Julie T. Millard

DNA modifying agents as tools for studying chromatin structure.

DNA modification leads to mutagenicity, carcinogenicity, and cytotoxicity. Because the majority of cellular DNA exists as chromatin, histone and other proteins may mediate DNA binding and bonding reactions in vivo. Chromatin modification by a wide variety of agents, including intercalators, groove binders, cross-linkers, and strand cleavers, is reviewed. Although the majority of agents demonstrate a preference for linker over core DNA, the sequence preference appears to be retained in most cases, suggesting that both the major and minor grooves in chromatin are quite accessible to small molecules. Furthermore, only a handful of agents demonstrate the 10-11 base pair periodicity of hydroxyl radical, strongly supporting a dynamic model of nucleosome structure.

Case-Study Investigation of Equine Maternity via PCR-RFLP: A Biochemistry Laboratory Experiment

A simple and robust biochemistry laboratory experiment is described that uses restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electrophoresis. An optional step of validating DNA extracts through realtime PCR can expand the experiment to three weeks. This experiment, which has an engaging and versatile scenario, provides students with exposure to key principles and techniques of molecular biology, bioinformatics, and evolution in a forensic context.

Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA

The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

Cross-linking by epichlorohydrin and diepoxybutane correlates with cytotoxicity and leads to apoptosis in human leukemia (HL-60) cells

&NA; The bifunctional alkylating agents epichlorohydrin (ECH) and diepoxybutane (DEB) have been linked to increased cancer risks in industrial workers. These compounds react with DNA and proteins, leading to genotoxic effects. We used the comet assay to monitor formation of cross‐links in HL‐60 cells treated with ECH, DEB, and the structurally related anti‐cancer drug mechlorethamine (HN2). We report a time‐ and dose‐dependent cytotoxicity that correlated with cross‐linking activity, following the order HN2 > DEB > ECH. The rate of cross‐link repair also varied with drug, with ECH‐induced lesions the fastest to repair. High drug doses led to the formation of saturating amounts of HN2 cross‐links that were repaired inefficiently. DEB and ECH produced fewer overall cross‐links, but some were also resistant to repair. These persistent cross‐links may activate cell‐cycle arrest to allow repair of damage, with prolonged arrest triggering apoptosis. Quantitative reverse transcription polymerase chain reaction experiments revealed that treatment of HL‐60 cells with DEB and ECH results in up‐regulation of several genes involved in the intrinsic (mitochondrial) apoptosis pathway, including BAX, BAK1, CASP‐9, APAF‐1, and BCL‐2. These findings contribute to our understanding of the principles underlying the carcinogenic potentials of these xenobiotics. HighlightsCytotoxicity paralleled cross‐linking in HL‐60 cells for the drugs of interest.This is the first report of epichlorohydrin (ECH) interstrand cross‐links in cells.At low drug doses, the cross‐links had differential rates of repair.Higher drug doses led to persistent cross‐links that were resistant to repair.ECH and diepoxybutane activated the intrinsic apoptosis pathway in HL‐60 cells.

Changes in apoptotic gene expression induced by the DNA cross-linkers epichlorohydrin and diepoxybutane in human cell lines

Real time quantitative reverse transcription PCR was used to monitor changes in apoptotic gene expression after treating cells with the DNA cross-linkers epichlorohydrin (ECH) and diepoxybutane (DEB). This article presents the data obtained from application of the comparative CT method to the amplification of twelve apoptotic genes in human MCF10-A cells and eight genes in HUVEC cells. Further insight regarding the significance of these data can be found in “Cross-linking by epichlorohydrin and diepoxybutane correlates with cytotoxicity and leads to apoptosis in human leukemia (HL-60) cells” (Le et al., 2018) [1].

Abstract 4904: Mechanisms of cytotoxicity of bifunctional epoxide cross-linking agents

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The discovery that bifunctional alkylating agents have antitumor activity led to the development of cancer chemotherapy over 50 years ago. While these compounds form a variety of cellular lesions, DNA interstrand cross-links are believed to be the most lethal, impeding both replication and expression of the genetic material. We are characterizing the mechanisms by which diepoxybutane (DEB) and the structurally related compound epichlorohydrin (ECH) exert their cytotoxic effects in cultured cells. Our first goal is to determine the relationship between interstrand cross-linking and cytotoxicity. We are assaying cross-linking ability in cultured cells using Hoechst 33258 to determine the amount of duplex DNA following alkaline denaturation. Only cross-linked DNA reanneals rapidly and interacts with the dye, which is highly fluorescent when bound to duplex DNA. Preliminary results in chicken 6C2 and human HL60 cells suggest a correlation between cross-linking and LD50 values for these compounds. Our second goal is to characterize the pathways by which these compounds induce apoptosis. Reverse-transcriptase real-time PCR analysis of HL-60 cells treated with DEB and ECH under conditions determined to induce apoptosis suggests up-regulation of several key genes involved in the mitochondrial apoptotic pathway, including BAK1, BAX, DIABLO, PUMA and APAF1. Finally, we are identifying the covalent structure of the ECH-cross-linked lesion via electrospray LC/MS. Characterization of the DNA modifications induced by these agents is an important step in understanding how these compounds exert their cytotoxic effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4904. doi:10.1158/1538-7445.AM2011-4904

Abstract 1695: Mechanisms of cytotoxicity of diepoxybutane, epichlorohydrin, and (1-chloroethenyl) oxirane

The bifunctional alkylating agents are an important class of drugs for the treatment of cancer, psoriasis, and some anemias. These compounds are widely believed to exert their cytotoxic effects through reactions with biological nucleophiles, with DNA interstrand cross-links considered to be the most lethal lesions. Indeed, for many families of bifunctional alkylators, DNA interstrand cross-linking correlates with cytotoxicity. Moreover, apoptotic potential may play a role in antitumor chemotherapeutic utility. We have established previously that diepoxybutane (DEB) and the structurally related compounds epichlorohydrin (ECH) and 1-(chloroethenyl)oxirane (CEO) form DNA interstrand cross-links in vitro. With the goal of elucidating the molecular and cellular mechanisms by which this family of epoxides exerts its cytotoxic effects, we are determining the relationships between interstrand cross-linking, LD50 values, and apoptotic potentials for DEB, ECH, and CEO in chicken 6C2 and human HL-60 cells. Preliminary results in 6C2 cells suggest that cytotoxicity follows the order DEB > CEO > ECH. Furthermore, flow cytometry with Annexin V-FITC/PI dual staining has revealed that the apoptotic potentials follow the order DEB >> ECH > CEO. Reverse-transcriptase real-time PCR analysis of HL-60 cells treated with DEB under conditions known to induce apoptosis suggests up-regulation of several key genes involved in the mitochondrial apoptotic pathway, including BAK1, BAX, DIABLO, and APAF1. Further work is underway to characterize the pathways by which ECH and CEO exert their apoptotic effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1695.