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[Cancer Research 62, 4048-4053, July 15, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

The Association of Death-associated Protein Kinase Hypermethylation with Early Recurrence in Superficial Bladder Cancers1

Yasuhiro Tada, Morimasa Wada2, Ken-ichi Taguchi, Yasushi Mochida, Naoko Kinugawa, Masazumi Tsuneyoshi, Seiji Naito and Michihiko Kuwano

Departments of Medical Biochemistry [Y. T., M. W., Y. M., M. K.], Anatomical Pathology [K. T., M. T.], Medical Information [N. K.], and Urology [S. N.], Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanisms for bladder carcinogenesis and the development of recurrentbladder cancer remain unclear. Aberrant methylation of the 5' CpG island is thought to play an important role in the inactivation of the tumor suppressor genes in cancer. To study whether specific or bulk hypermethylation predicts intrabladder recurrence, we determined the frequency of aberrant promoter hypermethylation of seven genes, hMLH1, O6-methylguanine-DNA-methyltransferase (MGMT), p16, Von Hippel-Lindau (VHL), death-associated protein kinase (DAP-kinase), glutathione S-transferase P1 (GST-P1) and E-cadherin in 55 superficial bladder cancers and 5 normal urothelial epithelia by methylation-specific PCR (MSP). These patients of superficial bladder cancer had been followed prospectively by cystoscopy. Simultaneous hypermethylation of three genes or more among the seven genes was observed in 2 (7%) of 30 patients in the nonrecurrence group and 7 (28%) of 25 patients in the recurrence group. There was a significant concordance between the number of methylated genes and the development of recurrence (P = 0.012). In particular, the recurrence rate for 24 months was 88% for hypermethylation of DAP-kinase and 28% for nonmethylation of DAP-kinase. Hypermethylation of DAP-kinase is, therefore, a strong indicator of the superficial bladder cancer associated with a high recurrence rate (P < 0.001; hazards ratio, 7.01). Our results suggest that hypermethylation of DAP-kinase might be a useful prognostic marker for disease recurrence in superficial bladder cancers.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the majority of patients, bladder cancer is superficial at initial diagnosis. Currently, superficial papillary bladder cancers are treated conservatively, whereas muscle-invasive bladder cancers are treated more aggressively. After TUR3 of superficial bladder cancer, patients are monitored by cystoscopy for its invasiveness at regular intervals, and they receive intravesical prophylactic intense instillation chemotherapy. Recurrence of superficial bladder cancer is seen in more than 50% of patients (1, 2, 3) . Clinical and histopathological factors that might assist in the prediction of tumor recurrence and the progression of bladder cancer have been studied, and tumor grade has been identified as a good predictor of recurrence (1, 2, 3) . The development of any useful diagnostic marker for the prediction of tumor recurrence would facilitate more effective management of this cancer.

Fewer mutations of tumor suppressor genes occur in superficial bladder cancers than in invasive bladder cancers (4) . Alterations of the DNA methylation pattern have been recognized as common changes in human cancers (5) . A plausible pathway for the global hypermethylation has been presented in colon cancer, which is characterized by simultaneous methylation of multiple CpG islands, including several known genes such as p16 and hMLH1 (6 , 7) . Aberrant methylation of the normally unmethylated CpG island, which is located in or near the promoter region of many genes, has been associated with transcriptional inactivation of cancer-related genes including hMLH1, MGMT, p16, VHL, DAP-kinase, and E-cadherin and in various human cancers (8 , 9) .

The inactivation of the tumor suppressor gene p16 by promoter hypermethylation has been described in the primary superficial bladder cancers (10) . Methylation analysis of a mismatch repair gene, hMLH1, a detoxification gene, GST-P1 and a cell adhesion gene, E-cadherin, has been demonstrated in various malignancies including colon, gastric, and prostate tumors. MGMT is a DNA repair gene for guanosine methyl adducts that is frequently inactivated in lymphomas and colon, lung, and brain tumors (11) . Aberrant methylation of the CpG island promoter region is also associated with inactivation of the VHL tumor suppressor gene in 20% of clear cell renal carcinomas (8) . The inactivation of DAP-kinase by hypermethylation in the promoter CpG region has been demonstrated in cells derived from human breast, urinary bladder, and renal cell carcinomas (12) and in clinical samples of primary lung cancer (13) and B-cell malignancies (14) . DAP-kinase, a pro-apoptotic calcium-regulated serine/threonine kinase, is involved in IFN-{gamma}-induced apoptosis. However, the clinical significance of these observations is limited. Moreover, the clinical significance of the aberrant methylation of hMLH1, MGMT, VHL, DAP-kinase, GST-P1, and E-cadherin genes in superficial bladder cancers has not been examined.

We previously reported the inverse correlation between MDR1 gene expression and methylation status of its promoter region in bladder cancer tissues, and we observed the weak correlation between the development of recurrence and hypermethylation of the MDR1 promoter region in untreated superficial bladder cancers (15) . In our present study, we have analyzed the methylation status of various cancer-related genes such as hMLH1, MGMT, p16, VHL, DAP-kinase, GST-P1, and E-cadherin genes in superficial bladder cancers. We examined for any possible association of methylation status of these seven genes with recurrence, because knowledge of such an association could be useful in the effective management of this cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor Samples, Normal Control, and DNA Extraction.
Between June 1991 and December 2000, 55 Japanese male and female patients with superficial bladder cancer (Ta, T1) underwent TUR of a bladder tumor in the Department of Urology, Kyushu University Hospital, Fukuoka, Japan. Median age at the time of diagnosis was 68 years (range, 24–86); the male: female ratio was 5.7:1. Clinical stage (Ta, T1) was assigned according to the unified TNM systems (16) . Clinical grade (grade 1, grade 2, grade 3) was assessed as defined by the Japanese Urological Association and the Japanese Society of Pathology (17) . We examined each patient’s 24-month clinical follow-up, including a cystoscopic examination every 3 months, from all of the patients that were diagnosed with superficial bladder cancer at the time of methylation analysis. A recurrence was defined as the presence of histologically proven bladder cancer at a positive cystoscopy after a complete previous TUR. All of the patients received the cystoscopic examination after a month of a complete TUR, and urologists confirmed that none of the patients had disease recurrence in the bladder. The samples of normal urothelial epithelium were obtained from the ureter and renal pelvis of patients with renal cell carcinoma who underwent radical nephrectomy. The normal peripheral blood was obtained from healthy volunteers. The samples of tumor tissue and tissue from normal controls were obtained after patients provided informed consent. The tumor and normal control tissue samples were frozen in liquid nitrogen and stored at -80°C until DNA extraction. DNA was isolated from the tissues of patients using the Easy DNA kit (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s protocol.

Bisulfite Modification and MSP.
We used the CpGenome DNA Modification kit (Invitrogen Corporation) for bisulfite treatment according to the manufacturer’s protocol. MSP was carried out essentially as described previously (18) and was based on the principle that treating DNA with bisulfite would result in the conversion of unmethylated cytosine residues into uracil and subsequent PCR using primers specific for either the methylated or the modified unmethylated sequence. PCR reactions were performed in a 10-µl reaction volume, using previously published conditions (18) and 0.5 units of Taq polymerase (Applied Biosystems, Foster, CA). The amplifications were carried out in a Thermal cycler (Takara Shuzo Co., Kyoto, Japan) for 32 cycles using an non-methyl-detected primer pair or 35 cycles using a methyl-detected primer pair (30 s at 95°C, variable temperatures according to the primer, and 30 s at 72°C) and a final 10-min extension at 72°C. The PCR products were electrophoresed on 3% agarose gel and stained with SYBER Green I (Molecular Probes, Eugene, OR), and they were then analyzed by scanner and FLA2000 image analysis (Fuji Film, Tokyo, Japan).

MSP was performed to examine the methylation at the promoter of hMLH1, MGMT, p16, VHL, DAP-kinase, GST-P1, and E-cadherin. The primer sequences for p16, VHL, E-cadherin (18) , hMLH1 (19) , MGMT (11) , DAP-kinase (14) , and GST-P1 (9) genes were described previously. Hypermethylations of each gene were defined as occurring when methylated molecules were detected using a methyl-detected primer pair. Nonmethylations of each gene were defined as occurring when only unmethylated molecules were detected using MSP analysis.

Bisulfite Sequencing Analysis.
The primer sequences and annealing temperature for Sequence 1 and 2 reactions are described in Table 1Citation . The PCR products were purified using the DNA and Gel Band Purification kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and cloned into pGMT-Easy vector (Promega Co., Madison, WI). Plasmid DNA was extracted from individual clones by alkaline lysis plasmid minipreparation. The inserted PCR fragments of more than 12 individual clones obtained from each sample were sequenced with both T7 forward primer and SP6 reverse primer using ABI Prism Dye Terminator Cycle sequencing kit (Perkin-Elmer, Boston, MA) and ABI Prism 377 DNA sequencer (Perkin-Elmer).


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  		Table 1 Primer sequences and PCR conditions for bisulfite sequencing

 
Immunohistochemistry.
The immunohistochemistry used in this study was performed as described previously with minor modifications (20) . In brief, immunohistochemical observations were performed on deparaffinized sections using the peroxidase-labeled streptavidin-biotin technique with the Histofine SAB-PO kit (Nichirei, Tokyo, Japan). DAP-kinase was detected using monoclonal antibody against DAP-kinase (BD Biosciences, San Jose, CA). The working dilution was 1:100. For the purpose of antigen retrieval, the sections were placed in plastic jars containing 0.1% Tween 20 in citrate buffer (pH 6.0) for 15 min at 100°C in a microwave oven. The negative controls consisted of substituting mouse normal serum for the primary antibodies to verify the possibility of false-positive responses from the secondary antibody.

Statistical Analysis.
Recurrence-free survival curves were calculated using Kaplan-Meier methods, and a log-rank test was used for the analysis. Multivariate analysis was performed using Cox’s proportional hazards model. Fischer’s exact test was used to analyze the methylation status of the DAP-kinase gene in relation to the expression of the gene. The Mann-Whitney t test was used for differences in medians of age. All of the probability values (Ps) of <0.05 were considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MSP Analysis in Superficial Bladder Cancer.
Representative results of MSP analysis on the seven genes, hMLH1, MGMT, p16, VHL, DAP-kinase, GST-P1, and E-cadherin, in superficial bladder cancer are presented in Fig. 1Citation . Fig. 2Citation summarizes the promoter hypermethylation frequency of each gene in the samples of superficial bladder cancers and normal controls, which were obtained from the ureter and renal pelvis of patients with renal cell carcinoma who underwent radical nephrectomy. The nonmethylation status of each gene could be determined when unmethylated molecules alone were detected by MSP analysis. Alternatively, hypermethylation status can be monitored by noting the presence of the band amplified using methylated sequence-specific primers. We found that 73% (40 of 55) of superficial bladder cancers exhibited hypermethylation of the promoter in at least one gene: 41% of tumors had one methylated gene, 15% of the tumors had two methylated genes, 5% of tumors had three methylated genes, and 9% of the tumors had more than three methylated genes (Fig. 2)Citation . Hypermethylation was detected in 13% for hMLH1, 17% for MGMT, 11% for p16, 4% for VHL, 29% for DAP-kinase, 13% for GST-P1, and 48% for E-cadherin in 55 superficial bladder cancers.



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  		Fig. 1. Methylation analysis of seven genes in superficial bladder cancers by MSP. At the left of each panel, the gene studied. Lane U, amplified product with primers recognizing an unmethylated sequence; lane M, amplified product with primers recognizing a methylated sequence. Normal peripheral blood lymphocytes (NL) were used as a negative control. In vitro methylated DNA (IVD) was used as a positive control for methylation. H2O,water blanks; L, 100-base marker. The PCR product sizes of all of the genes are summarized in Table 1Citation .

 


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  		Fig. 2. Summary of methylation of hMLH1, MGMT, p16, VHL, DAP-kinase, GST-P1, and E-cadherin in superficial bladder cancer tissues. Black boxes, samples that are methylated. White boxes, samples that are not methylated. (N.D), not determined.

 
Correlations between Recurrence in Bladder and Methylation Status.
We examined whether the development of recurrence in the bladder would be associated with the overall aberrant methylation at first diagnosis. A recurrence was defined as the presence of histologically proven bladder cancer at a positive cystoscopy after a complete previous TUR. The patients carrying tumors that showed simultaneous aberrant methylation of more genes were expected to develop recurrent tumors more easily. The fraction of recurrent patients from among patients who showed simultaneous hypermethylation of 0, 1, 2, 3, 4, and 5 genes were 33, 41, 50, 67, 75, and 100%, respectively. Simultaneous hypermethylation of three or more genes was observed in 28% of the patients in the recurrence group (n = 25) and in only 7% of the patients in the no-recurrence group (n = 30; Fig. 3ACitation ). Moreover, all of the genes were unmethylated in only 17% of patients in the recurrence group, and all were unmethylated in 33% of the patients in the no-recurrence group (Fig. 2)Citation . Seven (77%) of nine patients who showed simultaneous hypermethylation of three or more genes developed recurrence (P = 0.012; Fig. 3ACitation ).



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  		Fig. 3. Association of recurrence with simultaneous hypermethylation of three or more genes (A) and aberrant hypermethylation of DAP-kinase (B). The Kaplan-Meier method was used to determine the percentage without recurrence and the log-rank test was used to compare the recurrence-free curves between two groups. A, the percentage without recurrence for patients with three or more methylated genes (3<=) versus patients with less than three methylated genes (<3) at primary diagnosis, respectively. B, the percentage without recurrence for patients with DAP-kinase hypermethylation (Hyper) versus patients with nonmethylation (Non) at primary diagnosis.

 
We next examined whether hypermethylation of any specific gene would correlate with recurrence. Hypermethylation of hMLH1, MGMT, p16, VHL, DAP-kinase, GST-P1, and E-cadherin was detected in 7, 14, 10, 0%, 7, 13, and 48, respectively, of tumor tissues associated with no recurrence (n = 30) and in 20, 20, 12, 8, 56, 12, and 48%, respectively, of tumor tissues associated with recurrence (n = 25). Among these various genes, the methylation status of DAP-kinase was significantly different in the recurrence group versus the no-recurrence group (Fig. 2)Citation . We further analyzed the relationship between recurrence rate for 24 months after complete TUR and the methylation status of the DAP-kinase gene (Fig. 3B)Citation . Fourteen (88%) of 16 patients with hypermethylation of the DAP-kinase gene had tumor recurrence within 15 months. 28 (71%) of 39 patients who showed nonmethylation of the DAP-kinase gene had no disease recurrence for 24 months (P < 0.001; Fig. 3BCitation ).

We performed multivariate analysis using Cox’s proportional hazards model to assess the most informative factors among Grade and T stage and DAP-kinase-methylation for recurrence in bladder (Table 2)Citation . We found that the most important prognostic factor for recurrence was the methylation status of DAP-kinase (P < 0.001; hazards ratio, 7.01). Other factors were not found to create significant risk for the model. Hypermethylation of the DAP-kinase gene, thus, appeared as a strong indicator of the development of recurrence.


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  		Table 2 Contribution of various potential prognostic factors to recurrence-free survival by Cox’s proportional hazards model in 55 superficial bladder cancers

 
Correlation between Methylation Status and Expression of the DAP-kinase Gene in Superficial Bladder Cancer.
We examined the methylation status at the two regions of the DAP-kinase gene in tumor samples and normal tissues by bisulfite genomic sequencing analysis to confirm the results obtained by MSP analysis (Fig. 4)Citation . We selected two regions at promoter because it has been well established that expression is suppressed by hypermethylated promoter region, but not at coding region (21 , 22) . Sequence 1 region is located at the upstream region of the transcriptional start site, and Sequence 2 region is located at exon 1 and intron 1, which is a noncoding region. The methylation status of DAP-kinase in both of the Sequence 1 and 2 regions analyzed by bisulfite sequencing were found to be well correlated with the results obtained by MSP. Degree of methylation measured by bisulfite sequencing analysis at almost all of the CpG sites of the Sequence 1 and 2 regions were 0% in the tumor samples of 91BT-15, 92BT-15, and 92-19, which were unmethylated, using MSP. In contrast, almost all of the CpG sites of that region were heavily methylated in the tumor sample of 97BT-23, and all of the CpG sites of the Sequence 1 and 2 regions were methylated under 25% in the sample of 93BT-16, whereas tumor samples of both 97BT-23 and 93BT-16 were methylated using MSP. The MSP analysis, thus, appeared to detect a low level of methylation in the promoter of DAP-kinase (Fig. 4)Citation .



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  		Fig. 4. Methylation maps of the DAP-kinase CpG island. Summary of bisulfite genomic sequencing analysis for normal controls and superficial bladder cancers. A, short vertical bars, the CpG sites and GpC sites. Bent arrow, the transcription start site; straight arrow below the line, the start site of translation. MSP, Sequence 1, and Sequence 2, the location of each analysis. B, on the left, the sample number; numbered across the top, CpG sites from the start site of transcription. On the right, the results using MSP analysis; +, hypermethylation; -, nonmethylation. {circ}, 0% methylation; , 1–25% methylation; , 26–50% methylation; , 51–75% methylation; {bullet}, 76–100% methylation.

 
We also evaluated the correlation between the methylation status and the expression of DAP-kinase in superficial bladder cancer using immunohistochemistry (Fig. 5Citation ; Table 3Citation ). Fig. 5ACitation shows the immunostaining data of DAP-kinase in a noncancerous region that showed nonmethylation of DAP-kinase using MSP analysis. Normal epithelium in a noncancerous region showed positive staining (Fig. 5A)Citation . The tumor sample of 93BT-11 that showed the nonmethylation of DAP-kinase using MSP analysis showed positive staining in cancer cells (Fig. 5B)Citation . The same results were obtained in other samples that showed the nonmethylation of DAP-kinase using MSP analysis (Table 3)Citation . In contrast, almost none of the cancer cells were stained with anti-DAP-kinase antibody in the tumor sample of 93BT-16 that had the hypermethylation of DAP-kinase using MSP and bisulfite sequencing analysis (Fig. 5C)Citation . Two cases of the positive expression of DAP-kinase gene by immunohistochemistry showed hypermethylation of DAP-kinase gene (Table 3)Citation . These two cases were 99BT-4 and 00BT-2 (Fig. 2)Citation . The patient 99BT-4 had no recurrence in the bladder. We examined the degree of DAP-kinase methylation in Sequence 1 and 2 regions using bisulfite sequencing in these two cases. There appeared to be methyl-cytosine in almost none of the CpG sites of Sequence 1and 2 regions in these two cases, when we independently examined 12 clones in each case (data not shown). The expressions of DAP-kinase using immunostaining, thus, appeared to be well correlated with the results of MSP and bisulfite sequencing analysis in the Sequence 1and 2 regions.



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  		Fig. 5. Examples of DAP-kinase immunostaining in two representative cases of superficial bladder cancers and noncancerous region. A, normal urothelial epithelium in a noncancerous region showed positive staining. B, cancer cells show a diffuse and strong, positive staining (93BT-11). C, less than 50% of cancer cells can be regarded as positive (93BT-16). The strong staining of lymphocytes provided a useful internal positive control for preservation of DAP-kinase immunogenicity. A, B, and C, x400.

 

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  		Table 3 The correlation between DAP-kinase expression and methylation status of DAP-kinase in superficial bladder cancers

 
Previous studies reported that the hypermethylation of DAP-kinase gene was related to the aging (23 , 24) . We examined whether the methylation status of DAP-kinase gene was correlated with the patient’s age (Table 4)Citation . However, there appeared to be no significant relationship between methylation status of DAP-kinase and the aging in superficial bladder cancer.


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  		Table 4 Methylation status of DAP-kinase gene and patient’s age

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of methylation status of each gene using MSP analysis suggested that aberrant methylations were important for the tumorigenesis of superficial bladder cancer. Our results showing hypermethylation of p16 and E-cadherin in 11 and 48% of bladder cancers, respectively, are in agreement with the results described in previous reports (25 , 26) . GST-P1 was methylated in 7 (13%) of 55 patients in our study, and altered methylation of the gene has also been reported in prostate, breast, and renal cancers (9) . However, the methylation status of p16, GST-P1, and E-cadherin in the original tumors was not clearly associated with early recurrence. We first found the promoter hypermethylation of hMLH1, MGMT, VHL, and DAP-kinase genes in superficial bladder cancer. These genes are associated with an increased risk of recurrence, and the bulk methylations of the seven genes appeared to be associated with recurrence. In particular, we noted that 14 (88%) of 16 patients with hypermethylation of DAP-kinase had the recurrence of bladder tumor. The number of clinical samples showing hypermethylation of DAP-kinase gene was 16. The P using log-rank test was very small, and the hazards ratio and confidence interval using multivariate analysis were, thus, impressive. Although independent studies with larger scale are required to validate usefulness before clinical application, the methylation status of DAP-kinase could be useful as a molecular marker in the diagnosis of bladder cancer and during follow-up treatment after complete TUR.

The gene encoding DAP-kinase was initially isolated as a positive mediator of apoptosis induced by IFN-{gamma} (27) . DAP-kinase also participates in apoptosis induced by other ligands such as TNF-{alpha} and Fas, and it is also involved in the p53-dependent apoptosis pathway (28) . Relevant studies have previously reported that the promoter hypermethylation detected by only MSP analysis is associated with down-regulation of DAP-kinase expression in cancer cells and clinical samples of B-cell malignancy (12 , 14) . We observed a closer association of hypermethylation in the Sequence 1 and 2 regions and the MSP region of the DAP-kinase with the suppression of the expression.

It is generally accepted that the mechanisms of recurrence in bladder cancer develop through an accumulation of the genetic and epigenetic changes in tumor suppressor genes and drug resistance-related genes, and/or shedding cells from original tumors are equipped to become implanted in the bladder epithelium (29 , 30) , but it remains unclear how the methylation status of the DAP-kinase gene is involved in the recurrence of bladder cancer. The survival of the malignant cells through the down-regulation of the DAP-kinase gene and the subsequent increase in intrabladder shedding might increase the risk of recurrence. Alternatively, the decreased DAP-kinase expression could not eliminate premalignant cells by apoptosis that had been plausibly developed under the exposure to chemical carcinogens in the bladder, and the down-regulation of DAP-kinase expression, thus, might promote tumorigenesis, resulting in an increased risk of another tumor developing after a complete TUR. In the present study, all tumor tissues with hypermethylation of the DAP-kinase gene showed early recurrence within 15 months, and, moreover, the hypermethylation of the DAP-kinase gene was observed in the cancerous region but not in the noncancerous region, when seven clinical specimens were examined.4 These results suggest that the above-mentioned first possibility might be preferentially involved in the close association of hypermethylation of the DAP-kinase gene and tumor recurrence.

The MSP method would be useful for molecular diagnosis because it is simple, rapid, cost effective, and sensitive, allowing the rapid examination of multiple markers and samples (18) . Molecular markers for bladder cancer may further provide useful information for clinical decision making. Our results suggest that the frequency of the uncomfortable, invasive, and expensive diagnostic procedure used to detect bladder cancer could be reduced considerably if patients could be identified because of nonmethylation of multiple genes including the DAP-kinase gene.

In conclusion, treatment strategies may also be determined based on these molecular markers used as part of evidence-based medical treatment. Aberrant methylation frequently occurs in superficial cancers. Hypermethylation of multiple genes, especially the DAP-kinase gene, might be a strong indicator of a high recurrence rate in cases of superficial bladder cancer.


   ACKNOWLEDGMENTS
 
We thank Hirohumi Koga, Koji Okumura, and Akira Yokomizo in Kyushu University for helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the Second-Term Comprehensive Ten-Year Strategy for Cancer Control from the Ministry of Health and Welfare. Back

2 To whom requests for reprints should be addressed, at the Department of Medical Biochemistry, Graduate School of Medical Science, 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 Back

3 The abbreviations used are: TUR, transurethral resection; DAP-kinase, death-associated protein kinase; GST-P1, glutathione S-transferase P1; VHL, Von Hippel-Lindau; MGMT, O6-methylguanine-DNA-methyltransferase; MSP, methylation-specific PCR. Back

4 Y. Tada, M. Wada, and M. Kuwano, unpublished data. Back

Received 11/26/01. Accepted 5/16/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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