Download Evaluation of tumor suppressor gene expressions and aberrant

Mol Biol Rep (2013) 40:5921–5929
DOI 10.1007/s11033-013-2699-8
Evaluation of tumor suppressor gene expressions and aberrant
methylation in the colon of cancer-induced rats: a pilot study
Veronika Polakova Vymetalkova • Luca Vannucci • Vlasta Korenkova •
Pavel Prochazka • Jana Slyskova • Ludmila Vodickova • Vendula Rusnakova
Ludovit Bielik • Monika Burocziova • Pavel Rossmann • Pavel Vodicka
•
Received: 28 March 2013 / Accepted: 14 September 2013 / Published online: 25 September 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Altered expression and methylation pattern of
tumor suppressor and DNA repair genes, in particular
involved in mismatch repair (MMR) pathway, frequently
occur in primary colorectal (CRC) tumors. However, little
is known about (epi)genetic changes of these genes in
precancerous and early stages of CRC. The aim of this pilot
study was to analyze expression profile and promoter
methylation status of important tumor suppressor and DNA
repair genes in the early stages of experimentally induced
colorectal carcinogenesis. Rats were treated with azoxymethane (AOM), dextran sodium sulphate (DSS) or
with their combination, and sacrificed 1 or 4 months posttreatment period. The down-regulation of Apc expression
in left colon, detectable in animals treated with DSS–AOM
and sacrificed 1 month after the end of treatment, represents most early marker of the experimental colorectal
carcinogenesis. Significantly reduced gene expressions
V. Polakova Vymetalkova P. Prochazka J. Slyskova L. Vodickova L. Bielik P. Vodicka (&)
Institute of Experimental Medicine, Academy of Sciences of the
Czech Republic, Videnska 1083, 14200 Prague 4,
Czech Republic
e-mail: [email protected]
V. Polakova Vymetalkova P. Prochazka J. Slyskova L. Vodickova L. Bielik P. Vodicka
First Faculty of Medicine and General Teaching Hospital,
Institute of Biology and Medical Genetics, Charles University,
Prague, Czech Republic
L. Vannucci M. Burocziova P. Rossmann
Institute of Microbiology, Academy of Sciences of the Czech
Republic, Prague, Czech Republic
V. Korenkova V. Rusnakova
Institute of Biotechnology, Academy of Sciences of the Czech
Republic, Prague, Czech Republic
were also found in 5 out of 7 studied MMR genes (Mlh1,
Mlh3, Msh3 Pms1, Pms2), regarding the sequential
administration of DSS–AOM at 4 months since the treatment. Strong down-regulation was also discovered for Apc,
Apex1, Mgmt and TP53. Tumors developed in rectumsigmoid region displayed significantly lower Apc and Pms2
expressions. The decreased expression of studied genes
was not in any case associated with aberrant methylation of
promoter region. Present data suggest that down-regulation
of Apc and MMR genes are prerequisite for the development of CRC. In this study we addressed for the first time
early functional alterations of tumor suppressor genes with
underlying epigenetic mechanisms in experimentally
induced CRC in rats.
Keywords Colorectal cancer Rats mRNA
expression Mismatch repair genes Promoter
methylation
Introduction
Colorectal cancer (CRC), an example of multistage human
carcinogenesis, is hallmarked by a sequence of events
during which normal colonic epithelium gradually transforms into carcinoma tissue, often via the development of
colorectal adenomas [1]. Genomic sequencing of human
CRCs has revealed frequent somatic mutations and deletions in a spectrum of tumor-related genes [1, 2].
Recently, epigenetic factors have been implicated in the
development of polyps and CRC. Alterations of gene
expression, often due to epigenetic inactivation of Apc and
DNA mismatch repair (MMR) genes by aberrant DNA
methylation, may be involved in the progression and
metastasis of CRC [3–5]. However, the causal role of
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aberrant DNA methylation in the process of cancer initiation and promotion is presently poorly understood [6]. It
has been postulated that the aberrant hypermethylation of
tumor suppressor genes can result in their transcriptional
silencing (lower mRNA expression), a mechanism through
which DNA methylation is believed to promote cancer
formation. There is lack of information about the precise
timing of these epigenetic alterations in the transition from
normal colon epithelium to malignant cells through the
adenoma-carcinoma sequence. The lack of mechanistic
data linking the promoter methylation with ultimate gene
expression levels in human colon carcinogenesis [6, 7]
highlights the need to closely examine this phenomenon in
controlled animal experiments.
A commonly used model to investigate the development
of colon cancer in rodents is administration of azoxymethane (AOM) [6]. AOM is a potent carcinogen causing a
high incidence of colon cancer in rodents [8]. Colon cancer
also represents the most serious complication associated
with chronic inflammatory bowel disease (IBD) [9]. The
risk of CRC increases with the extent and duration of the
IBD, and putative mechanisms of chronic or repeated
mucosal inflammation in CRC tumorigenesis have recently
been proposed [10, 11]. To explore an association of IBD
with CRC, the most frequently used model is the induction
of colon inflammation in mice and rats by dextran sodium
sulphate (DSS) administration [12]. The DSS induces
chronic colitis, which ultimately results in epithelial
malignant neoplasia [10, 11]. Recently, Lin and collaborators identified changes in DNA methylation associated
with the two major IBD subtypes, Crohn’s disease and
ulcerative colitis [13].
In our study of early stages of experimentally-induced
CRC, we have analyzed mRNA expression levels and
possible epigenetic alterations of tumor suppressor candidate genes Apc, Mlh1, Mlh3, Msh2, Msh3, Pms1, Pms2,
Exo1, Mgmt, Apex1, p16, TP53, and Cdh1 in conventional
(CV) rats. Animals were treated with AOM and/or DSS to
discern possible environment-independent markers of the
CRC evolution. The aim of this pilot study was to characterize mRNA expressions in tumor suppressor genes
during various time points in the early stages of experimental CRC development.
Materials and methods
Animals
The study was conducted on inbred male Wistar-AVN rats
([70 generations, at the starting of the experiments the
animals were 6–8 weeks old), obtained from the Institute
of Physiology, Academy of Sciences of the Czech Republic
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Mol Biol Rep (2013) 40:5921–5929
(ASCR) v.v.i., Prague, Czech Republic and reared under
standard conditions in the animal facility of the Institute of
Microbiology, ASCR v.v.i, Prague, Czech Republic. Rats
received standard diet and free water intake. All animal
experiments were approved by the Laboratory Animal Care
and Use Committee of the Institute of Microbiology v.v.i.,
ASCR, according to the Animal Protection Act of the
ASCR and the European Convention for the Care and Use
of Laboratory Animals.
Generation of AOM-induced tumors and DDS-induced
colitis
A total of 24 animals were used in this pilot experiment.
Animals were divided into four sub-groups according to the
treatment: six untreated control animals, six animals treated with AOM (Sigma-Aldrich, St. Louis, MO, USA), six
animals treated with DSS (MP Biomedicals, Santa Ana,
CA, USA), and six animals treated with both DSS and
AOM in sequence.
DSS (sterilized 3 % solution in drinking water) was
supplied orally ad libitum for about 10 days until appearance of acute symptoms of colitis (anal edema, diarrhea
and rectal bleeding). The animals received a total of three
courses with 1 week interruption between them (the average time for recovery from the acute symptoms). A second
group received AOM (9 mg/kg body weight, subcutaneously), administered once a week for 4 consecutive weeks.
In the DSS–AOM group, three courses of DSS treatment
were followed by AOM treatment performed once a week
for 4 weeks.
The study was designed to follow the development of
early stages of carcinogenesis, therefore three animals in
each of four sub-groups were sacrificed respectively at 1
and 4 months after the end of treatment. AOM induces
bulky cancers in a period of 6–8 months. Characteristics of
tumor development and appearance in the rat model of
colorectal carcinogenesis have been described in our earlier
studies [14–16].
Tissue harvesting
At the sacrifice, a complete autopsy was performed and the
large bowel was totally removed, longitudinally opened in
all its length and washed from stools. Then, colonic
mucosa was collected by gently scraping with a glass slide.
Samples of colonic mucosa (right and left colon) were
collected into cryocentrifuge tubes, snap-frozen and stored
at -80 °C. Presence of tumors was macroscopically evaluated and representative sampling of colon and tumors was
performed before the removal of the mucosa, for regular
histological diagnosis. Samples were fixed in formalin 5 %
and then embedded in paraffin to prepare 4 lm sections to
Mol Biol Rep (2013) 40:5921–5929
be stained with hematoxylin-eosin and pricrosirius (staining for collagen fibers) according to the standard protocols.
‘‘In blind’’ evaluation of the stained sections was performed by an expert pathologist.
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Table 1 Primers used in the MSP analysis and for verification of
methylation by MS-HRM
Gene
Tm (°C)
Primers
Apc
58.37
MF: AGGTTTTGGTTTTAGTTTTTGAGAC
59.47
MR: TAAAAATTCCACAACTTCGATACG
57.95
UF: AGGTTTTGGTTTTAGTTTTTGAGAT
57.31
UR: AAAAATTCCACAACTTCAATACATT
60.8
HRM F: GTGGATGTTTGATATGAATAGATGTT
60.2
HRM R: AACAACCCATACCCTACCTCTA
59.19
59.80
MF: GTTGTAGGTATGTGGAGGGTTTTAC
MR: ACAACGTAACCTCCAAAACGAT
58.40
UF: GTAGGTATGTGGAGGGTTTTATGA
58.83
UR: TACACAACATAACCTCCAAAACAAT
58.43
MF: GGGTATATAACGTTAGGTTAAACGG
59.53
MR: ACAAAAAACTCCATACTACTCCGAA
54.55
UF: GGGTATATAATGTTAGGTTAAATGG
59.13
UR: TATCTACAAAAAACTCCATACTACTCCAA
59.67
MF: GTCGAGTTTTGAGGTCGTAGTAAAC
59.95
MR: TCTCTAACCAATAAAAACGAAAACG
57.60
UF: GTTGAGTTTTGAGGTTGTAGTAAATG
57.81
UR: TCTAACCAATAAAAACAAAAACACC
59.19
MF: CGGGTTATGTTTGTGTGTGTATC
59.02
MR: CACTCCTCACTCCTAAACTACGAA
58.73
UF: AGTGGGTTATGTTTGTGTGTGTATT
58.89
60.4
UR: CCACTCCTCACTCCTAAACTACAA
HRM F: TAGTTTAGGAGTGAGGAGTGGAG
DNA and RNA isolation and the quality control
Total RNA and DNA were isolated using AllPrep DNA/
RNA mini kit according to the manufacturer protocol
(Qiagen, Czech Republic). Quantity and purity of both
RNA and DNA were measured using ASP-3700 Microvolume UV–Vis Spectrophotometer (Avans-Biotechnology, Taiwan). OD260/280 ratios for all samples were
between 1.8 and 2.0. After the isolation DNA and RNA
were stored at -80 °C. RNA integrity number (RIN) was
measured using capillary electrophoresis performed on
Agilent Bioanalyzer 2100, with RNA 6000 Nano Assay
(Agilent Technologies, Palo Alto, CA). RINs of all collected samples were not lower than 6.5 and thus the samples were appropriate for further analyses.
Cdh1
p16
Mlh1
Methylation-specific PCR (MSP)
Msh2
Methylation in promoter region was analyzed in 7 genes,
namely in tumor suppressors Apc and Cdh1, DNA repair
genes Mlh1, Msh2, Pms2 and Mgmt and cell cycle regulator p16.
Isolated genomic DNA from leukocytes was treated
with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) in order to generate a positive control for
methylation analysis. As reference samples we used two
aliquots of genomic DNA, the former methylated as above
and the latter without enzymatic SssI methylation. Sodium
bisulfite conversion of DNA was performed using the
Epitect Bisulfite Kit (Qiagen, Valencia, CA, USA) following the producer’s protocol. MSP analysis of bisulfitetreated DNA was carried out using the methylated and
unmethylated primer sets (Table 1), which were designed
using MethPrimer software (http://www.urogene.org/
methprimer/). MSP reactions were performed as previously described [17].
Methylation-sensitive high resolution melting
(MS-HRM)
MS-HRM was performed in order to verify positive findings obtained by MSP for Apc and Msh2 genes, as well as
to quantify the extent of cytosine methylation.
Real-time PCR followed by HRM was carried out by
high-performance Eco Real-Time PCR system (Illumina,
Inc., San Diego, CA, USA). Primers specific for bisulfiteconverted DNA (Table 1) were designed using Methyl
Primer Express Software v1.0 (Applied Biosystems, Foster
Pms2
Mgmt
60.5
HRM R: CTACCCTTCCACAATAATCCAT
58.52
MF: GTAGTTAATGGGTGGTTTAGGAGAC
58.43
MR: ACCGAAAACTCTATAAAAAATACGC
58.09
UF: GTAGTTAATGGGTGGTTTAGGAGAT
57.25
UR: AATACCAAAAACTCTATAAAAAATACACC
57.58
MF: TTGTTTATTAGGTTTCGTTTTACGT
57.77
MR: ATCTCCCTAAACTCTAAACTTCGAC
53.65
UF: TTGTTTATTAGGTTTTGTTTTATGT
55.77
UR: ATCTCCCTAAACTCTAAACTTCAAC
Tm melting temperature, MF methylated primers forward, MR
methylated primers reverse, UF unmethylated primers forward, UR
unmethylated primers reverse, HRM F primers forward designed for
MS-HRM, HRM R primers reverse optimized for MS-HRM
City, CA, USA). The reaction mixture in final volume of
10 lL consisted of 10 ng of bisulfite converted template
DNA, 19 EpiTect HRM Master Mix (Qiagen) and
300 nmol/L of each primer. PCR was initiated by incubation step at 95 °C for 5 min, followed by 50 cycles at
95 °C for 10 s in case of Apc, and 57 or 58 °C for 20 s in
case of Msh2, and finalized by 72 °C for 10 s. Melting
thermal profile was set up according to the manufacturer’s
recommendation (Qiagen). For each assay, a standard
dilution series of Rat High Methylated DNA control and
Rat Low Methylated DNA control (EpigenDx, Worcester,
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MA, USA) was run in parallel to assess the quantitative
properties and sensitivity of the assay. Fluorescence data
were converted into melting peaks by the Eco Software
(Illumina, Ver. 3.0.16.0).
in the mastermix was used ROX. Data were collected using
BioMark Data Collection Software 2.1.1., built
20090519.0926 (Fluidigm, USA). Expression experiments
were performed following MIQE guidelines [18].
Gene expression analysis
Data pre-processing and analysis
Reverse transcription
Obtained data were inspected for the correct baseline and
threshold and exported from BioMark Real-Time PCR
Analysis software (Fluidigm, USA). Data set contained
tested samples, interplate calibrator, NTC, positive control
and reference assays. Data were preprocessed in Genex
software (MultiD, version 5.1). Missing data were filled
with highest value in gene column ?1, which represented
one half of the lowest measured expression. All values
higher than value of NTC were removed. Positive control
was checked for positivity in all assays. The Grub’s test for
identification of outliers was performed on mean values of
technical replicates and outliers were removed. Interplate
calibration within all 5 plates was performed. Samples,
which were negative for at least one of their reference
gene, were removed from analysis (3 colon samples). Each
data set was normalized with appropriate reference genes:
Gapdh, Ubc. At the end, data were recalculated to relative
quantities with the lowest expression being set to 1, and
transformed to log2 scale. Assays and samples with more
than 12 % of missing data were removed from the dataset.
It concerns particularly Cdh1 gene.
Complementary DNA (cDNA) was synthesized from 1 lg
of total RNA by using RevertAidTM First strand cDNA
synthesis kit (MBI Fermentas, Vilnius, Lithuania) with
random hexamer primers in a final volume of 40 lL following manufacturer’s instructions. cDNA was stored at
-20 °C. Before use, cDNA was diluted ten times.
BioMarkTM qPCR analysis and gene specific
preamplification
In all tested samples, target gene assays together with
selected reference genes (described below), were run using
qPCR high throughput platform BioMarkTM HD System
(Fluidigm, USA). As reference genes, Gapdh and Ubc were
selected on the basis of a geNormTM reference gene
selection kit of 12 genes with PerfectProbeTM and analyzed
by both Genorm and Normfinder algorithms (GenEx Professional, MultiD Analyses AB, Göteborg, Sweden). The
cut off value for the level of stability of expression M was
0.15 and average pairwise variation was 2/3. All samples
were preamplified because of the need of highly concentrated cDNA for use on 48.48 Gene Expression Dynamic
Arrays (Fluidigm, USA). Preamplification was performed
with TaqmanÒ PreAmp Master Mix (Applied Biosystems)
with 2.5 lL of transcribed cDNA according to the manufacturer protocol in volume of 10 lL per reaction. Preamplified cDNA was stored at -20 °C. Samples were
distributed in four 48.48 Gene Expression Dynamic Arrays
(Fluidigm, USA). Each chip included no template control
(NTC), positive control and interplate calibrator. The interplate calibrator was a mixture of rat qPCR products. Each
sample was run in triplicate. Five microlitre sample premix
consisted of 2.25 lL of 59 diluted preamplified cDNA,
0.25 lL of 209 GE sample loading reagent (Fluidigm,
USA) and 2.5 lL of Taqman universal mastermix II
without UNG (Applied Biosystems). Each sample premix
was combined with 5 lL assay premix, which was
assembled from FAM-MGB custom designed 209
PrimerdesignÒ gene expression assay (Primer Design Ltd.,
Southampton, UK) and 29 Assay loading reagent (Fluidigm, USA). The reaction volume for a single qPCR
reaction was 6.7 nL. The thermal protocol for the qPCR
reaction was: 95 °C for 10 min followed by 45 cycles 15 s
long at 95 , and 50 °C for 60 s. As a passive reference dye
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Statistical analysis
Investigated parameters were normally distributed in the
study population (by Kolmogorov–Smirnov test). The
relationships between variables of interest at the bivariate
level were studied by means of two-tailed t test. All statistical tests were conducted at a 95 % confidence level;
expression data were corrected for multiple testing.
Results
Tumor occurrence
All animals treated with DSS had developed colitis with
typical symptoms. Macroscopically, the colon mucosa
showed a more pronounced vascular design, hypertrophy
of the lymphatic plaques, as a sign of immune activation by
persistent inflammatory stimulation. Microscopic examination showed typical inflammatory infiltrate in the mucosa
and submucosa, hypotrophy of crypts and increase in collagen fiber accumulation (fibrosis), well evident at
4 months. Malignancies were not detected either macroscopically or, in the taken samples, microscopically.
Mol Biol Rep (2013) 40:5921–5929
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We did not observe any tumor development in animals
treated only with AOM, irrespectively to the sacrifice time,
neither macroscopically nor microscopically, while aberrant crypts were present at the 4 month (not shown).
Animals treated with DSS and AOM in sequence and
sacrificed 1 month after the end of treatment resulted
negatively for colonic tumors at the inspection and
microscopy. However, at the fourth month evaluation, all
animals treated with DSS–AOM sequence had developed
multiple polypoid tumors of various dimensions (ranging
from 1 up to 6 mm in diameter and in a number from 10 up
to 21 per rat). They were located exclusively in the lowest
tract of left colon including the rectum-sigmoid tract.
Microscopy confirmed the presence of infiltrating adenocarcinomas in a chronic colitis context.
Gene expression
Gene expression profile was analyzed for 12 tumor suppressor genes, including Apc, p16, TP53, involved in cell
cycle regulation, Mlh1, Mlh3, Msh2, Msh3, Pms1, Pms2,
and Exo1 involved in DNA MMR pathway, and Mgmt and
Apex1 genes, acting in direct reversal and base excision
repair pathways.
We did not observe any significant differences in
expression of investigated genes in colon tissue of animals
treated with AOM, irrespective of observation time (the
overall gene expression data are summarized in Tables 2, 3).
In animals sacrificed 4 months after the end of DSS
treatment, approximately twofold decrease in expressions
of Pms2 in right colon mucosa and of Mlh1 in left colon
mucosa have been recorded (Table 3).
The only significant change in the expression profile in
animals treated with the combination of DSS–AOM and
sacrificed 1 month after the treatment was striking (nearly
30-fold) decrease in the expression of Apc in the left colon.
Regarding the animals treated with the combination of
DSS–AOM and sacrificed 4 months since the end of
treatment, a significant (more than twofold) decrease of
mRNA levels of Apc gene was observed in the right colon.
Table 2 Expression levels of tumor suppressor genes in the colon of animals sacrificed after 1 month (all expressions expressed as log2)
Gene
Mlh1
Tissue
Control
AOM
Mean ± SD
Mean ± SD
DSS
P
Mean ± SD
AOM ? DSS
P
Mean ± SD
P
RCM
6.98 ± 0.50
7.82 ± 0.53
0.570
8.27 ± 0.95
0.353
7.63 ± 1.22
0.776
LCM
6.40 ± 1.82
7.05 ± 1.34
0.857
7.01 ± 0.25
0.877
7.32 ± 0.42
0.761
Mlh3
RCM
11.54 ± 0.16
11.84 ± 0.59
0.873
11.79 ± 0.21
0.933
11.61 ± 0.85
0.998
LCM
10.62 ± 0.65
11.04 ± 0.97
0.776
10.97 ± 0.26
0.844
10.58 ± 0.01
0.999
Msh2
RCM
2.49 ± 0.53
3.64 ± 0.81
0.491
2.93 ± 0.32
0.937
3.66 ± 1.85
0.539
LCM
2.75 ± 1.15
3.27 ± 0.85
0.659
2.36 ± 0.40
0.879
2.74 ± 0.05
0.999
Msh3
RCM
LCM
2.47 ± 0.93
2.92 ± 1.29
3.27 ± 0.59
3.12 ± 0.80
0.819
0.981
2.50 ± 0.29
2.14 ± 0.20
0.999
0.556
3.70 ± 2.45
1.81 ± 0.33
0.652
0.389
Pms1
RCM
1.46 ± 0.47
2.64 ± 0.51
0.679
1.70 ± 0.47
0.995
3.59 ± 2.90
0.369
LCM
2.61 ± 1.41
2.14 ± 1.02
0.894
1.35 ± 0.44
0.347
1.52 ± 0.13
0.529
Pms2
RCM
1.65 ± 0.97
2.64 ± 0.50
0.784
1.83 ± 0.48
0.998
3.60 ± 2.88
0.439
LCM
3.27 ± 1.87
2.38 ± 0.77
0.894
1.48 ± 0.37
0.198
1.23 ± 0.20
0.190
Exo1
RCM
4.61 ± 2.58
7.07 ± 0.58
0.546
4.96 ± 0.21
0.933
7.66 ± 4.35
0.466
LCM
7.41 ± 2.54
6.44 ± 0.72
0.770
5.50 ± 0.75
0.334
5.83 ± 0.05
0.547
Mgmt
RCM
1.91 ± 0.49
2.57 ± 0.80
0.947
1.62 ± 0.76
0.996
3.54 ± 3.56
0.677
LCM
3.12 ± 2.54
3.05 ± 0.66
0.999
1.30 ± 0.61
0.877
0.32 ± 0.45
0.168
Apex1
RCM
1.55 ± 1.15
2.76 ± 0.76
0.646
1.84 ± 0.40
0.992
3.51 ± 2.48
0.395
LCM
2.85 ± 1.87
2.30 ± 1.14
0.899
1.79 ± 0.28
0.592
2.12 ± 0.03
0.845
Cdkn2a (p16)
RCM
9.24 ± 1.83
9.97 ± 2.46
0.995
4.86 ± 6.87
0.627
11.59 ± 5.20
0.896
LCM
12.11 ± 3.52
10.55 ± 1.44
0.881
7.99 ± 1.85
0.335
6.62 ± 5.87
0.223
TP53
RCM
5.49 ± 1.51
7.27 ± 1.28
0.411
7.00 ± 0.40
0.595
7.80 ± 1.85
0.305
Apc
LCM
RCM
6.09 ± 2.02
1.70 ± 0.08
6.92 ± 1.80
1.70 ± 0.19
0.842
0.963
6.06 ± 0.69
1.09 ± 0.46
0.999
0.914
7.09 ± 0.14
2.28 ± 2.62
0.813
0.923
LCM
2.10 ± 0.70
1.45 ± 0.55
0.405
1.07 ± 0.49
0.130
0.33 – 0.12
0.024
P value was used for comparison of means between control and treated animals
RCM right colon mucosa, LCM left colon mucosa
Significant P-values (P \ 0.05) are in bold
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Table 3 Expression levels of tumor suppressor genes in the respective colon tissues and tumors of animals sacrificed after 4 months (all
expressions expressed as log2)
Gene
Mlh1
Tissue
Control
AOM
Mean ± SD
Mean ± SD
DSS
P
AOM ? DSS
Mean ± SD
P
Mean ± SD
Tumors
P
Mean ± SD
P
RCM
7.92 ± 0.31
8.13 ± 0.59
0.958
8.09 ± 0.33
0.983
8.11 ± 0.83
0.968
–
LCM
8.47 ± 0.33
8.51 ± 0.25
0.998
7.21 – 0.60
0.022
5.98 – 0.26
0.001
8.37 ± 0.71
0.831
Mlh3
RCM
LCM
12.10 ± 0.16
11.93 ± 0.14
12.08 ± 0.31
11.00 ± 2.27
0.999
0.749
11.94 ± 0.81
10.65 ± 0.64
0.966
0.633
10.67 – 0.47
10.57 ± 0.01
0.036
0.592
–
10.48 – 0.80
0.014
Msh2
RCM
3.86 ± 0.38
4.29 ± 0.46
0.680
4.22 ± 0.85
0.804
3.84 ± 0.32
0.999
–
LCM
4.22 ± 0.11
4.45 ± 1.05
0.972
3.00 ± 1.07
0.327
3.24 ± 0.73
0.479
4.70 – 0.26
Msh3
RCM
3.84 ± 0.23
3.55 ± 0.22
0.599
3.32 ± 0.47
0.281
2.86 – 0.19
0.028
–
LCM
3.81 ± 0.17
3.68 ± 1.15
0.992
2.80 ± 0.24
0.335
2.32 ± 0.06
0.124
2.85 – 0.31
Pms1
RCM
3.57 ± 0.18
3.71 ± 0.21
0.931
3.61 ± 0.65
0.997
2.58 – 0.26
0.043
–
LCM
3.51 ± 0.32
3.98 ± 0.63
0.520
3.03 ± 0.07
0.602
1.82 – 0.56
0.020
3.10 ± 0.29
Pms2
RCM
4.50 ± 0.10
4.17 ± 0.30
0.381
3.29 – 0.20
0.007
2.58 – 0.26
0.001
–
0.017
0.008
0.078
LCM
3.59 ± 0.29
4.10 ± 0.63
0.471
3.03 ± 0.07
0.481
1.82 – 0.56
0.020
3.10 – 0.29
Exo1
RCM
8.51 ± 0.30
8.26 ± 0.88
0.948
6.90 ± 0.75
0.107
6.69 – 0.41
0.050
–
LCM
7.37 ± 0.61
7.85 ± 0.90
0.927
6.92 ± 2.43
0.953
4.82 ± 0.68
0.127
7.40 ± 0.69
Mgmt
RCM
5.50 ± 0.33
4.62 ± 0.90
0.542
3.08 ± 0.92
0.061
2.72 – 0.89
0.024
–
LCM
3.65 ± 0.56
4.61 ± 0.38
0.599
3.29 ± 2.41
0.969
3.11 ± 0.38
0.907
2.83 ± 0.80
Apex1
RCM
3.34 ± 0.11
3.68 ± 0.22
0.415
3.00 ± 0.11
0.470
2.49 – 0.39
0.028
–
3.35 ± 0.25
10.85 ± 0.40
3.67 ± 0.70
10.39 ± 1.13
0.773
0.993
2.96 ± 0.49
8.22 ± 3.50
0.748
0.607
1.32 – 0.36
7.37 ± 3.37
0.011
0.968
3.96 ± 0.44
–
0.051
Cdkn2a (p16)
LCM
RCM
LCM
8.49 ± 1.89
10.18 ± 1.32
0.798
8.38 ± 4.89
0.999
6.78 ± 3.30
0.855
9.58 ± 1.88
0.393
TP53
RCM
7.92 ± 0.39
8.76 ± 0.11
0.399
8.44 ± 0.56
0.752
7.70 ± 1.00
0.963
–
LCM
8.64 ± 0.29
8.32 ± 1.61
0.963
8.15 ± 0.15
0.915
5.02 – 0.77
0.018
8.65 ± 0.89
Apc
RCM
3.00 ± 0.59
2.59 ± 0.14
0.671
2.10 ± 0.65
0.225
1.37 – 0.52
0.022
–
LCM
2.77 ± 0.15
2.59 ± 0.67
0.963
1.78 ± 0.45
0.240
2.05 ± 0.91
0.453
0.59 – 0.30
0.036
0.953
0.144
0.982
0.0001
P value was used for comparison of means between corresponding tissues from control and treated animals or those from tumors, respectively
RCM right colon mucosa, LCM left colon mucosa
Significant P-values (P \ 0.05) are in bold
In the same animals, significant down-regulation of gene
expression was found in 5 out of 7 studied DNA MMR
genes, in comparison with control untreated rats (Table 3).
As expected, the down-regulation of DNA MMR genes
Mlh3, Msh3, Pms1 and Pms2 occurred predominantly in
the right colon mucosa. Interestingly, Mlh1 was significantly down-regulated in the left colon mucosa as well, so
were Pms1 and Pms2, respectively. The expression level of
Mgmt gene was fourfold lower in the right colon, two- to
sixfold down-regulation of Apex1 was observed in both the
right and left colon. A significant threefold down-regulation of the TP53 gene was recorded only in the left colon
when compared to right colon and to control animals.
Some genes, such as Apc, Mlh3, Msh3 and Pms2 were
further reduced in their transcription level in tumor tissue,
when compared to healthy tissue from the same colon
sequence. Only Msh2 was observed to be increased in
tumor samples (Table 3).
123
Methylation status
Methylation status was analyzed by MSP and all samples
providing positive signals (Apc and Msh2 genes) were reanalyzed by MS-HRM in order to obtain higher specificity.
None of the samples showed any aberrant methylation in
the promoter region of analyzed genes.
Discussion
The etiology of sporadic CRC is polygenic [19]. Currently,
functional variability in genes responsible for DNA repair
mechanisms and control of the cell cycle in the presence of
carcinogen-mediated cell damage is believed to be an
attractive mechanism for explaining interindividual variation in CRC susceptibility [2].
Mol Biol Rep (2013) 40:5921–5929
Analyses of phenotype concordance in monozygotic
twin CRC cases suggest that inherited susceptibility
underlies 35 % of all CRCs. However, only 6 % of CRCs
occur in the context of a known high-penetrance cancer
predisposition syndrome, such as Familial Adenomatous
Polyposis or Lynch Syndrome with characteristic mutations in DNA MMR genes Mlh1, Msh2, Msh6, or Pms2
[20]. Although, alterations of gene function, often due to
epigenetic inactivation of relevant tumor suppressor genes
by aberrant DNA methylation, may be involved in the
onset, progression and metastasis of CRC [3–5], most of
the genetic risks for CRC remain unknown.
In the present study we addressed for the first time early
functional alterations of tumor suppressor genes with
underlying epigenetic mechanisms in experimentally
induced CRC in rats.
The considerable down-regulation of the key tumor
suppressor gene Apc in the left colon of animals treated
with the combination of DSS–AOM and sacrificed 1 month
after the treatment suggests its critical role in early stages
of carcinogenesis. In later stages of experiment, Apc gene
expression in the non-malignant left colon non-significantly returns to the control levels, but in arising tumors
and in the right colon significantly decreases. These results
suggest different pathways leading to colon cancer in both
bowel segments with preferential early Apc down-regulation in the distal colon and MMR deficient tumors in the
proximal (right) colon, as described by [21]. Although Apc
inactivation has important role in evolution of precancerous adenomas and in tumorigenesis, colorectal neoplasias
may also occur irrespectively of Apc inactivation or
deregulation of Wnt pathway signaling, with possible
contribution of colonic bacterial environment [22]. In our
case, the Apc down-regulation was apparently not due to
methylation of Apc promoter region. However, aberrant
methylation of Apc gene is not very frequent (around
20 %) in human gastrointestinal cancers [23, 24]. Contemporary study hypothesizes that the lack of gross genetic
alterations (such as mutations, loss of heterozygosity, copy
number changes etc.) may not necessarily underlie Apc
down-regulation in animal model [25]. Apparently, mechanisms of early colorectal carcinogenesis in controlled
environment, animal models require further investigation.
Significantly reduced gene expression was found in 5
out of 7 studied MMR genes in relation to the combined
DSS–AOM treatment. The aberrant expression of DNA
mismatch genes occurred predominantly in the right colon
mucosa and these findings are in accord with the human
studies [26]. However, down-regulations in Mlh1 (the
major DNA MMR gene), Pms1 and Pms2 in the left colon
are in agreement with the finding of Svrcek et al. [27], who
described heterogeneous MMR defects (with no preference
to proximal colon), involving Mlh1, Msh2, Msh6 and Pms2
5927
in patients with IBD. Alterations in expression profiles of
MMR genes may be associated with a cascade of changes
in inflammatory cells (such as a production of reactive
oxygen and nitrogen species) which can modify regulation
of tumor suppressor genes, transcription factors, or signaling proteins [28].
The expression level of Mgmt gene, which is responsible
for the removal of methyl moieties on O6-position of
guanine, was four-fold lower in the right colon, indirectly
suggesting the role of the suppression of this repair
mechanism in right-sided colon carcinogenesis. Interestingly, Apex1 gene, which encodes apurinic site endonuclease and is involved in base excision repair, was downregulated two-to six-fold in both colonic segments. Apex1,
in cooperation with Ogg1, is also responsible for the
removal of oxidative DNA damage, often induced by
inflammatory processes. The suppression of Apex1
expression, more pronounced in the left colon, may be an
additional contributing factor in multifactorial colorectal
carcinogenesis.
Interestingly, the sequential treatment with DSS–AOM
with a sacrifice at 4 months was characterized by a significant three-fold down-regulation of the important tumor
suppressor gene, TP53 in the left colon. Apparently, a
relative accumulation of oxidative DNA damage (as a
consequence of sequential DSS–AOM treatment) due to
incomplete DNA repair may escape the surveillance by
TP53, a key gene regulating cell cycle, cell survival and
involved in DNA damage sensing, thus giving rise to
double-strand breaks and subsequent chromosomal instability. On the other hand, in human CRC Miladi-Abdennadher et al. [29] observed TP53 over-expression, which
was significantly correlated with distal (left) tumor location. It was also demonstrated in recent review by Van
Wezel et al. [30] that a loss of 17p (harboring TP53) in
human CRC is associated with predominant distal location.
Whereas, Perraud et al. [31] detected that TP53 protein
expression decreased progressively with CRC stage, suggesting that this protein is important marker of advanced
tumor stages.
We did not find any aberrant methylation in promoter
regions of any investigated tumor suppressor genes by using
two independent methods, as recommended by Kristensen
et al. [32]. The lack of aberrant methylation even in the
tumor tissues represents an interesting observation, since
gene silencing based on aberrant methylation of promoters
in tumor suppressor genes is an important mechanism in
carcinogenesis [19]. Nevertheless, the recent clinical studies
in humans CRC patients indeed showed that only a small
fraction of patients have a hypermethylation in any particular tumor suppressor genes; varying between 15 % for
RASSF1A and around 29 % for p14 and p16 and 27 % for
Apc genes [33]. However, the interaction between the
123
5928
Mol Biol Rep (2013) 40:5921–5929
epigenetic characteristics and inflammation in the tumor
development must be further investigated. For instance,
absence of hypermethylation in p16 and Mlh1 tumor suppressor genes was observed in association with colon cancer
arising on the inflammatory background [34, 35].
The results of our pilot study imply different pathways
leading to colon cancer in both bowel segments with
preferential early Apc down-regulation in the distal colon
and MMR deficient tumors in the proximal (right) colon, as
described by [21]. Due to the lack of altered tumor suppressor gene expressions in purely chemical carcinogenesis
(AOM treatment), further attention shall be dedicated to
the interactions between the inflammatory and bacterial
factors in the cell transformation process as it appears
driving force of the carcinogenic processes. A lack of
association between methylation in tumor suppressor genes
(representing rather low-frequency events) and their corresponding expressions could be due to the current design
of the study. Above aspect emerges therefore as a main
limitation of the present pilot study.
Acknowledgments The study was supported by grant GAAV
IAA500200917, IRC MBU RVO 61388971 and for IEM, GA CR P304/
11/P715, AVOZ50390703, AVOZ 50390512 and AV0Z50520701.
Conflict of interest
of interest.
The authors declare that there are no conflicts
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