Energy metabolism and T-cell-mediated cytolysis. I. Synergism between inhibitors of respiration and glycolysis.

The energy requirements for T-cell-mediated cytolysis have been investigated. Cytolytic thymus-derived lymphocytes (CTL) were generated in vitro in mixed leukocyte cultures and assayed for cytotoxicity on 51Cr-labeled mastocytoma target cells. Cytolysis was only slightly reduced in the absence of exogenous glucose (less than 5 micrometer) or under conditions of extreme hypoxia (less than 0.2 micrometer oxygen). Furthermore, neither the glucose analogues 2-deoxy-D-glucose and 5-thio-D-glucose nor the respiratory antagonists sodium azide and 2,4-dinitrophenol were very effective inhibitors of cytolysis when used individually. However, these glucose analogues were highly effective in inhibiting cytolysis in the absence of oxygen, and the respiratory antagonists inhibited cytolysis to a much greater extent in the absence of glucose. In addition, synergistic effects were observed when the glycolytic and respiratory inhibitors were combined. Taken together, these results indicate that T-cell-mediated cytolysis is an energy-dependent process which can be supported by either oxidative or glycolytic energy pathways.

Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53.

Significant numbers of myocytes die by apoptosis during myocardial infarction. The molecular mechanism of this process, however, remains largely unexplored. To facilitate a molecular genetic analysis, we have developed a model of ischemia-induced cardiac myocyte apoptosis in the mouse. Surgical occlusion of the left coronary artery results in apoptosis, as indicated by the presence of nucleosome ladders and in situ DNA strand breaks. Apoptosis occurs mainly in cardiac myocytes, and is shown for the first time to be limited to hypoxic regions during acute infarction. Since hypoxia-induced apoptosis in other cell types is dependent on p53, and p53 is induced by hypoxia in cardiac myocytes, we investigated the necessity of p53 for myocyte apoptosis during myocardial infarction. Myocyte apoptosis occurs as readily, however, in the hearts of mice nullizygous for p53 as in wild-type littermates. These data demonstrate the existence of a p53-independent pathway that mediates myocyte apoptosis during myocardial infarction.

Unusual oxygen concentration dependence of toxicity of SR-4233, a hypoxic cell toxin.

Toxicity from drugs activated by bioreductive metabolism has been suggested as a means to eliminate the treatment resistance caused by hypoxic tumor cells. In general, drugs have been selected to maximize the hypoxic cytotoxicity ratio [exposure (drug concentration x time) in air:exposure in nitrogen] to cause equal toxicity. On this basis, two recently developed drugs have very similar characteristics; an aziridine derivative of misonidazole (RSU1069) and a benzotriazine di-N-oxide (SR4233). The oxygen dependence of the toxic response has not previously been characterized. This report shows that the toxicity from SR4233 extends over a much greater range of oxygen concentrations than does that of RSU1069. Furthermore, unlike all previous drugs studied, the toxicity of SR4233 does not level off at high oxygen concentrations, but continues to decrease as the oxygen concentration increases. For 1 mM oxygen (the solubility of oxygen in medium at 37 degrees C equilibrated with 100% oxygen and water vapor) the toxicity from SR4233 is at least 2000-fold less than that for hypoxia. Modeling the effect of oxygen on combined radiation and toxicity shows that radiation plus SR4233 should be much more effective in eliminating hypoxic cells than radiation plus RSU1069. The unusual oxygen dependence of toxicity by SR4233 may indicate a unique biochemical activation process.

Detection of hypoxic cells by monoclonal antibody recognizing 2-nitroimidazole adducts.

Hypoxic cells in tissue pose many medical problems, and there is a need for more accurate measurements of tissue hypoxia. However, measurement of the pO2 and the extent of hypoxia within normal and tumor tissue have proven difficult. One of the most sensitive of the currently available methodologies involves the oxygen-dependent metabolic activation of nitroheterocyclic drugs, leading to adducts between the drugs and cellular macromolecules. Limitations of the present drugs and adduct-detection methods prompted the present studies. A pentafluorinated derivative [EF5; 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetam ide] of etanidazole was synthesized with the expectation of lessening some of the non-oxygen-dependent variability in adduct formation observed previously with other nitroaromatic compounds. EF5-protein conjugates, prepared by radiochemical reduction, were found to be immunogenic and allowed the development of monoclonal antibodies. One of these antibodies, ELK2-4, has been characterized and found to be highly specific for the EF5 adducts whether produced radiochemically or by cellular bioreductive metabolism. 9L rat glioma cells pretreated with EF5 under hypoxic, compared with aerobic, conditions were readily discriminated immunochemically using fluorochrome-conjugated secondary antibodies which recognize the ELK2-4 antibody subtype (IgG1). Similarly, the central region of multicenter spheroids, composed of EMT6 mouse mammary sarcoma cells, was selectively visualized by immunohistochemistry after the spheroids were incubated for 4 h in 0.5 mM EF5. Tumor biopsy, preparation, and immunohistochemical staining 24 h after treatment of tumor-bearing animals with drug also demonstrated high contrast regions within EMT6 mouse or Morris 7777 hepatoma rat tumors. The use of this new compound and its highly specific monoclonal antibody may allow elucidation of bioreductive metabolism of the nitroheterocyclics and significantly improve technologies for the quantitation of tissue pO2.

Distribution of misonidazole adducts in 9L gliosarcoma tumors and spheroids: implications for oxygen distribution.

The 9L rat gliosarcoma is a widely used experimental brain tumor which has an unusual radiation response. The radiation sensitivity of cells in subcutaneous tumors has been shown to be intermediate between those of aerobic and hypoxic 9L cells in vitro, and cells in spheroids and intracranial tumors appear to be as sensitive as fully aerobic cells. The hypoxic marker misonidazole was used to investigate the distribution of oxygen in these 9L systems. In vitro calibration of binding of [3H]-misonidazole as a function of oxygen concentration demonstrated an inverse relationship similar to those of several other experimental tumors. In autoradiograms of sections from tumors labeled in vivo the grain density rose gradually from the periphery of the tumor to the center. Over millimeter distances the distribution of grains was remarkably uniform, in contrast to the substantial variation reported for several other tumors. The grain density as a function of distance from capillaries was essentially constant. Several interpretations of this result are possible, including the postulate that intermittent blood flow occurs in all 9L tumor capillaries, which results in the majority of the binding of misonidazole occurring during the periods of transient hypoxia. Cells adjacent to the necrotic center in 9L spheroids and the rare necrotic regions in tumors appeared to be severely hypoxic, based on the quantity of misonidazole they bound. Spheroid "cure" experiments demonstrated that these cells were not clonogenic.

Depletion of tumor oxygenation during photodynamic therapy: detection by the hypoxia marker EF3 [2-(2-nitroimidazol-1[H]-yl)-N-(3,3,3-trifluoropropyl)acetamide ].

Photodynamic therapy (PDT) of tumors can create hypoxia when oxygen is depleted by photochemical consumption or the oxygen supply is compromised by microvascular damage. However, oxygen is a requirement for PDT, and hypoxia during illumination can lead to poorer tumor response. As such, sensitive methods of quantifying tumor oxygen and evaluating its distribution may help in the development and optimization of treatment protocols. In this study, the hypoxia marker EF3 [2-(2-nitroimidazol-1[H]-yl)-N-(3,3,3-trifluoropropyl)acetam ide] was used to evaluate the oxygenation of PDT-treated radiation-induced fibrosarcoma tumors. Tumor-bearing mice were administered Photofrin (5 mg/kg) 24 h before PDT illumination at 75 mW/cm2, 135 J/cm2 (30 min). EF3 (52 mg/kg) was injected either within 3 min before PDT illumination, with tumor excision at the conclusion of illumination, or within 3 min after illumination, with tumor excision 30 min later. Control animals received EF3 alone, EF3 plus Photofrin, or EF3 plus illumination. After tumor disaggregation, staining with a fluorochrome-conjugated monoclonal antibody, and flow cytometric analysis, control tumors demonstrated an averaged median fluorescence intensity (+/- SE) of 17.1 +/- 2.8. EF3 binding significantly (P = 0.007) increased during PDT to a median fluorescence intensity of 48.9 +/- 8.3. In the 30 min after PDT, EF3 binding returned to control levels (median, 18.3 +/- 3.3). To evaluate the oxygen concentrations corresponding to these fluorescence intensities, an in vitro standard curve was created based on the in vivo exposure conditions. From this curve, the oxygen tensions of tumors exposed to EF3 under control conditions, during PDT, or after PDT were calculated to be 3.1-5.3, 1.2-2.4, and 3.0-5.2 mm Hg, respectively. Detection of EF3 binding using a monoclonal antibody correlated well with direct detection of binding using a radioactive assay. EF3 binding was linear with drug incubation for times from 1.5 to 60 min. Overall, this work demonstrates that hypoxia during PDT illumination of radiation-induced fibrosarcoma tumors can be detected by the hypoxia marker EF3. Hypoxia during illumination can be labeled separately from that found before or after PDT. Tissue oxygen tensions corresponding to EF3 binding levels can be calculated.

Induction of apoptosis at different oxygen tensions: evidence that oxygen radicals do not mediate apoptotic signaling.

Apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathways. Furthermore, it has been proposed that Bcl-2 acts to inhibit apoptosis induced by a wide variety of stimuli by preventing the production of oxygen-derived free radicals. Since the generation of oxygen free radicals is dependent upon oxygen concentration, this hypothesis would lead to the prediction that the concentration of oxygen should affect the induction of apoptosis. In order to test this prediction, we have examined the induction of apoptosis in T-lymphoma cell lines S49.1 and WEHI 7.1 by dexamethasone and by withdrawal of serum from myc-immortalized fibroblasts in 95% oxygen, atmospheric oxygen (20%), and hypoxic conditions of up to 125-fold less oxygen. Culture in 95% oxygen induced apoptosis in all cells tested, confirming that oxidative damage can lead to apoptosis. However, for one cell line, WEHI 7.1, hypoxia also induced apoptosis. Furthermore, for the other cell lines tested, induction of apoptosis by either dexamethasone or by serum withdrawal was not affected by hypoxia. These results are not consistent with the hypothesis that apoptosis is mediated via oxygen-generated free radical formation.

2-Nitroimidazole (EF5) binding predicts radiation resistance in individual 9L s.c. tumors.

The presence of hypoxic tumor cells is known to be an important cause of radiation treatment resistance in vivo. The ability to predict the presence and extent of hypoxic cells in individual tumors would allow the addition of specific "antihypoxia"-based treatment regimes. Hypoxia can be monitored by measuring the binding of 2-nitroimidazoles. We have tested the hypothesis that binding of EF5, a fluorinated derivative of the 2-nitroimidazole, Etanidazole, can predict radioresistance in individual tumors. Fischer rats bearing 9L s.c. tumors were given injections i.v. with EF5 3 h before irradiation and tumor harvest. Tumor cells were dissociated for flow cytometric analysis and plating efficiency studies. EF5 binding was detected via monoclonal antibodies conjugated to the orange emitting dye, Cy3. In air breathing rats, for a given radiation dose, a large amount of variation in plating efficiency was seen. However, there was minimal variability of the plating efficiency for tumors irradiated in euthanized animals (hypoxic tumors; correlation coefficient for the fitted curve = 0.93) and in cells dissociated from tumors and irradiated in suspension (correlation coefficient for the fitted curve = 0.99), suggesting that varying sensitivity to the cell disaggregation technique was not responsible. In contrast, a good correlation between the relative radiation resistance or hypoxic survival and EF5 binding of "moderately" hypoxic cells in air breathing rats was identified using these techniques. In these 9L s.c. tumors, intertumor variation in oxygenation accounted for most of the range in individual tumor radiation response, and this was found to be independent of tumor size. This study provides evidence for the application of EF5 binding with monoclonal antibody detection as an in vivo predictive assay of individual tumor hypoxia and resultant therapy resistance.

Oxygen dependence of binding of misonidazole to rodent and human tumors in vitro.

Misonidazole, a 2-nitroimidazole, has been shown to form metabolically induced adducts to cellular molecules at a very high rate in the absence of oxygen, and this rate decreases substantially as the oxygen concentration increases. Thus, it has considerable potential as a marker for hypoxic, radiation resistant cells in tumors. The dependence of the rate of adduct formation (binding) on oxygen concentration was studied for EMT6/Ed, Walker 256, and Dunning R3327-AT rodent tumors and for two human colon carcinomas, a human melanoma, and a human breast carcinoma. Fragments of these tumors were incubated with [14C]- or [3H]misonidazole in vitro at several oxygen concentrations and the quantity of misonidazole bound was determined from autoradiographs as a function of distance from the surfaces of the fragments. The Km of binding inhibition (oxygen concentration for half-maximal binding) for the tumors varied by a factor of 10. The range was centered on the range of values reported for the Km of cellular radioresistance (oxygen concentration for half-maximal radioresistance). The patterns of binding at depth within the tumor fragments indicated that gradients of cellular waste products and nutrients other than oxygen had minimal effects on binding. All tumors were capable of metabolizing oxygen to levels sufficiently low to yield the maximal binding rate, but the distance of penetration of oxygen varied, indicating a range of at least 4 in rates of oxygen consumption. The ratio of misonidazole bound by stromal tissue versus tumor cells ranged from 0.9 for a colon tumor to 0.3 for a breast tumor. These properties of misonidazole binding indicate that it should be a good marker for radiobiological hypoxia in tumors, providing adequate controls can be performed.

Interleukin 2 expression by tumor cells alters both the immune response and the tumor microenvironment.

Microenvironmental conditions within solid tumors can have marked effects on the growth of the tumors and their response to therapies. The disorganized growth of tumors and their attendant vascular systems tends to result in areas of the tumors that are deficient in oxygen (hypoxic). Cells within these hypoxic areas are more resistant to conventional therapies such as radiation and chemotherapy. Here, we examine the hypoxic state of EMT6 mouse mammary tumors and the location of host cells within the different areas of the tumors to determine whether such microenvironmental conditions might also affect their ability to be recognized by the immune system. Hypoxia within tumors was quantified by flow cytometry and visualized by immunohistochemistry using a monoclonal antibody (ELK3-51) against cellular adducts of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetam ide (EF5), a nitroimidazole compound that binds selectively to hypoxic cells. Thy-1+ cells, quantified using a monoclonal antibody, were found only in the well-oxygenated areas. The location of these Thy-1+ cells was also examined in EMT6 tumors that had been transfected with the gene for interleukin-2 (IL-2) because these tumors contain greatly increased numbers of host cells. Surprisingly, we found that IL-2-transfected tumors had significantly decreased hypoxia compared to parental tumors. Furthermore, using the fluorescent dye Hoechst 33342, an in vivo marker of perfused vessels, combined with immunochemical staining of PECAM-1 (CD31) as a marker of tumor vasculature, we found increased vascularization in the IL-2-transfected tumors. Thus, expression of IL-2 at the site of tumor growth may enhance tumor immunity not only by inducing the generation of tumor-reactive CTLs but also by allowing increased infiltration of activated T cells into the tumors.

Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker.

We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide], a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitroimidazole and produce immunologically detectable adducts of the EF5 compound. Northern blotting analyses demonstrated that VEGF165 is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression. This study demonstrates that VEGF expression is up-regulated in response to hypoxia and in the microenvironments found in human multicellular tumor spheroids. This investigation also illustrates the utility of the EF5 binding in multi-cellular tumor spheroids as a means of studying the expression and regulation of hypoxia-inducible genes.

Tamoxifen induces hypoxia in MCF-7 xenografts.

Tamoxifen is widely used as an adjunct therapy for breast cancer. We hypothesized that hypoxia develops in tumors as a result of tamoxifen treatment because tamoxifen has been reported to be antiangiogenic and thrombogenic. MCF-7 breast tumors were grown under estrogenic stimulation in 4-6-week-old CD-1 nu/nu female mice. When the tumors were approximately 5 mm in diameter, 17beta-estradiol pellets were replaced with either placebo or tamoxifen-containing pellets. Two days later, tissue oxygenation was measured using immunohistochemical detection of binding of the 2-nitroimidazole EF5. Intravascular oxygen partial pressures were measured noninvasively by oxygen-dependent quenching of phosphorescence of an injected dye that is excited by light pulses. Tamoxifen treatment increased hypoxia in the tumors, as measured by EF5 binding (P = 0.01 by Mann-Whitney test). This observation was not dependent on the presence of tamoxifen-induced necrosis. Intravascular oxygen partial pressures were lower in tumors relative to surrounding normal tissue in tamoxifen-treated tumors as compared to placebo-treated tumors. In vitro, tamoxifen did not modify the oxygen-dependent metabolism of EF5, indicating that the increased EF5 binding in tamoxifen-treated tumors reflects a physiological decrease in tissue oxygenation. The clinical significance of these observations is discussed in the context of the sequencing of tamoxifen with other therapies, and in light of recent data suggesting that hypoxia may be associated with genetic changes resulting in a more aggressive tumor phenotype.

The farnesyltransferase inhibitor L744,832 reduces hypoxia in tumors expressing activated H-ras.

Many tumors contain extensive regions of hypoxia. Because hypoxic cells are markedly more resistant to killing by radiation, repeated attempts have been made to improve the oxygenation of tumors to enhance radiotherapy. We have studied the oxygenation of tumor xenografts in nude mice after treatment with the farnesyltransferase inhibitor L744,832. Hypoxia was assessed by measuring the binding of the hypoxic cell marker pentafluorinated 2-nitroimidazole. We show that xenografts from two tumor cell lines with mutations in H-ras had markedly improved oxygenation after farnesyltransferase treatment. In contrast, xenografts from two tumors without ras mutations had equivalent hypoxia regardless of treatment. The effect on tumor oxygenation could be detected at 3 days and remained after 7 days of treatment. These results indicate that treatment with farnesyltransferase inhibitors can alter the oxygenation of certain tumors and suggest that such treatment might be useful in the radiosensitization of these tumors.

Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12.

The role of angiogenesis inhibition in the antitumor activity of recombinant murine interleukin 12 (rmIL-12) was studied in K1735 murine melanomas, the growth of which is rapidly and markedly suppressed by rmIL-12 treatment. On the basis of the prediction that tumor ischemia should result from therapeutic angiogenesis inhibition, tumor cell hypoxia was evaluated as a marker of ischemia using the EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide] approach. This method measures intracellular binding of the nitroimidazole EF5, which covalently binds to cellular macromolecules selectively under hypoxic conditions. Whereas 1 week of rmIL-12 treatment effectively inhibited K1735 cell-induced angiogenesis in Matrigel neovascularization assays, 2 weeks of treatment were needed before severe tumor cell hypoxia was detected in K1735 tumors. The hypoxia that developed was regional and localized to tumor areas distant from blood vessels. The great majority of severely hypoxic tumor cells were apoptotic, and in vitro studies indicated that the degree of hypoxia present within treated tumors was sufficient to trigger K1735 apoptosis. Tumor cell apoptosis was also prevalent in the first week of rmIL-12 treatment when few cells were hypoxic. In vitro studies indicated that this non-hypoxia-related apoptosis was induced directly by IFN-gamma produced in response to rmIL-12 administration. These studies reveal that rmIL-12 controls K1735 tumors initially by IFN-gamma-induced apoptosis and later by hypoxia-induced apoptosis. They also establish hypoxia as an expected result of tumor angiogenesis inhibition and a mediator of its therapeutic effect.

Detection of hypoxia in human squamous cell carcinoma by EF5 binding.

Localization and quantitation of 2-nitroimidazole drug binding in low pO2 tumors is a technique that can allow the assessment of hypoxia as a predictive assay. EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] is such a drug, and it has been shown to be predictive of radiation response in rodent tumors. Using fluorescence immunohistochemical techniques, we provide data on the presence, distribution, and levels of EF5 binding as a surrogate for hypoxia in human head and neck and uterine cervix squamous cell cancers (SCCs). Six patients with SCC were studied. Four patients had head and neck tumors, and two had uterine cervix cancers. The incubation of fresh tissue cubes in EF3 under hypoxic conditions ("reference binding") demonstrated that all tumors were capable of binding drug, and that this binding varied by a factor of 2.9-fold (174.5-516.1) on an absolute fluorescence scale. In the five patients treated at the lowest drug doses (9 mg/kg), in situ binding was quantitatable. For all six patients, the maximum rate of in situ binding varied by a factor of 6.7 between the lowest and highest binding tumor (24.8-160.3) on an absolute fluorescence scale. In tumors with high binding regions, intratumoral heterogeneity was large, extending from minimal fluorescence (<1%) up to 88.6% of reference binding. In tumors with minimal binding, there was little intratumoral heterogeneity. These studies demonstrate substantial heterogeneity of in situ binding between and within individual squamous cell tumors.

Hypoxia and VEGF mRNA expression in human tumors.

High expression of circulating plasma vascular endothelial growth factor (VEGF) in patients with cancer is an indicator of poor treatment response. Similarly, hypoxia in tumors, as measured by oxygen needle electrodes, has been found to predict for tumor-treatment failure. These two predictors may be related because hypoxia is a potent stimulator of VEGF expression in vitro. However, the demonstration of a relationship between hypoxia and VEGF in human tumors has, to date, been indirect or even negative. The purpose of this study was to test whether this unexpected result was caused by factors unique to human tumors, or whether the prior results could have been influenced by the known complexities of VEGF regulation. Therefore, we undertook a direct assessment of VEGF induction in human tumors using in situ hybridization and compared its distribution with that of hypoxia, as measured by the distribution of adducts of the hypoxia marker EF5. The distribution of both markers was assessed in relationship to the distribution of blood vessels, as measured by antibodies to CD31. Our hypothesis was that VEGF mRNA and hypoxia would colocalize, assuming that detectability of the former was not limiting. Four squamous cell carcinomas, three sarcomas and one glioblastoma multiforme were studied. When VEGF mRNA signal was detectable, its maxima colocalized with regional maxima of EF5 binding. The strongest levels of both signals were sometimes adjacent to regions of tissue necrosis. However, we were unable to predict absolute levels of EF5 binding based on absolute levels of VEGF mRNA. Conversely, for all tumors studied, regions with relatively low levels of EF5 binding had relatively low or undetectable VEGF mRNA. We found moderate EF5 binding in some keratinized cells but VEGF mRNA was not expressed by these differentiated cells. The paradigm that hypoxia and VEGF expression are linked in human tumors is supported by the data presented herein. A better understanding of the biology behind VEGF expression, including its modulation by hypoxia, is important for optimizing its use as a prognostic indicator and/or modulating its presence with biologic therapies.

The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide.

Instability of hydrogen peroxide solutions was noted during the experimental exposure of human cells in culture to hydrogen peroxide in experiments designed to study the production and repair of DNA single-strand breaks. A hydrogen peroxide concentrate was diluted into culture medium, which was then added to experimental cell cultures at various times, with all cultures assessed for DNA damage at 2 h. Only cells treated by the first addition had observable DNA damage. This result was unexpected since these cells had had the maximum repair time. It was determined that the hydrogen peroxide had been eliminated by the culture medium. To determine the mechanism of this elimination, 200 microM hydrogen peroxide was added to various cell culture components, and the solutions were assayed for hydrogen peroxide after 1 h at 37 degrees C. Although most components (except the balanced salts) showed some hydrogen peroxide degradation, it was found that sodium pyruvate was most effective, by a wide margin, in eliminating hydrogen peroxide and its toxic effects. This was confirmed by addition of pyruvate to balanced salt solutions or buffers, and observing the same elimination of hydrogen peroxide. We subsequently found a few earlier reports describing the decarboxylation reaction between hydrogen peroxide and pyruvate, but no kinetic measurements have been published and there seems to be no general appreciation for the very high efficiency of this reaction. The present work presents a preliminary assessment of the importance of pyruvate in the study of hydrogen peroxide and other reactive oxygen species in mammalian cell culture.

Monoclonal antibodies differentially reactive with native and reductively modified Bowman-Birk protease inhibitor.

Bowman-Birk protease inhibitor (BBI) is a potent anticarcinogen that suppresses malignant transformation at nanomolar concentrations. Small amounts of BBI in its native form can be measured by immunoassay using specific monoclonal antibodies (MAbs); however, the MAbs currently available are not capable of detecting BBI metabolites in human body fluids. To develop new reagents for the study of BBI exposure and pharmacokinetics, we produced four MAbs, designated 3B6, 3E3, 4H8 and 5G2, from hybridomas derived from a mouse immunized with reductively modified BBI. The epitopes recognized by the four MAbs were characterized using BBI in its native form or modified by different methods. MAb 3B6 reacted with native BBI. Partial reduction of BBI with 720 Gy of gamma radiation in an oxygen-free solution of 100 mM formate increased the reactivity of BBI with 3B6; however, extensive reduction of BBI with 100 mM DL-dithiothreitol (DTT) completely abolished this antigenic reactivity. In contrast, the other three MAbs reacted with BBI molecules that had been reduced either with 720 Gy of radiation in formate solution or with DTT. Alkylation of the radiochemically reduced BBI with N-ethylmaleimide further increased the reactivity of BBI with 3E3, 4H8 and 5G2, possibly by preventing the formation of new disulfide bonds within the BBI molecules. The binding of 4H8 and 5G2 to BBI antigen was inhibited by the binding of 3E3, and vice versa. Thus, the epitopes recognized by 3E3, 4H8 and 5G2 are probably located close to one another on the reduced BBI molecules. These three MAbs were able to react with BBI metabolites in urine samples collected from volunteers after oral administration of BBI. The ability of these MAbs to detect BBI metabolites indicates that BBI may be reductively modified in vivo and these MAbs may be useful reagents for monitoring the uptake of BBI into human tissues in cancer chemoprevention studies with BBI.

Binding of misonidazole to V79 spheroids and fragments of Dunning rat prostatic and human colon carcinomas in vitro: diffusion of oxygen and reactive metabolites.

Differences were noted previously in the binding of 14C-Misonidazole (MISO) to V79 and EMT6 spheroids when incubated at low oxygen levels. While binding to the viable cells in EMT6 spheroids was uniform, in V79 spheroids the binding rate increased gradually with distance from the spheroid surface. Further data reported here indicate that the Km for the inhibition of binding by oxygen is lower in V79 than EMT6 spheroids, so that part of the non-uniformity of binding to V79 spheroids can be explained by diffusion of small amounts of oxygen through the entire rim of viable cells. Diffusion of reactive metabolites of MISO out of the spheroid previously was considered an unlikely explanation. Further evidence to support this interpretation is presented here. Patterns of binding of 3H-MISO to Dunning and human colon carcinomas are presented which are consistent with the interpretation that most of the reactive metabolites are confined to the cell in which they are produced. This conclusion is based on a substantial difference in the rate of binding to adjacent stromal and tumor elements.

Misonidazole: inter-related factors affecting cytotoxicity.

A detailed investigation was undertaken of reported modifiers of the toxicity towards hypoxic cells of misonidazole. The modifiers tested were medium type, cell type, cell density, concentration of misonidazole, addition of serum, addition of sulfhydryl, addition of oxygen and pH. The latter 5 modifiers were found to be most important and were studied in many of the possible combinations. Serum has its greatest protective effect at low concentration (e.g. 0.5 mM) of misonidazole. In the absence of serum, the (log10) survival curve for misonidazole toxicity can be described mathematically as a function of time by a shoulder (DQ) inversely related to misonidazole concentration, and a slope (1/D0) related directly to log10 misonidazole concentration. Sulfhydryl's (cysteamine) protective effect dominates at high concentrations of misonidazole. The protective action of SH can change to potentiative in the absence of serum or at high pH. Addition of oxygen results in overall protection but no relative changes in the effect of the other modifiers. Drugs like ascorbate and disulfide may only potentiate toxicity to the level found in the absence of serum.