George S Watts

Interests

Research

Diagnosis of infectious pathogens by whole genome sequencing.

Courses

Scholarly Contributions

Journals/Publications

• Bixby, B., Vrba, L., Lenka, J., Oshiro, M., Watts, G. S., Hughes, T., Erickson, H., Chopra, M., Knepler, J. L., Knox, K. S., Jarnagin, L., Alalawi, R., Kala, M., Bernert, R., Routh, J., Roe, D. J., Garland, L. L., Futscher, B. W., & Nelson, M. A. (2023).

  Cell-Free DNA Methylation Analysis as a Marker of Malignancy in Pleural Fluid

  . Scientific Reports. doi:10.21203/rs.3.rs-3390107/v1
• Futscher, B. W., Gavini, H., Golconda, U., Hammad, H., Hu, C., Menashi, E., Nelson, M. T., Oshiro, M. M., Pennington, D. J., Roe, D. J., Shroff, R. T., Vrba, L., & Watts, G. S. (2022).

  Liquid biopsy, using a novel DNA methylation signature, distinguishes pancreatic adenocarcinoma from benign pancreatic disease

  . Clinical Epigenetics. doi:10.1186/s13148-022-01246-2
• Vrba, L., Futscher, B. W., Oshiro, M., Watts, G. S., Menashi, E., Hu, C., Hammad, H., Pennington, D. R., Golconda, U., Gavini, H., Roe, D. J., Shroff, R. T., & Nelson, M. A. (2022). Liquid biopsy, using a novel DNA methylation signature, distinguishes pancreatic adenocarcinoma from benign pancreatic disease. Clinical epigenetics, 14(1), 28.
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  We tested the ability of a novel DNA methylation biomarker set to distinguish metastatic pancreatic cancer cases from benign pancreatic cyst patients and to monitor tumor dynamics using quantitative DNA methylation analysis of cell-free DNA (cfDNA) from blood samples. The biomarkers were able to distinguish malignant cases from benign disease with high sensitivity and specificity (AUC = 0.999). Furthermore, the biomarkers detected a consistent decline in tumor-derived cfDNA in samples from patients undergoing chemotherapy. The study indicates that our liquid biopsy assay could be useful for management of pancreatic cancer patients.
• Aras, S., Cannon, D. K., Harris, P. R., Hurwitz, B. L., Konhilas, J. P., Koppinger, M. P., Lopez-Pier, M. A., Skaria, R., Slepian, M. J., & Watts, G. S. (2021).

  An adaptable and non-invasive method for tracking Bifidobacterium animalis subspecies lactis 420 in the mouse gut

  . Journal of Microbiological Methods. doi:10.1016/j.mimet.2021.106302
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  Probiotic strains from the Bifidobacterium or Lactobacillus genera improve health outcomes in models of metabolic and cardiovascular disease. Yet, underlying mechanisms governing these improved health outcomes are rooted in the interaction of gut microbiota , intestinal interface, and probiotic strain. Central to defining the underlying mechanisms governing these improved health outcomes is the development of adaptable and non-invasive tools to study probiotic localization and colonization within the host gut microbiome . The objective of this study was to test labeling and tracking efficacy of Bifidobacterium animalis subspecies lactis 420 (B420) using a common clinical imaging agent, indocyanine green (ICG). ICG was an effective in situ labeling agent visualized in either intact mouse or excised gastrointestinal (GI) tract at different time intervals. Quantitative PCR was used to validate ICG visualization of B420, which also demonstrated that B420 transit time matched normal murine GI motility (~8 hours). Contrary to previous thoughts, B420 did not colonize any region of the GI tract whether following a single bolus or daily administration for up to 10 days. We conclude that ICG may provide a useful tool to visualize and track probiotic species such as B420 without implementing complex molecular and genetic tools. Proof-of-concept studies indicate that B420 did not colonize and establish residency align the murine GI tract. Workflow diagram of ICG-B420 incubation to murine administration followed by ICG fluorescence visualization and qPCR quantitation. • Bifidobacterium or Lactobacillus genera improve outcomes in diseased heart models. • Indocyanine green (bio)fluorescently labeled Bifidobacterium 420 in vitro and in vivo . • Contrary to previous thoughts, the probiotic did not colonize any region of the gut.
• Lopez-Pier, M. A., Koppinger, M. P., Harris, P. R., Cannon, D. K., Skaria, R. S., Hurwitz, B. L., Watts, G., Aras, S., Slepian, M. J., & Konhilas, J. P. (2021). An adaptable and non-invasive method for tracking Bifidobacterium animalis subspecies lactis 420 in the mouse gut. Journal of microbiological methods, 189, 106302.
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  Probiotic strains from the Bifidobacterium or Lactobacillus genera improve health outcomes in models of metabolic and cardiovascular disease. Yet, underlying mechanisms governing these improved health outcomes are rooted in the interaction of gut microbiota, intestinal interface, and probiotic strain. Central to defining the underlying mechanisms governing these improved health outcomes is the development of adaptable and non-invasive tools to study probiotic localization and colonization within the host gut microbiome. The objective of this study was to test labeling and tracking efficacy of Bifidobacterium animalis subspecies lactis 420 (B420) using a common clinical imaging agent, indocyanine green (ICG). ICG was an effective in situ labeling agent visualized in either intact mouse or excised gastrointestinal (GI) tract at different time intervals. Quantitative PCR was used to validate ICG visualization of B420, which also demonstrated that B420 transit time matched normal murine GI motility (~8 hours). Contrary to previous thoughts, B420 did not colonize any region of the GI tract whether following a single bolus or daily administration for up to 10 days. We conclude that ICG may provide a useful tool to visualize and track probiotic species such as B420 without implementing complex molecular and genetic tools. Proof-of-concept studies indicate that B420 did not colonize and establish residency align the murine GI tract.
• Hurwitz, B. L., & Watts, G. S. (2020).

  Metagenomic Next-Generation Sequencing in Clinical Microbiology

  . Clinical Microbiology Newsletter. doi:10.1016/j.clinmicnews.2020.03.004
• Watts, G. S., & Hurwitz, B. L. (2020). Metagenomic Next-Generation Sequencing in Clinical Microbiology. Clinical Microbiology Newsletter, 42(7). doi:https://doi.org/10.1016/j.clinmicnews.2020.03.004
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  Invited Editorial
• Armstrong, D. G., Cranmer, L. D., Deng, W., Hu, C. K., Hurwitz, B. L., Menashi, E., Ponsero, A. J., Reed, S., Slepian, M. J., Thornton, J., Watts, G. S., & Youens-Clark, K. (2019).

  Identification and quantitation of clinically relevant microbes in patient samples: Comparison of three k-mer based classifiers for speed, accuracy, and sensitivity

  . PLOS Computational Biology. doi:10.1371/journal.pcbi.1006863
• Cranmer, L. D., Hurwitz, B. L., Thornton, J., Watts, G. S., & Youens-Clark, K. (2019).

  Speed, accuracy, sensitivity and quality control choices for detecting clinically relevant microbes in whole blood from patients

  . bioRxiv. doi:10.1101/549477
• Watts, G. S. (2019). Speed, accuracy, sensitivity and quality control choices for detectiing clinically relevant microbes in whole blood from patients. PLoS One Computational Biology.
• Yeager, A. M., Yeager, A. M., Watts, G. S., Nawrocki, S. T., Lander, E. M., & Boiles, A. R. (2018). Paraneoplastic Leukemoid Reaction Associated with Increased Levels of and Tumor Overexpression of Receptors for G-CSF, GM-CSF, and IL-6: A Clinico-Pathological-Molecular Study. Blood, 132(Supplement 1), 4945-4945. doi:10.1182/blood-2018-99-115736
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  Abstract Introduction: Paraneoplastic leukemoid reaction (PLR) comprises 10% of leukemoid reactions among patients with solid tumors, especially those with pulmonary malignancy and metastatic disease. Defined as a white blood cell (WBC) count of >50 x 109/L with mature, non-clonally derived neutrophils, and without tumor involvement in the bone marrow, PLR is associated with a poor prognosis. Systemic inflammation promotes tumor growth and metastasis; however, the mechanisms underlying PLR are not well elucidated. We performed a comprehensive clinico-pathological-molecular analysis of cytokines and gene expression in bone marrow, metastatic tumor, and serum from a 76-year-old man with metastatic poorly differentiated non-small cell lung cancer (NSCLC), hyperleukocytosis (WBC 146.5 x 109/L) and extreme neutrophilia (96% neutrophils; absolute neutrophil count 140.6 x 109/L). Methods: After informed consent, we conducted an extensive clinical evaluation of the patient's neutrophilic hyperleukocytosis. We examined the patient's peripheral blood, serum and bone marrow via histologic examination, flow cytometry, cytogenetics, and fluorescent in situ hybridization (FISH) following established protocols. We evaluated levels of 12 cytokines (G-CSF, GM-CSF, IFN-gamma, IL-1a, -2, -4, -6, -8, -10, -12, and -17a, and TNF-a) in serum by enzyme-linked immunosorbent assay (ELISA; Quantikine, R&D Systems). We compared gene expression of 30 cytokines and their receptors (CSF2/R, CSF3/R, IFN-gamma/R1, IL-1A/B/R1/RN, IL-2/RA/RB/RG, IL-4/R, IL-6/R, CXCL8/CXCR1, IL-10/RA, IL-12A/RB2, IL17/RA, and TNF-a/RSF1A/1B) on paraffin-fixed samples of the patient's NSCLC and on an age- and gender-matched sample of NSCLC from a patient without PLR (Geneticist Inc. Biorepository), using real-time polymerase chain reaction (PCR; RT2 Profiler, QIAGEN). Results: We confirmed the diagnosis of PLR after an extensive evaluation did not show any infectious or clonal myeloproliferative process. The peripheral blood smear showed marked leukocytosis, composed mainly of mature neutrophils and mild absolute monocytosis without circulating blasts or atypical cells; there was also a normochromic, normocytic anemia (hemoglobin 11.9 g/dL, hematocrit 35.7%, and MCV 94 fl) and mild thrombocytopenia (platelets 148 x 109/L). Flow cytometric analysis of peripheral blood showed granulocyte predominance (98.8% of the events) and no blast population. Bone marrow showed 40-50% cellularity, trilineage hematopoiesis, and myeloid: erythroid ratio 14.3, without dysplasia, increased blasts and metastatic cancer. FISH analysis with extended acute leukemia panel probe showed no malignancy. Cytogenetics showed 46, XY, and PCR studies were negative for mutations of JAK2 V617F and CSF3R. Serum levels of IFN-G, IL-2, IL-4, IL-10, IL-12, and IL-17a were modestly elevated relative to normal values (3.1- to 6.3-fold increase), while level of IL-1a was decreased (0.7 normal level). In contrast, the serum levels of GM-CSF (40.06 pg/mL), G-CSF (1880.63 pg/mL), and IL-6 (361.91 pg/mL) were all markedly elevated above normal by 48.2-fold, 40.1-fold, and 72.4-fold, respectively. When compared with control non-PLR NSCLC tissue, the patient's tumor showed 3-fold overexpression of the G-CSF receptor, 13.3-fold overexpression of the GM-CSF receptor, and 1.7-fold overexpression of the IL-6 receptor. However, neither PLR nor control NSCLC samples showed increased expression of genes for those cytokines (Table 1). Conclusion: In this comprehensive mechanistic analysis of PLR, we have shown that the metastatic NSCLC tumor overexpressed genes for receptors for G-CSF, GM-CSF, and IL-6, but did not overexpress the genes for those cytokines. Significantly elevated serum levels of G-CSF, GM-CSF and IL-6, synthesized from non-tumor tissues, caused hyperleukocytosis. We hypothesize that an autocrine positive feedback loop, in which these cytokines led to autostimulation of their respective aberrantly expressed receptors on tumor cells, resulted in tumor proliferation as well as off-target stimulation of granulocytopoiesis and corresponding PLR. Disclosures No relevant conflicts of interest to declare.
• Cranmer, L. D., Dhingra, D., Hurwitz, B. L., Metzger, G. S., Oshiro, M. M., Slepian, M. J., Watts, G. S., Wolk, D. M., & Youens-Clark, K. (2017).

  16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria

  . Journal of Applied Microbiology, 123(6), 1584-1596. doi:10.1111/jam.13590
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  Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer.Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture.Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare.We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.
• Watts, G. S., Youens-Clark, K., Slepian, M. J., Wolk, D. M., Oshiro, M. M., Metzger, G. S., Dhingra, D., Cranmer, L. D., & Hurwitz, B. L. (2017). 16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria. Journal of applied microbiology, 123(6), 1584-1596.
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  Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer.
• Watts, G. S. (2016).

  Whole genome sequencing for identification of pathogens from whole blood: Febrile neutropenia and beyond

  . Journal of Infectious Diseases and Therapy. doi:10.4172/2332-0877.c1.017
• Harshman, D. K., McLain, J. E., Rao, B. M., Watts, G. S., & Yoon, J. Y. (2015).

  Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

  . Science Advances, 1(8). doi:10.1126/sciadv.1400061
• Harshman, D. K., Rao, B. M., McLain, J. E., Watts, G. S., & Yoon, J. Y. (2015). Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief. Science advances, 1(8), e1400061.
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  Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.
• Jandova, J., Mason, C. J., Pawar, S. C., & Watts, G. S. (2015).

  Fn14 receptor promotes invasive potential and metastatic capacity of non-small lung adenocarcinoma cells through the up-regulation of integrin α6

  . Neoplasma. doi:10.4149/neo_2015_006
• Jandova, J., Mason, C. J., Pawar, S. C., & Watts, G. S. (2015). Fn14 receptor promotes invasive potential and metastatic capacity of non-small lung adenocarcinoma cells through the up-regulation of integrin α6. Neoplasma, 62(1), 41-52.
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  Lung cancer is one of the leading cause of cancer-related death around the world with the majority of diagnoses being non-small cell lung cancer (NSCLC). Given the poor survival rate and efficacy of current therapy for NSCLC, there is a need to identify and develop new therapeutic targets for treatment. We have observed significantly up-regulated levels of Fn14 in clinical samples of lung cancer relative to normal adjacent tissue. However, the functional role of Fn14 in these tumors is not understood yet. We used RT-PCR to establish the Fn14 expression profile in various NSCLC cell lines. Using isogenic variants of H460 NSCLC cell line with low, intermediate and high Fn14 expression as a cellular model, we determined that increased levels of integrin α6 in cells over-expressing Fn14 is suggestive of an important role of α6β1-fn14 interactions in motility of lung carcinoma and formation of metastases. Enhanced levels of Fn14 correlated with higher tumor cell migration and invasion in an MMP-1 dependent manner. Cells over-expressing Fn14 showed increased in vivo tumor formation with metastatic capacity to lymph nodes, lungs and liver. Thus, this research may be a step toward developing improved treatment strategies for NSCLC by improved detection and inhibition of metastases.
• Lebeau, L., Nagle, R. B., Cranmer, L. D., Zhu, W., Watts, G. S., Sheth, G., Ring, A., Novak, P., Nokes, B., Nagle, R. B., Mineyev, N., Lebeau, L., Lang, J. E., Futscher, B. W., Fuchs, L., & Cranmer, L. D. (2015). Characterization of a novel radiation-induced sarcoma cell line.. Journal of surgical oncology, 111(6), 669-82. doi:10.1002/jso.23860
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  Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models exist to facilitate studies of RIS..We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a RIS..Short tandem repeat (STR) profiling of UACC-SARC1 was virtually identical to its parental tumor. Immunohistochemistry (IHC) analysis of the tumor and immunocytochemistry (ICC) analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. Comparative genomic hybridization (CGH) provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. NOD/SCID gamma mouse xenografts demonstrated tumor formation and metastasis. Clonogenicity assays demonstrated that 90% of single cells produced viable colonies. NOD/SCID gamma mice produced useful patient-derived xenografts for orthotopic or metastatic models..Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. UACC-SARC1 is an aneuploid cell line with complex genomics lacking common oncogenes or tumor suppressor genes as drivers of its biology. The UACC-SARC1 cell line will enable further studies of the drivers of RIS.
• Armstrong, D. G., Cucher, D., Giovinco, N. A., Hurwitz, B. L., Rankin, T. M., & Watts, G. S. (2014).

  Three-dimensional printing surgical instruments: are we there yet?

  . Journal of Surgical Research. doi:10.1016/j.jss.2014.02.020
• Brothman, A. R., Chin, K., Fuchs, L., Futscher, B. W., Garbe, J. C., Jackson, M. W., LaBarge, M. A., Novák, P., Sputova, K., Stampfer, M. R., Vrba, L., & Watts, G. S. (2014).

  Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations

  . Cell Cycle. doi:10.4161/15384101.2014.954456
• Burgess, J. L., Harber, P., Lu, Z., Lutz, E., Meza-Montenegro, M. M., Watts, G. S., & White, A. E. (2014).

  Environmental Arsenic Exposure and Microbiota in Induced Sputum

  . International Journal of Environmental Research and Public Health. doi:10.3390/ijerph110202299
• Garbe, J. C., Vrba, L., Sputova, K., Fuchs, L., Novak, P., Brothman, A. R., Jackson, M., Chin, K., LaBarge, M. A., Watts, G., Futscher, B. W., & Stampfer, M. R. (2014). Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations. Cell cycle (Georgetown, Tex.), 13(21), 3423-35.
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  Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16(INK4); replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agents are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional "passenger" errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of "passenger" genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.
• Lebeau, L., Nagle, R. B., Cranmer, L. D., Zhu, W., Watts, G. S., Sheth, G., Novak, P., Nokes, B., Nagle, R. B., Mineyev, N., Lebeau, L., Lang, J. E., Futscher, B. W., Fuchs, L., & Cranmer, L. D. (2014). Abstract 1200: Characterization of a novel radiation-induced sarcoma cell line. Cancer Research, 74, 1200-1200. doi:10.1158/1538-7445.am2014-1200
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  Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models currently exist to facilitate studies of RIS. Methods: We derived a primary human cell line, UACC-SARC1, from a RIS. Short tandem repeat (STR) genotyping was used to confirm that this cell line was propagated from the tumor. Further characterization of this cell line involved comparing 24 markers using immunocytochemistry (ICC) to immunohistochemistry (IHC) of the tumor, a Matrigel invasion assay, karyotyping of the cell line, comparative genomic hybridization (CGH), DNA sequencing using the Ion AmpliSeq Cancer panel and in vivo mouse xenografts after subcutaneous injection of UACC-SARC1 in immunodeficient mice. Results: STR profiling of UACC-SARC1 was virtually identical to its parental tumor. IHC analysis of the tumor and ICC analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. CGH provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. SCID mice xenografts exhibited tumor formation but resulting tumors failed to metastasize. Further xenografts with NOD SCID gamma (NSG) mice are planned. The doubling time of UACC-SARC1 was 28.3 hours. Conclusion: Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. Citation Format: Julie E. Lang, Brandon Nokes, Grishma Sheth, Petr Novak, Laura Fuchs, George S. Watts, Bernard W. Futscher, Neal Mineyev, Weizhu Zhu, Lauren LeBeau, Ray Nagle, Lee Cranmer. Characterization of a novel radiation-induced sarcoma cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1200. doi:10.1158/1538-7445.AM2014-1200
• Rankin, T. M., Giovinco, N. A., Cucher, D. J., Watts, G., Hurwitz, B., & Armstrong, D. G. (2014). Three-dimensional printing surgical instruments: are we there yet?. The Journal of surgical research, 189(2), 193-7.
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  The applications for rapid prototyping have expanded dramatically over the last 20 y. In recent years, additive manufacturing has been intensely investigated for surgical implants, tissue scaffolds, and organs. There is, however, scant literature to date that has investigated the viability of three-dimensional (3D) printing of surgical instruments.
• White, A. G., Watts, G. S., Lu, Z., Meza-Montenegro, M. M., Lutz, E. A., Harber, P., & Burgess, J. L. (2014). Environmental arsenic exposure and microbiota in induced sputum. International journal of environmental research and public health, 11(2), 2299-313.
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  Arsenic exposure from drinking water is associated with adverse respiratory outcomes, but it is unknown whether arsenic affects pulmonary microbiota. This exploratory study assessed the effect of exposure to arsenic in drinking water on bacterial diversity in the respiratory tract of non-smokers. Induced sputum was collected from 10 subjects with moderate mean household water arsenic concentration (21.1 ± 6.4 ppb) and 10 subjects with low household water arsenic (2.4 ± 0.8 ppb). To assess microbiota in sputum, the V6 hypervariable region amplicons of bacterial 16s rRNA genes were sequenced using the Ion Torrent Personal Genome Machine. Microbial community differences between arsenic exposure groups were evaluated using QIIME and Metastats. A total of 3,920,441 sequence reads, ranging from 37,935 to 508,787 per sample for 316 chips after QIIME quality filtering, were taxonomically classified into 142 individual genera and five phyla. Firmicutes (22%), Proteobacteria (17%) and Bacteriodetes (12%) were the main phyla in all samples, with Neisseriaceae (15%), Prevotellaceae (12%) and Veillonellacea (7%) being most common at the genus level. Some genera, including Gemella, Lactobacillales, Streptococcus, Neisseria and Pasteurellaceae were elevated in the moderate arsenic exposure group, while Rothia, Prevotella, Prevotellaceae Fusobacterium and Neisseriaceae were decreased, although none of these differences was statistically significant. Future studies with more participants and a greater range of arsenic exposure are needed to further elucidate the effects of drinking water arsenic consumption on respiratory microbiota.
• Brown, K. E., Case, A. J., Coleman, M. C., Cyr, A. R., Domann, F. E., Futscher, B. W., McCormick, M. L., Spitz, D. R., & Watts, G. S. (2013).

  Maintenance of mitochondrial genomic integrity in the absence of manganese superoxide dismutase in mouse liver hepatocytes

  . Redox biology. doi:10.1016/j.redox.2013.01.001
• Cyr, A. R., Brown, K. E., McCormick, M. L., Coleman, M. C., Case, A. J., Watts, G. S., Futscher, B. W., Spitz, D. R., & Domann, F. E. (2013). Maintenance of mitochondrial genomic integrity in the absence of manganese superoxide dismutase in mouse liver hepatocytes. Redox biology, 1, 172-7.
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  Manganese superoxide dismutase, encoded by the Sod2 gene, is a ubiquitously expressed mitochondrial antioxidant enzyme that is essential for mammalian life. Mice born with constitutive genetic knockout of Sod2 do not survive the neonatal stage, which renders the longitudinal study of the biochemical and metabolic effects of Sod2 loss difficult. However, multiple studies have demonstrated that tissue-specific knockout of Sod2 in murine liver yields no observable gross pathology or injury to the mouse. We hypothesized that Sod2 loss may have sub-pathologic effects on liver biology, including the acquisition of reactive oxygen species-mediated mitochondrial DNA mutations. To evaluate this, we established and verified a hepatocyte-specific knockout of Sod2 in C57/B6 mice using Cre-LoxP recombination technology. We utilized deep sequencing to identify possible mutations in Sod2 (-/-) mitochondrial DNA as compared to wt, and both RT-PCR and traditional biochemical assays to evaluate baseline differences in redox-sensitive pathways in Sod2 (-/-) hepatocytes. Surprisingly, no mutations in Sod2 (-/-) mitochondrial DNA were detected despite measurable increases in dihydroethidium staining in situ and concomitant decreases in complex II activity indicative of elevated superoxide in the Sod2 (-/-) hepatocytes. In contrast, numerous compensatory alterations in gene expression were identified that suggest hepatocytes have a remarkable capacity to adapt and overcome the loss of Sod2 through transcriptional means. Taken together, these results suggest that murine hepatocytes have a large reserve capacity to cope with the presence of additional mitochondrial reactive oxygen species.
• Beyer, T. E., Jandova, J., Meuillet, E. J., & Watts, G. S. (2012).

  The matrix protein CCN1/CYR61 is required for α_Vβ₅-mediated cancer cell migration

  . Cell Biochemistry and Function, 30(8), 687-695. doi:10.1002/cbf.2853
• Chen, X., Chen, H. D., Dvorak, K., Hill, K. A., Kruszynski, R., Narendran, N., Roesly, H. B., & Watts, G. S. (2012).

  The decreased expression of Beclin-1 correlates with progression to esophageal adenocarcinoma: the role of deoxycholic acid

  . American Journal of Physiology-gastrointestinal and Liver Physiology, 302(8), G864-G872. doi:10.1152/ajpgi.00340.2011
• Cranmer, L. D., Watts, G. S., Oshiro, M. M., & Cranmer, L. D. (2012). Abstract 1641: Concordance between mutations identified by bar-coded multiplexed sequencing of BRAF, NRAS, and KIT in formalin fixed (FFPE) tissue from melanoma patients using the Ion Torrent Personal Genome Machine (PGM) and a real-time PCR assay. Cancer Research, 72, 1641-1641. doi:10.1158/1538-7445.am2012-1641
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  Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The standard chemotherapy for metastatic melanoma is dacarbazine, however, progression free survival with dacarbazine is a dismal two months. Efforts to improve the treatment of melanoma have focused on specific molecular defects following the discovery that the majority of patients have a mutation in one of three members of the BRAF-MAPK signaling pathway: KIT, BRAF, and RAS. Recent clinical trials have shown promise for drugs that target the molecular defects in melanoma, including the recently approved mutant BRAF inhibitor vemurafenib. As new drugs that target specific mutations move through clinical trials, the opportunity to perform precision medicine is becoming a reality. To deliver on the promise shown by the new generation of drugs it will be necessary to detect specific mutations in patients so they can be matched with the right drug. To this end, we have used an Ion Torrent Personal Genome Machine to sequence the six most commonly mutated codons in melanoma (BRAF 600, NRAS 12 and 13, NRAS 61, KIT 576, and KIT 642) from formalin fixed paraffin embedded tissue of late stage melanoma patients. All six codons of interest were sequenced in forty-six patients and mutations identified by comparison to normal controls. The A375 cell line, containing the known mutation V600E in BRAF was used as a positive control. Samples were sequenced in a multiplexed format using bar coding of the amplicons which allowed 14 patients and two controls to be sequenced simultaneously on each chip. Results were confirmed by real time RT-PCR assay specific for the BRAF V600E mutation. We demonstrate the feasibility of using rapid low cost next-generation sequencing to provide robust detection of mutations in fixed patient samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1641. doi:1538-7445.AM2012-1641
• Jandova, J., Beyer, T. E., Meuillet, E. J., & Watts, G. S. (2012). The matrix protein CCN1/CYR61 is required for α(V)β5-mediated cancer cell migration. Cell biochemistry and function, 30(8), 687-95.
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  CYR61 is one of the six proteins of the CCN family of proteins known to play diverse roles in angiogenesis, cellular proliferation, survival, migration and wound healing. However, the specific function of CYR61 in cancer is unclear, and the literature remains controversial. We used quantitative real-time PCR to establish the expression profile of CYR61 and integrin α(V)β5 in three non-small cell lung cancer, five colorectal cancer, one breast cancer and one oesophageal squamous carcinoma cell lines. We showed that the levels of CYR61 were significantly increased in oesophageal squamous carcinoma cell line along with the enhanced levels of α(V)β5 integrin. Further, we investigated whether tumour cell-secreted CYR61 can facilitate cell migration by interacting with the α(V)β5 integrin. Using tumour cell lines with low, intermediate and high CYR61 expression and their isogenic variants as a cellular model, we determined that integrin α(V)β5 expressed on these tumour cells is required for cell migration. Moreover, we showed that the modulation of expression levels of CYR61 in these cancer cells affected their capacity for migration. These results represent an advance to the understanding of the role of CYR61 and α(V)β5 integrin as proteins that cooperate to mediate cancer cell migration.
• Roesly, H. B., Khan, M. R., Chen, H. D., Hill, K. A., Narendran, N., Watts, G. S., Chen, X., & Dvorak, K. (2012). The decreased expression of Beclin-1 correlates with progression to esophageal adenocarcinoma: the role of deoxycholic acid. American journal of physiology. Gastrointestinal and liver physiology, 302(8), G864-72.
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  Beclin-1 has a central role in the regulation of autophagy. Barrett's esophagus (BE) is associated with a significantly increased risk for the development of esophageal adenocarcinoma (EAC). In the current study, we evaluated the role of Beclin-1 and autophagy in the EAC. Biopsies obtained from patients with BE and EAC, tissues from a rat model of BE and EAC, and esophageal cell lines were evaluated for the expression of Beclin-1 by immunohistochemistry, immunoblotting, or RT-PCR. Since reflux of bile acids is important in EAC, we also evaluated the effect of exposure to deoxycholic acid (DCA) on autophagy and Beclin-1 expression. Beclin-1 expression was high in squamous epithelium and nondysplastic BE, whereas its expression was low in dysplastic BE and EAC. The same pattern of expression was observed in rat tissues and in esophageal cell lines. Normal esophageal epithelium and HET-1A cells (derived from normal squamous epithelium) show high levels of Beclin-1, but lower levels of Beclin-1 were found in BE and EAC cell lines (CP-A, CP-C, and OE33). Acute exposure to DCA led to increased Beclin-1 expression and increased autophagy as evaluated by electron microscopy and counting percentage of GFP-LC3-positive BE cells with punctate pattern. In contrast, chronic exposure to DCA did not result in the alteration of Beclin-1 levels or autophagy. In summary, these data suggest that autophagy is initially activated in response to bile acids, but chronic exposure to bile acids leads to decreased Beclin-1 expression and autophagy resistance.
• Delamere, N. A., Dvorak, K., Goldman, D. S., Goldman, A., Khailova, L., Shahidullah, M., & Watts, G. S. (2010).

  A novel mechanism of acid and bile acid-induced DNA damage involving Na⁺/H⁺exchanger: implication for Barrett's oesophagus

  . Gut. doi:10.1136/gut.2010.213686
• Dvorak, K., Watts, G. S., Shahidullah, M., Goldman, A., Dvorak, K., Delamere, N. A., & Chen, H. (2010). S1626 A Novel Mechanism of Bile Acid Induced Damage: Relevance to Esophageal Carcinogenesis. Gastroenterology, 138(5), S-241. doi:10.1016/s0016-5085(10)61096-7
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  on autophagy in BE cells. Methods: Barrett's esophagus CP-A cells were treated for 10 minutes with medium acidified to pH 4 and/or 0.2mM hydrophobic bile acid cocktail following 170 minutes incubation in normal medium. As a positive control for autophagy CP-A cells were also exposed to rapamycin or amino acid starvation. Autophagy was assessed by evaluating the LC3II/LC3I ratio, by quantification of cells showing the punctuate pattern of LC3-GFP and by transmission electronmicroscopy (TEM). In addition, we also determined the localization and expression of proautophagy protein Beclin1 and Lamp1, a specific marker of autophagic vacuole maturation, in the cells exposed to different treatments by confocal microsopy and western blot analysis. Results: Our results indicate that acidified medium in combination with bile acids induces autophagy in BE cells as demonstrated by the increased conversion of LC3-I (the cytosolic form) into LC3-II (membrane bound), the increased expression of Beclin 1 and the formation of typical autophagosomes by TEM. Furthermore, 53.5±4.0% of cells transfected with GFP-LC3 showed a punctuated pattern after exposure to acidified medium and bile acids, while only 17.4±2.5% of untreated cells showed this pattern. Supporting this, we observed an apparent expansion of the lysosomal compartment in cells treated with acidified medium and bile acids compared with untreated cells as visualized with the lysosomal-specific marker, Lamp1. No significant increase in these markers was detected in the cells exposed to 0.2mM bile acid cocktail or to medium acidified to pH 4 alone. Rapamycin and amino acid starvation induced similar changes in these autophagic markers as acid in combination with bile acids. Conclusion: The autophagic pathway is activated in response to stressful condition. Here we report that acute exposure of BE cells to hydrophobic bile acids in combination with acid induce autophagy, which may play a key role in the survival of BE cells exposed to refluxate and progression to cancer.
• Goldman, A., Shahidullah, M., Goldman, D., Khailova, L., Watts, G., Delamere, N., & Dvorak, K. (2010). A novel mechanism of acid and bile acid-induced DNA damage involving Na+/H+ exchanger: implication for Barrett's oesophagus. Gut, 59(12), 1606-16.
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  Barrett's oesophagus is a premalignant disease associated with oesophageal adenocarcinoma. The major goal of this study was to determine the mechanism responsible for bile acid-induced alteration in intracellular pH (pH(i)) and its effect on DNA damage in cells derived from normal oesophagus (HET1A) or Barrett's oesophagus (CP-A).
• Bernstein, H., Bernstein, C., Dvorak, K., Garewal, H. S., Holubec, H., Jenkins, G. J., Payne, C. M., Prasad, A., Ramsey, L., Sampliner, R. E., & Watts, G. S. (2009).

  Expression of Bile Acid Transporting Proteins in Barrett's Esophagus and Esophageal Adenocarcinoma

  . The American Journal of Gastroenterology. doi:10.1038/ajg.2008.85
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  Barrett's esophagus (BE) is a metaplastic lesion characterized by replacement of the normal squamous epithelium by columnar intestinal epithelium containing goblet cells. It is speculated that this process is an adaptation to protect cells from components of refluxate, such as gastric acid and bile acids. In contrast to the normal squamous epithelium, enterocytes of the distal ileum are adapted to transport bile acids from the intestinal lumen. Several bile acid transporters are utilized for effective removal of bile acids, including the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP), and the multidrug-resistant protein 3 (MRP3). We hypothesized that one of the possible functions of newly arising metaplastic epithelium, in the esophagus, is to transport bile acids. Our major goal was to evaluate the expression of bile acid transporters in normal squamous epithelium, BE with different grades of dysplasia, and esophageal adenocarcinoma (EAC).A total of 101 patients were included in this study. Immunohistochemistry (IHC) and reverse transcriptase (RT)-PCR were used to detect the expression of these transporters at the mRNA and protein levels.Our immunohistochemical studies showed that all three bile acid transporters are expressed in BE glands, but not in squamous epithelium. ASBT was found in the apical border in BE biopsies. The highest frequency of ASBT expression was in patients with nondysplastic BE (9 of 15, 60%), and a progressive loss of ASBT was observed through the stages of dysplasia. ASBT was not detected in EAC (0 of 15). IBABP staining was observed in the cytoplasm of BE epithelial surface cells. Expression of IBABP was found in 100% of nondysplastic BE (14 of 14), in 93% of low-grade dysplasia (LGD, 15 of 16), in 73% of high-grade dysplasia (HGD, 10 of 14), and in 33% of EAC (5 of 15). MRP3 was expressed in the basolateral membrane in 93% of nondysplastic BE (13 of 14), in 60% of LGD (10 of 16), and in 86% of HGD (11 of 13). Only weak MRP3 staining was detected in EAC biopsies (5 of 15, 33%). In addition, RT-PCR studies showed increased expression of mRNA coding for ASBT (6.1x), IBABP (9.1x), and MRP3 (2.4x) in BE (N=13) compared with normal squamous epithelium (N=15). Significantly increased mRNA levels of IBABP (10.1x) and MRP3 (2.5x) were also detected in EAC (N=21) compared with normal squamous epithelium.We found that bile acid transporters expression is increased in BE tissue at the mRNA and protein levels and that expression of bile acid transporter proteins decreased with progression to cancer.
• Dvorak, K., Watts, G. S., Ramsey, L., Holubec, H., Payne, C. M., Bernstein, C., Jenkins, G. J., Sampliner, R. E., Prasad, A., Garewal, H. S., & Bernstein, H. (2009). Expression of bile acid transporting proteins in Barrett's esophagus and esophageal adenocarcinoma. The American journal of gastroenterology, 104(2), 302-9.
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  Barrett's esophagus (BE) is a metaplastic lesion characterized by replacement of the normal squamous epithelium by columnar intestinal epithelium containing goblet cells. It is speculated that this process is an adaptation to protect cells from components of refluxate, such as gastric acid and bile acids. In contrast to the normal squamous epithelium, enterocytes of the distal ileum are adapted to transport bile acids from the intestinal lumen. Several bile acid transporters are utilized for effective removal of bile acids, including the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP), and the multidrug-resistant protein 3 (MRP3). We hypothesized that one of the possible functions of newly arising metaplastic epithelium, in the esophagus, is to transport bile acids. Our major goal was to evaluate the expression of bile acid transporters in normal squamous epithelium, BE with different grades of dysplasia, and esophageal adenocarcinoma (EAC).
• DeGeest, K., Domann, F. E., Futscher, B. W., Holtan, N., Rose, S. E., & Watts, G. S. (2008).

  DNA methylation changes in ovarian cancer are cumulative with disease progression and identify tumor stage

  . BMC Medical Genomics. doi:10.1186/1755-8794-1-47
• Dvorak, K., Watts, G. S., Sampliner, R. E., Ramsey, L., Payne, C. M., Holubec, H., Garewal, H. S., Dvorak, K., Bernstein, H., & Bernstein, C. (2008). M1957 Expression of Bile Acid Transporting Proteins in Barrett's Esophagus and Esophageal Adenocarcinoma. Gastroenterology, 134(4), A-439. doi:10.1016/s0016-5085(08)62050-8
• Futscher, B. W., Jensen, T. J., Kim, C., Novák, P., Oshiro, M. M., & Watts, G. S. (2008).

  Agglomerative Epigenetic Aberrations Are a Common Event in Human Breast Cancer

  . Cancer Research, 68(20), 8616-8625. doi:10.1158/0008-5472.can-08-1419
• Futscher, B. W., Junk, D. J., Martinez, J. D., Oshiro, M. M., Vrba, L., & Watts, G. S. (2008).

  Different Mutant/Wild-Type p53 Combinations Cause a Spectrum of Increased Invasive Potential in Nonmalignant Immortalized Human Mammary Epithelial Cells

  . Neoplasia. doi:10.1593/neo.08120
• Junk, D. J., Vrba, L., Watts, G. S., Oshiro, M. M., Martinez, J. D., & Futscher, B. W. (2008). Different mutant/wild-type p53 combinations cause a spectrum of increased invasive potential in nonmalignant immortalized human mammary epithelial cells. Neoplasia (New York, N.Y.), 10(5), 450-61.
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  Aberrations of p53 occur in most, if not all, human cancers. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Immortalized human mammary epithelial cells resemble the earliest forms of aberrant breast tissue growth but do not express many malignancy-associated phenotypes. We created a model of human mammary epithelial tumorigenesis by infecting hTERT-HME1 immortalized human mammary epithelial cells expressing wild-type p53 with four different mutant p53 constructs to determine the role of p53 mutation on the evolution of tumor phenotypes. We demonstrate that different mutant/wild-type p53 heterozygous models generate loss of function, dominant negative activity, and a spectrum of gain of function activities that induce varying degrees of invasive potential. We suggest that this model can be used to elucidate changes that occur in early stages of human mammary epithelial tumorigenesis. These changes may constitute novel biomarkers or reveal novel treatment modalities that could inhibit progression from primary to metastatic breast disease.
• Novak, P., Jensen, T., Oshiro, M. M., Watts, G. S., Kim, C. J., & Futscher, B. W. (2008). Agglomerative epigenetic aberrations are a common event in human breast cancer. Cancer research, 68(20), 8616-25.
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  Changes in DNA methylation patterns are a common characteristic of cancer cells. Recent studies suggest that DNA methylation affects not only discrete genes, but it can also affect large chromosomal regions, potentially leading to LRES. It is unclear whether such long-range epigenetic events are relatively rare or frequent occurrences in cancer. Here, we use a high-resolution promoter tiling array approach to analyze DNA methylation in breast cancer specimens and normal breast tissue to address this question. We identified 3,506 cancer-specific differentially methylated regions (DMR) in human breast cancer with 2,033 being hypermethylation events and 1,473 hypomethylation events. Most of these DMRs are recurrent in breast cancer; 90% of the identified DMRs occurred in at least 33% of the samples. Interestingly, we found a nonrandom spatial distribution of aberrantly methylated regions across the genome that showed a tendency to concentrate in relatively small genomic regions. Such agglomerates of hypermethylated and hypomethylated DMRs spanned up to several hundred kilobases and were frequently found at gene family clusters. The hypermethylation events usually occurred in the proximity of the transcription start site in CpG island promoters, whereas hypomethylation events were frequently found in regions of segmental duplication. One example of a newly discovered agglomerate of hypermethylated DMRs associated with gene silencing in breast cancer that we examined in greater detail involved the protocadherin gene family clusters on chromosome 5 (PCDHA, PCDHB, and PCDHG). Taken together, our results suggest that agglomerative epigenetic aberrations are frequent events in human breast cancer.
• Watts, G. S., Futscher, B. W., Holtan, N., Degeest, K., Domann, F. E., & Rose, S. L. (2008). DNA methylation changes in ovarian cancer are cumulative with disease progression and identify tumor stage. BMC medical genomics, 1, 47.
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  Hypermethylation of promoter CpG islands with associated loss of gene expression, and hypomethylation of CpG-rich repetitive elements that may destabilize the genome are common events in most, if not all, epithelial cancers.
• Berens, M. E., Bhattacharyya, A. K., Montgomery, E. A., Nelson, M. A., Sampliner, R. E., Tran, N. L., & Watts, G. S. (2007).

  Identification of Fn14/TWEAK receptor as a potential therapeutic target in esophageal adenocarcinoma

  . International Journal of Cancer, 121(10), 2132-2139. doi:10.1002/ijc.22898
• Goulet, A. C., Lord, J. L., Nelson, M. A., & Watts, G. S. (2007).

  Profiling of selenomethionine responsive genes in colon cancer by microarray analysis

  . Cancer Biology & Therapy. doi:10.4161/cbt.6.4.3813
• Goulet, A., Watts, G., Lord, J. L., & Nelson, M. A. (2007). Profiling of selenomethionine responsive genes in colon cancer by microarray analysis. Cancer biology & therapy, 6(4), 494-503.
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  High-selenium containing yeast is being evaluated in clinical trials against colon polyp recurrence. However, the molecular targets for the anticancer effects of selenium remain unclear. Previous studies by our group demonstrated that selenomethionine-induced growth arrest appears to be mediated by activation of ERK and subsequent phosphorylation of RSK and histone H3. These results suggest that selenomethionine can alter gene expression. In the present study, we have used cDNA microarrays to determine whether gene expression differences exist in HCT116 colon cancer cells treated with selenomethionine. These experiments reveal statistically significant expression changes for 50 genes. Genes we found to increase with selenomethionine treatment include KLK6, ATOX1, SGK, GJB2, DAP-1, PLAU, VIM, DPYSL2, STC2 and PXN. Conversely, genes downregulated by selenomethionine include PRKACB, LIM, DEPP, MYC, CDH5, ELF3, VSNL1, SAT and EGLN3. Further analysis of those genes using chromatin immunoprecipitation experiments showed that phosphorylated histone H3 on serine 10 bound to the GJB2 promoter (connexin 26) or the serum glucocorticoid kinase promoter is increased with selenomethionine treatment. Cells overexpressing CX26 or DAP-1 displayed a reduced number of colonies which suggests that these two genes could play a functional role in the growth inhibitory effects of selenomethionine. These data support the notion that selenomethionine-induced growth inhibition is associated with global changes in gene expression. They also demonstrate that selenomethionine can modify chromatin state to alter gene transcription. Finally, our studies provide a practical foundation for the further development of biomarkers to monitor the efficacy of selenomethionine in clinical trials.
• Watts, G. S., Tran, N. L., Berens, M. E., Bhattacharyya, A. K., Nelson, M. A., Montgomery, E. A., & Sampliner, R. E. (2007). Identification of Fn14/TWEAK receptor as a potential therapeutic target in esophageal adenocarcinoma. International journal of cancer. Journal international du cancer, 121(10), 2132-9.
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  Given the poor survival rate and efficacy of current therapy for esophageal adenocarcinoma (EAC), there is a need to identify and develop new therapeutic targets for treatment. Microarray analysis (Affymetrix U133A GeneChips, Robust Multi-Chip Analysis) was used to expression profile 11 normal squamous and 18 Barrett's esophagus biopsies, 7 surgically resected EACs and 3 EAC cell lines. Two hundred transcripts representing potential therapeutic targets were identified using the following criteria: significant overexpression in EAC by analysis of variance (p = 0.05, Benjamini Hochberg false discovery rate); 3-fold increase in EAC relative to normal and Barrett's esophagus and expression in at least 2 of the 3 EAC cell lines. From the list of potential targets we selected TNFRSF12A/Fn14/TWEAK receptor, a tumor necrosis factor super-family receptor, for further validation based on its reported role in tumor cell survival and potential as a target for therapy. Fn14 protein expression was confirmed in SEG-1 and BIC-1 cell lines, but Fn14 was not found to affect tumor cell survival after exposure to chemotherapeutics as expected. Instead, a novel role in EAC was discovered in transwell assays, in which modulating Fn14 expression affected tumor cell invasion. Fn14's potential as a therapeutic target was further supported by immunohistochemistry on a tissue microarray of patient samples that showed that Fn14 protein expression increased with disease progression in EAC.
• Futscher, B. W., Gerner, E. W., Ignatenko, N. A., Marton, L. J., Stringer, D. E., Watts, G. S., & Yerushalmi, H. F. (2006).

  Pharmacogenomics of the Polyamine Analog 3,8,13,18-tetraaza-10,11-[(E)-1,2-cyclopropyl] eicosane Tetrahydrochloride, CGC-11093, in the Colon Adenocarcinoma Cell Line HCT1161

  . Technology in Cancer Research & Treatment, 5(6), 553-564. doi:10.1177/153303460600500602
• Futscher, B. W., Jensen, T., Kim, C., Klimecki, W. T., Nouzova, M., Novak, P., Oshiro, M. M., Watts, G. S., & Wozniak, R. J. (2006).

  Epigenetic Inactivation of the HOXA Gene Cluster in Breast Cancer

  . Cancer Research, 66(22), 10664-10670. doi:10.1158/0008-5472.can-06-2761
• Ignatenko, N. A., Yerushalmi, H. F., Watts, G. S., Futscher, B. W., Stringer, D. E., Marton, L. J., & Gerner, E. W. (2006). Pharmacogenomics of the polyamine analog 3,8,13,18-tetraaza-10,11-[(E)-1,2-cyclopropyl]eicosane tetrahydrochloride, CGC-11093, in the colon adenocarcinoma cell line HCT1161. Technology in cancer research & treatment, 5(6), 553-64.
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  Polyamine analogs are known to inhibit tumorigenesis at least in part by mimicking some of the regulatory roles of natural polyamines. To begin the identification of those signaling pathways that are involved in differential cellular responses to the synthetic conformationally restricted polyamine analog CGC-11093, we conducted gene expression profiling, proteomic, and genome-wide DNA methylation and histone acetylation analyses of the HCT116 colon adenocarcinoma cell line after treatment with this analog. Gene expression analysis was performed using Affymetrix GeneChip human genome U133 Plus 2.0 arrays. Changes in protein expression were evaluated using 2D polyacrylamide gels followed by LCMS/MS. DNA methylation was measured using 6,800 element CpG island microarrays. Treatment of cells with CGC-11093 at concentrations ranging from 0.1 to 10 microM caused inhibition of cell growth and metabolic activity, but only minimally affected cell viability. Gene expression analysis showed concentration-dependent effects of CGC-11093 on the DNA/RNA binding transcription factor, cell cycle, signaling, transport, cytoskeletal/structural, and serine protease genes. Functional gene analysis revealed distinct expression patterns related to inhibition of cell cycle control, TGF beta signaling, proteasome and RNA polymerase pathways, upregulation of the aminoacyl-tRNA synthesis pathway, and perturbations in the MAPK and Wnt signaling pathways. Microarray results were validated for selected genes with real time RT PCR. Proteomics analysis showed correlative changes in the expression of proteins involved in the regulation of proteasome function (proteasome subunit Y) and tRNA synthesis. CGC-11093 treatment did not produce any detectable changes in DNA methylation or histone acetylation in cells. This study validates specific target pathways for a specific conformationally restricted polyamine analog and suggests the utility of combined gene and DNA methylation microarrays along with proteomic analyses as a useful approach to the evaluation of the mechanisms of action of anticancer drugs.
• Li, H., Watts, G. S., Oshiro, M. M., Futscher, B. W., & Domann, F. E. (2006). AP-2alpha and AP-2gamma are transcriptional targets of p53 in human breast carcinoma cells. Oncogene, 25(39), 5405-15.
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  Activating enhancer-binding protein 2alpha (AP-2alpha) and activating enhancer-binding protein 2gamma (AP-2gamma) are transcription factors that bind GC-rich consensus sequences and regulate the expression of many downstream genes. AP-2alpha and AP-2gamma interact with p53 both physically and functionally. Expression microarray results in human breast carcinoma cells with forced p53 expression revealed AP-2gamma as a putative transcriptional target of p53. To confirm and extend these findings we measured the effects of forced p53 expression in human breast carcinoma cells by real-time reverse transcription-PCR, Western blotting, electrophoretic gel mobility shift assays, promoter reporter, chromatin immunoprecipitation and chromatin accessibility assays. Wild-type p53 expression rapidly induced not only AP-2gamma but also AP-2alpha mRNA. The subsequent increase in these proteins led to increased AP-2 DNA-binding and transactivating activity. Candidate p53-binding sites were identified in the AP-2alpha and AP-2gamma promoters. p53 binding to these cis-elements in vivo was also observed, together with a relaxation of chromatin structure in these regions. Finally, expression of either AP-2alpha or gamma inhibited growth of human breast carcinoma cells in vitro. Taken together, our findings indicate that these AP-2 genes are targets for transcriptional activation by p53 and suggest that AP-2 proteins may mediate some of the downstream effects of p53 expression such as inhibition of proliferation.
• Novak, P., Jensen, T., Oshiro, M. M., Wozniak, R. J., Nouzova, M., Watts, G. S., Klimecki, W. T., Kim, C., & Futscher, B. W. (2006). Epigenetic inactivation of the HOXA gene cluster in breast cancer. Cancer research, 66(22), 10664-70.
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  Using an integrated approach of epigenomic scanning and gene expression profiling, we found aberrant methylation and epigenetic silencing of a small neighborhood of contiguous genes-the HOXA gene cluster in human breast cancer. The observed transcriptional repression was localized to approximately 100 kb of the HOXA gene cluster and did not extend to genes located upstream or downstream of the cluster. Bisulfite sequencing, chromatin immunoprecipitation, and quantitative reverse transcription-PCR analysis confirmed that the loss of expression of the HOXA gene cluster in human breast cancer is closely linked to aberrant DNA methylation and loss of permissive histone modifications in the region. Pharmacologic manipulations showed the importance of these aberrant epigenetic changes in gene silencing and support the hypothesis that aberrant DNA methylation is dominant to histone hypoacetylation. Overall, these data suggest that inactivation of the HOXA gene cluster in breast cancer may represent a new type of genomic lesion-epigenetic microdeletion. We predict that epigenetic microdeletions are common in human cancer and that they functionally resemble genetic microdeletions but are defined by epigenetic inactivation and transcriptional silencing of a relatively small set of contiguous genes along a chromosome, and that this type of genomic lesion is metastable and reversible in a classic epigenetic fashion.
• Alsobrook, J. P., Kaysser-Kranich, T., Lockner, R. J., Palaniappan, C., Sendera, T. J., Shippy, R., & Watts, G. S. (2004).

  Performance evaluation of commercial short-oligonucleotide microarrays and the impact of noise in making cross-platform correlations

  . BMC Genomics. doi:10.1186/1471-2164-5-61
• Gerner, E. W., Ignatenko, N. A., Skovan, B. A., Stringer, D., Watts, G. S., & Zhang, H. (2004).

  The chemopreventive agent ?-difluoromethylornithine blocks Ki-ras-dependent tumor formation and specific gene expression in Caco-2 cells

  . Molecular Carcinogenesis. doi:10.1002/mc.20008
• Ignatenko, N. A., Zhang, H., Watts, G. S., Skovan, B. A., Stringer, D. E., & Gerner, E. W. (2004). The chemopreventive agent alpha-difluoromethylornithine blocks Ki-ras-dependent tumor formation and specific gene expression in Caco-2 cells. Molecular carcinogenesis, 39(4), 221-33.
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  Mutation of the Kirsten-ras (Ki-ras) proto-oncogene occurs frequently in colorectal cancers. alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), inhibits Ki-ras transformation and colon tumorigenesis in carcinogen-treated animal models by mechanisms yet to be elucidated. Caco-2 cells transfected with an activated Ki-ras, but not parental cells, formed tumors in severe combined immunodeficient (SCID) mice. DFMO treatment (2% in drinking water) prevented tumor growth. Gene expression profiling was performed to identify Ki-ras-and DFMO-dependent patterns of gene expression. Microarray results were validated with real-time or semi-quantitative RT-PCR and/or Western blot analysis. Genes upregulated in Caco-2 cells expressing an activated Ki-ras encoded cytoskeletal-, transport-, protease-, and gap junction-associated proteins. These genes are important for normal development and maintenance of colonic epithelial tissue. Caco-2 cells transfected with an activated Ki-ras displayed increased expression of the integrin alpha 1 (INGA1) and enhanced cell migration on laminin. These parameters were unaffected by DFMO, but Ki-ras-dependent migration was inhibited by INGA1 antibodies. Other Ki-ras-dependent, but DFMO-independent, genes included transglutaminase (TGase) and kallikrein 6 (KLK6). Ki-ras-transfected cells also expressed increased levels of connexin43 (Cx43) (RNA and protein), tight junction protein, and endothelin 1. DFMO reversed these increases. The results indicated that the Ki-ras oncogene caused changes in experimental cell migration and cell-cell communication genes and that some of these changes could be reversed by DFMO.
• Shippy, R., Sendera, T. J., Lockner, R., Palaniappan, C., Kaysser-Kranich, T., Watts, G., & Alsobrook, J. (2004). Performance evaluation of commercial short-oligonucleotide microarrays and the impact of noise in making cross-platform correlations. BMC genomics, 5, 61.
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  Despite the widespread use of microarrays, much ambiguity regarding data analysis, interpretation and correlation of the different technologies exists. There is a considerable amount of interest in correlating results obtained between different microarray platforms. To date, only a few cross-platform evaluations have been published and unfortunately, no guidelines have been established on the best methods of making such correlations. To address this issue we conducted a thorough evaluation of two commercial microarray platforms to determine an appropriate methodology for making cross-platform correlations.
• Wozniak, R. J., Watts, G. S., Watterson, S. J., Oshiro, M. M., Junk, D. J., Futscher, B. W., & Domann, F. E. (2004). The acetyltransferase p300/CBP-associated factor is a p53 target gene in breast tumor cells.. Neoplasia (New York, N.Y.), 6(3), 187-94. doi:10.1593/neo.3292
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  p300/CBP-associated factor (PCAF) is a coactivator of the tumor suppressor, p53. PCAF participates in p53's transactivation of target genes through acetylation of both bound p53 and histones within p53 target promoters. Using microarrays, we discovered that PCAF itself is induced by p53 in a panel of breast tumor cell lines. Two p53 mutant breast tumor cell lines, BT-549 and UACC-1179, were chosen for further study of PCAF induction by wild-type p53. PCAF induction following adenoviral transduction of p53 expression was confirmed with real-time polymerase chain reaction in a time course experiment. Chromatin immunoprecipitation experiments then showed that PCAF induction was associated with increased p53 binding to the PCAF promoter, which contains p53 consensus-binding sites. PCAF induction by p53 activity was further demonstrated in wild-type p53 MCF10A cells when PCAF expression was induced following activation of endogenous wild-type p53 with doxorubicin in a dose- and time-dependent manner. Furthermore, the doxorubicin-induced increase in PCAF expression was blocked by pretreatment of the MCF10A cells with siRNA (small interfering RNA) targeted against p53 mRNA. Taken together, the results show that PCAF expression can be induced by wild-type p53.
• Domann, F. E., Futscher, B. W., Junk, D. J., Muñoz-Rodríguez, J. L., Oshiro, M. M., Watts, G. S., & Wozniak, R. J. (2003).

  Mutant p53 and aberrant cytosine methylation cooperate to silence gene expression

  . Oncogene. doi:10.1038/sj.onc.1206545
• Gandolfi, A. J., Vaught, S., Watts, G. S., & Zheng, X. H. (2003).

  Low-level arsenite induced gene expression in HEK293 cells☆

  . Toxicology. doi:10.1016/s0300-483x(03)00025-8
• Oshiro, M. M., Watts, G. S., Wozniak, R. J., Junk, D. J., Munoz-Rodriguez, J. L., Domann, F. E., & Futscher, B. W. (2003). Mutant p53 and aberrant cytosine methylation cooperate to silence gene expression. Oncogene, 22(23), 3624-34.
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  p53 is an important transcriptional regulator that is frequently mutated in cancer. Gene-profiling experiments of breast cancer cells infected with wt p53 revealed both MASPIN and desmocollin 3 (DSC3) to be p53-target genes, even though both genes are silenced in association with aberrant cytosine methylation of their promoters. Despite the transcriptional repression of these genes by aberrant DNA methylation, restoration of p53 resulted in the partial reactivation of both genes. This reactivation is a result of wt p53 binding to its consensus DNA-binding sites within the MASPIN and DSC3 promoters, stimulating histone acetylation, and enhancing chromatin accessibility of their promoters. Interestingly, wt p53 alone did not affect the methylation status of either promoter, suggesting that p53 itself can partially overcome the repressive barrier of DNA methylation. Pharmacologic inhibition of DNA methylation with 5-aza-2'-deoxycytidine in combination with restoration of wt p53 status resulted in a synergistic reactivation of these genes to near-normal levels. These results suggest that cancer treatments that target both genetic and epigenetic facets of gene regulation may be a useful strategy towards the therapeutic transcriptional reprogramming of cancer cells.
• Zheng, X. H., Watts, G. S., Vaught, S., & Gandolfi, A. J. (2003). Low-level arsenite induced gene expression in HEK293 cells. Toxicology, 187(1), 39-48.
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  Chronic, low-level exposure to arsenic frequently results in skin, lung, bladder, and kidney cancer. Since arsenic is primarily excreted via the kidney, this study focused on this target tissue. Gene array was used as a sensitive low-level monitor of the impact of arsenic on this target tissue. Arsenite [As(III)] was chosen as the chemical species of arsenic since As(III) species are touted as the cellular toxic form of arsenic. Human embryonic kidney cell line HEK293 cells were incubated with 1, 10, and 25 microM arsenite [As(III)] for 6 or 24 h. Total RNA from treated and control cells was isolated, reverse transcribed, and labeled with Cy3 or Cy5, and hybridized to a human cDNA microarray. Hybridizations were performed four times using independent total RNA preparations to ensure reproducibility. Raw data from 10 and 25 microM treated cells exposed for 6 h was normalized within, and between, hybridizations followed by identification of genes affected by arsenite exposure based on practical significance (2-fold change up or down) and reproducibility (affected in four of six measurements). In these studies, 20 genes (HMOX1, MT1E, or FOSL1, etc.) were up-regulated, and 19 genes (MYC, JAK1, or CENPE, etc.) were down-regulated. Genes identified at 10 and 25 microM arsenic exposure were then examined after 1 microM treatment for 6 or 24 h. Expression of affected genes showed a dose-dependent (1-25 microM) trend that was apparently not time-dependent (6 vs. 24 h). The affected genes indicate that even this realistic, low-level arsenite exposure was recognized by the HEK293 cells (e.g. metallothionein genes) and produced an oxidative stress (e.g. heme oxygenase gene). These affected genes were characterized as stress response genes, proto-oncogene, signaling molecules, transcription factors, chemokine receptors, proteolytic enzymes, ESTs, and unknown genes. These findings imply that arsenite induces complex cellular injury and the cellular adaptation to As(III) is associated with alterations in the expression of many genes.
• Bernstein, H., Bernstein, C., Craven, M. T., Crowley, C., Crowley-Weber, C. L., Dvorakova, K., Futscher, B. W., Garewal, H. S., Gleason-Guzman, M., Payne, C. M., Waltmire, C. N., & Watts, G. S. (2002).

  Development and molecular characterization of HCT-116 cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate

  . Carcinogenesis. doi:10.1093/carcin/23.12.2063
• Crowley-Weber, C. L., Payne, C. M., Gleason-Guzman, M., Watts, G. S., Futscher, B., Waltmire, C. N., Crowley, C., Dvorakova, K., Bernstein, C., Craven, M., Garewal, H., & Bernstein, H. (2002). Development and molecular characterization of HCT-116 cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate. Carcinogenesis, 23(12), 2063-80.
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  Evidence from live cell bioassays shows that the flat mucosa from patients with colon cancer exhibits resistance to bile salt-induced apoptosis. Three independent cell lines derived from the colonic epithelial cell line HCT-116 were selected for resistance to bile salt-induced apoptosis. These cell lines were developed as tissue culture models of apoptosis resistance. Selection was carried out for resistance to apoptosis induced by sodium deoxycholate (NaDOC), the bile salt found in highest concentrations in human fecal water. Cultures of HCT-116 cells were serially passaged in the presence of increasing concentrations of NaDOC. The resulting apoptosis resistant cells were able to grow at concentrations of NaDOC (0.5 mM) that cause apoptosis in a few hours in unselected HCT-116 cells. These cells were then analyzed for changes in gene expression. Observations from cDNA microarray, 2-D gel electrophoresis/MALDI-mass spectroscopy, and confocal microscopy of immunofluorescently stained preparations indicated underexpression or overexpression of numerous genes at either the protein or mRNA level. Genes that may play a role in apoptosis and early stage carcinogenesis have been identified as upregulated in these cell lines, including Grp78, Bcl-2, NF-kappaB(p50), NF-kappaB(p65), thioredoxin peroxidase (peroxiredoxin) 2, peroxiredoxin 4, maspin, guanylate cyclase activating protein-1, PKCzeta, EGFR, Ras family members, PKA, PI(4,5)K, TRAF2 and BIRC1 (IAP protein). Under-expressed mRNAs included BNIP3, caspase-6, caspase-3 and serine protease 11. NF-kappaB was constitutively activated in all three resistant cell lines, and was responsible, in part, for the observed apoptosis resistance, determined using antisense oligonucleotide strategies. Molecular and cellular analyses of these resistant cell lines has suggested potential mechanisms by which apoptosis resistance may develop in the colonic epithelium in response to high concentrations of hydrophobic bile acids that are associated with a Western-style diet. These analyses provide the rationale for the development of hypothesis-driven intermediate biomarkers to assess colon cancer risk on an individual basis.
• Barrera, J., Bowden, G. T., Calaluce, R., Cress, A. E., Fitchmun, M., Futscher, B. W., Gleason-Guzman, M., Hao, J., Isett, R., Kunkel, M., Nagle, R. B., Schmelz, M., & Watts, G. S. (2001).

  Laminin-5-mediated gene expression in human prostate carcinoma cells

  . Molecular Carcinogenesis. doi:10.1002/1098-2744(200102)30:2<119::aid-mc1020>3.0.co;2-n
• Calaluce, R., Kunkel, M. W., Watts, G. S., Schmelz, M., Hao, J., Barrera, J., Gleason-Guzman, M., Isett, R., Fitchmun, M., Bowden, G. T., Cress, A. E., Futscher, B. W., & Nagle, R. B. (2001). Laminin-5-mediated gene expression in human prostate carcinoma cells. Molecular carcinogenesis, 30(2), 119-29.
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  Interactions between extracellular matrix (ECM) proteins and prostate carcinoma cells provide a dynamic model of prostate tumor progression. Previous work in our laboratory showed that laminin-5, an important member of a family of ECM glycoproteins expressed in the basal lamina, is lost in prostate carcinoma. Moreover, we showed that the receptor for laminin-5, the alpha6beta4 integrin, is altered in prostate tumors. However, the genes that laminin-5 potentially regulates and the significance of its loss of expression in prostate cancer are not known. We selected cDNA microarray as a comprehensive and systematic method for surveying and examining gene expression induced by laminin-5. To establish a definitive role for laminin-5 in prostate tumor progression and understand the significance of its loss of expression, we used a cDNA microarray containing 5289 human genes to detect perturbations of gene expression when DU145 prostate carcinoma cells interacted with purified laminin-5 after 0.5, 6, and 24 h. Triplicate experiments showed modulations of four, 61, and 14 genes at 0.5, 6, and 24 h, respectively. Genes associated with signal transduction, cell adhesion, the cell cycle, and cell structure were identified and validated by northern blot analysis. Protein expression was further assessed by immunohistochemistry. Mol. Carcinog. 30:119-129, 2001.
• Futscher, B. W., Gleason-Guzman, M., Isett, R., Kunkel, M., Salmon, S. E., & Watts, G. S. (2001).

  cDNA microarray analysis of multidrug resistance: doxorubicin selection produces multiple defects in apoptosis signaling pathways.

  . Journal of Pharmacology and Experimental Therapeutics.
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  Doxorubicin plays an important role in the treatment of leukemias, lymphomas, and a variety of carcinomas. Tumor cell resistance to doxorubicin is often associated with expression of the multidrug resistance gene MDR1, which codes for the drug efflux pump P-glycoprotein, and a multidrug-resistant phenotype. Evidence from multiple sources suggests, however, that additional genes besides MDR1 are involved in development of multidrug resistance. To identify genes involved in the multidrug resistance phenotype, we created a 5760-gene cDNA microarray to search for differentially expressed genes between the human multiple myeloma cell line RPMI 8226 and its doxorubicin-selected sublines 8226/Dox6 and 8226/Dox40, both of which express MDR1 and are multidrug-resistant. The cDNA microarray results identified a set of differentially expressed genes, which included MDR1 as expected. Thirty Northern analyses were used to confirm the results of the cDNA microarrays; comparison with the microarray results showed a 90% agreement between the two techniques. Within the set of differentially expressed genes identified by the cDNA microarrays, 29 were of particular interest as they can participate in apoptotic signaling, particularly as mediated by ceramide and the mitochondrial permeability transition. The functional importance of these changes in gene expression is supported by their explanation of the 8226/Dox cell lines' cross-resistance to substances that are not P-glycoprotein substrates, such as Fas/CD95 ligand and staurosporine. We conclude that doxorubicin selection led to changes in gene expression that reduce the apoptotic response to death-inducing stimuli and thus contribute to the multidrug resistance phenotype.
• Watts, G. S., Futscher, B. W., Isett, R., Gleason-Guzman, M., Kunkel, M. W., & Salmon, S. E. (2001). cDNA microarray analysis of multidrug resistance: doxorubicin selection produces multiple defects in apoptosis signaling pathways. The Journal of pharmacology and experimental therapeutics, 299(2), 434-41.
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  Doxorubicin plays an important role in the treatment of leukemias, lymphomas, and a variety of carcinomas. Tumor cell resistance to doxorubicin is often associated with expression of the multidrug resistance gene MDR1, which codes for the drug efflux pump P-glycoprotein, and a multidrug-resistant phenotype. Evidence from multiple sources suggests, however, that additional genes besides MDR1 are involved in development of multidrug resistance. To identify genes involved in the multidrug resistance phenotype, we created a 5760-gene cDNA microarray to search for differentially expressed genes between the human multiple myeloma cell line RPMI 8226 and its doxorubicin-selected sublines 8226/Dox6 and 8226/Dox40, both of which express MDR1 and are multidrug-resistant. The cDNA microarray results identified a set of differentially expressed genes, which included MDR1 as expected. Thirty Northern analyses were used to confirm the results of the cDNA microarrays; comparison with the microarray results showed a 90% agreement between the two techniques. Within the set of differentially expressed genes identified by the cDNA microarrays, 29 were of particular interest as they can participate in apoptotic signaling, particularly as mediated by ceramide and the mitochondrial permeability transition. The functional importance of these changes in gene expression is supported by their explanation of the 8226/Dox cell lines' cross-resistance to substances that are not P-glycoprotein substrates, such as Fas/CD95 ligand and staurosporine. We conclude that doxorubicin selection led to changes in gene expression that reduce the apoptotic response to death-inducing stimuli and thus contribute to the multidrug resistance phenotype.
• Watts, G. S., Tye, D., Gleason-guzman, M., & Futscher, B. W. (2001). Microarray analysis of a human multiple myeloma cell line selected with melphalan or doxorubicin identifies a set of commonly affected genes. Nature Genetics, 27(4), 94-95. doi:10.1038/87356
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  Microarray analysis of a human multiple myeloma cell line selected with melphalan or doxorubicin identifies a set of commonly affected genes
• Wei, J. S., Watts, G. S., Trent, J. M., Ross, K. R., Mcdonough, W. S., Mariani, L., Hoelzinger, D. B., Giese, A., Demuth, T., Coons, S. W., Berens, T. J., Berens, M. E., & Beaudry, C. (2001). Glioma cell motility is associated with reduced transcription of proapoptotic and proliferation genes: a cDNA microarray analysis.. Journal of neuro-oncology, 53(2), 161-76. doi:10.1023/a:1012253317934
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  Microarray analysis of complementary DNA (cDNA) allows large-scale, comparative, gene expression profiling of two different cell populations. This approach has the potential for elucidating the primary transcription events and genetic cascades responsible for increased glioma cell motility in vitro and invasion in vivo. These genetic determinants could become therapeutic targets. We compared cDNA populations of a glioma cell line (G112) exposed or not to a motility-inducing substrate of cell-derived extracellular matrix (ECM) proteins using two sets of cDNA microarrays of 5,700 and 7,000 gene sequences. The data were analyzed considering the level and consistency of differential expression (outliers) and whether genes involved in pathways of motility, apoptosis, and proliferation were differentially expressed when the motility behavior was engaged. Validation of differential expression of selected genes was performed on additional cell lines and human glioblastoma tissue using quantitative RT-PCR. Some genes involved in cell motility, like tenascin C, neuropilin 2, GAP43, PARG1 (an inhibitor of Rho), PLCy, and CD44, were over expressed; other genes, like adducin 3y and integrins, were down regulated in migrating cells. Many key cell cycle components, like cyclin A and B, and proliferation markers, like PCNA, were strongly down regulated on ECM. Interestingly, genes involved in apoptotic cascades, like Bcl-2 and effector caspases, were differentially expressed, suggesting the global down regulation of proapoptotic components in cells exposed to cell-derived ECM. Overall, our findings indicate a reduced proliferative and apoptotic activity of migrating cells. cDNA microarray analysis has the potential for uncovering genes linking the phenotypic aspects of motility, proliferation, and apoptosis.
• Feldmann, K. A., Xu, W., Watts, G. S., Walbot, V., Pierson, E. A., Macas, J., Galbraith, D. W., Feyereisen, R., Feldmann, K. A., Deyholos, M. K., & Bohnert, H. J. (1999). Implementing microarrays for the study of plant genomics. Nature Genetics, 23(3), 45-45. doi:10.1038/14307
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  Microarrays provide the means for the large-scale analysis of gene expression patterns in living organisms. My laboratory is part of three federally funded projects that are directed toward an understanding of the regulation of gene expression in higher plants.
• Watts, G. S., Salmon, S. E., Kunkel, M., Guzman, M., Futscher, B. W., & Epner, E. (1999). Gene expression profiles associated with MDR1 expression in a doxorubicin-resistant human multiple myeloma cell line. Nature Genetics, 23(3), 45-45. doi:10.1038/14306
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  Gene expression profiles associated with MDR1 expression in a doxorubicin-resistant human multiple myeloma cell line
• Costello, J. F., Dalton, W. S., Futscher, B. W., Peng, Y. M., Pieper, R. O., & Watts, G. S. (1997).

  Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene

  . Molecular and Cellular Biology, 17(9), 5612-5619. doi:10.1128/mcb.17.9.5612
• Watts, G. S., Pieper, R. O., Costello, J. F., Peng, Y. M., Dalton, W. S., & Futscher, B. W. (1997). Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene. Molecular and cellular biology, 17(9), 5612-9.
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  O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.
• Futscher, B. W., & Watts, G. S. (1995).

  Detecting differences in 5-methylcytosine using restriction enzyme isoschizomers: an endogenous control for complete digestion

  . Nucleic Acids Research. doi:10.1093/nar/23.22.4740
• Watts, G. S., & Futscher, B. W. (1995). Detecting differences in 5-methylcytosine using restriction enzyme isoschizomers: an endogenous control for complete digestion. Nucleic acids research, 23(22), 4740-1.

Proceedings Publications

• Lenka, J., Bixby, B., Watts, G., Oshiro, M., Trina, H., Erickson, H., Futscher, B., Garland, L., & Mark, M. (2023).

  A Pilot Study of Diagnostic Value of Sentinel-10 Liquid Biopsy Assay for the Detection of Malignancy in Pleural Fluid

  . In American Thoracic Society.
• Watts, G. S., Su, H., Li, J., Huang, Z., & Chen, H. (2007). Large-scale regulatory network analysis from microarray data: modified Bayesian network learning and association rule mining. In Decision Support Systems, 43, 1207-1225.
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  We present two algorithms for learning large-scale gene regulatory networks from microarray data: a modified information-theory-based Bayesian network algorithm and a modified association rule algorithm. Simulation-based evaluation using six datasets indicated that both algorithms outperformed their unmodified counterparts, especially when analyzing large numbers of genes. Both algorithms learned about 20% (50% if directionality and relation type were not considered) of the relations in the actual models. In our empirical evaluation based on two real datasets, domain experts evaluated subsets of learned relations with high confidence and identified 20-30% to be "interesting" or "maybe interesting" as potential experiment hypotheses.