Laura J. Trudel

The rational design of nitric oxide selectivity in single-walled carbon nanotube near infrared fluorescence sensors for biological detection

A major challenge in the synthesis of nanotube or nanowire sensors is to impart selective analyte binding through means other than covalent linkages, which compromise electronic and optical properties. We synthesized a 3,4-diaminophenyl-functionalized dextran (DAP-dex) wrapping for single-walled carbon nanotubes (SWNTs) that imparts rapid and selective fluorescence detection of nitric oxide (NO), a messenger for biological signalling. The near-infrared (nIR) fluorescence of SWNT(DAP-dex) is immediately and directly bleached by NO, but not by other reactive nitrogen and oxygen species. This bleaching is reversible and shown to be caused by electron transfer from the top of the valence band of the SWNT to the lowest unoccupied molecular orbital of NO. The resulting optical sensor is capable of real-time and spatially resolved detection of NO produced by stimulating NO synthase in macrophage cells. We also demonstrate the potential of the optical sensor for in vivo detection of NO in a mouse model.

In vivo biosensing via tissue-localizable near-infrared-fluorescent single-walled carbon nanotubes

Single-walled carbon nanotubes (SWNT) are particularly attractive for biomedical applications, because they exhibit a fluorescent signal in a spectral region where there is minimal interference from biological media. Although SWNT have been used as highly-sensitive detectors for various molecules, their use as in vivo biosensors requires the simultaneous optimization of various parameters, including biocompatibility, molecular recognition, high fluorescence quantum efficiency and signal transduction. Here we demonstrate that a polyethylene glycol ligated copolymer stabilizes near infrared fluorescent SWNT sensors in solution, enabling intravenous injection into mice and the selective detection of local nitric oxide (NO) concentration with a detection limit of 1 μM. The half-life for liver retention is 4 hours, with sensors clearing the lungs within 2 hours after injection, thus avoiding a dominant route of in vivo nanotoxicology. After localization within the liver, it is possible to follow the transient inflammation using NO as a marker and signalling molecule. To this end, we also report a spatial-spectral imaging algorithm to deconvolute fluorescence intensity and spatial information from measurements. Finally, we show that alginate encapsulated SWNT can function as an implantable inflammation sensor for in vivo NO detection, with no intrinsic immune reactivity or other adverse response, for more than 400 days. These results open new avenues for the use of such nanosensors in vivo for biomedical applications.

Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2-mediated suppression of mTORC1.

Significance NO exposure triggered an ATM-mediated damage response in breast cancer cells involving activation of the LKB1 and TSC2 tumor suppressors, repression of mTORC1, ULK phosphorylation, and increased autophagic flux. The associated loss of cell viability indicates that autophagy can function as a cytotoxic response to nitrosative stress in tumor cells. Collectively, the data identify a nitrosative-stress signaling pathway that regulates autophagy. A more comprehensive understanding of signaling pathways regulating autophagy holds promise for developing new therapeutic approaches compromising prosurvival autophagic pathways that enable tumor cells to evade therapy, or promoting prodeath autophagic pathways that kill cancer cells. Reactive intermediates such as reactive nitrogen species play essential roles in the cell as signaling molecules but, in excess, constitute a major source of cellular damage. We found that nitrosative stress induced by steady-state nitric oxide (NO) caused rapid activation of an ATM damage-response pathway leading to downstream signaling by this stress kinase to LKB1 and AMPK kinases, and activation of the TSC tumor suppressor. As a result, in an ATM-, LKB1-, TSC-dependent fashion, mTORC1 was repressed, as evidenced by decreased phosphorylation of S6K, 4E-BP1, and ULK1, direct targets of the mTORC1 kinase. Decreased ULK1 phosphorylation by mTORC1 at S757 and activation of AMPK to phosphorylate ULK1 at S317 in response to nitrosative stress resulted in increased autophagy: the LC3-II/LC3-I ratio increased as did GFP-LC3 puncta and acidic vesicles; p62 levels decreased in a lysosome-dependent manner, confirming an NO-induced increase in autophagic flux. Induction of autophagy by NO correlated with loss of cell viability, suggesting that, in this setting, autophagy was functioning primarily as a cytotoxic response to excess nitrosative stress. These data identify a nitrosative-stress signaling pathway that engages ATM and the LKB1 and TSC2 tumor suppressors to repress mTORC1 and regulate autophagy. As cancer cells are particularly sensitive to nitrosative stress, these data open another path for therapies capitalizing on the ability of reactive nitrogen species to induce autophagy-mediated cell death.

Nitric oxide-induced genotoxicity, mitochondrial damage, and apoptosis in human lymphoblastoid cells expressing wild-type and mutant p53

Nitric oxide (NO•) is mutagenic and, under appropriate conditions of exposure, also induces apoptosis in many in vitro and in vivo experimental models. Biochemical and cellular mechanisms through which NO• induces apoptosis are incompletely understood, but involve p53/mitochondria-dependent signaling pathways. In this study, we exposed human lymphoblastoid cells harboring either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) to NO•, delivered by diffusion through Silastic tubing. Cells were exposed for 2 h at constant rates of 100–533 nM/s, similar to levels estimated to occur in vivo in inflamed tissues. DNA double-strand breaks and fragmentation detected 8–48 h after NO• treatment were more extensive in TK6 cells than in WTK-1 cells, whereas NO•-induced mutant fractions in both HPRT and TK1 genes were significantly lower in TK6 cells than in WTK-1 cells (P < 0.01–0.05). Treatment of TK6 cells with NO• caused extensive apoptosis, but this response was delayed and greatly reduced in magnitude in WTK-1 cells. Mitochondrial membrane depolarization and cytochrome c release were induced in both cell types. However, elevation of apoptotic protease-activating factor-1 (Apaf-1) protein and reduction of X-chromosome-linked inhibitor of apoptosis (XIAP) protein were observed only in TK6 cells. These results indicate that p53 status is an important modulator of NO•-induced mutagenesis and apoptosis, and suggest that levels of the Apaf-1 and XIAP proteins, but not mitochondrial depolarization and cytochrome c release, are regulated by p53 in these human lymphoblastoid cells. Thus, Apaf-1 and XIAP may play important roles in the regulation of p53-mediated apoptotic responses.

Antibody-antigen binding in organic-solvents

We describe, for the first time, the action of antibodies in anhydrous organic solvents. It has been demonstrated that the binding of a hapten, 4-aminobiphenyl, to the immobilized monoclonal antibody 2E11 is strong and specific not only in water but also in a variety of non-aqueous media. Further, the strength of interaction between antibody and hapten has been related to the hydrophobicity of the solvent: the more hydrophobic the solvent, the weaker the protein-ligand interaction.

Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production

The objective of this study was to compare monoclonal antibody production in hollow fiber bioreactor systems and murine ascites to determine the feasibility of the bioreactor system as a potential alternative to the use of mice. Three hybridoma cell lines were grown in each of three different hollow fiber bioreactor systems and in groups of 20 mice. Mice were primed with 0.5 ml pristane intraperitoneally 14 days prior to inoculation of 1X10(6) hybridoma cells. Each mouse was tapped a maximum of three times for collection of ascites. Ascites volumes and daily clinical observations were recorded. Bioreactors were harvested three times weekly for 65 day and were monitored by cell counts, cell viability and media glucose consumption. Time and materials logs were maintained. The total quantity of monoclonal antibody produced in 20 mice versus the mean production for the three different bioreactors in 65 days was as follows: cell line 2B11, 455 mg vs. 168 mg; cell line 3C9, 446 mg vs. 565 mg; and cell line RMK, 997 mg vs. 1023 mg. Mean monoclonal antibody concentration ranged from 4.07 to 8.37 mg/ml in murine ascites, and from 0.71 to 11.10 mg/ml in hollow fiber bioreactor system. Although time and material costs were generally greater for the bioreactors, these results suggest that hollow fiber bioreactor system merit further investigations as potentially viable in vitro alternatives to the use of mice for small scale (< 1 g) monoclonal antibody production.

Apoptotic Signaling Pathways Induced by Nitric Oxide in Human Lymphoblastoid Cells Expressing Wild-Type or Mutant p53

Loss of p53 function by inactivating mutations results in abrogation of NO*induced apoptosis in human lymphoblastoid cells. Here we report characterization of apoptotic signaling pathways activated by NO* in these cells by cDNA microarray expression and immunoblotting. A p53-mediated transcriptional response to NO* was observed in p53-wild-type TK6, but not in closely related p53-mutant WTK1, cells. Several previously characterized p53 target genes were up-regulated transcriptionally in TK6 cells, including phosphatase PPM1D (WIP1), oxidoreductase homolog PIG3, death receptor TNFRSF6 (Fas/CD95), and BH3-only proteins BBC3 (PUMA) and PMAIP1 (NOXA). NO* also modulated levels of several gene products in the mitochondria-dependent and death-receptor-mediated apoptotic pathways. Inhibitors of apoptosis proteins X-chromosome-linked inhibitor of apoptosis, cellular inhibitor of apoptosis protein-1, and survivin were significantly down-regulated in TK6 cells, but not in WTK1 cells. Smac release from mitochondria was induced in both cell types, but release of apoptosis-inducing factor and endonuclease G was detected only in TK6 cells. Fas/CD95 was increased, and levels of the antiapoptotic proteins Bcl-2 and Bcl-x/L were reduced in TK6 cells. Activation of procaspases 3, 8, 9, and 10, as well as Bid and poly(ADP-ribose) polymerase cleavage, were observed only in TK6 cells. NO* treatment did not alter levels of death receptors 4 and 5, Fas-associated death domain or proapoptotic Bax and Bak proteins in either cell line. Collectively, these data show that NO* exposure activated a complex network of responses leading to p53-dependent apoptosis via both mitochondrial and Fas receptor pathways, which were abrogated in the presence of mutant p53.

Nitric oxide activation of Keap1/Nrf2 signaling in human colon carcinoma cells

The transcription factor NF-E2-related nuclear factor 2 (Nrf2) regulates expression of genes that protect cells from oxidative damage. Here, we characterized nitric oxide (•NO)-induced Nrf2–Kelch-like ECH-associated protein 1 (Keap1) signaling and its role in counteracting •NO-induced apoptosis of human colon cancer HCT116 cells. Nrf2 was localized in the cytoplasm in control cells; •NO triggered its rapid nuclear accumulation, transcriptional activation, and up-regulation of HO-1, NQO1, and GCL, but not GST A4 and P1 subunits. Nrf2 accumulation in the nucleus was also associated with enhanced transcription and posttranscriptional modifications. (S)-nitrosation of Keap1 may contribute to nuclear accumulation of Nrf2 by facilitating its dissociation from Keap1, thus initiating •NO-mediated Nrf2–Keap1 signaling. •NO-mediated induction of ARE-dependent genes occurred well before apoptosis, as judged by caspase 3 activation. Collectively, these results show that the Nrf2–Keap1 signaling pathway mediates protective cellular responses to mitigate •NO-induced damage and may contribute to the relative resistance of HCT116 to •NO-induced cytotoxicity.

Aflatoxin B1-DNA Adduct Formation and Mutagenicity in Livers of Neonatal Male and Female B6C3F1 Mice

Exposure to genotoxic chemicals at a young age increases cancer incidence later in life. Aflatoxin B(1) (AFB(1)) is a potent genotoxin that induces hepatocellular carcinoma (HCC) in many animal species and in humans. Whereas adult mice are insensitive to aflatoxin-induced carcinogenesis, mice treated with AFB(1) shortly after birth develop a high incidence of HCC in adulthood. Furthermore, the incidence of HCC in adult male mice treated as infants is much greater than in females, reasons for which are unclear. In this study, treatment with AFB(1) produced similar levels of DNA damage and mutations in the liver of newborn male and female gpt delta B6C3F1 mice. Twenty-four hours after dosing with AFB(1) (6 mg/kg), the highly mutagenic AFB(1)-FAPY adduct was present at twice the level of AFB(1)-N(7)-guanine in liver DNA of males and females. A multiple dose regimen (3 × 2 mg/kg), while delivering the same total dose, resulted in lower AFB(1) adduct levels. Mutation frequencies in the gpt transgene in liver were increased by 20- to 30-fold. The most prominent mutations in AFB(1)-treated mice were G:C to T:A transversions and G:C to A:T transitions. At this 21-day time point, no significant differences were found in mutation frequency or types of mutations between males and females. These results show that infant male and female B6C3F1 mice experience similar amounts of DNA damage and mutation from AFB(1) that may initiate the neoplastic process. The gender difference in the subsequent development of HCC highlights the importance of elucidating additional factors that modulate HCC development.

Endogenously produced nitric oxide mitigates sensitivity of melanoma cells to cisplatin.

Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.