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Barault L.,Harvard University | Ellsworth R.E.,Foundation Medicine | Harris H.R.,Harvard University | Valente A.L.,Windber Research Institute | And 2 more authors.
PLoS ONE | Year: 2013

There is growing interest in identifying surrogate tissues to identify epimutations in cancer patients since primary target tissues are often difficult to obtain. Methylation patterns at imprinted loci are established during gametogenesis and post fertilization and their alterations have been associated with elevated risk of cancer. Methylation at several imprinted differentially methylated regions (GRB10 ICR, H19 ICR, KvDMR, SNRPN/SNURF ICR, IGF2 DMR0, and IGF2 DMR2) were analyzed in DNA from leukocytes and mammary tissue (normal, benign diseases, or malignant tumors) from 87 women with and without breast cancer (average age of cancer patients: 53; range: 31-77). Correlations between genomic variants and DNA methylation at the studied loci could not be assessed, making it impossible to exclude such effects. Methylation levels observed in leukocyte and mammary tissue DNA were close to the 50% expected for monoallellic methylation. While no correlation was observed between leukocyte and mammary tissue DNA methylation for most of the analyzed imprinted genes, Spearman's correlations were statistically significant for IGF2 DMR0 and IGF2 DMR2, although absolute methylation levels differed. Leukocyte DNA methylation levels of selected imprinted genes may therefore serve as surrogate markers of DNA methylation in cancer tissue.

Rummel S.,Windber Research Institute | Shriver C.D.,U.S. National Institutes of Health | Ellsworth R.E.,Foundation Medicine
BMC Medical Genetics | Year: 2012

Background: To date, evaluation of the association of the ABO blood group and breast cancer has yielded mixed results. SNP rs505922, located within the first intron of the ABO gene, has been associated with the adenocarcinoma subtype of pancreatic cancer. To evaluate the association between genetic variation in the ABO blood group and risk of breast cancer, rs505922 was genotyped in 629 Caucasian women with invasive breast cancer, representing a variety of clinical and pathological tumor types.Methods: Genomic DNA was isolated from blood. TaqMan SNP assay C_2253769_10 was used to determine genotypes for each patient at rs505922. Statistical analysis was performed using chi-square analysis using a P-value <0.05 to define significance.Results: Genotypes were generated for 100% of the 629 patients in this study. Allele and genotype frequencies did not vary significantly for age at diagnosis, tumor stage, size or grade, hormone, HER2 or lymph node status, intrinsic subtype, tumor type or patient outcome.Conclusions: Allele frequencies for rs505922 did not differ between women with breast cancer and published HapMap frequencies from women of European descent. Further stratification into different tumor phenotypes also failed to reveal an association between rs505922 and any clinical characteristics. Together, these data suggest that the minor allele of rs505922 and the resulting non-O blood types are not associated with increased risk or less favorable tumor characteristics or prognosis in breast cancer. © 2012 Rummel et al.; licensee BioMed Central Ltd.

Paul S.,Bethesda University | Paul S.,University of Toledo | Traver M.K.,Bethesda University | Kashyap A.K.,Bethesda University | And 6 more authors.
Science Signaling | Year: 2014

Antigen-mediated stimulation of the T cell receptor (TCR) triggers activation of nuclear factor κB (NF-κB), a key transcriptional regulator of T cell proliferation and effector cell differentiation. TCR signaling to NF-κB requires both the Carma1-Bcl10-Malt1 (CBM) complex and the inhibitor of κB (IκB) kinase (IKK) complex; however, the molecularmechanisms connecting the CBM complex to activation of IKK are incompletely defined. We found that the active IKK complex is a component of a TCR-dependent cytosolic Bcl10-Malt1 signalosome containing the adaptor protein p62, which forms in effector T cells. Phosphorylated IκBα and NF-κB were transiently recruited to this signalosome before NF-κB translocated to the nucleus. Inhibiting the activity of the kinase TAK1 or IKK blocked the phosphorylation of IKK, but not the formation of p62-Bcl10-Malt1 clusters, suggesting that activation of IKK occurs after signalosome assembly. Furthermore, analysis of T cells from p62-deficient mice demonstrated that the p62-dependent clustering of signaling components stimulated activation of NF-κB in effector T cells. Thus, TCR-stimulated activation of NF-κB requires the assembly of cytosolic p62-Bcl10-Malt1-IKK signalosomes, which may ensure highly regulated activation of NF-κB in response to TCR engagement.

Pastwa E.,Medical University of Lodz | Poplawski T.,University of Lodz | Lewandowska U.,Medical University of Lodz | Somiari S.B.,Windber Research Institute | And 2 more authors.
International Journal of Biochemistry and Cell Biology | Year: 2014

The combination of etoposide and cisplatin represents a common modality for treating of glioma patients. These drugs directly and indirectly produce the most lethal DNA double-stand breaks (DSB), which are mainly repaired by non-homologous DNA end joining (NHEJ). Drugs that can specifically inhibit the kinase activity of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the major component of NHEJ, are of special interest in cancer research. These small molecule inhibitors can effectively enhance the efficacy of current cancer treatments that generate DNA damage. In this study, we investigated the effect of DNA-PKcs inhibitor, wortmannin, on the cytotoxic mechanism of etoposide and cisplatin in MO59K and MO59J human glioblastoma cell lines. These cell lines are proficient and deficient in DNA-PKcs, respectively. Wortmannin synergistically increased the cytotoxicity of cisplatin and etoposide, when combined, in NHEJ-proficient MO59K cells. Surprisingly, wortmannin sensitizing effect was also observed in DNA-PKcs-deficient MO59J cells. These data suggest that wortmannin sensitization to etoposide and cisplatin in human glioma cells is mediated by inhibition of not only DNA-PKcs activity but other enzymes from PI3-K family, e.g. ATM and ATR. A concentration-dependent increase in etoposide and cisplatin-induced DSB levels was potentiated by inhibitor in both cell lines. Moreover, drug-induced accumulation in the G2/M checkpoint and S-phase was increased by wortmannin. Wortmannin significantly inhibited drug-induced DSB repair in MO59 cells and this effect was more pronounced in MO59J cells. We conclude that the mechanism of wortmannin potentiation of etoposide and cisplatin cytotoxicity involves DSBs induction, DSBs repair inhibition, G2/M checkpoint arrest and inhibition of not only DNA-PKcs activity. © 2014 Published by Elsevier Ltd.

Rummel S.,Windber Research Institute | Varner E.,Windber Research Institute | Shriver C.D.,U.S. National Institutes of Health | Ellsworth R.E.,Foundation Medicine
Breast Cancer Research and Treatment | Year: 2013

Triple-negative breast cancer (TNBC) is characterized by aggressive behavior and poor prognosis. While >50 % of patients with inherited BRCA1 mutations have TNBC, the prevalence of BRCA1 mutations in patients with TNBC remains unclear. Deciphering the relationship between BRCA1 and TNBC is critical to understanding the etiology of TNBC, leading to improved patient counseling and treatment. All female patients with TNBC enrolled in the Clinical Breast Care Project were identified. Genomic DNA was isolated from blood and the exonic regions of the BRCA1 gene were amplified and sequenced. Sequence data was analyzed and mutations identified using Sequencher 4.10.1. Of the 190 women with TNBC, genomic DNA was available for 182. Seventy percent of patients were considered high-risk for having a BRCA1 mutation based on the National Comprehensive Cancer Network criteria. Clinically relevant mutations were detected in 16 (9 %) patients ranging in age from 26 to 69 years at diagnosis. Six of these patients were diagnosed >50 years. The C61G mutation was found in three Caucasian women diagnosed >40 years, while six African-American women had mutations, including the 943ins10 West African founder mutation. Upon conclusion, causative BRCA1 mutations were detected in 9 % of TNBC patients, including patients without significant family histories and/or diagnosed at a later age. The mutation frequency in patients <60 years was 11.2-18.3 % in those patients with significant risk factors and 4.6 % in those without, while in patients >60 years, the mutation frequency was 3.5-7.7 % in patients with risk factors, 2.3 % in those without. Thus, evaluation of additional risk factors in both patients younger and older than 60 years should improve the identification of TNBC patients benefiting from genetic testing of BRCA1. © 2012 Springer Science+Business Media New York.

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