In silico Selection of Amplification Targets for Rapid Polymorphism Screening in Ebola Virus Outbreaks.

To achieve maximum transmission chain tracking in the current Ebola outbreak, whole genome sequencing (WGS) has been proposed to provide optimal information. However, WGS remains a costly and time-intensive procedure that is poorly suited for the large numbers of samples being generated, especially under severe time and work-environment constraints as in the present DRC outbreak. To better prepare for future outbreaks, where an apparent single outbreak may actually represent overlapping outbreaks caused by independent variants, and where rapid identification of emerging new transmission chains will be essential, a more practical method would be to amplify and sequence genomic areas that reveal the highest information to differentiate EBOV variants. We have identified four highly informative polymorphism PCR sequencing targets, suitable for rapid tracing of transmission chains and identification of new sources of Ebola outbreaks, an approach which will be far more practical in the field than WGS.

Ebolavirus comparative genomics.

The 2014 Ebola outbreak in West Africa is the largest documented for this virus. To examine the dynamics of this genome, we compare more than 100 currently available ebolavirus genomes to each other and to other viral genomes. Based on oligomer frequency analysis, the family Filoviridae forms a distinct group from all other sequenced viral genomes. All filovirus genomes sequenced to date encode proteins with similar functions and gene order, although there is considerable divergence in sequences between the three genera Ebolavirus, Cuevavirus and Marburgvirus within the family Filoviridae. Whereas all ebolavirus genomes are quite similar (multiple sequences of the same strain are often identical), variation is most common in the intergenic regions and within specific areas of the genes encoding the glycoprotein (GP), nucleoprotein (NP) and polymerase (L). We predict regions that could contain epitope-binding sites, which might be good vaccine targets. This information, combined with glycosylation sites and experimentally determined epitopes, can identify the most promising regions for the development of therapeutic strategies.This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness.

The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA Master(PLUS) HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Real-time PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs.

Truncation of histone H2A's C-terminal tail, as is typical for Ni(II)-assisted specific peptide bond hydrolysis, has gene expression altering effects.

Nickel(II), capable of transforming cells and causing tumors in humans and animals, has been previously shown by us to mediate hydrolytic truncation of histone H2A's C-terminal tail by 8 amino acids in both cell-free and cell culture systems. Since H2A's C-tail is involved in maintaining chromatin structure, such truncation might alter this structure and affect gene expression. To test the latter possibility, we transfected cultured T-REx 293 human embryonic kidney cells with plasmids expressing either wild type (wt) or truncated (q) histone H2A proteins, which were either untagged or N-terminally tagged with fluorescent proteins. Each histone variant was found to be incorporated into chromatin at 24 and 48 hr post-transfection. Cells transfected with the untagged plasmids were tested for gene expression by microarray and real-time PCR. Evaluation of the results for over 21,000 genes using the multidimensional scaling and hierarchical clustering methods revealed significant differences in expression of numerous genes between the q-H2A and wt-H2A transfectants. Many of the differentially expressed genes, including BAZ2A, CLDN18, CYP51A1, GFR, GIPC2, HMGB1, IRF7, JAK3, PSIP1, and VEGF, are cancer-related genes. The results thus demonstrate the potential of q-H2A to contribute to the process of carcinogenesis through epigenetic mechanisms.

Effects of o-aminoazotoluene on liver regeneration and p53 activation in mice susceptible and resistant to hepatocarcinogenesis.

The susceptibility to hepatocellular carcinoma (HCC) varies greatly within human populations in response to environmental risk agents. The mechanisms underlying differential susceptibility are still largely unknown and need to be clarified to improve HCC chemoprevention and therapeutic treatment. Inbred rodent strains with established predispositions for hepatocarcinogenesis offer the opportunity to identify intrinsic susceptibility and resistance factors. Previously, we have characterized mouse strains showing differential susceptibility to o-aminoazotoluene (OAT) and established that susceptibility does not result from OAT metabolism or genotoxicity in the livers of resistant and susceptible mice. In this study we have found that OAT differently affects hepatocyte proliferation in mice after partial hepatectomy (PH). OAT inhibited hepatocyte proliferation by 60-80% in the livers of susceptible mice, whereas resistant mice showed less than 15% inhibition. The inhibition resulted in significant delay of hepatic mass recovery in susceptible mice. OAT induced p53 stabilization and transcriptional activation in response to carcinogen treatment to the same degree in both, susceptible and resistant mice. Taken together, our data support inhibition of hepatocyte proliferation as a major cause for increased mouse susceptibility to hepatocarcinogenesis, and acceleration of functional liver recovery may offer a way to increase resistance to hepatic neoplasms. These results may have relevance to clinical observations of HCCs and implications for HCC chemoprevention and treatment.

Structure-Based Design of Anticancer Prodrug PABA/NO.

Glutathione S-transferase (GST) is a superfamily of detoxification enzymes, represented by GSTalpha, GSTmu, GSTpi, etc. GSTalpha is the predominant isoform of GST in human liver, playing important roles for our well being. GSTpi is overexpressed in many forms of cancer, thus presenting an opportunity for selective targeting of cancer cells. Our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GSTpi-overexpressing cancer cells yielded PABA/NO, which exhibited anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin (Findlay et al. Mol. Pharmacol. 2004, 65, 1070-1079). Here, we present the details on structural modification, molecular modeling, and enzymatic characterization for the design of PABA/NO. The design was efficient because it was on the basis of the reaction mechanism and the structures of related GST isozymes at both the ground state and the transition state. The ground-state structures outlined the shape and property of the substrate-binding site in different isozymes, and the structural information at the transition-state indicated distinct conformations of the Meisenheimer complex of prodrugs in the active site of different isozymes, providing guidance for the modifications of the molecular structure of the prodrug molecules. Two key alterations of a GSTalpha -selective compound led to the GSTpi-selective PABA/NO.

Down-regulation of insulin-like growth factor binding protein-5 (IGFBP-5): novel marker for cervical carcinogenesis.

To better understand the underlying pathways of cervical carcinogenesis, cDNA microarray analysis was performed on 2 sets of squamous cell carcinomas (SCCs) and their adjacent normal squamous epithelia. Consistently altered expression was detected for 32 genes. Real-time RT-PCR analysis was conducted on a selected subset of these genes (S100A2, GPC4, p72, IGFBP-5, TRIM2 and NAB2) for 14 additional SCCs and 10 normal epithelia. This found that, of the 6 candidate genes, only the insulin-like growth factor binding protein-5 (IGFBP-5) mRNA was generally and significantly under-expressed in SCCs (p < 0.001). All normal cervical epithelia (30 of 30) stained positively for IGFBP-5 protein, with 70% showing strong staining, whereas 65% (17/26) of SCC had complete loss of IGFBP-5, and only 8% (2/26) SCC retained strong expression (p < 0.001). Immunohistochemistry of premalignant cervical intraepithelial neoplasia (CIN) lesions shows a significantly weaker or negative staining in advanced CIN3 lesions compared with normal squamous epithelia (p = 0.001). This is the first study to show that down-regulation of IGFBP-5 protein correlates with cervical carcinogenesis and does so at a preneoplastic stage.

PABA/NO as an anticancer lead: analogue synthesis, structure revision, solution chemistry, reactivity toward glutathione, and in vitro activity.

PABA/NO is a diazeniumdiolate of structure Me(2)NN(O)=NOAr (where Ar is a 5-substituted-2,4-dinitrophenyl ring whose 5-substituent is N-methyl-p-aminobenzoic acid). It has shown activity against human ovarian cancer xenografts in mice rivaling that of cisplatin, but it is poorly soluble and relatively unstable in water. Here we report structure-based optimization efforts resulting in three analogues with improved solubility and stability in aqueous solution. We sought to explain PABA/NO's physicochemical uniqueness among these four compounds, whose aminobenzoic acid precursors differ structurally only in the presence or absence of the N-methyl group and/or the position of the carboxyl moiety (meta or para). Studies revealed that PABA/NO's N-methyl-p-aminobenzoic acid substituent is bound to the dinitrobenzene ring via its carboxyl oxygen while the other three are linked through the aniline nitrogen. This constitutes a revision of the previously published PABA/NO structure. All four analogues reacted with GSH to produce bioactive nitric oxide (NO), but PABA/NO was the most reactive. Consistent with PABA/NO's potent suppression of A2780 human ovarian cancer xenograft growth in mice, it was the most potent of the four in the OVCAR-3 cell line.

Deamidation of peptides in aerobic nitric oxide solution by a nitrosative pathway.

Hydrolytic deamidation of asparagine (Asn) and glutamine (Gln) residues to aspartate (Asp) and glutamate (Glu), respectively, can have significant biological consequences. We hypothesize that a wholly different mechanism of deamidation might occur in the presence of aerobic nitric oxide (NO). Accordingly, we examined the deamidating ability of aerobic NO toward three model peptides, 2,4-dinitrophenyl (DNP)-Pro-Gln-Gly, Lys-Trp-Asp-Asn-Gln, and Ser-Glu-Asn-Tyr-Pro-Ile-Val, incubating them with the NO-generating compound, Et(2)N[N(O)NO]Na (DEA/NO, 30-48 mM), in aerobic, pH 7.4, buffer at 37 degrees C. DNP-Pro-Glu-Gly was detected after 2 h, while Lys-Trp-Asp-Asp-Gln, Lys-Trp-Asp-Asn-Glu, and Ser-Glu-Asp-Tyr-Pro-Ile-Val were detected within 10 min, accumulating in apparent yields of up to approximately 10%. In the latter case, tyrosine nitration was also observed, producing the expected nitrotyrosine residue. DEA/NO solutions preincubated to exhaust the NO before the peptides were added did not induce detectable deamidation. The data demonstrate that aerobic NO exposures can lead to nitrosative deamidation of peptides, a pathway that differs from the established hydrolytic deamidation mechanism in several key respects: the by-products of the former are molecular nitrogen and an acid (HONO) while that of the latter is a base (NH(3)); the nitrosative path can in principle proceed in the absence of water molecules; Asn is much more easily deamidated than Gln in the hydrolytic pathway, while the two amino acid residues were deamidated to a similar extent by exposure to NO in the presence of oxygen.

Overexpression of LAMP3/TSC403/DC-LAMP promotes metastasis in uterine cervical cancer.

LAMP3 (DC-LAMP, TSC403, CD208) was originally isolated as a gene specifically expressed in lung tissues. LAMP3 is located on a chromosome 3q segment that is frequently amplified in some human cancers, including uterine cervical cancer. Because two other members of the LAMP family of lysosomal membrane glycoproteins, LAMP1 and LAMP2, were previously implicated in potentially modulating the interaction of vascular endothelial and cancer cells, we hypothesized that LAMP3 might also play an important part in metastasis. To clarify the metastatic potential of LAMP3 in cervical cancers, we transfected a LAMP3 expression vector into a human uterine cervical cancer cell line, TCS. In an in vitro invasion assay, the migration of LAMP3-overexpressing TCS cells was significantly higher than in control TCS cells. In an in vivo metastasis assay, distant metastasis was detected in 9 of 11 LAMP3-overexpressing TCS cell-injected mice and in only 1 of 11 control mice. Histologic study showed that LAMP3-overexpressing cells readily invaded into the lymph-vascular space. In clinical samples, quantitative real-time reverse transcription-PCR (RT-PCR) analyses showed that LAMP3 mRNA was significantly up-regulated in 47 of 47 (100%) cervical cancers and in 2 of 15 (13%) cervical intraepithelial neoplasias, compared with a low level of LAMP3 mRNA expressed in normal uterine cervixes. Interestingly, high LAMP3 expression was significantly correlated with the overall survival of patients with stage I/II cervical cancers. These findings indicate that LAMP3 overexpression is associated with an enhanced metastatic potential and may be a prognostic factor for cervical cancer.

Altered protein expression in endometrial carcinogenesis.

We have discovered several protein biomarkers that are altered during carcinogenesis of the human uterine endometrium. Proteins prepared from 19 endometrial carcinomas (Group A), and 20 normal endometria obtained from benign gynecological conditions (Group B), were investigated by Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). Two proteins, EC1 and EC2, were consistently expressed differentially. EC1 had an increased level of expression in carcinomas (P<0.001), while EC2 was expressed at a lower level (P=0.004). The isoelectric points of EC1 and EC2 were approximately pH 5.0 and 7.0, respectively. These proteins are thus potential biomarkers for detection of endometrial carcinoma.

Inhibition of prolidase activity by nickel causes decreased growth of proline auxotrophic CHO cells.

Occupational exposure to nickel has been epidemiologically linked to increased cancer risk in the respiratory tract. Nickel-induced cell transformation is associated with both genotoxic and epigenetic mechanisms that are poorly understood. Prolidase [E.C.3.4.13.9] is a cytosolic Mn(II)-activated metalloproteinase that specifically hydrolyzes imidodipeptides with C-terminal proline or hydroxyproline and plays an important role in the recycling of proline for protein synthesis and cell growth. Prolidase also provides free proline as substrate for proline oxidase, whose gene is activated by p53 during apoptosis. The inhibition of prolidase activity by nickel has not yet been studied. We first showed that Ni(II) chloride specifically inhibited prolidase activity in CHO-K1 cells in situ. This interpretation was possible because CHO-K1 cells are proline auxotrophs requiring added free proline or proline released from added Gly-Pro by prolidase. In a dose-dependent fashion, Ni(II) inhibited growth on Gly-Pro but did not inhibit growth on proline, thereby showing inhibition of prolidase in situ in the absence of nonspecific toxicity. Studies using cell-free extracts showed that Ni(II) inhibited prolidase activity when present during prolidase activation with Mn(II) or during incubation with Gly-Pro. In kinetic studies, we found that Ni(II) inhibition of prolidase varied with respect to Mn(II) concentration. Analysis of these data suggested that increasing concentrations of Mn(II) stabilized the enzyme protein against Ni(II) inhibition. Because prolidase is an important enzyme in collagen metabolism, inhibition of the enzyme activity by nickel could alter the metabolism of collagen and other matrix proteins, and thereby alter cell-matrix and cell-cell interactions involved in gene expression, genomic stability, cellular differentiation, and cell proliferation.

Correlation between p14(ARF)/p16(INK4A) expression and HPV infection in uterine cervical cancer.

The CDKN2A locus on human chromosome 9p21 encodes two tumor suppressors, p14(ARF) and p16(INK4A), which enhance the growth-suppressive functions of the retinoblastoma (Rb) and the p53 proteins, respectively. Conversely, the E6 and E7 oncoproteins of the high-risk human papillomaviruses (HPVs) causally associated with carcinogenesis of the uterine cervix contributes to tumor development by inactivating p53 and Rb. Nevertheless, a correlation between expression of p14(ARF)/p16(INK4A) and HPV infection in uterine cervix is less clear. To clarify this, we examined 25 cervical cancers and 11 normal uterine cervixes. HPV was detected in 21 of 25 cervical cancers (84%) and their subtype was determined by PCR-RFLP. Quantitative real-time RT-PCR assays showed overexpression of p14(ARF) mRNA in all 21 HPV-positive cases (100%). p16(INK4A) mRNA was overexpressed in 17 cases of the HPV-positive cases (81%). In four HPV-negative cancers, reduced expression of p14(ARF) mRNA was detected in two cases (50%) and reduced p16(INK4A) mRNA in three cases (75%). Our data indicate that the overexpression of p14(ARF) and p16(INK4A) strongly associates with HPV-positive cervical cancers and that reduced expression of p14(ARF) and p16(INK4A) correlates with HPV-negative cervical cancers. These findings may indicate that impaired p14(ARF) and p16(INK4A) mRNA expression contribute to tumor development in HPV-negative cervical cancers by failure to support p53 and Rb instead of their inactivation by HPV E6 and E7.

Nitric oxide prodrugs and metallochemotherapeutics: JS-K and CB-3-100 enhance arsenic and cisplatin cytolethality by increasing cellular accumulation.

Development of chemotherapeutic resistance is a major cause of pharmacologic failure in cancer treatment. One mechanism of resistance in tumor cells is the overexpression of glutathione S-transferases (GSTs) that serve two distinct roles in the development of drug resistance via the formation of glutathione conjugates with drugs for their cellular efflux, and the inhibition of the mitogen-activated protein kinase pathway. To target GST-based resistance to chemotherapeutics, a series of nitric oxide (NO)-releasing diazeniumdiolates was synthesized and shown to release NO on reaction with GST and/or glutathione. Two diazeniumdiolates, JS-K [O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] and CB-3-100 [O(2)-(2,4-dinitrophenyl) 1-[4-(N,N-diethylcarboxamido)piperazin-1-yl]diazen-1-ium-1,2-diolate], were studied on their ability in reversing arsenic and cisplatin resistance in a rat liver cell line that is tumorigenic and shows acquired tolerance to arsenic and cisplatin, with overexpression of GSTs. The enhanced cytolethality produced by the NO donors was accompanied by increased accumulation of arsenic and platinum within cells and by enhanced activation of mitogen-activated protein kinase members c-jun-NH-kinase and extracellular signal-regulated kinase. Our data indicate that JS-K and CB-3-100 are promising lead compounds for the possible development of a novel class of adjuvant chemotherapeutic agents potentially capable of reversing arsenic and cisplatin resistance in certain tumor cells.

Tumor cell responses to a novel glutathione S-transferase-activated nitric oxide-releasing prodrug.

We have used structure-based design techniques to introduce the drug O(2)-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino) phenyl] 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), which is efficiently metabolized to potentially cytolytic nitric oxide by the pi isoform of glutathione S-transferase, an enzyme expressed at high levels in many tumors. We have used mouse embryo fibroblasts (MEFs) null for GSTpi (GSTpi(-/-)) to show that the absence of GSTpi results in a decreased sensitivity to PABA/NO. Cytotoxicity of PABA/NO was also examined in a mouse skin fibroblast (NIH3T3) cell line that was stably transfected with GSTpi and/or various combinations of gamma-glutamyl cysteine synthetase and the ATP-binding cassette transporter MRP1. Overexpression of MRP1 conferred the most significant degree of resistance, and in vitro transport studies confirmed that a GSTpi-activated metabolite of PABA/NO was effluxed by MRP1 in a GSH-dependent manner. Additional studies showed that in the absence of MRP1, PABA/NO activated the extracellular-regulated and stress-activated protein kinases ERK, c-Jun NH(2)-terminal kinase (JNK), and p38. Selective inhibition studies showed that the activation of JNK and p38 were critical to the cytotoxic effects of PABA/NO. Finally, PABA/NO produced antitumor effects in a human ovarian cancer model grown in SCID mice.

JS-K, a glutathione/glutathione S-transferase-activated nitric oxide donor of the diazeniumdiolate class with potent antineoplastic activity.

We have previously shown that nitric oxide (NO) inhibits growth and induces differentiation and apoptosis in acute myeloid leukemia cells, with the HL-60 human myeloid leukemia line being particularly sensitive to NO-mediated cytolysis. With the goal of identifying a prodrug that can target NO to the leukemia cells without inducing NO-mediated systemic hypotension, we have screened a series of O(2)-aryl diazeniumdiolates designed to be stable at physiological pH but to release NO upon reaction with glutathione. O(2)-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) proved to be the most active antiproliferative agent among those tested in HL-60 cells, with an IC(50) of 0.2-0.5 microM. After 5 days of exposure to 0.5 micro M JS-K, HL-60 cells had differentiated and acquired some of the phenotypic features of normal monocytes. One- to 2-day treatment with JS-K at concentrations of 0.5-1 microM resulted in apoptosis induction in a concentration- and caspase-dependent manner. JS-K also inhibited the growth of solid tumor cell lines but to a lesser extent than HL-60 cells. JS-K was administered i.v. to nonobese diabetic-severe combined immune deficient mice at doses of up to 4 micromol/kg without inducing significant hypotension. The growth of s.c. implanted HL-60 cells was reduced by approximately 50% when the mice received i.v. injections three times/week with 4 micromol/kg boluses of JS-K. Histological examination of tumor explants from JS-K-treated animals revealed extensive necrosis. Similar results were seen with s.c. human prostate cancer (PPC-1) xenografts. Our data indicate that JS-K is a promising lead compound for the possible development of a novel class of antineoplastic agents.