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Departments of Medical Biochemistry [Y. T., M. W., T. H., J. N., M. K.], Urology [K. K., S. N.], and Medical Informatics [N. K.], Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582; and Department of Urology [M. N.], Faculty of Medicine, Kagoshima University, Kagoshima 890-8520, Japan
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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The MDR1 gene/P-gp is expressed not only in various normal human tissues or organs (9) but also in various malignant tumors (10 , 11) . To develop MDR1/P-gp as a diagnostic marker for additional therapeutic improvement with fewer side effects, one critical question regards how and when the MDR1 gene is specifically expressed in human malignancies during clinical courses. Gene rearrangements at the 5'-flanking region of the MDR1 gene cause gene activation in multidrug- resistant breast and colon cancer cells as well as in human lymphomas, but analysis of the molecular basis for the gene rearrangements in clinical samples remains to be performed (12 , 13) . The Y-box (inverted CCAAT box) binding protein (YB-1) has an essential role in human MDR1 gene transcriptional activation in cultured cancer cells in the presence or absence of genotoxic stress (14 , 15) . Intracellular localization of YB-1 in either the cytoplasm or the nucleus appears to be critical for P-gp expression in some human malignant tumors such as breast cancers, osteosarcoma, and ovarian cancers (16, 17, 18) . On the other hand, the degree of methylation at CpG sites on the MDR1 promoter also plays a key role in MDR1 gene expression. The presence or absence of methylation at CpG sites is closely associated with transcriptional activation of the MDR1 gene in various cultured cell lines (19 , 20) . Overexpression of the MDR1 gene is inversely correlated with DNA methylation at CpG sites at the 5'-flanking region in chronic lymphocytic leukemias (21) and acute myeloid leukemias (22) . These findings suggest that increased expression of the MDR1 gene/P-gp in some human malignancies is induced as a result of changes in either the intracellular localization of YB-1 or methylation of the MDR1 promoter.
Overexpression of P-gp has been found in human bladder cancer cells selected by drug resistance against P-gp-targeting drugs (23, 24, 25) . In patients with bladder cancers, expression of P-gp is often increased after chemoradiotherapeutic treatment (26) . Chemotherapeutic treatments such as systemic chemotherapy (methotrexate, vincristine, doxorubicin, and cisplatin combined treatment) have been effective for advanced bladder cancer, and prophylactic intravesical instillation chemotherapy using anthracyclines (doxrubicin, epirubicin, and terarubicin) has been effective for preventing recurrence of superficial bladder cancer. However, patient responses against advanced bladder cancers are still of relatively short duration, and the recurrence of superficial bladder cancers are still often observed even after intensive intravesical chemotherapy. In this study, we examined whether the degree of methylation at the promoter site is associated with MDR1 gene expression in bladder cancers and also whether chemotherapeutic treatment affects both the degree of methylation at the MDR1 promoter and MDR1 gene expression in bladder cancers. The degree of methylation at the MDR1 promoter is discussed as well as P-gp expression in the presence or absence of chemotherapeutic treatment in bladder cancers.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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shows the clinicopathegenic characteristics of the 51 clinical samples.
All of the patients with residual bladder cancer had received systemic
chemotherapy, i.e., combined treatment using methotrexate,
vincristine, doxorubicin, and cisplatin, or their recurrent tumors had
received prophylactic intravesical instillation chemotherapy with
either doxorubicin, epirubicin, or terarubicin before surgery. Tumor
tissue samples were obtained under an Institutional Review
Board-approved protocol, with subjects providing informed consent.
Tumor samples were frozen in liquid nitrogen and were stored at
-80°C until RNA and DNA extraction. SNK57 cells derived from human
bladder cancer were cultivated as previously described
(27)
.
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Semiquantitative RT-PCR Analysis.
The PCR in this study was a modification of a previously described
method (22)
. The PCR primers were
5'-CACGTGGTTGGAAGCTAACC-3' and 5'GAAGG CCAGAGCATAAGATGC-3' for the
human MDR1 gene and 5'-GTGGAGCATTCAGACTTGTCTTTCAGC-3'
and 5'TTCACTC AATCCAAATGCGGCATCTTC-3' for human
ß2-microglobulin. To quantify human
MDR1 and human ß2-microglobulin mRNA
using RT-PCR (28
, 29)
, the patients’ cDNA samples were
diluted serially in water, from 50 to 3 ng/µl for the human
MDR1 gene and from 25 to 0.04 ng/µl for the human
ß2-microglobulin mRNA, and were mixed to a
final volume of 5 µl with 1 µM primer pairs
and 1 U of Taq DNA polymerase. The PCR products were separated by
electrophoresis on 3% agarose gels, which were then stained with
SYBER Green I (Molecular Probe, Eugene, OR) and were examined by
means of FLA2000 (Fuji Film, Tokyo, Japan) and image analysis (FLA2000;
Fuji Film). We repeated the assay at least twice to confirm its
reproducibility. We accurately detected MDR1 gene expression
under the condition of an average error of ±18% in each sample.
RNase Protection Assay.
The RNase protection assay in this study was a modification of a
previously described method (22)
. Transcripts originating
from the MDR1 promoter, referred to as the "downstream
promoter," protect 130- and 134-bp fragments of the RNA probe. A
324-nucleotide sequence of the same probe was protected by
MDR1 transcripts originating at an "upstream promoter"
(29)
. Twenty-five µg of RNA was hybridized using 2 x 105 cpm of antisense RNA probe. We evaluated
the MDR1 mRNA levels standardized by the
ß2-microglobulin expression. The
ß2-microglobulin cDNA expression was measured
using a 206-bp cDNA fragment from human
ß2-microglobulin cDNA subcloned into the pGEM-T
vector, and was linearized using PstI.
Immunohistochemistry.
The immunohistochemistry used in this study was a modification of
a previously described method (17)
. In brief, bladder
tumors were fixed in 10% formalin and were embedded in paraffin.
Histological sections were stained with H&E. P-gp was detected using
two kinds of monoclonal antibody, C219 (Centacor, Malvern, PA)
and JSB-1 (Sanbio, Uden, the Netherlands).
Quantitative PCR-based Methylation Analysis.
The PCR used in this study was a modification of a previously described
method (22)
. In brief, 3 µg each of control and DNA were
digested by 300 U of MspI (Fermentas MBI, Vilnius,
Lithuania) or HpaII (Takara Shuzo, Kyoto, Japan) at
37°C for 16 h,
volume of 0.6 M
Tris (pH 7.5) and 1.5 M NaCl were added, and the mixture was then
digested by 30 U of PstI (Nippon Gene) at 37°C for 8 h. To analyze the degree of methylation of the MDR1 5'CpG
promoter region, restriction-digested DNA was analyzed using PCR in 5
µl reactions containing 1 µM each of sense
and antisense primers and 1 U of Taq DNA polymerase. The PCR products
were separated by electrophoresis on 3% agarose gels, which were then
stained with SYBER Green and were analyzed by scanner and image
analysis (FLA2000 image; Fuji Film).
Southern Blot Analysis.
The degree of methylation of MspI/HpaII sites was
investigated by separating genomic DNA that had been digested by
HpaII and PstI on a 2.0% NuSieve 3:1 gel and by
transferring the DNA fragments to a nylon filter (Hybond
N+; Amersham). A 978-bp
PstI-PstI fragment of the promoter region of
MDR1 was used as a probe (20)
. This probe
contained five MspI/HpaII sites (see
"Results").
Statistical Analysis.
Mann-Whitney tests, KruskalWallis tests, and multiple comparisons
were used to compare two or more groups. Piecewise linear regression
analysis was performed to examine the relationship between the
expression and degree of methylation of the MDR1 gene
(30)
. Computations were carried out using BMDP statistical
software on a SPARK Station 20 (Los Angeles, CA).
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Results |
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A). Different dilutions of cDNA were tested within the
exponential range, depending on the level of MDR1 mRNA
(Figs. 1, A and B)
. The diluted series of cDNA
from patient 1 (Pt 1) and SNK57 cells were used as templates
for the PCR reaction using MDR1 primer pairs.
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and C, shows the results of the
FLA2000 quantitative analysis of the PCR products as a function of the
dilution rate of input RNA. The relative mRNA levels of the
MDR1 gene were calculated on the basis of the dilution rate
at the exponential range and were normalized by dividing by the
relative ß2-microglobulin mRNA level. The
ß2-microglobulin mRNA levels were almost the
same (Fig. 1
D). The MDR1 mRNA levels were
4.2-fold higher in patient 1 (Pt 1) than in SNK57 cells
(Fig. 1
B).
We also performed an RNase protection assay to evaluate the mRNA level
of MDR1, with the results correlating well with the results
by RT-PCR for higher expression samples (Fig. 2)
. For instance, MDR1 expression levels in patients
(Pt) 2, 4, 6, 8, 9, 7, 3–1, 5, and 3–2 were 1.2, 1.2, 1.2,
1.7, 4.7, 6.2, 8.3, 11, and 20, respectively, using RT-PCR analysis and
were 1, 2, 2, 3, 5, 6, 8, 22, and 30, respectively, using the RNase
protection assay (Fig. 2)
. The MDR1 transcripts of KB-C1
cells overexpressing MDR1 that were used as a positive
control were detected as 130/134- and 324-bp fragments as reported by
Mickley et al. (12)
, whereas only
130/134-bp fragments were detected in all of the patients.
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and B, shows the immunostaining data of P-gp in
one patient (patient 5) who showed an MDR1 expression value
of 11 as determined by semi-quantitative RT-PCR. In contrast, cells
were not stained with anti-P-gp antibody in one patient (patient 2) who
had an expression value of 1.2 (Fig. 3, C and D)
.
These immunohistochemical findings were represented by using anti-P-gp
antibody C219 (Fig. 3, A and C)
and JSB-1 (Fig. 3, B and D)
. We also analyzed P-gp expression
immunohistochemically in other patients who had undergone total
cystectomy, using two P-gp antibodies, C219 and JSB-1.
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. The MDR1 gene overexpression after chemotherapeutic
treatment thus might be a prognostic factor indicating additional
recurrence.
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, whereas primer pair MC2 was used as a positive control to assess the
quality of source genomic DNA (Fig. 5)
. By contrast, TPI5 that crosses
the MspI/HpaII site, which is never methylated,
at the triosephosphate isomerase gene promoter region was used as a
negative control (Fig. 5)
.
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. By contrast, we observed only completely digested fragments of 322
and 401 bp in patients 3–1, 13, and 3–2, who had a degree of
methylation of 21, 16, and 5%, respectively (Fig. 6, B)
. The
results obtained using the PCR assay correlated well with the results
of Southern blot analysis for heavily methylated patients. However, we
did not detect a low level of methylation using Southern blot analysis,
although we detected a low level of methylation using the PCR assay.
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shows the correlation between the degree of methylation at CpG sites
analyzed using MM2 primer pairs (MM2 sites, Fig. 5
) and MM4 primer
pairs (MM4 sites, Fig. 5
). No obvious correlation was shown between the
degree of methylation at MM2 sites and at MM4 sites statistically in
the whole. We detected hypermethylation at MM2 sites with
hypomethylation at MM4 sites in some patients, hypomethylation at MM2
sites with hypermethylation at MM4 sites in other patients, and hyper-
or hypomethylation at both sites in other patients (Fig. 7)
. The degree
of methylation at two HpaII clusters, MM2 and MM4 sites,
thus appears to be altered independently. Our results indicate that the
degree of methylation at both MM2 and MM4 sites should be included in
the evaluation of methylation conditions at the MDR1
promoter (see next section). We also analyzed separately the
relationship between the degree of methylation at MM2 sites and at MM4
sites in light of the types of therapy used for each primary tumor,
residual tumor, and recurrent tumor. However, we could not find any
correlation between them.
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/
sites, or (b) 728- and (d) 455-bp bands that indicated a hypomethylated
site with hypermethylated sites (/ and/or 
) in any samples
(Fig. 6, A and B)
. Although we did not
distinguish and because of the short distance, the
HpaII / site tended to be hypomethylated, and the
site tended to be hypermethylated. Furthermore, all of the expected
bands of 492, 546, 602, and 656 bp were observed in each patient,
depending on the degree of methylation of sites and 
(Fig. 6)
.
The degree of methylation of the HpaII sites of and
thus appears to shift randomly, consistent with the results obtained
using PCR assay (Figs. 6
and 7)
. The order of the sites that were
demethylated appeared to proceed from site / to and and
then to , as MDR1 expression increased in patients
undergoing chemotherapeutic treatment (Fig. 6)
.
Correlation between MDR1 Gene Expression and Degree
of Methylation at the MDR1 Promoter Region.
We next examined if MDR1 expression is correlated with the
degree of methylation at the promoter region. Using Southern blot
analysis, we determined that MDR1 expression correlated
inversely with the degree of methylation except in several clinical
samples of untreated primary tumors that showed a low MDR1
expression and hypomethylation. Using RT-PCR analysis, the level of
MDR1 expression in patients with untreated primary tumors
(n = 23) was lower than 5.0, and the level of
methylation assessed at both MM2 sites and MM4 sites varied from 2.2 to
115%, which was the sum of the methylation values at MM2 sites and at
MM4 sites (Fig. 7)
. MDR1 expression was low in untreated
primary tumors, regardless of the degree of methylation.
We then analyzed the correlation between the degree of methylation and
expression of the MDR1 gene in patients after the completion
of chemotherapeutic treatment. Figure 8
shows the correlation between MDR1 gene expression and
degree of methylation of 5'CpG sites at the MDR1 promoter
region as assessed by MM2 primer pairs together with MM4 primer pairs.
Similar correlations were obtained when the degree of methylation was
assessed either at MM2 sites alone or MM4 sites alone (data not shown).
We also evaluated the degree of methylation at the MM2 and MM4 sites by
calculation: a x MM2 + b x
MM4, where a = 3.1 and b = 1.2;
the maximum correlation between the expression of the MDR1
gene and the degree of methylation was -0.7515 and P < 0.001. With a = 1.00 and b = 1.00,
the correlation was -0.7263 and P < 0.001. The
results obtained by these two calculations were similar, and we
presented the result obtained by introducing a = 1.00
and b = 1.00, rather than a = 3.1 and
b = 1.2.
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These results strongly indicate an inverse correlation between expression and the degree of methylation of promoter CpG sites of the MDR1 gene in bladder cancer after chemotherapeutic treatment.
Alteration of the Degree of Methylation during the Clinical Course.
Knowing if the degree of methylation affects the expression of the MDR1
gene during a clinical course for bladder cancer is important. We
defined the cutoff point as 33.5%, which was the median value of the
degree of methylation at the promoter region. We evaluated three groups
of patients undergoing a clinical course for treatment of bladder
cancer. In the first group, composed of patients with untreated primary
tumors with intravesical recurrence, 9 (82%) of 11 showed
hypermethylation, whereas the median value of the MDR1 gene
expression was 0.9. The second patient group had recurrent tumors after
intravesical chemotherapy, with a median MDR1 expression
value of 4.2 (n = 16) and 8 (50%) of 16 patients
showing hypermethylation. The number of patients with a hypermethylated
MDR1 promoter thus decreased from 82 to 50% during the
interval between intravesical chemotherapy and recurrence. The third
group used systemic chemotherapy. The median value of MDR1
expression was 6.8 (n = 12) and 2 (17%) patients of 12
showed hypermethylation. Again, the MDR1 promoter was demethylated, and
MDR1 expression increased after systemic chemotherapy
compared with before the treatment.
Samples for each patient before and after chemotherapeutic treatment
were available from four patients. In all of the cases, the degree of
methylation decreased, and MDR1 expression increased during
the clinical course lasting from the first detection of the untreated
primary tumor to tumor recurrence or residual tumor (Table 3)
. In cases 1 and 2 (see Table 3
), MDR1 gene expression was
very low when the promoter region was hypermethylated in primary
tumors. In recurrent tumors after intravesical chemotherapy,
MDR1 gene expression increased, and the promoter region was
hypomethylated (Table 3)
. In case 3, MDR1 gene expression
increased 8.0-fold in residual tumors after systematic chemotherapy,
whereas the degree of methylation decreased only slightly to 60% of
the initial level. In case 4, the residual tumors after systemic
chemotherapy showed a 2.4-fold increase in MDR1 mRNA levels
compared with MDR1 mRNA levels at initial recurrence after
intravesical chemotherapy. Thus, about a threefold decrease in the
degree of methylation appeared in residual tumor after systemic
chemotherapy in case 4.
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Discussion |
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We then asked what determines MDR1 overexpression, and we
found an inverse correlation between MDR1 overexpression and
the degree of methylation at CpG sites of the MDR1 promoter
region. Alteration of the degree of methylation at the promoter region
of MDR1 appears to progress in two steps during a clinical
course. In the majority of patients with bladder cancer, the
MDR1 gene is de novo methylated during
carcinogenesis from the hypomethylation level in normal bladder tissue
(data not shown) by means of increased DNA methyltransferase activity
or by other mechanisms (32)
. Thus, demethylation occurs
during chemotherapeutic treatment, and the MDR1 gene is
overexpressed after chemotherapeutic treatment attributable to cytoxic
stress caused by anticancer drugs and/or the selection of cancer cells
with hypomethylation and a high MDR1 expression. We found
that the degree of methylation changed from the hypermethylation level
to the hypomethylation level, which was accompanied by overexpression
of the MDR1 gene, in all of the patients whose
specimens were available both before and after chemotherapeutic
treatment (Table 3)
.
Regardless of a slight degree of site specificity, such as sites
/ and for methylation and/or demethylation (see Fig. 5
A), the degree of methylation of any CpG site appeared to
correlate inversely with MDR1 expression. Moreover, we found
not only a lesser degree of methylation at any specific site or a
lesser fraction of cells with hypermethylation but also a smaller
number of sites with hypermethylation and more MDR1
expression. In our previous study (21)
, the degree of
methylation detected by MM2 primer pairs at sites / and 21 bp
adjacent to the YB-1 binding site correlates inversely with the
MDR1 expression in acute myeloid leukemias, but such an
inverse correlation is not observed when determined by MM4 primer
pairs. In this study, we found again that sites / were highly
susceptible to demethylation or were highly protected from methylation
during overexpression of the MDR1 gene. Transcription
factors protect the promoter from methylation (33)
.
Consistent with this fact, Sp1 elements may prevent the spread of
methylation (34
, 35)
. In contrast to the results obtained
in acute myeloid leukemias (21)
, in the present study the
altered degree of methylation status of other sites such as and
as detected by MM4 primer pairs also correlates with MDR1
expression in chemotreated bladder cancers. The degree of methylation
of putative cis-acting elements at or near sites and
in intron 1 may affect the MDR1 expression. Alternatively,
the hypermethylation status expands from sites and to sites
/ and , resulting in overall hypermethylation at the promoter
region (see Fig. 5
A), including the YB-1 binding site, and
inhibition of MDR1 expression. The expansion of the
methylated level at CpG sites with alteration of the chromatin
structure may be a mechanism for transcriptional inhibition both in
in vivo and in vitro systems (19
, 36
, 37)
.
In conclusion, MDR1 gene overexpression might be a prognostic factor for intravesical recurrence, and the degree of methylation in the MDR1 promoter region appears to be associated closely with MDR1 gene expression. The hypomethylation status of the MDR1 promoter might be a necessary condition for increasing MDR1 mRNA levels, as well as for developing a multidrug resistant phenotype, in patients with bladder cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Ministry of
Education, Science, Sports, and Culture of Japan, and Second-Term
Comprehensive Ten-Year Strategy for Cancer Control from the Ministry of
Health and Welfare, and CREST (Core Reseach for Evolutional Science and
Technology) of Japan Science and Technology Corporation (to
J. S. T.). 
2 To whom requests for reprints should be
addressed, at Department of Medical Biochemistry, Graduate School of
Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan. Phone: 81-92-642-6100; Fax: 81-92-642-6203;
E-mail: wada{at}biochem1.med.kyushu-u.ac.jp
3 The abbreviations used are: P-gp,
P-glycoprotein; MRP, multidrug resistance protein; RT, reverse
transcription; YB-1, Y-box binding protein.
Received 6/27/00; revised 9/14/00; accepted 9/14/00.
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, Pt 1; 
, SNK57.
, the position of exons 1 and 2.
B, representative results of quantitative PCR analysis.
The corresponding region and size of PCR products for the primer pairs
MM2, MM4, MC2, and TPI5 are shown. C, plots of the
density of the bands analyzed by FLA2000 image. Results for PCR primer
pairs MM2 (
); and TPI5 (x).
, patient group with residual
tumors after systemic chemotherapy; • the patient group with
recurrent tumors after intravesical chemotherapy. Solid
line, the gradual linear regression. The value of the degree of
methylation status at which the slope changes significantly is
17.5%.