Janene Pierce

Organ-specific roles for transcription factor NF-κB in reovirus-induced apoptosis and disease

Reovirus induces apoptosis in cultured cells and in vivo. In cell culture models, apoptosis is contingent upon a mechanism involving reovirus-induced activation of transcription factor NF-kappaB complexes containing p50 and p65/RelA subunits. To explore the in vivo role of NF-kappaB in this process, we tested the capacity of reovirus to induce apoptosis in mice lacking a functional nfkb1/p50 gene. The genetic defect had no apparent effect on reovirus replication in the intestine or dissemination to secondary sites of infection. In comparison to what was observed in wild-type controls, apoptosis was significantly diminished in the CNS of p50-null mice following reovirus infection. In sharp contrast, the loss of p50 was associated with massive reovirus-induced apoptosis and uncontrolled reovirus replication in the heart. Levels of IFN-beta mRNA were markedly increased in the hearts of wild-type animals but not p50-null animals infected with reovirus. Treatment of p50-null mice with IFN-beta substantially diminished reovirus replication and apoptosis, which suggests that IFN-beta induction by NF-kappaB protects against reovirus-induced myocarditis. These findings reveal an organ-specific role for NF-kappaB in the regulation of reovirus-induced apoptosis, which modulates encephalitis and myocarditis associated with reovirus infection.

Warm hepatic ischemia-reperfusion promotes growth of colorectal carcinoma micrometastases in mouse liver via matrix metalloproteinase-9 induction

Surgical resection remains the best treatment for colorectal metastases isolated to the liver; however, 5-year survival rates following liver resection are only 40% to 50%, with liver recurrence being a significant reason for treatment failure. The ischemia-reperfusion (I/R) injury incurred during liver surgery can lead to cellular dysfunction and elevations in proinflammatory cytokines and matrix metalloproteinases (MMP). In rodents, I/R injury to the liver has been shown to accelerate the outgrowth of implanted tumors. The mechanism for increased tumor growth in the setting of liver I/R injury is unknown. To investigate the effect of I/R on tumor growth, an experimental model was used whereby small hepatic metastases form after 28 days. Mice subjected to 30 min of 70% liver ischemia at the time of tumor inoculation had significantly larger tumor number and volume, and had elevated MMP9 serum and liver tissue MMP9 as evidenced by zymography and quantitative real-time PCR. Mice treated with doxycycline, a broad-spectrum MMP inhibitor, had reduced MMP9 levels and significantly smaller tumor number and volume in the liver. MMP9-null mice were used to determine if the effects of doxycycline were due to the absence of stromal-derived MMP9. The MMP9-null mice, with or without doxycycline treatment, had reduced tumor number and volume that was equivalent to wild-type mice treated with doxycycline. These findings indicate that hepatic I/R-induced elevations in MMP9 contribute to the growth of metastatic colorectal carcinoma in the liver and that postresection MMP9 inhibition may be clinically beneficial in preventing recurrence following hepatic surgery.

Silencing of TLR4 Decreases Liver Tumor Burden in a Murine Model of Colorectal Metastasis and Hepatic Steatosis

BackgroundThe relationship between obesity and cancer has become of particular interest due to the rapidly growing prevalence of overweight individuals. Obesity predisposes individuals to the development of hepatic steatosis and is an independent risk factor for several neoplasms. Toll-like receptor 4 (TLR4) is the innate receptor for endotoxin, and steatotic livers are known to be sensitive to endotoxin. TLR4 signaling has been shown to have proneoplastic effects in vitro due to its effect on immune surveillance. Thus far, studies have predominantly focused on the effect of tumor-cell-derived TLR4 without regard to host TLR4 signaling.ResultsIn the present study we show that steatotic livers have increased expression of TLR4. Obese animals developed higher metastatic tumor burden in the liver than lean controls regardless of the presence or absence of intact host TLR4. After silencing TLR4 expression using RNAi in the mouse colon cancer cell line MC38, there was a significant decrease in metastatic tumor burden within the liver of obese animals.ConclusionsThese findings demonstrate that steatotic livers have increased susceptibility to metastatic tumor growth and that silencing tumor cell TLR4 reduces metastatic tumor burden in steatotic liver.

Identification of an NF-κB-Dependent Gene Network in Cells Infected by Mammalian Reovirus

ABSTRACT Reovirus infection activates NF-κB, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-κB elicits this cellular response. To identify host genes activated by NF-κB following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IκBα (mIκBα) under the control of an inducible promoter. Induction of mIκBα inhibited the activation of NF-κB and blocked the expression of NF-κB-responsive genes. RNA extracted from infected and uninfected cells was used in high-density oligonucleotide microarrays to examine the expression of constitutively activated genes and reovirus-stimulated genes in the presence and absence of an intact NF-κB signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIκBα. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-κB-regulated genes could be grouped into four distinct gene clusters that were temporally regulated. Gene ontology analysis identified biological processes that were significantly overrepresented in the reovirus-induced genes under NF-κB control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-κB-dependent gene networks induced by other stimuli, including respiratory syncytial virus, Epstein-Barr virus, tumor necrosis factor alpha, and heart disease, revealed a number of common components, including CCL5/RANTES, CXCL1/GRO-α, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-κB-directed expression of cellular genes that activate death signaling pathways in infected cells.

Modulation of mitochondrial calcium management attenuates hepatic warm ischemia-reperfusion injury.

Hepatic warm ischemia and reperfusion (IR) injury occurs in many clinical situations and has an important link to subsequent hepatic failure. The pathogenesis of this injury involves numerous pathways, including mitochondrial‐associated apoptosis. We studied the effect of mitochondrial calcium uptake inhibition on hepatic IR injury using the specific mitochondrial calcium uptake inhibitor, ruthenium red (RR). Rats were subjected to 1 hour of 70% warm hepatic ischemia following RR pretreatment or vehicle injection. Sham‐operated animals served as controls. Analysis was performed at 15 minutes, 1 hour, 3 hours, or 6 hours after reperfusion. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations were determined. Terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling (TUNEL) staining was performed to assess apoptosis, and hepatocellular necrosis was semiquantitated from hematoxylin and eosin–stained tissue sections. RR pretreatment significantly decreased both AST and ALT serum levels after 6 hours of reperfusion (AST: 1,556 ± 181 U/L vs. 597 ± 121 U/L, P = 0.005; ALT: 1,118 ± 187 U/L vs. 294 ± 39 U/L, P = 0.005). Apoptosis was observed within 15 minutes of reperfusion in vehicle‐pretreated animals and peaked after 3 hours of reperfusion (98 ± 21 cells/high‐power field [hpf]). Apoptosis was inhibited at all time points by RR pretreatment. Histologic evidence of necrosis was not observed prior to 3 hours of reperfusion (23% ± 4%), and maximal necrosis was observed after 6 hours of reperfusion (26% ± 1% percent area). RR pretreatment significantly decreased the necrotic percent area at both the 3‐hour and the 6‐hour time points (4.2% ± 2%; 3.7% ± 1%, respectively). Hepatic IR injury resulted in both apoptotic and necrotic cell death, which were attenuated by RR pretreatment. In conclusion, these observations implicate mitochondrial calcium uptake in the pathogenesis of hepatic IR injury. (Liver Transpl 2005;11:663–668.)

Mitochondrial Calcium Uptake Regulates Cold Preservation‐Induced Bax Translocation and Early Reperfusion Apoptosis

Mitochondrial calcium (mCa + 2) overload occurs during cold preservation and is an integral part of mitochondrial‐dependent apoptotic pathways. We investigated the role of mCa + 2 overload in cell death following hypothermic storage using HepG2 cells stored in normoxic‐hypothermic (4 °C) or hypoxic (< 0.1% O2)‐hypothermic Belzer storage solution. Cells were stored for 6 h, with or without 10 μM ruthenium red (mCa + 2 uniporter inhibitor) followed by rewarming in oxygenated media at 37 °C. Cytoplasmic cytochrome c levels were studied by Western analysis and by fluorescent microscopy after transfection of cytochrome c‐GFP expression plasmid. Immunofluorescence determined the intracellular, spatio‐temporal distribution of Bax, and TUNEL staining was used to evaluate cell death after 180 min of rewarming. Caspase activation was evaluated using Western analysis and a caspase 3 activity assay. Bax translocation, cytochrome c release, and early rewarming cell death occurred following hypothermic storage and were exacerbated by hypoxia. Caspase 3 activation did not occur following hypothermic storage. Blockade of mCa + 2 uptake prevented Bax translocation, cytochrome c release, and early rewarming cell death. These studies demonstrate that mCa + 2 uptake during hypothermic storage, both hypoxic and normoxic, contributes to early rewarming apoptosis by triggering Bax translocation to mitochondria and cytochrome c release.

Mitochondrial P2Y-Like receptors link cytosolic adenosine nucleotides to mitochondrial calcium uptake.

ATP is a known extracellular ligand for cell membrane purinergic receptors. Intracellular ATP can work also as a regulatory ligand via binding sites on functional proteins. We report herein the existence of P2Y1‐like and P2Y2‐like receptors in hepatocyte mitochondria (mP2Y1 and mP2Y2), which regulate mCa2+ uptake though the uniporter. Mitochondrial P2Y1 activation stimulates mCa2+ uptake; whereas, mP2Y2 activation inhibits mCa2+ uptake. ATP acts preferentially on mP2Y2 receptors, while ADP and AMP‐PNP stimulate both the mP2Y1 and mP2Y2. PPADS inhibits ADP stimulated mP2Y1‐mediated mCa2+ uptake. In addition, UTP, a selective P2Y2 agonist, strongly inhibits mCa2+ uptake. The newly discovered presence and function of these receptors is significant because it explains increased mCa2+ uptake in the setting of low cytosolic [ATP] and, therefore, establishes a mechanism for direct feedback in which cytosolic [ATP] governs mitochondrial ATP production through regulation of mCa2+ uptake.

Real-time spectroscopic assessment of thermal damage: implications for radiofrequency ablation

Radiofrequency ablation (RFA) is an evolving technology used to treat unresectable liver tumors. Currently, there is no accurate method to determine RFA margins in real-time during the procedure. We hypothesized that fiber-optic based spectroscopic monitoring system could detect thermal damage from RFA in real-time. Fluorescence (F) and diffuse reflectance (Rd) spectra were continuously acquired from within the expected ablation zone during canine hepatic RFA using a fiber-optic microinterrogation probe (MIP). The F and Rd spectral feedback were continuously monitored and ablations were stopped based on changes in spectra alone. After each ablation, the MIP tract was marked with India ink and the ablation zone was excised. The relationship of the MIP to the zone of ablation was examined grossly and microscopically. F and Rd spectral changes occurred in three characteristic phases as the ablation zone progresses past the MIP. Phase 1 indicates minimal deviation from normal lives. Phase 2 occurs as the MIP lies within the hemorrhagic zone of the ablated tissue. Phase 3 correlates with complete tissue coagulation. The absolute magnitude of spectral change correlates with the gross and histologic degree of thermal damage. Optical spectroscopy is a technology that allows real-time detection of thermal tissue damage. In this study, both F and Rd spectroscopy accurately defined the advancing hemorrhagic edge of the zone of ablation and the central coagulation zone. These results suggest that F and Rd spectroscopy can be used to create a real-time feedback system to accurately define RFA margins.

Race Disparities in Wilms Tumor Incidence and Biology

BACKGROUND Wilms tumor (WT) is thought to arise in children of Black African ancestry with greater frequency than in Whites. To clarify the biological basis for race disparities in WT, we first verified that Black children residing in Tennessee have an increased incidence of WT, and second, established molecular profiles in WT that are specific to race. MATERIALS AND METHODS To assess race disparities in WT epidemiology, the Tennessee Cancer Registry (TCR) was queried for all in-state patients less than 20 y of age and registered between 1999 and 2008. To explore race disparities in WT biology, six Black and four White WT specimens acquired in Tennessee were analyzed using imaging mass spectrometry (IMS). RESULTS TCR data show that Black children are over-represented among WT patients (29%) relative to all other childhood cancers (18.5%; P = 0.01). WT ranked the fifth most common cancer diagnosis among Blacks, but ninth among Whites. The diagnosis of WT occurred 79% more frequently among Blacks (n = 28) than Whites (n = 69; P = 0.01), and proportionally more Blacks tended to present with distant disease. Although overall survival from WT was not statistically different between Blacks (92.9%) and Whites (94.0%), Black males showed the lowest survival (85%; P = 0.21). IMS analysis identified peptide spectra from both WT blastema and stroma that independently classify specimens according to race with greater than 80% accuracy. CONCLUSIONS In Tennessee, Black children appear more susceptible than Whites to develop WT. Race-specific molecular profiles can be determined that may help to clarify pathways of Wilms tumorigenesis and the biological basis for race disparities in WT incidence and biology.

Mitochondrial calcium transport is regulated by P2Y1‐ and P2Y2‐like mitochondrial receptors

Ischemia‐reperfusion injury remains a major clinical problem in liver transplantation. One contributing factor is mitochondrial calcium (mCa2+) overload, which triggers apoptosis; calcium also regulates mitochondrial respiration and adenosine 5′‐triphosphate (ATP) production. Recently, we reported the presence of purinergic P2Y1‐ and P2Y2‐like receptor proteins in mitochondrial membranes. Herein, we present an evaluation of the functional characteristics of these receptors. In experiments with isolated mitochondria, specific P2Y1 and P2Y2 receptors ligands: 2‐methylthio‐adenosine 5′‐diphosphate (2meSADP) and uridine 5′‐triphosphate (UTP), respectively, were used, and mitochondrial calcium uptake was measured. 2meSADP and UTP had a maximum effect at concentrations in the range of the known P2Y1 and P2Y2 receptors. The P2Y inhibitor phosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS) blocked the effects of both ligands. The phospholipase C (PLC) antagonist U73122 inhibited the effect of both ligands while its inactive analog U73343 had no effect. These data strongly support the hypothesis that mitochondrial Ca2+ uptake is regulated in part by adenine nucleotides via a P2Y‐like receptor mechanism that involves mitochondrial PLC activation. J. Cell. Biochem. 99: 1165–1174, 2006.