Promoter hypermethylation of tissue specific tumor supressor genes and point mutation in K-ras, c-myc proto-oncogenes in urinary (transitional cell) bladder carcinoma

doi:10.4236/health.2010.28128

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Vol.2, No.8, 850-856 (2010)

doi:10.4236/health.2010.28128

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:

ozdemir615@yahoo.com

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.

ABSTRACT

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-

cinogenesis.

Keywords: C-myc; K-ras; Ki-67; Urinary Bladder;

Urothelial Neoplasms; Promoter Hypermethylation;

Tumor Supressor Genes

1. INTRODUCTION

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

851

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-

chemically.

2. MATERIALS AND METHODS

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

Evaluation

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

852

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

significant.

3. RESULTS

3.1. Clinicopathologic Data and Follow-Up

Knowledge

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).

Variables

Ki-67, LI

(mean% ±

Std.deviation)

P value in relation to

Ki-67 expression

c-myc

(mean% ±

Std.deviation)

P value in relation to

c-myc expression

WHO/ISUPGrade

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

0.0001

11.00 ± 8.24

11.85 ± 10.05

9.13 ± 8.71

7.09 ± 7.37

0.261

pT stage

pTa (n = 36)

pT1 (n = 20

pT2 (n = 27)

8.91 ± 7.77

19.45 ± 10.03

32.48 ± 9.79

0.0001

10.55 ± 10.10

6.80 ± 5.21

7.70 ± 7.32

0.203

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.

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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

papilloma.

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

854

Table 2. Promoter hypermethylation status of distinct tissue

specific tumor supressor genes according to the tumoral

grades.

WHO/ISUP Grade

Total = 83

Tumor

Supressor

Type

Epigenetic

Profile Papillom

(n = 6)

UN of

LMP

(n = 14)

LGUC

(n = 22)

HGUC

(n = 41)

Unmethylated,

active gene

100

Partially

methyleted-

heterozygous

inactive gene

SFRP2

Fully methyleted,

inactive gene

Unmethylated,

active gene

100

Partially

methyleted-

heterozygous

inactive gene

p16

Fully methyleted,

inactive gene

Unmethylated,

active gene

100

Partially

methyleted-

heterozygous

inactive gene

DAPK1

Fully methyleted,

inactive gene

Unmethylated,

active gene

100

Partially

methyleted-

heterozygous

inactive gene

HIC1

Fully methyleted,

inactive gene

Unmethylated,

active gene

Partially

methyleted-

heterozygous

inactive gene

MGMT

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).

4. DISCUSSION

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

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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.

5. ACKNOWLEDGEMENTS

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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