Download in serum of prostate cancer patients and its clinical relevance

The Prostate
Promoter Hypermethylation of GSTP1, AR,
and14-3 -3s in Serum of Prostate Cancer Patients
and its Clinical Relevance
Jochen Reibenwein,1 Dietmar Pils,1 Peter Horak,1 Birgit Tomicek,2
Gregor Goldner,2 Nina Worel,3 Katarzyna Elandt,1 and Michael Krainer1*{
1
Division of Oncology, Department of Internal Medicine I, Medical University of Vienna,Vienna, Austria
2
Department of Radiotherapyand Radiobiology, Medical University of Vienna,Vienna, Austria
3
Department of Bloodgroup Serologyand Transfusion Medicine, Medical University of Vienna,Vienna, Austria
OBJECTIVE. Hypermethylation of tumor suppressor genes (TSG) is thought to play an
important role in tumorigenesis of prostate cancer. The main focus of research was the detection
of TSG hypermethylation in cancer tissue. Our aim was to evaluate the feasibility of detection of
hypermethylated genes in serum of prostate cancer patients and its correlation with
clinicopathological parameters.
METHODS. One hundred twenty-five serum samples from 62 patients with hormone
refractory prostate cancer (HRPC), 14 patients with early disease, and 49 healthy controls were
examined. After DNA extraction and sodium-bisulfite treatment, conventional methylationspecific PCR (MSP) was performed for glutathione S-transferase P1 (GSTP1), androgen receptor
(AR), and 14-3-3s.
RESULTS. In serum of HRCP patients, frequency of GSTP1, AR, and 14-3-3s hypermethylation was 32.2, 40.3, and 86.6%, respectively. In serum of patients with early disease frequency
of GSTP1, AR, and 14-3-3s, hypermethylation was 21.4, 35.7, and 85.7%. In healthy
controls, frequency of GSTP1, AR, and 14-3-3s hypermethylation was 0, 26.5, and 55.1%,
respectively. There was a significant increase of frequency of TSG hypermethylation for GSTP1
and 14-3-3s in HRPC patients, in comparison with healthy controls. GSTP1 hypermethylation
in HRPC patients was significantly correlated with differentiation of cancer and metastatic
disease.
CONCLUSIONS. Hypermethylation of TSG can be detected in serum of prostate cancer
patients. Some hypermethylated TSG can be detected in serum of healthy controls. GSTP1
was not detectable in controls and correlated significantly with Gleason score and stage of
disease. Therefore, this gene may be a promising new tool in prostate cancer diagnosis.
Prostate # 2006 Wiley-Liss, Inc.
KEY WORDS:
prostate cancer; hypermethylation; serum; CpG island
INTRODUCTION
Prostate cancer has become the most commonly
diagnosed cancer among men and the third leading
cancer-related cause of death after lung/bronchus and
colon/rectum in western countries [1]. Detection of
early-stage prostate cancer offers the opportunity to a
therapy with curative intent, whereas for advanced
stages only palliative treatment remains as available
option. A mainstay in early prostate cancer detection is
the measurement of PSA in serum. Since the beginning
of the PSA era, a remarkable change in prostate cancer
2006 Wiley-Liss, Inc.
{
Professor of Medicine.
*Correspondence to: Michael Krainer, MD, Department of Internal
Medicine I, Medical University of Vienna, Waehringer Guertel 18-20,
A-1090 Vienna, Austria. E-mail: [email protected]
Received 20 September 2006; Accepted 4 October 2006
DOI 10.1002/pros.20533
Published online in Wiley InterScience
(www.interscience.wiley.com).
2
Reibenwein et al.
detection has occurred, leading to a significant
increase of newly diagnosed, nonpalpable cancers [2].
Nevertheless, an elevated PSA level is not specific to
prostate cancer and can be caused by manifold
nonmalignant pathologies like prostatic inflammation
and benign prostatic hypertrophy [3]. Hence, an
additional set of noninvasive screening markers is
needed for the detection of early prostate cancer.
Epigenetic changes like hypermethylation of CpG
islands promoters of tumor suppressor genes (TSG)
have been described as characteristics of malignant
cells [4]. Hypermethylated tumor-specific DNA can be
detected in body fluids like plasma, serum, urine, and
ejaculate of prostate cancer patients [5–8]. The origin
and the mechanism of release of this cell-free DNA is
still not well known. Cell-free DNA may be released
due to lysis of circulating cancer cells, tumor necrosis,
tumor apoptosis, or active release, respectively [9].
One of the most frequent hypermethylated TSG
found in serum of prostate cancer patients is GSTP1,
which belongs to a family of detoxifier enzymes
involved in DNA damage repair [10]. Hypermethylation of GSTP1 was detected by the use of conventional
MSP in 36–72% of plasma/serum samples of prostate
cancer patients, but it was not detectable in noncancerous controls [6,11]. Despite controversial data
whether GSTP1 serves as a real TSG or just as a
‘‘caretaker’’ gene, leading to increased sensitiveness to
oxidative DNA damage when loss of function [12], it is
a promising marker for prostate cancer screening.
Progress of prostate cancer is usually associated
with loss of androgen sensitivity by the tumor. This
represents a critical step in prostate cancer development.
Hypermethylation of the androgen receptor (AR)
and consequently, its downregulation is thought to be a
possible mechanism for the loss of androgen sensitiveness [13]. Hypermethylation of the AR has already
been described in prostate cancer tissue and cell lines
[14,15], but there are no reports of detection of AR
hypermethylation in body fluids of prostate cancer
patients.
14-3-3s plays a critical role in signal transduction
pathways and cell-cycle regulation [16]. In response to
DNA damage, 14-3-3s enforces a G2/M arrest by
inhibiting the cyclinB1-cdc2 complex from entering the
nucleus. This allows DNA repair before cell-cycle
progression [17]. Due to its function, 14-3-3s is
considered as a TSG in different cancers, including
prostate cancer [18]. Loss of or weak 14-3-3s expression
was reported as a very frequent event in prostate
cancer, seen in up to 97% of cases [19]. Downregulation
of 14-3-3s expression seems to be significantly
associated with 14-3-3s CpG methylation [20]. However, 14-3-3s hypermethylation can also be seen at a
The Prostate DOI 10.1002/pros
very high frequency in benign prostate pathologies like
benign prostate hypertrophy (BPH) [21]. No reports of
detection of 14-3-3s hypermethylation in body fluids of
cancer patients have been published so far.
The aim of our study was to detect hypermethylated
TSG in serum of prostate cancer patients and to
correlate the methylation status, with clinicopathological parameters.
METHODS
Patients and Sample Collection
Serum samples from 62 patients with hormone
refractory prostate cancer (HRPC) were examined in
this study (HRPC group). All patients of the HRCP
group were recruited consecutively from April 2003 to
November 2005 for a prospective clinical trial for HRPC
at the Department of Internal Medicine I, Division of
Oncology, Medical University of Vienna, and received
a chemotherapy, consisting of intravenous administration of docetaxel. Fourteen patients with histologically
confirmed primary prostate cancer were additionally
enrolled in this study (RT group). All RT patients were
recruited consecutively from March 2006 to June 2006
at the Department of Radiooncology and Radiobiology,
Medical University of Vienna, for radiation therapy of
early-stage prostate cancer, and received a primary
external beam radiotherapy with a maximum dose of
70–74 Gy (Table I). Forty-nine healthy male individuals
served as controls. Serum samples were collected
consecutively in May 2006 at the Department of
Transfusion Medicine, Medical University of Vienna,
during voluntary blood donation after informed consent was obtained.
All patients gave informed consent, according to the
criteria of the ethics committee of the Medical University of Vienna.
One milliliter of serum was collected from each
patient, prior to individual therapy. DNA was
extracted from serum using the QIAamp1 DNA Mini
Kit, according to the manufacturer’s instructions
(Quiagen, Germany, Hilden). After extraction, the
DNA level was measured using the PicoGreen1
dsDNA Quantification Kit (Molecular Probes, The
Netherlands, Leiden).
Bisulf|teTreatment
In brief, after denaturation with 3 M NaOH, DNA
was treated over 4 hr with 100 ml of a solution of 2.5 M
sodium disulfite and 6.25 mM hydrochinone. For
purification of bisulfite-treated DNA, the QIAquick1
PCR Purification Kit Protocol (Quiagen) was used
according to the manufacturer’s instructions.
Promoter Hypermethylation of GSTP1, AR, and14 -3 -3s
TABLE I. Patient Characteristics
Age (years)
Median
Range
Gleason score
2–4
5–7
8–10
Missing
Grade
G1
G2
G3
Missing
Gleason score/grade
Valid
Missing
Pathologic stage
T1
T2
T3
T4
Missing
Bone metastasis
Yes
No
Missing
LN metastasis
Yes
No
Missing
LN and bone metastasis
Yes
No
Missing
PSA (ng/ml)
Median
Range
DNA (ng/ml)
Median
Range
HRPC group
(n ¼ 62)
RT group
(n ¼ 14)
67
50–83
70 (ns)
58–76
1
12
18
31
0
12
1
1
1
8
6
47
0
1
0
13
45
17
14
0
4
1
13
11
11
9
5
0
0
0
44
11
7
0
14
0
19
24
19
0
14
0
13
42
7
0
14
0
57.9
0.01–3,779
6.0 (P < 0.001)
0.7–13.5
14.4
1.9–136.3
19.3 (ns)
6.4-32.7
ns, not significant.
and negative controls were added to all MSP reactions.
MSP products were loaded on 3% agarose gels,
prestained with ethidium bromide, separated electrophoretically, and visualized under ultraviolet illumination.
Statistical Analysis
For comparison of age distribution, PSA level and
DNA level between the different groups Mann–
Whitney U tests were performed. Correlations between
the TSG methylation status and clinicopathological
parameters were analyzed using the chi-square test and
Fisher exact test as appropriate. All statistical analyses
were performed with SPSS 13.0 (SPSS, Inc., Chicago, IL,
2004). P-values below 0.05 were considered as statistical significant. P-values were corrected for multiple
testing.
RESULTS
Our study population consisted of 62 patients with
hormone refractory prostate cancer (HRPC group),
14 patients with early-stage prostate cancer (RT
group), and 49 healthy male controls. Table I shows
the clinicopathological and demographic features of
the patients. Median PSA level was 57.9 ng/ml in the
HRPC group and 6.0 ng/ml in the RT group (P < 0.001).
In the RT group, all patients had early-stage prostate
cancer, whereas metastatic disease was diagnosed in
81% of patients in the HRPC group. The remaining
patients of the HRPC group had PSA rise only. No
significant difference was seen between the two groups
for age distribution and amount serum DNA level.
Forty-nine healthy male served as controls. The
methylation status of GSTP1, AR, and 14-3-3s in the
serum of all three groups was assessed by conventional
MSP (Table II). Representative examples of the results
TABLE II. Frequencies of TSGPromoter
Hypermethylation in Serum of HRCP Patients,
RTX Patients, and Controls
GSTP1
n/N
Methylation-Specif|c PCR
Methylation-specific PCR (MSP) was performed
using primers and conditions selected to especially
amplify bisulfite-treated promoter DNA for the gene
of interest. All oligo sequences and MSP conditions
are available upon request. The positive control was
normal human DNA treated with Sss I methyl
transferase before bisulfite modification. The negative
control was distilled water without template. Positive
The Prostate DOI 10.1002/pros
3
AR
%
a
HRPC group 20/62
RT group
3/14b
Controls
0/49
32.3
21.4
0
n/N
25/62
5/14
13/49
14-3-3s
%
40.3
35.7
26.5
n/N
%
c
33/38 86.8
12/14 85.7
27/49 55.1
All P-values corrected for multiple testing.
a
Significant differences between HRPC group and controls
(P < 0.001).
b
Significant differences between RT group and controls
(P ¼ 0.03).
c
Significant differences between HRPC group and controls
(P ¼ 0.006).
4
Reibenwein et al.
of the electrophoresis of MSP products are shown in
Figure 1. For GSTP1, the frequency of hypermethylation was 32.2, 21.4, and 0 in the HRCP group, RT
group, and controls, respectively. A highly significant
difference in the frequency of GSTP1 methylation was
seen between the HRPC group and controls (P < 0.001)
and between the RT group and controls (P ¼ 0.006),
respectively. For AR, the incidence of methylation in
the three groups was 40.3, 35.7, and 26.5%, respectively.
No significant increase of AR methylation frequency
between the groups was discovered. For 14-3-3s the
frequency of methylation was 86.8, 85.7, and 55.1%,
respectively. A significant difference in the methylation
frequency of 14-3-3s was seen between the HRPC
group and controls (P ¼ 0.03). GSTP1 hypermethylation was significantly associated with differentiation of
cancer in the HRPC group. Eleven of 24 patients (46%)
with Gleason score 8 or WHO Grade ¼ 3 showed
GSTP1 hypermethylation, compared to 2/22 patients
(9%) with Gleason score <8 or WHO Grade 2
(P ¼ 0.03). Moreover, a significant correlation of GSTP1
hypermethylation and presence of metastases were
found in the HRPC group. Ten of 19 patients (53%) with
lymph node metastases showed GSTP1 hypermethylation, in comparison to 7/38 patients (18%) without
metastases of the lymph nodes (P ¼ 0.02). Furthermore,
8/13 patients (62%) with lymph node metastases as
well as bone metastases presented GSTP1 hypermethylation, compared to 4/20 patients (20%) with bone
metastases only (P ¼ 0.03). No correlation was found
between the methylation status of the samples and age,
pathologic stage, presence of bone metastases,
response to treatment, overall survival, PSA level,
and DNA level.
Fig. 1. Representative examples of MSP analyses of the three
methylatedgenes14 -3-3s, AR, and GSTP1in serum of nineprostate
cancer patients. Amplification of the unmethylated form of14 -3-3s
wasusedascontrolforDNAintegrity.Thepositive controlisnormal
human DNA treated with Sss I methyl transferase before bisulfite
modification. The negative control is distilled water without
template.
The Prostate DOI 10.1002/pros
DISCUSSION
We examined the methylation status of the three
genes GSTP1, AR, and 14-3-3s in serum of prostate
cancer patients.
GSTP1 is an intensively investigated putative TSG in
prostate cancer. Hypermethylation of GSTP1 is the
most common somatic epigenetic alteration reported in
prostate cancer. Frequency of GSTP1 hypermethylation
in prostate cancer tissue varies from 36 to >90%
[22–24]. In nonmalignant tissues, GSTP1 hypermethylation seems to be a very rare event. It was found to be
hypermethylated in BPH in less than 5% [3,14,23].
In prostatic intraepithelial neoplasia, a potential
precursor of prostate cancer, frequency varies between
30 and 70% [14,25]. Only a few reports about GSTP1
hypermethylation in serum of cancer patients have
been published so far. The detection rate of GSTP1
hypermethylation in serum or plasma samples of
patients, with clinically localized prostate cancer
ranged from 12 to 36% [6,26]. In serum or plasma of
a study cohort, including patients with clinically
localized disease as well as advanced prostate cancer,
GSTP1 hypermethylation was described in 72% [11].
No GSTP1 hypermethylation was found in serum of
healthy controls or of patients with BPH [11,26]. In our
study, GSTP1 hypermethylation was found in the
HRPC group in 32% and in the RT group in 21% of
serum samples. According to the literature, no GSTP1
hypermethylation was detectable in serum of controls.
There are inconsistent reports about correlation of
GSTP1 hypermethylation in prostate cancer with
clinicopathological parameters. Maruyama et al.
reports a significant correlation of GSTP1 hypermethylation in prostate cancer tissue with Gleason score >7
and with PSA levels >8 ng/ml [22]. Other investigators
did not find such a correlation [23,24,27]. In a multivariate analysis of 55 men with clinically localized
prostate cancer, GSTP1 hypermethylation in serum
was the most significant predictor for PSA recurrence
[26]. We found a significant correlation of GSTP1
hypermethylation with Gleason score 8 or WHO
Grade ¼ 3 (P ¼ 0.03). Furthermore, a significant correlation with metastatic disease was seen (P ¼ 0.03).
Moreover, the presence of lymph node metastases
correlated significantly with GSTP1 hypermethylation
(P ¼ 0.02). Bone metastases only did not correlate
with GSTP1 hypermethylation. As far as we know, this
is the first report about correlation of clinicopathological parameters, with methylation status in body
fluids of prostate cancer patients. GSTP1 hypermethylation is not detectable in healthy controls. Hence,
it seems to be a promising screening marker for
prostate cancer in tissue and serum samples. Further
efforts are needed to be done to establish GSTP1 as an
Promoter Hypermethylation of GSTP1, AR, and14 -3 -3s
additional diagnostic tool both in prostate tissue and
body fluids.
The progression from androgen sensitive to HRPC is
a critical step in the natural course of prostate cancer
and displays a fatal watershed in the individual
medical history of most patients. Hypermethylation
of the AR promoter downregulates AR expression and
is thought to be one mechanism for development of
HRPC, with a methylation frequency of 20% in primary
prostate cancer tissues and of 28% in HRPC tissues [28].
Interestingly, Yamanaka et al. reported a decreasing
AR methylation frequency of 50% for stage A, 24% for
stage B, 10% for stage C, and 6% for stage D prostate
cancer, respectively [14]. No reports about AR methylation in serum of prostate cancer patients have been
published so far. In our present study, AR hypermethylation in the HRPC group, in the RT group, and in
controls was seen in 40, 36, and 27%, respectively.
Thus, because of the presence of AR methylation in
healthy male controls, there must be other reasons for
AR methylation beside carcinogenesis. It has been
demonstrated that many genes are methylated as a
function of age [29], but in our study, no association
of methylation status and age was found (data not
shown).
Loss of 14-3-3s expression is a very frequent event,
related to progression from normal epithelium to PIN
and invasive prostate cancer. In normal prostate
epithelium, expression of 14-3-3s was moderate or
strong in all samples, whereas 90% of PIN and 97% of
invasive prostate cancer showed negative or weak
expression, respectively [19]. Downregulation of 14-33s expression was seen to be significantly correlated
with CpG island methylation in prostate cancer [20].
However, 14-3-3s hypermethylation is not a carcinogenesis-specific event. Hypermethylation was detectable in 99% of prostate cancer, 100% of PIN, and 100% of
BPH tissue samples, respectively [21]. Hypermethylation of 14-3-3s can also be seen in normal lymphoid
cells [30]. In our present study, 14-3-3s hypermethylation was found in serum samples of the HRPC group,
the RT group, and controls in 87, 86, and 55%,
respectively. The difference between HRPC group
and controls was statistically significant (P ¼ 0.006).
14-3-3s was not methylated as a function of age, so
there must be other reasons for hypermethylation of
14-3-3s in healthy controls.
Recapitulating, MSP is a feasible method for detection of hypermethylated TSG in serum of prostate
cancer patients. GSTP1 seems to be a promising
candidate for further development to an additional
diagnostic tool for prostate cancer, based on prostate
tissues and serum samples. More intensive research
for mechanisms of hypermethylation of genes, besides
carcinogenesis and aging, is essential for underThe Prostate DOI 10.1002/pros
5
standing epigenetic alterations in cancer and establishing new approaches in cancer diagnosis and screening.
REFERENCES
1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, Thun MJ.
Cancer statistics, 2006. CA Cancer J Clin 2006;56:106–130.
2. Brawer MK. Prostate cancer: Epidemiology and screening.
Prostate Cancer Prostatic Dis 1999;2:2–6.
3. Bastian PJ, Ellinger J, Wellmann A, Wernert N, Heukamp LC,
Muller SC, von RA. Diagnostic and prognostic information in
prostate cancer with the help of a small set of hypermethylated
gene loci. Clin Cancer Res 2005;11:4097–4106.
4. Jones PA, Baylin SB. The fundamental role of epigenetic events in
cancer. Nat Rev Genet 2002;3:415–428.
5. Boddy JL, Gal S, Malone PR, Harris AL, Wainscoat JS.
Prospective study of quantitation of plasma DNA levels in the
diagnosis of malignant versus benign prostate disease. Clin
Cancer Res 2005;11:1394–1399.
6. Jeronimo C, Usadel H, Henrique R, Silva C, Oliveira J, Lopes C,
Sidransky D. Quantitative GSTP1 hypermethylation in bodily
fluids of patients with prostate cancer. Urology 2002;60:1131–
1135.
7. Papadopoulou E, Davilas E, Sotiriou V, Koliopanos A, Aggelakis
F, Dardoufas K, Agnanti NJ, Karydas I, Nasioulas G. Cell-free
DNA and RNA in plasma as a new molecular marker for prostate
cancer. Oncol Res 2004;14:439–445.
8. Goessl C, Muller M, Straub B, Miller K. DNA alterations in body
fluids as molecular tumor markers for urological malignancies.
Eur Urol 2002;41:668–676.
9. Stroun M, Maurice P, Vasioukhin V, Lyautey J, Lederrey C,
Lefort F, Rossier A, Chen XQ, Anker P. The origin and
mechanism of circulating DNA. Ann N Y Acad Sci 2000;906:
161–168.
10. Henderson CJ, Wolf CR. Disruption of the glutathione transferase pi class genes. Methods Enzymol 2005;401:116–135.
11. Goessl C, Krause H, Muller M, Heicappell R, Schrader M,
Sachsinger J, Miller K. Fluorescent methylation-specific
polymerase chain reaction for DNA-based detection of
prostate cancer in bodily fluids. Cancer Res 2000;60:5941–
5945.
12. Nelson WG, De Marzo AM, Deweese TL, Lin X, Brooks JD, Putzi
MJ, Nelson CP, Groopman JD, Kensler TW. Preneoplastic
prostate lesions: An opportunity for prostate cancer prevention.
Ann N Y Acad Sci 2001;952:135–144.
13. Suzuki H, Ueda T, Ichikawa T, Ito H. Androgen receptor
involvement in the progression of prostate cancer. Endocr Relat
Cancer 2003;10:209–216.
14. Yamanaka M, Watanabe M, Yamada Y, Takagi A, Murata T,
Takahashi H, Suzuki H, Ito H, Tsukino H, Katoh T, Sugimura Y,
Shiraishi T. Altered methylation of multiple genes in carcinogenesis of the prostate. Int J Cancer 2003;106:382–387.
15. Kinoshita H, Shi Y, Sandefur C, Meisner LF, Chang C, Choon A,
Reznikoff CR, Bova GS, Friedl A, Jarrard DF. Methylation of the
androgen receptor minimal promoter silences transcription in
human prostate cancer. Cancer Res 2000;60:3623–3630.
16. Fu H, Subramanian RR, Masters SC. 14-3-3 proteins: Structure,
function, and regulation. Annu Rev Pharmacol Toxicol 2000;40:
617–647.
17. Chan TA, Hermeking H, Lengauer C, Kinzler KW, Vogelstein B.
14-3-3Sigma is required to prevent mitotic catastrophe after
DNA damage. Nature 1999;401:616–620.
6
Reibenwein et al.
18. Lodygin D, Hermeking H. The role of epigenetic inactivation of
14-3-3sigma in human cancer. Cell Res 2005;15:237–246.
metastatic human prostate cancer. Cancer Res 2004;64:1975–
1986.
19. Cheng L, Pan CX, Zhang JT, Zhang S, Kinch MS, Li L, Baldridge
LA, Wade C, Hu Z, Koch MO, Ulbright TM, Eble JN. Loss of 14-33sigma in prostate cancer and its precursors. Clin Cancer Res
2004;10:3064–3068.
25. Brooks JD, Weinstein M, Lin X, Sun Y, Pin SS, Bova GS, Epstein JI,
Isaacs WB, Nelson WG. CG island methylation changes near the
GSTP1 gene in prostatic intraepithelial neoplasia. Cancer
Epidemiol Biomarkers Prev 1998;7:531–536.
20. Mhawech P, Benz A, Cerato C, Greloz V, Assaly M, Desmond JC,
Koeffler HP, Lodygin D, Hermeking H, Herrmann F, Schwaller J.
Downregulation of 14-3-3sigma in ovary, prostate and endometrial carcinomas is associated with CpG island methylation. Mod
Pathol 2005;18:340–348.
26. Bastian PJ, Palapattu GS, Lin X, Yegnasubramanian S, Mangold
LA, Trock B, Eisenberger MA, Partin AW, Nelson WG.
Preoperative serum DNA GSTP1 CpG island hypermethylation
and the risk of early prostate-specific antigen recurrence
following radical prostatectomy. Clin Cancer Res 2005;11:
4037–4043.
27. Bastian PJ, Ellinger J, Schmidt D, Wernert N, Wellmann A,
Muller SC, von RA. GSTP1 hypermethylation as a molecular
marker in the diagnosis of prostatic cancer: Is there a correlation
with clinical stage, Gleason grade, PSA value or age? Eur J Med
Res 2004;9:523–527.
21. Henrique R, Jeronimo C, Hoque MO, Carvalho AL, Oliveira J,
Teixeira MR, Lopes C, Sidransky D. Frequent 14-3-3 sigma
promoter methylation in benign and malignant prostate lesions.
DNA Cell Biol 2005;24:264–269.
22. Maruyama R, Toyooka S, Toyooka KO, Virmani AK, ZochbauerMuller S, Farinas AJ, Minna JD, McConnell J, Frenkel EP, Gazdar
AF. Aberrant promoter methylation profile of prostate cancers
and its relationship to clinicopathological features. Clin Cancer
Res 2002;8:514–519.
23. Singal R, Ferdinand L, Reis IM, Schlesselman JJ. Methylation of
multiple genes in prostate cancer and the relationship with
clinicopathological features of disease. Oncol Rep 2004;12:631–
637.
24. Yegnasubramanian S, Kowalski J, Gonzalgo ML, Zahurak M,
Piantadosi S, Walsh PC, Bova GS, De Marzo AM, Isaacs WB,
Nelson WG. Hypermethylation of CpG islands in primary and
The Prostate DOI 10.1002/pros
28. Nakayama T, Watanabe M, Suzuki H, Toyota M, Sekita N,
Hirokawa Y, Mizokami A, Ito H, Yatani R, Shiraishi T. Epigenetic
regulation of androgen receptor gene expression in human
prostate cancers. Lab Invest 2000;80:1789–1796.
29. Ahuja N, Issa JP. Aging, methylation and cancer. Histol
Histopathol 2000;15:835–842.
30. Bhatia K, Siraj AK, Hussain A, Bu R, Gutierrez MI. The tumor
suppressor gene 14-3-3 sigma is commonly methylated in
normal and malignant lymphoid cells. Cancer Epidemiol
Biomarkers Prev 2003;12:165–169.