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Quantitative Assessment of AKAP12 Promoter Methylation in Human Prostate Cancer Using Methylation-sensitive High-resolution Melting: Correlation With Gleason Score
Basic and Translational Science Quantitative Assessment of AKAP12 Promoter Methylation in Human Prostate Cancer Using Methylation-sensitive High-resolution Melting: Correlation With Gleason Score Weiwei Liu, Jian Gong, Jinghui Hu, Tingting Hu, Yaofei Sun, Junhua Du, Chuanyu Sun, Ming Guan, Haowen Jiang, and Yuan Lu OBJECTIVES
METHODS
RESULTS
CONCLUSIONS
To quantitatively investigate the A kinase anchoring protein 12 (AKAP12) gene promoter methylation and its association with clinicopathologic variables in human prostate cancer (PCa). The AKAP12 gene has shown reduced expression and marked hypermethylation in a variety of cancers. The percentage levels of DNA methylation were measured in 78 PCa, 22 benign prostatic hyperplasia, and 22 normal adjacent tissue samples using an AKAP12 methylation-sensitive high-resolution melting assay. AKAP12 gene expression was also examined in 4 human prostate carcinoma cell lines, PC-3, DU145, LNCaP, and 22RV1, using quantitative reverse transcriptase-polymerase chain reaction and methylation-sensitive high-resolution melting analysis and after DNA methyltransferase inhibition with 5-aza-2=-deoxycytidine. Methylation (⬎1%) of the AKAP12 promoter region was present in 47 (60.2%) of the 78 PCa, 5 (22.7%) of the 22 benign prostatic hyperplasia, and 2 (9.1%) of the 22 adjacent normal tissue samples. AKAP12 methylation was significantly greater in the PCa than in the benign prostatic hyperplasia or adjacent tissue samples (P ⬍ .01). AKAP12 methylation was significantly greater in the PCa samples with higher Gleason scores (P ⫽ .03); however, no correlation was found with age, pT category, or serum prostate-specific antigen level. Reverse transcriptase-polymerase chain reaction demonstrated that PC-3 and DU-145 cells expressed AKAP12 RNA and LNCaP and 22RV1 did not. The AKAP12 locus was methylated in the LNCaP and 22RV1 cells. Treatment of LNCaP cells with 5-aza-2=-deoxycytidine markedly decreased the methylation levels and increased the expression of AKAP12. The results of the present study have demonstrated that AKAP12 promoter methylation is a frequent event in human PCa. AKAP12 methylation represents a potential molecular biomarker for predicting the malignancy of PCa. UROLOGY 77: 1006.e1–1006.e7, 2011. © 2011 Elsevier Inc.
P
rostate cancer (PCa) has been rapidly increasing in China and is currently recognized as one of the principal medical problems facing the male population. The incidence of PCa in Shanghai has been estimated to have increased from 1.8-2.4/100 000 in 1990 Weiwei Liu and Jian Gong contributed equally to this work. This study was supported by the National Natural Science Foundation (grant 81001059), the Natural Science Foundation of Shanghai (grant 09ZR1405300), a grant from the Shanghai Medical Key Discipline, and the Science and Technology Commission of Shanghai Municipality (grant 074119519). From the Departments of Laboratory Medicine and Urology, Huashan Hospital, Fudan University Shanghai Medical College, Shanghai, China Reprint requests: Yuan Lu, Professor, Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College Fudan University, 12 Central Urumqi Road, Shanghai 200040 China. E-mail: [email protected] Submitted: June 18, 2010, accepted (with revisions): December 5, 2010
© 2011 Elsevier Inc. All Rights Reserved
to 4.5-7.7/100 000 in 2000 and to about 10.0/100 000 in 2004.1 Furthermore, because screening for PCa using prostate-specific antigen determination and digital rectal examination are not routine practice in China, most Chinese patients with newly diagnosed PCa will already be symptomatic and have metastatic disease.2 The A kinase anchor protein 12 (AKAP12/gravin) was first isolated as a protein recognized by the serum from patients with myasthenia gravis.3 It is an A kinase anchoring protein (AKAP) that belongs to a family of scaffold proteins and organizes protein kinase A and C.4 It also is an important regulator of the 2-adrenergic receptor complex, which controls cell signaling, cell adhesion, mitogenesis, and differentiation.5 DNA hypermethylation of the AKAP12 promoter region and the 0090-4295/11/$36.00 1006.e1 doi:10.1016/j.urology.2010.12.010
resulting underexpression of the corresponding gene has been noted in a variety of human cancers, including gastric cancer, esophageal cancer, and lung cancer and in myeloma cells and myeloid malignancies.5-9 Downregulation of AKAP12 expression suggests that inactivation of AKAP12 expression could be linked to oncogenesis. A previous study found that the SSeCKS/gravin (the rodent ortholog of human AKAP12) protein and RNA levels were severely reduced in human and rat PCa cell lines. Primary site tumors that progressed lost regulated SSeCKS expression. SSeCKS/gravin expression was detected in benign human prostatic lesions and well-differentiated carcinoma, but not in undifferentiated lesions with a Gleason score (GS) ⬎6. These data suggest a role for the loss of SSeCKS/gravin expression in the metastatic progression of PCa.10 We hypothesized that inactivation of the AKAP12 gene through CpG methylation could be responsible for the pathogenesis and progression of PCa. To investigate this possibility, we analyzed the promoter methylation status of the AKAP12 gene in PCa samples and related these findings to the clinical and pathologic outcomes. The study used methylation-sensitive high-resolution melting (MS-HRM), which was originally developed for single nucleotide polymorphism genotyping.11 MS-HRM operates on the principle that bisulfite-treated DNA templates with different methylcytosine contents can be resolved by melting analysis owing to the differences in melting temperatures.12 HRM has also recently been proposed as a rapid and sensitive technique for the assessment of DNA methylation.12-18 We applied MSHRM technology to the detection and quantification of AKAP12 methylation in PCa samples.
MATERIAL AND METHODS Samples and Clinical Characteristics The tissue samples from 78 patients with PCa and 22 patients with benign prostatic hyperplasia (BPH) were obtained from Huashan Hospital, Fudan University. Non-neoplastic prostatic tissue samples were obtained from 22 patients with PCa as controls. All tissue specimens were frozen immediately after surgery and stored at ⫺80°C for additional analysis. All the tumors were confirmed to contain ⬎80% tumor cells by histologic examination of sequential sections. All patients provided written informed consent, and the Ethics Committee of Huashan Hospital approved the study.
Cell Culture Conditions and 5-Aza-2=-Deoxycytidine Treatment In addition, 4 human PCa cell lines, PC-3, DU145, LNCaP, and 22RV1, were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in Roswell Park Memorial Institute 1640 medium and F12K (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO), and incubated in 5% carbon dioxide at 37°C. The effects of the demethylating agent, 5-aza-2=-deoxycytidine (5-aza-dC), on the AKAP12 gene expression were inves1006.e2
tigated in cultured PCa cell lines. The LNCaP cells were treated with 1 mol/L 5-aza-dC (Sigma-Aldrich) for 72 hours, following a previously described protocol.19 The cells were treated with 5-aza-dC daily, and the medium was replaced on treatment. Control wells received only the medium with vehicle. The cells were harvested at the end of the treatment and used for DNA and RNA analysis. cDNA from these cell lines was used to measure the difference in AKAP12 mRNA expression levels before and after 5-aza-dC treatment, using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).
RNA Extraction and RT-PCR Total RNA was extracted from each cell line using Trizol (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. First-strand cDNAs were generated from the purified mRNA using the SuperScript III First-Strand Synthesis System for RT-PCR and the oligo(dT) primer (Invitrogen). Reverse transcriptase reactions were performed using 1 g total RNA. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal reference gene. AKAP12 was amplified on the same plate as glyceraldehyde 3-phosphate dehydrogenase, using the HotStarTaq Plus Master Mix (Qiagen, Hilden, Germany) or Sybr Green PCR Master Mix (Qiagen). The RT-PCR primers were performed, as previously described.20
DNA Extraction and Bisulfite Treatment DNA was isolated from the tissue samples and cell lines using the QIAamp DNA minikit (Qiagen), according to the manufacturer’s instructions. The DNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). One microgram of DNA was treated with sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research, Orange, CA), according to the manufacturer’s instructions.
MS-HRM and Evaluation Real-time PCR followed by HRM was performed in a Rotor Gene 6000 (Corbett Research, Sydney, Australia). The AKAP12 MS-HRM assay and evaluation were performed as previously described.20 In brief, the reaction mixture consisted of 100 ng of bisulfite-modified template, 1 ⫻ PCR Master Mix plus (Qiagen), 200 nmol/L of each primer (as described previously20), and 5 mol/L of SYTO(R) 9 green fluoresce (Invitrogen), in a final volume of 20 mL. The reaction cycle started with 1 cycle at 95°C for 5 minutes for enzyme activation, followed by 42 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension step at 72°C for 7 minutes. The MS-HRM analyses were performed at the temperature ramping and fluorescence acquisition settings recommended by the manufacturer, ramping from 75° to 90°C, increasing by 0.1°C/s. Universal methylated DNA (Chemicon, Millipore, Billerica, MA) and DNA from peripheral blood mononuclear cells of healthy volunteers were used as fully methylated positive and unmethylated references, respectively. These 2 controls were mixed in ratios of 0%, 1%, 5%, 10%, 20%, 40%, 60%, 80%, and 100% methylated to unmethylated template after bisulfite modification to create a range of methylated and unmethylated allele dilutions. These standards were included in each experiment. All samples were analyzed in duplicate. The sensitivity, linearity, and reproducibility of the UROLOGY 77 (4), 2011
Figure 1. Verification of AKAP12 MS-HRM assay using serial dilutions of methylated DNA. (A) Amplification plots obtained with serial dilutions of methylated DNA (from 0% to 100%) as template. (B) Melting curve of standard dilutions used to test specificity of assay. (C) AKAP12 MS-HRM profiles of serial dilutions. (D) Differential graph of each HRM profile normalized against unmethylated DNA (0%). (E) Fluorescence values obtained at melting temperature for serial dilutions of methylated DNA (from 0% to 100%). (F) Values of fluorescence plotted against percentage of methylation for each dilution to generate typical standard curve.
AKAP12 MS-HRM assay were determined as previously described.20
Statistical Analysis The Mann-Whitney U test was used to compare the AKAP12 methylation in the PCa, BPH, and adjacent normal tissue samples. The Mann-Whitney U test was also used to compare AKAP12 methylation with the clinicopathologic features in the PCa samples. The Student t test was used to compare the values obtained after 5-aza-dC treatment with those from the corresponding control experiments. P ⬍ .05 was considered statistically significant.
RESULTS Sensitivity, Linearity, and Reproducibility Of AKAP12 MS-HRM Assay The AKAP12 MS-HRM assay was established in our previous study.20 In brief, the assay sensitivity was tested by analyzing the consistency of the normalized melting UROLOGY 77 (4), 2011
profiles derived from the samples with the different methylated/unmethylated ratios. The obtained amplification plots indicated that all dilutions were amplified with comparable Cycle threshold (Ct) values (Fig. 1A). The specificity of the assay was determined from the melting curve of the standard dilutions (Fig. 1B). The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA in a background of unmethylated DNA (Fig. 1C). A differential graph was generated by normalizing each HRM profile against unmethylated DNA (0% methylated; Fig. 1D). The differential analysis generated peaks of variable heights because of the differences in fluorescence among the dilutions. The values for the differential fluorescence peaks were obtained using the Rotor Gene 6000 software (Fig. 1E). These values were then plotted against the dilution factor to generate a linear calibration curve (Fig. 1F). The intra- and interassay variability as a coefficient of variation of the MSHRM assay were tested by comparing the methylation 1006.e3
Figure 2. Expression and methylation status of AKAP12 in 4 prostate cancer cell lines (PC-3, DU-145, LNCaP, and 22RV1). (A) Expression of AKAP12 (165 bp) in 4 prostate cancer cell lines analyzed using RT-PCR. Expression of glyceraldehyde 3-phosphate dehydrogenase (226 bp) analyzed as internal control. (B) Differential fluorescence signals of 4 prostate cancer cell lines (PC-3, DU-145, LNCaP, and 22RV1) normalized against unmethylated control. (C) Restoration of AKAP12 expression by 5-aza-dC. Expression of AKAP12 gene in PCa cell line LNCaP after exposure to demethylating agent 5-aza-dC (1 mol/L) for 72 hours. Expression level represented as x-fold increases over untreated control cell. Bars indicate standard error of 3 independent experiments. *P ⬍ .05. (D) Differential fluorescence signals of LNCaP cells before and after exposure to 5-aza-dC for 72 hours normalized against unmethylated control.
results obtained from 4 results of the same assay and 4 independent assays performed on different days. According to the methylation levels, the intra-assay variability was 6.05%-9.10%, and the interassay variability was 14.50%-17.17% in the present study. The performance of the AKAP12 MS-HRM assay was validated and compared with the methylation-specific PCR assay for the AKAP12 promoter region, as previously described.20 Methylation levels of ⬍5% were undetectable by the methylation-specific PCR assay (data not shown). AKAP12 Expression and Promoter Methylation and Effects Of 5-Aza-Dc in Pca Cell Lines RT-PCR showed that the PCa PC-3 and DU-145 cell lines expressed AKAP12 RNA (Fig. 2A) and LNCaP and 22RV1 did not. The AKAP12 locus was methylated in LNCaP and 22RV1 cells (Fig. 2B). However, the percentage of methylation differed between the 2 cell lines. LNCaP cells showed extensive methylation (84.6%), and 22RV1 showed less methylation (13.2%). The expression level of AKAP12 mRNA was increased 9.3-fold after 5-aza-dC treatment in LNCaP cells (Fig. 2C). The AKAP12 methylation levels were reduced from 84.6% to 8.51% (Fig. 2D) after treatment with 5-aza-dC. These 1006.e4
results suggest that demethylation restored AKAP12 gene expression in PCa cell lines.
Clinicopathologic Correlations With AKAP12 Promoter Methylation in Prostatic Tissues The level of DNA methylation was detected in 78 PCa, 22 BPH, and 22 normal adjacent tissue samples using the AKAP12 MS-HRM assay. A total of 47 (60.2%) of the 78 PCa samples, 5 (22.7%) of the 22 BPH samples, and 2 (9.1%) of the 22 adjacent tissue samples showed ⬎1% methylation of the AKAP12 promoter region. The median methylation value of AKAP12 in 78 PCa tissues was 15.4% (range 1.0%-82.1%). Figure 3A presents the AKAP12 MS-HRM results of 3 representative samples (PCa, BPH, and adjacent tissues samples). The MannWhitney U test revealed that AKAP12 methylation was significantly greater in PCa tissues than in the BPH or adjacent normal tissues (Fig. 3B, P ⬍ .01, for both). However, a significant difference was also seen in AKAP12 methylation between the BPH and adjacent normal tissue samples (P ⫽ .04). Within the PCa tissue samples, no correlations were found between age, pT category, or serum prostate-speUROLOGY 77 (4), 2011
Figure 3. AKAP12 MS-HRM curves showing methylation status of PCa samples. (A) Differential fluorescence signals of 3 prostate samples (PCa, BPH, and adjacent normal tissue) normalized against unmethylated control. (B) Association between prostatic tissue group and concentration of methylated DNA. Table 1. Comparison of human AKAP12 methylation stratified by patient characteristics
Variable
Patients (n)
Total Gleason score 4-7 8-10 pT stage pT1-T2 pT3-T4 Age (y) ⬍70 ⬎70 Preoperative serum PSA (ng/mL) ⬍10 ⬎10
AKAP12 Methylation (%) P Median (Range) Value
78
15.4 (1.0-82.1)
39 39
9.8 (1.0-66.2) 22.2 (7.8-82.1)
40 38
18.7 (1.0-69.8) 11.9 (2.6-82.1)
50 28
17.8 (1.0-61.4) 11.1 (2.6-82.1)
.03 .27 .51 .38 29 49
12.3 (1.0-69.3) 17.2 (2.6-82.1)
AKAP12, A kinase anchoring protein 12; PSA, prostate-specific antigen.
cific antigen level and methylation status (Table 1). The degree of AKAP12 methylation increased as the GS increased (P ⫽ .03). The AKAP12 methylation levels were 1%-20% in 24 (51.1%), 20%-60% in 18 (38.3%), and 60%-100% in 5 (10.6%) of the 47 PCa samples.
COMMENT Accumulating evidence has indicated that DNA hypermethylation in the AKAP12 promoter region and concurrent underexpression of the gene occurs in a variety of human cancers. One study found that SSeCKS/gravin protein and RNA levels were severely reduced in human and Rat PCa cell lines. They could be detected in benign human prostatic lesions and well-differentiated carcinoma but not in undifferentiated lesions with a GS ⬎6, suggesting a role for the loss of SSeCKS/gravin expression in the metastatic progression of human PCa.10 However, transcriptional regulation of AKAP12 gene expression UROLOGY 77 (4), 2011
through epigenetic mechanisms has not been investigated in PCa. Furthermore, the relationships between AKAP12 methylation levels and clinicopathologic variables in PCa remain to be elucidated. Promoter methylation of cancer-related genes is a frequent event in PCa21,22 and is a promising tool for early cancer detection.22 We sought to determine the promoter methylation status of AKAP12 in tissue specimens from primary PCa, BPH, and normal adjacent tissue, as well as in 4 PCa cell lines, using MS-HRM, a highly sensitive and specific method for the detection and quantitation of DNA methylation. The AKAP12 promoter was hypermethylated in PCa (P ⬍ .01), providing evidence to support the AKAP12 epigenetic-mediated silencing hypothesis. AKAP12 methylation levels were also significantly associated with the GS. The methylation levels were significantly greater in GS 8-10 tumors, indicating that AKAP12 methylation could be a useful marker for monitoring the progression of carcinogenesis in those with PCa. It is possible that the increased level of methylation might be related to the progressive accumulation of cells with AKAP12 promoter methylation during prostate carcinogenesis in these patients. AKAP12-mediated suppression of tumor growth and metastatic colonization can be achieved through direct or indirect interactions with multiple proteins involved in apoptosis, angiogenesis, and associated signaling pathways in cancer cells.23,24 These included several signaling molecules that participate in cell proliferation and cytoskeletal organization, as well as protein kinase C, protein kinase A, cyclin D, calmodulin, and vascular endothelial growth factor.10,25,26 These functions of AKAP12 mean that neoplastic cells might obtain a growth advantage through AKAP12 silencing, explaining the high prevalence of promoter methylation at this locus in PCa specimens. Additional studies are needed to determine the basis for this observation. Regarding the PCa cell lines, LNCaP cells showed extensive methylation at the AKAP12 locus, and 22RV1 1006.e5
cells displayed much lower methylation rates. Furthermore, AKAP12 gene expression was increased 9.3-fold in the LNCaP cells after treatment with the demethylating agent, 5-aza-dC. The mechanisms responsible for the downregulation of AKAP12 expression could differ between PCa cell lines, although the level of promoter methylation was lower than that in LNCaP cells. These discrepant results might be explained by other factors, such as histone deacetylase. The AKAP12 promoter was methylated in 22.7% of the BPH samples, a proliferative benign lesion of the prostate. This level of methylation was significantly lower than that of the PCa tissues (P ⬍ .01). These results support the hypothesis that AKAP12 acts as a tumor suppressor gene. However, immunohistochemistry for AKAP12 was not performed in the BPH specimens in the present series; therefore, no direct comparisons could be made. Nonetheless, previous studies reported AKAP12 promoter methylation in normal tissues and showed that this epigenetic alteration failed to completely abrogate gene expression.10 The principle of MS-HRM analysis is that PCR products generated from bisulfite-treated DNA templates with different methylcytosine contents have different melting temperatures, which can be resolved by melting analysis in a thermal cycler coupled with a fluorometer.18,27 Quantification can be performed by interpolation on a standard curve generated from serial dilutions of methylated and unmethylated DNA. The efficiency of bisulfite conversion was tested by sequencing cloned PCR fragments derived from bisulfite-treated CpGenome Universal Methylated DNA (Zymo Research, Irvine, CA), and all the CpG sites were shown to be fully methylated. The sensitivity of detection with MS-HRM has been reported to be equivalent to that of the MethyLight assay, with both assays providing reproducible results at levels as low as 0.1% methylation.14 In our study, AKAP12 MS-HRM could reproducibly detect 1% methylated DNA in a background of unmethylated DNA, which methylationspecific PCR was able to reproducibly detect 10% methylation in our previously described study.20 After changing the cutoff value to ⱕ5%, the AKAP12 methylation was still significantly greater in the PCa tissues than in the BPH specimens (P ⬍ .01). The reason for setting the cutpoint at ⬎1% was to highlight the high sensitivity of the quantitative MS-HRM assay in detecting the AKAP12 promoter methylation. However, MS-HRM analysis demonstrated important advantages over the MethyLight assay used in our previous study.20 We have concluded that AKAP12 promoter methylation is a frequent event in human PCa and represents a potential molecular biomarker for predicting the malignancy of PCa. References 1. Liu ZY, Sun YH, Xu CL, et al. Age-specific PSA reference ranges in Chinese men without prostate cancer. Asian J Androl. 2009;11: 100-103.
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2. Dai B, Kong YY, Ye DW, et al. Human epidermal growth factor receptor type 2 protein expression in Chinese metastatic prostate cancer patients correlates with cancer specific survival and increases after exposure to hormonal therapy. Asian J Androl. 2008; 10:701-709. 3. Gordon T, Grove B, Loftus JC, et al. Molecular cloning and preliminary characterization of a novel cytoplasmic antigen recognized by myasthenia gravis sera. J Clin Invest. 1992;90:992-999. 4. Nauert JB, Klauck TM, Langeberg LK, et al. An autoantigen recognized by serum from myasthenia gravis patients, is a kinase scaffold protein. Curr Biol. 1997;7:52-62. 5. Tessema M, Willink R, Do K, et al. Promoter methylation of genes in and around the candidate lung cancer susceptibility locus 6q2325. Cancer Res. 2008;68:1707-1714. 6. Jin Z, Hamilton JP, Yang J, et al. Hypermethylation of the AKAP12 promoter is a biomarker of Barrett’s-associated esophageal neoplastic progression. Cancer Epidemiol Biomarkers Prev. 2008; 17:111-117. 7. Choi MC, Jong HS, Kim TY, et al. AKAP12/gravin is inactivated by epigenetic mechanism in human gastric carcinoma and shows growth suppressor activity. Oncogene. 2004;23:7095-7103. 8. Flotho C, Paulun A, Batz C, et al. AKAP12, a gene with tumour suppressor properties, is a target of promoter DNA methylation in childhood myeloid malignancies. Br J Haematol. 2007;138:644650. 9. Heller G, Schmidt WM, Ziegler B, et al. Genome-wide transcriptional response to 5-aza-2=-deoxycytidine and trichostatin A in multiple myeloma cells. Cancer Res. 2008;68:44-54. 10. Xia W, Unger P, Miller L, et al. The Src-suppressed C kinase substrate, SSeCKS, is a potential metastasis inhibitor in prostate cancer. Cancer Res. 2001;61:5644-5651. 11. Wittwer CT, Reed GH, Gundry CN, et al. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem. 2003;49(6 Pt 1):853-860. 12. Balic M, Pichler M, Strutz J, et al. High quality assessment of DNA methylation in archival tissues from colorectal cancer patients using quantitative high-resolution melting analysis. J Mol Diagn. 2009;11:102-108. 13. Wojdacz TK, Dobrovic A, Algar EM. Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting. Hum Mutat. 2008;29: 1255-1260. 14. Wojdacz TK, Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and highthroughput assessment of methylation. Nucleic Acids Res. 2007;35: e41. 15. White HE, Hall VJ, Cross NC. Methylation-sensitive high-resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman syndromes. Clin Chem. 2007;53:1960-1962. 16. Kristensen LS, Mikeska T, Krypuy M, et al. Sensitive melting analysis after real time-methylation specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection. Nucleic Acids Res. 2008;36:e42. 17. Snell C, Krypuy M, Wong EM, et al. BRCA1 promoter methylation in peripheral blood DNA of mutation negative familial breast cancer patients with a BRCA1 tumour phenotype. Breast Cancer Res. 2008;10:R12. 18. Dahl C, Guldberg P. High-resolution melting for accurate assessment of DNA methylation. Clin Chem. 2007;53:18771878. 19. Liu W, Guan M, Su B, et al. Quantitative assessment of AKAP12 promoter methylation in colorectal cancer using methylation-sensitive high resolution melting: Correlation with dukes’ stage. Cancer Biol Ther. 2010;9:862-871. 20. Liu W, Guan M, Su B, et al. Rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylationsensitive high resolution melting (MS-HRM) analysis: application in colorectal cancer. Clin Chim Acta. 2010;411:940-946.
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21. Phe V, Cussenot O, Roupret M. Methylated genes as potential biomarkers in prostate cancer. BJU Int. 2010;105:1364-1370. 22. Hoque MO. DNA methylation changes in prostate cancer: current developments and future clinical implementation. Expert Rev Mol Diagn. 2009;9:243-257. 23. Li HZ, Gao Y, Zhao XL, et al. Effects of Raf kinase inhibitor protein expression on metastasis and progression of human breast cancer. Mol Cancer Res. 2009;7:832-840. 24. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis. 2000;21: 485-495.
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25. Lin X, Nelson P, Gelman IH. SSeCKS, a major protein kinase C substrate with tumor suppressor activity, regulates G(1)¡S progression by controlling the expression and cellular compartmentalization of cyclin D. Mol Cell Biol. 2000;20:7259-7272. 26. Lin X, Gelman IH. Reexpression of the major protein kinase C substrate, SSeCKS, suppresses v-src-induced morphological transformation and tumorigenesis. Cancer Res. 1997;57:23042312. 27. Wojdacz TK, Dobrovic A. Melting curve assays for DNA methylation analysis. Methods Mol Biol. 2009;507:229-240:229-240.
1006.e7
METHODS
RESULTS
CONCLUSIONS
To quantitatively investigate the A kinase anchoring protein 12 (AKAP12) gene promoter methylation and its association with clinicopathologic variables in human prostate cancer (PCa). The AKAP12 gene has shown reduced expression and marked hypermethylation in a variety of cancers. The percentage levels of DNA methylation were measured in 78 PCa, 22 benign prostatic hyperplasia, and 22 normal adjacent tissue samples using an AKAP12 methylation-sensitive high-resolution melting assay. AKAP12 gene expression was also examined in 4 human prostate carcinoma cell lines, PC-3, DU145, LNCaP, and 22RV1, using quantitative reverse transcriptase-polymerase chain reaction and methylation-sensitive high-resolution melting analysis and after DNA methyltransferase inhibition with 5-aza-2=-deoxycytidine. Methylation (⬎1%) of the AKAP12 promoter region was present in 47 (60.2%) of the 78 PCa, 5 (22.7%) of the 22 benign prostatic hyperplasia, and 2 (9.1%) of the 22 adjacent normal tissue samples. AKAP12 methylation was significantly greater in the PCa than in the benign prostatic hyperplasia or adjacent tissue samples (P ⬍ .01). AKAP12 methylation was significantly greater in the PCa samples with higher Gleason scores (P ⫽ .03); however, no correlation was found with age, pT category, or serum prostate-specific antigen level. Reverse transcriptase-polymerase chain reaction demonstrated that PC-3 and DU-145 cells expressed AKAP12 RNA and LNCaP and 22RV1 did not. The AKAP12 locus was methylated in the LNCaP and 22RV1 cells. Treatment of LNCaP cells with 5-aza-2=-deoxycytidine markedly decreased the methylation levels and increased the expression of AKAP12. The results of the present study have demonstrated that AKAP12 promoter methylation is a frequent event in human PCa. AKAP12 methylation represents a potential molecular biomarker for predicting the malignancy of PCa. UROLOGY 77: 1006.e1–1006.e7, 2011. © 2011 Elsevier Inc.
P
rostate cancer (PCa) has been rapidly increasing in China and is currently recognized as one of the principal medical problems facing the male population. The incidence of PCa in Shanghai has been estimated to have increased from 1.8-2.4/100 000 in 1990 Weiwei Liu and Jian Gong contributed equally to this work. This study was supported by the National Natural Science Foundation (grant 81001059), the Natural Science Foundation of Shanghai (grant 09ZR1405300), a grant from the Shanghai Medical Key Discipline, and the Science and Technology Commission of Shanghai Municipality (grant 074119519). From the Departments of Laboratory Medicine and Urology, Huashan Hospital, Fudan University Shanghai Medical College, Shanghai, China Reprint requests: Yuan Lu, Professor, Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College Fudan University, 12 Central Urumqi Road, Shanghai 200040 China. E-mail: [email protected] Submitted: June 18, 2010, accepted (with revisions): December 5, 2010
© 2011 Elsevier Inc. All Rights Reserved
to 4.5-7.7/100 000 in 2000 and to about 10.0/100 000 in 2004.1 Furthermore, because screening for PCa using prostate-specific antigen determination and digital rectal examination are not routine practice in China, most Chinese patients with newly diagnosed PCa will already be symptomatic and have metastatic disease.2 The A kinase anchor protein 12 (AKAP12/gravin) was first isolated as a protein recognized by the serum from patients with myasthenia gravis.3 It is an A kinase anchoring protein (AKAP) that belongs to a family of scaffold proteins and organizes protein kinase A and C.4 It also is an important regulator of the 2-adrenergic receptor complex, which controls cell signaling, cell adhesion, mitogenesis, and differentiation.5 DNA hypermethylation of the AKAP12 promoter region and the 0090-4295/11/$36.00 1006.e1 doi:10.1016/j.urology.2010.12.010
resulting underexpression of the corresponding gene has been noted in a variety of human cancers, including gastric cancer, esophageal cancer, and lung cancer and in myeloma cells and myeloid malignancies.5-9 Downregulation of AKAP12 expression suggests that inactivation of AKAP12 expression could be linked to oncogenesis. A previous study found that the SSeCKS/gravin (the rodent ortholog of human AKAP12) protein and RNA levels were severely reduced in human and rat PCa cell lines. Primary site tumors that progressed lost regulated SSeCKS expression. SSeCKS/gravin expression was detected in benign human prostatic lesions and well-differentiated carcinoma, but not in undifferentiated lesions with a Gleason score (GS) ⬎6. These data suggest a role for the loss of SSeCKS/gravin expression in the metastatic progression of PCa.10 We hypothesized that inactivation of the AKAP12 gene through CpG methylation could be responsible for the pathogenesis and progression of PCa. To investigate this possibility, we analyzed the promoter methylation status of the AKAP12 gene in PCa samples and related these findings to the clinical and pathologic outcomes. The study used methylation-sensitive high-resolution melting (MS-HRM), which was originally developed for single nucleotide polymorphism genotyping.11 MS-HRM operates on the principle that bisulfite-treated DNA templates with different methylcytosine contents can be resolved by melting analysis owing to the differences in melting temperatures.12 HRM has also recently been proposed as a rapid and sensitive technique for the assessment of DNA methylation.12-18 We applied MSHRM technology to the detection and quantification of AKAP12 methylation in PCa samples.
MATERIAL AND METHODS Samples and Clinical Characteristics The tissue samples from 78 patients with PCa and 22 patients with benign prostatic hyperplasia (BPH) were obtained from Huashan Hospital, Fudan University. Non-neoplastic prostatic tissue samples were obtained from 22 patients with PCa as controls. All tissue specimens were frozen immediately after surgery and stored at ⫺80°C for additional analysis. All the tumors were confirmed to contain ⬎80% tumor cells by histologic examination of sequential sections. All patients provided written informed consent, and the Ethics Committee of Huashan Hospital approved the study.
Cell Culture Conditions and 5-Aza-2=-Deoxycytidine Treatment In addition, 4 human PCa cell lines, PC-3, DU145, LNCaP, and 22RV1, were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in Roswell Park Memorial Institute 1640 medium and F12K (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO), and incubated in 5% carbon dioxide at 37°C. The effects of the demethylating agent, 5-aza-2=-deoxycytidine (5-aza-dC), on the AKAP12 gene expression were inves1006.e2
tigated in cultured PCa cell lines. The LNCaP cells were treated with 1 mol/L 5-aza-dC (Sigma-Aldrich) for 72 hours, following a previously described protocol.19 The cells were treated with 5-aza-dC daily, and the medium was replaced on treatment. Control wells received only the medium with vehicle. The cells were harvested at the end of the treatment and used for DNA and RNA analysis. cDNA from these cell lines was used to measure the difference in AKAP12 mRNA expression levels before and after 5-aza-dC treatment, using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).
RNA Extraction and RT-PCR Total RNA was extracted from each cell line using Trizol (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. First-strand cDNAs were generated from the purified mRNA using the SuperScript III First-Strand Synthesis System for RT-PCR and the oligo(dT) primer (Invitrogen). Reverse transcriptase reactions were performed using 1 g total RNA. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal reference gene. AKAP12 was amplified on the same plate as glyceraldehyde 3-phosphate dehydrogenase, using the HotStarTaq Plus Master Mix (Qiagen, Hilden, Germany) or Sybr Green PCR Master Mix (Qiagen). The RT-PCR primers were performed, as previously described.20
DNA Extraction and Bisulfite Treatment DNA was isolated from the tissue samples and cell lines using the QIAamp DNA minikit (Qiagen), according to the manufacturer’s instructions. The DNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). One microgram of DNA was treated with sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research, Orange, CA), according to the manufacturer’s instructions.
MS-HRM and Evaluation Real-time PCR followed by HRM was performed in a Rotor Gene 6000 (Corbett Research, Sydney, Australia). The AKAP12 MS-HRM assay and evaluation were performed as previously described.20 In brief, the reaction mixture consisted of 100 ng of bisulfite-modified template, 1 ⫻ PCR Master Mix plus (Qiagen), 200 nmol/L of each primer (as described previously20), and 5 mol/L of SYTO(R) 9 green fluoresce (Invitrogen), in a final volume of 20 mL. The reaction cycle started with 1 cycle at 95°C for 5 minutes for enzyme activation, followed by 42 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension step at 72°C for 7 minutes. The MS-HRM analyses were performed at the temperature ramping and fluorescence acquisition settings recommended by the manufacturer, ramping from 75° to 90°C, increasing by 0.1°C/s. Universal methylated DNA (Chemicon, Millipore, Billerica, MA) and DNA from peripheral blood mononuclear cells of healthy volunteers were used as fully methylated positive and unmethylated references, respectively. These 2 controls were mixed in ratios of 0%, 1%, 5%, 10%, 20%, 40%, 60%, 80%, and 100% methylated to unmethylated template after bisulfite modification to create a range of methylated and unmethylated allele dilutions. These standards were included in each experiment. All samples were analyzed in duplicate. The sensitivity, linearity, and reproducibility of the UROLOGY 77 (4), 2011
Figure 1. Verification of AKAP12 MS-HRM assay using serial dilutions of methylated DNA. (A) Amplification plots obtained with serial dilutions of methylated DNA (from 0% to 100%) as template. (B) Melting curve of standard dilutions used to test specificity of assay. (C) AKAP12 MS-HRM profiles of serial dilutions. (D) Differential graph of each HRM profile normalized against unmethylated DNA (0%). (E) Fluorescence values obtained at melting temperature for serial dilutions of methylated DNA (from 0% to 100%). (F) Values of fluorescence plotted against percentage of methylation for each dilution to generate typical standard curve.
AKAP12 MS-HRM assay were determined as previously described.20
Statistical Analysis The Mann-Whitney U test was used to compare the AKAP12 methylation in the PCa, BPH, and adjacent normal tissue samples. The Mann-Whitney U test was also used to compare AKAP12 methylation with the clinicopathologic features in the PCa samples. The Student t test was used to compare the values obtained after 5-aza-dC treatment with those from the corresponding control experiments. P ⬍ .05 was considered statistically significant.
RESULTS Sensitivity, Linearity, and Reproducibility Of AKAP12 MS-HRM Assay The AKAP12 MS-HRM assay was established in our previous study.20 In brief, the assay sensitivity was tested by analyzing the consistency of the normalized melting UROLOGY 77 (4), 2011
profiles derived from the samples with the different methylated/unmethylated ratios. The obtained amplification plots indicated that all dilutions were amplified with comparable Cycle threshold (Ct) values (Fig. 1A). The specificity of the assay was determined from the melting curve of the standard dilutions (Fig. 1B). The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA in a background of unmethylated DNA (Fig. 1C). A differential graph was generated by normalizing each HRM profile against unmethylated DNA (0% methylated; Fig. 1D). The differential analysis generated peaks of variable heights because of the differences in fluorescence among the dilutions. The values for the differential fluorescence peaks were obtained using the Rotor Gene 6000 software (Fig. 1E). These values were then plotted against the dilution factor to generate a linear calibration curve (Fig. 1F). The intra- and interassay variability as a coefficient of variation of the MSHRM assay were tested by comparing the methylation 1006.e3
Figure 2. Expression and methylation status of AKAP12 in 4 prostate cancer cell lines (PC-3, DU-145, LNCaP, and 22RV1). (A) Expression of AKAP12 (165 bp) in 4 prostate cancer cell lines analyzed using RT-PCR. Expression of glyceraldehyde 3-phosphate dehydrogenase (226 bp) analyzed as internal control. (B) Differential fluorescence signals of 4 prostate cancer cell lines (PC-3, DU-145, LNCaP, and 22RV1) normalized against unmethylated control. (C) Restoration of AKAP12 expression by 5-aza-dC. Expression of AKAP12 gene in PCa cell line LNCaP after exposure to demethylating agent 5-aza-dC (1 mol/L) for 72 hours. Expression level represented as x-fold increases over untreated control cell. Bars indicate standard error of 3 independent experiments. *P ⬍ .05. (D) Differential fluorescence signals of LNCaP cells before and after exposure to 5-aza-dC for 72 hours normalized against unmethylated control.
results obtained from 4 results of the same assay and 4 independent assays performed on different days. According to the methylation levels, the intra-assay variability was 6.05%-9.10%, and the interassay variability was 14.50%-17.17% in the present study. The performance of the AKAP12 MS-HRM assay was validated and compared with the methylation-specific PCR assay for the AKAP12 promoter region, as previously described.20 Methylation levels of ⬍5% were undetectable by the methylation-specific PCR assay (data not shown). AKAP12 Expression and Promoter Methylation and Effects Of 5-Aza-Dc in Pca Cell Lines RT-PCR showed that the PCa PC-3 and DU-145 cell lines expressed AKAP12 RNA (Fig. 2A) and LNCaP and 22RV1 did not. The AKAP12 locus was methylated in LNCaP and 22RV1 cells (Fig. 2B). However, the percentage of methylation differed between the 2 cell lines. LNCaP cells showed extensive methylation (84.6%), and 22RV1 showed less methylation (13.2%). The expression level of AKAP12 mRNA was increased 9.3-fold after 5-aza-dC treatment in LNCaP cells (Fig. 2C). The AKAP12 methylation levels were reduced from 84.6% to 8.51% (Fig. 2D) after treatment with 5-aza-dC. These 1006.e4
results suggest that demethylation restored AKAP12 gene expression in PCa cell lines.
Clinicopathologic Correlations With AKAP12 Promoter Methylation in Prostatic Tissues The level of DNA methylation was detected in 78 PCa, 22 BPH, and 22 normal adjacent tissue samples using the AKAP12 MS-HRM assay. A total of 47 (60.2%) of the 78 PCa samples, 5 (22.7%) of the 22 BPH samples, and 2 (9.1%) of the 22 adjacent tissue samples showed ⬎1% methylation of the AKAP12 promoter region. The median methylation value of AKAP12 in 78 PCa tissues was 15.4% (range 1.0%-82.1%). Figure 3A presents the AKAP12 MS-HRM results of 3 representative samples (PCa, BPH, and adjacent tissues samples). The MannWhitney U test revealed that AKAP12 methylation was significantly greater in PCa tissues than in the BPH or adjacent normal tissues (Fig. 3B, P ⬍ .01, for both). However, a significant difference was also seen in AKAP12 methylation between the BPH and adjacent normal tissue samples (P ⫽ .04). Within the PCa tissue samples, no correlations were found between age, pT category, or serum prostate-speUROLOGY 77 (4), 2011
Figure 3. AKAP12 MS-HRM curves showing methylation status of PCa samples. (A) Differential fluorescence signals of 3 prostate samples (PCa, BPH, and adjacent normal tissue) normalized against unmethylated control. (B) Association between prostatic tissue group and concentration of methylated DNA. Table 1. Comparison of human AKAP12 methylation stratified by patient characteristics
Variable
Patients (n)
Total Gleason score 4-7 8-10 pT stage pT1-T2 pT3-T4 Age (y) ⬍70 ⬎70 Preoperative serum PSA (ng/mL) ⬍10 ⬎10
AKAP12 Methylation (%) P Median (Range) Value
78
15.4 (1.0-82.1)
39 39
9.8 (1.0-66.2) 22.2 (7.8-82.1)
40 38
18.7 (1.0-69.8) 11.9 (2.6-82.1)
50 28
17.8 (1.0-61.4) 11.1 (2.6-82.1)
.03 .27 .51 .38 29 49
12.3 (1.0-69.3) 17.2 (2.6-82.1)
AKAP12, A kinase anchoring protein 12; PSA, prostate-specific antigen.
cific antigen level and methylation status (Table 1). The degree of AKAP12 methylation increased as the GS increased (P ⫽ .03). The AKAP12 methylation levels were 1%-20% in 24 (51.1%), 20%-60% in 18 (38.3%), and 60%-100% in 5 (10.6%) of the 47 PCa samples.
COMMENT Accumulating evidence has indicated that DNA hypermethylation in the AKAP12 promoter region and concurrent underexpression of the gene occurs in a variety of human cancers. One study found that SSeCKS/gravin protein and RNA levels were severely reduced in human and Rat PCa cell lines. They could be detected in benign human prostatic lesions and well-differentiated carcinoma but not in undifferentiated lesions with a GS ⬎6, suggesting a role for the loss of SSeCKS/gravin expression in the metastatic progression of human PCa.10 However, transcriptional regulation of AKAP12 gene expression UROLOGY 77 (4), 2011
through epigenetic mechanisms has not been investigated in PCa. Furthermore, the relationships between AKAP12 methylation levels and clinicopathologic variables in PCa remain to be elucidated. Promoter methylation of cancer-related genes is a frequent event in PCa21,22 and is a promising tool for early cancer detection.22 We sought to determine the promoter methylation status of AKAP12 in tissue specimens from primary PCa, BPH, and normal adjacent tissue, as well as in 4 PCa cell lines, using MS-HRM, a highly sensitive and specific method for the detection and quantitation of DNA methylation. The AKAP12 promoter was hypermethylated in PCa (P ⬍ .01), providing evidence to support the AKAP12 epigenetic-mediated silencing hypothesis. AKAP12 methylation levels were also significantly associated with the GS. The methylation levels were significantly greater in GS 8-10 tumors, indicating that AKAP12 methylation could be a useful marker for monitoring the progression of carcinogenesis in those with PCa. It is possible that the increased level of methylation might be related to the progressive accumulation of cells with AKAP12 promoter methylation during prostate carcinogenesis in these patients. AKAP12-mediated suppression of tumor growth and metastatic colonization can be achieved through direct or indirect interactions with multiple proteins involved in apoptosis, angiogenesis, and associated signaling pathways in cancer cells.23,24 These included several signaling molecules that participate in cell proliferation and cytoskeletal organization, as well as protein kinase C, protein kinase A, cyclin D, calmodulin, and vascular endothelial growth factor.10,25,26 These functions of AKAP12 mean that neoplastic cells might obtain a growth advantage through AKAP12 silencing, explaining the high prevalence of promoter methylation at this locus in PCa specimens. Additional studies are needed to determine the basis for this observation. Regarding the PCa cell lines, LNCaP cells showed extensive methylation at the AKAP12 locus, and 22RV1 1006.e5
cells displayed much lower methylation rates. Furthermore, AKAP12 gene expression was increased 9.3-fold in the LNCaP cells after treatment with the demethylating agent, 5-aza-dC. The mechanisms responsible for the downregulation of AKAP12 expression could differ between PCa cell lines, although the level of promoter methylation was lower than that in LNCaP cells. These discrepant results might be explained by other factors, such as histone deacetylase. The AKAP12 promoter was methylated in 22.7% of the BPH samples, a proliferative benign lesion of the prostate. This level of methylation was significantly lower than that of the PCa tissues (P ⬍ .01). These results support the hypothesis that AKAP12 acts as a tumor suppressor gene. However, immunohistochemistry for AKAP12 was not performed in the BPH specimens in the present series; therefore, no direct comparisons could be made. Nonetheless, previous studies reported AKAP12 promoter methylation in normal tissues and showed that this epigenetic alteration failed to completely abrogate gene expression.10 The principle of MS-HRM analysis is that PCR products generated from bisulfite-treated DNA templates with different methylcytosine contents have different melting temperatures, which can be resolved by melting analysis in a thermal cycler coupled with a fluorometer.18,27 Quantification can be performed by interpolation on a standard curve generated from serial dilutions of methylated and unmethylated DNA. The efficiency of bisulfite conversion was tested by sequencing cloned PCR fragments derived from bisulfite-treated CpGenome Universal Methylated DNA (Zymo Research, Irvine, CA), and all the CpG sites were shown to be fully methylated. The sensitivity of detection with MS-HRM has been reported to be equivalent to that of the MethyLight assay, with both assays providing reproducible results at levels as low as 0.1% methylation.14 In our study, AKAP12 MS-HRM could reproducibly detect 1% methylated DNA in a background of unmethylated DNA, which methylationspecific PCR was able to reproducibly detect 10% methylation in our previously described study.20 After changing the cutoff value to ⱕ5%, the AKAP12 methylation was still significantly greater in the PCa tissues than in the BPH specimens (P ⬍ .01). The reason for setting the cutpoint at ⬎1% was to highlight the high sensitivity of the quantitative MS-HRM assay in detecting the AKAP12 promoter methylation. However, MS-HRM analysis demonstrated important advantages over the MethyLight assay used in our previous study.20 We have concluded that AKAP12 promoter methylation is a frequent event in human PCa and represents a potential molecular biomarker for predicting the malignancy of PCa. References 1. Liu ZY, Sun YH, Xu CL, et al. Age-specific PSA reference ranges in Chinese men without prostate cancer. Asian J Androl. 2009;11: 100-103.
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2. Dai B, Kong YY, Ye DW, et al. Human epidermal growth factor receptor type 2 protein expression in Chinese metastatic prostate cancer patients correlates with cancer specific survival and increases after exposure to hormonal therapy. Asian J Androl. 2008; 10:701-709. 3. Gordon T, Grove B, Loftus JC, et al. Molecular cloning and preliminary characterization of a novel cytoplasmic antigen recognized by myasthenia gravis sera. J Clin Invest. 1992;90:992-999. 4. Nauert JB, Klauck TM, Langeberg LK, et al. An autoantigen recognized by serum from myasthenia gravis patients, is a kinase scaffold protein. Curr Biol. 1997;7:52-62. 5. Tessema M, Willink R, Do K, et al. Promoter methylation of genes in and around the candidate lung cancer susceptibility locus 6q2325. Cancer Res. 2008;68:1707-1714. 6. Jin Z, Hamilton JP, Yang J, et al. Hypermethylation of the AKAP12 promoter is a biomarker of Barrett’s-associated esophageal neoplastic progression. Cancer Epidemiol Biomarkers Prev. 2008; 17:111-117. 7. Choi MC, Jong HS, Kim TY, et al. AKAP12/gravin is inactivated by epigenetic mechanism in human gastric carcinoma and shows growth suppressor activity. Oncogene. 2004;23:7095-7103. 8. Flotho C, Paulun A, Batz C, et al. AKAP12, a gene with tumour suppressor properties, is a target of promoter DNA methylation in childhood myeloid malignancies. Br J Haematol. 2007;138:644650. 9. Heller G, Schmidt WM, Ziegler B, et al. Genome-wide transcriptional response to 5-aza-2=-deoxycytidine and trichostatin A in multiple myeloma cells. Cancer Res. 2008;68:44-54. 10. Xia W, Unger P, Miller L, et al. The Src-suppressed C kinase substrate, SSeCKS, is a potential metastasis inhibitor in prostate cancer. Cancer Res. 2001;61:5644-5651. 11. Wittwer CT, Reed GH, Gundry CN, et al. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem. 2003;49(6 Pt 1):853-860. 12. Balic M, Pichler M, Strutz J, et al. High quality assessment of DNA methylation in archival tissues from colorectal cancer patients using quantitative high-resolution melting analysis. J Mol Diagn. 2009;11:102-108. 13. Wojdacz TK, Dobrovic A, Algar EM. Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting. Hum Mutat. 2008;29: 1255-1260. 14. Wojdacz TK, Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and highthroughput assessment of methylation. Nucleic Acids Res. 2007;35: e41. 15. White HE, Hall VJ, Cross NC. Methylation-sensitive high-resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman syndromes. Clin Chem. 2007;53:1960-1962. 16. Kristensen LS, Mikeska T, Krypuy M, et al. Sensitive melting analysis after real time-methylation specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection. Nucleic Acids Res. 2008;36:e42. 17. Snell C, Krypuy M, Wong EM, et al. BRCA1 promoter methylation in peripheral blood DNA of mutation negative familial breast cancer patients with a BRCA1 tumour phenotype. Breast Cancer Res. 2008;10:R12. 18. Dahl C, Guldberg P. High-resolution melting for accurate assessment of DNA methylation. Clin Chem. 2007;53:18771878. 19. Liu W, Guan M, Su B, et al. Quantitative assessment of AKAP12 promoter methylation in colorectal cancer using methylation-sensitive high resolution melting: Correlation with dukes’ stage. Cancer Biol Ther. 2010;9:862-871. 20. Liu W, Guan M, Su B, et al. Rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylationsensitive high resolution melting (MS-HRM) analysis: application in colorectal cancer. Clin Chim Acta. 2010;411:940-946.
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21. Phe V, Cussenot O, Roupret M. Methylated genes as potential biomarkers in prostate cancer. BJU Int. 2010;105:1364-1370. 22. Hoque MO. DNA methylation changes in prostate cancer: current developments and future clinical implementation. Expert Rev Mol Diagn. 2009;9:243-257. 23. Li HZ, Gao Y, Zhao XL, et al. Effects of Raf kinase inhibitor protein expression on metastasis and progression of human breast cancer. Mol Cancer Res. 2009;7:832-840. 24. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis. 2000;21: 485-495.
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25. Lin X, Nelson P, Gelman IH. SSeCKS, a major protein kinase C substrate with tumor suppressor activity, regulates G(1)¡S progression by controlling the expression and cellular compartmentalization of cyclin D. Mol Cell Biol. 2000;20:7259-7272. 26. Lin X, Gelman IH. Reexpression of the major protein kinase C substrate, SSeCKS, suppresses v-src-induced morphological transformation and tumorigenesis. Cancer Res. 1997;57:23042312. 27. Wojdacz TK, Dobrovic A. Melting curve assays for DNA methylation analysis. Methods Mol Biol. 2009;507:229-240:229-240.
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