Clinical Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Christoph, F.
Right arrow Articles by Schrader, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Christoph, F.
Right arrow Articles by Schrader, M.
Clinical Cancer Research Vol. 12, 5040-5046, September 1, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Promoter Hypermethylation Profile of Kidney Cancer with New Proapoptotic p53 Target Genes and Clinical Implications

Frank Christoph1, Steffen Weikert1, Carsten Kempkensteffen1, Hans Krause1, Martin Schostak1, Jens Köllermann2, Kurt Miller1 and Mark Schrader1

Authors' Affiliations: 1 Department of Urology, Charité, Universitätsmedizin Berlin, Berlin, Germany and 2 Department of Urology, Klinikum Fulda gAG, Fulda, Germany

Requests for reprints: Frank Christoph, Department of Urology, Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. Phone: 0049-30-8445-2575; E-mail: frank.christoph{at}charite.de.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Risk stratification of renal cell carcinoma is based on the histopathologic classification. Promoter hypermethylation as a mechanism of gene inactivation in renal cell carcinoma has been shown for only a small number of genes. We examined the usefulness of quantitative methylation analysis with a new set of p53 target genes for determining the clinical outcome and aggressiveness of the tumor disease.

Experimental Design: The genes selected were APAF-1, CASPASE-8, DAPK-1, IGFBP-3, and PML. The tissue samples analyzed were taken from tumor specimens obtained from 90 consecutive patients with clear cell renal carcinoma and from 20 normal kidney specimens. Quantitative methylation analysis of CpG sites in the promoter region was done by methylation-specific real-time PCR and the normalized index of methylation (NIM) was determined for each sample.

Results: Hypermethylation of the promoter region was common for APAF-1 (97%) and DAPK-1 (41%) but not for IGFBP-3 (3%), PML (3%), or CASP-8 (0%). The tumor patients had a median follow-up of 55 months. A correlation was found between the methylation level of APAF-1 and tumor size and nodal status, but not for tumor stage, grade, and age of patient. Kaplan-Meier analysis was able to identify patients with a higher risk of recurrence and tumor-related death by using APAF-1 (≥56% NIM) and DAPK-1 (≥10% NIM) methylation levels. In multivariate analysis, APAF-1 and DAPK-1 methylation levels were independent prognostic markers for metastatic disease and death from renal cell carcinoma.

Conclusions: Our findings indicate that promoter hypermethylation of APAF-1 and DAPK-1 is a marker of aggressive renal cell carcinoma and provides independent prognostic information on disease outcome.

Renal cell carcinoma is the third most common urogenital malignancy, with an estimated 35,000 new cases diagnosed in the United States each year (1). Metastases are detectable in nearly one fourth of the patients at presentation and develop during the follow-up in another 50%. Surgical removal of solitary metastatic masses is recommended. However, in patients with multiple metastatic lesions, palliative immunochemotherapy maintains stable disease in <10% with a median survival of 5 to 7 months (2). Because efficient therapeutic strategies are limited in advanced metastatic renal cell carcinoma, it is important to pinpoint clinical markers that can identify patients at higher risk for metastatic disease. This would enable risk stratification and possibly early induction of adjuvant therapy.

Cytogenetic alterations are frequently found in renal cell carcinoma and have been described for a variety of histologic subtypes. These alterations may consist in mutations of the VHL gene (3p25) or the loss of chromosome 3p as well as in trisomy of chromosomes 7 and 17, which is seen in papillary renal cell carcinoma (3, 4). A common mechanism of gene inactivation is hypermethylation of normally unmethylated CpG dinucleotides (CpG islands) in the promoter region of a gene. Promoter hypermethylation has proved to be frequent in human cancer involving genes that function as tumor suppressors or control signaling pathways (5). Hypermethylation panels have recently been used for renal cell carcinoma, and some genes such as the VHL gene, RASSF1A, and Timp-3 have proved to be frequently methylated within their promoter region (6). In addition, the cell adhesion protein {gamma}-catenin was recently found to be hypermethylated and to correlate with the aggressiveness of the tumor disease (7). Because tumor suppressor genes are essential for growth regulation and tumor growth inhibition, their epigenetic inactivation could have fatal consequences. The mechanisms by which p53 is regulated in renal cell carcinoma are not yet entirely understood. Mutations of the p53 gene, as frequently seen in cervical or bladder carcinoma, are not common (8). We therefore suggested that a set of p53 target genes mediating p53-induced apoptosis is eventually altered by epigenetic mechanisms. All genes selected contain p53 binding sites in their downstream region. The genes APAF-1 and DAPK-1 show varying degrees of promoter methylation in malignant melanoma, lymphoblastic leukemia (APAF-1), and bladder cancer (DAPK-1; refs. 9–11). The genes CASPASE-8, IGFBP-3, and PML have not yet been evaluated for promoter methylation in tumor disease. We also hypothesized that the methylation level of the genes investigated depends on the histopathologic tumor classification and may be associated with a poor prognosis of renal cell carcinoma. The methylation status of the functional promoter of these genes was therefore analyzed by highly sensitive real-time quantitative PCR with special reference to the clinical outcome of renal cell carcinoma patients.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Specimen collection and DNA extraction. Tissue specimens were obtained from 90 patients (61 males, 29 females) who had primary clear cell renal cell carcinoma and underwent radical nephrectomy. Normal kidney specimens were obtained from 10 patients who underwent nephrectomy for a nonmalignant tumor disease like hydronephrosis and from 10 patients in whom normal kidney tissue was taken distant from the tumor. The tissue samples were immediately shock-frozen after surgical resection of the tumor or normal tissue and stored in liquid nitrogen at –80°C. Before further processing, samples were serially sectioned, stained with H&E, and examined by a uropathologist (J.K.) to ensure that slices used for subsequent DNA extraction predominantly contained malignant tissue of the respective tumor. The areas with the highest neoplastic cell content (>80%) were selected and microdissected under microscopic control using a sterile needle. Histopathologic staging and grading followed the tumor-node-metastasis (TNM) classification of the International Union against Cancer (1997). All patients signed a consent form approved by the Committee on Human Rights in Research at our institution. Patients' characteristics are specified in Table 1 .


View this table:
[in this window]
[in a new window]
  		Table 1. Patient characteristics

 
DNA preparation. DNA was isolated from the tissue of patients using the BioRobot EZ1 workstation according to the instructions of the manufacturer (Qiagen, Hilden, Germany). The DNA concentration was determined by spectrophotometry and its integrity was checked by 1.5% gel electrophoresis.

Bisulfite modification and methylation-specific PCR. We used the MethylEasy DNA Bisulfite Modification Kit for bisulfite treatment according to the protocol of the manufacturer (Human Genetic Signature, Sydney, Australia). Methylation analysis was done by fluorescence-based real-time PCR using TaqMan hybridization probes with the Light Cycler Instrument (Roche, Mannheim, Germany). Each primer set contained at least two CpG sites and, except for DAPK-1 (5' untranslated region), was located within the promoter region of the gene. PCR consisted of initial denaturation for 10 minutes at 95°C, followed by 50 denaturation cycles with individual annealing temperatures and times (APAF-1, 60°C, 20 seconds; CASP-8, 64°C, 30 seconds; DAPK-1, 58°C, 20 seconds; IGFBP-3, 60°C, 15 seconds; MyoD, 59°C, 15 seconds) and final denaturation for 40 seconds at 72°C. PCR was also done for non-CpG-containing regions of MyoD, which served as the control gene (sense, 5'-CCAACTCCAATTCCCCTCTCTAT; antisense, 5'-TGATTAATTTAGATTGGGTTTAGAGAAGGA; TaqMan, 6FAM-5'-TCCCTTCCTATTCCTAAATCCAACCTAAATACCTCCXT-PH; accession no. NM_002478). Primer sequences were used for APAF-1 (sense, 5'-TTTCGGGTAAAAGGGATAGAATTAGA; antisense, 5'-AAAAATCTTCCCGACCTATAACGC; TaqMan, 6FAM-5'-ATACCGCTACGACACCTCAAATCTTCGC-TMR; accession no. AB070829), for CASP-8 (sense, 5'-TAGGGGATTCGGAGATTGCGA; antisense, 5'-AAACCGTATATCTACATTCGAAACGA; TaqMan, 6FAM-5'-CCCGCTCCACCCTTTCCTAACACCA-TMR; accession no. AY291598), for DAPK-1 (sense, 5'-TCGTCGTCGTTTCGGTTAGTT; antisense, 5'-TCCCTCCGAAACGCTATCG; TaqMan, 6FAM-5'-CGACCATAAACGCCAACGCCG-TMR; accession no. NM_004938), for IGFBP-3 (sense, 5'-GAGATTTTATTTCGAGAGCGGAAG; antisense, 5'-GAACACCTACTCCTCGTACTCCACGC; TaqMan, 6FAM-5'-AAACCCGTCACCTTATCGTCTACAAAAACC-TMR; accession no. NM_0010133), and for PML (sense,: 5'-TACGAAGTAGTGTTAGTGTGAACGG; antisense, 5'-ACCGAACGAAATCCTACTAAAACCTC; TaqMan, 6FAM-5'-AAACCCCAACTTAATTTCGATTCTCGATTT-TMR; accession no. NM_0010133). Commercially available methylated human genomic DNA served as the fully methylated reference sample (Chemicon International, Temecula CA). All PCR reactions were run in duplicate for samples and controls. Fully methylated reference DNA was used to generate a standard curve with cycle threshold (Ct) values from each PCR reaction plotted against copy numbers. Thus, Ct values from each PCR reaction were first related to copy numbers. The next step was to calculate the percentage of the methylated target gene as the ratio of the copy number of the methylated gene of interest to that of the reference sample of fully methylated DNA, both normalized to the copy number of the control gene. This yielded the normalized index of methylation (NIM). Occasionally, the percentage of methylation was >100% in cases of aneuploidy at the gene locus of interest.

Statistical analysis. Two-tailed statistical analysis was done with SPSS computer software (version 12, SPSS, Inc., Chicago, IL). The Mann-Whitney, Kruskal-Wallis, and Spearman {rho} tests were done to statistically evaluate the methylation level of individual genes in relation to the patients' tumor stage, grade, and size as well as to their age and nodal status. The prognostic values of the methylation levels of the different genes investigated were assessed by univariate and multivariate analyses using the Kaplan-Meier method and the Cox proportional hazard model. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
We examined the hypermethylation status of a panel of five normally unmethylated tumor suppressor p53 target genes. The overall promoter hypermethylation prevalence of the genes investigated was 97% (87 of 90) for APAF-1, 41% (37 of 90) for DAPK-1, 3% (3 of 90) for IGFBP-3, 3% (3 of 90) for PML, and 0% (0 of 90) for CASP-8. Promoter methylation was found in only 4 (1 in DAPK-1 and 3 in APAF-1) of the 20 normal renal tissue samples and only at low levels with a maximum NIM of 3% for DAPK-1 and 1.5% for APAF-1. The methylation levels were evaluated according to the methylation prevalence and the methylation level expressed by the NIM. The less frequently (n = 3) methylated genes IGFBP-3 and PML had median methylation levels of 7% for IGFBP-3 in a pT1 G3 and a pT2 G3 tumor and 4% for PML in two pT1 G3 tumors.

The prevalence and level of gene methylation in relation to tumor stage and grade. The methylation prevalence of APAF-1 ranged from 93% (pT1) to 100% (pT2 and ≥pT3); it was 94% in G1/2 and 100% in G3 tumors. DAPK-1 had a methylation prevalence of 57% for pT1, 68% for pT2, and 71% for ≥pT3 tumors. DAPK-1 methylation was found in 60% of the G1/2 and 61% of the G3 tumors. The median APAF-1 methylation level (NIM) was higher in ≥pT3 (70%) than in pT2 (50.5%) or pT1 tumors (39%). The median NIM level of APAF-1 tended to correlate positively with tumor stage (Table 2 ). The individual methylation levels of DAPK-1 were very heterogeneous with median levels of 2.1% in pT1 tumors, 2.4% in pT2 tumors, and 7.2% in ≥pT3 tumors. No relevant correlation was detected between the stage and the methylation level. Comparing the different methylation levels according to the tumor grade disclosed a NIM of 38% in G1/2 and 56% in G3 tumors for APAF-1. The trend towards a statistically significant correlation was found for APAF-1 (P = 0.06) but not for DAPK-1, where the median NIM was 2% for both G1/2 and G3 tumors (P = 0.9).


View this table:
[in this window]
[in a new window]
  		Table 2. Percentage of methylation (NIM) and tumor stage/grade

 
The prevalence and level of gene methylation in relation to tumor size, nodal status, and age of patient. The APAF-1 (DAPK-1) methylation prevalence was 88% (63%) in tumors <4 cm and 96% (71%) in larger ones (TNM stage pT1a tumors are <4 cm). The NIM of APAF-1 was significantly lower in smaller tumors (<4 cm) than in those >4 cm (36% versus 59%; P = 0.004). The NIM of DAPK-1 was 1% in the smaller tumors and 3% in the larger ones (P = 0.36). Evaluation of the nodal status of a tumor and its relation to the methylation prevalence and the NIM revealed that 64 of 71 (90%) node-negative (N–) and all 19 (100%) node-positive (N+) tumors showed methylation of the APAF-1 gene. The DAPK-1 gene had a methylation prevalence of 64% (46 of 71) for the N– and 89% (17 of 19) for the N+ tumors. The NIM of APAF-1 was 48% for the N– tumors and 60% for the N+ tumors (P = 0.07). DAPK-1 had a NIM of 2% for the N– and 10% for the N+ tumors (P = 0.13). The NIM of APAF-1 and DAPK-1 did not correlate significantly with the age of patient (Spearman correlation coefficient, 0.1 and 0.08). When patients were grouped according to a cutoff age of 60 years, the prevalence for APAF-1 (DAPK-1) was 84% (76%) in younger patients and 96% (66%) in older ones. The NIM of APAF-1 was 40% in the younger group and 53% in the older one (P = 0.08), whereas that of DAPK-1 was 1% and 2%, respectively (P = 0.4). Further details are given in Table 3 .


View this table:
[in this window]
[in a new window]
  		Table 3. Median percentage of NIM (SD) and tumor characteristics

 
Correlation between methylation level, tumor recurrence, and death from renal cell carcinoma in univariate analysis. To address the question of whether the NIM can help to predict tumor recurrence or death from tumor disease, the recurrence-free and the disease-specific survival rates were evaluated in relation to the different methylation levels by Kaplan-Meier analysis. The first step was to group tumors according their median methylation level, which was 51% for APAF-1 and 2% for DAPK-1. Tumors were thus separated into low and high NIM groups (<51% or ≥51% NIM or 2% NIM, respectively). According to the log-rank test, recurrence-free survival was significantly associated with APAF-1 (P = 0.0007) and DAPK-1 (P = 0.01) methylation levels. The second step involved optimization by receiver operating characteristic analysis with a methylation cutoff level of 56% for APAF-1 and 10% for DAPK-1. Grouping was done as mentioned above. Using the same cutoff levels, univariate analysis with Kaplan Meier estimates showed that a higher NIM of APAF-1 (≥56%) and DAPK-1 (≥10%) was predictive of recurrence-free (P = 0.00001 for APAF-1; P = 0.0001 for DAPK-1) and overall survival (P = 0.00001 for APAF-1; P = 0.0006 for DAPK-1) in renal cell carcinoma patients. See Fig. 1A to D .


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1. A, recurrence free survival in patients with low versus high APAF-I NIM (≥56%). B, overall survival in patients with low versus high APAF-l NIM (≥56%). C, recurrence-free survival in patients with low versus high DAPK-1 NIM (≥10%). D, overall survival in patients with low versus high DAPK-1 NIM (≥10%).

 
Correlation between methylation level, node-negative status, and tumor recurrence and death from renal cell carcinoma. Next we focused on metachronous metastasis and tumor-related death in the subgroup of the 71 patients with N– tumors. Twelve (17%) of these patients later developed metastases and died of metastatic disease. Using the same APAF-1 and DAPK-1 methylation cutoff levels (after receiver operating characteristic analysis), the log-rank test showed a better recurrence-free (P = 0.0003 for APAF-1; P = 0.012 for DAPK-1) and overall survival (P = 0.0003 for APAF-1; P = 0.003 for DAPK-1) in the low NIM group. See Fig. 2A to D .


Figure 2
View larger version (19K):
[in this window]
[in a new window]
  		Fig. 2. A, recurrence free survival in N– patients with low versus high APAF-1 NIM (≥56%). B, overall survival in N– patients with low versus high APAF-1 NIM (≥56%). C, recurrence free survival in N– patients with low versus high DAPK-1 NIM (≥10%). D, overall survival in N– patients with low versus high DAPK-1 NIM (≥10%).

 
Multivariate analysis of APAF-1 and DAPK-1 methylation levels. A multivariate analysis model adjusted for tumor stage, grade, and size >4 cm and the methylation level (NIM) of the genes APAF-1 (≥56%) and DAPK-1 (≥10%) showed that the APAF-1 and DAPK-1 methylation levels (high methylation levels) were independent methylation-related prognostic factors for metachronous metastatic disease (APAF-1: 95% confidence interval, 1.63-10.71; hazard ratio, 4.18; P = 0.003; DAPK-1: 95%, confidence interval, 1.57-9.14; hazard ratio, 3.79; P = 0.003). Both APAF-1 and DAPK-1 NIM levels were also independent prognostic factors for tumor-related death (APAF-1: 95% confidence interval, 1.54-9.15; hazard ratio, 3.76; P = 0.003; DAPK-1: 95% confidence interval, 1.31-6.49; hazard ratio, 2.92; P = 0.009).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Using a methylation panel might be helpful for better risk stratification in renal cell carcinoma. Unfortunately, few of the genes recently investigated are frequently methylated in the promoter region. In renal cell carcinoma, different studies have shown promoter methylation prevalence of 45%, 58%, and 83% for the genes RASSF1A, Timp-3, and {gamma}-catenin (6, 7). Quantification of the observed promoter methylation by applying the NIM has revealed that the number of methylated cells can vary within different tumor entities. Gonzalgo et al. (12) have shown differentiation of tumor subtypes (e.g., papillary and clear cell renal cell carcinoma) using NIM with subsequent stratification after methylation analysis of RASSF1A.

Our study shows for the first time that, in a specific sample, the methylation level in the promoter region of a specific gene increases in relation to clinicopathologic features such as tumor size and nodal status, but not in relation to the common staging or grading system. Moreover, it has the potential to predict tumor recurrence and thus also the malignant potential of a tumor. Using quantitative real-time methylation-specific PCR, we showed a higher median methylation level in the node-positive tumor group than in the node-negative one. In the node-negative subgroup, patients with tumor recurrence or tumor-related death also had higher APAF-1 and DAPK-1 methylation levels. Kaplan-Meier analysis identified patients with a higher risk of recurrence or death from renal cell carcinoma. Moreover, multivariate analysis showed that APAF-1 and DAPK-1 methylation levels are independent prognostic factors for tumor recurrence and survival in renal cell carcinoma. Thus, methylation levels of the antiapoptotic protein-activating factor (APAF-1) and the death-associated protein kinase (DAPK-1) seem to be reliable tools for risk stratification in clinical practice.

The role of APAF-1 methylation and its inactivation have been described in malignant melanoma, where the p53 mutation rate is low (9). This tumor also displays high chemoresistance, and APAF-1 inactivation by promoter methylation was suggested as the factor responsible for the inability of cells to undergo apoptosis. In our study, the prevalence and extent of APAF-1 methylation were essentially independent of tumor stage and grade, thus indicating that this event occurs at an early stage. However, the finding that the APAF-1 promoter methylation level is an independent prognostic factor for metastatic recurrence and death from renal cell carcinoma supports the hypothesis that loss of APAF-1 function may be related to a more aggressive phenotype with a higher risk of metastatic disease. To date, the correlation between APAF-1 promoter methylation, mRNA down-regulation, and lower APAF-1 protein levels has only been shown by in vitro experiments applying demethylating agents in malignant melanoma and leukemic cell lines (9, 10). The trend towards an early tumor relapse was observed in patients with higher promoter methylation levels of APAF-1, but the sample size was small in this subgroup. The question of whether APAF-1 methylation correlates with a higher rate of resistance to immunotherapy remains to be elucidated and, thus, also the functional relationship between promoter methylation and transcriptional or translational silencing.

Silencing of death-associated protein kinase by promoter methylation has been showed for a variety of tumors with a frequency ranging from 15% in colorectal to 58% in bladder cancer (13, 14). DAP kinase is involved in the p53-dependent apoptosis pathway and was first identified as a mediator of IFN-{gamma}-induced apoptosis after IFN-{gamma} treatment (15–17). DAPK-1 promoter methylation was less common in our study (41%), and we could not find a stage- or grade-dependent methylation level (except for G1/2 versus G3 tumors in the ≥pT3 group). However, the relation to a higher recurrence risk, as shown by Tada et al. (11) in superficial transitional cell carcinoma of the bladder, could be detected for patients in the higher NIM group. Our data also show that DAPK-1 NIM levels have the potential to identify patients with a higher risk of tumor-related death. In multivariate analysis, DAPK-1 was also shown to be an independent prognostic factor for tumor recurrence and tumor-related death. Given the fact that DAPK-1 inactivation protects cells from IFN-{gamma}-induced apoptosis, the assumption that patients with low DAPK-1 methylation levels respond better to immunochemotherapy with standard IFN and interleukin treatment could not be confirmed but is the subject of ongoing studies. Moreover, the relation between DAPK-1 promoter methylation and subsequent mRNA down-regulation or lower protein levels could shed light on the regulation of this suppressor gene. Interestingly, a recent publication by Wethkamp et al. (18) suggests that DAPK-1 is regulated at the posttranslational level because DAPK enzyme activity was found to be low even in the presence of high mRNA and protein levels. Finally, the DAPK-1 methylation level was elevated in tumor tissue compared with normal renal tissue, which evidenced less frequent methylation and a rather low median methylation level (<2%). Nevertheless, the fact that DAPK-1 methylation occurred in less than half the patients (41%) and at NIM levels of 2% to 26% limits its use as a potential methylation marker for risk estimation. The question of whether it determines the treatment response to IFN therapy should be further evaluated in prospective studies.

The potential importance of PML loss in the development of nonhematologic malignancies is now emerging (19). PML is a direct p53 target gene involved in several biological processes, especially the induction of apoptosis (20). The best-characterized PML inducer is IFN, and PML-deficient cells do not respond appropriately to IFN stimulation. Because PML inactivation has been found to correlate with a poor prognosis in leukemia, our suggestion was that epigenetic silencing could be an inactivation mechanism and also correlated with a poor prognosis or resistance to immunotherapy (21). Unfortunately, the PML promoter was methylated in only 2% of the samples. Nevertheless, this is the first study that investigates PML promoter methylation and excludes epigenetic silencing as a common inactivation mechanism in renal cell carcinoma.

Another finding was the low frequency of IGFBP-3 methylation (2%). Lower IGFBP-3 plasma levels have been associated with metastatic risk, cancer progression, and survival in bladder cancer (22). IGFBP-3 also acts as a p53 effector gene regulating cell growth and apoptosis (23). As already observed for PML, IGFBP-3 methylation was only detected in 2% of the samples, suggesting the existence of regulatory mechanisms other than promoter hypermethylation.

The last candidate gene, CASP-8, was not found to be methylated in its promoter region. This is surprising in view of the fact that CASP-8 methylation was detected in 90% of medulloblastoma samples by nonquantitative methylation-specific PCR (24). Restoration of CASP-8 mRNA expression after demethylation by 5'-aza-2'-deoxycytidine has been described in human brain tumor cells (25). In renal cell carcinoma, regulation of CASP-8 expression seems to underlie other mechanisms.

In conclusion, our study disclosed promoter hypermethylation in a selected number of p53 target genes. It is not the promoter methylation prevalence but the relative number of methylated cells in a specific sample—represented by the NIM—that may correlate well with the phenotype and aggressiveness of a tumor. Our findings showed this correlation for APAF-1 and DAPK-1 methylation levels. The detection of elevated methylation levels points to a higher risk of metastatic tumor disease or death from renal cell carcinoma. This applies especially to the node-negative tumor patients who have no macroscopic metastatic disease after surgery but will later develop metastasis. Thus, the APAF-1 and DAPK-1 methylation level could be a surrogate marker for risk estimation of tumor recurrence and death in renal cell carcinoma. Further research should clarify the question of whether nonmetastatic patients with higher APAF-1 and DAPK-1 methylation levels would benefit from the induction of adjuvant therapies to lower the risk of tumor recurrence. On the other hand, patients with lower methylation levels could benefit from a reduced frequency of postoperative chest X-rays and computed tomographies, which would lower their X-ray exposure and also decrease the economic effect of expensive follow-up procedures.


   Acknowledgments
 
We thank Waltraud Jekabsons, Petra von Kwiatkowski, and Antonia Maas for expert technical assistance and Dr. Joanne Weirowski for linguistic advice.


   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.

Received 1/23/06; revised 5/28/06; accepted 6/22/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, 2004. CA Cancer J Clin 2004;54:8–29.[Abstract/Free Full Text]
  2. Mickisch GH, Garin A, van Poppel H, de Prijck L, Sylvester R. Radical nephrectomy plus interferon-alfa-based immunotherapy compared with interferon alfa alone in metastatic renal-cell carcinoma: a randomised trial. Lancet 2001;358:966–70.[CrossRef][Medline]
  3. Gnarra JR, Tory K, Weng Y, et al. Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat Genet 1994;7:85–90.[CrossRef][Medline]
  4. Pavlovich CP, Schmidt LS, Phillips JL. The genetic basis of renal cell carcinoma. Urol Clin North Am 2003;30:437–54, vii.[CrossRef][Medline]
  5. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3:415–28.[Medline]
  6. Dulaimi E, De C II, Uzzo RG, et al. Promoter hypermethylation profile of kidney cancer. Clin Cancer Res 2004;10:3972–9.[Abstract/Free Full Text]
  7. Breault JE, Shiina H, Igawa M, et al. Methylation of the {gamma}-catenin gene is associated with poor prognosis of renal cell carcinoma. Clin Cancer Res 2005;11:557–64.[Abstract/Free Full Text]
  8. Soussi T, Dehouche K, Beroud C. p53 website and analysis of p53 gene mutations in human cancer: forging a link between epidemiology and carcinogenesis. Hum Mutat 2000;15:105–13.[CrossRef][Medline]
  9. Soengas MS, Capodieci P, Polsky D, et al. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 2001;409:207–11.[CrossRef][Medline]
  10. Jia L, Srinivasula SM, Liu FT, et al. Apaf-1 protein deficiency confers resistance to cytochrome c-dependent apoptosis in human leukemic cells. Blood 2001;98:414–21.[Abstract/Free Full Text]
  11. Tada Y, Wada M, Taguchi K, et al. The association of death-associated protein kinase hypermethylation with early recurrence in superficial bladder cancers. Cancer Res 2002;62:4048–53.[Abstract/Free Full Text]
  12. Gonzalgo ML, Yegnasubramanian S, Yan G, et al. Molecular profiling and classification of sporadic renal cell carcinoma by quantitative methylation analysis. Clin Cancer Res 2004;10:7276–83.[Abstract/Free Full Text]
  13. Satoh A, Toyota M, Itoh F, et al. DNA methylation and histone deacetylation associated with silencing DAP kinase gene expression in colorectal and gastric cancers. Br J Cancer 2002;86:1817–23.[CrossRef][Medline]
  14. Chan MW, Chan LW, Tang NL, et al. Frequent hypermethylation of promoter region of RASSF1A in tumor tissues and voided urine of urinary bladder cancer patients. Int J Cancer 2003;104:611–6.[CrossRef][Medline]
  15. Raveh T, Droguett G, Horwitz MS, DePinho RA, Kimchi A. DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation. Nat Cell Biol 2001;3:1–7.[CrossRef][Medline]
  16. Feinstein E, Druck T, Kastury K, et al. Assignment of DAP1 and DAPK-genes that positively mediate programmed cell death triggered by IFN-{gamma}-to chromosome regions 5p12.2 and 9q34.1, respectively. Genomics 1995;29:305–7.[CrossRef][Medline]
  17. Deiss LP, Feinstein E, Berissi H, Cohen O, Kimchi A. Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the {gamma} interferon-induced cell death. Genes Dev 1995;9:15–30.[Abstract/Free Full Text]
  18. Wethkamp N, Ramp U, Geddert H, et al. Expression of death-associated protein kinase during tumour progression of human renal cell carcinomas: Hypermethylation independent mechanisms of inactivation. Eur J Cancer 2006;42:264–74.[CrossRef][Medline]
  19. Li L, He D, He H, et al. Overexpression of PML induced apoptosis in bladder cancer cell by caspase dependent pathway. Cancer Lett 2006;236:259–68.[CrossRef][Medline]
  20. de Stanchina E, Querido E, Narita M, et al. PML is a direct p53 target that modulates p53 effector functions. Mol Cell 2004;13:523–35.[CrossRef][Medline]
  21. Gurrieri C, Nafa K, Merghoub T, et al. Mutations of the PML tumor suppressor gene in acute promyelocytic leukemia. Blood 2004;103:2358–62.[Abstract/Free Full Text]
  22. Shariat SF, Kim J, Nguyen C, et al. Correlation of preoperative levels of IGF-I and IGFBP-3 with pathologic parameters and clinical outcome in patients with bladder cancer. Urology 2003;61:359–64.[CrossRef][Medline]
  23. Rajah R, Valentinis B, Cohen P. Insulin-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-ß1 on programmed cell death through a p53- and IGF-independent mechanism. J Biol Chem 1997;272:12181–8.[Abstract/Free Full Text]
  24. Ebinger M, Senf L, Wachowski O, Scheurlen W. Promoter methylation pattern of caspase-8, P16INK4A, MGMT, TIMP-3, and E-cadherin in medulloblastoma. Pathol Oncol Res 2004;10:17–21.[Medline]
  25. Grotzer MA, Eggert A, Zuzak TJ, et al. Resistance to TRAIL-induced apoptosis in primitive neuroectodermal brain tumor cells correlates with a loss of caspase-8 expression. Oncogene 2000;19:4604–10.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Christoph, F.
Right arrow Articles by Schrader, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Christoph, F.
Right arrow Articles by Schrader, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online