Vol.2, No.8, 850-856 (2010)
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/
Openly accessible at
Promoter hypermethylation of tissue specific tumor
supressor genes and point mutation in K-ras, c-myc
proto-oncogenes in urinary (transitional cell)
bladder carcinoma
Oztürk Ozdemir1*, Esin Yildiz2, Semih Ayan3, Eylem Gul1, Gökhan Gokce3, Fazilet Yildiz1,
Binnur Koksal1
1Department of Medical Genetics, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey; *Corresponding Author:
2Department of Pathology, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey
3Department of Urology, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey
Received 8 November 2009; revised 26 December 2009; accepted 30 December 2009.
In a total of 83 UN specimens were investigated
for proto-oncogene mutations, tumor supressor
genes promoter methylation status and c-myc
and Ki-67 expression. Point mutations in c-myc
were detected in cases with high grade and
proliferation index. Mutated K-ras proto-onco-
gene profiles were detected in 17 (21%) tumoral
spiecemens that examined. Tumor specimens
were also showed hypermethylated promoter
domain for the SFRP2, MGMT tumor supressor
genes. These findings showed the combine ef-
fect of mutated c-myc and K-ras oncogene and
epigenetic inactivation of tissue specific tumor
supressor genes (TS) play a crucial role in tu-
mor progression and recurrence in UN car-
Keywords: C-myc; K-ras; Ki-67; Urinary Bladder;
Urothelial Neoplasms; Promoter Hypermethylation;
Tumor Supressor Genes
The urothelial (transitional cell) neoplasms (UNs) of the
urinary bladder are diseases of the entire urothelium
characterized and caused by multiple parameters. The
molecular events in cancer reveal that histopathologi-
cally identical tumors may arise through different mo-
lecular mechanisms such as instable genom, tumor sup-
pressor gene inactivation, proto-oncogene activation,
chromosomal rearrangements, point mutations, instabil-
ity of the microsatellite DNA resulting from deficient
DNA repair and multiple epigenetic alterations [1]. On-
coproducts that expressed from active oncogenes play
crucial role in eukaryotic cell transformation. The c-myc
proto-oncogene encodes a nuclear transcription factors
that playing an important role in the regulation of cell
proliferation, cell differentiation, cancer initiation and
progression [2]. K-ras is the other well known proto-
oncogene that encodes a cytoplasmic guanine nucleotide
binding protein and transduces external signals to the
nucleus. Acquired missense mutations in codons 12, 13
and 61, in or near the guanine nucleotide binding domain
of K-ras, have transforming potential upon the oncogene
by locking the RAS protein into a conformation that
promoted the continued stimulation of cell growth [3].
Mutational hot spots were reported in K-ras with high
frequency in variety of cancer types including lung,
breast, colorectal carcinoma (CRC) and pancreas cell
carcinomas [4-6]. As indicated by some researchers the
epigenetic alterations play important role in carcino-
genesis, tumor invasion and metastasis [7,8]. Tran-
sitional cell carcinomas of UNs have diverse molecular
and functional characteristics. In the last decade, aberra-
tions in DNA methylation patterns have been accepted to
be a common feature of human cancer [9]. Promoter
hypermethylation of CpG islands of some tumor sup-
pressor genes is strongly associated with tumor deve-
lopment and discriminated the tumors in a case and
organspecific manner. Tumor suppressor SFRP2 promoter
hypermethylation has been reported for some cancer
types such as bladder, breast, gastric, hepatocellular car-
cinoma and renal cell carcinomas [10-12]. In bladder
cancers, hypermethylation of the SFRP genes occurs
more often in active smokers and is a strong predictor of
poor patient survival [12]. Promoter hypermethylation of
O. Ozdemir et al. / HEALTH 2 (2010) 850-856
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/Openly accessible at
MGMT (O-6-methylguanine-DNA methyl transferase)
prevents the removal of groups at the O6 position of
guanine, leads to G-A transition in genome [13]. HIC1 is
a zinc finger transcriptional repressor activated by P53
and represses transcription of genes which activated by
E2F [14]. The other tumor suppressor gene, DAPK1
encodes a calcium calmoduline dependent protein kinase
and induces apoptosis positively [15]. Constitutive acti-
vation of K-ras and other oncogenes such as c-myc and
E2F stimulate DAPK1 activation [16,17].
In the current study, we aimed to investigate the effect
of mutated proto-oncogenes K-ras, c-myc and promoter
hypermethylation of some cancer-associated tumor sup-
pressor genes in urinary bladder carcinomas. The asso-
ciation between c-myc, Ki-67 expressions and tumoral
recurrence were also aimed to investigate immunohisto-
2.1. Patient and Biological Specimens
The current study was approved by the Research Ethical
Committee of Faculty of Medicine of Cumhuriyet Uni-
versity. The first part of the current study was designed
to investigate the correlation between c-myc and Ki-67
expressions and cell proliferation, tumor grade, stage
and recurrence. Fresh and paraffin-embedded tumoral
tissue samples from 83 patients underwent transurethral
resection (n = 78) and radical cystectomy (n = 5) at the
Department of Urology for UNs of the urinary bladder
between January 1997 and December 2008 were used in
the current study. Cases that were found to have new
tumoral growth in the cystoscopy after the 3 months
following the tumor resection were evaluated as recur-
rent tumors. The recurrent tumors in a high histological
grade and/or pathological stage were evaluated as pro-
gressive tumors. To detect the recurrence and/or pro-
gression in a total of 56 patients were followed up with
3-6 months of intervals for the first 3 years. Twenty
seven patients were lost during the current follow up.
The tumor samples were graded histologically based on
the WHO/ISUP and staged according to the TNM classi-
fication [15,16]. All fresh and paraffin-embedded speci-
mens were used for detecting promoter methylation
status for some target TS and point mutations in c-myc,
K-ras proto-oncogenes.
2.2. Immunostaining Procedure and
All fresh and paraffin-embedded samples were analised
by immunohistochemical staining procedure for Ki-67
and c-myc evaluation was performed with the avidin-
biotin-peroxidase (ABP) method. Immunostaining was
independently evaluated by three pathologist, and discor-
dances were resolved in a common reading. The samples
were fixed in 10% buffered formalin (pH = 7.0) and
embedded in parafin wax. Sections (5-μm) were used for
hematoxylin and eosin (H&E) staining for histological
evaluation, and for immunostaining with c-myc and
Ki-67 antibody. The immunohistochemical studies were
scored as follows. The Ki-67 Labeling Index (LI) and
c-myc were expressed as percentages of the Ki-67 and
c-myc positive cells by counting at least 1000 tumor
cells at 400X magnification.
2.3. SSCP Analysis
The PCR-SSCP (single strand conformation polymor-
phism) genotyping was done according to the basic pro-
tocol. Sections (10 μm thick) from all UN specimens
were used for genomic DNA isolation, in vitro gene am-
plification and epigenetic analysis. Total genomic DNA
was isolated by the nucleospin kit extraction technique
(Invitrogene, Germany) according to the basic DNA iso-
lation protocol with some modification [17]. Primers for
exon 2 of c-myc oncogene were obtained from MWG-
Biotech CimbH Paris/FRANCE. Amplifications of exon
2 of c-myc gene were performed in a volume of 50 l
reaction mixtures containing 200 M dNTPs (MBI,
Fermentas), 0.5 M primers, 30 ng template DNA, 10 ×
Taq DNA polymerase buffer, 1.5 U l Taq DNA poly-
merase (Boehringer, Mannheim) for 35 cycles in Am-
plitron I DNA Thermal Cycler (Thermolyne) under the
following conditions: denaturation at 96°C for 30 sec-
onds, annealing at 55°C for 30 seconds and extention at
72°C for 30 seconds.
2.4. Analysis of Mutation Status of the K-ras
Oncogene and Methylation Status of
the Promoter Region of TS Genes
After total genomic DNA isolation the target genes were
modified by sodium bisulfite technique and promoter
methylation status was determined. Direct in vitro am-
plification of codons 12 and 13 of proto-oncogene K-ras
and promoter methylation status for SFRP2, p16,
DAPK1, HIC1 and MGMT were performed by multi-
plex PCR based stripAssay reverse hybridisation analy-
sis (Vienalab). PCR was performed in a Perkin Elmer
9600 and the profile consisted of an initial melting step
of 2 minutes at 94°C; followed by 35 cycles of 30 sec-
onds at 94°C, 30 seconds at 61°C, and 30 seconds at 72°C;
and a final elongation step of 7 minutes at 72°C. The
mutation analysis was performed by stripAssay tech-
nique (Vienna Lab, StripAssay GmbH, Austria) which is
based on the reverse-hybridization principle automati-
cally. DNA methylation patterns in the promoter CpG
islands were determined in tumoral tissue samples by
methylation-specific PCR (MSP) following the bisul-
O. Ozdemir et al. / HEALTH 2 (2010) 850-856
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/Openly accessible at
fite modification of isolated genomic DNA.
2.5. Statistical Analysis
Statistical analysis of immunostaining findings was per-
formed by One-Way ANOVA (Kruskal Wallins test) for
tumor grade and stage, by Mann Whithney U test from
two-dependent-samples tests for recurrence and progres-
sion and/or to high grade and stage, and by Pearson rank
correlation to show the relationship between Ki-67 LI
and c-myc expression. The recorded data was independ-
ently processed by a biostatistician and performed using
SPSS software (RE10.0; SPSS, INC., Chicago, IL). Uni-
variate analysis was done using simple linear regression
analysis in relation to recurrence and progression. Mul-
tivariate analysis was done using multiple regression
analysis. A p value of < 0.05 was considered statistically
3.1. Clinicopathologic Data and Follow-Up
In a total of 83 UN tumors were examined in the current
study. Tumors grade were; 6 papilloma, 14 papillary
neoplasm of low malignant potential, 22 low grade pap-
illary carcinoma and 41 cases with high grade papillary
carcinoma. The stages of tumors that investigated were;
36 in pTa, 20 in pT1 and 27 cases were in pT2. Complete
follow-up was obtained for 56 (67.4%) of 83 UN pa-
tients. Forty four of 56 patients (78.5%) had recurrence.
Two (4.5%) of that reccurent 44 tumors had progression
into both high grade and stage and 42 tumors (95.5%)
were in high grade and/or stages.
3.2. Immunostaining and Statistical
Analysis of the Results
The c-myc and Ki-67 expression was not observed in
normal control tissues (n = 5). The Ki-67 expression was
detected in all tumoral tissues in different stage and
grades. Ki-67 antigen labelling was localized to the nu-
cleus with a fine, strong and homogenous brown granu-
larity (Figure 1). The relationships between progression
to high grade and/or stage with tumour grade (respec-
tively, p = 0.75, 0.06) and stage (respectively, p = 0.75,
0.06) were not statistically significant. But, Ki-67 LI
correlated with each one of tumor grade, stage, recur-
rence and/or progression and presence of invasion (p<
0.05). Positive staining for c-myc was defined as a nu-
clear and/or cytoplasmic staining pattern of epithelial
tumor cells, even if the staining was found diffusely or
focally in tumor cells (Figure 2). There was no statisti-
cally significant differences were found between c-myc
expression and each one of tumor grade, stage, recur-
rence and/or progression (p > 0.05), (Table 1). The
c-myc expression did not correlated with Ki-67 LI (r =
–0.06, p = 0.587; p > 0.05) as well. In univariate analysis,
higher tumor stage (p = 0.0001) and grade (p = 0.0001)
Table 1. The mean values of c-myc and Ki-67 expression in the cases with UN in relation to variables (with Kruskal Wallins test for
tumor grade and stage, Mann Whithney U test for tumoral recurrence, progression to high grade and/or stage).
Ki-67, LI
(mean% ±
P value in relation to
Ki-67 expression
(mean% ±
P value in relation to
c-myc expression
Papillom (n = 6)
UN of LMP (n = 14)
LGUC (n = 22)
HGUC (n = 41)
1.83 ± 0.40
5.07 ± 1.81
11.36 ± 3.82
30.60 ± 9.30
11.00 ± 8.24
11.85 ± 10.05
9.13 ± 8.71
7.09 ± 7.37
pT stage
pTa (n = 36)
pT1 (n = 20
pT2 (n = 27)
8.91 ± 7.77
19.45 ± 10.03
32.48 ± 9.79
10.55 ± 10.10
6.80 ± 5.21
7.70 ± 7.32
Tumoural recurrence in 56 patients
with follow up
Absent (n = 12)
Presence (n = 44)
4.00 ± 2.92
23.09 ± 14.09 0.0001 11.08 ± 7.64
8.59 ± 9.13 0.158
Progression to high grade in 44 tu-
mours with recurrence
Absent (n = 27)
Presence (n = 17)
2.50 ± 0.70
10.64 ± 3.85 0.023 11.50 ± 12.02
10.82 ± 11.53 0.842
Progression to high stage in 44 tu-
mours with recurrence
Absent (n = 34)
Presence (n = 10)
17.00 ± 14.84
24.88 ± 13.57 0.062 7.80 ± 9.19
8.82 ± 9.23 0.546
LI, labelling index; UN of LMP, urothelial neoplasm of low malignant potential; LGUC, low grade urothelial carcinoma; HGUC, high-grade urothe-
lial carcinoma.
O. Ozdemir et al. / HEALTH 2 (2010) 850-856
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Openly accessible at
were associated with higher recurrence ratio. In a multi-
variate analysis including the factors tumor grade and
stage, c-myc and Ki-67 expression, independent prog-
nostic value was seen tumor grade for recurrence (p
0.0001), the Ki-67 expression was statistically signifi-
cant (p 0.0001) for progression to high tumor grade.
No factor had independent prognostic value for progres-
sion to high tumor stage in the current results.
3.3. Analysis of Exon 2 of C-myc and Codon
12-13 of K-ras Proto-Oncogenes
The mechanism underlying c-myc expression was fur-
ther investigated in 54 bladder tumor samples. The exon
Figure 1. A fine, strong and homogenous brown granularity in
the nuclei of the tumoral cells with Ki-67 antibody (Avidin
Biotine Peroxidase tecnique, × 150). (a) High-grade papillary
carcinoma; (b) Low-grade papillary carcinoma; (c) Low ma-
lignant potential papillary neoplasm; (d) Papilloma.
Figure 2. A nuclear and/or cytoplasmic staining pattern of
epithelial tumor cells for c-myc antibody (Avidin Biotine Per-
oxidase tecnique, × 150); (a) Both nuclear and cytoplasmic
staining in high-grade urothelial carcinoma; (b) Low-grade
urothelial carcinoma; (c) Nuclear staining in low malignant
potential urothelial neoplasm; (d) Cytoplasmic staining in low
malignant potential urothelial neoplasm; (e) Nuclear staining in
2 of c-myc gene was found mutated in one case which is
in high grade and advanced stage of bladder cancer
(Figure 3, arrow). Mutated KRAS proto-oncogene pro-
files were detected in 17 (21%) tumoral specimens that
examined. Ten point mutations were detected in codon13
Asp (Figure 4, lane 1), 5 were in codon12 Val and 2
point mutations were in codon12 Asp.
3.4. Promoter Hypermethylation Profiles in
Tumor Suppressor Genes
Clinic, promoter hypermethylation profiles, histopa-
thological and molecular parameters in bladder cancer of
current 83 UNs samples showed a high sensitivity and a
specificity. Tumor specimens were showed fully pro-
moter hypermethylation for MGMT(66%), SFRP2(44%)
and partially inactivation for other tumor suppressor
genes that examined. In general no epigenetic alterations
Figure 3. SSCP profiles of exon 2 of c-myc proto-oncogene in
human bladder’s papillary urothelial (transitional cell) neo-
plasm tissues. Arrow indicates mutated single strand (ss). ds:
double strand.
C 1 2 3MC 1 2 3M
Figure 4. Shows epigenetically modified profiles of the TS
genes of SFRP2, p16, DAPK1, HIC1, MGMT and point muta-
tions in codon 12 and 13 of proto-oncogene K-ras in control
and tumoral tissue samples of the current UN samples.
O. Ozdemir et al. / HEALTH 2 (2010) 850-856
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Table 2. Promoter hypermethylation status of distinct tissue
specific tumor supressor genes according to the tumoral
Total = 83
Profile Papillom
(n = 6)
UN of
(n = 14)
(n = 22)
(n = 41)
active gene
inactive gene
Fully methyleted,
inactive gene
active gene
inactive gene
Fully methyleted,
inactive gene
active gene
inactive gene
Fully methyleted,
inactive gene
active gene
inactive gene
Fully methyleted,
inactive gene
active gene
inactive gene
Fully methyleted,
inactive gene
TS, tumor supressor; SFRP2, secreted frizzled-related protein 2; p16,
death-associated protein kinase 2; DAPK1, death-associated protein
kinase 1; HIC1 hypermethylated in cancer 1; MGMT, O-6- methyl-
guanine-DNA methyltransferase
were detected in papilloma and LMP type tumors but
heterogenous type alterations were detected in LGUC
tumors (Table 2). Tumor supressor p16, DAPK1 and
HIC1 were showed partially inactivation in HGUC tu-
mors but fully active profiles in papilloma and other two
tumors that were in early stages (Table 2). The current
results showed fully inactivation (homozygous of both
allels) of SFRP2 and MGMT genes in especially in UN
tumors that HGUC stage (Figure 4, lanes 1-3, Table 2).
Normal active gene profiles (unmethylated) were de-
tected in healthy control group tissue for all studied TS
genes(blood) and no point mutation was detected in
c-myc (Figure 3, lanes 1-4) and K-ras proto-oncogenes
(Figure 4, lane C).
UN of the bladder is the second most common tumors of
the genitourinary tract and causes the second highest
number of deaths of all genitourinary tumors [18]. The
risk of recurrence in these patients after transurethral
resection and intravesical chemotherapy and/or immu-
notherapy occures between 33-70% [19]. As many as
20-30% of these recurrent tumors was found to have
progressed to a higher histological grade and/or patho-
logical stage [19,20]. However, it is not possible to make
accurate assumptions on a patient’s clinical outcome in
regards to prognosis simply by considering the grade and
stage of the tumor. Latest progressions in epigenetic
methodologies and gene silencing techniques opened a
new area for the identification of epigenetic parameters,
which appears to be more useful for UNs and other tu-
moral cell diagnosis and prognosis. Previous studies
have shown an association between cell proliferation and
tumor grade, stage and prognosis in bladder carcinoma
[21-23]. Nowadays, biomarkers that reported for UNs
have not shown sufficient sensitivity and specificity in
routine clinical practice in particular, the high-risk patient
groups that are at risk of progression and recurrence [24].
In the present study, a significant correlation was found
between Ki-67 expression and tumor grade, stage, re-
currence and/or progression. As claimed by Kim and
Bae, the promoter silencing of TS genes is most impor-
tant phenomenon that plays tumor specific effect in uri-
nary bladder due to reverse epigenetic changes [24,25].
Marsit and co-workers were examined the relationship
between epigenetic silencing of three TS genes, p16
(INK4A), RASSF1A and PRSS3 in a population-based
study of human bladder cancer. They were reported that
the promoter hypermethylation status of each gene was
detected in approximately 30% of bladder cancers [10].
Our results confirmed the association between gene hy-
permethylation status and advance tumoral differenti-
ations. Tumoral specimen showed fully methyl pattern
O. Ozdemir et al. / HEALTH 2 (2010) 850-856
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profiles for the SFRP2, MGMT and partially hyper-
methylated profile for the HIC1, p16 and DAPK1 genes
in different ratios according to the tumor grade in the
current results (Table 2, Figure 4).
The current results also showed the structural mut-
ations in exon 2 of c-myc and codons 12, 13 of the K-ra s
proto-oncogenes in high grade and advanced stage of
UN tumors. No correlation was found between c-myc
structural mutation and overexpression and tumor grade,
stage, recurrence, progression and/or invasion in the
current results. With a high-throughput tissue specific
microarray analysis Zaharieva et al. showed that the
alterations of the c-myc gene was linked to genetically
unstable bladder cancers that are characterized by a high
histologic grade and/or invasion [26]. Using fluo-
rescence in situ hybridization (FISH), Mahdy et al.
observed a correlation bettween c-myc copy number gain
and bladder cancer grade and stage [27]. On the other
hand Schmitz-Dräger et al. were not reported any corre-
lation between c-myc overexpression and tumor grade
and stage in 185 paraffin-embedded urothelial tissue
specimens by using immunohistochemical analysis [28].
Our results confirm that UNs have a heterogeneous
cell profiles, multiple molecular factors are likely to be
involved in tumor recurrence and progression. It has also
been verified that combine molecular biomarkers such as
epigenetic alterations and proto-oncogene point mut-
ations widely occur at a late stage of the invasive HGPC
tumors. The fully and/or partially silencing through
promoter region of SFRP2 and MGMT have a potential
clinical implications as a UNs tumor progression and
good target for the development of new therapeutic ap-
proaches such as adjuvant therapies with demethylating
agents. So we claimed that the tumoral tissue specific
idendification extremely need for the patient's survival.
The current project was supported by basic research fund of Cumhuri-
yet University (CUBAP). Authors want to thank to A. Sahin, F. Bektaş
and Z. CINAR for their kindly technical assistance.
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