bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3880: Synthesis of complex oligonucleotide libraries enables flexible and precise high-resolution fluorescence imaging of cellular nucleic acids
Authors: Ach Robert A. , Yamada N. Alice , Tsang Peter , Scheffer-Wong Alicia , Bruhn Laurakay 
Published: June 2012

Abstract
Fluorescence in situ Hybridization (FISH) is a powerful technique for studying chromosome structure, organization, and stability. To date, the use of FISH has been largely dependent upon access to cloned template DNA for the generation of probes. Clones for specific regions may be unavailable, or the genomic region of interest may contain repetitive and other non-informative sequences which can be problematic for FISH analysis. We have leveraged our ability to chemically synthesize DNA in massively parallel reactions to produce libraries of oligonucleotides up to 200 bases in length that can be used to generate FISH probes. The sequences of the oligonucleotides in these libraries are selected in silico using empirically determined criteria so as to avoid repetitive elements or regions homologous to other non-targeted loci, thus targeting only the most informative elements in the region of the genome to be analyzed. We demonstrate that using our FISH technique, human genomic regions as small as 1.8 kb and as large as whole chromosomes can be visualized in both metaphase and interphase cells using the same simple assay protocol. In addition, specific and robust signals can readily be obtained for chromosomal regions rich in repeats and/or GC content. These libraries can also be used to determine the localization of cellular RNAs. Co-detection of both nuclear proteins by immunocytochemistry and fluorescent imaging of chromosome domains has also been demonstrated.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3880. doi:10.1158/1538-7445.AM2011-3880
Abstract 3883: Multiplexed formats for detection of SNPs for cell line authentication and cancer related mutations
Authors: Fang Rixun , Furtado Manohar R. 
Published: June 2012

Abstract
Rapid and accurate identification of SNPs and mutations are useful in ensuring that the correctly identified cell lines are being used in cancer research studies and for accurate identification of SNPs and indels in cancer cells. In this study we report on a method that uses nucleic acid isolation methods, multiplexed PCR to amplify up to 48 regions within the human genome followed by an oligonucleotide ligation assay that can detect SNPs, mutations and indels. The protocol takes about 4 hrs to complete and uses panels optimized for human cell identification and detection of mutations in EGFR and KRAS genes.
We typed the NCI-60 panel and several cell lines from the ATCC using two panels. One is a panel of SNPs that has been tested with samples from over 46 human populations to identify a set of SNPs that would discriminate globally. This SNP set has 48 markers with high heterozygosity (&gt;0.4) and low Fst values (&lt;0.06) across multiple human populations. The panel has a high power of discrimination. (Hum Genet; 127 (3) 315-324; 2010) comparable to that obtained with STR kits. A panel of 31 samples from closely related individuals within families was typed to test the power of discrimination. In another study, we applied this system to a cancer mutation study, in which an assay for detecting 29 EGFR and 5 KRAS gene mutations was developed. These mutations are frequently found in human cancers. DNA preparations from 48 ATCC and 60 NCI tumor cell lines were genotyped by this assay. These SNPs and mutations were further confirmed by the Sanger sequencing chemistry, showing 100% concordance between two technologies. The information generated from this study will be definitely useful when these cell lines are used as models for cancer study and cancer drug screening.
In conclusion, we have developed a sensitive, highly accurate, high throughput genotyping system. Studies have demonstrated its effectiveness for successful SNP/InDel detection and mutation screening. This system has been successfully used for oncogen mutation studies in cancer cell lines. Overall, the system can become a valuable tool for molecular and clinical diagnostics.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3883. doi:10.1158/1538-7445.AM2011-3883
Abstract 3884: DNA mismatch repair defects, MSI, does not play major role in colon carcinogenesis in young African Americans
Authors: Ashktorab Hassan , Lee Edward L. , Razjouan Hadi , Mohammed Roshila , Hassanzadeh Hassan , Qadwai Saad , Smoot Duane T. , Ali Waleed El-sayed Mohammed , Elnour Hassan , Sanderson Andrew , Horton Sara , Singleton Daniella , Brim Hassan 
Published: June 2012

Abstract
Back ground: African Americans (AAs) are known to display a higher rate and even earlier age of colorectal cancer (CRC) than in the general population. Genetically, 10-12% microsatellite instability (MSI) cases involved in CRC development. However, if it occurs below the age of 50 it could be suspicious of Lynch syndrome.
Aims and methods: We aimed to assess the role of MSI in pathogenesis of sporadic colorectal cancer in young African American. The CRC patients in this study were less than 50 years and referred to Howard University Hospital from 2005 to 2008. The medical records (n=50) as well as the pathology reports were reviewed. The MSI status was assessed in a multiplex PCR targeting 5 markers: BAT25, BAT26, NR21, NR27 and NR24. The PCR products were analysed in an Applied Biosystems Gene Scan 3130 machine. Samples with two or more instable markers were labeled MSI-H, with one instable marker: MSI-L and no stable marker: MSS. The presence or absence of expression of four DNA MMR proteins: MLH1, PMS2, MSH2 and MSH6 were evaluated using immunohistochemistry on a Tissue Microarray (TMA, n=26, duplicate) consisting (stage1: 3; stage 2: 6; stage3: 5; stage4: 4).TMA IHC staining were read by two different pathologists who were not aware of the patient clinical status and were scored thus in an objective manner. Two patients were MSI-L and one MSI-H. The response to 5-FU based therapy was reported.
Results: Twenty six patients [age, median: 44.5; age range: 26-49; male, 17 (65.4%)] were enrolled in the study. Seven out of 13 available data had family history of CRC. Fourteen CRC cases were left sided. MSI analysis revealed that out of 24 patients: one was MSI-H and two were MIS-L and the rest were MSS. The MSI high patient and one of the MSI low had family history of CRC. MLH1, PMS2, MSH2 and MSH6 were not expressed in 7 (26.9%), 1 (3.8%), 0 (0%) and 5 (19.2%) of tumors, respectively. IHC confirmed the MSI-H tumor. There was a recurrence with lung and brain metastases at 72 months in one of the MSI-L patients following adjuvant chemotherapy with 5 Fluorouracil and Leucovorin.
Discussion: Our results show that MSI is not a major pathway in tumorgenesis of sporadic CRCs in young AAs and we may need to study chromosomal instability or CpG Island methylator phenotype as alternative pathways which occur in African Americans and make them present the disease at younger age and advanced stage.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3884. doi:10.1158/1538-7445.AM2011-3884
Abstract 3886: The G-quadruplex induced DNA damage at telomeres is regulated by ATM
Authors: Islam Ashraful , Thoma Shelia D. , Miller Donald M. 
Published: June 2012

Abstract
Quadruplex-forming DNA sequences are present throughout the genome, including in the telomeres. Our group has shown that quadruplex-forming oligonucleotides selectively inhibit proliferation of transformed cells. One of these oligonucleotides, AS1411, is currently in a Phase IIb clinical trial. We have recently shown that the c-myc promoter quadruplex-forming sequence, Pu-27, also inhibits growth of malignant cells. We have hypothesized that one potential mechanism of this effect is competition for telomere-binding proteins. If this is correct, we would expect this effect to be mediated by the DNA repair response. Telomere integrity is maintained by the shelterin protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks. Like many other DNA damage response proteins, ATM has been shown to play a role in the maintenance of telomere integrity. We have shown that ATM deficient cells are more sensitive to growth inhibition by Pu-27 whereas ATM proficient cells were relatively resistant to Pu-27. Co-immunostaining with γ-H2AX and TRF1 revealed that the sensitivity to Pu-27 in ATM deficient cells was associated with phosphorylation of H2AX, an indicator of DNA double strand break and induction of TIFs (Telomere-dysfunction Induced Foci). There was induction of γ-H2AX and downregulation of shelterin proteins TRF2 and TRF1 in Pu-27 treated ATM deficient cells (as determined by western blotting). U937 leukemia cells, which were sensitive to Pu-27, became relatively resistant when they were stably transfected with a vector expressing a dominant negative mutant of the shelterin protein TRF2 (dn-TRF2). This suggests that Pu-27 may act by altering the telomere-shelterin complex. This idea was further substantiated by the fact that ALT (Alternative Lengthening of Telomere) cells were also relatively resistant to Pu27. A549 cells, which were sensitive to Pu-27, also showed abundant presence of TIF compared to untreated cells. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, this data suggests that ATM plays a pivotal role in Pu-27 induced DNA damage at telomere ends, which occurs in response to a Pu-27 induced alteration in telomere-shelterin homeostasis. It is likely that this response is induced in a p53 independent manner.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2011-3886
Abstract 3890: Regulation of mediator of DNA damage checkpoint 1 (MDC1) during mitosis
Authors: Yu Bing , Dalton William B. , Yang Vincent W. 
Published: June 2012

Abstract
Genetic instability is one of the hallmarks of human cancers. DNA double strand break (DSB), if improperly repaired, results in genetic instability and tumorigenesis. Previous research has established γH2AX as a bona fide marker of DSBs and the hierarchical foci assembly of DNA damage response (DDR) proteins at DSB sites is required for efficient repair. Recent findings from our group demonstrated that during either spontaneous or induced prolonged mitosis, cancer cells acquire DSBs which lead to further chromosomal abnormality [Dalton et al.(2007) Cancer Res. 67:11487]. Deciphering the mechanism of such DSB accumulation may provide new insights into the mechanism of genetic instability. We hypothesize that the increase of DSBs during prolonged mitosis is due to lack of efficient DDR. To this end, we focus on the protein Mediator of DNA damage Checkpoint 1 (MDC1), an important mediator in DSB repair. MDC1 directly binds to γH2AX and serves as a platform to accumulate/retain downstream DDR proteins at DSB sites and concomitantly initiates cell cycle arrest through its multiple interaction domains. We observed that during mitosis, many DDR proteins fail to colocalize with γH2AX in response to DSBs, while MDC1 exhibits a weakened interaction with γH2AX as shown by immunofluorescent microscopy. Inhibition of Cyclin-dependent kinase 1 (CDK1) activity, either by treatment with a specific CDK1 inhibitor RO-3306 or transient knockdown of CDK1 protein by siRNA, strengthened MDC1-γH2AX colocalization in colon cancer cells in mitosis. In vitro phosphorylation assay confirmed MDC1 as a direct substrate of CDK1. Moreover, a deletion mutant of MDC1 exhibits an increased colocalization with γH2AX compared to wild-type MDC1. Considering the published role of MDC1 as a positive regulator in metaphase-anaphase transition, we propose that phosphorylation of MDC1 by CDK1 promotes a partial disassembly of MDC1 from γH2AX foci during mitosis, in order for MDC1 to function as a modulator of mitotic progression.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2011-3890
Abstract 3891: Identification and quantification of DNA repair proteins by liquid chromatography/isotope-dilution tandem mass spectrometry using 15N-labeled whole proteins as internal standards
Authors: Dizdaroglu Miral , Jaruga Pawel , Reddy Prasad 
Published: June 2012

Abstract
Reduced DNA repair capacity is associated with increased risk for a variety of disease processes including carcinogenesis. Thus, DNA repair proteins have the potential to be used as important predictive, prognostic and therapeutic biomarkers in cancer and other diseases. The measurement of the expression level of these enzymes may be an excellent tool for this purpose. Mass spectrometry is becoming the technique of choice for the identification and quantification of proteins. We applied liquid chromatography/ isotope-dilution tandem mass spectrometry (LC-MS/MS) for the identification and quantification of DNA repair proteins human 8-oxoguanine-DNA glycosylase (hOGG1) and E. coli formamidopyrimidine DNA glycosylase (Fpg), which are involved in base-excision repair of DNA damage. We over-expressed, purified and characterized 15N-labeled analogues of these proteins to be used as internal standards. 15N-labeled whole proteins are ideal internal standards to ensure the accuracy of quantification of proteins by mass spectrometry. DNA glycosylase activities of 15N-labeled hOGG1 and 15N-labeled Fpg were determined and found to be essentially identical to those of their respective unlabeled counterparts, ascertaining that the 15N-labeling did not perturb their catalytic sites. hOGG1, Fpg and their 15N-labeled analogues were digested with trypsin and analyzed by LC-MS/MS. A large number of tryptic peptides of both proteins were identified on the basis of their full-scan mass spectra. These peptides matched the theoretical peptide fragments expected from the trypsin digestion and provided statistically significant protein scores that would unequivocally identify these proteins. We also recorded the product ion spectra (MS/MS spectra) of the tryptic peptides and defined the characteristic product ions. Mixtures of the analyte proteins and their 15N-labeled analogues as internal standards were analyzed by selected-reaction monitoring (SRM) on the basis of the previously identified product ions in the MS/MS spectra. The experimental conditions for these analyses were optimized. The methodology was successfully applied to the measurement of human OGG1 in human cells and Fpg in E.coli. The results obtained in this work suggest that the methodology developed would be highly suitable for the positive identification and accurate quantification of DNA repair proteins in vivo as potential biomarkers for cancer and other diseases.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3891. doi:10.1158/1538-7445.AM2011-3891
Abstract 3892: Discovery and analysis of COMMD1 in the DNA damage response network
Authors: Woods Nicholas T. , Nguyen Huey , Carvalho Marcelo , Li Xueli , Dapic Virna , Monteiro Alvaro 
Published: June 2012

Abstract
Spatially distinct domains impart functionality to the proteins and are often under evolutionary pressure to maintain sequence and/or structure. The BRCA1 C-terminal (BRCT) domain has an established role in cancer susceptibility and in the DNA damage response (DDR), as mutations in cases of familial breast cancer often result in a truncated protein lacking the two BRCT domains which are required for interactions with proteins such as p53, ABRAXAS, BACH1, and CtIP. BRCT domains can occur as singletons or as tandem domains (tBRCT) that function as a single cohesive structural unit involved in binding phosphorylated peptides targeted by PI3K-like proteins such as ATM in response to DNA damage. Using the Yeast Two-Hybrid system (Y2H) as well as Tandem Affinity Purification coupled to mass spectrometry (TAP-MS), we have built a domain centric protein-protein interaction network for a subset of tBRCT domains from BRCA1, TP53BP1, LIG4, PAXIP1, ECT2, BARD1, and MDC1. Evaluation of this network has revealed a number of interesting interactions and highlighted the interconnectivity potential found in these domains. Analysis of this network has revealed several interesting proteins that are highly interconnected with tBRCT domains, such as COMMD1 (an important regulator of copper homeostasis, sodium transport, NF-κB, and HIF-1), which has not yet been characterized to participate in the DDR, but was found to interact with the tBRCT domains of BARD1, BRCA1, and LIG4. Furthermore, an earlier Y2H screen conducted by our laboratory found COMMD1 as an interaction partner of CHK2. Therefore, we sought to determine the role of COMMD1 in response DNA damaging agents. Convincingly, the levels of COMMD1 are inversely related to apoptotic induction caused by cisplatin and doxorubicin. Furthermore, CHK2 signaling appears to control the ability of COMMD1 to enhance cisplatin induced apoptosis as wild type but not CHK2-/- HCT116 cells exhibited a repression in cisplatin induced apoptosis when COMMD1 was over expressed. These results indicate the importance of COMMD1 for cellular susceptibility to traditional chemotherapeutics. Currently, we are exploring the mechanisms by which COMMD1 in coordination with tBRCT domain containing proteins and CHK2 sensitizes cells to DNA damaging agents.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3892. doi:10.1158/1538-7445.AM2011-3892
Abstract 3893: Mechanism of radiosensitization by inhibitors of poly(ADP-ribose) polymerase (PARP)
Authors: Hegan Denise C. , Glazer Peter M. 
Published: June 2012

Abstract
Inhibitors of poly(ADP-ribose) polymerase (PARP) are in clinical trials for cancer therapy based on specific lethality to cancer cells deficient in BRCA1 or BRCA2. In addition, pre-clinical work has shown that PARP inhibition induces radiosensitization in cancer cells. To provide a basis for the clinical application of PARP inhibitors as radiosensitizers, we conducted mechanistic studies to elucidate the pathways by which PARP inhibitors influence radiation response.
Cells treated or not with PARP inhibitors and treated or not with siRNAs or other reagents to manipulate gene expression were assayed for radiation response by clonogenic survival assays. DNA double strand break repair was assayed using a chromosomally integrated GFP-based reporter construct. Although it was previously thought that PARP plays a role in DNA repair and radiation response primarily via the base excision repair (BER) pathway and recruitment of BER factors to sites of damage, we found that inhibitors of PARP can disrupt repair of double strand breaks (DSBs) by suppressing expression of BRCA1 and RAD51, key factors in homology-dependent repair (HDR) of DSBs. Treatment of human cancer cells with PARP inhibitors or with siRNAs targeted to PARP-1 yielded reduced levels of BRCA1 and RAD51. Radiosensitization by PARP inhibition was also seen in cells in which BER was already attenuated (via expression of a dominant negative variant of polymerase beta). Direct measurement of HDR using a GFP-based chromosomal reporter assay demonstrated reduced HDR in cells treated with PARP inhibitors. This work demonstrates that PARP inhibition can suppress the HDR pathway of DSB repair by down-regulation of BRCA1 and RAD51 expression. As such, it identifies a novel mechanism by which PARP regulates DNA repair and promotes radiosensitization, providing further rationale for therapeutic strategies combining PARP inhibition and radiation.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3893. doi:10.1158/1538-7445.AM2011-3893