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1.  Reduced Proficiency in Homologous Recombination Underlies the High Sensitivity of Embryonal Carcinoma Testicular Germ Cell Tumors to Cisplatin and Poly (ADP-Ribose) Polymerase Inhibition 
PLoS ONE  2012;7(12):e51563.
Testicular Germ Cell Tumors (TGCT) and patient-derived cell lines are extremely sensitive to cisplatin and other interstrand cross-link (ICL) inducing agents. Nevertheless, a subset of TGCTs are either innately resistant or acquire resistance to cisplatin during treatment. Understanding the mechanisms underlying TGCT sensitivity/resistance to cisplatin as well as the identification of novel strategies to target cisplatin-resistant TGCTs have major clinical implications. Herein, we have examined the proficiency of five embryonal carcinoma (EC) cell lines to repair cisplatin-induced ICLs. Using γH2AX staining as a marker of double strand break formation, we found that EC cell lines were either incapable of or had a reduced ability to repair ICL-induced damage. The defect correlated with reduced Homologous Recombination (HR) repair, as demonstrated by the reduction of RAD51 foci formation and by direct evaluation of HR efficiency using a GFP-reporter substrate. HR-defective tumors cells are known to be sensitive to the treatment with poly(ADP-ribose) polymerase (PARP) inhibitor. In line with this observation, we found that EC cell lines were also sensitive to PARP inhibitor monotherapy. The magnitude of sensitivity correlated with HR-repair reduced proficiency and with the expression levels and activity of PARP1 protein. In addition, we found that PARP inhibition strongly enhanced the response of the most resistant EC cells to cisplatin, by reducing their ability to overcome the damage. These results point to a reduced proficiency of HR repair as a source of sensitivity of ECs to ICL-inducing agents and PARP inhibitor monotherapy, and suggest that pharmacological inhibition of PARP can be exploited to target the stem cell component of the TGCTs (namely ECs) and to enhance the sensitivity of cisplatin-resistant TGCTs to standard treatments.
doi:10.1371/journal.pone.0051563
PMCID: PMC3520950  PMID: 23251575
2.  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
3.  Inactivation of the PRDM1/BLIMP1 gene in diffuse large B cell lymphoma 
PR domain containing 1 with zinc finger domain (PRDM1)/B lymphocyte–induced maturation protein 1 (BLIMP1) is a transcriptional repressor expressed in a subset of germinal center (GC) B cells and in all plasma cells, and required for terminal B cell differentiation. The BLIMP1 locus lies on chromosome 6q21-q22.1, a region frequently deleted in B cell lymphomas, suggesting that it may harbor a tumor suppressor gene. We report here that the BLIMP1 gene is inactivated by structural alterations in 24% (8 out of 34) activated B cell–like diffuse large cell lymphoma (ABC-DLBCL), but not in GC B cell–like (n = 0/37) or unclassified (n = 0/21) DLBCL. BLIMP1 alterations included gene truncations, nonsense mutations, frameshift deletions, and splice site mutations that generate aberrant transcripts encoding truncated BLIMP1 proteins. In all cases studied, both BLIMP1 alleles were inactivated by deletions or mutations. Furthermore, most non–GC type DLBCL cases (n = 20/26, 77%) lack BLIMP1 protein expression, despite the presence of BLIMP1 mRNA. These results indicate that a sizable fraction of ABC-DLBCL carry an inactive BLIMP1 gene, and suggest that the same gene is inactivated by epigenetic mechanisms in an additional large number of cases. These findings point to a role for BLIMP1 as a tumor suppressor gene, whose inactivation may contribute to lymphomagenesis by blocking post–GC differentiation of B cells toward plasma cells.
doi:10.1084/jem.20052204
PMCID: PMC2118216  PMID: 16492805
4.  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
5.  Characteristic promoter hypermethylation signatures in male germ cell tumors 
Molecular Cancer  2002;1:8.
Background
Human male germ cell tumors (GCTs) arise from undifferentiated primordial germ cells (PGCs), a stage in which extensive methylation reprogramming occurs. GCTs exhibit pluripotentality and are highly sensitive to cisplatin therapy. The molecular basis of germ cell (GC) transformation, differentiation, and exquisite treatment response is poorly understood.
Results
To assess the role and mechanism of promoter hypermethylation, we analyzed CpG islands of 21 gene promoters by methylation-specific PCR in seminomatous (SGCT) and nonseminomatous (NSGCT) GCTs. We found 60% of the NSGCTs demonstrating methylation in one or more gene promoters whereas SGCTs showed a near-absence of methylation, therefore identifying distinct methylation patterns in the two major histologies of GCT. DNA repair genes MGMT, RASSF1A, and BRCA1, and a transcriptional repressor gene HIC1, were frequently methylated in the NSGCTs. The promoter hypermethylation was associated with gene silencing in most methylated genes, and reactivation of gene expression occured upon treatment with 5-Aza-2' deoxycytidine in GCT cell lines.
Conclusions
Our results, therefore, suggest a potential role for epigenetic modification of critical tumor suppressor genes in pathways relevant to GC transformation, differentiation, and treatment response.
doi:10.1186/1476-4598-1-8
PMCID: PMC149411  PMID: 12495446
Germ cell tumor; promoter hypermethylation; MGMT; RASSF1A; BRCA1; gene expression

Results 1-5 (5)