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1.  Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors 
Molecular Cancer  2004;3:16.
Background
Male germ cell tumor (GCT) is a highly curable malignancy, which exhibits exquisite sensitivity to cisplatin treatment. The genetic pathway(s) that determine the chemotherapy sensitivity in GCT remain largely unknown.
Results
We studied epigenetic changes in relation to cisplatin response by examining promoter hypermethylation in a cohort of resistant and sensitive GCTs. Here, we show that promoter hypermethylation of RASSF1A and HIC1 genes is associated with resistance. The promoter hypermethylation and/or the down-regulated expression of MGMT is seen in the majority of tumors. We hypothesize that these epigenetic alterations affecting MGMT play a major role in the exquisite sensitivity to cisplatin, characteristic of GCTs. We also demonstrate that cisplatin treatment induce de novo promoter hypermethylation in vivo. In addition, we show that the acquired cisplatin resistance in vitro alters the expression of specific genes and the highly resistant cells fail to reactivate gene expression after treatment to demethylating and histone deacetylase inhibiting agents.
Conclusions
Our findings suggest that promoter hypermethylation of RASSF1A and HIC1 genes play a role in resistance of GCT, while the transcriptional inactivation of MGMT by epigenetic alterations confer exquisite sensitivity to cisplatin. These results also implicate defects in epigenetic pathways that regulate gene transcription in cisplatin resistant GCT.
doi:10.1186/1476-4598-3-16
PMCID: PMC420487  PMID: 15149548
2.  Current Management of Testicular Cancer 
Korean Journal of Urology  2013;54(1):2-10.
Germ cell tumors (GCTs) of the testis are rare, but are the most common cancer in young men. GCTs may consist of one predominant histologic pattern or may represent a mixture of multiple histologic types. For treatment purposes, two broad categories are recognized: 1) pure seminoma and 2) others, which together are termed nonseminomatous GCTs (NSGCTs). In general, seminoma tends to be less aggressive, to be diagnosed at an earlier stage, and to spread predictably along lymphatic channels to the retroperitoneum before spreading hematogenously to the lung or other organs. Compared with NSGCTs, seminoma is exquisitely sensitive to radiation therapy and platinum-based chemotherapy. NSGCTs are usually mixed tumors and teratoma often exists at the sites of metastasis with other GCT elements; cure often requires chemotherapy to kill the chemosensitive-components and surgery to remove the teratomatous components. The main factors contributing to excellent cure rates of GCTs are careful staging at diagnosis; adequate early treatment using chemotherapeutic combinations, with or without radiotherapy and surgery; and very strict follow-up and salvage therapy. We review several clinical studies and summarize the current trends in the management of GCTs.
doi:10.4111/kju.2013.54.1.2
PMCID: PMC3556548  PMID: 23362440
Neoplasms; Testis; Therapeutics
3.  Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes in head and neck squamous cell carcinoma: Pharmacological genome demethylation reduces proliferation of head and neck squamous carcinoma cells 
Oncology Reports  2012;27(4):1135-1141.
Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature of primary cancer cells. However, to date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined. In the present study, we analyzed the promoter methylation status of the genes mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of the three proteins was quantified by semi-quantitative immunohistochemical analysis. The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing. Proliferation was determined by Alamar blue staining. We found that the MGMT promoter in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated. The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples while in the examined cell line the MLH promoter was unmethylated. The RASSF1A promoter showed methylation in 13% of the tumor samples and in the cell line. MGMT expression in the group of tumor samples with a hypermethylated promoter was statistically significantly lower compared to the group of tumors with no measured hypermethylation of the MGMT promoter. After treatment of the cell line with the demethylating agent 5-Aza no demethylation of the methylated MGMT and RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results, however, increased numbers of unmethylated CpG islands in the promoter region of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05) reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this epigenetic inactivation of TSGs may play a role in the development of HNSCC. 5-Aza application resulted in partial demethylation of the MGMT and RASSF1A TSGs and reduced proliferation of the tumor cells suggesting further evaluation of 5-Aza for HNSCC treatment.
doi:10.3892/or.2012.1624
PMCID: PMC3583513  PMID: 22246327
O-6-methylguanine-DNA methyltransferase; mutL homolog 1; Ras association domain family member 1; tumor suppressor gene; head and neck squamous cell carcinoma; 5-azacytidine
4.  Profiling epigenetic inactivation of tumor suppressor genes in tumors and plasma from cutaneous melanoma patients 
Oncogene  2004;23(22):4014-4022.
Aberrant methylation of CpG islands in promoter regions of tumor suppressor genes (TSG) has been demonstrated in epithelial origin tumors. However, the methylation profiling of tumor-related gene promoter regions in cutaneous melanoma tumors has not been reported. Seven known or candidate TSGs that are frequently hyper-methylated in carcinomas were assessed by methylation-specific polymerase chain reaction (MSP) in 15 melanoma cell lines and 130 cutaneous melanoma tumors. Four TSGs were frequently hypermethylated in 86 metastatic tumor specimens: retinoic acid receptor-β2 (RAR-β2) (70%), RAS association domain family protein 1A (RASSF1A) (57%), and O6-methylguanine DNA methy-latransferase (MGMT) (34%), and death-associated protein kinase (DAPK) (19%). Hypermethylation of MGMT, RASSF1A, and DAPK was significantly lower in primary melanomas (n = 20) compared to metastatic melanomas. However, hypermethylation of RAR-β2 was 70% in both primary and metastatic melanomas. Cell lines had hypermethylation profiles similar to those of metastatic melanomas. The analysis of these four markers of metastatic tumors demonstrated that 97% had ≥1 gene(s) and 59% had ≥2 genes hypermethylated. The methylation of genes was verified by bisulfite sequencing. The mRNA transcripts could be re-expressed in melanoma cell lines having hypermethylated genes following treatment with 5′-aza 2′-deoxycytidine (5Aza-dC). Analysis of melanoma patients’ plasma (preoperative blood; n = 31) demonstrated circulating hypermethylated MGMT, RAR-β2, and RASSF1A DNA for at least one of the markers in 29% of the patients. Our findings indicate that the incidence of TSG hypermethylation increases during tumor progression. Methylation of TSG may play a significant role in cutaneous melanoma progression.
doi:10.1038/sj.onc.1207505
PMCID: PMC2856469  PMID: 15064737
MGMT; RAR-β2; RASSF1A; methylation; melanoma
5.  Clinical Implications of Promoter Hypermethylation in RASSF1A and MGMT in Retinoblastoma1 
Neoplasia (New York, N.Y.)  2005;7(3):200-206.
Abstract
We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the O6-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, and RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2′-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of theretinoblastoma samples, and the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced Reese-Ellsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma.
PMCID: PMC1501141  PMID: 15799820
Retinoblastoma; methylation; RASSF1A; MGMT; RB
6.  A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies  
PLoS Medicine  2006;3(12):e486.
Background
Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.
Methods and Findings
In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.
Conclusions
By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.
John Minna and colleagues report that a group of genes are commonly methylated in primary lung, breast, colon, and prostate cancer.
Editors' Summary
Background.
Tumors or cancers contain cells that have lost many of the control mechanisms that normally regulate their behavior. Unlike normal cells, which only divide to repair damaged tissues, cancer cells divide uncontrollably. They also gain the ability to move round the body and start metastases in secondary locations. These changes in behavior result from alterations in their genetic material. For example, mutations (permanent changes in the sequence of nucleotides in the cell's DNA) in genes known as oncogenes stimulate cells to divide constantly. Mutations in another group of genes—tumor suppressor genes—disable their ability to restrain cell growth. Key tumor suppressor genes are often completely lost in cancer cells. But not all the genetic changes in cancer cells are mutations. Some are “epigenetic” changes—chemical modifications of genes that affect the amount of protein made from them. In cancer cells, methyl groups are often added to CG-rich regions—this is called hypermethylation. These “CpG islands” lie near gene promoters—sequences that control the transcription of DNA into RNA, the template for protein production—and their methylation switches off the promoter. Methylation of the promoter of one copy of a tumor suppressor gene, which often coincides with the loss of the other copy of the gene, is thought to be involved in cancer development.
Why Was This Study Done?
The rules that govern which genes are hypermethylated during the development of different cancer types are not known, but it would be useful to identify any DNA methylation events that occur regularly in common cancers for two reasons. First, specific DNA methylation markers might be useful for the early detection of cancer. Second, identifying these epigenetic changes might reveal cellular pathways that are changed during cancer development and so identify new therapeutic targets. In this study, the researchers have used a systematic biological screen to identify genes that are methylated in many lung, breast, colon, and prostate cancers—all cancers that form in “epithelial” tissues.
What Did the Researchers Do and Find?
The researchers used microarray expression profiling to examine gene expression patterns in several lung cancer and normal lung cell lines. In this technique, labeled RNA molecules isolated from cells are applied to a “chip” carrying an array of gene fragments. Here, they stick to the fragment that represents the gene from which they were made, which allows the genes that the cells express to be catalogued. By comparing the expression profiles of lung cancer cells and normal lung cells before and after treatment with a chemical that inhibits DNA methylation, the researchers identified genes that were methylated in the cancer cells—that is, genes that were expressed in normal cells but not in cancer cells unless methylation was inhibited. 132 of these genes contained CpG islands. The researchers examined the promoters of 45 of these genes in lung cancer cells taken straight from patients and found that 31 of the promoters were methylated in tumor tissues but not in adjacent normal tissues. Finally, the researchers looked at promoter methylation of the eight genes most frequently and specifically methylated in the lung cancer samples in breast, colon, and prostate cancers. Seven of the genes were frequently methylated in both lung and breast cancers; four were extensively methylated in all the tumor types.
What Do These Findings Mean?
These results identify several new genes that are often methylated in four types of epithelial tumor. The observation that these genes are methylated in multiple independent tumors strongly suggests, but does not prove, that loss of expression of the proteins that they encode helps to convert normal cells into cancer cells. The frequency and diverse patterning of promoter methylation in different tumor types also indicates that methylation is not a random event, although what controls the patterns of methylation is not yet known. The identification of these genes is a step toward building a promoter hypermethylation profile for the early detection of human cancer. Furthermore, although tumors in different tissues vary greatly with respect to gene expression patterns, the similarities seen in this study in promoter methylation profiles might help to identify new therapeutic targets common to several cancer types.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030486.
US National Cancer Institute, information for patients on understanding cancer
CancerQuest, information provided by Emory University about how cancer develops
Cancer Research UK, information for patients on cancer biology
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence, background information and latest news about epigenetics
doi:10.1371/journal.pmed.0030486
PMCID: PMC1716188  PMID: 17194187
7.  CpG methylation of the FHIT, FANCF, cyclin-D2, BRCA2 and RUNX3 genes in Granulosa cell tumors (GCTs) of ovarian origin 
Molecular Cancer  2004;3:33.
Background
Granulosa cell tumors (GCTs) are relatively rare and are subtypes of the sex-cord stromal neoplasms. Methylation induced silencing in the promoters of genes such as tumor suppressor genes, DNA repair genes and pro-apoptotic genes is recognised as a critical factor in cancer development.
Methods
We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 5 gene promoters in 25 GCTs cases by methylation specific PCR and RT-PCR. In addition, the compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were also included as the control.
Results
Frequencies of methylation in GCTs were 7/25 (28 % for FHIT), 6/25 (24% for FNACF), 3/25 (12% for Cyclin D2), 1/25 (4% for BRCA2) and 14/25 (56%) in RUNX3 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed that overall promoter methylation was higher in more advanced stage of the disease. Promoter methylation was associated with gene silencing in GCT cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression.
Conclusions
These results may have implications in better understanding the underlying epigenetic mechanisms in GCT development, provide prognostic indicators, and identify important gene targets for treatment.
doi:10.1186/1476-4598-3-33
PMCID: PMC538268  PMID: 15574200
8.  Identification and Validation of a Gene Expression Signature That Predicts Outcome in Adult Men With Germ Cell Tumors 
Journal of Clinical Oncology  2009;27(31):5240-5247.
Purpose
Germ cell tumor (GCT) is the most common malignancy in young adult men. Currently, patients are risk-stratified on the basis of clinical presentation and serum tumor markers. The introduction of molecular markers could improve outcome prediction.
Patients and Methods
Expression profiling was performed on 74 nonseminomatous GCTs (NSGCTs) from cisplatin-treated patients (ie, training set) and on 34 similarly treated patients with NSGCTs (ie, validation set). A gene classifier was developed by using prediction analysis for microarrays (PAM) for the binary end point of 5-year overall survival (OS). A predictive score was developed for OS by using the univariate Cox model.
Results
In the training set, PAM identified 140 genes that predicted 5-year OS (cross-validated classification rate, 60%). The PAM model correctly classified 90% of patients in the validation set. Patients predicted to have good outcome had significantly longer survival than those with poor predicted outcome (P < .001). For the OS end point, a 10-gene model had a predictive accuracy (ie, concordance index) of 0.66 in the training set and a concordance index of 0.83 in the validation set. Dichotomization of the samples on the basis of the median score resulted in significant differences in survival (P = .002). For both end points, the gene-based predictor was an independent prognostic factor in a multivariate model that included clinical risk stratification (P < .01 for both).
Conclusion
We have identified gene expression signatures that accurately predict outcome in patients with GCTs. These predictive genes should be useful for the prediction of patient outcome and could provide novel targets for therapeutic intervention.
doi:10.1200/JCO.2008.20.0386
PMCID: PMC3651602  PMID: 19770384
9.  Management of good-risk metastatic nonseminomatous germ cell tumors of the testis: Current concepts and controversies 
Introduction/Methods:
Approximately 30% of nonseminomatous germ-cell tumors (NSGCT) of the testis present with metastatic disease. In 1997, the International Germ Cell Cancer Collaborative Group (IGCCCG) stratified all patients with metastatic NSGCT into various risk groups based on serum tumor markers and presence of visceral disease. We review the literature and present optimal stage-dependent management strategies in patients with favorable-risk metastatic NSGCT.
Results:
Primary chemotherapy (3 cycles BEP or 4 cycles EP) has been shown to be the preferred modality in patients with Clinical Stage IS (cIS) and in patients with bulky metastatic disease (≥CS IIb) due to their high risk of systemic disease and recurrence. Primary retroperitoneal lymph node dissection appears to be the most efficient primary therapy for retroperitoneal disease <2 cm (CS IIa), with adjuvant chemotherapy reserved for patients who are pathologically advanced (>5 nodes involved, single node > 2 cm) and for those who are non-compliant with surveillance regimens. Following primary chemotherapy, STM and radiographic evaluation are used to assess treatment response. For patients with normalization of STM and retroperitoneal masses < 1 cm, retroperitoneal lymph node dissection or observation with treatment at disease progression are considered options. Due to risk of teratoma or chemoresistant GCT, masses >1 cm and extra-retroperitoneal masses should be treated with surgical resection, which should be performed with nerve-sparing, if possible.
Conclusions:
In patients with favorable disease based on IGCCCG criteria, clinical stage, STM, and radiographic evaluation are used to guide appropriate therapy to provide excellent long-term cure rates (>92%) in patients with metastatic NSGCT.
doi:10.4103/0970-1591.60449
PMCID: PMC2878446  PMID: 20535293
Metastatic germ cell cancer; non-seminoma; RPLND; testicular cancer; testis
10.  Current Update of Management of Clinical Stage I Non Seminomatous Germ Cell Tumors of Testis 
The management of patients with testicular germ cell tumors (GCT) has evolved significantly over the past 30 years with cure rates approaching nearly 100% for low-stage disease and more than 80% for advanced disease. Controversy surrounds about ideal management of clinical stage I non seminomatous germ cell tumors (CS I NSGCT) of the testis due to multiple treatment options available with more or less equal efficacy. Nerve-sparing retroperitoneal lymph node dissection (RPLND), adjuvant chemotherapy with two cycles of bleomycin, etoposide, and cisplatin , or surveillance have all achieved long-term survival in nearly 100% of patients with clinical stage I NSGCT. Retroperitoneal lymph node dissection is still favoured as the therapy of choice for clinical stage I non-seminomatous germ cell tumors in many centres, but as risk factors for the primary tumor have become better understood, surveillance and risk-adapted therapy, including surveillance for low-risk patients and adjuvant chemotherapy for the high-risk group, is now being considered a therapeutic option. The objective of this study is to review current developments in the management of CS I NSGCT testis with emphasis on risk stratification and treatment recommendations.
doi:10.1007/s13193-012-0124-8
PMCID: PMC3392487  PMID: 23730098
Testis cancer; NSGCT stage 1; Surveillance; Chemotherapy; RPLND
11.  Sequential bilateral testicular tumours presenting with intervals of 20 years and more 
BMC Urology  2013;13:71.
Background
About 3 – 5% of all patients with testicular germ cell tumour (GCT) develop a contralateral cancer, the majority of which arise within 10–15 years. Little is known about the risk of second GCTs after more than two decades. Here we present 3 cases with very late presenting contralateral GCT and provide a summary of similar cases reported previously.
Case presentations
(1) This white Caucasian man underwent right-sided orchiectomy for a nonseminomatous GCT at the age of 22 years. Additional treatment consisted of retroperitoneal lymph node dissection (RPLND) and chemotherapy with 4 cycles of vinblastin / bleomycin. 36 years later, contralateral seminoma clinical stage 1 developed. Cure was achieved by orchiectomy. Histologically, testicular intraepithelial neoplasia (TIN; intratubular germ cell neoplasia) was detected in the tumour-surrounding tissue.
(2) This white Caucasian male had right-sided orchiectomy for nonseminomatous GCT at the age of 29 years. Pathological stage 1 was confirmed by RPLND. 25 years later, he received left sided orchiectomy for seminoma stage 1. Histologically, TIN was found in the tissue adjacent to seminoma. Two brothers had testicular GCT, too, one with bilateral GCT. (3) This 21 year old white Caucasian man underwent left-sided orchiectomy for nonseminomatous GCT. Pathological stage 1 was confirmed by RPLND. 21 years later, he received organ-preserving excision of a right-sided seminoma, followed by BEP chemotherapy for stage 3 disease. Histologically, TIN was found in the surrounding testicular tissue.
22 cases of bilateral GCT with intervals of 20 or more years have previously been reported, thereof three with intervals of more than 30 years, the longest interval being 40 years.
Conclusion
Apart from increased risks of cardiovascular diseases and non-testicular malignancies, patients with GCT face the specific probability of a second GCT in the long run. This risk persists life-long and is not eliminated by chemotherapy. Contralateral testicular biopsy can identify patients at risk by revealing precursor cells of GCT though false-negative biopsies may occur sporadically. However, in view of the multi-facetted late hazards of GCT patients, this minor surgical procedure might somewhat simplify the long-time care of these patients.
doi:10.1186/1471-2490-13-71
PMCID: PMC4028980  PMID: 24321309
Testicular germ cell neoplasms; Bilateral tumours; Testicular biopsy; Seminoma; Familial germ cell tumours
12.  DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines 
Molecular Cancer  2008;7:15.
Background
DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines.
Results
The relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by methylation-specific PCR, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity and expression of DNMT1, DNMT3a, and DNMT3b proteins) were examined in 12 breast cancer cell lines. Unsupervised cluster analysis of the expression of 64 methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A), whereas the low-frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein levels compared to normal breast epithelial cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Microarray data mining identified a strong cluster of primary breast tumors that express the hypermethylation signature defined by CDH1, CEACAM6, CST6, ESR1, LCN2, and SCNN1A. This subset of breast cancers represents 18/88 (20%) tumors in the dataset analyzed, and 100% of these tumors were classified as basal-like, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers.
Conclusion
These observations combine to strongly suggest that: (a) a subset of breast cancer cell lines express a hypermethylator phenotype, (b) the hypermethylation defect in these breast cancer cell lines is related to aberrant overexpression of DNMT activity, (c) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in tumor cells expressing this phenotype, and (d) the six-gene hypermethylator signature characterized in breast cancer cell lines defines a distinct cluster of primary basal-like breast cancers.
doi:10.1186/1476-4598-7-15
PMCID: PMC2246151  PMID: 18221536
13.  Resistance to Platinum-Containing Chemotherapy in Testicular Germ Cell Tumors Is Associated with Downregulation of the Protein Kinase SRPK11 
Neoplasia (New York, N.Y.)  2004;6(4):297-301.
Abstract
Male germ cell tumors (GCTs) are extremely sensitive to platinum-containing chemotherapy, with only 10% of patients showing therapy resistance. However, the biological basis of the high curability of disseminated GCTs by chemotherapy is still unknown. Recently, we demonstrated that the mammalian serine/arginine-rich protein-specific kinase 1 (SRPK1) is a cisplatin-sensitive gene, inactivation of which leads to cisplatin resistance. Because, in mammalians, the expression of SRPK1 is preferentially high in testicular tissues, cisplatin responsiveness of male GCTs might be associated with SRPK1 levels. In the present study, we monitored SRPK1 protein expression in a unique series of nonseminomatous GCTs by immunohistochemistry. Randomly selected GCTs (n = 70) and tumors from patients responding to standard chemotherapy (n = 20) generally showed strong SRPK1 staining. In contrast, expression in refractory GCTs (n = 20) as well as in GCTs from poor-prognosis patients responding to high-dose chemotherapy only (n = 11) was significantly lower (two-sided Wilcoxon rank sum test: P < .001). In conclusion, our data suggest that SRPK1 expression might be an important prognostic indicator for the chemoresponsiveness of nonseminomatous GCTs.
PMCID: PMC1502111  PMID: 15256051
Chemotherapy resistance; germ cell tumors; chemotherapy sensitivity; protein kinase SRPK1; immunohistochemistry
14.  Methylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma 
BMC Cancer  2004;4:65.
Background
Astrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma.
Methods
We used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients (The WHO grading:. I: 14, II: 15, III: 12 and IV: 12 cases, respectively). In addition, compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were included as the control.
Results
Seventeen genes (ABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14ARF, p15INK4b, p27KIP1, p57KIP2, RASSF1C, RB1, SURVIVIN, and VHL) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues (co-existence of the hypermethylated and demethylated alleles). Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1, and p16INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6,, MT1A, WT1, and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P < 0.001), while the IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P < 0.05). Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma.
Conclusions
Of the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma.
doi:10.1186/1471-2407-4-65
PMCID: PMC520749  PMID: 15367334
15.  Next generation sequencing analysis of platinum refractory advanced germ cell tumor sensitive to Sunitinib (Sutent®) a VEGFR2/PDGFRβ/c-kit/ FLT3/RET/CSF1R inhibitor in a phase II trial 
Background
Germ cell tumors (GCT) are the most common solid tumors in adolescent and young adult males (age 15 and 35 years) and remain one of the most curable of all solid malignancies. However a subset of patients will have tumors that are refractory to standard chemotherapy agents. The management of this refractory population remains challenging and approximately 400 patients continue to die every year of this refractory disease in the United States.
Methods
Given the preclinical evidence implicating vascular endothelial growth factor (VEGF) signaling in the biology of germ cell tumors, we hypothesized that the vascular endothelial growth factor receptor (VEGFR) inhibitor sunitinib (Sutent) may possess important clinical activity in the treatment of this refractory disease. We proposed a Phase II efficacy study of sunitinib in seminomatous and non-seminomatous metastatic GCT’s refractory to first line chemotherapy treatment (ClinicalTrials.gov Identifier: NCT00912912). Next generation targeted exome sequencing using HiSeq 2000 (Illumina Inc., San Diego, CA, USA) was performed on the tumor sample of the unusual responder.
Results
Five patients are enrolled into this Phase II study. Among them we report here the clinical course of a patient (Patient # 5) who had an exceptional response to sunitinib. Next generation sequencing to understand this patient’s response to sunitinib revealed RET amplification, EGFR and KRAS amplification as relevant aberrations. Oncoscan MIP array were employed to validate the copy number analysis that confirmed RET gene amplification.
Conclusion
Sunitinib conferred clinical benefit to this heavily pre-treated patient. Next generation sequencing of this ‘exceptional responder’ identified the first reported case of a RET amplification as a potential basis of sensitivity to sunitinib (VEGFR2/PDGFRβ/c-kit/ FLT3/RET/CSF1R inhibitor) in a patient with refractory germ cell tumor. Further characterization of GCT patients using biomarkers for clinical response and patient selection is warranted.
Trial registration
ClinicalTrials.gov Identifier: NCT00912912
doi:10.1186/s13045-014-0052-x
PMCID: PMC4237879  PMID: 25085632
Sunitinib; Germ cell tumor; Phase II trials; Targeted therapy; RET; EGFR; Next generation sequencing; Adolescent and young adult; Vascular endothelial growth factor receptor; VEGF; Unusual responder; Exceptional responder; Outlier responder
16.  Global DNA hypomethylation in intratubular germ cell neoplasia and seminoma, but not in nonseminomatous male germ cell tumors 
Alterations in methylation of CpG dinucleotides at the 5 position of deoxycytidine residues (5mC) are a hallmark of cancer cells, including testicular germ cell tumors. Virtually all testicular germ cell tumors are believed to be derived from intratubular germ cell neoplasia unclassified (IGCNU), which is thought to arise from primordial germ cells. Prior studies revealed that seminomas contain reduced levels of global DNA methylation as compared with nonseminomatous germ cell tumors. Smiraglia et al have proposed a model whereby seminomas arise from IGCNU cells derived from primordial germ cells that have undergone 5mC erasure, and nonseminomas arise from IGCNU cells derived from primordial germ cells that have already undergone de novo methylation after the original erasure of methylation and contain normal 5mC levels. Yet the methylation status of IGCNU has not been determined previously. We used immunohistochemical staining against 5mC to evaluate global methylation in IGCNU and associated invasive testicular germ cell tumors. Strikingly, staining for 5mC was undetectable (or markedly reduced) in the majority of IGCNU and seminomas, yet there was robust staining in nonseminomatous germ cell tumors. The lack of staining for 5mC in IGCNU and seminomas was also found in mixed germ cell tumors containing both seminomatous and nonseminomatous components. Lack of 5mC staining was not related to a lack of the maintenance methyltransferase (DNA methyltransferase 1) protein. We conclude that testicular germ cell tumors are derived in most cases from IGCNU cells that have undergone developmentally programmed 5mC erasure and that the degree of subsequent de novo methylation is most closely related to the differentiation state of the neoplastic cells. That is, IGCNU cells and seminoma cells remain unmethylated, whereas all other histological types appear to arise after de novo methylation.
doi:10.1038/modpathol.2008.127
PMCID: PMC4086525  PMID: 18622385
seminoma; nonseminomatous germ cell tumors; IGCNU; global methylation
17.  Molecular Characteristics of Malignant Ovarian Germ Cell Tumors and Comparison With Testicular Counterparts: Implications for Pathogenesis 
Endocrine Reviews  2013;34(3):339-376.
This review focuses on the molecular characteristics and development of rare malignant ovarian germ cell tumors (mOGCTs). We provide an overview of the genomic aberrations assessed by ploidy, cytogenetic banding, and comparative genomic hybridization. We summarize and discuss the transcriptome profiles of mRNA and microRNA (miRNA), and biomarkers (DNA methylation, gene mutation, individual protein expression) for each mOGCT histological subtype. Parallels between the origin of mOGCT and their male counterpart testicular GCT (TGCT) are discussed from the perspective of germ cell development, endocrinological influences, and pathogenesis, as is the GCT origin in patients with disorders of sex development. Integrated molecular profiles of the 3 main histological subtypes, dysgerminoma (DG), yolk sac tumor (YST), and immature teratoma (IT), are presented. DGs show genomic aberrations comparable to TGCT. In contrast, the genome profiles of YST and IT are different both from each other and from DG/TGCT. Differences between DG and YST are underlined by their miRNA/mRNA expression patterns, suggesting preferential involvement of the WNT/β-catenin and TGF-β/bone morphogenetic protein signaling pathways among YSTs. Characteristic protein expression patterns are observed in DG, YST and IT. We propose that mOGCT develop through different developmental pathways, including one that is likely shared with TGCT and involves insufficient sexual differentiation of the germ cell niche. The molecular features of the mOGCTs underline their similarity to pluripotent precursor cells (primordial germ cells, PGCs) and other stem cells. This similarity combined with the process of ovary development, explain why mOGCTs present so early in life, and with greater histological complexity, than most somatic solid tumors.
doi:10.1210/er.2012-1045
PMCID: PMC3787935  PMID: 23575763
18.  Tumor Suppressor Gene Inactivation during Cadmium-Induced Malignant Transformation of Human Prostate Cells Correlates with Overexpression of de Novo DNA Methyltransferase 
Environmental Health Perspectives  2007;115(10):1454-1459.
Background
Aberrant DNA methylation is common in carcinogenesis. The typical pattern appears to involve reduced expression of maintenance DNA methyltransferase, DNMT1, inducing genomic hypomethylation, whereas increased expression of de novo DNMT3a or 3b causes gene-specific hypermethylation.
Objectives
During cadmium-induced malignant transformation, an unusual pattern of genomic hypermethylation occurred that we studied to provide insight into the roles of specific DNMTs in oncogenesis.
Methods
Gene expression and DNA methylation were assessed in control and chronic cadmium-transformed prostate epithelial cells (CTPE) using reverse transcription–polymerase chain reaction (RT-PCR), Western blot analysis, methylation-specific PCR, and methyl acceptance assay.
Results
During the 10-weeks of cadmium exposure that induced malignant transformation, progressive increases in generalized DNMT enzymatic activity occurred that were associated with over-expression of DNMT3b without changes in DNMT1 expression. Increased DNMT3b expression preceded increased DNMT enzymatic activity. Procainamide, a specific DNMT1 inhibitor, reversed cadmium-induced genomic DNA hypermethylation. Reduced expression of the tumor suppressor genes, RASSF1A and p16, began about the time DNMT3b overexpression first occurred and progressively decreased thereafter. RASSF1A and p16 promoter regions were heavily methylated in CTPE cells, indicating silencing by hypermethylation, while the DNA demethylating agent, 5-aza-2′-deoxycytidine, reversed this silencing. DNMT1 inhibition only modestly increased RASSF1A and p16 expression in CTPE cells and did not completely reverse silencing.
Conclusions
These data indicate that DNMT3b overexpression can result in generalized DNA hypermethylation and gene silencing but that DNMT1 is required to maintain these effects. The pattern of genomic DNA hypermethylation together with up-regulation of DNMT3b may provide a unique set of biomarkers to specifically identify cadmium-induced human prostate cancers.
doi:10.1289/ehp.10207
PMCID: PMC2022656  PMID: 17938735
cadmium; carcinogenesis; DNA methylation; DNMT3b; p16; prostate; RASSF1A
19.  Mixed germ cell tumor of the testicle with ravdomuosarcomatous component: a case report 
Cases Journal  2009;2:9299.
Introduction
Testicular tumors can be classified as seminomatous and non-seminomatous germ-cell tumor (NSGCT) types. Mixed germ cell tumors contain more than one germ cell component and are much more common than any of the pure histologic forms representing 32%-60% of all germ cell tumors. The composition of these tumors varies. Here we present a rare case of a mixed germ cell tumor composed of seminoma, Yolk sack tumor and teratoma containing a sarcoma component of somatic type malignancy.
Case presentation
A 32-year-old Caucasian male presented with history of right-sided scrotal swelling since 6 months. Backache was present since 2 months and a history of right epididimitis was also present since 8 months. Alpha-Fetoprotein, beta-HCG and LDH values were found abmormal. USG of the scrotum revealed a large right testis swelling characterized by scarce cystic elements and calcifications. CT scan of the abdomen showed nodular metastasis involving the interaortocaval, precaval, and right para-aortic lymph nodes. The block of enlarged lymph nodes infiltrated the psoas muscle. The patient underwent right-sided high orchidectomy and was given chemotherapy of the BEP regimen. After the 2nd cycle the patient discontinued the chemotherapy and when he came for follow-up after a gap of 3 months, despite the normalisation in tumor markers values, the retroperitoneal mass was relapsed. CT scan of the chest showed multiple lung metastases.
Conclusion
More than 50% of germ-cell tumors include more than 2 basic germ-cell tumor types, with the exception of spermatocytic seminoma. About 90% of the patients with nonseminomatous tumors can achieve complete cure with aggressive chemotherapy and most of them can be cured. Although prognosis of testicular tumors depends largely on clinical stage, histological type and adhesion to the treatment influence the prognosis as well.
doi:10.1186/1757-1626-2-9299
PMCID: PMC2803963  PMID: 20062623
20.  Global Mapping of DNA Methylation in Mouse Promoters Reveals Epigenetic Reprogramming of Pluripotency Genes 
PLoS Genetics  2008;4(6):e1000116.
DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early embryo is thus crucial for transmission of pluripotency.
Author Summary
Large scale epigenetic reprogramming occurs in mammalian germ cells and the early embryo. The biological purpose of this reprogramming is largely unknown, although it has been suggested that it may be required for the embryonic genome to return to a pluripotent state. We carried out a genome-wide screen of promoter methylation in the mouse, comparing germ cells with pluripotent cells, multipotent cells, and more differentiated cell types. We find that promoter methylation is an epigenetic signature of developmental potency. Genes linked to pluripotency are generally hypomethylated in stem cells and hypermethylated (and silenced) in more differentiated cell types, and our genome-wide screen provides new candidates for the regulation of pluripotency. Importantly, germ cells resemble pluripotent cell types in that most promoters have been reprogrammed. However, a small group of key pluripotency regulators (including Nanog), are methylated in mature germ cells, presumably in order to suppress pluripotency at critical stages of germ cell differentiation. Indeed, methylation in these genes becomes reprogrammed after fertilisation so that the embryo can regain totipotency. This work, therefore, shows for the first time that epigenetic reprogramming is crucial for maintaining the pluripotency of germ and embryonic stem cells.
doi:10.1371/journal.pgen.1000116
PMCID: PMC2432031  PMID: 18584034
21.  Clinical Significance of Amyloid Precursor Protein in Patients with Testicular Germ Cell Tumor 
Advances in Urology  2013;2013:348438.
Introduction. The biological role of amyloid precursor protein (APP) is not well understood, especially in testicular germ cell tumors (TGCTs). Therefore, we aimed to investigate the immunoreactivity (IR) and expression of APP in TGCTs and evaluated its clinical relevance. Materials and Methods. We performed an analysis of immunohistochemistry and mRNA expression of APP in 64 testicular specimens and 21 snap-frozen samples obtained from 1985 to 2004. We then evaluated the association between APP expression and clinicopathological status in TGCTs. Results. Positive APP IR was observed in 9.8% (4/41) of seminomatous germ cell tumors (SGCTs) and 39.1% (9/23) of nonseminomatous germ cell tumors (NGCTs). NGCTs showed significantly more cases of positive IR (P = 0.00870) and a higher mRNA expression level compared with those of SGCTs (P = 0.0140). Positive APP IR was also significantly associated with α-fetoprotein (αFP) elevation (P = 0.00870) and venous invasion (P = 0.0414). Conclusion. We observed an elevated APP expression in TGCTs, especially in NGCTs. APP may be associated with a more aggressive cancer in TGCTs.
doi:10.1155/2013/348438
PMCID: PMC3639667  PMID: 23662100
22.  Re-expression of methylation-induced tumor suppressor gene silencing is associated with the state of histone modification in gastric cancer cell lines 
AIM: To identify the relationship between DNA hyper-methylation and histone modification at a hyperme-thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification.
METHODS: We used chromatin immunoprecipitation (ChIP) assay to assess the status of histone acetylation and methylation in promoter regions of the p16 and mutL homolog 1 (MLH1) genes in 2 gastric cancer cell lines, SGC-7901 and MGC-803. We used methylation-specific PCR (MSP) to evaluate the effect of 5-Aza-2’-deoxycytidine (5-Aza-dC), trichostatin A (TSA) or their combination treatment on DNA methylation status. We used RT-PCR to determine whether alterations of histone modification status after 5-Aza-dC and TSA treatment are reflected in gene expression.
RESULTS: For the p16 and MLH1 genes in two cell lines, silenced loci associated with DNA hypermethylation were characterized by histone H3-K9 hypoacetylation and hypermethylation and histone H3-K4 hypomethylation. Treatment with TSA resulted in moderately increased histone H3-K9 acetylation at the silenced loci with no effect on histone H3-K9 methylation and minimal effects on gene expression. In contrast, treatment with 5-Aza-dC rapidly reduced histone H3-K9 methylation at the silenced loci and resulted in reactivation of the two genes. Combined treatment with 5-Aza-dC and TSA was synergistic in reactivating gene expression at the loci showing DNA hypermethylation. Similarly, histone H3-K4 methylation was not affected after TSA treatment, and increased moderately at the silenced loci after 5-Aza-dC treatment.
CONCLUSION: Hypermethylation of DNA in promoter CpG islands is related to transcriptional silencing of tumor suppressor genes. Histone H3-K9 methylation in different regions of the promoters studied correlates with DNA methylation status of each gene in gastric cancer cells. However, histone H3-K9 acetylation and H3-K4 methylation inversely correlate with DNA methylation status of each gene in gastric cancer cells. Alteration of DNA methylation affects histone modification.
doi:10.3748/wjg.v13.i46.6166
PMCID: PMC4171225  PMID: 18069755
Gastric cancer; DNA hypermethylation; Histone methylation; Histone acetylation; p16; mutL homolog 1; 5-Aza-2’-deoxycytidine; Trichostatin A
23.  Zinc finger protein 382 is downregulated by promoter hypermethylation in pediatric acute myeloid leukemia patients 
Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are characteristic of AML. Zinc finger protein 382 (ZNF382) has been suggested to be a tumor suppressor gene possibly regulated by promoter hypermethylation in various types of human cancer. However, ZNF382 expression and methylation status in pediatric AML is unknown. In the present study, ZNF382 transcription levels were evaluated by quantitative reverse-transcription PCR. Methylation status was investigated by methylation-specific (MSP) PCR and bisulfate genomic sequencing (BGS). The prognostic significance of ZNF382 expression and promoter methylation was assessed in 105 cases of pediatric AML. The array data suggested that the ZNF382 promoter was hypermethylated in the AML cases examined. MSP PCR and BGS analysis revealed that ZNF382 was hypermethylated in leukemia cell lines. Furthermore, treatment with 5-aza-2′-deoxycytidine (5-Aza) upregulated ZNF382 expression in the selected leukemia cell lines. The aberrant methylation of ZNF382 was observed in 10% (2/20) of the control samples compared with 26.7% (28/105) of the AML samples. ZNF382 expression was significantly decreased in the 105 AML patients compared with the controls. Patients with ZNF382 methylation showed lower ZNF382 transcript levels compared with patients exhibiting no methylation. There were no significant differences in clinical characteristics or cytogenetic analysis between the patients with or without ZNF382 methylation. ZNF382 methylation correlated with minimal residual disease (MRD). Kaplan-Meier survival analysis revealed similar survival times in the samples with ZNF382 methylation, and multivariate analysis revealed that ZNF382 methylation was not an independent prognostic factor in pediatric AML. The epigenetic inactivation of ZNF382 by promoter hypermethylation can be observed in AML cell lines and pediatric AML samples. Therefore, our study suggests that ZNF382 may be considered a putative tumor suppressor gene in pediatric AML. However, further studies focusing on the mechanisms responsible for ZNF382 downregulation in pediatric leukemia are required.
doi:10.3892/ijmm.2014.1966
PMCID: PMC4214337  PMID: 25319049
zinc finger protein 382; pediatric acute myeloid leukemia; methylation; tumor suppressor
24.  Epigenetic silencing of genes and microRNAs within the imprinted Dlk1-Dio3 region at human chromosome 14.32 in giant cell tumor of bone 
BMC Cancer  2014;14:495.
Background
Growing evidence exists that the neoplastic stromal cell population (GCTSC) within giant cell tumors (GCT) originates from mesenchymal stem cells (MSC). In a previous study we identified a microRNA signature that differentiates between these cell types. Five differentially expressed microRNAs are located within the Dlk1-Dio3 region on chromosome 14. Aberrant regulation within this region is known to influence cell growth, differentiation and the development of cancer. The aim of this study was to elucidate the involvement of deregulations within the Dlk1-Dio3 region in GCT pathogenesis.
Methods
Quantitative gene and microRNA expression analyses were performed on GCTSCs and MSCs with or without treatment with epigenetic modifiers. Methylation analysis of differentially methylated regions was performed by bisulfite sequencing.
Results
In addition to microRNA silencing we detected a significant downregulation of Dlk1, Meg3 and Meg8 in GCTSCs compared to MSCs. DNA methylation analyses of the Meg3-DMR and IG-DMR revealed a frequent hypermethylation within the IG-DMR in GCTs. Epigenetic modification could restore expression of some but not all analyzed genes and microRNAs suggesting further regulatory mechanisms.
Conclusion
Epigenetic silencing of genes and microRNAs within the Dlk1-Dio3 region is a common event in GCTSCs, in part mediated by hypermethylation within the IG-DMR. The identified genes, micro RNAs and microRNA target genes might be valuable targets for the development of improved strategies for GCT diagnosis and therapy.
doi:10.1186/1471-2407-14-495
PMCID: PMC4101709  PMID: 25005035
Giant cell tumor; Mesenchymal stem cell; MicroRNA; Epigenetics; Methylation
25.  High DNA Methyltransferase 3B Expression Mediates 5-aza-deoxycytidine Hypersensitivity in Testicular Germ Cell Tumors 
Cancer research  2009;69(24):9360-9366.
Testicular germ cell tumors (TGCTs) are the most common solid tumors of 15–35 year old men. TGCT patients are frequently cured with cytotoxic cisplatin-based therapy. However, TGCT patients refractory to cisplatin-based chemotherapy have a poor prognosis, as do those having a late relapse. Pluripotent embryonal carcinomas (ECs) are the malignant counterparts to embryonic stem (ES) cells and are considered the stem cells of TGCTs. Here we demonstrate that human EC cells are highly sensitive to 5-aza-deoxycytidine (5-aza-CdR) as compared to somatic solid tumor cells. Decreased proliferation and survival with low nanomolar concentrations of 5-aza-CdR is associated with ATM activation, H2AX phosphorylation, increased expression of p21, and the induction of genes known to be methylated in TGCTs (MGMT, RASSF1A and HOXA9). Notably, 5-aza-CdR hypersensitivity is associated with markedly abundant expression of the pluripotency-associated DNA methyltransferase 3B (DNMT3B) as compared to somatic tumor cells. Knockdown of DNMT3B in EC cells results in substantial resistance to 5-aza-CdR, strongly indicating that 5-aza-CdR sensitivity is mechanistically linked to high levels of DNMT3B. Intriguingly, cisplatin-resistant EC cells retain an exquisite sensitivity to low dose 5-aza-CdR treatment and pretreatment of 5-aza-CdR re-sensitizes these cells to cisplatin-mediated toxicity. This re-sensitization is also partially dependent on high DNMT3B levels. These novel findings indicate that high expression of DNMT3B, a likely byproduct of their pluripotency and germ cell origin, sensitizes TGCT-derived EC cells to low dose 5-aza-CdR treatment.
doi:10.1158/0008-5472.CAN-09-1490
PMCID: PMC2795063  PMID: 19951990
DNMT3B; 5-aza-deoxycytidine; embryonal carcinoma; cisplatin; resistance; testicular cancer

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