Aiming to identify immune molecules with a novel function in cancer pathogenesis, we found the cluster of differentiation 177 (CD177), a known neutrophil antigen, expression to be positively correlated with relapse-free (RFS), metastasis-free (MFS) or overall survival (OS) in several solid cancers including those from breast, prostate, cervix, and lung. To study the role of CD177 in breast cancer, we generated a total body Cd177 knockout mouse. These mice had no profound phenotype at 3 - months of age or younger. The only phenotype found at this age was reduced peripheral neutrophil counts, but no difference in their ability to clear infections. Upon further analysis these mice developed an age dependent hyperproliferative mammary gland phenotype at 10 - months of age that was lost in mice 15 - months and older. Focusing on breast cancer, we found that CD177 is expressed in normal breast epithelial cells and is significantly reduced in invasive cancer. We found that CD177 suppresses breast cancer pathogenesis. To understand the mechanism behind CD177 mediated suppression of breast cancer, we performed mass spectrometry on the purified CD177 complex. Mass spectrometry and co-immunoprecipitation results revealed CD177 interacts with β-Catenin and glycolytic enzymes PFK, aldolase A, GAPDH and enolase-ɑ. Further studies revealed that mechanistically CD177 forms a complex with ECadherin and β-Catenin at adherens junctions. This physical interaction between CD177, E-Cadherin and β-Catenin prevents β-Catenin activation via the canonical WNT. We also found CD177 suppressed WNT/β-Catenin signaling independent of E-Cadherin with an unknown protein. Thus, we identified a novel protein complex involving CD177 and proteins from adherens junctions that can suppress cancer formation via inhibiting the WNT/β-Catenin signaling pathway, a key cellular biological process relevant to the oncogenesis of multiple cancer types and tissue development. The lack of WNT/β- Catenin signaling control explains how mice without CD177 develop hyperproliferation of mammary epithelium in the mouse mammary gland. Interestingly, this phenotype is lost with age, possibly due to a decrease in WNT/β-Catenin signaling resulting from a decrease in progesterone and estrogen. In addition to CD177’s role in the regulation of WNT/β-Catenin signaling we also identified that CD177 plays a role in cancer cell metabolism. Since metabolism plays a significant role in cancer and CD177 interacts with glycolytic enzymes, we sought to determine if CD177 plays a role in metabolism. CD177 appears to interact with glycolytic enzymes, PFK, aldolase A, GAPDH, and ɑ-enolase and ultimately suppresses their mRNA expression. Furthermore, we found novel localization of CD177 at the mitochondrion, thus providing a potential explanation as to how an extracellular membrane bound protein such as CD177 interacts with glycolytic enzymes. Metabolic analysis of CD177 expression on cancer cells revealed that CD177 leads to a decrease in glucose uptake and a slight decrease in basal glycolysis, but an increase in lactate concentration. Further metabolic profiling also revealed that CD177 expression results in a significant decrease in glycolytic capacity (ECAR). Expression of CD177 also resulted in a significant decrease in basal respiration, ATP production, maximal respiration, and spare capacity (OCR) as well as an increase in reactive oxygen species. These data reveal that CD177 plays a novel role in cancer cell metabolism.

Replicative and chronological lifespan are two different modes of cellular aging. Chronological lifespan is defined as the duration during which quiescent normal cells retain their capacity to re-enter the proliferative cycle. This study investigates whether changes in metabolism occur during aging of quiescent normal human fibroblasts (NHFs) and the mechanisms that regulate these changes. Bioenergetics measurements were performed in quiescent NHFs from younger (newborn, 3-d, 5-m, and 1-y) and older (58-y, 61-y, 63-y, 68-y, and 70-y) donors as well as NHFs from the same individual at different ages (29-y, 36-y, and 46-y). Results show significant changes in cellular metabolism during aging of quiescent NHFs: old NHFs exhibit significant decreases in glycolytic flux and lactate levels, and increases in oxygen consumption rate (OCR) and ATP levels compared to young NHFs. Results from Seahorse Mito Stress Test show that old NHFs with a lower Bioenergetic Health Index (BHI) are more prone to oxidative stress compared to young NHFs with a higher BHI. The increase in OCR in old NHFs is associated with a shift in mitochondrial dynamics more towards fusion. Genetic knock-down of mitofusin 1 (MFN1) and optic atrophy 1 (OPA1) in old NHFs decreased OCR and shifted metabolism more towards glycolysis. Downregulation of MFN1 and OPA1 also suppressed the radiation-induced increase in doubling time of NHFs. These results suggest that a metabolic shift from glycolysis in young to mitochondrial respiration in old NHFs occurs during the chronological lifespan, and MFN1 and OPA1 regulate this process. Age-associated metabolic reprogramming can also impact the age-related disease progression such as cancer. Recent evidence suggests a significant role of fibroblasts in pancreatic ductal adenocarcinoma (PDAC) stromal cellularity, metabolism, and therapy response. Considering PDAC being an age-related disease and a dismal 5-y survival of less than 9%, this study investigates whether stromal aging regulates PDAC progression. Results show that NHFs from older healthy individuals stimulate proliferation of PDAC cells compared to younger NHFs. Results from an in vivo study show that rate of tumor growth in xenografts of PDAC cells cultured with the old NHFs is significantly increased compared to the co-cultures of the young NHFs. In addition, decreased survival was also observed in mice carrying xenograft of co-culture of PDAC and the old NHFs compared to the young NHFs. Results from quantitative RT-PCR assays show that arachidonic acid lipoxygenase (ALOX12) expression decreased in PDAC, but increased in stromal fibroblasts in an age-dependent manner. Molecular inhibition of ALOX12 in the old NHFs suppressed PDAC proliferation in co-culture. These results show that aging of stromal fibroblasts aging promotes progression of PDAC, and identified ALOX12 and its metabolite 12-hydroxyeicosatetraenoic acid (12(S)-HETE) as critical regulators of PDAC proliferation. Taken together, findings from this project demonstrate that age-associated metabolic reprogramming of NHFs from glycolysis in young to mitochondrial respiration in old regulates fibroblasts-induced stimulation of proliferation of human PDAC. Importantly, results from this study are anticipated to contribute to the development of novel approaches targeting stromal aging for cancer prevention and therapy response.

No description available.

A scientific and cultural revolution occurred with the sequencing of the human genome. The information provided by this accomplishment has provided tools for researchers to test new ideas in silico and on the bench. In redox biology many of the genes, transcripts, proteins, and redox active species have been well characterized. However, the vast majority have not been quantitated in an absolute manner. This is a necessary step to provide the tools for mathematical modeling and systems biology approaches for predicting changes in the cellular redox environment and the biochemical and biological consequences. Here we demonstrate techniques for the absolute quantitation of human catalase, glutathione peroxidase, peroxiredoxin, thioredoxin, and superoxide dismutase within cells. These techniques can be parsed into two groups: detection of activity and detection of total amount of species. Methods for the absolute quantitation of active catalase, peroxiredoxins, and superoxide dismutase have been developed by utilizing specific characteristics of each enzyme. Catalase generates oxygen in the presence of hydrogen peroxide that can easily be detected with a Clark electrode (oxygen monitor); the data are fit to a single-exponential to determine the observed pseudo-first-order rate constant. From this the effective number of fully active catalase enzymes in the sample can be determined. Peroxiredoxin in the disulfide state can be reduced by thioredoxin; thioredoxin from E. coli loses fluorescence upon oxidation. The loss of fluorescence over time is mathematically fit to a single-exponential to determine the observed pseudo first-order rate constant from which the number of active enzymes can be determined. Using an inhibition assay to detect superoxide dismutase activity along with the rate constants at which superoxide reacts with the dismutase and the competing superoxide-reacting-indicator-molecule, the concentration of active superoxide dismutase can be determined. To detect the total amount of protein of an enzyme in a biological sample, an immunoassay was first implemented. This method utilized Bio-Plex® beads from Bio-Rad; however, it was problematic because the antibodies applied did not perform satisfactorily not allowing sufficient signal-to-noise to be deployed. Quantitative mass spectrometry was then implemented to detect total catalase, glutathione peroxidase 1, peroxiredoxin 2, and thioredoxin 1 in human red blood cells. With the absolute concentration of these enzymes and proteins along with data for oxygen consumption rates and peroxisomal hydrogen peroxide concentration for several cell lines, we hypothesize that a reasonable model of hydrogen peroxide and superoxide flux can be constructed. Quantitative data such as these provide the foundation for the new redox biology of the 21st century. Presented here is a roadmap for the obligatory first steps to dissect quantitatively the cellular and tissue metabolic pathways and redox networks that are the basis of all of biology.

Hydrogen sulfide (H2S) reduces ischemia reperfusion (IR) injury by stimulating adenosine triphosphate (ATP) sensitive potassium channels (KATP) [1-5]. Demonstrating H2S stimulation is unique to KATP, as other inwardly rectifying potassium (Kir) channels demonstrate inhibition or are unaffected [6]. We recently showed that H2S inhibits Kir2 and Kir3 by decreasing channel sensitivity to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) [6]. Here, we test the hypothesis that H2S regulation of Kir6.2, a pore-forming subunit of the KATP channel, is also dependent on PIP2. Using whole-cell patch-clamp we show that H2S increases the activity of Kir6.2 channels expressed in HEK-293 cells. To study the mechanism, we modulated PIP2 levels by expressing a light- activated phosphatase, or by including high levels of a water-soluble PIP2 analog in the patch pipette. The results suggest that H2S augmentation of Kir6.2 channel activity is increased when PIP2 levels are elevated.

Adeno-associated Virus (AAV) Rep proteins are multifunctional proteins that carry out various DNA transactions required for the life cycle of AAV. The Rep proteins have been found to be important for genome replication, gene regulation, site-specific integration and play an essential role in genome packaging. There are two main groups of Rep proteins: large and small Reps; both groups are SF3 helicase family members. During DNA packaging, studies have shown that the small Rep proteins are critical to produce fully packed particles. Using stopped-flow kinetic analysis, we show a significant difference in helicase activity between the small and large Rep proteins that support the notion that the small Rep proteins are the primary motor to package DNA due to more efficient motor activity. That leaves the large Rep proteins to serve a different role during packaging. In previous studies, we have shown that the large Rep proteins have the ability to change their oligomeric state depending on the nature of the DNA substrate. We can observe double octameric rings with single-stranded DNA (ssDNA) and heptameric complex with double-stranded DNA (dsDNA). To understand Rep protein structural plasticity, we solved a 6.96 Å cryo-EM structure of Rep68*/ssDNA complex illustrating that the formation of Rep octamer rings is dominated by interactions between their N-terminal origin-binding domain (OBD) using the same interface utilized to recognize dsDNA specifically. Our analysis of the structural data suggests that the double octameric ring structure is stabilized by ssDNA that bridges octameric rings together. The structure shows that the helicase domains are highly flexible and that ssDNA is present at the center of the ring. In addition, we have solved a preliminary 12 Å model of Rep68*/dsDNA complex showing a heptameric ring encircling a DNA molecule. Our structural and functional data offer insights to the various Rep-DNA scaffolds that can perform diverse functions during the AAV life cycle.

The enzyme DNA topoisomerase I is an essential enzyme that plays an important role in eukaryotic and prokaryotic cellular processes such as DNA replication, transcription, recombination and repair. Mycobacterium tuberculosistopoisomerase I (MtTOP1) is a validated drug target for antituberculosis treatment. Mycobacterial topoisomerase I regulates the topological constraints in chromosomes and helps in maintaining the growth of mycobacteria. The N- terminal domain (NTD) of mycobacterial topoisomerase I contains conserved catalytic domains that along with the active site Tyrosine are involved in cleaving and rejoining a single strand of DNA. Magnesium is required in DNA cleavage activity of type IA topoisomerases. The C-terminal domain (CTD) of mycobacterial topoisomerase I is divided into four subdomains (D5-D8) and a positively charged tail. Each subdomain has a GxxGPY sequence motif. The DNA binding, relaxation, cleavage, religation, catenation and decatenation ability of each subdomains of CTD were studied. The present study shows that each subdomain has its own characteristics. Subdomain D8 and D7 are responsible for maintaining the relaxation activity of mycobacterial topoisomerase I. Subdomain D5 is essential to maintain the DNA cleavage, religation, catenation and decatenation activity. A new crystal structure of MtTOP1-704t (amino acids A2-T704 containing NTD+D5 domains) was obtained. Structures with ssDNA substrate bound to the active site (Y342) in the presence and absence of Mg2+ were also investigated. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition position for cleavage of a specific phosphodiester linkage to form a covalent intermediate. Meanwhile, the enzyme/DNA complex with Mg2+ bound at active site may present the post- transition state for religation in the enzyme’s multiple-state DNA relaxation activity. The critical function of a strictly conserved glutamic acid in acid-base catalysis of the DNA cleavage step was also demonstrated by site-directed mutagenesis. The present work provides new functional insights into the more stringent requirement for DNA rejoining versus cleavage by type IA topoisomerase, and further establishes the potential for select interference of DNA rejoining via specific inhibitors.

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are known to elicit adverse health effects including skin toxicity and cancer to animals and humans. 4-Monochlorobiphenyl (PCB3), a low-chlorinated airborne PCB conger is present in human blood and the environment. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of PCB3, has been shown to induce oxidative stress and toxicity in human mammary and prostate epithelial cells. These studies were designed to investigate and characterize the cellular responses to 4-ClBQ in HaCaT human skin keratinocytes. We found that 4-ClBQ treatment increased cellular reactive oxygen species (ROS) production, inhibited cell proliferation, and induced toxicity in HaCaT cells. Results from a Human Antioxidant Mechanism PCR array and quantitative RT-PCR assay showed that the mRNA levels of antioxidant gene selenoprotein P (sepp1) and catalase were significantly downregulated by the treatment, which correlated with evident decreases in their protein levels and catalase enzymatic activity. Pharmacological (sodium selenite supplementation) and molecular (sepp1overexpression) manipulation of SEPP1 expression significantly suppressed 4-ClBQ induced oxidative stress and toxicity. Additional results demonstrated that decreased catalase expression was associated with an inhibition in transcriptional coactivator peroxisome proliferator activated receptor Γ coactivator 1α (PGC-1α) expression. Overexpression of pgc-1α restored catalase expression and activity and consequently protected HaCaT cells from 4-ClBQ induced oxidative stress and toxicity. Furthermore, results from metabolic flux analysis using Seahorse XF96 Analyzer showed that 4-ClBQ treatment increased extracellular acidification rate, proton production rate, and oxygen consumption rate, which were associated with increases in glucose uptake and in the expression of glucose metabolism regulatory gene hexokinase 2, pyruvate kinase M2, and glucose-6-phosphate dehydrogenase (G6PD). G6PD is the rate-limiting enzyme of the pentose phosphate pathway. The enhanced expression of G6PD correlated with an increase in cellular glutathione content; and inhibition of G6PD activity sensitized HaCaT cells to 4-ClBQ induced toxicity, suggesting that the protective function of the pentose phosphate pathway is active in 4-ClBQ treated cells. Interestingly, we also found that 4-ClBQ selectively and significantly decreased mitochondrial complex II subunits C (sdhc) and D (sdhd) mRNA expression and subsequently reduced complex II activity leading to metabolic oxidative stress and toxicity, which were significantly suppressed by overexpressing sdhc and sdhd in HaCaT cells. Taken together, findings from this project demonstrate that 4-ClBQ treatment increases ROS production through perturbing cellular metabolism and mitochondrial function and decreases antioxidant capacity by inhibiting SEPP1 and catalase expression in HaCaT cells. This imbalance due to increased mitochondrial prooxidant production and decreased antioxidant capacity leads to oxidative stress and toxicity. Importantly, antioxidant supplementation could abrogate 4-ClBQ induced toxicity, suggesting that antioxidants, especially nutrient-based manipulation of selenoproteins could be promising countermeasures for PCB induced adverse health effects in humans.

Manganese superoxide dismutase (MnSOD), is a mitochondria-localized antioxidant enzyme that scavenges superoxide anions generated in the respiratory chains, has been known to play an important role in the radioprotection, and function as a tumor suppressor gene in many types of cancer. Sirtuin 3 (Sirt3) is a mitochondrial NAD+-dependent deacetylase that regulates mitochondrial oxidative metabolism, MnSOD activity, and tumorigenesis. The current study was designed to examine the role of MnSOD and Sirt3 in regulating mitochondrial metabolism and steady-state levels of O2*- in thymocyte responses to ionizing radiation (IR) and lymphomagenesis. Loss of MnSOD in thymocytes resulted in the decreased levels of viability when mice were exposed to 0.1 or 1 Gy 137Cs radiation. In contrast, loss of Sirt3 did not affect thymocyte radiosensitivity or radiation-induced superoxide levels when mice were exposed to a single dose of 137Cs radiation (0.1 or 1 Gy). Interestingly, the Sirt3-/- thymocytes demonstrated a compromised ability to induce an adaptive response following whole body exposure to a 0.1 Gy dose of 137Cs radiation when challenged 4 h later with a 1.5 Gy dose of 137Cs radiation. Of note, both Sirt3-/- and MnSOD-/- thymocytes did not demonstrate increased T cell lymphoma in C57BL/6 mice at 15-18 months following low dose (0.1 Gy or 1 Gy) radiation exposure to low linear energy transfer (LET) 137Cs or high LET 300 MeV/n Silicon (67 kev/µM) irradiation. Interestingly, the loss of Sirt3 in Bax overexpressing transgenic thymocytes did cause significant alterations in mitochondrial oxidative metabolism including increased levels of superoxide, increased mitochondrial membrane potential, and increased oxygen consumption, as well as decreased levels of MnSOD activity and decreased steady-state levels of ATP. Sirt3 was also found to be upregulated in Lck-Bax transgenic pre-malignant thymocytes, and downregulation of Sirt3 was noted in lymphomas from Lck-Bax mice. Furthermore, a significant acceleration of thymic lymphoma development was observed in Lck-Bax transgenic animals lacking Sirt3. In conclusion, the data presented here support the hypothesis that increased levels of superoxide in thymocytes induced by loss of Sirt3 or MnSOD and/or exposure to low dose radiation was not sufficient to induce T cell lymphomas. However, loss of Sirt3 significantly accelerated thymic lymphoma formation induced in Lck-Bax overexpressing C57BL/6 mice, showing that Sirt3 could act as a tumor suppressor in this model system. These results also support the speculation that mitochondrial oxidative metabolism regulated by Sirt3 leads to increased steady-state levels of O2*- and may contribute to the promotion of carcinogenesis in the Lck-Bax model of T cell lymphoma development.

Pharmacological ascorbate treatment (P-AscH-, high-dose, intravenous vitamin C) results in a short-term increased flux of H2O2 that is preferentially cytotoxic to cancer cells vs. normal cells. We hypothesized that there may be a sustained effect (> 24 h) of P-AscH- that may contribute to cytotoxicity. P-AscH- significantly increased sustained oxygen consumption (OCR), DCFH-DA oxidation, and extracellular acidification (ECAR) in tumor lines with no change in non-tumorigenic cells. One possible source of this sustained ROS and OCR, the NADPH oxidase family of enzymes Dual Oxidase 1 and 2 (DUOX), which are epigenetically silenced by methylation in vitro and in vivo in PDAC, are up-regulated with P-AscH- treatment. Catalase pretreatment reversed the P-AscH--induced increases in DUOX, while DUOX inhibition partially rescues P-AscH- toxicity. Additionally, nutritional ascorbate is unable to mediate the increase in DUOX expression. Together these results suggest that P-AscH--induced toxicity may be enhanced by late metabolic and epigenetic shifts in tumor cells resulting in a feed-forward mechanism of H2O2 generation and induction of metabolic stress via enhanced DUOX expression and OCR. These data highlight a novel epigenetic mechanism of action for P-AscH-.