FLT1 activation in cancer cells promotes PARP-inhibitor resistance in breast cancer.

Acquired resistance to PARP inhibitors (PARPi) remains a treatment challenge for BRCA1/2-mutant breast cancer that drastically shortens patient survival. Although several resistance mechanisms have been identified, none have been successfully targeted in the clinic. Using new PARPi-resistance models of Brca1- and Bard1-mutant breast cancer generated in-vivo, we identified FLT1 (VEGFR1) as a driver of resistance. Unlike the known role of VEGF signaling in angiogenesis, we demonstrate a novel, non-canonical role for FLT1 signaling that protects cancer cells from PARPi in-vivo through a combination of cell-intrinsic and cell-extrinsic pathways. We demonstrate that FLT1 blockade suppresses AKT activation, increases tumor infiltration of CD8+ T cells, and causes dramatic regression of PARPi-resistant breast tumors in a T-cell-dependent manner. Moreover, PARPi-resistant tumor cells can be readily re-sensitized to PARPi by targeting Flt1 either genetically (Flt1-suppression) or pharmacologically (axitinib). Importantly, a retrospective series of breast cancer patients treated with PARPi demonstrated shorter progression-free survival in cases with FLT1 activation at pre-treatment. Our study therefore identifies FLT1 as a potential therapeutic target in PARPi-resistant, BRCA1/2-mutant breast cancer.

Study on the diffusion and deposition law of pore slurry in gangue filling zone based on CFD-DEM coupling.

In this study, the slurry diffusion in a cavity filled with coal gangue was studied by combining experimental and numerical simulation methods. By calibrating slurry and particle materials, the grouting process in coal gangue filling area is simulated successfully, and the change of slurry diffusion flow field and particle movement and settling process in different dimensions are deeply analyzed. Both experimental and numerical simulation results show that the particle settlement presents a bell-shaped curve, which is of great significance for understanding the particle movement and settlement behavior in the filling cavity. In addition, it is found that the grouting speed has a significant effect on the particle settlement during the slurry diffusion process. When the grouting speed increases from 0.1m /s to 0.2m /s, the particle settlement and diffusion range increases about twice. In the plane flow field, it is observed that the outward diffusion trend and speed of grouting are more obvious. It is worth noting that in the whole process of grouting, it is observed that with the increase of grouting distance and depth, both the velocity of slurry and particles show a trend of rapid initial decline and gradually slow down, and the flow velocity of slurry near the grouting outlet at a flow rate of 0.2m/s is 2-4 times that of 0.1m/s. This provides important enlightenment for the porous seepage effect at different grouting speeds.

Phase I trial of the TNF-α inhibitor certolizumab plus chemotherapy in stage IV lung adenocarcinomas.

We previously identified a chemotherapy-induced paracrine inflammatory loop that paradoxically mitigates the anti-tumor effect of chemotherapy and triggers metastatic propagation in breast and lung cancer models. Therefore, we sought to further validate and translate these findings into patient care by coupling the anti-TNF-α drug certolizumab pegol with standard cisplatin doublet chemotherapy. Here we first validate the anti-metastatic effect of certolizumab in a liver-metastatic Lewis Lung Carcinoma model. We then evaluate the safety, efficacy, and pharmacodynamic effects of certolizumab with cisplatin and pemetrexed in an open label Phase 1 clinical trial (NCT02120807) of eighteen adult patients with stage IV lung adenocarcinomas. The primary outcome is maximum tolerated dose. Secondary outcomes are response rate and progression-free survival (PFS); pharmacodynamic changes in blood and tumor are evaluated as a correlative outcome. There were nine partial responses among 16 patients evaluable (56%, 95% CI 30 to 80%). The median duration of response was 9.0 months (range 5.9 to 42.6 months) and median PFS was 7.1 months (95% CI 6.3 to NR). The standard 400 mg dose of certolizumab, added to cisplatin and pemetrexed, is well-tolerated and, as a correlative endpoint, demonstrates potent pharmacodynamic inhibition of peripheral cytokines associated with the paracrine inflammatory loop.

Targeting S100A9-ALDH1A1-Retinoic Acid Signaling to Suppress Brain Relapse in EGFR-Mutant Lung Cancer.

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib has significantly prolonged progression-free survival (PFS) in patients with EGFR-mutant lung cancer, including those with brain metastases. However, despite striking initial responses, osimertinib-treated patients eventually develop lethal metastatic relapse, often to the brain. Although osimertinib-refractory brain relapse is a major clinical challenge, its underlying mechanisms remain poorly understood. Using metastatic models of EGFR-mutant lung cancer, we show that cancer cells expressing high intracellular S100A9 escape osimertinib and initiate brain relapses. Mechanistically, S100A9 upregulates ALDH1A1 expression and activates the retinoic acid (RA) signaling pathway in osimertinib-refractory cancer cells. We demonstrate that the genetic repression of S100A9, ALDH1A1, or RA receptors (RAR) in cancer cells, or treatment with a pan-RAR antagonist, dramatically reduces brain metastasis. Importantly, S100A9 expression in cancer cells correlates with poor PFS in osimertinib-treated patients. Our study, therefore, identifies a novel, therapeutically targetable S100A9-ALDH1A1-RA axis that drives brain relapse. SIGNIFICANCE: Treatment with the EGFR TKI osimertinib prolongs the survival of patients with EGFR-mutant lung cancer; however, patients develop metastatic relapses, often to the brain. We identified a novel intracellular S100A9-ALDH1A1-RA signaling pathway that drives lethal brain relapse and can be targeted by pan-RAR antagonists to prevent cancer progression and prolong patient survival. This article is highlighted in the In This Issue feature, p. 873.

Aberrant Zip14 expression in muscle is associated with cachexia in a Bard1-deficient mouse model of breast cancer metastasis.

Nearly 80% of advanced cancer patients are afflicted with cachexia, a debilitating syndrome characterized by extensive loss of muscle mass and function. Cachectic cancer patients have a reduced tolerance to antineoplastic therapies and often succumb to premature death from the wasting of respiratory and cardiac muscles. Since there are no available treatments for cachexia, it is imperative to understand the mechanisms that drive cachexia in order to devise effective strategies to treat it. Although 25% of metastatic breast cancer patients develop symptoms of muscle wasting, mechanistic studies of breast cancer cachexia have been hampered by a lack of experimental models. Using tumor cells deficient for BARD1, a subunit of the BRCA1/BARD1 tumor suppressor complex, we have developed a new orthotopic model of triple-negative breast cancer that spontaneously metastasizes to the lung and leads to systemic muscle deterioration. We show that expression of the metal-ion transporter, Zip14, is markedly upregulated in cachectic muscles from these mice and is associated with elevated intramuscular zinc and iron levels. Aberrant Zip14 expression and altered metal-ion homeostasis could therefore represent an underlying mechanism of cachexia development in human patients with triple-negative breast cancer. Our study provides a unique model for studying breast cancer cachexia and identifies a potential therapeutic target for its treatment.

Upregulation of ZIP14 and Altered Zinc Homeostasis in Muscles in Pancreatic Cancer Cachexia.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer type in which the mortality rate approaches the incidence rate. More than 85% of PDAC patients experience a profound loss of muscle mass and function, known as cachexia. PDAC patients with this condition suffer from decreased tolerance to anti-cancer therapies and often succumb to premature death due to respiratory and cardiac muscle wasting. Yet, there are no approved therapies available to alleviate cachexia. We previously found that upregulation of the metal ion transporter, Zip14, and altered zinc homeostasis are critical mediators of cachexia in metastatic colon, lung, and breast cancer models. Here, we show that a similar mechanism is likely driving the development of cachexia in PDAC. In two independent experimental metastasis models generated from the murine PDAC cell lines, Pan02 and FC1242, we observed aberrant Zip14 expression and increased zinc ion levels in cachectic muscles. Moreover, in advanced PDAC patients, high levels of ZIP14 in muscles correlated with the presence of cachexia. These studies underscore the importance of altered ZIP14 function in PDAC-associated cachexia development and highlight a potential therapeutic opportunity for improving the quality of life and prolonging survival in PDAC patients.

Metastatic cancers promote cachexia through ZIP14 upregulation in skeletal muscle.

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-ß cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.

Bioactive lysophospholipids generated by hepatic lipase degradation of lipoproteins lead to complement activation via the classical pathway.

PURPOSE: We determined bioactivity of lysophospholipids generated by degradation of the low-density (LDL), very low-density (VLDL), and high-density (HDL) lipoproteins with hepatic lipase (HL), cholesterol esterase (CE), and lipoprotein-associated phospholipase A2 (Lp-PLA2). METHODS: The LDL, VLDL, and HDL were treated with HL, CE, and Lp-PLA2 after immobilization on plates, and complement activation studies were performed with diluted human serum. Complement component 3 (C3) fixation, a marker for complement activation, was determined with a monoclonal anti-human C3d antibody. Enzymatic properties of HL and CE were assayed with triglyceride and phosphatidylcholine substrates for triglyceride hydrolase and phospholipase A activities. The ARPE-19 cells were used for viability studies. RESULTS: The HL degradation of human lipoproteins LDL, VLDL, or HDL results in the formation of modified lipoproteins that can activate the complement pathway. Complement activation is dose- and time-dependent upon HL and occurs via the classical pathway. Enzymatic studies suggest that the phospholipase A1 activity of HL generates complement-activating lysophospholipids. C-reactive protein (CRP), known to simultaneously interact with complement C1 and complement factor H (CFH), further enhances HL-induced complement activation. The lysophospholipids, 1-Palmitoyl-sn-glycero-3-phosphocholine and 1-Oleoyl-sn-glycero-3-phosphocholine, can be directly cytotoxic to ARPE-19 cells. CONCLUSIONS: The HL degradation of lipoproteins, known to accumulate in the outer retina and in drusen, can lead to the formation of bioactive lysophospholipids that can trigger complement activation and induce RPE cellular dysfunction. Given the known risk associations for age-related macular degeneration (AMD) with HL, CRP, and CFH, this study elucidates a possible damage pathway for age-related macular degeneration (AMD) in genetically predisposed individuals, that HL activity may lead to accumulation of lysophospholipids to initiate complement activation, with CFH dysregulation exacerbating the effects of this process.

RAGE binds C1q and enhances C1q-mediated phagocytosis.

RAGE, the multiligand receptor of the immunoglobulin superfamily of cell surface molecules, is implicated in innate and adaptive immunity. Complement component C1q serves roles in complement activation and antibody-independent opsonization. Using soluble forms of RAGE (sRAGE) and RAGE-expressing cells, we determined that RAGE is a native C1q globular domain receptor. Direct C1q-sRAGE interaction was demonstrated with surface plasmon resonance (SPR), with minimum K(d) 5.6 µM, and stronger binding affinity seen in ELISA-like experiments involving multivalent binding. Pull-down experiments suggested formation of a receptor complex of RAGE and Mac-1 to further enhance affinity for C1q. C1q induced U937 cell adhesion and phagocytosis was inhibited by antibodies to RAGE or Mac-1. These data link C1q and RAGE to the recruitment of leukocytes and phagocytosis of C1q-coated material.

Ultrastructural features of retinal capillary basement membrane thickening in diabetic swine.

PURPOSE: To assess retinal capillary basement membrane thickening (BMT) in a swine model of type 1 diabetes. MATERIALS AND METHODS: Yorkshire pigs were rendered diabetic with streptozotocin and dyslipidemic with a high fat and cholesterol diet. At 18, 26, and 32 weeks of diabetes, the retina sections within 3 disc diameters from the optic disc were examined under transmission electron microscopy to evaluate the ultrastructural features of the capillary BM. Digital morphometric analysis was performed to measure BMT. RESULTS: Diabetic swine had significantly thicker retinal capillary BMs compared to controls. Pigs that sustained diabetes for longer periods or experienced severe diabetes tended to have more BMT. Those pigs that did not sustain glucose levels above 200 mg/dL did not demonstrate thicker retinal capillary BMs. Characteristic ultrastructural features of diabetic vasculopathy observed included rarefaction as an early stage of Swiss cheese cavitation, lamellation with multiplication of electron dense layers, and fibrillar materials within capillary BM. CONCLUSIONS: Diabetic Yorkshire pigs develop characteristic features of an early retinal microvasculopathy fairly rapidly and may serve as a higher-order animal model for studies of type 1 diabetes.

RAGE mediates podocyte injury in adriamycin-induced glomerulosclerosis.

In the kidney, the receptor for advanced glycation end products (RAGE) is principally expressed in the podocyte at low levels, but is upregulated in both human and mouse glomerular diseases. Because podocyte injury is central to proteinuric states, such as the nephrotic syndrome, the murine adriamycin nephrosis model was used to explore the role of RAGE in podocyte damage. In this model, administration of the anthracycline antibiotic adriamycin provokes severe podocyte stress and glomerulosclerosis. In contrast to wild-type animals, adriamycin-treated RAGE-null mice were significantly protected from effacement of the podocyte foot processes, albuminuria, and glomerulosclerosis. Administration of adriamycin induced rapid generation of RAGE ligands, and treatment with soluble RAGE protected against podocyte injury and glomerulosclerosis. In vitro, incubation of RAGE-expressing murine podocytes with adriamycin stimulated AGE formation, and treatment with RAGE ligands rapidly activated nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, via p44/p42 MAP kinase signaling, and upregulated pro-fibrotic growth factors. These data suggest that RAGE may contribute to the pathogenesis of podocyte injury in sclerosing glomerulopathies such as focal segmental glomerulosclerosis.

RAGE ligand upregulation of VEGF secretion in ARPE-19 cells.

PURPOSE: The importance of VEGF in stimulating neovascular age-related macular degeneration (AMD) is well-recognized, but the initiating factors that induce local upregulation of VEGF remain unclear. The current study was conducted to test the hypothesis that activation of RAGE (receptor for advanced glycation end products [AGEs]) by its ligands, including AGEs, amyloid-beta peptide (Abeta), and S100B/calgranulins, some of which are known components of drusen and Bruch's membrane deposits, modulate secretion of VEGF by retinal pigment epithelial (RPE) cells. METHODS: ARPE-19 cells were used for all experiments. The cells were transfected with constructs encoding a signal transduction mutant of human RAGE to assess the RAGE-dependence of intracellular signaling. VEGF secretion and gene expression were assessed by ELISA and quantitative real-time PCR. SDS-PAGE and size exclusion chromatography were performed to analyze the structural changes of S100B after oxidation of its thiol groups under denaturing and nondenaturing conditions, respectively. NF-kappaB activation was assessed via electrophoretic mobility shift assay (EMSA). The impact of the NF-kappaB inhibition was assessed by using parthenolide. RESULTS: ARPE-19 cells basally secreted VEGF under normal cell culture conditions. Immobilized ligands of RAGE increased VEGF secretion in a RAGE-dependent manner. In contrast, soluble AGE-BSA, fresh Abeta, and S100B were less effective in increasing VEGF secretion. Studies with Abeta demonstrated that oligomeric and surface-immobilized forms of Abeta, but not soluble monomeric forms of Abeta, were effective upregulators of VEGF secretion via RAGE. Oxidation of S100B's thiol groups resulted in the formation of oligomers that displayed distinct RAGE biological activity compared with the simple dimeric form. RAGE-mediated upregulation of VEGF secretion by ARPE-19 cells was largely dependent on NF-kappaB, as indicated by studies with parthenolide. CONCLUSIONS: Immobilized or oligomerized ligands for RAGE induce RPE cells to increase VEGF secretion. NF-kappaB plays a central role in RAGE-dependent RPE secretion of VEGF. In AMD, activation of the RAGE axis in RPE cells may contribute to upregulation of VEGF, potentially inciting or propagating neovascular macular disease.

Catalase enrichment using recombinant adenovirus protects alphaTN4-1 cells from H(2)O(2).

Since oxidative stress has been implicated in the development of numerous diseases including cataract, this laboratory has created and investigated the stress response of murine immortal lens epithelial cell lines (alphaTN4-1) conditioned to withstand lethal peroxide concentrations. Two of a group of antioxidative defense (AOD) enzymes found in such cells to have markedly enhanced activity are catalase (CAT) and GSH S-transferase alpha2 (GST). In order to determine if enrichment of one or both of these AODs is sufficient to protect alphaTN4-1 cells from lethal H(2)O(2) levels, these cells were infected with adenovirus vectors capable of expressing these AODs at a high level. With this system, gene enrichment and increased enzyme activity were observed with both CAT and GST vectors. The percentage of cells infected ranged from about 50 to 90% depending on the multiplicity of infection (MOI). CAT but not GST protected the cells from H(2)O(2) stress. The CAT activity was increased from 15- to 150-fold and even at the lower levels protected the cells from H(2)O(2) concentrations as high as 200 microM or more (H(2)O(2) levels which rapidly kill non-enriched cells). Even when only about 50% of the cell population is infected as judged by GFP infection, the entire population appeared to be protected based on cell viability. The CAT enrichment appears to protect other intracellular defense systems such as GSH from being depleted in contrast to non-enriched cell populations where GSH is rapidly exhausted. The overall results suggest that enriching the cellular CAT gene level with an appropriate recombinant viral vector may be sufficient to protect in vivo systems from peroxide stress.

Upregulation of RAGE and its ligands in proliferative retinal disease.

We sought to study the presence of the receptor for advanced glycation endproducts (RAGE) and its ligands, advanced glycation endproducts (AGEs), S100/calgranulins and amphoterin (high mobility group box 1 protein; HMGB1), in the vitreous cavity and epiretinal membranes (ERMs) of eyes of patients with proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). Undiluted vitreous specimens were collected from 30 eyes of 30 patients undergoing pars plana vitrectomy for repair of retinal detachment (RD) secondary to PDR (n = 15) or PVR (n = 15). The vitreous samples obtained from 10 eyes undergoing macular hole repair were used as controls. Epiretinal membranes were obtained from eight eyes with PDR and from 10 eyes with PVR. The levels of AGEs in the vitreous were measured using ELISA. The vitreous levels of soluble RAGE (sRAGE), S100/calgranulins and amphoterin were measured using Western blot analyses. The localization of RAGE and its ligands in ERMs was determined with immunohistochemistry. The vitreous levels of sRAGE were significantly increased in both PDR and PVR (p < or = 0.05) compared to control vitreous. In both PDR and PVR, the vitreous levels of AGEs (p < or = 0.01), S100/calgranulins (p < or = 0.05), and amphoterin (p < or = 0.01) were also elevated compared to control eyes. Expression of RAGE was detected in six of eight ERMs from eyes with PDR and eight of 10 ERMs from eyes with PVR. Many cells expressing RAGE also expressed vimentin, suggesting a glial cell origin. Ligands for RAGE were also detected in ERMs, with AGEs detected in five eyes with PDR and eight eyes with PVR. Similarly, S100 and amphoterin ERM expression was observed in six eyes with PDR; these ligands were also expressed in ERMs from eyes with PVR (8 and 7 cases, respectively). We conclude that RAGE and its ligands are increased in the vitreous cavity of eyes with PDR and PVR and are present in ERMs of eyes with these proliferative retinal disorders. These findings suggest a role for the proinflammatory RAGE axis in the pathogenesis of proliferative retinal diseases.

The RAGE axis in early diabetic retinopathy.

PURPOSE: The receptor for advanced glycation end products (AGEs) has been implicated in the pathogenesis of diabetic complications. This study was conducted to characterize the role of the RAGE axis in a murine model of nonproliferative diabetic retinopathy (NPDR). METHODS: The retinas of hyperglycemic, hyperlipidemic (HGHL, apolipoprotein E(-/-) db/db) mice were examined for the development of early retinal vascular lesions of NPDR and compared to littermates at 6 months of age. Neural function was assessed with electroretinography. Immunohistochemistry, real-time RT-PCR, autofluorescence, and ELISA studies were used to localize and quantify the AGE/RAGE axis. Soluble RAGE, a competitor of cellular RAGE for its ligands, was administered to assess the impact of RAGE blockade. RESULTS: Early inner retinal neuronal dysfunction, manifested by prolonged latencies of the oscillatory potentials and b-wave, was detected in hyperglycemic mice. HGHL mice exhibited accelerated development of acellular capillaries and pericyte ghosts compared with littermate control animals. AGEs were localized primarily to the vitreous cavity and internal limiting membrane (ILM) of the retina, where they were intimately associated with the footplates of RAGE-expressing Müller cells. AGE accumulation measured by ELISA was increased within the retinal extracellular matrix of hyperglycemic mice. AGE fluorescence and upregulation of RAGE transcripts was highest in the retinas of HGHL mice, and attenuation of the RAGE axis with soluble RAGE ameliorated neuronal dysfunction and reduced the development of capillary lesions in these mice. CONCLUSIONS: In early diabetic retinopathy, the RAGE axis, comprising the cellular receptor and its AGE ligands, is amplified within the retina and is accentuated along the vitreoretinal interface. Antagonism of the RAGE axis in NPDR reduces neurovascular perturbations, providing an important therapeutic target for intervention.

Comparison of characteristics of peroxide-conditioned immortal human lens-epithelial cell lines with their murine counterparts.

Previously, this laboratory has reported the characteristics of murine immortal lens-epithelial cells (alphaTN4-1) conditioned to survive either H2O2 or tertiary butyl hydroperoxide (TBOOH) stress. This communication now describes similar observations upon human HLE-B3 cells. It was found that the human cells are more sensitive to peroxides than their murine counterpart. Similar to the murine cells, conditioning to TBOOH endows the HLE-B3 cells with resistance to H2O2 but unlike the murine cells, conditioning to H2O2 gives the human cells resistance to TBOOH. Furthermore, while withdrawal of TBOOH stress from TBOOH-conditioned alphaTN4-1 cells causes a loss of resistance to this peroxide but not H2O2, with human cells resistance to both peroxides is retained. Examination of the antioxidative defense (AOD) enzyme activities show an extraordinary increase in catalase activity and significant augmentation of most other enzymes assayed in all conditioned human cell lines. In contrast, it was previously found that only catalase and glutathione-S-transferase have considerable increases in activity in the murine lines. However, in most cases, the AOD enzyme activity in murine-control cells is about 2-fold higher than in human control cells. The gene expression of human TBOOH-conditioned (Thum) and control (Chum) lines were also examined utilizing microarray analysis. Surprisingly, no significant change in gene expression was found for any of the prominent AOD enzymes. Such results differ from the response of murine cells where many AOD enzymes have increased expression. These observations suggest while the same AOD enzymes may be utilized in both murine and human lens-epithelial cells, the levels at which they are maintained and the manner in which they are recruited in response to stress may differ.

Mice lacking catalase develop normally but show differential sensitivity to oxidant tissue injury.

Catalase plays a major role in cellular antioxidant defense by decomposing hydrogen peroxide, thereby preventing the generation of hydroxyl radical by the Fenton reaction. The degree of catalase deficiency in acatalasemic and hypocatalasemic mice varies from tissue to tissue. They therefore may not be suitable for studying the function of this enzyme in certain models of oxidant-mediated tissue injury. We sought to generate a new line of catalase null mice by the gene targeting technique. The mouse catalase (Cat or Cas1) gene was disrupted by replacing parts of intron 4 and exon 5 with a neomycin resistance cassette. Homozygous Cat knockout mice, which are completely deficient in catalase expression, develop normally and show no gross abnormalities. Slices of liver and lung and lenses from the knockout mice exhibited a retarded rate in decomposing extracellular hydrogen peroxide compared with those of wild-type mice. However, mice deficient in catalase were not more vulnerable to hyperoxia-induced lung injury; nor did their lenses show any increased susceptibility to oxidative stress generated by photochemical reaction, suggesting that the antioxidant function of catalase in these two models of oxidant injury is negligible. Further studies showed that cortical injury from physical impact caused a significant decrease in NAD-linked electron transfer activities and energy coupling capacities in brain mitochondria of Cat knockout mice but not wild-type mice. The observed decrease in efficiency of mitochondrial respiration may be a direct result of an increase in mitochondrion-associated calcium, which is secondary to the increased oxidative stress. These studies suggest that the role of catalase in antioxidant defense is dependent on the type of tissue and the model of oxidant-mediated tissue injury.

The effect of stress withdrawal on gene expression and certain biochemical and cell biological properties of peroxide-conditioned cell lines.

Maturity onset cataract is a disease that afflicts >25% of the U.S. population over 65. Oxidative stress is believed to be a major factor in the development of this disease and peroxides are suspected to be prominent stressing agents. To elucidate mechanisms involved in the protection of cells against oxidative stress, immortal murine lens epithelial cells (alphaTN4-1) have been conditioned to survive lethal concentrations of either tertiary butyl hydroperoxide, TBOOH (a lipid peroxide prototype) (T cells), or H2O2 (H cells). It was found that T cells survived exposure to H2O2 but H cells were killed by TBOOH. In this communication, biological characteristics of the T cells are reported. It is shown that the T cell's ability to survive TBOOH is lost if the cells are grown in the absence of this peroxide (denoted as T- cells). By comparing the differential gene expression of 12,422 genes and ESTs from T and T- and the unconditioned control cells, 16 genes were found that may account for the loss of resistance to TBOOH. They include 5 glutathione-S-transferases, superoxide dismutase 1, zeta crystallin, a NADPH quinone reductase, as well as genes involved in detoxifying aldehydes, controlling iron metabolism, and degrading toxic lipoproteins.

Characteristics of tertiary butyl hydroperoxide and hydrogen peroxide conditioned cells withdrawn from peroxide stress.

This laboratory has recently reported the preparation of immortal lens epithelial cell lines conditioned to survive in concentrations of peroxide sufficient to cause cataract with in vitro lens culture conditions. The cell conditioning process takes many months during which time the peroxide concentration is gradually increased. It was found that while the acquired resistance to H2O2 was permanent, if tertiary butyl hydroperoxide (TBOOH) was used the resistance was lost within 6-8 weeks of the withdrawal of the peroxide. We now report that resistance is lost within a few days but can be regained within 48 hr. Furthermore, cells resistant to H2O2 while vulnerable to TBOOH could also be rapidly conditioned to tolerate TBOOH in a manner similar to the reconditioning of cells that had lost their TBOOH resistance. The results suggest that a history of exposure to certain oxidative stresses produces a change in cell biology which allows the cell to rapidly respond to the same or other stresses and survive.

Cluster analysis of genes with significant change in expression in cells conditioned to survive TBOOH.

Immortal murine lens epithelial cells, alphaTN4-1 have been conditioned to survive H2O2, H cells, or TBOOH, T cells, at concentrations that will cause cataract in vitro. Since H cells are killed by TBOOH but T cells survive H2O2, it is of interest to examine the gene expression of these cell lines. We now report the results of cluster analysis of genes whose expression is significantly changed by TBOOH. The analysis has revealed a small group of antioxidative defense genes that contribute to the survival of T and H cells when exposed to oxidative stress.