Collaborators

• R. Sobol, University of Pittsburgh Cancer Institute
• S. Bhatia, Harvard-MIT Division of Health Science and Technology
• P. Hammond, ChemE

Senior Postdoctoral Associate

• O. Kiraly

Graduate Students

• J. Ge, Research Assistant, BE
• M. Parrish, Research Assistant, BE
• M. Sukup Jackson, Research Assistant, BE
• D. Weingeist, Research Assistant, BE

Support Staff

• K. Reposa, Administrative Assistant II
• P. Mazzucato, Lab Technician

Publications

Engelward, B.P., Dreslin, A., Christensen, J., Huszar, D., Kurahara, C., and Samson, L.,  Repair deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation induced chromosome damage and cell killing, EMBO J., 15: 945-952 (1996).  PMCID: PMC450292

Engelward, B.P., Weeda, G., Wyatt, M.D., Broekhof, J.L.M., de Wit, J., Donker, I., Allan, J.M., Gold, B.,Hoeijmakers, J.H.J., and Samson, L.D., Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase, Proc. Natl. Acad. Sci. USA, 94: 13087-13092 (1997). PMCID: PMC24267

Engelward, B.P., Allan, J.M., Dreslin, A.J., Kelly, J.D., Wu, M.M., Gold, B., and Samson, L.D., A chemical and genetic approach together define the biological consequences of 3-methyladenine lesions in the mammalian genome, J. Biol. Chem., 273: 5412-5418 (1998).  PMID: 9479003

Hendricks, C.A., Razlog, M., Matsuguchi, T., Goyal, A., Brock, A.L., and Engelward, B.P., The S. cerevisiae Mag1 3-methyladenine DNA glycosylase modulates susceptibility to homologous recombination, DNA Repair, 1: 645-659 (2002).  PMID: 12509287

Hendricks, C.A.,  Almeida, K., Stitt, M.,  Jonnalagadda, V.,  Kerrison, F., Rugo, R.,Engelward,B.P.,Spontaneous mitotic homologous recombination at an EYFP direct repeat in transgenic mice, Proc. Natl. Acad. Sci. USA, 100: 6325-6330(2003).  PMCID: PMC164445

Kiziltepe, T., Yan, A., Dong, M., Jonnalagadda, V.S., Dedon, P.C., and Engelward, B.P., Delineation of the chemical pathways underlying nitric oxide-induced homologous recombination in mammalian cells, Chem. Biol., 3: 357-369 (2005).  PMID: 15797220

Jonnalagadda, V.S., Matsuguchi, T., and Engelward, B.P., Interstrand crosslink-induced homologous recombination carries an increased risk of deletions and insertions, DNA Repair, 4: 594-605 (2005).  PMID: 15811631

Rugo, R.E., Almeida, K.H., Hendricks, C.A., Jonnalagadda, V.S., and Engelward, B.P., A single acute exposure to a chemotherapeutic agent induces hyper-recombination in distantly descendant cells and in their neighbors, Oncogene, 25:5016-5025 (2005). PMID: 15856014

Wiktor-Brown, D.M., Hendricks, C.A., Olipitz, W., and Engelward, B.P., Age dependent accumulation of recombinant cells in the mouse pancreas revealed by in situ fluorescence imaging, Proc. Natl. Acad. Sci. U.S.A., 103:11862-11867 (2006).  PMCID: PMC1567667

Wiktor-Brown, D.M., Olipitz W., Hendricks C.A., Rugo R.E., and Engelward B.P., Tissue specific differences in the accumulation of sequence rearrangements with age, DNA Repair, 7:694-703 (2008).  PMCID: PMC3014828

Wiktor-Brown, D.M., Kwon H.S., Nam Y.S., So P.T., and Engelward B.P., Integrated one- and two-photon imaging platform reveals clonal expansion as a major driver of mutation load, Proc. Natl. Acad. Sci. U. S. A., 105:10314-10319 (2008).  PMCID: PMC2492490

Niu, Y., Wang H., Wiktor-Brown D.M., Rugo R., Shen H., Huq S., Engelward B.P., Epperly M., and Greenberger J.S., Irradiated esophageal cells are protected from radiation-induced recombination by MnSOD gene therapy, Rad. Res., 173:453-61 (2010).  PMCID:PMC2872095

Olipitz, W., Hembrador S., Davidson M., Yanch J.C. and Engelward B.P., Development and characterization of a novel variable low-dose rate irradiator for in vivo mouse studies, Health Phys. J., 98:727-34 (2010).  PMCID:PMC3020895

Wood, D.K., Weingeist D.M., Wu Y., Bhatia S.N., and Engelward B.P. Single cell trapping and DNA damage analysis using microwell arrays, Proc. Natl. Acad. Sci. U.S.A.,107:10008-13 (2010).  PMCID:PMC2890454

Rugo, R.E., Mutamba J.T., Mohan K.N., Yee T., Chaillet J.R., Greenberger J.S., and Engelward B.P.  Methyltransferases mediate cell memory of a genotoxic insult, Oncogene, 30: 751–756 (2011).  PMCID: PMC3044496

DNA damage has been found to play critical roles in cancer, aging, and heritable disease.  There is rising interest in studying DNA damage and repair kinetics in cells, but the lack of a robust, inexpensive, and high-throughput device for quantitative DNA damage analysis makes such investigations far from routine.  The single cell gel electrophoresis or “comet” assay is one of the best-established methods for detection of DNA lesions and strand breaks. Based on the principle that relaxed loops and fragments of damaged DNA migrate farther under the influence of an electrical field in agarose gel than undamaged DNA, the level of DNA damage can be assessed by measuring the relative amount of DNA migration. Although extremely versatile and inexpensive, the comet assay is restrained from wider acceptance due to its low throughput, poor reproducibility and laborious and potentially biased analysis methods. Through incorporation of microfabrication techniques, we have developed a microarray platform to perform high throughput single cell electrophoresis with improved consistency.  Different from randomly dispersed cells in the traditional comet assay, cells on our platform are patterned into spatially registered microscopic wells. These microwells are formed by direct stamping of hydrated gels with molds that contain patterned microposts fabricated through Su-8 photolithography (Figure 1). Cells are arrayed through passive settling into the microwells and can then undergo treatment and analysis. We have also developed software that utilizes the unique patterning feature to automatically analyze images with high accuracy and reproducibility (Figure 1). By sandwiching our patterned agarose gel between a bottomless 96-well plate and glass plate, we have transformed our assay into a multiwell version, referred to as “the CometChip.” This 96-well format enables simultaneous investigation of different chemical conditions among different cells samples, as well as analysis of repair kinetics. Importantly, the CometChip is compatible with standard automated liquid handling and imaging. The efficacy and increased throughput of the CometChip for DNA damage analysis is demonstrated by comparing an irradiation dose response to the traditional comet assay (Figure 2). All doses and replicates were assayed on a single CometChip in significantly shorter time and with less labor. Our research has demonstrated a significant technological advance to traditional methodology and opened countless possibilities in epidemiology and drug development applications.