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Diepoxybutane (DEB) is the most potent active metabolite of the environmental chemical 1,3-butadiene (BD). BD is a human carcinogen that exhibits multiorgan systems toxicity. Our previous studies demonstrated that the X-C motif chemokine ligand 1 (XCL1) gene expression was upregulated 3.3-fold in a p53-dependent manner in TK6 lymphoblasts undergoing DEB-induced apoptosis. The tumor-suppressor p53 protein is a transcription factor that regulates a wide variety of cellular processes, including apoptosis, through its various target genes. Thus, the objective of this study was to determine whether XCL1 is a novel direct p53 transcriptional target gene and deduce its role in DEB-induced toxicity in human lymphoblasts. We utilized the bioinformatics tool p53scan to search for known p53 consensus sequences within the XCL1 promoter region. The XCL1 gene promoter region was found to contain the p53 consensus sequences 5'-AGACATGCCTAGACATGCCT-3' at three positions relative to the transcription start site (TSS). Furthermore, the XCL1 promoter region was found, through reporter gene assays, to be transactivated at least threefold by wild-type p53 promoter in DEB-exposed human lymphoblasts. Inactivation of the XCL1 promoter p53-binding motif located at -2.579 kb relative to TSS reduced the transactivation function of p53 on this promoter in DEB-exposed cells by 97%. Finally, knockdown of XCL1 messenger RNA with specific small interfering RNA inhibited DEB-induced apoptosis in human lymphoblasts by 50%. These observations demonstrate, for the first time, that XCL1 is a novel DEB-induced direct p53 transcriptional target gene that mediates apoptosis in DEB-exposed human lymphoblasts.

Diepoxybutane (DEB) is the most potent active metabolite of butadiene, a regulated air pollutant. We previously reported the occurrence of DEB-induced, p53-dependent, mitochondrial-mediated apoptosis in human lymphoblasts. The present study investigated the role of the extracellular signal–regulated protein kinases 1 and 2 (ERK1/2) pathway in DEB-induced apoptotic signaling in exposed human lymphoblasts. Activated ERK1/2 and mitogen-activated protein (MAP) kinase/ERK1/2 kinase (MEK) levels were significantly upregulated in DEB-exposed human lymphoblasts. The MEK inhibitor PD98059 and ERK1/2 siRNA significantly inhibited apoptosis, ERK1/2 activation, as well as p53 and phospho-p53 (serine-15) levels in human lymphoblasts undergoing DEB-induced apoptosis. Collectively, these results demonstrate that DEB induces apoptotic signaling through the MEK-ERK1/2-p53 pathway in human lymphoblasts. This is the first report implicating the activation of the ERK1/2 pathway and its subsequent role in mediating DEB-induced apoptotic signaling in human lymphoblasts. These findings contribute towards the understanding of DEB toxicity, as well as the signaling pathways mediating DEB-induced apoptosis in human lymphoblasts.

Diepoxybutane (DEB) is the most potent active metabolite of the environmental chemical 1,3-butadiene (BD). BD is a known mutagen and human carcinogen and possesses multisystems organ toxicity. We previously reported the elevation of p53 in human TK6 lymphoblasts undergoing DEB-induced apoptosis. In this study, we have characterized the DEB-induced p53 accumulation and investigated the mechanisms by which DEB regulates this p53 accumulation. The elevation of p53 levels in DEB-exposed TK6 lymphoblasts and human embryonic lung (HEL) human fibroblasts was found to be largely due to the stabilization of the p53 protein. DEB increased the acetylation of p53 at lys-382, dramatically reduced complex formation between p53 and its regulator protein mdm2 and induced the phosphorylation of p53 at serines 15, 20, 37, 46, and 392 in human lymphoblasts. A dramatic increase in phosphorylation of p53 at serine 15 in correlation to total p53 levels was observed in DEB-exposed Ataxia Telangiectasia Mutated (ATM) proficient human lymphoblasts as compared to DEB-exposed ATM-deficient human lymphoblasts; this implicates the ATM kinase in the elevation of p53 levels in DEB-exposed cells. Collectively, these findings explain for the first time the mechanism by which p53 accumulates in DEB-exposed cells and contributes to the understanding of the molecular toxicity of DEB and BD. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:373–386, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20300

Diepoxybutane (DEB) is the most potent metabolite of the environmental chemical 1, 3-butadiene (BD), which is prevalent in petrochemical industrial areas. BD is a known mutagen and human carcinogen, and possesses multi-systems organ toxicity. We recently reported that DEB-induced cell death in TK6 lymphoblasts was due to the occurrence of apoptosis, and not necrosis. In this study, we investigated the molecular mechanisms responsible for DEB-induced apoptosis in these cells. Bax and Bak were found to be over-expressed and activated, and the mitochondrial trans-membrane potential was attenuated in cells undergoing DEB-induced apoptosis. Cytochrome c was depleted from the mitochondria of TK6 cells undergoing apoptosis, and was released into the cytosol in Jurkat-T lymphoblasts exposed to the same concentrations of DEB. Executioner caspase 3 was deduced to be activated by initiator caspase 9. DEB induced reactive oxygen species (ROS) formation, and the ROS scavenger N-acetyl-L-cysteine effectively blocked DEB-induced apoptosis in TK6 cells. Collectively, these results demonstrate that the mitochondrial apoptotic pathway is activated to mediate DEB-induced apoptosis in human TK6 lymphoblasts. These results further demonstrate that DEB-induced apoptosis is also mediated by the DEB-induced generation of ROS. This is the first report to examine the mechanism of DEB-induced apoptosis in human lymphoblasts.

1,3-butadiene (BD) causes genetic damage, including adduct formation, sister chomatid exchange, and point mutations. Previous studies have focused on the types of genetic damage and tumors found after long-term exposure of rodents to butadiene. This study examined the effect of the most active BD metabolite, butadiene diepoxide (BDO2), on cell cycle entry and progression in human lung fibroblasts (LU cells) with a normal diploid karyotype. Serum-arrested (G0) LU cells were exposed to BDO2 for 1 hr and stimulated to divide with medium containing 10% fetal bovine serum. The BDO2-treated LU cells were evaluated for cell cycle progression, nuclear localization of arrest mediators, mitotic index, and cellular proliferation. The BDO2-treated cells demonstrated a substantial inhibition of cell proliferation when treated with 100 μM BDO2 for 1 hr. No appreciable levels of apoptosis or mitotic figures were observed in the BDO2-treated cells through 96 hr posttreatment. Flow cytometric analysis revealed that the lack of proliferation in BDO2-treated LU cells was related to G1 arrest in about half of the cells and a delayed progression through S and G2 arrest in nearly all of the remaining cells. Both G1 and G2 arrest were prolonged and only a very small percentage of BDO2-treated cells were eventually able to replicate. Increased nuclear localization of both p53 and p21cip1 was observed in BDO2-treated cells, suggesting that the cell cycle arrest was p21cip1-mediated. These results demonstrate that BDO2 induces cell cycle perturbation and arrest even with short-term exposure that does not produce other pathologic cellular effects. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc.

Triple-negative breast cancers (TNBC) accounts for up to 20% of breast cancer cases. These tumors are negative for estrogen, progesterone, and HER2 receptors, which are markers used for diagnosis and treatment of other types of breast cancer. Clinically, TNBCs are notoriously aggressive, respond poorly to targeted therapies, have high rates of relapse, as well as poor prognosis. The molecular basis of TNBC oncogenesis is currently unknown, but it is possible that viral factors, such as Epstein-Barr Virus (EBV) miRNAs, play a role. EBV miRNAs have been implicated in the oncogenesis of several forms of cancer, including Burkitt’s lymphoma and nasopharyngeal carcinoma. The objective of this study, therefore, was to determine the differential expression of EBV miRNAs in TNBC tumors as compared to normal breast tissue. We conducted a comprehensive profiling of viral miRNAs in 48 TNBC tumors as compared to 15 control normal breast tissues, utilizing deep sequencing analysis software and publically available deep sequencing data. Four EBV miRNAs (BART18-3p, BART 8-5p, BART15, BART22) were significantly expressed in 11-17% of the TNBC tumors, and not in control normal breast tissue. Four novel putative EBV miRNAs were found to be differentially expressed in TNBC tumors as compared to control normal cells. Two of the novel putative EBV miRNAs were differentially expressed above 125 reads per million (RPM) in 20% and 27% of the TNBC tumors as compared to 0% and 6.6% of normal breast tissues, respectively. One novel putative EBV miRNA was expressed above 125 RPM in 83% of TNBC tumors as compared to 13% of normal breast tissue. One novel putative EBV miRNA was expressed above 125 RPM in 66% of the normal breast tissue as compared to 2% of the TNBCs. These putative EBV miRNAs localize within genomic regions of EBV, including the LMP-2 region. Ongoing work has so far computationally validated one of these miRNAs as a novel EBV miRNA; in vitro validation studies are in progress. This is the first report on the differential expression of EBV miRNAs in TNBC tumors. Since TNBC tumors are extremely heterogeneous, it is intriguing that 20% of TNBC tumors specifically express the same EBV miRNA. Our findings suggest that these differentially expressed EBV miRNAs may potentially play a role in the pathogenesis of TNBC. Citation Format: Alexander Blanchard, Tiffany Walls, Ramon Vidal, Stefan Bonn, Scott Harrison, Perpetua Muganda. Differential expression of Epstein Barr virus miRNAs in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1566. doi:10.1158/1538-7445.AM2017-1566

Triple-negative breast cancer (TNBC) accounts for 15-20% of the breast cancer cases. These tumors are heterogeneous and aggressive, fail to respond to targeted therapy, have poor prognosis, and a high risk of relapse. The molecular basis of this breast cancer subtype is currently unknown. Viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and human papillomavirus (HPV), are major suspects in the etiology of breast cancer. Since miRNAs have been implicated in the pathogenesis of breast cancer, including triple-negative breast cancer, we hypothesized that viral miRNAs may play a role in this aggressive disease. The objective of this project, therefore, was to determine the identity, prevalence, sequence variation, and differential expression of viral miRNAs in TNBC tumors as compared to control samples. We conducted a comprehensive profiling of viral miRNAs in 48 TNBC tumors as compared to 15 control normal breast tissues, utilizing deep sequencing analysis software and publically available deep sequencing data. Five novel putative HCMV miRNAs were found to be differentially expressed in TNBC tumors as compared to controls. Two of the putative miRNAs were differentially expressed in 60-79% of the TNBC tumors as compared to 0% of normal controls. Two additional putative miRNAs were expressed in 67-88% of TNBC tumors as compared to 25-31% of normal controls. One putative miRNA was expressed in 67% of TNBC tumors as compared to 88% of normal samples, while 1 putative miRNA in addition to HCMV-US25-1-3p displayed no significant differences. These putative HCMV miRNAs localize within genomic regions of HCMV that encode UL56 DNA packaging terminase subunit 2, single-stranded DNA-binding protein, noncoding RNA4.9, and enveloped glycoprotein M. Ongoing work has so far computationally validated one of these miRNAs as a novel HCMV miRNA. This is the first report on the differential expression of HCMV miRNAs in TNBC tumors. Since TNBC tumors are extremely heterogeneous, it is intriguing that 60-80% of TNBC tumors specifically express the same HCMV miRNA. Our findings suggest that these differentially expressed HCMV miRNAs may potentially play a role in the pathogenesis of TNBC. Citation Format: Tia Hudson, Scott Harrison, Dukka K C, Jan Prins, Perpetua M. Muganda. Differential expression of human cytomegalovirus microRNA in triple-negative breast cancer tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1120.

Cancer health disparities exist among different ethnicities and races. Various factors such as lifestyle, environmental exposure, genetics, and epigenetics are thought to play a role in the existence of these disparities. Viruses, which outnumber cells in the human body by 100-fold, have a major impact on human health. Viruses have been estimated to cause 15-20% of human cancers, and we expect that some of these oncogenic viruses may have the potential to impact health disparities. Next generation sequencing (NGS) technologies are being used to investigate novel virus-cancer associations and interactions, and several bioinformatics tools for the detection and analysis of virus sequences in human NGS data have recently become available. These tools have not been validated, however, for use in human cancers with extremely low levels of viral sequences. In this study, we have compared the sensitivity and specificity of READSCAN to a manually constructed analysis workflow in a common, commercial NGS web application. READSCAN is a freely available software application that utilizes automated “digital subtraction” to eliminate host reads and identify specific virus sequences in NGS data. The functionality of this tool was compared to a workflow constructed within the web interface for the commercially available NGS software Partek. This constructed workflow first subtracted human sequences and then aligned the remaining sequences against select viral genomes. The sensitivity of the various tools was compared using NGS data obtained from human cytomegalovirus (HCMV) infected cells. Specificity of the tools was determined by analyzing the same data set against the Epstein-Barr virus (EBV) and Human Papilloma virus type 16 (HPV-16). Partek and READSCAN detected 69.66% and 60.54% of the input number of reads as HCMV sequences. Under these conditions, neither bioinformatics tool detected EBV or HPV16 specific reads in the HCMV infected cell data. This indicated that both Partek and READSCAN were capable of readily detecting the presence of large amounts of virus reads in NGS data from infected cells in a specific manner. In order to test the ability of these tools to detect viral specific reads in NGS data from tumors, data obtained from the HPV16 positive cervical squamous cell carcinoma cell line SiHa were analyzed. Both Partek and READSCAN detected six reads out of 969,798 total reads as HPV16 sequences; EBV or HCMV sequences were not detected. Our results are in agreement with previously published observations for this SiHa cell line, in which five HPV16 specific reads were detected. While we are still in the process of testing additional bioinformatics tools and configurations, our results attest that the open–source bioinformatics tool READSCAN and the commercially available Partek are comparable in performance, but differ in utility. Partek is faster, has a user-friendly interface, and a knowledge of Linux commands is not required. READSCAN lacks these features, but it is free and has an algorithm more clearly proscribed in the literature for digital subtraction. Next steps include utilizing a wider range of digital subtraction software, evaluating a broader range of NGS data from demographically diverse origins, and investigating discordance as it is found between alternative tools and configuration parameters. Ultimately, an ensemble of tools and configuration contexts is expected to yield critical information on the role of viruses in cancer and associated health disparities Citation Format: Barbara Swanson, Scott Harrison, Dukka KC, Perpetua Muganda. Comparative analysis of bioinformatic tools for the detection of viral DNA sequences in tumor cells. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C04. doi:10.1158/1538-7755.DISP13-C04

Protein phosphorylation plays an important role in the regulation of cellular growth and proliferation and is thus thought to play a role in tumorigenesis. It has previously been reported that cells transformed by human cytomegalovirus (HCMV) contain two to four fold higher than normal levels of protein phosphorylation on serine and threonine residues, and two to six fold higher than normal levels of a casein kinase activity. We have now identified the major casein kinase activity found elevated in HCMV transformed cells as casein kinase type II; identification of this kinase was necessary in order to begin to define its role in HCMV mediated morphological transformation. Most of the differences in casein kinase II activity between normal and HCMV transformed cells were explained by differences in casein kinase II protein levels. This represents the first report concerning the elevation of casein kinase II activity in cells transformed by human cytomegalovirus.

Human cytomegalovirus (HCMV), like other DNA tumor viruses, induces morphological transformation of cells in vitro and stimulates host cell macromolecular synthesis in infected cells. Since other DNA tumor viruses, such as simian virus 40 and adenovirus, have previously been shown to interact with cellular protein p53, we investigated whether infection of cells by HCMV would modulate cellular p53 levels. Our results indicate that HCMV elevates cellular p53 levels on the order of 10- to 20-fold in infected fibroblasts. The induction of elevated p53 levels was dependent upon the presence of active virus and was prevented by neutralizing antibody. The induction of elevated p53 levels was determined not to be due to virus-receptor interactions or HCMV late events. The induction of elevated p53 levels commenced at immediate-early times of the HCMV multiplication cycle (6 h postinfection) and reached maximal levels by 24 h postinfection, before most of the HCMV DNA synthesis was initiated. HCMV immediate-early proteins were clearly shown to be responsible for elevating p53 levels in infected fibroblasts; expression of HCMV immediate-early region 1 and 2 proteins resulted in elevation of p53 levels in transfected human fibroblasts. This is the first report of increased p53 levels caused by HCMV in infected fibroblasts.

Ribonucleoprotein particles (RNPs) were extracted from monkey cell nuclei in media of low ionic strength. The rapidly labeled RNPs were comparable in terms of size, protein patterns and protein content to those extracted by sonication. The overnight labeled RNPs were homogenous (se diment ing at 6 0–6 5S and containing RNA of 30–31S), and appear to be a subset of pre-ribo-somal RNPs. This procedure produces nuclear RNPs free of contaminating chromatin. Nuclear RNPs (rapidly-labeled and overnight-labe1ed RNPs, extracted by either our procedure or by sonication), quantitatively precipitated in 10 mM MgCl2 when the concentration of monovalent cations was low. There was no detectable degradation of the RNA components, nor was there loss of enzymatic activity of an RNP associated protein kinase. Precipitation in Mg++ provides a rapid, gentle and convenient method of concentrating RNPs.

The examination of the regulation of the system of 3'-5' cyclic nucleotide monophosphates has only begun in cancer tissues. In human cancers, these studies are notably non-existent. However, in animal cancers, especially the Morris hepatomas, enough data has been gathered that, while risky, certain trends seem to begin to appear. Cyclic AMP is constant or lowered, while cyclic GMP is elevated in the fast growing hepatomas. Regulation of adenylate cyclase by protein hormones is reduced, while regulation by epinephrine may be increased. Binding of glucagon is decreased in the fast growing hepatomas. Guanylate cyclase, while being predominantly cytoplasmic in the normal liver, is predominantly membrane bound in the tumors. The liver enzyme is also readily stimulated by several chemical carcinogens. The cyclic GMP phosphodiesterases are decreased in these tumors; while the cAMP phosphodiesterases are increased. Although the cyclic nucleotide dependent protein kinases (histone as substrate) are altered in the hepatomas, observations of unique cyclic nucleotide binding proteins or cAMP independent protein kinases in cancer tissues may be of even greater significance for the development of or the maintenance of the neoplastic state of cells.