Clinical development of cancer therapeutics that target metabolism.

Glucose and glutamine metabolism in cancer cells are markedly elevated relative to non-transformed normal cells. This metabolic reprogramming enables the production of adenosine triphosphate and the anabolic precursors needed for survival, growth and motility. The recent observations that mutant oncogenic proteins and the loss of tumor suppressors activate key metabolic enzymes suggest that selective inhibition of these enzymes may yield effective cancer therapeutics with acceptable toxicities. In support of this concept, pre-clinical studies of small molecule antagonists of several metabolic enzymes in tumor-bearing mice have demonstrated reasonable therapeutic indices. We will review the rationale for targeting metabolic enzymes as a strategy to treat cancer and will detail the results of several recent clinical trials of metabolic inhibitors in advanced cancer patients.

6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27.

The control of glucose metabolism and the cell cycle must be coordinated in order to guarantee sufficient ATP and anabolic substrates at distinct phases of the cell cycle. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are well established regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent allosteric activator of 6-phosphofructo-1-kinase (Pfk-1). PFKFB3 is overexpressed in human cancers, regulated by HIF-1α, Akt and PTEN, and required for the survival and growth of multiple cancer types. Although most functional studies of the role of PFKFB3 in cancer progression have invoked its well-recognized function in the regulation of glycolysis, recent observations have established that PFKFB3 also traffics to the nucleus and that its product, F2,6BP, activates cyclin-dependent kinases (Cdks). In particular, F2,6BP stimulates the Cdk-mediated phosphorylation of the Cip/Kip protein p27 (threonine 187), which in turn results in p27's ubiquitination and proteasomal degradation. As p27 is a potent suppressor of the G1/S transition and activator of apoptosis, we hypothesized that the known requirement of PFKFB3 for cell cycle progression and prevention of apoptosis may be partly due to the ability of F2,6BP to activate Cdks. In this study, we demonstrate that siRNA silencing of endogenous PFKFB3 inhibits Cdk1 activity, which in turn stabilizes p27 protein levels causing cell cycle arrest at G1/S and increased apoptosis in HeLa cells. Importantly, we demonstrate that the increase in apoptosis and suppression of the G1/S transition caused by siRNA silencing of PFKFB3 expression is reversed by co-siRNA silencing of p27. Taken together with prior publications, these observations support a model whereby PFKFB3 and F2,6BP function not only as regulators of Pfk-1 but also of Cdk1 activity, and therefore serve to couple glucose metabolism with cell proliferation and survival in transformed cells.

Regulation of glycolytic and mitochondrial metabolism by ras.

High glucose uptake is a characteristic of most metastatic tumors and activation of Ras signaling in immortalized cells increases glycolytic flux into lactate, de novo nucleic acid synthesis and the tricarboxylic acid cycle, and increases NADH shuttling, oxygen consumption and uncoupling of ATP synthase from the proton gradient. Fructose-2,6- bisphosphate, C-Myc, HIF1α and AKT each have been found to be key regulators of glycolysis and to be controlled by Ras signaling, and there is abundant evidence for cross-talk between these regulators. The reprogramming of glycolytic and mitochondrial metabolism by Ras enables an integrated activation of energetic and anabolic pathways via the redox state of NADH that is required for the survival and growth of neoplastic cells in poorly vascularized tumors. Several small molecule antagonists specific for essential metabolic enzymes have been found to be selectively toxic to Ras-transformed cells as opposed to wild-type cells, indicating that this metabolic reprogramming and addiction may have utility for the development of anti-neoplastic agents.

A novel small molecule antagonist of choline kinase-α that simultaneously suppresses MAPK and PI3K/AKT signaling.

Choline kinase-α expression and activity are increased in multiple human neoplasms as a result of growth factor stimulation and activation of cancer-related signaling pathways. The product of choline kinase-α, phosphocholine, serves as an essential metabolic reservoir for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the production of lipid second messengers. Using in silico screening for small molecules that may interact with the choline kinase-α substrate binding domain, we identified a novel competitive inhibitor, N-(3,5-dimethylphenyl)-2-[[5-(4-ethylphenyl)-1H-1,2,4-triazol-3-yl]sulfanyl] acetamide (termed CK37) that inhibited purified recombinant human choline kinase-α activity, reduced the steady-state concentration of phosphocholine in transformed cells, and selectively suppressed the growth of neoplastic cells relative to normal epithelial cells. Choline kinase-α activity is required for the downstream production of phosphatidic acid, a promoter of several Ras signaling pathways. CK37 suppressed mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling, disrupted actin cytoskeletal organization, and reduced plasma membrane ruffling. Finally, administration of CK37 significantly decreased tumor growth in a lung tumor xenograft mouse model, suppressed tumor phosphocholine, and diminished activating phosphorylations of extracellular signal-regulated kinase and AKT in vivo. Together, these results further validate choline kinase-α as a molecular target for the development of agents that interrupt Ras signaling pathways, and indicate that receptor-based computational screening should facilitate the identification of new classes of choline kinase-α inhibitors.

Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling.

Choline is an essential anabolic substrate for the synthesis of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the synthesis of lipid signaling molecules. Nuclear magnetic resonance (NMR)-based metabolomic studies of human tumors have identified a marked increase in the intracellular concentration of phosphocholine relative to normal tissues. We postulated that the observed intracellular pooling of phosphocholine may be required to sustain the production of the pleiotropic lipid second messenger, phosphatidic acid. Phosphatidic acid is generated from the cleavage of phosphatidylcholine by phospholipase D2 and is a key activator of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT survival signaling pathways. In this study we show that the steady-state concentration of phosphocholine is increased by the ectopic expression of oncogenic H-Ras(V12) in immortalized human bronchial epithelial cells. We then find that small interfering RNA (siRNA) silencing of choline kinase expression in transformed HeLa cells completely abrogates the high concentration of phosphocholine, which in turn decreases phosphatidylcholine, phosphatidic acid and signaling through the MAPK and PI3K/AKT pathways. This simultaneous reduction in survival signaling markedly decreases the anchorage-independent survival of HeLa cells in soft agar and in athymic mice. Last, we confirm the relative importance of phosphatidic acid for this pro-survival effect as phosphatidic acid supplementation fully restores MAPK signaling and partially rescues HeLa cells from choline kinase inhibition. Taken together, these data indicate that the pooling of phosphocholine in cancer cells may be required to provide a ready supply of phosphatidic acid necessary for the feed-forward amplification of cancer survival signaling pathways.

Ras transformation requires metabolic control by 6-phosphofructo-2-kinase.

Neoplastic cells transport large amounts of glucose in order to produce anabolic precursors and energy within the inhospitable environment of a tumor. The ras signaling pathway is activated in several cancers and has been found to stimulate glycolytic flux to lactate. Glycolysis is regulated by ras via the activity of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFK2/FBPase), which modulate the intracellular concentration of the allosteric glycolytic activator, fructose-2,6-bisphosphate (F2,6BP). We report herein that sequential immortalization and ras-transformation of mouse fibroblasts or human bronchial epithelial cells paradoxically decreases the intracellular concentration of F2,6BP. This marked reduction in the intracellular concentration of F2,6BP sensitizes transformed cells to the antimetabolic effects of PFK2/FBPase inhibition. Moreover, despite co-expression of all four mRNA species (PFKFB1-4), heterozygotic genomic deletion of the inducible PFKFB3 gene in ras-transformed mouse lung fibroblasts suppresses F2,6BP production, glycolytic flux to lactate, and growth as soft agar colonies or tumors in athymic mice. These data indicate that the PFKFB3 protein product may serve as an essential downstream metabolic mediator of oncogenic ras, and we propose that pharmacologic inhibition of this enzyme should selectively suppress the high rate of glycolysis and growth by cancer cells.

A role for peripheral blood fibrocytes in Lyme disease?

It is proposed that peripheral blood fibrocytes will be a new and important player in the pathogenesis of Lyme disease. Peripheral blood fibrocytes are a circulating leukocyte subpopulation that: (a) express collagen; (b) are an abundant source of cytokines, chemoattractants and growth factors; and (c) are able to recruit and activate naive T-cells and memory T-cells. We predict that peripheral blood fibrocytes will represent a new and important antigen-presenting cell which will play an important role in directing the immune response from the pathogenic Th1 to the protective Th2 response cell in Borrelia infections.

Inhibition of macrophage migration inhibitory factor (MIF) tautomerase and biological activities by acetaminophen metabolites.

The cytokine macrophage migration inhibitory factor (MIF) has emerged to be an important regulator of the inflammatory response and is critically involved in the development of septic shock, arthritis, and glomerulonephritis. Although the biological activities of MIF are presumed to require a receptor-based mechanism of action, the protein is also a tautomerase and has a catalytically active N-terminal proline that is invariant in structurally homologous bacterial isomerases. This observation raises the possibility that MIF may exert its biological action via an enzymatic reaction. Physiologically relevant substrates for MIF have not been identified, nor have site-directed mutagenesis studies consistently supported the requirement for a functional catalytic site. Small molecule inhibitors of MIF's isomerase activity also have been developed, but none have been shown yet to inhibit MIF biological activity. We report herein that the iminoquinone metabolite of acetaminophen, N-acetyl-p-benzoquinone imine (NAPQI), inhibits both the isomerase and the biological activities of MIF. The reaction between NAPQI and MIF is covalent and produces a NAPQI-modified MIF species with diminished cell binding activity and decreased recognition by anti-MIF mAb. These data are consistent with a model by which the NAPQI reacts with the catalytic Pro-1 of MIF to disrupt the integrity of epitope(s) critical to MIF's biological activity and point to the importance of the catalytic domain, but not the catalytic activity per se, in MIF function. These results also point to a powerful approach for the design of small molecule inhibitors of MIF based on interaction with its catalytic site and constitute an example of a pharmacophore capable of irreversibly inhibiting the action of a proinflammatory cytokine.

New strategies for ER admissions.

Henry Ford Hospital, Detroit, operates a Level 1 emergency department. Last year, nearly 89,000 adults and children were treated; one-third were brought in with life-threatening conditions. This year, the emergency department expects to handle more than 100,000 patients. Patients with nonemergency problems sometimes wait four to six hours before being seen by a physician. Others get discouraged and leave.

Peripheral blood fibrocytes: mesenchymal precursor cells and the pathogenesis of fibrosis.

Peripheral blood fibrocytes are a novel population of cells that rapidly enter sites of tissue injury and contribute to connective scar formation. Fibrocytes display a distinct cell surface phenotype (CD34+/CD45+/collagen I+), and are an abundant source of cytokines and growth factors that function to attract and activate inflammatory and connective tissue cells. Fibrocytes also are specialized to activate naïve T cells against foreign antigen, and may play a critical role in the initiation of cognate immunity during the earliest phases of tissue injury. Recently, immunohistochemical studies of fibrotic tissues have shown that fibrocytes co-localize to areas of connective tissue matrix deposition. Peripheral blood fibrocytes thus may participate in the generation of excessive fibroses associated with various autoimmune disorders involving a persistent T-cell activation, as occurs in scleroderma.

An essential role for macrophage migration inhibitory factor (MIF) in angiogenesis and the growth of a murine lymphoma.

BACKGROUND: Macrophage migration inhibitory factor (MIF) has been shown to counterregulate glucocorticoid action and to play an essential role in the activation of macrophages and T cells in vivo. MIF also may function as an autocrine growth factor in certain cell systems. We have explored the role of MIF in the growth of the 38C13 B cell lymphoma in C3H/HeN mice, a well-characterized syngeneic model for the study of solid tumor biology. MATERIALS AND METHODS: Tumor-bearing mice were treated with a neutralizing anti-MIF monoclonal antibody and the tumor response assessed grossly and histologically. Tumor capillaries were enumerated by immunohistochemistry and analyzed for MIF expression. The effect of MIF on endothelial cell proliferation was studied in vitro, utilizing both specific antibody and antisense oligonucleotide constructs. The role of MIF in angiogenesis also was examined in a standard Matrigel model of new blood vessel formation in vivo. RESULTS: The administration of anti-MIF monoclonal antibodies to mice was found to reduce significantly the growth and the vascularization of the 38C13 B cell lymphoma. By immunohistochemistry, MIF was expressed predominantly within the tumor-associated neovasculature. Cultured microvascular endothelial cells, but not 38C13 B cells, produced MIF protein and required its activity for proliferation in vitro. Anti-MIF monoclonal antibody also was found to markedly inhibit the neovascularization response elicited by Matrigel implantation. CONCLUSION: These data significantly expand the role of MIF in host responses, and suggest a new target for the development of anti-neoplastic agents that inhibit tumor neovascularization.

An inducible gene product for 6-phosphofructo-2-kinase with an AU-rich instability element: role in tumor cell glycolysis and the Warburg effect.

Cancer cells maintain a high glycolytic rate even in the presence of oxygen, a phenomenon first described over 70 years ago and known historically as the Warburg effect. Fructose 2,6-bisphosphate is a powerful allosteric regulator of glycolysis that acts to stimulate the activity of 6-phosphofructo-1-kinase (PFK-1), the most important control point in mammalian glycolysis. The steady state concentration of fructose 2,6-bisphosphate in turn depends on the activity of the enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2, 6-bisphosphatase, which is expressed in several tissue-specific isoforms. We report herein the identification of a gene product for this enzyme that is induced by proinflammatory stimuli and which is distinguished by the presence of multiple copies of the AUUUA mRNA instability motif in its 3'-untranslated end. This inducible gene for PFK-2 is expressed constitutively in several human cancer cell lines and was found to be required for tumor cell growth in vitro and in vivo. Inhibition of inducible PFK-2 protein expression decreased the intracellular level of 5-phosphoribosyl-1-pyrophosphate, a product of the pentose phosphate pathway and an important precursor for nucleic acid biosynthesis. These studies identify a regulatory isoenzyme that may be essential for tumor growth and provide an explanation for long-standing observations concerning the apparent coupling of enhanced glycolysis and cell proliferation.

Interaction of Borrelia burgdorferi with peripheral blood fibrocytes, antigen-presenting cells with the potential for connective tissue targeting.

BACKGROUND: Borrelia Burgdorferi has a predilection for collagenous tissue and can interact with fibronectin and cellular collagens. While the molecular mechanisms of how B. burgdorferi targets connective tissues and causes arthritis are not understood, the spirochetes can bind to a number of different cell types, including fibroblasts. A novel circulating fibroblast-like cell called the peripheral blood fibrocyte has recently been described. Fibrocytes express collagen types I and III as well as fibronectin. Besides playing a role in wound healing, fibrocytes have the potential to target to connective tissue and the functional capacity to recruit, activate, and present antigen to CD4(+) T cells. MATERIALS AND METHODS: Rhesus monkey fibrocytes were isolated and characterized by flow cytometry. B. burgdorferi were incubated with human or monkey fibrocyte cultures in vitro and the cellular interactions analyzed by light and electron microscopy. The two strains of B. burgdorferi studied included JD1, which is highly pathogenic for monkeys, and M297, which lacks the cell surface OspA and OspB proteins. RESULTS: In this study, we demonstrate that B. burgdorferi binds to both human and monkey (rhesus) fibrocytes in vitro. This process does not require OspA or OspB. In addition, the spirochetes are not phagocytosed but are taken into deep recesses of the cell membrane, a process that may protect them from the immune system. CONCLUSIONS: This interaction between B. burgdorferi and peripheral blood fibrocytes provides a potential explanation for the targeting of spirochetes to joint connective tissue and may contribute to the inflammatory process in Lyme arthritis.

MIF expression in the rat brain: implications for neuronal function.

BACKGROUND: The mediator known historically as macrophage migration inhibitory factor (MIF) has been identified recently as being released into the circulation by the anterior pituitary gland as a consequence of stress or during a systemic inflammatory response. Macrophages and T cells also secrete MIF, both in response to proinflammatory factors or upon stimulation with glucocorticoids. Once released, MIF "overrides" or counterregulates the immunosuppressive effects of steroids on cytokine production and immune cellular activation. To further investigate the biology of MIF and its role in the neuroendocrine system, we have studied the regional and cellular expression of MIF in brain tissue obtained from normal rats and rats administered LPS intracisternally. MATERIALS AND METHODS: Rat brain sections were analyzed by immunohistochemistry utilizing an affinity-purified, anti-MIF antibody raised to recombinant MIF, and by in situ hybridization using a digoxigenin-labeled, antisense MIF cRNA probe. The kinetics of MIF mRNA expression in brain were compared with that of IL-1, IL-6, and TNF-alpha by RT-PCR of total brain RNA. The cerebrospinal fluid content of MIF and TNF-alpha proteins was analyzed by Western blotting and ELISA. RESULTS: A strong baseline expression pattern for MIF was observed in neurons of the cortex, hypothalamus, hippocampus, cerebellum, and pons. By in situ hybridization, MIF mRNA was found predominantly in cell bodies whereas MIF protein was detected mostly within the terminal fields associated with neurons. There was a marked pattern of MIF immunoreactivity within the mossy fibers of the dentate gyrus and dendrites of the hippocampal CA3 field. These structures have been shown previously to be involved in glucocorticoid-induced tissue damage within the hippocampus, suggesting an association between MIF and targets of glucocorticoid action. The intracisternal injection of LPS increased MIF mRNA and protein expression in brain and MIF immunoreactivity was due in part to infiltrating monocytes/macrophages. MIF protein also was found to be rapidly released into the cerebrospinal fluid. This response corresponded with that of LPS-induced cytokine release and MIF mRNA expression increased in a distribution that colocalized in large part with that of TNF-alpha, IL-1 beta, and IL-6. CONCLUSION: The significant levels of baseline and inducible MIF expression in the brain and its regional association with glucocorticoid action underscore the importance of this mediator as a physiological regulator of the inflammatory stress response and further define its role within the neuroendocrine system.

Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes.

We recently described a novel population of blood-borne cells, termed fibrocytes, that display a distinct cell surface phenotype (collagen+/CD13+/CD34+/CD45+), rapidly enter sites of tissue injury, and contribute to scar formation. To further characterize the role of these cells in vivo, we examined the expression of type I collagen and cytokine mRNAs by cells isolated from wound chambers implanted into mice. Five days after chamber implantation, CD34+ fibrocytes but not CD14+ monocytes or CD90+ T cells expressed mRNA for type I collagen. Fibrocytes purified from wound chambers also were found to express mRNA for IL-1beta, IL-10, TNF-alpha, JE/MCP, MIP-1alpha, MIP-1beta, MIP-2, PDGF-A, TGF-beta1, and M-CSF. The addition of IL-1beta (1-100 ng/ml), a critical mediator in wound healing, to fibrocytes isolated from human peripheral blood induced the secretion of chemokines (MIP-1alpha, MIP-1beta, MCP-1, IL-8, and GRO alpha), hemopoietic growth factors (IL-6, IL-10, and macrophage-CSF), and the fibrogenic cytokine TNF-alpha. By contrast, IL-1beta decreased the constitutive secretion of type I collagen as measured by ELISA. Additional evidence for a role for fibrocytes in collagen production in vivo was obtained in studies of livers obtained from Schistosoma japonicum-infected mice. Mouse fibrocytes localized to areas of granuloma formation and connective matrix deposition. We conclude that fibrocytes are an important source of cytokines and type I collagen during both the inflammatory and the repair phase of the wound healing response. Furthermore, IL-1beta may act on fibrocytes to effect a phenotypic transition between a repair/remodeling and a proinflammatory mode.

The peripheral blood fibrocyte is a potent antigen-presenting cell capable of priming naive T cells in situ.

Recent studies have identified a novel population of blood-borne cells, termed fibrocytes, that have a distinct cell surface phenotype (collagen+/CD13(+)/CD34(+)/CD45(+)), rapidly enter sites of tissue injury, and synthesize connective tissue matrix molecules. We found by flow cytometry that purified human fibrocytes express each of the known surface components that are required for antigen presentation, including class II major histocompatability complex molecules (HLA-DP, -DQ, and -DR), the costimulatory molecules CD80 and CD86, and the adhesion molecules CD11a, CD54, and CD58. Human fibrocytes induced antigen-presenting cell-dependent T cell proliferation when cultured with specific antigen and this proliferative activity was significantly higher than that induced by monocytes and nearly as high as that induced by purified dendritic cells. Mouse fibrocytes also were found to express the surface components required for antigen presentation and to function as potent APCs in vitro. Mouse fibrocytes pulsed in vitro with the HIV-proteins p24 or gp120 and delivered to a site of cutaneous injury were found to migrate to proximal lymph nodes and to specifically prime naive T cells. These data suggest that fibrocytes play an early and important role in the initiation of antigen-specific immunity.

Migration inhibitory factor expression in experimentally induced endotoxemia.

Macrophage migration inhibitory factor (MIF) is an important constituent of the host response to stress and infection and is the first mediator that has been identified to be released from immune cells upon stimulation with glucocorticoids. MIF also has been shown to be secreted from the anterior pituitary gland, monocytes/macrophages, and T cells activated by various proinflammatory stimuli. Once released, MIF acts to counter-regulate the inhibitory effect of glucocorticoids on inflammatory cytokine production. To characterize more precisely the role of MIF in the host response to infection, we undertook a systematic analysis of MIF expression in various organs of the rat after endotoxin (lipopolysaccharide) administration. MIF protein and mRNA were analyzed by immunohistochemistry and in situ hybridization, respectively. MIF was found to be expressed constitutively in organs such as the lung, liver, kidney, spleen, adrenal gland, and skin. Significant quantities of MIF protein were detected preformed in various cell types and appeared to be released as a consequence of endotoxemia. In virtually all tissues examined, the loss of MIF protein 6 hours after lipopolysaccharide administration was accompanied by the induction of MIF mRNA and, at 24 hours, by the restoration of immunoreactive, intracellular MIF. The constitutive production of MIF by several cell and tissue types together with its rapid release from intracellular pools distinguishes MIF from other cytokines or hormonal mediators and significantly expands the physiological role of this unique counter-regulator of glucocorticoid action.

An essential regulatory role for macrophage migration inhibitory factor in T-cell activation.

The protein known as macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered and was described 30 years ago to be a T-cell-derived factor that inhibited the random migration of macrophages in vitro. A much broader role for MIF has emerged recently as a result of studies that have demonstrated it to be released from the anterior pituitary gland in vivo. MIF also is the first protein that has been identified to be secreted from monocytes/macrophages upon glucocorticoid stimulation. Once released, MIF acts to "override" or counter-regulate the suppressive effects of glucocorticoids on macrophage cytokine production. We report herein that MIF plays an important regulatory role in the activation of T cells induced by mitogenic or antigenic stimuli. Activated T cells produce MIF and neutralizing anti-MIF antibodies inhibit T-cell proliferation and interleukin 2 production in vitro, and suppress antigen-driven T-cell activation and antibody production in vivo. T cells also release MIF in response to glucocorticoid stimulation and MIF acts to override glucocorticoid inhibition of T-cell proliferation and interleukin 2 and interferon gamma production. These studies indicate that MIF acts in concert with glucocorticoids to control T-cell activation and assign a previously unsuspected but critical role for MIF in antigen-specific immune responses.

Bacterial glutathione: a sacrificial defense against chlorine compounds.

Aerobic organisms possess a number of often overlapping and well-characterized defenses against common oxidants such as superoxide and hydrogen peroxide. However, much less is known of mechanisms of defense against halogens such as chlorine compounds. Although chlorine-based oxidants may oxidize a number of cellular components, sulfhydrl groups are particularly reactive. We have, therefore, assessed the importance of intracellular glutathione in protection of Escherichia coli cells against hydrogen peroxide, hypochlorous acid, and chloramines. Employing a glutathione-deficient E. coli strain (JTG10) and an otherwise isogenic glutathione-sufficient E. coli strain (AB1157), we find that glutathione-deficient organisms are approximately twice as sensitive to killing by both hydrogen peroxide and chlorine compounds. However, the mode of protection by glutathione in these two cases appears to differ: exogenous glutathione added to glutathione-deficient E. coli in amounts equal to those which would be present in a similar suspension of the wild-type bacteria fully restored resistance of glutathione-deficient bacteria to chlorine-based oxidants but did not change resistance to hydrogen peroxide. Furthermore, in protection against chlorine compounds, oxidized glutathione is almost as effective as reduced glutathione, implying that the tripeptide and/or oxidized thiol undergo further reactions with chlorine compounds. Indeed, in vitro, 1 mol of reduced glutathione will react with approximately 3.5 to 4.0 mol of hypochlorous acid. We conclude that glutathione defends E. coli cells against attack by chlorine compounds and hydrogen peroxide but, in the case of the halogen compounds, does so nonenzymatically and sacrificially.