laboratories reported the protective effects of lifetime ingestion of soy protein isolate (SPI) on azoxymethane (AOM)-induced colon cancer in rats [8]. Similarly, the effect of whey protein hydrolysate (WPH) in the diet to reduce colon tumor incidence has been reported by us and others [911]. Several hypotheses have been proposed to account for soy and whey protein-induced anti-tumorigenesis. For example, soy isoflavones have been proposed to play a key role in soy's anti-cancer functions [12]. Yanagihara et al., among others, reported that genistein inhibits colon cancer cell proliferation and stimulates apoptosis in vitro [1315]. However, subcutaneous administration of genistein to mice did not confirm these in vitro effects [16]. Holly et al. reported that soy sphingolipids inhibit colonic cell proliferation, and suggested that this may partially account for its anticancer benefits [17]. Other reports indicate that soy diets inhibit tumorigenesis by regulating the synthesis or activities of specific proteins. For example, Rowlands et al. reported that dietary soy and whey proteins down-regulate expression of liver and mammary gland phase I enzymes involved in carcinogen activation [18]. Elevated activities of phase II detoxification enzymes were reported in soy-fed rats [19, 20]. Such dietary effects may result in lower tissue concentrations of activated carcinogen. The anticancer properties of whey proteins have been ascribed to their ability to elevate cellular levels of the antioxidant glutathione [21, 22]. Moreover, the whey protein, α-lactalbumin, inhibits proliferation of mammary epithelial cells in vitro [11]. The anticancer properties of whey may also relate to its immune system-enhancing actions [23].

Despite extensive research, there is no consensus for anti-cancer mechanism(s) of soy and whey, which will undoubtedly involve multiple interrelated processes, pathways and many components. Many of the same molecular and biochemical changes underlying human colon cancer are observed in the azoxymethane (AOM)-induced rat colon cancer model [24]. Moreover, previous studies suggest a different molecular etiology for tumors of the proximal and distal colon in this model and in human colon [24, 25]. Differential dietary effects on proximal vs. distal colon DNA damage were noted [26] and Westernization of the human diet is thought to have favored a shift of tumors from distal to more proximal locations [27]. Thus, region-specific localization of dietary effects on colon tumorigenesis is an important factor to consider in any molecular analysis of CRC. Here, we use Affymetrix high-density oligonucleotide microarrays to determine the expression profiles of non-tumor (i.e., normal) tissue in proximal colons (PC) of rats, subjected to lifetime diets containing casein (CAS, control diet), soy protein isolate (SPI), or whey protein hydrolysate (WPH) and which were administered AOM to induce tumors. We hypothesized that genes whose expression contributes to anti-tumorigenesis would be regulated in parallel by SPI and WPH; in addition, changes unique to each diet might also be apparent.

Results

Validation of the microarray approach

Quality control steps ensured that the RNA used for microarray and real-time RT-PCR analysis was of high quality. These steps included evaluation of the RNA with the RNA 6000 Nano Assay and assessment of the cRNA hybridization to GeneChips by comparison of data obtained for probe sets representative of 5' and 3' ends of control genes. All RNA samples had an A260/280 absorbance ratio between 1.9 and 2.1. The ratio of 28S to 18S rRNA was very close to 2 on RNA electropherograms, and signal ratios below 3 were noted for 3' vs. 5' probe sets for β-actin and glyceraldehyde-3-phosphate dehydrogenase (per Affymetrix user guidelines) after hybridization.

Total false change rates (TFC) were determined following Affymetrix-recommended guidelines [28], except that the inter-chip comparisons used cRNA targets made in parallel starting from the same RNA pool. Inter-chip variability, measured as TFC%, was 0.25% – 0.6% and well below the suggested 2% cutoff (Table 1). These values confirmed the fidelity and reproducibility of the microarray procedures used. Unsupervised nearest-neighbor hierarchical clustering identified differences in proximal colon gene expression profiles of CAS, SPI and WPH groups (Figs. 1 and 2), indicating that the type of dietary protein has a major effect on gene expression in normal proximal colon tissue of AOM-treated rats. Interestingly, the overall gene expression profiles for SPI and WPH groups were more similar to each other than each was to the CAS group (Fig. 1A).
Table 1

Inter-chip variability

Diet group

Number of arrays

TFC (%)*

CAS

3

0.252 ± 0.138

WPH

3

0.369 ± 0.025

SPI

3

0.570 ± 0.165

*TFC (Total false change) = false change rate (decreased category) + false change rate (increased category), as described in ref. 28; TFC reported as mean ± SEM, TFC should be no more than 2% (Affymetrix).

Figure 1

Hierarchical clustering of proximal colon gene expression profiles. A. Clustering of nine PC global gene expression profiles (8799 genes); n = 3 profiles each for CAS, SPI and WPH. Each cell represents the expression level of an individual gene in each sample (green = low expression, black = middle expression, red = high expression). The dendrogram reflects the extent of relatedness of different profiles; the shorter branch-point of the SPI and WPH trees indicates the greater similarity between these profiles. B. Clustering of 18 global comparative expression profiles including 9 of SPI vs. CAS and 9 of WPH vs. CAS profiles. Each row in the heat map represents the relative expression level of a given gene across all comparisons (red = up regulated, black = unchanged, green = down regulated).

Figure 2

Hierarchical clustering of 211 differentially expressed genes in either SPI or WPH. The differential expression data are taken only from the pairwise comparison analysis, with CAS profiles used as baseline. Each cell in the heat map represents the relative expression level of a given gene in an individual comparison analysis (red = up regulated, black = unchanged, green = down regulated). The dendrogram reflects the relatedness of different profiles.

Differentially expressed genes

Multiple filtering criteria were applied to the microarray data set so as to identify differentially expressed colon transcripts in rats fed SPI, WPH or CAS; results are reported only for transcripts that passed all three analytical filters used: DMT t-test, SAM and pair-wise comparison survival methods. Among the 8799 genes and ESTs examined with the rat U34A array, we identified 31 induced and 49 repressed genes in proximal colons of SPI-fed rats, whereas 44 induced and 119 repressed genes were detected in WPH-fed rats (Tables 2, 3, 4, 5). Interestingly, more down- than up-regulated genes were noted for both SPI and WPH. Additionally, 37 genes were co-repressed, whereas only two were co-induced by SPI and WPH (Table 6). More than 90% of identified genes in WPH and SPI animals showed the same direction of change relative to CAS. This is visually apparent in the hierarchical clustering output (Fig. 2).
Table 2

Down-regulated genes in rats fed with WPH diet*

Category and Gene Name

Probe Set GB Accession No.

Fold Change

P value

Cell adhesion

   

   Embigin

AJ009698

-6.57

0

   Cadherin 17

L46874

-4.8

0.036

   Cadherin

X78997

-3.36

0.004

   Protein tyrosine phosphatase

M60103

-2.64

0.004

   Cytokeratin-8

S76054

-2.71

0

   Trans-Golgi network integral membrane protein TGN38

X53565

-4.92

0.012

   Tumor-associated calcium signal transducer 1

AJ001044

-9.37

0.001

   Claudin-3

AJ011656

-7.55

0.02

   Claudin-9

AJ011811

-5.12

0

Cell cycle/growth control

   

   Mapk6

M64301

-2.61

0.003

   Epithelial membrane protein 1

Z54212

-4.67

0.015

   Glucagon

K02813

-7.73

0.005

   Peptide tyrosine-tyrosine (YY)

M17523

-4.56

0.001

   Src related tyrosine kinase

U09583

-3.31

0.033

   FGF receptor activating protein

U57715

-4.25

0.002

   Cyclin D1

D14014

-1.97

0.001

   Neu oncogene

X03362

-2.61

0.017

Defense/immunity protein

   

   Seminal vesicle secretion protein iv

J00791

-5.35

0.001

   Putative cell surface antigen

U89744

-5.22

0.008

   Decay accelerating factor GPI

AF039583

-6.12

0

   Beta defensin-1

AF093536

-26.78

0.001

Detoxification/antioxidation

   

   Glutathione S-transferase

J02810

-5.17

0

   Glutathione S-transferase Yb

X04229

-9.33

0

 

J03914

-2.43

0.002

   Glutathione S-transferase, alpha 1

K01932

-3.07

0.002

   Glutathione transferase, subunit 8

X62660

-6.42

0.001

   Glutathione S-transferase Yc1

S72505

-3.69

0.004

   Glutathione S-transferase Yc2

S72506

-21.38

0.008

   N-acetyltransferase 1

U01348

-4.64

0.003

   Cytochrome P450CMF1b

J02869

-8.23

0.001

   Cytochrome P450 4F4

U39206

-6.43

0.004

   Cytochrome P450 monooxygenase

U39943

-2.82

0.011

   Cytochrome P450 pseudogene

U40004

-2.87

0

   Cytochrome P450 3A9

U46118

-6.91

0

   Cytochrome P450IVF

M94548

-5.78

0.002

   Cytochrome P450, subfamily 51

U17697

-2.07

0.005

   Alcohol dehydrogenase

M15327

-2.06

0.025

   Aldehyde dehydrogenase

M23995

-10.56

0.035

 

AF001898

-2.72

0.004

   D-amino-acid oxidase

AB003400

-13.69

0

   3-methylcholanthrene-inducible UDP-glucuronosyltransferase

S56937

-9

0

   UDP-glucuronosyltransferase

D38062

-3.17

0.005

 

D38065

-3.29

0.002

   UDP glycosyltransferase 1

D83796

-6.87

0

 

J02612

-6.58

0

 

J05132

-4.03

0

Metabolism

   

   Meprin 1 alpha

S43408

-3.82

0.014

   Brain serine protease bsp1

AJ005641

-4.42

0.007

   Cystathionine gamma-lyase

D17370

-3.05

0.002

   Cathepsin S

L03201

-2.62

0

   Meprin beta-subunit

M88601

-5

0.004

   Disintegrin and metalloprotease domain 7

X66140

-11.91

0

   Fucosyltransferase 1

AB006137

-4.96

0.001

   Fucosyltransferase 2

AB006138

-7.97

0.017

   UDP-glucose:ceramide glycosyltransferase

AF047707

-2.86

0.007

   Type II Hexokinase

D26393

-2.7

0.001

   Hexokinase II

S56464

-4.45

0.007

   CDP-diacylglycerol synthase

AB009999

-4.66

0

   Carboxylesterase precursor

AB010635

-5.29

0.002

   Fatty acid Coenzyme A ligase

AB012933

-2.5

0.041

   3beta-HSD

L17138

-3.27

0

   11-beta-hydroxylsteroid dehydrogenase type 2

U22424

-3

0.001

   Peroxiredoxin 6

AF014009

-3.55

0.01

   Platelet phospholipase A2

X51529

-3.25

0.001

Ligand binding/carrier

   

   Carnitine transporter

AB017260

-3.95

0.005

   Chloride channel (ClC-2)

AF005720

-5.69

0.002

   Putative potassium channel

AF022819

-4.84

0

   Mitochondrial dicarboxylate carrier

AJ223355

-3.55

0.009

   Aquaporin 3

D17695

-7.83

0

   Na_H_Exchanger

L11236

-9.81

0.003

   Angiotensin/vasopressin receptor (AII/AVP)

M85183

-3.3

0.002

   H+, K+-ATPase

M90398

-13.87

0

   Intestinal fatty acid binding protein

K01180

-7.29

0.001

   Apolipoprotein A-I precursor

M00001

-3.45

0.023

   Apolipoprotein A-I

J02597

-2.47

0.003

   Sodium-hydrogen exchange protein-isoform 3

M85300

-7.36

0.004

   Liver fatty acid binding protein

V01235

-2.62

0

   Sodium transporter

X59677

-3.8

0

   Cation transporter

X78855

-3.62

0.003

   ATP-binding cassette

AB010467

-3.89

0.004

   Methionine adenosyltransferase II, alpha

J05571

-2.91

0.007

   Phenylalanine hydroxylase

M12337

-7.43

0

   Carbonic anhydrase IV

S68245

-4.28

0.011

Signal transduction

   

   B7 antigen

X76697

-170.95

0.002

   CD24 antigen

U49062

-3.08

0

   Chemokine CX3C

AF030358

-5.04

0.011

   Itmap1

AF022147

-7.5

0.001

   HCNP

E05646

-2.5

0.001

   Brain glucose-transporter protein

M13979

-2.97

0.019

   Protein kinase C delta

M18330

-2.48

0.002

   Guanylate cyclase 2C

M55636

-4.58

0.003

   A2b-adenosine receptor

M91466

-2.8

0.04

   Guanylate cyclase activator 2A

M95493

-4.18

0.005

   Phospholipase C beta-3

M99567

-2.57

0.018

   Tm4sf3

Y13275

-3.33

0

   Phospholipase D

AB000778

-2.71

0.009

   BEM-2

D45413

-6.41

0.015

   Sgk

L01624

-3.93

0

Stress response/apoptosis

   

   Prostaglandin D synthetase

J04488

-43.11

0.009

   GTP cyclohydrolase I

M58364

-3.26

0.014

Structure proteins

   

   Chromogranin B (Chgb)

AF019974

-2.56

0.005

   Intestinal mucin

M76740

-5.09

0.002

   Muc3

U76551

-11.07

0.006

   Mucin-like protein

M81920

-11.97

0.001

   Myosin 5B

U60416

-3.94

0

   Keratin 18

X81448

-3.23

0.004

   Keratin 19

X81449

-2.69

0.001

   ZG-16p protein

Z30584

-4.43

0.002

   Plasmolipin

Z49858

-7.2

0

   Cytokeratin 21

M63665

-4.96

0

   Syndecan

S61865

-3.3

0.006

   Claudin 3

M74067

-6.68

0.01

Transcription factor/regulator

   

   Hepatocyte nuclear factor 3 gamma

AB017044

-6.96

0

   Apolipoprotein B mRNA editing protein

L07114

-2.34

0

   DNA-binding inhibitor

L23148

-4.1

0.01

   Kruppel-like factor 4 (gut)

L26292

-3.08

0.017

Others

   

   Prolactin receptor

M74152

-3.26

0.014

   LOC286964

U89280

-2.96

0.003

   Ckmt1

X59737mRNA

-2.65

0.025

   Arginase II

U90887

-23.69

0

   Deleted in malignant brain tumors 1

U32681

-3.47

0.002

   3' end GAA-triplet repeat

L13025

-2.73

0.001

   Polymeric immunoglobulin receptor

L13235

-2.93

0.004

*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), and SAM (Stanford). Gene expression profiles from CAS animals were used as control. P value and fold change are based on DMT analysis; whereas final genes listed met all of the analytical criteria as described in Methods.

Table 3

Up-regulated genes in rats fed with WPH diet*

Category and Gene Name

Probe Set GB Accession No.

Fold Change

P value

Cell adhesion

   

   Fibronectin

X05834

2.3

0

   EGF-containing fibulin-like extracellular matrix protein 1

D89730

2.17

0.004

Cell cycle/growth control

   

   Somatostatin

M25890

2.72

0.001

   Somatostatin-14

K02248

3.87

0.009

   APEG-1

U57097

3.24

0.002

Defense/immunity protein

   

   IgG gamma heavy chain

M28670

2.21

0.009

   T-cell receptor beta chain

X14319

2.14

0

   Adipsin

M92059

3.21

0

Ligand binding/carrier

   

   Angiotensin receptor

M86912

2.75

0.017

   Calretinin

X66974

2.52

0.005

   Purkinje cell protein 4

M24852

3.06

0.001

   Secretogranin III

U02983

2.77

0.005

   Secretogranin II

M93669

2.84

0.001

   Aquaporin 1

X67948

3.4

0.008

   Cacna2d1

M86621

2.84

0

   Retinol-binding protein

M10934

2.17

0.018

Metabolism

   

   Lipoprotein lipase

L03294

2.72

0

   Ubiquitin carboxyl-terminal hydrolase

D10699

3

0.003

Signal transduction

   

   Thy-1 protein

X02002

2.89

0.002

   CD3 gamma-chain

S79711

3.28

0.002

   Synapsin

M27925

3.94

0.001

   Alpha-actinin-2 associated LIM protein

AF002281

2.74

0.009

   RESP18

L25633

2.74

0.033

   T3 delta protein

X53430

2.75

0.003

   Protein phosphatase inhibitor-1

J05592t

2.6

0.009

   CART protein

U10071

2.16

0.001

   Neuroendrocrine protein

M63901

3.7

0.006

   Protein kinase C-binding protein Zeta1

U63740

3.14

0.003

   cannabinoid receptor 1

X55812

2.17

0.002

   Guanylyl cyclase A

J05677

3.18

0.007

   Tachykinin 1

X56306

2.36

0.036

   Protein tyrosine phosphatase

L19180

2.47

0.041

   Argininosuccinate synthetase

X12459

4.69

0.004

Stress response/apoptosis

   

   Small inducible cytokine

Y08358

3.35

0.029

Structure proteins

   

   Fast myosin alkali light chain

L00088

4.52

0.03

   Light molecular-weight neurofilament

AF031880

2.41

0

   Neurofilament protein middle

Z12152

2.97

0.006

   Alpha-tubulin

V01227

2.25

0

   Peripherin

AF031878

2.82

0.007

Transcription factor/regulator

   

   snRNP

M29293

2.11

0.004

   snRNP-associated polypeptide

X73411

3.33

0.002

Others

   

   C1-13 gene product

X52817

3.17

0

   ND5, ND6

S46798

2.31

0.015

   Sensory neuron synuclein

X86789

2.84

0

*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), and SAM (Stanford). Gene expression profiles from CAS animals were used as control. P value and fold change are based on the DMT analysis; whereas listed genes met all of the analytical criteria as described in Methods.

Table 4

Down-regulated genes in rats fed with SPI diet*

Category and Gene Name

Probe Set GB Accession No.

Fold Change

P value

Cell adhesion

   

   Embigin

AJ009698

-5.13

0.001

Cell Cycle/growth control

   

   FGF receptor activating protein 1

U57715

-5.59

0.002

   BEST5 protein

Y07704

-2.37

0.003

   Peptide tyrosine-tyrosine (YY)

M17523

-3.91

0.002

   Glucagon gene

K02813

-6.58

0.002

   Epithelial membrane protein-1

Z54212

-3.47

0.017

   Neu oncogene

X03362

-1.58

0.05

Defense/immunity protein

   

   Beta defensin-1

AF068860

-42.16

0.001

 

AF093536

-10.2

0

Detoxification/antioxidation

   

   Glutathione S-transferase

J02810

-7.14

0

   Glutathione S-transferase Yb

X04229

-11.71

0.001

   Glutathione S-transferase, alpha 1

K01932

-4.18

0.004

   Glutathione S-transferase Yc1

S72505

-5.23

0.001

   Glutathione S-transferase Yc2

S72506

-5.27

0.012

 

S82820

-3.45

0.006

   Cytochrome P450 4F4 (CYP4F4)

U39206

-6.52

0.002

   Cytochrome P450CMF1b

J02869

-4.12

0.002

   Cytochrome P450 (CYP4F1)

M94548

-2.88

0.002

   1-Cys peroxiredoxin

Y17295

-2.55

0.002

   Metallothionein

M11794

-2.92

0.006

   D-amino-acid oxidase

AB003400

-5.42

0

   Peroxiredoxin 6

AF014009

-3.07

0.008

   Phenylalanine hydroxylase

M12337

-10.99

0.001

Metabolism

   

   Dipeptidase

L07315

-3.08

0.001

   Meprin beta-subunit

M88601

-3.27

0.001

   Disintegrin and metalloprotease domain 7

X66140

-14.03

0

Ligand binding/carrier

   

   Carnitine transporter

AB017260

-3.81

0.003

   Chloride channel (ClC-2)

AF005720

-3.26

0.001

   Putative potassium channel

AF022819

-2.69

0.001

   Mitochondrial dicarboxylate carrier

AJ223355

-2.54

0.01

   Aquaporin 3

D17695

-4.13

0

   Intestinal fatty acid binding protein

K01180

-4.43

0.005

   Na_H_Exchanger

L11236

-4.47

0.002

   H+, K+-ATPase

M90398

-2.52

0.001

   Carbonic anhydrase IV

S68245

-4.28

0.005

   Sodium transporter

X59677

-3.4

0

   Phosphatidylethanolamine binding protein

X75253

-2.69

0

Signal transduction

   

   B7 antigen

X76697

-170.95

0.002

   HCNP

E05646

-3.38

0

   Itmap1

AF022147

-7.97

0.005

   Guanylate cyclase activator 2A

M95493

-3.28

0.006

   Sgk

L01624

-2.76

0

Stress response/apoptosis

   

   Prostaglandin D synthetase

J04488

-45.8

0.01

Structure proteins

   

   Muc3

U76551

-3.56

0.01

   Intestinal mucin

M76740

-3.31

0.006

   Mucin-like protein

M81920

-3

0

   Plasmolipin

Z49858

-2.92

0.003

Transcription factor/regulator

   

   Testis specific X-linked gene

X99797

-6.91

0.003

Others

   

   Arginase II

U90887

-3.22

0

   3-phosphoglycerate dehydrogenase

X97772

-4.15

0.017

   Aldehyde dehydrogenase family 1

AF001898

-3.93

0.004

*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), and SAM (Stanford). Gene expression profiles from CAS animals were used as control. P value and fold-change are based on the DMT analysis; whereas genes listed above met all of the analytical criteria as described in Methods.

Table 5

Up-regulated genes in rats fed with SPI diet*

Category and Gene Name

Probe Set GB Accession No.

Fold Change

P value

Cell adhesion

   

   Collagen alpha1 type I

Z78279

2.49

0

   Secreted phosphoprotein 1

M14656

111.39

0.006

   Matrix metalloproteinase 13

M60616

24.34

0.002

   Regenerating islet

M62930

193.08

0.011

Defense/immunity protein

   

   Ig gamma-2a chain

L22654

115.17

0.001

   Ig gamma heavy chain

M28670

3.22

0

   Ig germline kappa-chain C-region

M18528

2.48

0.038

   Ig light-chain

U39609

2.63

0.021

   Fc-gamma

M32062

4.72

0.017

Detoxification

   

   Glutathione S-transferase 1

J03752

2.86

0

   Glutathione-S-transferase,alpha type2

K00136

2.56

0.009

   UDP glucuronosyltransferase

D38066

2.83

0.014

Metabolism

   

   Matrix metalloproteinase 7

L24374

3.63

0.02

   lysozyme

rc_AA892775

2.77

0

   Matrix metalloproteinase 12

X98517

11.8

0.013

   Mitochondrial carbamyl phosphate synthetase I

M12335

59.25

0.001

   Aldolase B, exon 9

X02291

8.7

0.01

   Aldolase B, exon 2

X02284

2.71

0.001

Signal transduction

   

   MHC class II antigen RT1.B-1 beta-chain

X56596

2.55

0.001

   CD3 gamma-chain

S79711

4.51

0.001

Ligand binding/carrier

   

   Intracellular calcium-binding protein

L18948

28.29

0.014

   Retinol binding protein II

M13949

5.11

0.001

   Apolipoprotein B

M27440

6.47

0.024

   Apolipoprotein A-I

J02597

2.49

0.004

   Iron ion transporter

AF008439

18.78

0.008

Stress response/apoptosis

   

   Heme oxygenase

J02722

9.66

0.002

   JE product

X17053

3.52

0.001

   Pancreatitis-associated protein

M98049

68.39

0.004

   Pancreatitis associated protein III

L20869

15.35

0

   Reg protein

E01983

30.25

0.001

Others

   

   Histamine N-tele-methyltransferase

S82579

6.17

0.04

*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), and SAM (Stanford). Gene expression profiles from CAS animals were used as control. P value and fold-change are based on the DMT analysis; whereas final listed genes met all of the analytical criteria as described in the Methods.

Table 6

Genes co-regulated with WPH and SPI diet*

Category and Gene Name

Probe Set GB Accession No.

Fold Change in WPH

P value

Fold Change in SPI

P value

Down-regulated genes

     

   Embigin

AJ009698

-6.57

0

-5.13

0.001

   Epithelial membrane protein 1

Z54212

-4.67

0.015

-3.47

0.017

   Glucagon

K02813

-7.73

0.005

-6.58

0.002

   Peptide tyrosine-tyrosine (YY)

M17523

-4.56

0.001

-3.91

0.002

   FGF receptor activating protein

U57715

-4.25

0.002

-5.59

0.002

   Neu oncogene

X03362

-2.61

0.017

-1.58

0.05

   CD52 antigen

X76697

-170.95

0.002

-170.95

0.002

   Beta defensin-1

AF068860

-54.48

0.001

-42.16

0.001

   Glutathione S-transferase

J02810

-5.17

0

-7.14

0

   Glutathione S-transferase Yb

X04229

-9.33

0

-11.71

0.001

   Glutathione S-transferase, alpha 1

K01932

-3.07

0.002

-4.18

0.004

   Glutathione S-transferase Yc1

S72505

-3.69

0.004

-5.23

0.001

   Glutathione S-transferase Yc2

S72506

-21.38

0.008

-5.27

0.012

   Cytochrome P450CMF1b

J02869

-8.23

0.001

-4.12

0.002

   Cytochrome P450 4F4

U39206

-6.43

0.004

-6.52

0.002

   Cytochrome P450IVF

M94548

-5.78

0.002

-2.88

0.002

   D-amino-acid oxidase

AB003400

-13.69

0

-5.42

0

   Meprin beta-subunit

M88601

-5

0.004

-3.27

0.001

   Disintegrin and metalloprotease domain 7

X66140

-11.91

0

-14.03

0

   Carnitine transporter

AB017260

-3.95

0.005

-3.81

0.003

   Chloride channel (ClC-2)

AF005720

-5.69

0.002

-3.26

0.001

   Putative potassium channel

AF022819

-4.84

0

-2.69

0.001

   Mitochondrial dicarboxylate carrier

AJ223355

-3.55

0.009

-2.54

0.01

   Aquaporin 3

D17695

-7.83

0

-4.13

0

   Na_H_Exchanger

L11236

-9.81

0.003

-4.47

0.002

   H+, K+-ATPase

M90398

-13.87

0

-2.52

0.001

   Fatty acid binding protein 1

K01180

-7.29

0.001

-4.43

0.005

   Sodium transporter

X59677

-3.8

0

-3.4

0

   Carbonic anhydrase IV

S68245

-4.28

0.011

-4.28

0.005

   Itmap1

AF022147

-7.5

0.001

-7.97

0.005

   HCNP

E05646

-2.5

0.001

-3.38

0

   Guanylate cyclase activator 2A

M95493

-4.18

0.005

-3.28

0.006

   Sgk

L01624

-3.93

0

-2.76

0

   Prostaglandin D synthetase

J04488

-43.11

0.009

-45.8

0.01

   Mucin 3

M76740

-5.09

0.002

-3.31

0.006

   Mucin-like protein

M81920

-11.97

0.001

-3

0

   Plasmolipin

Z49858

-7.2

0

-2.92

0.003

Up-regulated genes

     

   Ig gamma heavy chain

M28670

2.21

0.009

3.22

0

   CD3 gamma-chain

S79711

3.28

0.002

4.51

0.001

*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), and SAM (Stanford). Gene expression profiles from CAS animals were used as control. P value and fold change are based on the DMT analysis; whereas final listed genes met all of the analytical criteria described in Methods.

Gene expression: effects of WPH

As based on Gene Ontology (GO) annotations, the 44 up-regulated and 119 down-regulated genes of the WPH group belong to multiple functional categories including cell adhesion (n = 10), cell cycle and growth control (n = 10), detoxification (n = 17), defense and immunity (n = 7), signal transduction (n = 29), transcriptional regulation (n = 6), metabolism (n = 19), ligands and carriers (n = 27), cell death (n = 3), structural proteins (n = 16), and others (Tables 2 &3). The fold change for up-regulated genes ranged between 2.1 [small nuclear ribonucleoparticle-associated protein (snRNP)] to 4.7 (argininosuccinate synthetase), whereas down-regulated genes exhibited fold changes between 2.0 (cyclin D1) and 171 (CD52 antigen).

Lifetime ingestion of WPH affected the expression of xenobiotic metabolism-related enzymes including several of the cytochrome P450s and glutathione S-transferases, alcohol dehydrogenase (ADH), and UDP-glucuronosyltransferase. Cytochrome P450 enzymes and ADH are considered to play key roles in activation of the proximate carcinogen from AOM [29]. Down-regulation of expression of Phase I detoxification enzymes by WPH might therefore diminish AOM-induced DNA adducts and genomic instability. Consistent with results from a study in which whey proteins inhibited cell proliferation in vitro [11], lifetime feeding of WPH was associated with changes in expression of genes involved in cell cycle control and proliferation; cyclin D1, neu oncogene, mapk6, glucagon, and peptide YY (PYY) genes were down-regulated, whereas the expression of somatostatin, a growth-inhibitory peptide was induced. WPH altered expression of genes involved in cellular defense. Induced genes included Ig gamma heavy chain, adipsin, and T-cell receptor beta chain, whereas expression of the antibacterial peptide beta defensin-1 and seminal vesicle secretion protein IV (SVS IV) were down-regulated. About 20% of WPH-affected genes are involved in cell signaling; these include guanylate cyclase 2C, protein kinase C delta, and synapsin. Additionally, genes encoding ligands or membrane channels [i.e., chloride channel, intestinal fatty acid binding protein (I-FABP), apoliprotein A-I (Apo-AI), Na+, K+-ATPase, and sodium transporter] were down-regulated by WPH, whereas calretinin and retinol binding protein (RBP) levels were increased.

Gene expression: effects of SPI

Colon genes, whose mRNA expression was affected by ingestion of SPI, fell into multiple functional categories including cell adhesion (n = 4), cell cycle and growth control (n = 6), detoxification (n = 18), defense and immunity (n = 6), signal transduction (n = 4), transcriptional regulation (n = 1), metabolism (n = 8), ligands and carrier proteins (n = 17), cell death proteins (n = 5), and structural proteins (n = 3) (Tables 4 &5).

Relative abundance of numerous transcripts was changed in the same direction by WPH and SPI (Fig. 2). However, some exceptions were noted. For example, mRNA encoding Apo-AI was down-regulated by WPH, but elevated by SPI. Apo-AI is the major determinant of the capacity of HDL particles to promote cholesterol efflux and this protein is associated with the inhibition of atherosclerosis [30]. However, the impact of differential response of Apo-AI to WPH and SPI on anti-tumorigenesis is unknown.

Confirmation of differential gene expression

We performed quantitative real-time RT-PCR on selected genes to confirm the microarray results. Based upon known associations with cell proliferation or differentiation, 14 genes were chosen for further study. Included in this group was BTEB2; this gene was not present on the microarrays but was included in RT-PCR analysis due to its significant expression in intestine and involvement in cell proliferation [see discussion]. As shown in Figure 3, eight genes were confirmed to be differentially expressed: these included the gastrointestinal hormone genes PYY (12.9-fold down-regulated in WPH fed rats; P = 0.004), glucagon (17.8-fold down-regulated in WPH fed rats; P = 0.005), and somatostatin (3.92-, 2.65-fold up-regulated in SPI and WPH fed rats; P = 0.05, P = 0.025, respectively); cyclin D1 (1.6-, 2.4-fold down-regulated in SPI and WPH fed rats; P = 0.033, P = 0.001, respectively); BTEB2 (1.9-, 6.7-fold down-regulated in SPI and WPH fed rats; P = 0.024, P < 0.001, respectively); c-neu proto-oncogene (2.5-, 4.1-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively); the colonocyte differentiation marker I-FABP (2.9-, 4.0-fold down-regulated in SPI and WPH fed rats; P = 0.023, P = 0.01, respectively); and the mucin, MUC3 (2.78-, 4.05-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively). Differential expression of five other genes was not confirmed statistically, due to individual animal variation in the transcript levels; however, the mean-fold changes for mRNA abundance were greater than two and in agreement with the corresponding microarray results for these genes. Only one of the selected genes – retinol binding protein (RBP), failed to exhibit greater than a 2-fold change (in the predicted direction) at the mRNA level by real-time RT-PCR.
Figure 3

Quantitative real-time RT-PCR verification of microarray results. RNA used for real-time RT-PCRs was from the same animals (n = 7 per diet group) whose RNAs comprised the pools for microarray analysis. Values are mean ± SEM and were analyzed by one-way ANOVA, *P < 0.05, SPI or WPH vs. CAS.

Serum somatostatin (Sst)

As shown in Fig. 4, circulating Sst concentration was significantly higher in rats fed WPH and SPI. Colonic Sst protein content in colon homogenates was below the limit of detection of the assay used (data not shown).
Figure 4

Diet effects on serum Sst concentration. Values are mean ± SEM. One-way ANOVA. *P < 0.05, SPI or WPH vs. CAS.

Discussion

The type of dietary protein(s) can markedly affect the onset and/or progression of CRC [31]. Epidemiological and animal studies have found that dietary soy and whey proteins decrease the incidence of certain tumors, including those of the colon and rectum [6, 7, 3235]. Using the AOM-treated male Sprague Dawley rat model, we previously found that lifetime feeding of SPI led to a ~ 76% lower incidence of AOM-induced colon tumors compared to rats lifetime-fed CAS [8]. Additionally, in the same studies, a ~ 46% lower incidence of colon tumors was found in WPH-fed compared to CAS-fed rats [9]. The molecular mechanism(s) by which these dietary proteins reduce the incidence of chemically-induced colon tumors is unclear, although several mechanisms and putative bio-active factors have been proposed [1124]. The present study has now identified genes that are differentially expressed as a function of these diets and which serve to highlight potential pathways for dietary protection from carcinogenesis.

The ability to simultaneously analyze a large number of different mRNAs makes microarrays very appealing for identifying genes and gene families whose expression is altered by diet [36, 37]. We focused on the 'normal' colon tissue since we are interested in genes that are differentially regulated by diet and which act in anti-oncogenic fashion in pre-cancerous tissues. We limited our analysis to the proximal colon since several studies have suggested that the molecular etiology of proximal and distal colon tumors differs [25, 26] and proximal colon tumors have become more prevalent with Westernization of the diet and aging of the population [27]. We chose to include colonic smooth muscle with the mucosa since: a) the former tissue layer interacts with the latter to influence its growth and function, and b) we could monitor all colonic genes affected by diet. However, one potential caveat to this strategy is the 'dilution' effect that may have been imposed on the more rare mucosal transcripts. Another caveat is that no information is obtained regarding where the differentially expressed transcripts occur. In this regard, however, we have confirmed by immuno-histochemistry that I-FABP is expressed predominantly in the inter-cryptal surface epithelium of colons from AOM-treated rats (Fig. 5). Our study used a sample size of three (per diet group) which balanced the costs for the experimental reagents with the minimum number required for statistical analysis. The quantitative PCR analyses provided confirmation that the filtering strategies used yielded bona-fide differentially expressed transcripts.
Figure 5

Immuno-histochemistry for I-FABP in colons from AOM-treated rats. Panels A and B are sections from CAS and WPH-fed animals, respectively. Arrows point to the strong areas of staining for I-FABP in the inter-cryptal surface epithelium (overall intensity of staining is greater for CAS than for WPH).

Only two transcripts were induced by both SPI and WPH; whereas 37 transcripts were repressed by both SPI and WPH. This suggests that the cancer-protective actions of the two diets are generally associated with repression of colonic genes that facilitate tumorigenesis. An alternative explanation is that CAS induces genes that facilitate colon cancer development when compared to SPI and WPH. It is also likely that SPI and WPH diets act in unique ways to inhibit tumorigenesis. Irregardless, our results indicate that the nature of the dietary protein can profoundly affect colon gene expression profiles. Thus, gene expression profiling studies of colons should account for potential confounding effects of diet.

Dietary factors in SPI or WPH inhibit cell proliferation and induce apoptosis among other biological actions [11, 13]. In the present study, we identified cyclin D1 gene and the neu proto-oncogene as being repressed in proximal colon by SPI and WPH. Cyclin D1 is a key regulator of cell cycle progression [38], and a target of β-catenin, a protein whose abnormal accumulation in the nucleus is strongly linked to the development of multiple tumor types, including those of the colon [39]. Aberrantly increased expression of cyclin D1 in colon epithelial cells contributes to their abnormal proliferation and tumorigenicity [40, 41]. Similarly, the oncogenic and cellular growth-promoting activities of the HER-2/neu proto-oncogene are well known [42]. HER-2/neu, a tyrosine kinase receptor for neu-differentiation factor, is expressed in normal colonic epithelium and is up-regulated in adenomatous polyps of the colon [43]. The down regulation of cyclin D1 and c-neu mRNA abundance by SPI and WPH may at least partly explain their anti-tumorigenic properties. Similarly, Krüppel-like transcription factors have been linked to cell growth and tumorigenesis. BTEB2 (also known as Krüppel-like factor 5, KLF5, or intestinal KLF) was reported to enhance intestinal epithelia cell colony formation, cyclin D1 transcription, and cell proliferation [44]. Consistent with our results for cyclin D1, colonic BTEB2 mRNA expression was down-regulated by SPI and WPH. Aquaporin 3, a water channel highly expressed in colonic epithelium, was down-regulated by SPI and WPH. Aquaporins are thought to be induced early in colon cancer and to facilitate oncogenesis [45], therefore, dietary repression of such genes may additionally contribute to anti-tumorigenesis. The results for I-FABP and MUC3 indicated 3–4 fold decreases in transcript abundance in proximal colons of rats on SPI or WPH diets. These particular results are not easily reconciled with decreased tumorigenesis in SPI and WPH groups, since both genes are highly expressed in the normal differentiated colonic epithelium and are likely to be under-expressed in adenomas and adenocarcinomas [46]. Perhaps, these represent diet-modulated genes that are not direct participants in anti-tumorigenesis.

Gastrointestinal hormones regulate a myriad of intestinal functions including motility, absorption, digestion, cell proliferation and death, and immune response [47]. The microarray and real-time RT-PCR assays identified inductive effects of SPI and WPH on somatostatin mRNA and protein abundance. These results implicate this gene product in autocrine and paracrine mechanisms underlying colon cancer protection by SPI and WPH since somatostatin is a well-known anti-proliferative agent for colon tumor cells [48, 49]. This hormone is also a negative regulator of angiogenesis [50]; this is predicted to counter tumorigenesis. It is possible that the small decrements in rat growth rates observed with lifetime SPI or WPH diets [8, 9] are a consequence of this increased circulating somatostatin level. We also found decreased abundance of mRNAs encoding peptide YY (PYY) and glucagon in colons of WPH-fed rats. PYY gene expression in human colon tumors is much reduced relative to the adjacent normal tissue [51]; however, chemically-induced colon tumors in rats generally exhibit higher overall expression of PYY due to increased prevalence of PYY-positive cells, compared to normal mucosa [52, 53]. PYY stimulates proliferation of intestinal epithelium [54]; therefore, an inhibition of PYY expression by dietary WPH may contribute to colon cancer-protective actions. A similar scenario might apply to colon glucagon gene expression, as this growth stimulatory peptide for colon cancer cells [55] was inhibited by WPH at the level of colon mRNA abundance.

Our data highlighted other aspects of diet and colon gene expression that warrant further study. For example, the B7 antigen (also known as CD52) mRNA was strongly down-regulated by SPI or WPH. The corresponding protein is normally expressed at high levels on cell membranes of T and B lymphocytes and monocytes; infusion with anti-CD52 antibody leads to systemic depletion of T cells [56]. The lower abundance of this transcript in non-tumor colon tissue of rats on SPI or WPH diets may reflect fewer numbers of immune cells in this tissue, as compared to CAS-fed animals. One possible interpretation of this data is that the 'normal' tissue of the CAS group has manifested an immune response, perhaps in response to increased tumorigenicity relative to SPI or WPH groups. Such an interpretation raises the prospect of a functional immuno-editing mechanism [57] occurring in this model of colon cancer and an indirect effect of diet on lymphocyte populations (via presence of tumors or tumor precursors) in the colon. An alternative mechanism is that dietary protein can directly affect the populations of lymphocytes resident in the colon, which in turn, may affect tumorigenesis. A related observation was the enhanced abundance of CD3 gamma chain transcripts in colons of SPI and WPH animals. The protein encoded by this transcript helps mediate T cell antigen receptor engagement and signaling [58]; its decreased abundance in colonic T cells of CAS-fed animals may indicate a specific immune defect [59] occurring in the CAS-fed animals after exposure to carcinogen and thereby contributing to enhanced tumorigenesis in this group.

Several microarray studies of human paired normal colon vs. colon tumors have been published [6064]. Comparison of the present results for normal colon tissue of AOM-treated rats on different diets to the published studies for human CRC identified only a small number of common differentially expressed genes and/or gene families in common (data not shown). This small number is probably due to the fact that our study did not examine colon tumors; rather we focused on 'normal' colon tissue. In this regard, it will be interesting to examine the expression profiles of colons from animals not treated with AOM so as to more specifically correlate transcripts with diet and cancer phenotype. This study has illuminated a number of genes and gene families that may act as dietary protein-dependent modulators of oncogenesis in the rat colon. Additional studies that specifically address the functional involvement of these genes in cancer-prevention via dietary means are required to confirm the postulated roles.

Conclusions

We have identified genes in rat colon that are differentially expressed, as a consequence of altered dietary protein, during AOM-induced oncogenesis. These are candidates for genes that sub-serve the anti-cancer effects of dietary SPI and WPH in this tissue.

Methods

Rats, diets and carcinogen treatment

The animals whose colons were used in the present study have been previously described [8, 9]. Time-mated [gestation day (GD) 4] Sprague-Dawley rats were purchased from Harlan Industries (Indianapolis, IN), housed individually and allowed ad libitum access to water and pelleted food. Rats were randomly assigned to one of three semi-purified isocaloric diets made according to the AIN-93G diet formula [65] and which differed only by protein source: a) CAS (New Zealand Milk Products, Santa Rosa, CA), b) WPH (New Zealand Milk Products, Santa Rosa, CA) or c) SPI (Dupont Protein Technologies, St. Louis, MO). Offspring were weaned to the same diet as their mothers and were fed the same diets throughout the study. At 90 days of age, male offspring received s.c. injections of 15 mg/kg AOM (Ash Stevens, Detroit, MI) in saline once a week for 2 weeks. Forty weeks later, rats were euthanized, and the colon (cecum to anus) was divided into two equal segments (proximal and distal), opened longitudinally, and examined for tumors. We found that both WPH and SPI significantly decreased the colon tumor incidence [data published in [8, 9]]. A representative non-tumor segment of each proximal colon (PC) was frozen in liquid nitrogen and stored at -80°C for later use. Animal care and handling were in accordance with the Institutional Animal Care & Use Committee guidelines of the University of Arkansas for Medical Sciences.

RNA processing

Total RNA was isolated from rat proximal colons (n = 7 for each of CAS, SPI and WPH diets) using TRIzol reagent (Invitrogen, Carlsbad, CA), and further purified with the RNeasy Mini Kit (QIAGEN, Valencia, CA). To remove contaminating DNA, on-column DNA digestion with RNase-Free DNase (QIAGEN) was performed. Integrity of isolated RNAs was confirmed using the RNA 6000 Nano LabChip kit with the Agilent 2100 Bioanalyzer System (Agilent Biotechnologies, Palo Alto, CA). To reduce errors due to biological variability, RNA samples were pooled as proposed by Bakay et al [66]. Pooled RNA (equal amounts of RNA from each of 7 animals; 8 ug total) was used for cDNA synthesis using a T7-(deoxythymidine)24 primer and Superscript II (Life Technologies, Inc., Gaithersburg, MD). The resulting cDNA was used with the ENZO BioArray High Yield RNA Transcript labeling kit (ENZO, Farmingdale, NY) to synthesize biotin-labeled cRNA. The cRNA was purified on RNeasy spin columns (QIAGEN) and subjected to chemical fragmentation (size range of 35 to 200 bp). Three replicate cRNA targets were made in parallel starting from each RNA pool.

Microarray procedures

Ten ug of cRNA was hybridized for 16 hours to an Affymetrix (Santa Clara, CA) rat U34A GeneChip (3 chips used per diet group), followed by incubations with streptavidin-conjugated phycoerythrin, and then with polyclonal anti-streptavidin antibody coupled to phycoerythrin. Following washing, GeneChips were scanned using an Agilent GeneArray laser scanner. Images were analyzed using Affymetrix MAS 5.0 software. Bacterial sequence-derived probes on the arrays served as external controls for hybridization, whereas the housekeeping genes β-actin and GAPDH served as endogenous controls and for monitoring the quality of the RNA target. To compare array data between GeneChips, we scaled the average of the fluorescent intensities of all probes on each array to a constant target intensity of 500.

Bioinformatics

To validate the microarray procedure for our samples, unsupervised nearest-neighbor hierarchical clustering (Spotfire, Somerville, MA) was performed on gene expression data. The inter-chip variability test also was performed as specified in the Affymetrix data analysis manual [28]. To identify colon genes differentially expressed with SPI or WPH (control: CAS diet), multiple criteria were applied; final results are reported only for transcripts that passed all three analytical steps described below. Firstly, the t-test feature of DMT (Affymetrix) was used to identify genes whose expression was regulated (induced/repressed with P < 0.05) by SPI or WPH, and signal fold changes (FC) for these genes were calculated. Secondly, microarray data were analyzed using 'Significance of Analysis of Microarrays' (SAM, Stanford) to identify significant changes in gene expression among diet groups [67], using a false discovery rate (FDR) cutoff of 0.5%. Lastly, a pair-wise comparison survival (3 × 3) method was used to identify differentially expressed transcripts [68]. In brief, the three replicate expression profiles obtained for SPI colons were iteratively compared with the three CAS profiles (latter as baseline) in MAS 5.0 (Affymetrix), generating nine comparisons in total. Transcripts with a log ratio greater than or equal to 1 (≥2 fold change), which increased (I) in nine of nine comparisons, and which were expressed above background (i.e., called as Present) in all three SPI GeneChips, were considered to be up-regulated by SPI. Transcripts with a log ratio less than or equal to -1, were decreased (D) in nine of nine comparisons, and expressed above background (Present) in all three CAS chips were considered to be down-regulated by SPI. WPH-regulated genes were similarly identified. Genes that were independently identified by all three approaches comprised the final reported lists of differentially expressed genes (Tables 2, 3, 4, 5, 6).

Validation of gene expression by quantitative real-time RT-PCR

One μg of total RNA from each of the 21 individual proximal colons (which comprised the original pools for the microarray experiment) was reverse-transcribed using random hexamers and MultiScribe Reverse Transcriptase in a two-step RT-PCR reaction (Applied Biosystems, Foster City, CA). Primers (Table 7) were designed using 'Primer Express' (Applied Biosystems) and were selected to yield a single amplicon; this was verified by dissociation curves and/or analysis in agarose gels. SYBR Green real-time PCR was performed with an ABI Prism 7000 Sequence Detector. Thermal cycling conditions included pre-incubation at 50°C for 2 min, 95°C for 10 min followed by 40 PCR cycles at 95°C for 15 sec and 60°C for 1 min. The relative transcript levels for each gene were calculated using the relative standard curve method (User Bulletin #2, Applied Biosystems) and normalized to the house-keeping gene β-actin. Data are reported as mean ± SEM of n = 7 animals per dietary group. Significant differences between diet groups were determined by one-way ANOVA (P < 0.05).
Table 7

Primer sequences for real-time RT-PCR

Gene

Forward primer

Reverse primer

Accession no.

Beta-actin

5'-GACGGTCAGGTCATCACTATCG-3'

5'-ACGGATGTCAACGTCACACTTC-3'

NM_031144

I-FABP

5'-AGGAAGCTTGGAGCTCATGACA-3'

5'-TCCTTCCTGTGTGATCGTCAGTT-3'

K01180

Neu Oncogene

5'-GTGGTCGTTGGAATCCTAATCAA-3'

5'-CCTTCCTTAGCTCCGTCTCTTTTA-3'

X03362

PYY

5'-AGGAGCTGAGCCGCTACTATGC-3'

5'-TTCTCGCTGTCGTCTGTGAAGA-3'

M17523

Glucagon

5'-TGGTGAAAGGCCGAGGAAG-3'

5'-TGGTGGCAAGGTTATCGAGAA-3'

K02813

Somatostatin

5'-GGAAACAGGAACTGGCCAAGT-3'

5'-TGCAGCTCCAGCCTCATCTC-3'

K02248

PAP III

5'-AAGAGGCCATCAGGACACCTT-3'

5'-CACTCCCATCCACCTCTGTTG-3'

L20869

CYP4F1

5'-CCAAGTGGAAACGGTTGATTTC-3'

5'-TCCTGGCAGTTGCTGTCAAAG-3'

M94548

GST

5'-ACTTCCCCAATCTGCCCTACTTA-3'

5'-CGAATCCGCTCCTCCTCTGT-3'

X04229

Cyclin D1

5'-TCAAGTGTGACCCGGACTGC-3'

5'-ACTTCCCCTTCCTCCTCGGT-3'

D14014

Beta defensin-1

5'-TCTTGGACGCAGAACAGATCAATA-3'

5'-TCCTGCAACAGTTGGGCTATC-3'

AF093536

H+, K+-ATPase

5'-ATTCCGCATCCCTAGACAACG-3'

5'-TCTTACTAAAGCTGGCCATGATGTT-3'

M90398

Prostaglandin D synthetase

5'-CAAGCTGGTTCCGGGAGAAG-3'

5'-TTGGTCTCACACTGGTTTTTCCTTA-3'

J04488

RBP

5'-TCGTTTCTCTGGGCTCTGGTAT- 3'

5'-TTCCCAGTTGCTCAGAAGACG-3'

M10934

Muc3

5'-AAGGTGTGAGGAAGTGATGGAGA-3'

5'-GCAGAGACCGTCGGCTTTATC-3'

U76551

BTEB1

5'-ACACTGGTCACCATCGCCAA-3'

5'-GGACTCGACCCAGATTCGGT-3'

NM_057211

BTEB2

5'-CTACTTTCCCCCATCACCACC-3'

5'-GAATCGCCAGTTTCGAAGCA-3'

AB096709

Serum Sst

Rat serum Sst content (15 animals from each diet) was determined using the somatostatin-28 EIA kit purchased from Phoenix Pharmaceuticals Corporation (Belmont, California).

Declarations

Acknowledgements

We thank Dr. Rosalia C.M. Simmen and Dr. Rick Helm for insightful comments on the manuscript and Amanda L. Linz for performing the I-FABP immuno-histochemistry. Supported by USDA CRIS 6251-5100-002-06S.

Authors’ Affiliations

(1)
Arkansas Children's Nutrition Center
(2)
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences

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© Xiao et al; licensee BioMed Central Ltd. 2005

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