Optimizing assessment of CD30 expression in Hodgkin lymphoma by controlling for low expression.

Since the approval of brentuximab vedotin (BV), assessment of CD30 status by immunohistochemistry gained increasing importance in the clinical management of patients diagnosed with CD30-expressing lymphomas, including classical Hodgkin lymphoma (CHL). Paradoxically, patients with low or no CD30 expression respond to BV. This discrepancy may be due to lack of standardization in CD30 staining methods. In this study, we examined 29 cases of CHL and 4 cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) for CD30 expression using a staining protocol that was designed to detect low CD30 expression levels, and an evaluation system similar to the Allred scoring system used for breast cancer evaluation. For CHL, 10% of cases had low scores and 3% were CD30 negative, with 3 cases in which the majority of tumor cells showed very weak staining. Unexpectedly, one of four cases of NLPHL was positive. We demonstrate intra-patient heterogeneity in CD30 expression levels and staining patterns in tumor cells. Three CHL cases with weak staining may have been missed without the use of control tissue for low expression. Thus, standardization of CD30 immunohistochemical staining with use of known low-expressing controls may aid in proper CD30 assessment and subsequent therapeutic stratification of patients.

Thiol regulation by Mn porphyrins, commonly known as SOD mimics.

Superoxide dismutases play an important role in human health and disease. Three decades of effort have gone into synthesizing SOD mimics for clinical use. The result is the Mn porphyrins which have SOD-like activity. Several clinical trials are underway to test the efficacy of these compounds in patients, particularly as radioprotectors of normal tissue during cancer treatment. However, aqueous chemistry data indicate that the Mn porphyrins react equally well with multiple redox active species in cells including H2O2, O2•-, ONOO-, thiols, and ascorbate among others. The redox potential of the Mn porphyrins is midway between the potentials for the oxidation and reduction of O2•-. This positions them to react equally well as oxidants and reductants in cells. The result of this unique chemistry is that: 1) the species the Mn porphyrins react with in vivo will depend on the relative concentrations of the reactive species and Mn porphyrins in the cell of interest, and 2) the Mn porphyrins will act as catalytic (redox cycling) agents in vivo. The ability of the Mn porphyrins to catalyze protein S-glutathionylation means that Mn porphyrins have the potential to globally modulate cellular redox regulatory signaling networks. The purpose of this review is to summarize the data that indicate the Mn porphyrins have diverse reactions in vivo that are the basis of the observed biological effects. The ability to catalyze multiple reactions in vivo expands the potential therapeutic use of the Mn porphyrins to disease models that are not SOD based.

Acute pain alters P-glycoprotein-containing protein complexes in rat cerebral microvessels: Implications for P-glycoprotein trafficking.

P-glycoprotein (PgP) is the major drug efflux pump in human cerebral microvessels. PgP prevents pathogens, toxins and therapeutic drugs from entering the CNS. Understanding the molecular regulation of PgP activity will suggest novel mechanisms to improve CNS drug delivery. Previously, we found that during peripheral inflammatory pain (PIP) (3 h after λ carrageenan injection in the rat paw), PgP traffics to the cortical microvessel endothelial cell plasma membrane concomitant with increased PgP activity. In the current study, we measured the changes in composition of PgP-containing protein complexes after PIP in rat microvessel isolates. We found that a portion of the PgP is contained in a multi-protein complex that also contains the caveolar proteins CAV1, SDPR, PTRF and PRKCDBP. With PIP, total CAV1 bound to PgP was unchanged; however, phosphorylated CAV1 (Y14P-CAV1) in the complex increased. There were few PgP/CAV1 complexes relative to total PgP and CAV1 in the microvessels suggesting CAV1 bound to PgP is unlikely to affect total PgP activity. However, both PgP and CAV1 trafficked away from the nucleus in response to PIP. These data suggest that P-CAV1 bound to PgP potentially regulates PgP trafficking and contributes to the acute PgP activity increase after a PIP stimulus.

Loss of Blood-Brain Barrier Integrity in a KCl-Induced Model of Episodic Headache Enhances CNS Drug Delivery.

Cortical spreading depression (CSD) in the CNS is suggested as a common mechanism contributing to headache. Despite strong evidence for CNS involvement in headache disorders, drug development for headache disorders remains focused on peripheral targets. Difficulty in delivering drugs across the blood-brain barrier (BBB) may partially account for this disparity. It is known, however, that BBB permeability is increased during several CNS pathologies. In this study, we investigated BBB changes in response to KCl-induced CSD events and subsequent allodynia in rats. Cortical KCl injection in awake, freely moving rats produced facial allodynia with peak intensity between 1.5 and 3 h and CSD induction within 0.5-2 h postinjection. Brain perfusion of 14C-sucrose as a marker of BBB paracellular permeability revealed increased leak in the cortex, but not brainstem, beginning 0.5 h post-KCl injection and resolving within 6 h; no changes in tight junction (TJ) proteins occludin or claudin-5 expression were observed. Acute pretreatment with topiramate to inhibit CSD did not prevent the increased BBB paracellular permeability. CNS delivery of the abortive anti-migraine agent sumatriptan was increased in the cortex 1.5 h post-KCl injection. Surprisingly, sumatriptan uptake was also increased in the brainstem following CSD induction, suggesting regulation of active transport mechanisms at the BBB. Together, these results demonstrate the ability of CSD events to produce transient, time-dependent changes in BBB permeability when allodynia is present and to mediate access of clinically relevant therapeutics (i.e., sumatriptan) to the CNS.

Radiation-Mediated Tumor Growth Inhibition Is Significantly Enhanced with Redox-Active Compounds That Cycle with Ascorbate.

AIMS: We aim here to demonstrate that radiation (RT) enhances tumor sensitization by only those Mn complexes that are redox active and cycle with ascorbate (Asc), thereby producing H2O2 and utilizing it subsequently in protein S-glutathionylation in a glutathione peroxidase (GPx)-like manner. In turn, such compounds affect cellular redox environment, described by glutathione disulfide (GSSG)/glutathione (GSH) ratio, and tumor growth. To achieve our goal, we tested several Mn complexes of different chemical and physical properties in cellular and animal flank models of 4T1 breast cancer cell. Four other cancer cell lines were used to substantiate key findings. RESULTS: Joint administration of cationic Mn porphyrin (MnP)-based redox active compounds, MnTE-2-PyP5+ or MnTnBuOE-2-PyP5+ with RT and Asc contributes to high H2O2 production in cancer cells and tumor, which along with high MnP accumulation in cancer cells and tumor induces the largest suppression of cell viability and tumor growth, while increasing GSSG/GSH ratio and levels of total S-glutathionylated proteins. Redox-inert MnP, MnTBAP3- and two other different types of redox-active Mn complexes (EUK-8 and M40403) were neither efficacious in the cellular nor in the animal model. Such outcome is in accordance with their inability to catalyze Asc oxidation and mimic GPx. INNOVATION: We provided here the first evidence how structure-activity relationship between the catalytic potency and the redox properties of Mn complexes controls their ability to impact cellular redox environment and thus enhance the radiation and ascorbate-mediated tumor suppression. CONCLUSIONS: The interplay between the accumulation of cationic MnPs and their potency as catalysts for oxidation of Asc, protein cysteines, and GSH controls the magnitude of their anticancer therapeutic effects.

Chronic morphine exposure potentiates p-glycoprotein trafficking from nuclear reservoirs in cortical rat brain microvessels.

The rates of opioid prescription and use have continued to increase over the last few decades resulting in a greater number of opioid tolerant patients. Treatment of acute pain from surgery and injury is a clinical challenge for these patients. Several pain management strategies including prescribing increased opioids are used clinically with limited success; all currently available strategies have significant limitations. Many opioids are a substrate for p-glycoprotein (p-gp), an efflux transporter at the blood-brain barrier (BBB). Increased p-gp is associated with a decreased central nervous system uptake and analgesic efficacy of morphine. Our laboratory previously found that acute peripheral inflammatory pain (PIP) induces p-gp trafficking from the nucleus to the luminal surface of endothelial cells making up the BBB concomitant with increased p-gp activity and decreased morphine analgesic efficacy. In the current study, we tested whether PIP-induced p-gp trafficking could contribute to decreased opioid efficacy in morphine tolerant rats. A 6-day continuous dosing of morphine from osmotic minipumps was used to establish morphine tolerance in female rats. PIP induced p-gp trafficking away from nuclear stores showed a 2-fold increase in morphine tolerant rats. This observation suggests that p-gp trafficking contributes to the decreased morphine analgesic effects in morphine tolerant rats experiencing an acute pain stimulus. Attenuating p-gp trafficking during an acute pain stimulus could improve pain management by increasing the amount of opioid that could reach CNS analgesic targets and decrease the need for the dose escalation that is a serious challenge in pain management.

The opioid epidemic: a central role for the blood brain barrier in opioid analgesia and abuse.

Opioids are currently the primary treatment method used to manage both acute and chronic pain. In the past two to three decades, there has been a surge in the use, abuse and misuse of opioids. The mechanism by which opioids relieve pain and induce euphoria is dependent on the drug crossing the blood-brain barrier and accessing the central nervous system. This suggests the blood brain barrier plays a central role in both the benefits and risks of opioid use. The complex physiological responses to opioids that provide the benefits and drive the abuse also needs to be considered in the resolution of the opioid epidemic.

P-glycoprotein traffics from the nucleus to the plasma membrane in rat brain endothelium during inflammatory pain.

P-glycoprotein (PgP), a drug efflux pump in blood-brain barrier endothelial cells, is a major clinical obstacle for effective central nervous system drug delivery. Identifying PgP regulatory pathways that can be exploited clinically is critical for improving central nervous system drug delivery. We previously found that PgP activity increases in rat brain microvessels concomitant with decreased central nervous system drug delivery in response to acute peripheral inflammatory pain. In the current study, we tested the hypothesis that PgP traffics to the luminal plasma membrane of the microvessel endothelial cells from intracellular stores during peripheral inflammatory pain. Using immunofluorescence microscopy, we detected PgP in endothelial cell nuclei and in the luminal plasma membrane in control animals. Following peripheral inflammatory pain, luminal PgP staining increased while staining in the nucleus decreased. Biochemical analysis of nuclear PgP content confirmed our visual observations. Peripheral inflammatory pain also increased endothelial cell luminal staining of polymerase 1 and transcript release factor/cavin1 and serum deprivation response protein/cavin2, two caveolar scaffold proteins, without changing caveolin1 or protein kinase C delta binding protein/cavin3 location. Our data (a) indicate that PgP traffics from stores in the nucleus to the endothelial cell luminal membrane in response to peripheral inflammatory pain; (b) provide an explanation for our previous observation that peripheral inflammatory pain inhibits central nervous system drug uptake; and (c) suggest a novel regulatory mechanism for PgP activity in rat brain.

Identification of P-glycoprotein co-fractionating proteins and specific binding partners in rat brain microvessels.

Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P-glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood-brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP-containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post-translationally regulated at the BBB. The goal of the current study was to identify proteins that co-localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co-localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co-fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post-translational regulation of PgP activity at the BBB.

Manganese (III) meso-tetrakis N-ethylpyridinium-2-yl porphyrin acts as a pro-oxidant to inhibit electron transport chain proteins, modulate bioenergetics, and enhance the response to chemotherapy in lymphoma cells.

The manganese porphyrin, manganese (III) meso-tetrakis N-ethylpyridinium-2-yl porphyrin (MnTE-2-PyP(5+)), acts as a pro-oxidant in the presence of intracellular H2O2. Mitochondria are the most prominent source of intracellular ROS and important regulators of the intrinsic apoptotic pathway. Due to the increased oxidants near and within the mitochondria, we hypothesized that the mitochondria are a target of the pro-oxidative activity of MnTE-2-PyP(5+) and that we could exploit this effect to enhance the chemotherapeutic response in lymphoma. In this study, we demonstrate that MnTE-2-PyP(5+) modulates the mitochondrial redox environment and sensitizes lymphoma cells to antilymphoma chemotherapeutics. MnTE-2-PyP(5+) increased dexamethasone-induced mitochondrial ROS and oxidation of the mitochondrial glutathione pool in lymphoma cells. The combination treatment induced glutathionylation of Complexes I, III, and IV in the electron transport chain, and decreased the activity of Complexes I and III, but not the activity of Complex IV. Treatment with the porphyrin and dexamethasone also decreased cellular ATP levels. Rho(0) malignant T-cells with impaired mitochondrial electron transport chain function were less sensitive to the combination treatment than wild-type cells. These findings suggest that mitochondria are important for the porphyrin's ability to enhance cell death. MnTE-2-PyP(5+) also augmented the effects of 2-deoxy-D-glucose (2DG), an antiglycolytic agent. In combination with 2DG, MnTE-2-PyP(5+) increased protein glutathionylation, decreased ATP levels more than 2DG treatment alone, and enhanced 2DG-induced cell death in primary B-ALL cells. MnTE-2-PyP(5+) did not enhance dexamethasone- or 2DG-induced cell death in normal cells. Our findings suggest that MnTE-2-PyP(5+) has potential as an adjuvant for the treatment of hematologic malignancies.

P-glycoprotein trafficking as a therapeutic target to optimize CNS drug delivery.

The primary function of the blood-brain barrier (BBB)/neurovascular unit is to protect the central nervous system (CNS) from potentially harmful xenobiotic substances and maintain CNS homeostasis. Restricted access to the CNS is maintained via a combination of tight junction proteins as well as a variety of efflux and influx transporters that limits the transcellular and paracellular movement of solutes. Of the transporters identified at the BBB, P-glycoprotein (P-gp) has emerged as the transporter that is the greatest obstacle to effective CNS drug delivery. In this chapter, we provide data to support intracellular protein trafficking of P-gp within cerebral capillary microvessels as a potential target for improved drug delivery. We show that pain-induced changes in P-gp trafficking are associated with changes in P-gp's association with caveolin-1, a key scaffolding/trafficking protein that colocalizes with P-gp at the luminal membrane of brain microvessels. Changes in colocalization with the phosphorylated and nonphosphorylated forms of caveolin-1, by pain, are accompanied by dynamic changes in the distribution, relocalization, and activation of P-gp "pools" between microvascular endothelial cell subcellular compartments. Since redox-sensitive processes may be involved in signaling disassembly of higher-order structures of P-gp, we feel that manipulating redox signaling, via specific protein targeting at the BBB, may protect disulfide bond integrity of P-gp reservoirs and control trafficking to the membrane surface, providing improved CNS drug delivery. The advantage of therapeutic drug "relocalization" of a protein is that the physiological impact can be modified, temporarily or long term, despite pathology-induced changes in gene transcription.

Mitochondria and redox homoeostasis as chemotherapeutic targets.

Characteristics of cancer cells include a more oxidized redox environment, metabolic reprogramming and apoptosis resistance. Our studies with a lymphoma model have explored connections between the cellular redox environment and cancer cell phenotypes. Alterations seen in lymphoma cells made resistant to oxidative stress include: a more oxidized redox environment despite increased expression of antioxidant enzymes, enhanced net tumour growth, metabolic changes involving the mitochondria and resistance to the mitochondrial pathway to apoptosis. Of particular importance, the cells show cross-resistance to multiple chemotherapeutic agents used to treat aggressive lymphomas. Analyses of clinical and tumour data reveal the worst prognosis when patients' lymphomas have gene expression patterns consistent with the most oxidized redox environment. Lymphomas from patients with the worst survival outcomes express increased levels of proteins involved in oxidative phosphorylation, including cytochrome c. This is consistent with these cells functioning as metabolic opportunists. Using lymphoma cell models and primary lymphoma cultures, we observed enhanced killing using genetic and drug approaches which further oxidize the cellular redox environment. These approaches include increased expression of SOD2 (superoxide dismutase 2), treatment with a manganoporphyrin that oxidizes the glutathione redox couple, or treatment with a copper chelator that inhibits SOD1 and leads to peroxynitrite-dependent cell death. The latter approach effectively kills lymphoma cells that overexpress the anti-apoptotic protein Bcl-2. Given the central role of mitochondria in redox homoeostasis, metabolism and the intrinsic pathway to apoptosis, our studies support the development of new anti-cancer drugs to target this organelle.

The copper chelator ATN-224 induces caspase-independent cell death in diffuse large B cell lymphoma.

Bcl-2 and other anti-apoptotic proteins are associated with defective caspase-dependent apoptotic pathways, resulting in chemoresistance. We have previously shown that ATN-224, a copper chelator drug, induces cell death in murine thymic lymphoma cells transfected with Bcl-2. In the current study, we tested whether ATN-224 was effective in diffuse large B cell lymphoma (DLBCL) cells, which have increased anti­apoptotic proteins through translocation or amplification. We found that nanomolar concentrations of ATN-224 induced cell death in DLBCL cells independent of Bcl-2, Bcl-xL or Mcl-1 status. ATN-224 treatment resulted in mitochondrial dysfunction, release of apoptosis-inducing factor (AIF) and induction of caspase-independent cell death. In addition, ATN-224 degraded Mcl-1 and enhanced the effect of the BH3 mimetic ABT-263. These findings indicate that ATN-224 has potential as a therapeutic for the treatment of DLBCL. Induction of caspase­independent cell death in apoptosis­resistant DLBCL would provide a therapeutic alternative for the treatment of refractory disease.

The copper chelator ATN-224 induces peroxynitrite-dependent cell death in hematological malignancies.

Chemoresistance due to oxidative stress resistance or upregulation of Bcl-2 contributes to poor outcome in the treatment of hematological malignancies. In this study, we utilize the copper-chelator drug ATN-224 (choline tetrathiomolybdate) to induce cell death in oxidative stress-resistant cells and cells overexpressing Bcl-2 by modulating the cellular redox environment and causing mitochondrial dysfunction. ATN-224 treatment decreases superoxide dismutase 1 (SOD1) activity, increases intracellular oxidants, and induces peroxynitrite-dependent cell death. ATN-224 also targets the mitochondria, decreasing both cytochrome c oxidase (CcOX) activity and mitochondrial membrane potential. The concentration of ATN-224 required to induce cell death is proportional to SOD1 levels, but independent of Bcl-2 status. In combination with doxorubicin, ATN-224 enhances cell death. In primary B-cell acute lymphoblastic leukemia patient samples, ATN-224 decreases the viable cell number. Our findings suggest that ATN-224's dual targeting of SOD1 and CcOX is a promising approach for treatment of hematological malignancies either as an adjuvant or as a single agent.

Mitochondrial adaptations to oxidative stress confer resistance to apoptosis in lymphoma cells.

Acquired resistance to drugs commonly used for lymphoma treatment poses a significant barrier to improving lymphoma patient survival. Previous work with a lymphoma tissue culture model indicates that selection for resistance to oxidative stress confers resistance to chemotherapy-induced apoptosis. This suggests that adaptation to chronic oxidative stress can contribute to chemoresistance seen in lymphoma patients. Oxidative stress-resistant WEHI7.2 cell variants in a lymphoma tissue culture model exhibit a range of apoptosis sensitivities. We exploited this phenotype to test for mitochondrial changes affecting sensitivity to apoptosis in cells made resistant to oxidative stress. We identified impaired release of cytochrome c, and the intermembrane proteins adenylate kinase 2 and Smac/DIABLO, indicating inhibition of the pathway leading to permeabilization of the outer mitochondrial membrane. Blunting of a glucocorticoid-induced signal and intrinsic mitochondrial resistance to cytochrome c release contributed to both points of resistance. The level of Bcl-2 family members or a difference in Bim induction were not contributing factors. The extent of cardiolipin oxidation following dexamethasone treatment, however, did correlate with apoptosis resistance. The differences found in the variants were all proportionate to the degree of resistance to glucocorticoid treatment. We conclude that tolerance to oxidative stress leads to mitochondrial changes that confer resistance to apoptosis.

Mitochondria are the primary source of the H(2)O(2) signal for glucocorticoid-induced apoptosis of lymphoma cells.

Glucocorticoids are a class of steroid hormones commonly used for the treatment of hematological malignancies due to their ability to induce apoptosis in lymphoid cells. An understanding of the critical steps in glucocorticoid-induced apoptosis is required to identify sources of drug resistance. Previously, we found that an increase in hydrogen peroxide is a necessary signal for glucocorticoid-induced apoptosis. In the current study, we found that mitochondria are the source of the signal. Glucocorticoid treatment inhibited Complex I and Complex III of the electron transport chain (ETC). Mitochondrial matrix reactive oxygen species (ROS) increased concomitantly with the oxidation of the mitochondrial glutathione pool. Treatment with Tiron, a superoxide scavenger, inhibited the signal. This suggests that the hydrogen peroxide signal originates as superoxide from the mitochondria and is metabolized to hydrogen peroxide. An inability to generate mitochondrial oxidants in response to glucocorticoids could cause drug resistance.

Lymphoma cells with increased anti-oxidant defenses acquire chemoresistance.

Chronic inflammation increases lymphoma risk. Chronic inflammation exposes cells to increased reactive oxygen species (ROS). Constant exposure to ROS selects for oxidative stress-resistant cells with upregulated anti-oxidant defense enzymes. The impact of oxidative stress resistance on the redox biology and chemotherapy response in lymphoma has not been rigorously tested. To measure the effect of antioxidant defense enzyme upregulation in lymphoid cells, we created oxidative stress-resistant WEHI7.2 thymic lymphoma cell variants. We selected a population of WEHI7.2 cells for resistance to hydrogen peroxide and constructed catalase-overexpressing WEHI7.2 transfectants. The WEHI7.2 variants had: i) increased catalase and total superoxide dismutase activities; ii) an altered GSSG/2GSH redox potential; iii) a more oxidized NADP(+)/NADPH pool; and iv) increased phase 2 enzymes, NAD(P)H:quinone oxidoreductase and glutathione S-transferases µ and π. Regression analysis showed a correlation between the GSSG/2GSH redox potential and the increased phase 2 enzyme activities. As predicted from the anti-oxidant defense enzyme profile, the variants were more resistant to the oxidants hydrogen peroxide and paraquat. The variants exhibited resistance to the common lymphoma chemotherapeutics, cyclophosphamide, doxorubicin, vincristine and glucocorticoids. These data indicate that chronic ROS exposure results in lymphoid cells with multiple changes in their redox biology and a chemoresistance phenotype. These data further suggest that lymphomas that arise at the site of chronic inflammation develop chemoresistance due to a combination of drug detoxification and removal of ROS.

Manganese porphyrin, MnTE-2-PyP5+, Acts as a pro-oxidant to potentiate glucocorticoid-induced apoptosis in lymphoma cells.

Using current chemotherapy protocols, over 55% of lymphoma patients fail treatment. Novel agents are needed to improve lymphoma survival. The manganese porphyrin, MnTE-2-PyP(5+), augments glucocorticoid-induced apoptosis in WEHI7.2 murine thymic lymphoma cells, suggesting that it may have potential as a lymphoma therapeutic. However, the mechanism by which MnTE-2-PyP(5+) potentiates glucocorticoid-induced apoptosis is unknown. Previously, we showed that glucocorticoid treatment increases the steady state levels of hydrogen peroxide ([H(2)O(2)](ss)) and oxidizes the redox environment in WEHI7.2 cells. In the current study, we found that when MnTE-2-PyP(5+) is combined with glucocorticoids, it augments dexamethasone-induced oxidative stress however, it does not augment the [H(2)O(2)](ss) levels. The combined treatment depletes GSH, oxidizes the 2GSH:GSSG ratio, and causes protein glutathionylation to a greater extent than glucocorticoid treatment alone. Removal of the glucocorticoid-generated H(2)O(2) or depletion of glutathione by BSO prevents MnTE-2-PyP(5+) from augmenting glucocorticoid-induced apoptosis. In combination with glucocorticoids, MnTE-2-PyP(5+) glutathionylates p65 NF-κB and inhibits NF-κB activity. Inhibition of NF-κB with SN50, an NF- κB inhibitor, enhances glucocorticoid-induced apoptosis to the same extent as MnTE-2-PyP(5+). Taken together, these findings indicate that: 1) H(2)O(2) is important for MnTE-2-PyP(5+) activity; 2) Mn-TE-2-PyP(5+) cycles with GSH; and 3) MnTE-2-PyP(5+) potentiates glucocorticoid-induced apoptosis by glutathionylating and inhibiting critical survival proteins, including NF-κB. In the clinic, over-expression of NF-κB is associated with a poor prognosis in lymphoma. MnTE-2-PyP(5+) may therefore, synergize with glucocorticoids to inhibit NF-κB and improve current treatment.

Hydrogen peroxide signaling is required for glucocorticoid-induced apoptosis in lymphoma cells.

Glucocorticoid-induced apoptosis is exploited clinically for the treatment of hematologic malignancies. Determining the required molecular events for glucocorticoid-induced apoptosis will identify resistance mechanisms and suggest strategies for overcoming resistance. In this study, we found that glucocorticoid treatment of WEHI7.2 murine thymic lymphoma cells increased the steady-state [H(2)O(2)] and oxidized the intracellular redox environment before cytochrome c release. Removal of glucocorticoids after the H(2)O(2) increase resulted in a 30% clonogenicity; treatment with PEG-CAT increased clonogenicity to 65%. Human leukemia cell lines also showed increased H(2)O(2) in response to glucocorticoids and attenuated apoptosis after PEG-CAT treatment. WEHI7.2 cells that overexpress catalase (CAT2, CAT38) or were selected for resistance to H(2)O(2) (200R) removed enough of the H(2)O(2) generated by glucocorticoids to prevent oxidation of the intracellular redox environment. CAT2, CAT38, and 200R cells showed a 90-100% clonogenicity. The resistant cells maintained pERK survival signaling in response to glucocorticoids, whereas the sensitive cells did not. Treating the resistant cells with a MEK inhibitor sensitized them to glucocorticoids. These data indicate that: (1) an increase in H(2)O(2) is necessary for glucocorticoid-induced apoptosis in lymphoid cells, (2) increased H(2)O(2) removal causes glucocorticoid resistance, and (3) MEK inhibition can sensitize oxidative stress-resistant cells to glucocorticoids.

A strategy for full interrogation of prognostic gene expression patterns: exploring the biology of diffuse large B cell lymphoma.

BACKGROUND: Gene expression profiling yields quantitative data on gene expression used to create prognostic models that accurately predict patient outcome in diffuse large B cell lymphoma (DLBCL). Often, data are analyzed with genes classified by whether they fall above or below the median expression level. We sought to determine whether examining multiple cut-points might be a more powerful technique to investigate the association of gene expression with outcome. METHODOLOGY/PRINCIPAL FINDINGS: We explored gene expression profiling data using variable cut-point analysis for 36 genes with reported prognostic value in DLBCL. We plotted two-group survival logrank test statistics against corresponding cut-points of the gene expression levels and smooth estimates of the hazard ratio of death versus gene expression levels. To facilitate comparisons we also standardized the expression of each of the genes by the fraction of patients that would be identified by any cut-point. A multiple comparison adjusted permutation p-value identified 3 different patterns of significance: 1) genes with significant cut-point points below the median, whose loss is associated with poor outcome (e.g. HLA-DR); 2) genes with significant cut-points above the median, whose over-expression is associated with poor outcome (e.g. CCND2); and 3) genes with significant cut-points on either side of the median, (e.g. extracellular molecules such as FN1). CONCLUSIONS/SIGNIFICANCE: Variable cut-point analysis with permutation p-value calculation can be used to identify significant genes that would not otherwise be identified with median cut-points and may suggest biological patterns of gene effects.