Biomedical subjects

C J Koch

Publications and source records attributed to C J Koch.

At least 37 records · Page 2Linked to original sources

PubMed2000

Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells.

PURPOSE: We investigated the effect of protein- and non protein-thiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation and its relevance in the survival of CHO cells. MATERIALS AND METHODS: We used mutant cells null for glucose 6 phosphate dehydrogenase (G6PD) activity since reducing equivalents, required for reduction of oxidized thiols, are typically generated through G6PD regulated production of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (ME). The concentrations of the intracellular and extracellular non-protein thiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell survival was determined by clonogenic assay. The induction and rejoining of DSB in cells was quantitated by Pulse Field Gel Electrophoresis after exposure to ionizing radiation. RESULTS: Much lower bioreduction of HEDS was found in the G6PD deficient mutants (E89) than in the wild-type cells (K1). A 1 h treatment of E89 cells with HEDS produced almost complete depletion of non-protein thiol (NPSH) and a 26% decrease in protein thiols. Only minor changes were found under similar conditions with K1 cells. When exposed to gamma radiation in the presence of HEDS, the G6PD null mutants exhibited a higher cell killing and decreased rate and extent of rejoining of DSB than were observed in K1 cells. Moreover, when the G6PD deficient cells were transfected with the gene encoding wild-type G6PD (A1A), they recovered close to wild-type cellular thiol status, cell survival and DSB rejoining. CONCLUSIONS: These results suggest that a functioning oxidative pentose phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.

Animals↗

PubMed2000

Tissue hypoxia inhibits prostaglandin and nitric oxide production and prevents ductus arteriosus reopening.

Regulation of ductus arteriosus (DA) tension depends on a balance between oxygen-induced constriction and PG and nitric oxide (NO)-mediated relaxation. After birth, increasing Pa(O(2)) produces DA constriction. However, as the full-term ductus constricts, it develops severe tissue hypoxia in its inner vessel wall (oxygen concentration <0.2%). We used isolated rings of fetal lamb DA to determine why the constricted ductus does not relax and reopen as it becomes hypoxic. We used a modification of the 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide (EF5) technique (Clyman RI, Chan CY, Mauray F, Chen YQ, Cox W, Seidner SR, Lord EM, Weiss H, Wale N, Evan SM, and Koch CJ. Pediatr Res 45: 19-29, 1999) to determine mean tissue oxygen concentration. A decrease in the ductus' mean tissue oxygen concentration from 1.4 to 0.1% lowers the isometric tone of the ductus by 15 +/- 10% of its maximal active tension (the maximal tension that can be produced by the ductus). Although decreases in oxygen concentration diminish ductus tension, most of the vasoconstrictor tone in the ductus is independent of ambient oxygen concentration. This oxygen-independent tone is equivalent to 64 +/- 10% of the maximal active tension. At mean tissue oxygen concentrations >0.2%, endogenous PGs and NO inhibit more than 40% of the active tension developed by the ductus. However, when tissue oxygen concentrations drop below 0.2%, the constitutive relaxation of the ductus by endogenous PGs and NO is lost. In the absence of PG and NO production, tension increases to a level normally observed only after treatment of the ductus with indomethacin and nitro-L-arginine methyl ester (inhibitors of PG and NO production). Therefore, under conditions of severe hypoxia (tissue oxygen concentration <0.2% oxygen), the loss of PG- and NO-mediated relaxation more than compensates for the loss of oxygen-induced tension. We hypothesize that this increased ductus tone enables the vessel to remain closed as it undergoes tissue remodeling.

Animals↗

PubMed2000

Noninvasive detection of tumor hypoxia using the 2-nitroimidazole [18F]EF1.

UNLABELLED: The noninvasive assessment of tumor hypoxia in vivo is under active investigation because hypoxia has been shown to be an important prognostic factor for therapy resistance. Various nuclear medicine imaging modalities are being used, including PET imaging of 18F-containing compounds. In this study, we report the development of 18F-labeled EF1 for noninvasive imaging of hypoxia. EF1 is a 3-monofluoro analog of the well-characterized hypoxia marker EF5, 2(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetami de, which has been used to detect hypoxia in tumor and nontumor systems using immunohistochemical methods. METHODS: We have studied 2 rat tumor types: the hypoxic Morris 7777 (Q7) hepatoma and the oxic 9LF glioma tumor, each grown in subcutaneous sites. PET studies were performed using a pharmacological dose of nonradioactive carrier in addition to [18F]EF1 to optimize and assess drug biodistribution. After PET imaging of the tumor-bearing rats, tissues were obtained for gamma-counting of the 18F in various tissues and immunohistochemical detection of intracellular drug adducts in tumors. In one pair of tumors, Eppendorf needle electrode studies were performed. RESULTS: [18F]EF1 was excreted dominantly through the urinary tract. The tumor-to-muscle (T/M) ratio of [18F]EF1 in the Q7 tumors was 2.7 and 2.4 based on PET studies and 2.1, 2.5, and 3.0 based on gamma-counting of the tissues (n = 3). In contrast, the T/M ratio of [18F]EF1 in the 9LF glioma tumor was 0.8 and 0.5 based on PET studies and 1.0, 1.2, and 1.4 based on gamma-counting of the tissues (n = 3). Immunohistochemical analysis of drug adducts for the two tumor types agreed with the radioactivity analysis. In the Q7 tumor, substantial heterogeneous binding was observed throughout the tumor, whereas in the 9LF tumor minimal binding was found. CONCLUSION: [18F]EF1 is an excellent radiotracer for noninvasive imaging of tumor hypoxia.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

Decreased brain infarct following focal ischemia in mice lacking the transcription factor E2F1.

E2F1+/- mice subjected to 2 h middle cerebral artery occlusion developed an infarct of 77.0 +/- 3.2 mm3 (mean +/- s.e.m., n = 15) in the ischemic hemisphere after 24 h reperfusion. A significantly smaller infarct of 58.8 +/- 4.8 mm3 (n = 15; p < 0.01) was found in E2F1-/- animals. Both deficient and normal mice had similar cerebral angioarchitecture and intra-ischemic decreases in regional blood flow. Similar areas of hypoxia in both groups of ischemic animals were demonstrated directly by immunohistochemical detection of nitroimidazole adducts. It was concluded that all animals received the same ischemic insult, yet the subsequent damage was different in the mutant mice. This is the first indication that the E2F1 gene plays a role in ischemic death of post-mitotic neurons.

Animals↗

PubMed1999

Detection of hypoxic cells with the 2-nitroimidazole, EF5, correlates with early redox changes in rat brain after perinatal hypoxia-ischemia.

The hypoxia-dependent activation of nitroheterocyclic drugs by cellular nitroreductases leads to the formation of intracellular adducts between the drugs and cellular macromolecules. Because this covalent binding is maximal in the absence of oxygen, detection of bound adducts provides an assay for estimating the degree of cellular hypoxia in vivo. Using a pentafluorintated derivative of etanidazole called EF5, we studied the distribution of EF5 adducts in seven-day-old rats subjected to different treatments which decrease the level of oxygen in the brain. EF5 solution was administered intraperitoneally 30 min prior to each treatment. The effect of acute and chronic hypoxia on EF5 adduct formation (binding) was studied in the brain of newborn rats exposed to global hypoxia (8% O2 for 30, 90 or 150 min) and in the brain of chronically hypoxic rat pups with congenital cardiac defects (Wistar Kyoto). The effect of combined hypoxia-ischemia was investigated in rat pups subjected to right carotid coagulation and concurrent exposure to 8% O2 for 30, 90 or 150 min. Brains were frozen immediately at the end of each treatment. Using a Cy3-conjugated monoclonal mouse antibody (ELK3-51) raised against EF5 adducts, hypoxic cells within brain regions were visualized by fluorescence immunocytochemistry. Brains from controls or vehicle-injected animals showed no EF5 binding. Notably, brains from animals which were chronically hypoxemic as a result of congenital cardiac defects also showed no EF5 binding. A short exposure (30 min) to hypoxia or to combined hypoxia-ischemia resulted in increased background stain and few scattered cells with low-intensity immunostaining. Acute hypoxia exposure of at least 90-150 min, which in this age animal does not result in frank cellular damage, produced patchy areas of low- to moderate-intensity fluorescence scattered throughout the brain. In contrast, 90-150 min of hypoxia-ischemia was associated with intense immunofluorescence in the hemisphere ipsilateral to the carotid occlusion, with a pattern similar to that reported previously for the histological damage seen in this model. This study provides a sensitive method for the evaluation of the level of oxygen depletion in brain tissue after neonatal hypoxia-ischemia at times much earlier than any method demonstrates apoptotic or necrotic cell death Since the level of in vivo formation of macromolecular adducts of EF5 depends on the degree of oxygen depletion in a tissue, intracellular EF5 binding may serve as a useful marker of regional cellular vulnerability and redox state after brain injury resulting from hypoxia-ischemia.

Acute Disease↗

PubMed1999

Synthesis of new hypoxia markers EF1 and [18F]-EF1.

We report on the preparation of a hypoxia marker 2-(2-nitroimidazol-1[H]-yl)-N-(3-fluoropropyl)acetamide (EF1) and its 18F analog, 2-(2-nitroimidazol-1[H]-yl)-N- (3-[18F]fluoropropyl)acetamide ([18F]-EF1). Two methods for the preparation of 3-fluoropropylamine, the EF1 side chain, are described. [18F]-EF1 was prepared with a radiochemical yield of 2% by nucleophilic substitution of bromine in 2-(2-nitroimidazol-1[H]-yl)-N-(3-bromopropyl)acetamide (EBr1) by carrier-added 18F in DMSO at 120 degrees C. Our results demonstrate the preparation of clinically relevant amounts of [18F]-EF1 for use as a non-invasive hypoxia marker with detection using positron emission tomography (PET).

Biomarkers↗

PubMed1999

Quantification of tumour vasculature and hypoxia by immunohistochemical staining and HbO2 saturation measurements.

Despite the possibility that tumour hypoxia may limit radiotherapeutic response, the underlying mechanisms remain poorly understood. A new methodology has been developed in which information from several sophisticated techniques is combined and analysed at a microregional level. First, tumour oxygen availability is spatially defined by measuring intravascular blood oxygen saturations (HbO2) cryospectrophotometrically in frozen tumour blocks. Second, hypoxic development is quantified in adjacent sections using immunohistochemical detection of a fluorescently conjugated monoclonal antibody (ELK3-51) to a nitroheterocyclic hypoxia marker (EF5), thereby providing information relating to both the oxygen consumption rates and the effective oxygen diffusion distances. Third, a combination of fluorescent (Hoechst 33342 or DiOC7(3)) and immunohistological (PECAM-1/CD31) stains is used to define the anatomical vascular densities and the fraction of blood vessels containing flow. Using a computer-interfaced microscope stage, image analysis software and a 3-CCD colour video camera, multiple images are digitized, combined to form a photo-montage and revisited after each of the three staining protocols. By applying image registration techniques, the spatial distribution of HbO2 saturations is matched to corresponding hypoxic marker intensities in adjacent sections. This permits vascular configuration to be related to oxygen availability and allows the hypoxic marker intensities to be quantitated in situ.

Animals↗

PubMed1999

Mechanism of copper-catalyzed autoxidation of cysteine.

The kinetics of copper-catalyzed autoxidation of cysteine and its derivatives were investigated using oxygen consumption, spectroscopy and hydroxyl radical detection by fluorescence of a coumarin probe. The process has complex two-phase kinetics. During the first phase a stoichiometric amount of oxygen (0.25 moles per mole of thiol) is consumed without production of hydroxyl radicals. In the second reaction phase excess oxygen is consumed in a hydrogen peroxide-mediated process with significant *OH production. The reaction rate in the second phase is decreased for cysteine derivatives with a free aminogroup and increased for compounds with a modified aminogroup. The kinetic data suggest the catalytic action of copper in the form of a cysteine complex. The reaction mechanism consists of two simultaneous reactions (superoxide-dependent and peroxide-dependent) in the first phase, and peroxide-dependent in the second phase. The second reaction phase begins after oxidation of free thiol. This consists of a Fenton-type reaction between cuprous-cysteinyl complex and following oxidation of cysteinyl radical to sulfonate with the consumption of excessive oxygen and significant production of hydroxyl radicals.

Catalase↗

PubMed1999

Permanent anatomic closure of the ductus arteriosus in newborn baboons: the roles of postnatal constriction, hypoxia, and gestation.

Permanent closure of the ductus arteriosus requires loss of cells from the muscle media and development of neointimal mounds, composed in part of proliferating endothelial cells. We hypothesized that postnatal ductus constriction produces hypoxia of the inner vessel wall; we also hypothesized that hypoxia might lead to cell death and the production of vascular endothelial cell growth factor (VEGF), a hypoxia-inducible growth factor that stimulates endothelial proliferation. We mapped the distribution of hypoxia in newborn baboons and correlated it with the appearance of cell death (TUNEL technique), VEGF expression, and endothelial proliferation (proliferating cell nuclear antigen expression). In the full-term baboon (n=10), the ductus was functionally closed on Doppler examination by 24 h after delivery. Regions of the ductus where the lumen was most constricted were associated with moderate/intense hypoxia; VEGF expression was increased in the hypoxic muscle media, and luminal endothelial cells, adjacent to the hypoxic media, were proliferating. Cells in the most hypoxic regions of the ductus wall were undergoing DNA fragmentation. In contrast, regions of the ductus with mild degrees of hypoxia had no evidence of cell death, VEGF expression, or endothelial proliferation. Cell death and endothelial proliferation seemed to be limited to regions of the full-term ductus experiencing moderate/intense hypoxia. In the premature baboon (67% gestation) (n=24), only 29% closed their ductus by Doppler examination before d 6. None of the premature baboons, including those with a closed ductus by Doppler, had evidence of moderate/intense hypoxia; also, there was no evidence of cell death, VEGF expression, endothelial proliferation, or neointima formation by d 6. Therefore, the premature ductus is resistant to developing hypoxia, even when its lumen is constricted; this may make it susceptible to later reopening.

Animals↗

PubMed1999

Effect of oxygen on radiation-induced DNA damage in isolated nuclei.

In intact mammalian cells, ionizing radiation causes substantially less damage to DNA in the absence of oxygen than in the presence of oxygen. In contrast, when DNA is isolated (usually from viruses) and irradiated in solution, the absence of oxygen does not lead to a decrease in damage unless low-molecular-weight thiols are also present. We investigated an intermediate condition: that of DNA irradiated in isolated nuclei. Using an HPLC-based assay of thiols with electrochemical detection, we have determined that the nuclear isolation procedure leads to the elimination of virtually all low-molecular-weight thiols (predominantly glutathione and cysteine). Thus it was our expectation that the thiol-depleted state would concurrently eliminate the OER, and thereby mimic the isolated DNA system, while retaining structural characteristics of chromosomal DNA. We evaluated radiation-induced DNA damage in isolated nuclei by measuring single-strand breaks using alkaline elution and by measuring double-strand breaks using neutral elution and pulsed-field gel electrophoresis. Despite the removal of low-molecular-weight thiol compounds, the oxygen dependence of radiation-induced damage more closely paralleled that of whole cells than that of DNA in solution. Thus damage of DNA irradiated in isolated nuclei is dependent on oxygen.

Animals↗

PubMed1999

Hypoxia induces renal tubular epithelial cell apoptosis in chronic renal disease.

Renal tubular atrophy characterizes chronic progressive renal disease, but the molecular mechanisms of renal tubular cell (RTC) deletion are unclear. Because glomerular sclerosis leads to impaired peritubular blood flow, we tested the hypothesis that chronic hypoxia contributes to RTC apoptosis. Tubule hypoxia in mice with progressive renal disease (Os/+) was assessed by injecting EF5, a nitroimidazole compound that preferentially binds to cells undergoing anaerobic metabolism. Hypoxic tubules, as determined by direct immunofluorescence with anti-EF5 antibodies, were identified in kidneys from Os/+ mice, but not in age-matched controls (+/+) at 12 weeks, coincident with the onset of glomerular pathology. Hypoxia can cause apoptosis, but apoptotic RTCs were rare and equivalent in number in 12 week Os/+ and +/+ kidneys. However, by 16 weeks apoptotic RTCs were significantly more frequent in Os/+ versus +/+ mice, demonstrating that tubule hypoxia preceded RTC apoptosis. Importantly, apoptotic RTCs co-localized to hypoxic, but not normoxic tubules, indicating that tubular atrophy may result from hypoxic stimulation of RTC apoptosis. We have previously demonstrated enhanced, diffuse expression of the Fas apoptosis receptor in Os/+ tubules, providing a potential intermediary between hypoxia and apoptosis. To determine whether hypoxia stimulates Fas-dependent apoptosis, RTCs were cultured within a hypoxia chamber or in the presence of the cyanide analog, sodium azide. Both in vitro hypoxic conditions stimulated RTC plasma membrane Fas expression, and caused RTC apoptosis upon ligation with agonistic Fas antibodies. The data suggest that in the context of progressive renal disease, chronic hypoxia stimulates Fas-dependent RTC apoptosis, which represents the first definitive link between hypoxia and tubular atrophy. We believe that hypoxic induction of RTC apoptosis provides a unifying mechanism for the pathogenesis of tubular atrophy, and this paradigm identifies novel targets for chronic renal failure therapy.

Animals↗

PubMed1998

The measurement of bioreductive capacity of tumor cells using methylene blue.

PURPOSE: Methylene blue (MB) can be used as an intracellular electron acceptor. The purpose of this study was to demonstrate the usefulness of MB for the determination of total bioreductive capacity of cell suspensions. METHODS AND MATERIALS: We measured oxygen consumption by Clark electrode and pentose cycle activity by release of 14CO2 from 1-14C-glucose. RESULTS: Methylene blue catalyzes the reaction of intracellular reductants NADPH, NADH, and reduced glutathione (GSH) with oxygen, causing the production of hydrogen peroxide. The reaction rate correlates with the negative charge of molecule (NADPH(-4) > NADH(-2) > GSH(-1)), suggesting that reaction with positively charged oxidized MB is the limiting step of the reaction. In a cellular system MB causes the electron flow from cellular endogenous substrates to oxygen. It is activated by the disruption of the NADP+/NADPH ratio due to several processes. These are direct oxidation of NADPH and GSH, the GSH peroxidase catalyzed reaction of GSH with H2O2, followed by NADPH oxidation by oxidized glutathione (GSSG). This results in increased cellular oxygen consumption and stimulation of the oxidative limb of pentose cycle (PC) in the presence of MB. The cellular effect of MB differs from other electron accepting drugs. Diamide and tert-butylhydroperoxide act as direct oxidants, while MB is an electron carrier to oxygen. Accordingly, MB shows the highest effect on PC activation and oxygen consumption. CONCLUSIONS: Our results indicate that MB may be used for the determination of the total bioreductive capacity of the cells, measured by oxygen consumption and PC activation.

Carbon Dioxide↗

PubMed1998

Radiation-sensitive tyrosine phosphorylation of cellular proteins: sensitive to changes in GSH content induced by pretreatment with N-acetyl-L-cysteine or L-buthionine-S,R-sulfoximine.

PURPOSE: At relatively high concentrations, ie., > 20 mM, N-acetyl-L-cysteine (NAC) scavenges reactive oxygen species produced by ionizing radiation in aqueous solution. Therefore, the ability of NAC to block signal transduction reactions in vivo, has lead to the suggestion that ROS are necessary for the normal propagation of these signals. In this paper we investigate the mechanism by which NAC alters signal transduction in whole cells. RESULTS: Exposing CHO-K1 cells to ionizing radiation results in elevated pp59fyn kinase activity. Moreover, we observe changes in the phosphotyrosine content of multiple cellular proteins, including one prominent phosphotyrosyl protein with a Mr of 85 kDa. Both the radiation-induced changes in pp59fyn kinase activity and the changes in phosphotyrosine content of pp85 were not affected by exposing K1 cells to NAC during the time of irradiation, suggesting that ROS generated extracellularly are not involved in the radiation-induced changes observed in phosphotyrosyl proteins. We also demonstrate that the cell membrane is an effective barrier against negatively charged NAC. Therefore, it seems unlikely that NAC's ability to block signal transduction reactions is related to scavenging of ROS intracellularly. Chronic exposure, ie., 1 h, to 20 mM NAC lead to a twofold elevation in GSH levels and resulted in a 17% decrease in the phosphotyrosine content of pp85 after exposure to 10 Gy. Moreover, pretreatment with L-buthionine-S,R-sulfoximine (BSO) decreased GSH levels and resulted in elevated phosphotyrosine levels in pp85 isolated from irradiated CHO-K1 cells. CONCLUSIONS: Since many signaling molecules contain redox sensitive cysteine residues that regulate enzyme activity, we suggest that the effects of NAC on radiation-induced signal transduction are due to its ability to alter the intracellular reducing environment, and not related to direct scavenging of ROS.

Acetylcysteine↗

PubMed1998

Extravascular diffusion of tirapazamine: effect of metabolic consumption assessed using the multicellular layer model.

PURPOSE: Hypoxia-selective cytotoxic agents, like tirapazamine (TPZ), must diffuse considerable distances in tumors to reach their target cell population. This study uses a new three-dimensional tissue culture model, in which cells are grown as multicellular layers (MCL), to investigate whether metabolic consumption of TPZ is sufficiently rapid to compromise its extravascular diffusion in tumors. METHODS AND MATERIALS: V79-171b and MGH-U1 cells were grown as MCL to thicknesses of approximately 120 and 360 microm respectively. The extent of hypoxia in MCL, as assessed by EF5 binding, was modulated by altering gas-phase O2 content, and flux of TPZ through MCL was investigated by high-performance liquid chromatography (HPLC). Data were fitted to a diffusion-reaction mathematical model to determine the diffusion coefficient of TPZ in the MCL (DM) and the rate of its metabolic consumption under anoxia. These parameters were used to simulate TPZ transport in tumors. RESULTS: The flux of TPZ through well-oxygenated MCL (equilibrated with 95% O2) was well fitted as Fickian diffusion without reaction, with a D(M) of 7.4 x 10(-7) cm2s(-1) (12-fold lower than in culture medium) for V79 and 1.3 x 10(-6) cm2s(-1) for MGH-U1 MCL. Flux of TPZ was suppressed under anoxia, and fitting the data required inclusion of a reaction term with a rate constant for metabolic consumption of TPZ of 0.52 min(-1) for V79 and 0.31 min(-1) for MGH-U1 MCL. These transport parameters would translate into a 43% or 30% decrease respectively in TPZ exposure, as a result of drug metabolism, in the center of a slab of anoxic tissue 100 microm in thickness. CONCLUSIONS: MCL cultures provide an in vitro model for investigating the interaction between metabolic consumption and diffusion of bioreductive drugs. If rates of diffusion and metabolism similar to those measured in V79 and MGH-U1 MCL apply in tumors, then cells in large confluent regions of hypoxia would be partially protected by failure of TPZ penetration. Simulation of extravascular transport of TPZ-like bioreductive drugs demonstrates that the optimum metabolic rate constant is determined by two competing requirements: it should be high enough to ensure potent cytotoxicity under hypoxia, yet low enough that penetration is not severely compromised.

Animals↗

PubMed1998

Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis.

As a result of deprivation of oxygen (hypoxia) and nutrients, the growth and viability of cells is reduced. Hypoxia-inducible factor (HIF)-1alpha helps to restore oxygen homeostasis by inducing glycolysis, erythropoiesis and angiogenesis. Here we show that hypoxia and hypoglycaemia reduce proliferation and increase apoptosis in wild-type (HIF-1alpha+/+) embryonic stem (ES) cells, but not in ES cells with inactivated HIF-1alpha genes (HIF-1alpha-/-); however, a deficiency of HIF-1alpha does not affect apoptosis induced by cytokines. We find that hypoxia/hypoglycaemia-regulated genes involved in controlling the cell cycle are either HIF-1alpha-dependent (those encoding the proteins p53, p21, Bcl-2) or HIF-1alpha-independent (p27, GADD153), suggesting that there are at least two different adaptive responses to being deprived of oxygen and nutrients. Loss of HIF-1alpha reduces hypoxia-induced expression of vascular endothelial growth factor, prevents formation of large vessels in ES-derived tumours, and impairs vascular function, resulting in hypoxic microenvironments within the tumour mass. However, growth of HIF-1alpha tumours was not retarded but was accelerated, owing to decreased hypoxia-induced apoptosis and increased stress-induced proliferation. As hypoxic stress contributes to many (patho)biological disorders, this new role for HIF-1alpha in hypoxic control of cell growth and death may be of general pathophysiological importance.

Animals↗

PubMed1998

A model of wound healing in chronically radiation-damaged rat skin.

The aim of this investigation was to develop a model for studying the chronic effects of radiation on wound healing in the rat. Six months after rats received a single radiation exposure of 20 Gy, a random-pattern dorsal skin flap was elevated. Two weeks after the flap was elevated, irradiated animals showed diminished scar formation and wound breaking strength, as compared with controls (P < 0.05). The effect of hyperbaric oxygen treatment was investigated in some rats who received 20 sessions at 2.4 atmospheres absolute for 90 min daily, 5 days per week, prior to flap elevation and 10 sessions after creation of the flap. Treated animals showed a trend toward improvements in wound breaking strength and scar formation (P = 0.06). A reproducible model of chronic radiation damage in the rat was established. Further studies involving investigations at times more that 2 weeks post-wounding are needed.

Animals↗

PubMed1998

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.

Animals↗