Biological activities of partially purified elastase produced by Flavobacterium meningosepticum

Injection of viable cells of Flavobacterium meningosepticum at doses of 10(6) colony-forming units/eye caused ophthalmia in rabbits' corneas. Elastase, free of protease activity, was partially purified from broth cultures of F. meningosepticum ATCC12535 and TMS516. The optimum pH of the elastase activity was about 8.0. The activity was suppressed by Ni, Zn, Co, EDTA, and o-phenanthroline, and accelerated by Cu and Fe. The minimum dose of the elastase causing ophthalmia in the eyes of rabbits was 5 micrograms/eye (0.065 elastase units). The minimum lethal dose of the elastase for suckling mice by intracerebral injection was 0.4 micrograms (0.0052 elastase units)/suckling mouse, and its minimum lethal dose for adult mice by intraperitoneal injection was 740 micrograms/mouse (9.52 elastase units).     

Protease priming of neutrophil superoxide production. Effects on membrane lipid order and lateral mobility.

Phagocyte superoxide (O2-) response is primed by a variety of physiologic compounds including the neutrophil secretory proteases cathepsin G and elastase. To study whether protease priming of neutrophil O2- response is related to changes in membrane physical state, we examined enzyme effects on the order and lateral mobility of lipid probes in intact neutrophil membranes. Exposure to cathepsin G (5 micrograms/ml) or elastase (10 micrograms/ml) caused a significant decrease in fluorescence anisotropy of the probe trimethylammonium diphenylhexatriene in neutrophil plasma membranes (0.279 to 0.256 for cathepsin G, 0.274 to 0.256 for elastase, p less than 0.02 for both), indicating a decrease in phospholipid chain order in the surface membrane bilayer. Cathepsin G and elastase also caused significant increases in membrane lipid lateral mobility as measured by excimer formation of the fluorescent probe 1-pyrenedecanoic acid (for cathepsin G, a 107% increase, and for elastase, a 44% increase in excimer/monomer fluorescence ratio, p less than 0.001). Enzyme effects on membrane structure were dependent on intact proteolytic activity, and were cell specific; the proteases had no effect on lipid order or lateral mobility in liposomes. In corollary studies, the possible association between the physical state of the polymorphonuclear leukocyte membrane and O2- generation was analyzed with the membrane modifying compounds, linoleic acid, ethanol, and cholesterol. Cell exposure to linoleic acid (1 microM) caused a significant decrease in lipid order and an increase in lipid lateral mobility along with increased O2- production to N-formyl-Met-Leu-Phe (fMLP) (191%) and phorbol myristate acetate (PMA) (39%), p less than 0.02 for each. 3 mM ethanol also augmented O2- response to fMLP (31%) and PMA (48%) and caused a significant decrease in lipid order, but did not affect lipid lateral mobility. Treatment with cholesteryl hemisuccinate (100 micrograms/ml) resulted in increased lipid order and decreased lipid lateral mobility, as well as decreased neutrophil superoxide response to fMLP (-61%, p less than 0.001) and PMA (-50%, p less than 0.02). We then examined whether modulation of membrane physical state may explain the mechanism of action of a known priming agent by studying the effects of low concentrations of a diacylglycerol. Cells treated with 10 microM 1-oleoyl-2-acetyl-sn-glycerol had a greater than 8-fold increase in superoxide response to fMLP (p less than 0.001) while demonstrating a significant decrease in lipid order (0.289 to 0.281, p less than 0.01) and a 50% increase in lipid lateral mobility (p less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)     

Fermentation production ofL-Homoserine byCorynehacterium sp. and its possible use in the preparation of threonine and lysine

A mutant Corynebacterium sp. requiring threonine and cultivated for 3 d in a medium containing 15% sucrose, 8% corn-steep and 50 micrograms biotin per litre accumulated 14.5 g L-homoserine per litre. The possibility of fermenting the homoserine obtained for threonine and lysine production was investigated.     

Interaction between human leukocyte elastase and chondroitin sulfate

Chondroitin sulfate (Structum) interacts with human leukocyte elastase, a potent mediator of articular cartilage degradation, producing a partial inhibition of the enzyme activity (60% at saturation). Kinetically, the inhibition mechanism can be classified as simple intersecting, hyperbolic noncompetitive and is almost identical to that found earlier for similar compounds. The best inhibitory activity of chondroitin sulfate was found in fractions having at the same time a high proportion of chondroitin-6-sulfate relative to the corresponding 4-isomer and a high molecular mass. Thus, a fraction with high Mr and containing 92% of isomer 6 inhibited leukocyte elastase with Ki = 1.8 micrograms/ml, whereas a fraction with low Mr and almost equal composition of the 4- and 6-isomer had Ki = 140 micrograms/ml. Ki for unfractionated chondroitin sulfate was 3.4 micrograms/ml. It is suggested, that the modulation of the extracellular activity of cartilage-degrading enzymes by cartilage-derived factors may explain, at least in part, the beneficial effects of some therapeutically used chondroprotective agents.     

A radioimmunoassay for duck serum ferritin

Development and optimization of a radioimmunoassay for duck serum ferritin described. 125I-labelled ferritin and rabbit anti-ferritin antibody are used together with goat anti-rabbit gamma globulin as separating agent for the bound and free fractions. The assay has a working range of up to 500 micrograms of ferritin per litre and a sample requirement of 50 microliter of serum. The assay requires a 24 h period and has a sensitivity of 10 micrograms of ferritin per litre.     

Stimulation of secretion into human and feline airways by Pseudomonas aeruginosa proteases

We have investigated the effect of elastase and alkaline protease from Pseudomonas aeruginosa on airway secretion into the trachea of anesthetized cats and from human bronchial mucosa in vitro. Secretory macromolecules were radiolabeled biosynthetically with two precursors in the cat, [3H]glucose and [35S]sulfate, and with [35S]-sulfate only in human tissue. Both enzymes (2.6 x 10(-9) to 1.3 x 10(-6)M elastase and 8 x 10(-9) to 2.4 x 10(-6)M alkaline protease) released radiolabeled macromolecules in a concentration-dependent manner from the two preparations. Purified elastase, 1.3 x 10(-6)M, released radiolabeled macromolecules (delta 3H = +397 +/- 72%, delta 35S 225 +/- 40% over control, P less than 0.001) and periodic acid-Schiff- (PAS) reactive glycoconjugates (delta PAS = +4.1 +/- 0.96 micrograms/min or +102 +/- 20%; P less than 0.01) from cat trachea, as did alkaline protease, 2.4 x 10(-6)M (delta 3H = +356 +/- 57%, delta 35S = +176 +/- 25%, delta PAS = +7.5 +/- 1.3 micrograms/min or 194 +/- 36%, P less than 0.001). Increases in 3H exceeded those of 35S, suggesting surface epithelium as the main source of secretion. Inhibition of enzyme activity abolished secretory effects. Both enzymes also stimulated secretion from human bronchus (e.g., with elastase, 1.3 x 10(-6)M: delta 35S = +331 +/- 67%, delta PAS = +4.3 +/- 0.92 micrograms/min or +131 +/- 24%, P less than 0.001; with alkaline protease, 2.4 x 10(-6)M: delta 35S = +220 +/- 67%, delta PAS = +12.7 +/- 3.2 micrograms/min or +575 +/- 245%, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.     

Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation.

The ability of various peptides cleaved by plasmin from human fibrinogen and fibronectin or fibrinogen- and fibronectin- related synthetic peptides to induce histamine release from mast cells and collagenase and elastase from PMN-leukocytes was examined. Low molecular weight fibrinogen degradation products showed dose dependent secretion of collagenase. These peptides (mol. wt. 1.4 kD) at the concentration of 10(-5) M released about 47% of collagenase and 13% of elastase. Synthetic fibrinopeptides A and B had a similar strong collagenase releasing potency and also released histamine from mast cells. Peptides from plasmin digestion of fibronectin containing cell attachment site with sequence Arg-Gly-Asp-Ser and also synthetic peptide reproducing this amino-acid sequence at the concentration of 1000 micrograms/ml released about 50% of collagenase and 55% of elastase from PMN-leukocytes. Moreover peptides containing cell attachment and gelatin binding site induced histamine release from mast cells. The association of fibrinogen and fibronectin degradation with activation of mast cells may motivate the treatment with antihistaminic drugs of all pathological conditions where the intensive protein degradation takes place.     

Human tissue-type plasminogen activator. Production in continuous serum-free cell culture and rapid purification.

A simplified procedure for the production and purification of human tissue-type plasminogen activator (t-PA) is described. Bowes-melanoma cells were maintained in continuous serum-free culture. The cell nutrient consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with insulin (5 mg/litre), transferrin (5 mg/litre), progesterone (1 nM), cortisol (10 nM), aprotinin (2 X 10(4) units/litre) and a mixture of trace elements. t-PA accumulated in the culture medium at a rate of 40 units/day per ml and was harvested every third day. Cell losses during each harvest, leading to a steady decline of enzyme yields, were compensated for by treating the cells with 5% (v/v) fetal-bovine serum in DMEM every 6-8 weeks. t-PA was rapidly purified by a combination of cation-exchange chromatography and gel filtration. The procedure yielded mainly single-chain t-PA of a specific activity of 80 000 to 100 000 units/mg.     

Regulation of enzyme release from human polymorphonuclear leukocytes: further evidence for the independent regulation of granule subpopulations

Addition of 5-20 mM LiCl to purified human polymorphonuclear leukocytes led to the release of lysozyme, the specific granule constituent, but not the release of elastase which is in azurophilic granules. In contrast, 2.5-10 micrograms cytochalasin D/mL induced the release of both lysozyme and elastase. Addition of lipopolysaccharide to leukocytes did not induce enzyme release but primed cells for enhanced release induced by cytochalasin D. Lipopolysaccharide also primed cells for enhanced release of lysozyme by either N-formylmethionylleucylphenylalanine (fMLP) or Li+ but did not prime cells for elastase release by these stimuli. In contrast, fMLP + cytochalasin D interacted synergistically, leading to enhanced elastase release but not lysozyme release from the cells. Additional experiments with combinations of secretagogues and lipopolysaccharide yielded results consistent with the hypothesis that specific granules and subpopulations of azurophilic granules are under separate regulation and, thus, may be influenced by separate elements of intracellular second messenger systems.     

Effect of hypercapnic acidosis on induction of arrhythmias by catecholamines in cat papillary muscles.

The effect of changes in PCO2 upon induction of arrhythmias in cat papillary muscles was studied. The average norepinephrine (NE) dose necessary to produce spontaneous contractions in muscles stimulated at rates of 10/min was higher at high PCO2. Whereas 2 100 +/- 295 X 10(-8) mol/litre of NE was necessary during acidosis, only 824 +/- 295 X 10(-8) mol/litre was necessary to produce spontaneous contractions in alkalosis. In quiescent muscles, the necessary doses in acidosis and alkalosis were 2 209 +/- 531 X 10(-8) and 518 +/- 159 X 10(-8) mol/litre respectively. With isoproterenol 458 +/- 84 X 10(-8) mol/litre was necessary to reach the end point at high PCO2, whereas only 131 +/- 52 X 10(-8) mol/litre was required at low PCO2. The lower sensitivity to catecholamine-induced arrhythmias with hypercapnic acidosis does not appear to be related to the re-uptake of the neurotransmitter by the nerve ending since it is also present with isoproterenol.     

Proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins.

Bac5 and Bac7, antibiotics of the bactenecin (proline/arginine-rich peptide) family, are stored as proforms in the large granules of bovine neutrophils [Zanetti, M., Litteri, L., Gennaro, R., Horstmann, H. and Romeo, D. (1990) J. Cell Biol. 111, 1363-1371]. These proforms have been purified to homogeneity from granule extracts by immunoaffinity and reverse-phase chromatography. While mature bactenecins efficiently kill Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium with minimal inhibitory concentrations of 6-12 micrograms/ml, proBac5 and proBac7 do not affect the growth of the same microorganisms, even at 500 micrograms/ml. Previous investigations have suggested that the conversion of probactenecins into mature antimicrobial peptides is catalyzed by a neutral serine protease stored in the azurophil granules. Purified proBac5 and proBac7 were thus treated with elastase, cathepsin G or proteinase 3, which constitute the pool of neutral serine proteases of the azurophils, and the reaction products were identified by Western blot analysis, mass spectrometry, and N-terminal sequence analysis. Of the three proteases, only elastase is able to catalyze the stepwise cleavage of probactenecins into the corresponding mature peptides, which have the same mass, N-terminal sequence and antibiotic activity of authentic Bac5 and Bac7. These results point to the importance of cooperation between azurophils and large granules in mounting a defense reaction.     

Conjugated dienes in lipids of apolipoprotein B containing lipoproteins of normal and type 2 (non-insulin-dependent) diabetic patients.

Conjugated dienes present in the fatty acyl chains of cholesterol esters and triglycerides associated with plasma apolipoprotein B containing lipoproteins of normal and Type 2 (non-insulin-dependent) diabetic patients (n = 17) have been analysed using second derivative electronic absorption spectroscopy. Characteristic spectral patterns for both normal subjects and Type 2 diabetic patients were observed. Cis, trans and trans, trans conjugated dienes in cholesterol esters of lipoprotein B of Type 2 patients and normal subjects were found to be 41.74 +/- 0.51 mg/litre, 8.20 +/- 0.20 mg/litre (p less than 0.01) and 24.70 +/- 0.33 mg/litre, 9.22 +/- 0.06 mg/litre (p less than 0.01), respectively. Levels of these dienes in triglyceride fraction were 21.21 +/- 0.52 mg/litre, 7.72 +/- 0.02 mg/litre (p greater than 0.05) and 15.49 +/- 0.36 mg/litre, 7.91 +/- 0.11 mg/litre (p greater than 0.05), respectively.     

Medium improvement by orthogonal array designs for cholesterol oxidase production by Rhodococcus equi No. 23

Medium improvement for the production of cholesterol oxidase (CO, EC 1.1.3.6) by Rhodococcus equi No. 23 was investigated using an orthogonal array design in two steps. Results revealed that yeast extract, Tween 80 and zinc sulphate had positive effects on CO production, but magnesium sulphate had an inhibitory effect. In addition, interaction between cholesterol and sodium chloride also had a significant effect on enzyme production. The improved medium consisted of 2·0 g/litre cholesterol, 8·0 g/litre yeast extract, 1·0 g/litre NH₄Cl, 1·0 g/litre NaCl, 0·50 g/litre KH₂PO₄, 0·25 g/litre Na₂HPO₄, 0·10 g/litre -valine, 0·15 g/litre -tyrosine, 0·15 g/litre MgSO₄·7H₂O, 0·01 g/litre ZnSO₄·7H₂O, 0·10 g/litre FeSO₄·7H₂O and 4·0 ml/litre Tween 80. CO production at 60 h (about 0·24 units/ml) was about four-fold greater than with the control medium.     

Isolation and partial characterization of canine epidermal growth factor/urogastrone.

Canine epidermal growth factor (EGF)/urogastrone was partially purified from dog urine by fractional precipitation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose DE-52, gel filtration with Sephadex G-50, and a second DE-52 chromatography, to yield receptor-competing activity equivalent to 13 micrograms of standard mouse EGF/litre of starting urine. The purification was monitored by a competitive radioreceptor assay using fixed monolayers of A431 cells. The partially purified canine EGF/urogastrone demonstrated a growth-stimulating activity in 3T3 mouse fibroblast cells as potent as mouse EGF. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one major peptide component with an Mr similar to that of mouse EGF, and two minor peptides of slightly higher Mr. The major peptide component was isolated after reduction and its amino acid composition was determined.     

Neutrophil-mediated increased permeability of microcarrier-cultured endothelial monolayers: a model for the in vitro study of neutrophil-dependent mediators of vasopermeability.

Changes in the permeability of human endothelial monolayers in response to activated human neutrophils were examined in a novel, in vitro model of vasopermeability changes. Microcarrier-cultured human umbilical vein endothelial monolayers were used in a system that responds to histamine. Human neutrophils did not increase Evans Blue staining of the endothelium-covered microcarriers if added alone or if added with the neutrophil-dependent mediator of vasopermeability, formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM). In contrast, neutrophils, added to the endothelial cells in a ratio as low as 2.5:1 caused time-dependent increases in microcarrier staining if pretreated with cytochalasin B (5 micrograms/mL) before addition with FMLP. Neutrophil cell-free releasate and purified human sputum elastase also caused concentration-related increases in Evans Blue staining of the endothelial-covered microcarriers and these effects were inhibited by the elastase inhibitor methoxysuccinyl-alanyl-alanyl-prolyl-valyl chloromethyl ketone. This compound also inhibited neutrophil-mediated endothelial permeability increases. The microcarrier-cultured human endothelial monolayer system rapidly detects permeability alterations of endothelial monolayers in response to activated human neutrophils. This model is a potentially useful screening assay for the development of therapeutic agents, directed at neutrophil degranulation or degranulation products, for the control of inflammatory vasopermeability abnormalities.     

Lipid peroxidation and antielastase activity in the lung under oxidant stress: role of antioxidant defenses

The role of lipid peroxidation in the inactivation of alpha 1-protease inhibitor (alpha 1-PI) in the alveolar lining fluid of human subjects has been examined under oxidant stress. Exposure to nitrogen dioxide (NO2) at 4 ppm for 3 h resulted in a significant increase in the amount of lipid peroxidation products in the alveolar lining fluid, with conjugated dienes the predominant species. Four-week supplementation with vitamins C and E before NO2 exposure markedly decreased the levels of conjugated dienes (control 804 +/- 103 pmol/micrograms total phospholipids vs. vitamin-supplemented 369 +/- 58, P = 0.003). Malondialdehydes, although detectable in the lavage fluid, contributed little to the total amount of lipid peroxidation products, and the levels were comparable in both groups. NO2 exposure in the absence of vitamin supplementation caused a significant decrease in the elastase inhibitory capacity (EIC) of the alveolar lining fluid in the control group but not in the vitamin-supplemented group [control 3.67 +/- 0.32 micrograms alpha 1-PI/micrograms porcine pancreatic elastase (PPE) vs. vitamin-supplemented 2.75 +/- 0.17, P less than 0.03]. The vitamin-supplemented group had a lower level of conjugated dienes and a higher EIC. Conversely, the control group had higher levels of conjugated dienes and a lower EIC in their lavage fluid. These studies demonstrate that lipid peroxidation occurs as an early event during oxidant exposure in the lungs of normal subjects. The appearance of lipid peroxidation products in the lavage fluid is associated with a decrease in the EIC of the alveolar lining fluid. Vitamins C and E diminish lipid peroxidation and preserve the EIC of the lower respiratory tract fluid during oxidant stress.     

Reactive oxygen species and elastase mediate lung permeability after acid aspiration.

Acid aspiration leads to increased neutrophil (PMN) oxidative metabolism, an event associated with lung leukosequestration and permeability increase. Neutropenia protected the vascular barrier function against acid injury. This study tests whether active oxygen species and elastase (which are presumably released by adherent PMNs) affect the microvascular barrier. Anesthetized rats underwent tracheostomy and insertion of a cannula into a lung segment. This was followed by localized instillation of 0.1 N HCl (n = 18) or saline (n = 18). Sequestration of PMNs in acid-aspirated and nonaspirated segments was 77 and 46 PMNs/high-power field (HPF), respectively, which was higher than control values of 11 and 8 PMNs/10 HPF in saline-aspirated and nonaspirated regions (P less than 0.05). Acid aspiration was associated with increased protein concentration in bronchoalveolar lavage (BAL) fluid to 3,550 and 2,900 micrograms/ml in the aspirated and nonaspirated lungs, respectively, which were higher than control values of 420 and 400 micrograms/ml (P less than 0.05). Acid aspiration also led to increased lung wet-to-dry weight ratios (W/D) of 6.6 and 5.4, which were higher than control values of 3.4 and 3.3 (P less than 0.05). Intravenous treatment of rats (n = 18) 90 min after aspiration with scavengers of reactive oxygen species, superoxide dismutase (1,500 U/kg), and catalase (5,000 U/kg), both conjugated to polyethylene glycol, did not reduce PMN sequestration but attenuated acid aspiration-induced increase in protein accumulation in BAL fluid in the aspirated and nonaspirated segments (990 and 610 micrograms/ml) as well as the increased lung W/D (4.6 and 4.0; all P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein C degradation in vitro by neutrophil elastase.

Purified protein C is completely degraded into small peptides by in vitro incubation with purified elastase. Protein C is a rather sensitive substrate as degradation is already accomplished by low elastase concentrations (molar enzyme-to-substrate ratio 1:510) and short incubation periods (5 min-60 min). Protein C in a PPSB coagulation factor concentrate is equally degraded and similar split products are detected by blotting techniques. The protein C activity (measured by a chromogenic substrate) is faster reduced by elastase than the protein C concentration (measured by an ELISA). Incubation of normal plasma with high elastase concentrations (5.7 nmol/ml plasma) results in reduction of the protein C band while no split products are detectable. The pathophysiologic significance of the effects of elastase on protein C remains to be elucidated.     

Enhanced release of oxygen metabolites by monocyte-derived macrophages exposed to proteolytic enzymes: activity of neutrophil elastase and cathepsin G

Macrophages at sites of inflammation are exposed to proteolytic enzymes derived from neutrophils, platelets, clotting factors, complement, and damaged tissues. To investigate the possible effect of proteases on the plasma membrane-mediated oxidative metabolic response of macrophages in inflammatory sites, cultured human monocyte-derived macrophages were treated in vitro with proteolytic enzymes and were then assayed for their ability to release superoxide anion (O2-) and hydrogen peroxide (H2O2) in response to stimuli. Macrophages pretreated for 1 to 20 min with trypsin, chymotrypsin, pronase, or papain, 0.1 to 200 micrograms/ml, released up to 3.5-times more O2- and H2O2 than did control (untreated) cells. This enhanced production of oxygen metabolites was observed by using either phorbol myristate acetate or opsonized zymosan as the stimulus. Macrophages were also "primed" for enhanced O2- release (2.3-fold) by pretreatment with a subfraction of granules extracted from human neutrophils. This subfraction contained primarily elastase and cathepsin G. Similar enhancement was observed with 60 ng/ml or purified human neutrophil cathepsin G (2.2-fold) and with 20 micrograms/ml of purified neutrophil elastase (3.3-fold). Priming by these neutrophil proteases could be blocked by specific inhibitors of their proteolytic activity. These results suggest that macrophages involved in an inflammatory response might be rapidly primed by proteases released from degranulating neutrophils. Primed macrophages could mount a more effective oxidative metabolic response to microorganisms or tumor cells, but might also cause greater tissue damage.