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Molecular Cancer
Open Access
Research
Role of promoter hypermethylation in Cisplatin treatment
response of male germ cell tumors
Sanjay Koul
†1
, James M McKiernan
†2
, Gopeshwar Narayan
1
,
Jane Houldsworth
3,4
, Jennifer Bacik
5
, Deborah L Dobrzynski
4
,
Adel M Assaad
1
, Mahesh Mansukhani
1
, Victor E Reuter
6
, George J Bosl
4
,
Raju SK Chaganti
3,4
and Vundavalli VVS Murty*
1,7
Address:
1
Department of Pathology, College of Physicians & Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032, USA,
2
Department of Urology, College of Physicians & Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032, USA,
3
The Cell
Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA,
4
Department of Medicine, Memorial Sloan-Kettering
Cancer Center, New York, NY 10021, USA,
5
Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York,
NY 10021, USA,
6
Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA and
7
Institute for Cancer
Genetics, College of Physicians & Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032, USA
Email: Sanjay Koul - sk1276@columbia.edu; James M McKiernan - jmm23@columbia.edu; Gopeshwar Narayan - gn110@columbia.edu;
Jane Houldsworth - houldswj@mskcc.org; Jennifer Bacik - bacik1@MSKCC.ORG; Deborah L Dobrzynski - dobrzynd@MSKCC.ORG;
Adel M Assaad - aa2071@columbia.edu; Mahesh Mansukhani - mm322@columbia.edu; Victor E Reuter - reuterv@mskcc.org;
George J Bosl - boslg@mskcc.org; Raju SK Chaganti - chagantr@mskcc.org; Vundavalli VVS Murty* - vvm2@columbia.edu
* Corresponding author †Equal contributors
Abstract
Background: Male germ cell tumor (GCT) is a highly curable malignancy, which exhibits exquisite
sensitivity to cisplatin treatment. The genetic pathway(s) that determine the chemotherapy
sensitivity in GCT remain largely unknown.
Results: We studied epigenetic changes in relation to cisplatin response by examining promoter
hypermethylation in a cohort of resistant and sensitive GCTs. Here, we show that promoter
hypermethylation of RASSF1A and HIC1 genes is associated with resistance. The promoter
hypermethylation and/or the down-regulated expression of MGMT is seen in the majority of
tumors. We hypothesize that these epigenetic alterations affecting MGMT play a major role in the
exquisite sensitivity to cisplatin, characteristic of GCTs. We also demonstrate that cisplatin
treatment induce de novo promoter hypermethylation in vivo. In addition, we show that the
acquired cisplatin resistance in vitro alters the expression of specific genes and the highly resistant
cells fail to reactivate gene expression after treatment to demethylating and histone deacetylase
inhibiting agents.
Conclusions: Our findings suggest that promoter hypermethylation of RASSF1A and HIC1 genes
play a role in resistance of GCT, while the transcriptional inactivation of MGMT by epigenetic
alterations confer exquisite sensitivity to cisplatin. These results also implicate defects in epigenetic
pathways that regulate gene transcription in cisplatin resistant GCT.
Published: 18 May 2004
Molecular Cancer 2004, 3:16
Received: 06 May 2004
Accepted: 18 May 2004
This article is available from: http://www.molecular-cancer.com/content/3/1/16
© 2004 Koul et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.
Molecular Cancer 2004, 3 http://www.molecular-cancer.com/content/3/1/16
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Background
Adult male germ cell tumors (GCTs) are considered to be
a model system for a curable malignancy because of their
exquisite sensitivity to cisplatin (CDDP)-based combina-
tion (cisplatin, etoposide, with or without bleomycin)
chemotherapy. Histologically, GCTs present as a germ cell
(GC)-like undifferentiated seminoma (SGCT) or a differ-
entiated nonseminoma (NSGCT). NSGCTs display com-
plex differentiation patterns that include embryonal,
extra-embryonal, and somatic tissue types [1]. Further-
more, embryonal lineage teratomas differentiate into var-
ious somatic cell types that may undergo malignant
transformation to epithelial, mesenchymal, neurogenic,
or hematologic tumors [2]. Seminomas are exquisitely
sensitive to radiation therapy while NSGCTs are highly
sensitive to treatment with CDDP-based chemotherapy.
Despite this sensitivity to chemotherapy, 20–30% of met-
astatic tumors are refractory to initial treatment, requiring
salvage therapy and accounting for high mortalitiy. Such
patients are treated with high dose and experimental
chemotherapy protocols [3]. The underlying molecular
basis of this exquisite drug responsiveness of GCT remains
to be fully understood [4].
Little is known about the genetic basis of chemotherapy
response in GCT. Studies have previously identified that
TP53 mutations and gene amplification may play a role in
GCT resistance [5,6]. It has also been recently shown that
microsatellite instability is associated with the treatment
resistance in GCT [7]. An epigenetic alteration by pro-
moter hypermethylation that plays a role in inactivating
tumor suppressor genes in a wide-variety of cancers also
has been shown to occur in GCT [8-10]. We previously
showed the absence of promoter hypermethylation in
SGCT and acquisition of unique patterns of promoter
hypermethylation in NSGCT [8]. However, the role of
such epigenetic changes in GCT resistance and sensitivity
remains unknown.
In the present study, we evaluated the status of hyper-
methylation in 22 gene promoters in 39 resistant and 31
sensitive NSGCTs. We found that RASSF1A and HIC1 pro-
moter hypermethylation was associated with highly resist-
ant tumors. Evidence was also obtained suggesting that
promoter hypermethylation is induced against the initial
CDDP treatment and that this hypermethylation plays a
crucial role in further treatment response. We show that
changes in the patterns of gene expression occur during
the in vitro acquisition of a highly refractory tumor to
CDDP, which irreversibly affects the response to demeth-
ylating and histone deacetylase inhibiting agents.
Results
Promoter hypermethylation in relation to chemotherapy
resistance and sensitivity
Based on our previous observations in GCT, we studied 22
gene promoters for hypermethylation in 70 NSGCTs
derived from 60 patients [8]. Promoter hypermethylation
was found in nine of 22 genes examined. One or more
genes were methylated in 41 (59%) tumors. The fre-
quency of hypermethylation for each of the genes was:
RASSF1A (35.7%), HIC1 (31.9%), BRCA1 (26.1%), APC
(24.3%), MGMT (20%), RARB (5.7%), FHIT (5.7%),
FANCF (5.7%), and ECAD (4.3%). This frequency was
similar to our previously published observations on unse-
lected patients with NSGCTs [8].
The frequency of overall promoter hypermethylation (one or
more of the 22 genes methylated) was similar in the sensitive
(18 of 29 patients; 62%) and resistant (21 of 31 patients;
68%) tumors. However, the frequency of promoter hyper-
methylation of individual genes differed between sensitive
and resistant tumors. RASSF1A (52% in resistant vs. 28% in
sensitive) and HIC1 (47% in resistant vs. 24% in sensitive)
genes showed higher frequency of promoter hypermethyla-
tion in resistant tumors (Table 2, Fig. 1). These differences
were not statistically significant due to small number of
tumors studied. However, the differences were more pro-
nounced when the sensitive and highly resistant tumors were
compared (discussed below). On the other hand, the sensitive
tumors exhibited higher frequency of promoter hypermethyl-
ation compared to resistant tumors in MGMT (31% vs. 13%)
and RARB (14% vs. 0%; P = 0.05) (Table 2, Fig. 1). Other
genes that exhibited frequent hypermethylation showed no
significant differences (APC, 24% vs. 29%; BRCA1, 31% vs.
30%) between the sensitive and resistant groups. These data,
thus, suggest that promoter hypermethylation of RASSF1A
and HIC1 is associated with chemotherapy resistance pheno-
type, while promoter hypermethylation of MGMT and RARB
genes is commonly seen in sensitive tumors.
Table 1: Histologic and phase characteristics of sensitive and
resistant NSGCTs
Sensitive
(N = 31)
Resistant
(N = 39)
Histology
Teratoma 14 15
Embryonal carcinoma 6 2
Yolk sac tumor 3 6
Mixed tumor/malignant transformation 8 16
Phase at tissue collection
Primary untreated (P) 1 5
Metastatic untreated (M1) 12 4
One regimen of chemotherapy (C1) 18 14
Two regimens of chemotherapy (C2) - 8
Three or more regimens of
chemotherapy (C3)
-8
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CDDP treatment induces de novo promoter
hypermethylation in vivo
To assess the effect of CDDP-treatment on promoter
hypermethylation, we examined tumor tissues that were
collected at different phases of resistance (Table 1). The
frequency of hypermethylation at different phases was: P,
16.7%; M1, 37.5%; C1, 75%; C2, 62.5%, and C3, 62.5%.
Tumors from patients who underwent one or more regi-
mens of chemotherapy (C1, C2, or C3 phases) exhibited
a significantly (P = 0.001) higher (34 of 48 patients; 71%)
frequency of promoter hypermethylation compared to
those from untreated (P and M1) (7 of 22 tumors; 32%)
patients after adjusting for sensitive/resistance status. The
frequency of promoter hypermethylation was also
significantly higher in tumors from treated patients when
sensitive and resistant groups were analyzed separately (P
≤ 0.02). The differences in overall promoter hypermethyl-
ation between untreated tumors (P/M1; 32%) and C1
tumors (75%) were highly significant (P = 0.004), while
the differences between untreated tumors and C2/C3
Promoter hypermethylation in patients with sensitive and resistant GCTs in response to cisplatin combination chemotherapyFigure 1
Promoter hypermethylation in patients with sensitive and resistant GCTs in response to cisplatin combination chemotherapy.
RASSF1A and HIC1 genes showed frequent methylation in resistant tumors, while MGMT and RARB promoters were more
commonly methylated in sensitive tumors.
Table 2: Frequency of promoter methylation of individual genes in sensitive and resistant NSGCT
Gene Sensitive
1
(N = 29) (%) Resistant
2
(N = 31) (%) P-value
APC 7 (24) 9 (29) 0.77
BRCA1
3
9 (31) 9 (30) 1.0
ECAD 1 (3) 2 (6) 1.0
FANCF 2 (7) 2 (6) 1.0
FHIT 2 (7) 2 (6) 1.0
HIC1
3
7 (24) 14 (47) 0.10
MGMT 9 (31) 4 (13) 0.12
RARB 4 (14) 0 0.05
RASSF1A 8 (28) 16 (52) 0.07
1
Consists of tumors, with or without retroperitoneal lymph node, sensitive for one cycle of chemotherapy
2
Consists of tumors, with or without
retroperitoneal lymph node, required of two or more cycles of chemotherapy, all resistant tumors, and all patients died of disease
3
Only 30
resistant tumors studied for methylation status
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Table 3: Promoter hypermethylation in various phases of treatment in NSGCT
Gene Phase
1
p-value
3
P/M1 (N = 22) C1 (N = 32) C2/C3 (N = 16) P/M1 vs C1 P/M1 vs C2/C3
APC 2 (9.1) 8 (25) 7 (43.8) 0.25 0.03
BRCA1
2
4 (18.2) 12 (37.5) 2 (13.3) 0.22 1.0
HIC1
2
3 (13.6) 11 (34.4) 8 (53.5) 0.17 0.09
MGMT 0 11 (34.4) 3 (18.8) 0.003
4
RARB 0 4 (12.5) 0 0.14
4
RASSF1A 5 (22.7) 10 (31.3) 10 (62.5) 0.72 0.09
1
See Table 1 for definition of phase
2
Only 15 tumors studied in C2/C3 phase
3
Adjusted for sensitive/resistant status
4
Model cannot be fit due to
sparse data; Fisher's exact p-value given
Comparisons of promoter hypermethylation frequencies in different phases of treated patients with GCTsFigure 2
Comparisons of promoter hypermethylation frequencies in different phases of treated patients with GCTs. Phase definitions
are shown in Table 1. P, untreated primary tumor; M1, untreated metastatic tumor; C1, one regimen of chemotherapy; C2/C3,
two or more regimens of chemotherapy. Promoter methylation of RASSF1A, HIC1, and APC genes was significantly high in
resistant tumors. The MGMT, BRCA1, and RARB genes show higher frequency of promoter methylation in tumors exposed to
one cycle of chemotherapy.
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tumors (62.5%) were less pronounced (P = 0.09). These
data, thus, suggest that the higher frequency of promoter
hypermethylation seen in treated tumors may be due in
response to CDDP treatment. This de novo increase in
promoter hypermethylation was evident in most analyzed
genes. Comparison among tumors that were not treated
(P/M1), those that were treated with one cycle of chemo-
therapy (C1) and those that were treated with two or more
cycles (C2/C3) showed notable differences for APC,
MGMT, HIC1, RARB and RASSF1A (Table 3). Promoter
hypermethylation of RASSF1A, HIC1, and APC genes was
higher in the treated tumors, with highly resistant (C2/
C3) tumors exhibiting the highest incidence (Fig. 2).
However, this trend was different for BRCA1, MGMT, and
RARB genes. These genes exhibited either no methylation
or low frequency of hypermethylation in untreated
tumors, while the C1 tumors had the highest incidence of
promoter hypermethylation (Fig. 2). However, this was
decreased or absent in highly resistant (C2/C3) tumors.
These data, thus, strongly suggest that promoter hyper-
methylation was induced in response to the first exposure
to CDDP and the tumors harboring promoter hypermeth-
ylation responded differently to further treatment in a
gene specific manner. Thus, our data indicate that the
tumors with promoter hypermethylation of RASSF1A,
HIC1, and APC resulted in failure to respond to further
treatment, while the tumors harboring promoter hyper-
methylation of MGMT, RARB, and BRCA1 responded
favorably.
Since we previously showed that yolk sac tumor (YST)
exhibit higher frequency of hypermethylation compared
to other histologic types among the genes tested [8], we
included histology as an additional covariate in the above
analyses in an attempt to account for histological differ-
ences in promoter hypermethylation. We found that the
differences in overall promoter hypermethylation
between untreated and treated tumors were no longer
significant when adjusted for histology (data not shown).
The small number of observations in each histological
group, however, prevents us from making any meaningful
conclusions from this analysis. Although the data is indic-
ative of histologic differences in promoter methylation,
further analysis of gene specific promoter hypermethyla-
tion on a larger panel of tumors is needed to satisfactorily
address this issue.
No effect of CDDP treatment in vitro on promoter
methylation
Since we showed CDDP treatment induces promoter
hypermethylation in tumors in vivo, we wanted to test
whether a similar phenomenon occurs in vitro. To investi-
gate this, we exposed four NSGCT cell lines to two differ-
ent concentrations of CDDP for various time periods as
described in the methods. The specimens from which the
169A, 218A, and 240A cell lines derived were also
included in the panel of tumors studied for hypermethyl-
ation. In addition, two independent clones derived from
833K-E and 240A as D1 and D4 resistant cells (see mate-
rials and methods) were also examined. We did not detect
changes in promoter hypermethylation in 18 (APC,
GSTP1, BRCA1, DAPK, p16, p14, MGMT, APAF1,
RASSF1A, HIC1, RB, TIMP3, FANCF, RARB, CDH1, TP53,
FHIT, and MLH1) genes examined. These results clearly
indicate that CDDP treatment in vitro, within the tested
concentrations, does not cause promoter hypermethyla-
tion. However, we found methylation of BRCA1,
RASSF1A, or HIC1gene promoters in three cell lines but
not in their corresponding primary tumors (Fig. 3). The
genes methylated in cultured tumor cells were the same
genes that were also frequently methylated in NSGCT
patients. The gene promoters that did not exhibit frequent
methylation in primary tumors were not methylated in
cultured tumor cells.
Loss of activation of gene expression to inhibitors of
methylation and histone deacetylation in acquired CDDP
resistance in vitro
To further examine the effect of CDDP treatment in vitro,
we then studied expression of MGMT, HIC1, and FANCF,
the genes that were either promoter hypermethylated or
down regulated in GCT, in D1 and D4 clones derived
from the cell lines 833K-E and 240A.
MGMT is a DNA repair enzyme that protects cells against
the effects of alkylating agents by removing adducts
formed at the O
6
position of guanine in DNA [11]. Tumor
sensitivity to alkylating agents has been shown to depend
on MGMT expression [12]. Resistant tumors are generally
Promoter methylation changes in vitro in GCT cell linesFigure 3
Promoter methylation changes in vitro in GCT cell lines.
Appearance of de novo promoter hypermethylation in cell
lines. T, tumor; CL, cell line; CDDP, cisplatin-treated cell line.
M, methylated DNA; U, unmethylated DNA; Cell lines exam-
ined are shown below. Note absence of methylation in pri-
mary tumor in both the cell lines and appearance of novel
methylated allele in cell line DNA. Note both alleles of
RASSF1A are methylated in 218A cell line.
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shown to express high levels of the MGMT gene [12]. To
examine the role of MGMT in in vitro-acquired CDDP
resistance of GCT, we studied mRNA levels in 833K-E and
240A cell lines that harbored methylated promoters. Both
showed low levels of detectable mRNA by RT-PCR. How-
ever, only the 833K-E showed reactivation of expression
upon treatment with 5-Aza-C or TSA, while these agents
had no effect on the 240A cell line. The levels of MGMT
mRNA in D1 and D4 refractory cells were either remained
at the levels similar to untreated cells in D1 cells or were
slightly increased in D4 cells in both the cell lines (Fig. 4).
The latter is consistent with the role of MGMT in effi-
ciently repairing DNA adducts in resistant cells and this
could be due to partial demethylation of the promoters in
highly resistant D4 cells in these cell lines. Concordant
with demethylation of promoter in D4 cells, these cells
fail to up-regulate gene expression after 5-Aza-C or TSA
treatment (Fig. 4).
The HIC1 gene showed hypermethylated promoters in
both 833K-E and 240A cell lines. Analysis of mRNA
showed that only 240A cell line exhibited a detectable
level of expression, while it was absent in the 833K-E cell
line. HIC1 was reactivated after treatment with 5-Aza-C or
TSA in 833K-E, while the treatment had no effect on 240A
cell line (Fig. 4). However, both the D1 and D4 clones
derived from these cell lines showed complete lack of
expression of HIC1 and failed to respond to either 5-Aza-
C or TSA (Fig. 4).
The FANCF gene belongs to the family of six Fanconi ane-
mia proteins that facilitate mono-ubiquitinylation of
FANCD2, which plays a role in a large multimeric protein
complex required for DNA repair [13]. Acquired CDDP
resistance in ovarian carcinoma correlates with subtle
methylation/demethylation of FANCF promoter leading
to the suggestion that demethylation of this gene causes
CDDP resistance [14]. Here, we examined the FANCF
gene expression in the development of in vitro CDDP
resistance. The FANCF promoter was not methylated in
both 833K-E and 240A cells and low levels of mRNA
expression were found in both. However, both cell lines
showed an up-regulated expression of FANCF mRNA after
5-Aza-C or TSA treatment. Although the levels of FANCF
expression in D1 cells of 833K-E remain at the levels in
untreated cells, the D1 cells of 240A showed an elevated
level of mRNA (Fig. 4). However, the D4 resistant clones
from both cell lines showed a decrease in expression com-
pared to untreated cells and lost the ability to respond to
5-Aza-C or TSA (Fig. 4). Analysis of a control gene did not
affect the pattern of expression in relation to in vitro
CDDP resistance in these cells (Fig. 4).
Taken together, the results obtained from all three genes
studied here indicate that changes in gene expression
occur in the development of low to high refractory CDDP
resistance. Highly resistant clones fail to respond to
demethylating or histone deacetylase inhibiting agents in
activating gene expression suggesting that irreversible
changes occur in a pathway that control gene
transcription.
MGMT is partially methylated and down regulated in most
GCTs
We showed earlier promoter hypermethylation of MGMT
in 21% NSGCT and complete lack of or down-regulated
gene expression by RT-PCR in 96% of tumors [8]. To
examine whether the down-regulated RNA levels reflected
in decreased protein, we performed an immunohisto-
chemical analysis of MGMT on a tissue array containing
18 SGCTs and 18 NSGCTs. The MGMT expression was
absent in 33 (91.7%) tumors. The remaining 3 tumors
(two yolk sac tumor and one immature teratoma) were
weakly positive compared to the controls (data not
shown). Thus, the combined data on analyses of RT-PCR
from our previous study and the levels of protein reported
here showed down-regulated levels of MGMT expression
in all histologic types of GCT. The cells from GCT have
been reported to exhibit reduced efficiency in the removal
of CDDP-induced adducts [15,16]. Concordantly, more
RT-PCR analysis of gene expression in relation to CDDP-induced resistance in GCT cell linesFigure 4
RT-PCR analysis of gene expression in relation to CDDP-
induced resistance in GCT cell lines. Aza, 5-Aza-2'-deoxycyti-
dine; TSA, trichostatin; D1, low refractory CDDP resistant
cells; D4, high refractory CDDP resistant cells; SEPT6, septin
6. Actin (empty arrow) was used as an internal control. Filled
arrowhead indicates the specific gene. Septin 6 gene was
used as another control.
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than 85% of metastatic GCT can be cured with chemo-
therapy [3]. Since GCTs exhibit either promoter hyper-
methylation or down-regulated gene expression of the
MGMT gene in most tumors, we suspected that this gene
may play a role in CDDP sensitivity. Our previous data
suggest that down-regulated expression of MGMT occurs
by other mechanisms, in addition to complete promoter
hypermethylation. We previously ruled out mutational
inactivation of this gene in GCT [8]. The MSP method
only detects complete hypermethylation of CpG islands
and partial methylation will not be identified by this
method. Since the down regulated MGMT expression can
be reactivated after cellular exposure to 5-aza-C in
unmethylated GCT cell lines, we reasoned that partial
methylation might exist. To test this, we sequenced 98
CpG methylation sites spanning the promoter of MGMT
in genomic DNA from two normal testes, one methylated
cell line, seven NSGCTs and five SGCTs which have
unmethylated promoters by MSP (Fig. 5). Normal testes
showed a few random methylated CpGs in a small
number of clones. The MSP positive Tera-2 cell line had
dense methylation of the entire MGMT promoter. All
seven NSGCTs and five SGCTs showed partial methyla-
tion of the promoter region at specific CpG residues (Fig.
5). Three (T-228A, T-186B, and T-288A) of the seven
NSGCTs studied were also classified as resistant GCTs. The
frequency of methylated residues varied within clones
derived from the same tumor and between tumors. The
present data, thus, suggest, but does not prove, that partial
methylation of MGMT promoter accounts for down-regu-
lated gene expression in GCT. Overall, these results indi-
cate a role of MGMT promoter hypermethylation state,
either complete or partial, in determining the exquisite
sensitivity to CDDP in GCT.
Discussion
The molecular mechanisms that determine the curability
of GCT to CDDP-based combination chemotherapy are
unclear [17-20]. Understanding the genetic basis of this
exquisite sensitivity could lead to the development of a
more effective treatment for resistant tumors. A number of
genetic mechanisms for CDDP resistance, such as
enhanced adduct repair, drug inactivation, or tolerance to
DNA damage, have been proposed [21].
We and others previously reported that epigenetic altera-
tions in the promoters of specific genes occur in NSGCT
[8-10,22]. We also showed that promoter hypermethyla-
tion was associated with gene repression in NSGCT and
this down regulated expression is reactivated upon
demethylation suggesting a potential role for epigenetic
changes in GCT biology [8]. These results prompted us to
examine the possible involvement of epigenetic changes
in chemo-sensitivity and resistance in GCT. To achieve
this, we investigated epigenetic changes in resistant and
sensitive NSGCTs and found a high incidence of promoter
hypermethylation of RASSF1A and HIC1 in resistant
tumors, while promoter hypermethylation of MGMT and
RARB genes was associated with sensitive tumors.
RASSF1A has shown to be epigenetically inactivated in a
wide variety of tumor types suggesting a major role for this
gene in cancer [23]. In the present study, we demonstrated
that a higher frequency of resistant tumors carry promoter
methylation compared to sensitive GCT, suggesting that
RASSF1A hypermethylation is associated with the resist-
ance phenotype. RASSF1A represents a long isoform of
human RASSF1 gene, which encodes a diacylglycerol
(DAG)-binding domain at the NH
2
terminus, a RAS-asso-
ciation domain at COOH terminus, which interacts with
the XPA protein. RASSF1A gene functions as a negative
regulator of cell growth [23].
Hic1encoding a zinc finger transcription factor acts as a
tumor suppressor gene [24]. HIC1 is silenced by promoter
hypermethylation in several types of human cancer [25].
We found a higher frequency of resistant tumors harbor-
ing HIC1 promoter hypermethylation.
On the other hand, we showed promoter hypermethyla-
tion of MGMT and RARB genes associated with CDDP
sensitivity. MGMT gene encodes O(6)-methylguanine-
DNA methyltransferase and plays an important role in
removing DNA adducts formed by alkylating agents [11].
Epigenetic silencing of MGMT has been shown to confer
enhanced sensitivity on cancer cell to alkylating agents,
while the lack of methylation and high-levels of protein
expression contribute to drug-resistance phenotype
[12,26,27]. We showed here that either complete or par-
tial methylation of MGMT occurs in a majority of GCT.
These data suggest that the complete promoter methyla-
tion of MGMT plays a role in favorable response to CDDP
treatment. However, the demonstration here of partial
methylation in most GCTs provides a possible mecha-
nism for down-regulated expression of MGMT, which is
commonly seen in this tumor. These results, thus, support
the view that the epigenetic alteration in MGMT may be a
factor in the exquisite sensitivity of GCT to CDDP. Such a
model provides opportunities to alter MGMT pathway
and chemosensitize relapsed tumor to CDDP.
Retinoids control gene transcription by activating retinoic
acid receptors (RAR∝, β and γ) and retinoid X receptors
(RXR∝, β and γ). Expression of these receptors regulates
organogenesis, organ homeostasis, cell growth, and differ-
entiation and death [28]. It is well established that
changes in expression of RARs play a major role in cancer
development and response of tumor cells to treatment of
all-trans retinoic acid (ATRA). A number of premalignant
lesions and cancers have been shown to exhibit a loss of
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expression of RARB due to promoter hypermethylation
[28]. In the present study, we found RARB promoter
hypermethylation only in sensitive NSGCTs. The data,
therefore, suggest that RARB down-regulation may favor
response to CDDP treatment. The mechanisms regulated
by RARB responsive genes in GCT require further studies
to understand the role of this gene in chemotherapy
response.
Analysis of promoter methylation by bisulphite sequencing in GCTFigure 5
Analysis of promoter methylation by bisulphite sequencing in GCT. CpG methylation status in 5 independent clones sequenced
is shown for each tumor. Shown on top by vertical lollypops connected to horizontal line are CpGs examined on the 5' pro-
moter region. Site of MSP primer is indicated. Shown below is the CpG sequence information for each tumor. Tumor numbers
are shown on left. Methylation status is indicated in parenthesis next to the tumor. Filled circles indicate methylated CpG sites
and empty circles indicate unmethylated CpGs. NSGCT, nonseminomatous germ cell tumor; SGCT, seminomatous germ cell
tumor.
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Previous studies indicated that tumor cells exposed to
anticancer agents induce DNA hypermethylation resulting
in the silencing of genes that play role in drug metabolism
and resistance [29]. Human tumor cells exposed to high
concentrations of CDDP in vitro induces alterations in 5-
methyl Cytosine (5-mCyt) [30]. Similarly, in vivo exposure
of bone marrow cells to cytosine arabinoside (araC) alone
or a combination of hydroxyurea, VP-16 and araC also
result in a several-fold increase of 5-mCyt content in
leukemic blasts ([30]. Thus, the exposure of tumor cells to
cytotoxic chemotherapy agents in vitro and in vivo causes
an induction of DNA hypermethylation. In the present
study, we examined whether CDDP treatment in vivo
causes such a hypermethylation in GCT by studying spe-
cific gene promoters. Our results suggest that initial
CDDP treatment in tumors induces promoter hypermeth-
ylation of certain gene promoters such as MGMT, RARB,
and BRCA1 (Fig. 2). This induction of methylation in
these genes hypersensitize the tumor to further treatment,
while tumors that had promoter hypermethylation of
RASSF1A, HIC1, and APC are selected upon further treat-
ment to develop drug resistance (Figs. 1 and 2). Such a
model of CDDP-induced non-random hypermethylation
can predict response to further treatment and allows spe-
cific gene targeted therapeutic approaches for resistant
GCTs. Hypermethylation of RASSF1A, HIC1, and APC
genes provides a plausible mechanism for the propensity
of these tumors to CDDP resistance, and demethylation
could result in restoration of hypersensitivity. Well docu-
mented evidence in certain tumor types suggest that drug
resistance disrupts general mechanisms of chemosensitiv-
ity by targeting mutations and gene amplifications [31].
Here, we demonstrate that epigenetic alterations in spe-
cific genes also play a role in chemosensitivity to CDDP in
GCT. The present data, thus, suggest that specific gene pro-
moter hypermethylation induced by drugs may serve as
prognostic indicator of treatment response in NSGCT. In
view of the biological relevance of DNA methylation,
CDDP-induced hypermethylation shown here in GCTs
will have clinical significance in drug-response
phenotypes.
CDDP-induced promoter hypermethylation in tumor
cells might set in motion a cascade of ectopic gene expres-
sion events that might release tumor from normal home-
ostatic controls. These changes include deamination of 5-
methyl cytosine in CpG causing genetic instability (i.e.,
mutations), transducing epigenetic changes into genetic
alterations, or inactivation of methylated genes. To test
the latter possibility, we tested gene expression in four dif-
ferent clones from two highly resistant cell lines. We could
not reactivate the gene expression by exposure to the
demethylating agent 5-Aza-C or histone deacetylase
inhibitor TSA, implying that a common epigenetic and/or
genetic mechanisms that regulate transcriptional activa-
tion of hypermethylated genes was affected in highly
resistant cells rather than simple methylation changes in
specific gene promoters.
The cytotoxic effectiveness of CDDP against tumor cell is
believed to be mediated through the formation of DNA
adducts, which inhibit DNA replication and transcription
[32,33]. Cisplatin primarily forms intra-strand GpG cross-
links, which are removed by neucleotide excision repair
(NER) [34]. Highly regulated steps involving a number of
proteins coordinate the NER in human cells. One hypoth-
esis to explain the hypersensitivity of GCT to CDDP is that
there is a deficiency in one or more components of this
repair machinery [35]. Recently, it has been shown that
elevated testis-specific high-mobility group (ts-HMG)
DNA-binding proteins may enhance sensitivity to CDDP
[34]. Our results suggest a potential molecular mecha-
nism of CDDP-induced transcriptional inactivation of
genes prone to hypermethylation. The CDDP exposure
may cause genetic damage that might sequester essential
proteins from their designated function such as elements
of DNA repair pathways. The results presented here sup-
port the notion that epigenetic mechanisms play a role in
CDDP-response in a gene specific manner. As cellular
response to CDDP treatment in GCT is believed to be a
complex process, future studies to address this issue need
to examine both epigenetic and genetic alterations.
Conclusions
Our studies provide evidence that the RASSF1A and HIC1
inactivation by promoter hypermethylation play a role in
NSGCT resistance and may serve as markers for the iden-
tification of resistant tumors. The epigenetic alterations in
MGMT may be an important factor in conferring the
exquisite sensitivity of GCT to CDDP. Although the
molecular mechanisms of GCT resistance are unclear cur-
rently, our findings of epigenetic alterations in the
RASSF1A, HIC1, MGMT, and RARB genes may serve as
prognostic indicators of CDDP-related treatment
response and provide molecular targets of therapy to
chemo-sensitize the resistant tumors. In view of the bio-
logical relevance of DNA methylation, CDDP-induced
hypermethylation shown here in GCTs will have clinical
significance in drug-response phenotypes and provides
opportunities to modulate pathways controlled by these
genes.
Methods
Tumor specimens and stratification of chemotherapy
resistance and sensitivity
Tumor tissues were identified by retrospective review of
GCT specimens obtained during diagnostic evaluation at
the Memorial Sloan-Kettering Cancer Center, New York,
between 1987 and 1999. Patients were identified based
on known response and resistance to chemotherapy. A
Molecular Cancer 2004, 3 http://www.molecular-cancer.com/content/3/1/16
Page 10 of 12
(page number not for citation purposes)
total of 70 GCT specimens from 60 patients comprised
the study cohort. The sensitive tumors consisted of 31 tis-
sues obtained from 29 patients that were relapse-free for
more than two years as a result of chemotherapy alone or
in combination with surgery. The resistant panel com-
prised of 39 tumors from 31 patients, with or without ret-
roperitoneal lymph node metastasis, who either did not
respond to one or more cycles of CDDP-based chemo-
therapy or responded and then relapsed, or died of disease
after any number of cycles of treatment. Table 1 summa-
rizes the histologic and phase characteristics of sensitive
and resistant patients. Additionally, 36 unselected GCTs
evaluated at Columbia University Medical Center were
also studied.
Cell lines and drug treatment
Four NSGCT cell lines, an established 833K-E and three
cell lines (169A, 240A and 218A) described by us earlier,
were grown in high-glucose DMEM medium containing
15% fetal bovine serum, L-glutamine, and penicillin-
streptomycin [36]. Sub-cultured cells after 24 hr were
treated with the specific drugs at different concentrations
and time periods. Cells in logarithmic phase were exposed
to CDDP at 0.5 µM and 1.0 µM concentrations for 2 h and
24 hr, at which time drug was removed, and fresh culture
medium was added. The CDDP-treated cells were contin-
ued to grow for 2, 4, and 7 days to clonally expand. We
have also derived CDDP-refractory cells from 833K-E and
240A cell lines by growing for 21 and 16 days, respec-
tively. Two independent clones derived from each of these
cell lines were designated as 833K-E/C10, 833K-E/C13,
240A/C4, and 240A/C10. After further expansion, these
cells were designated as D1-resistant cells for one time
point drug treatment. These D1 cells were further treated
serially with increasing concentrations (1.5 µM to 4.5 µM)
of CDDP and were grown in culture for more than 90
days. The final passage cells were designated as D4 cells
for four time points of drug selection. Untreated and the
CDDP-resistant cell lines were exposed to demethylating
agent 5-Aza-2' deoxycytidine (5-Aza-C) (Sigma) for five
days at a concentration of 2.5 µM and trichostatin (TSA)
at 250 nM for the last 24 hours or a combination of both.
Methylation Specific PCR (MSP) and gene expression
Genomic DNA was treated with sodium bisulphite as pre-
viously described [8]. Placental DNA treated in vitro with
SssI methyltransferase (New England Biolabs, Beverly,
MA) and similarly treated normal lymphocyte DNA were
used as controls for methylated and unmethylated tem-
plates, respectively. The primers used for methylated and
unmethylated-specific PCR have been either described
previously [8] or are available from authors upon request.
PCR products were run on 2% agarose gels and visualized
after ethidium bromide staining.
Gene expression was assessed on total RNA isolated from
four normal testes, a commercially purchased normal tes-
tis RNA (Clontech, Palo Alto, CA) and the cell lines
described above. Reverse transcription was performed
using random primers and the Pro-STAR first strand RT-
PCR kit (Stratagene, La Jolla, CA). A semi-quantitative
analysis of gene expression in replicate experiments was
performed using 26 to 28 cycles of multiplex RT-PCR with
β-actin (ACTB) as a control and gene specific primers
spanning at least 2 exons whenever possible. The gene
primers used have either been described previously [8] or
are available from authors. The PCR products were run on
1.5% agarose gels, visualized by ethidium bromide stain-
ing and quantitated using the Kodak Digital Image Analy-
sis System (Kodak, New Haven, CT).
Bisulphite sequencing
Bisulphite-treated DNA was amplified with primers
designed to amplify both methylated and unmethylated
DNA. Two sets of primers were designed to cover the
entire promoter region of the MGMT gene. The first set of
primers was MGMT-cl-F3 5'-AGGATTTGAGAAAAGTAA-
GAGAG-3' and MGMT-cl-R3 5'-ATT-
TAACAAACTAAAACACAAAACC-3', and the second set of
primers was MGMT-cl-F4 5'-TTTTTTTGTTTTTTTTAG-
GTTTT-3' and MGMT-cl-R4 5'-CAAACACCAACCAT-
AATAACCAA-3'. PCR products were sub-cloned into
pCR2.1-TOPO (Invitrogen) and DNA isolated from 15 to
20 clones for each tumor was sequenced.
Tissue microarray and immunohistochemical analysis
A panel of 36 unselected formalin-fixed, paraffin-embed-
ded tissue specimens from 18 NSGCTs and 18 SGCTs was
used to construct a tissue array (Beecher Instruments, Sil-
ver Spring MD). Representative areas of the biopsy were
chosen to construct a 14 × 8 tissue array. Four micron-
thick sections on the array were immuno-stained
following deparaffinization and antigen retrieval using
citrate buffer at pH6.0. The primary antibody against
MGMT was obtained from NeoMarkers (Fremont, CA).
The antibodies were detected with the Envision plus
(DAKO, Carpenteria, CA) system, using diaminobenzi-
dine as a chromogen. Tumors were considered positive for
MGMT when cells showed brown nuclear staining. Inter-
stitial and intravascular lymphocytes, as well as spermato-
gonia of any residual seminiferous tubules were used as
internal controls.
Statistical analyses
Comparisons for the analyses of sensitive vs. resistant
tumors were done via Fisher's exact test. For the eight cases
that contributed multiple specimens, the specimen with
the greatest number of methylated genes was used. Exact
logistic regression was used for the phase comparisons
with the analyses adjusted for sensitive/resistant status
Molecular Cancer 2004, 3 http://www.molecular-cancer.com/content/3/1/16
Page 11 of 12
(page number not for citation purposes)
and histology where noted. Here multiple specimens
from the same patient were included. None of the p-val-
ues were adjusted for multiple comparisons due to the
exploratory nature of the analysis.
Authors' contributions
SK carried out the methylation, cloning, sequencing and
gene expression analysis. JMM participated in selection of
tumor specimens, isolation of DNA and RNA. GN partic-
ipated in the analysis of gene expression. JH coordinated
the selection of tumors, tissue culture, isolation of
genomic DNA and RNA. JB performed statistical analysis.
DLB participated in obtaining the follow up on patients.
AMA and MM participated in the preparation of tissue
array and gene expression analysis. VER participated in
histologic diagnosis. GJB was responsible for referring the
patients and clinical information. RSKC and VVVSM have
conceived and coordinated the study. All authors read and
approved the final manuscript.
Acknowledgements
This work was supported by the NIH grant CA75925 (VVVSM), funds from
Lance Armstrong Foundation (VVVSM and JH), Tom Green's Nuts Cancer
Fund of California Community Foundation (VVVSM), and the Herbert Irv-
ing Comprehensive Cancer Center, Columbia University (JMM and
VVVSM).
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