bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 302: High throughput drug screening with engineered tumor spheroids
Authors: Lemmo Stephanie , Aefi Ehsan , Luker Gary , Tavana Hossein 
Published: August 2015

Abstract
Physiologically relevant, in vitro cell-based models are important tools to identify effective anticancer drugs. Three-dimensional (3D) clusters of cancer cells, called tumor spheroids, mimic key characteristics of avascular tumors such as diffusion limitations and hypoxia, and hence serve as a relevant model for compound screening. Nevertheless, current spheroid formation techniques face difficulties to generate large number of uniform sized spheroids, conveniently maintain them in culture and biochemically analyze them using commercially available tools. To address this need, we developed a robotic, high throughput technology for three-dimensional culture of cancer cells that is simple to implement, produces consistent size spheroids in standard microwell plates, and allows convenient robotic media exchange and drug addition, and analysis of cellular responses using microplate readers. The technology is based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous phase are robotically dispensed into a microwell containing the immersion aqueous phase. Cells remain within the drop at the bottom of the microwell and form a viable spheroid.
We demonstrate the robust performance of this technology by evaluating the effect of two clinically used drugs against biologically aggressive skin cancer and triple negative breast cancer (TNBC) cells. Skin cancer spheroids treated with paclitaxel and cisplatin show dose-dependent decrease in cell viability, but with a greater resistance compared to the monolayer culture (2D) of cells (paclitaxel: LD50,3D = 178.5 nM, LD50,2D = 22.1 nM and cisplatin: LD50,3D = 131.7 μM and LD50,2D = 29.2 μM. TNBC cell monolayer and spheroids treated with cisplatin displayed a similar LD50 value of ∼13.1 μM. However, unlike the monolayer culture of TNBC cells that showed a dose-response to paclitaxel with LD50,2D = 8.0 nM, TNBC spheroids showed resistance to this drug, even at micromolar-scale concentrations, and maintained a high viability.
In conclusion, our tumor spheroid technology enables quick and efficient generation of relevant tumor models in standard microplates to conveniently treat with chemical compounds and analyze. This technology allows investigating the efficacy of drug compounds against cancer cells and reveals cellular responses not captured with widely-used monolayer cultures. The ease of conducting and analyzing high throughput experiments will substantially reduce labor and cost, and expedite discovery of effective anti-cancer compounds with a physiologic tumor model.
Citation Format: Stephanie Lemmo, Ehsan Aefi, Gary Luker, Hossein Tavana. High throughput drug screening with engineered tumor spheroids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 302. doi:10.1158/1538-7445.AM2015-302
Abstract 3020: Homology-directed repair in mouse mammary tissue
Authors: Kass Elizabeth , Moynahan Mary Ellen , Jasin Maria 
Published: August 2015

Abstract
Numerous factors are linked to breast cancer risk including reproductive history, early exposure to radiation, age and genetic background. Defects in homology-directed repair (HDR)_considered the most accurate of the three major pathways for repairing double-strand breaks (DSBs) in DNA in mammalian cells_can lead to genomic instability and are associated with tumor predisposition. Mutations in multiple genes involved in HDR are linked to breast cancer susceptibility including BRCA1 and BRCA2. The mammary gland is highly influenced by its hormonal environment and undergoes significant changes during pregnancy, lactation and involution, but many questions remain about how the DNA damage response in mammary tissue, and specifically DNA DSB repair, is impacted by these developmental changes.
A widely used reporter for studying DSB repair by HDR is DR-GFP in which direct repeats of two defective GFP genes are induced to recombine by I-SceI endonuclease cleavage of one of the repeats, resulting in a functional GFP gene. We have recently generated DR-GFP containing mice that express I-SceI under the control of a tetracycline-inducible promoter. Induction of I-SceI by doxycycline administration ex vivo results in substantially increased HDR in primary cultures of mammary epithelial cells compared to transient transfection experiments.
To gain insight into the effects of breast cancer predisposing mutations on HDR in mammary epithelial cells, the I-SceI/DR-GFP mice were crossed to mice carrying hypomorphic mutations in Brca1 (Brca1tr/tr) or Brca2 (Brca2ex27/ex27). An approximately 3-fold reduction in HDR was observed in primary mammary epithelial cells from virgin Brca1tr/tr I-SceI DR-GFP mice compared to littermate controls, while an 8-fold reduction was observed in primary mammary epithelial cells from virgin Brca2ex27/ex27 mice. Primary ear fibroblasts from these animals showed similarly reduced HDR levels.
In order to understand how DSB repair processes in the mammary gland are affected by developmental changes we are assessing HDR at different stages of mammary gland development in vivo. We observe a significant increase in HDR relative to overall repair in mammary tissue from mice at mid-pregnancy compared to virgin littermates. We are now attempting to determine if there is a relationship between repair proficiency, lineage specificity of mammary epithelial cells and fate after involution in mammary tissue from normal and BRCA mutant mice.
Citation Format: Elizabeth Kass, Mary Ellen Moynahan, Maria Jasin. Homology-directed repair in mouse mammary tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3020. doi:10.1158/1538-7445.AM2015-3020
Abstract 3021: FoxP1 increases nucleotide excision repair capacity through activating xeroderma pigmentosum group C in human breast cancer
Authors: Kim Haesung , Kim Lee-Su , Lee Jae-yong , Kim Jeong-Hyeon , Lee Jeong-Min , Kim Chea-Ha 
Published: August 2015

Abstract
Breast cancer is the most common cancer in the women. Recent study revealed that nucleotide excision repair (NER) deficiency might play an important role in the cause of sporadic breast cancer, showing significant reduction of five NER gene products including xeroderma pigmentosum group C (XPC) in human breast cancer tissue. Thus, XPC inactivation might be related to occurrence and progression of breast cancer.
FoxP1 is a member of the forkhead O family of transcription factors which have a broad range of functions. FoxP1 is considered as a putative tumor suppressor in breast cancer because of its ability to cause apoptosis. The relationship between FoxP1 and XPC has been yet to be known in breast cancer.
In our data, Western blot analysis of 8 human primary tumors showed that the high expression of FoxP1 was correlated with the high level of XPC. In three established mammary epithelial cell lines (MCF-7, MDA-MB-231, 4T1), FoxP1 expression in MCF-7 was higher than in MDA-MB-231 and 4T1. Similar to the result of primary tumors, the expression of XPC was also high in MCF-7 and low in MDA-MB-231 and 4T1. Next, we found that the level of XPC decreased with the knockdown of FoxP1 compared to control in MCF-7. On the other hand, the ectopic expression of FoxP1 in MDA-MB-231 and 4T1 elevated XPC expression for protein level. In real time RT-PCR, the level of XPC mRNA was the same as in Western blot analysis. We tested whether NER activity is dependent on FoxP1. In vitro NER activity increased by overexpression of FoxP1 in MB-231. Sh-FoxP1 transfected MCF-7 cell shows reduced in vitro NER activity. In addition, immunostaining assay showed that XPC expression was much accumulated in the nucleus of MDA-MB-231 with overexpression of FoxP1 than in control.Taken together, these data suggested FoxP1 could up-regulate NER activity by activating XPC in breast cancer. However, the way how FoxP1 can regulate XPC remained to be cleared. To clarify the mechanism, we are performing biological interaction between FoxP1 and XPC in both breast cancer cell lines and human primary breast tumors.
Citation Format: Haesung Kim, Lee-Su Kim, Jae-yong Lee, Jeong-Hyeon Kim, Jeong-Min Lee, Chea-Ha Kim. FoxP1 increases nucleotide excision repair capacity through activating xeroderma pigmentosum group C in human breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3021. doi:10.1158/1538-7445.AM2015-3021
Abstract 3023: Chromatin accessibility underlies the tumor suppressor role of BAF (mSWI/SNF) complexes in many human cancers
Authors: Hodges Courtney , Hargreaves Diana C. , Miller Erik L. , Crabtree Gerald R. 
Published: August 2015

Abstract
Recent exome sequencing studies of human cancer indicate that ∼20% of malignancies have mutations in the subunits of the BAF (mSWI/SNF) chromatin remodeling complex, implicating the complex as a major tumor suppressor. However, the mechanisms of tumor suppression remain uncertain. To understand how defective BAF subunits contribute to cancer, we analyzed the genomic states of 5,659 patient samples derived from 22 tumor types, and compared samples containing genetic defects of the BAF complex against samples containing no such defects. We show that in the majority of cancer types, tumors with BAF subunit defects have significantly higher mutation counts than cancers of the same tissue lacking BAF defects. By reconstructing the mutational processes active in each patient sample, we find that excess mutations in BAF-deficient cancers represent the signatures of defective mismatch repair, cytidine deamination, UV-damage response, DNA end joining, Pol epsilon function, and other enzymatic processes acting on DNA. In four cancer types where ChIP-seq tracks were available for the healthy cell type of origin, we tracked the locations of over 280,000 mutations, and find that excess mutations in BAF-deficient tumors are biased towards regions of elevated H3K36me3 and low H3K27me3 in their corresponding non-cancerous tissues. We hypothesized that these mutations resulted from inaccessibility for repair and maintenance factors when a BAF subunit is defective. We tested this hypothesis using topoisomerase IIa binding as model for processes that require enzymatic access to DNA, and found that rapid conditional deletion of the ATPase Brg led to an increase in genome-wide deposition of H3K27me3 at Brg-dependent topoisomerase IIa sites from an initial low state. Our data indicate that the tumor-suppressive functions of BAF complexes arise largely from the requirement for BAF complexes to maintain genomic stability by providing access to DNA for repair and maintenance mechanisms.
Note: This abstract was not presented at the meeting.
Citation Format: Courtney Hodges, Diana C. Hargreaves, Erik L. Miller, Gerald R. Crabtree. Chromatin accessibility underlies the tumor suppressor role of BAF (mSWI/SNF) complexes in many human cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2015-3023
Abstract 3026: Gastric cancer associated variant of DNA polymerase Beta (Leu22Pro) induce genomic instability and cellular transformation
Authors: Kidane Dawit , Rozacky Jenna , B. Sweasy Joann 
Published: August 2015

Abstract
DNA polymerase beta (Pol β) is a key enzyme for the protection of oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of pol β, is responsible for removal of the 5′ phosphate group and the polymerase domain at subsequent steps fills the gaps. Previously, we demonstrated that gastric cancer-associated variant of pol β (Leu22Pro (L22P)) lacks dRP lyase function in-vitro. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which the normal function of dRP lyase of DNA polymerase beta is critical to maintain global replication fork rates in normal gastric epithelial cells. We report that replication fork rates in L22P expressing cells are on average half of those observed with wild-type cells. In addition, we show that L22P variant promotes replication dependent double strand breaks that may likely responsible for chromosomal aberrations. These data may implicate normal function of dRP lyase is critical to stabilize replication fork stall and prevent replication fork collapse to DNA double strand breaks.
Citation Format: Dawit Kidane, Jenna Rozacky, Joann B. Sweasy. Gastric cancer associated variant of DNA polymerase Beta (Leu22Pro) induce genomic instability and cellular transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3026. doi:10.1158/1538-7445.AM2015-3026
Abstract 3028: Tolerance and intolerance of proximal thymine glycol in DNA double-strand break repair by nonhomologous end joining
Authors: Bafail Duaa , Chalasani Sri Lakshmi , Al mohaini Mohammed , Akopiants Konstantin , Povirk Lawrence F. 
Published: August 2015

Abstract
Double-strand breaks (DSBs) induced by ionizing radiation are often accompanied by ancillary oxidative base damage that could prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, plasmid DSB substrates were constructed with two blunt ends, one having the oxidatively modified nonplanar base thymine glycol at the first, second or third positions from the 3′ terminus (Tg1, Tg2 and Tg3, respectively). End joining was examined in extracts of Bustel fibroblasts, wherein end joining is completely dependent on addition of exogenous recombinant XLF, and therefore reflects classical nonhomologous end joining. Tg at the third position had almost no effect on end-joining even when present on both ends of the break. However, the Tg1 and Tg2 substrates yielded tenfold to twentyfold less head-to-tail joins and no detectable head-to-head joins, suggesting that Tg as the terminal or penultimate base is a serious barrier to NHEJ and an absolute barrier when present at both ends. For all substrates, Tg-containing ligation products predominated at early times, and remarkably, a 3′ terminal Tg was in some cases directly ligated to an undamaged blunt end. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised in the extracts, but largely if not exclusively after DSB ligation. For all substrates, accurate blunt end ligation without end trimming predominated. However addition of excess Artemis nuclease to the extracts resulted in a slightly shorter ligation product, and a slight increase in yield, for the Tg2 substrate only. For the Tg1 substrate, there was a distinct 2-hr delay between addition of substrate and the detection of ligated products, while the unmodified and Tg3 products showed a much shorter delay of ∼30 min. Overall, the results suggest that Tg can be a major impediment to DSB repair when present as the terminal or penultimate base, being relatively resistant to both trimming and ligation.
Citation Format: Duaa Bafail, Sri Lakshmi Chalasani, Mohammed Al mohaini, Konstantin Akopiants, Lawrence F. Povirk. Tolerance and intolerance of proximal thymine glycol in DNA double-strand break repair by nonhomologous end joining. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3028. doi:10.1158/1538-7445.AM2015-3028
Abstract 3029: Role of eIF3a expression in cellular sensitivity to radiation treatment
Authors: Tumia Rima A. 
Published: August 2015

Abstract
Translation initiation involving eukaryotic translation initiation factors (eIFs) is a crucial mechanism in controlling gene expression. eIF3a has been shown previously to regulate protein synthesis and cellular response to cisplatin treatment. Its expression has also been shown to negatively associate with prognosis. In this study, we tested a hypothesis that eIF3a regulates synthesis of DNA repair proteins, which in turn regulates cellular response to ionizing radiation (IR) treatments. We found that eIF3a overexpression sensitizes cellular response to IR while its knockdown causes resistance to IR. We also found that eIF3a over-expression increases IR-induced DNA damage and decreases Non-Homologous End Joining (NHEJ) repair activity by suppressing expression level of NHEJ repair proteins such as DNAPKcs. Consistently, eIF3a knockdown decreases IR-induced DNA damage and increases Non-Homologous End Joining (NHEJ) repair activity by enhancing expression level of NHEJ repair proteins. Together, we conclude that eIF3a plays an important role in cellular response to DNA-damaging treatments by regulating synthesis of DNA repair proteins and, thus, eIIF3a likely plays an important role in the outcome of cancer patients treated with DNA-damaging strategies.
Note: This abstract was not presented at the meeting.
Citation Format: Rima A. Tumia. Role of eIF3a expression in cellular sensitivity to radiation treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3029. doi:10.1158/1538-7445.AM2015-3029
Abstract 3030: De novo engineering of chromosomal amplifications in human cells
Authors: Lauring Josh 
Published: August 2015

Abstract
Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to recurrent amplification events are lacking. Moreover, some common amplification events, such as that at 8p11-12 in breast cancer, harbor multiple candidate driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our general strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We chose regions recurrently amplified at high frequency in breast cancer, including 8p11-12, 17q12, and 11q13-14. After confirmation of successful gene targeting, the targeted cells are subjected to selective pressure directed at the amplifiable marker. Spontaneous DNA breakage events lead to varying degrees of co-amplification of linked loci near the targeting site. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using either the E. coli inosine monophosphate dehydrogenase enzyme IMPDH or a mutant dihydrofolate reductase gene as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR. Genome-wide array comparative genomic hybridization confirmed that amplification had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including “sawtooth” patterns, distal copy number loss suggesting a breakage-fusion-bridge mechanism, and large continuous regions of copy number gain. Amplification was confirmed by FISH. RT-PCR and immunoblotting demonstrated overexpression of selected amplified transcripts and proteins. In summary, we have used genome engineering techniques to create focal chromosomal amplification events de novo in human cell lines. These amplifications share many of the features of amplifications observed in primary human cancers. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a system to model oncogene cooperativity in amplifications harboring multiple candidate driver genes.
Citation Format: Josh Lauring. De novo engineering of chromosomal amplifications in human cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3030. doi:10.1158/1538-7445.AM2015-3030
Abstract 3034: A high-content screen to identify novel chromosome instability genes
Authors: Thompson Laura L. , Baergen Allison , Lichtensztejn Zelda , McManus Kirk J. 
Published: August 2015

Abstract
Chromosome instability (CIN) is defined as an increase in the rate at which whole chromosomes or large chromosomal fragments are gained or lost. It is a characteristic of virtually all cancer types that is frequently observed in highly aggressive, drug resistant tumors. Despite this, the majority of human CIN genes have yet to be elucidated, highlighting the need for studies aimed at identifying the defective genes that underlie CIN. In this study we developed image-based approaches capable of detecting CIN-associated phenotypes following RNAi-based silencing of candidate CIN genes. The first assay involves quantifying nuclear areas following silencing, where changes in mean nuclear area relative to controls act as a surrogate marker of CIN. The second approach monitors micronucleus (MN) formation where increases in the number of micronuclei are indicative of CIN. These assays were employed in a high-content screen of 164 human candidate CIN genes in two unrelated cell lines, HT1080 and hTERT. In HT1080, the nuclear area and MN enumeration assays identified 43 and 83 putative CIN genes, respectively. In hTERT, the nuclear area and MN assays identified 55 and 48 putative CIN genes, respectively. Preliminary data collected through Western blotting, mitotic spreads and flow cytometry, has provided evidence to support the validation of a subset of these putative CIN genes (e.g. SKP1), as bona fide human CIN genes. Identifying novel CIN genes will provide critical insights into CIN and tumorigenesis, as well as identify potential targets that could be exploited for the development of superior therapeutic strategies.
Citation Format: Laura L. Thompson, Allison Baergen, Zelda Lichtensztejn, Kirk J. McManus. A high-content screen to identify novel chromosome instability genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3034. doi:10.1158/1538-7445.AM2015-3034