Patent Application: US-24805894-A

Abstract:
a signaling pathway is identified that involves the activation of phospholipase a2 and protein kinase c in human cells , which in turn confers x - ray induction of the tumor necrosis factor α gene . inhibition of phospholipase a2 abolishes radiation - mediated arachidonate production , as well as the subsequent activation of protein kinase c and tnf gene expression . these phospholipase a2 inhibitors may be used to ameliorate the adverse - effects of radiotherapy associated with tnf production .

Description:
the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . hl - 60 cells in logarithmic growth were incubated for 16 h in rpmi 640 medium supplemented with 1 . 0 mg / ml fatty acid free bovine serum albumin and 0 . 2 μci / ml [ 3 h ] arachidonic acid as described ( spriggs et al ., 1990 , godfrey et al ., 1987 ). mepacrine 20 μm , bpb 10 μm , dexamethasone 1 μm , or pentoxifylline ( 3 , 7 - dimethyl - 1 -( 5 - oxo - hexyl )- xanthine , hoffman - la roche , basel , switzerland ) 1 mm were added to labeled cells 1 hr prior to irradiation with 10 gy at 1 gy / min . at the indicated times , a 1 ml aliquot of supernatant was assayed by the addition of hydrofluor ™ and scintillation counting . dag release from irradiated cells was quantified as previously described ( wright et al ., 1988 ). serum deprived hl - 60 cells were pelleted and irradiated ( 10 gy at 2 . 5 gy / sec ) and 100 % methanol was added immediately on ice . extracts were dried under n 2 and resuspended into cardiolipin and n - octylglucoside and bacterial dag kinase in the presence of 32 p - atp . cellular dag was quantified by using a known concentration of synthetic dag reacted with dag kinase . bradykinin was used as a positive control and resulted in a 3 . 0 ± 0 . 4 fold increase in dag , while x - irradiation produced dag levels 1 . 2 ± 0 . 4 as compared to untreated controls . at cells were grown to confluence and serum deprived for 24 hours . medium was aspirated , cells were washed with pbs and then γ - irradiated with 10 . 8 gy using a cobalt - 60 source ( gammacell 220 ) at a dose rate of 2 . 7 gy / second . protein was extracted on ice at 15 second intervals following irradiation by the addition of 0 . 4 ml of lysis buffer ( 20 μm tris / hcl , ph 7 . 5 , 0 . 5 mm edta , 0 . 5 mm egta , and 2 - mercaptoethanol 10 mm ( tem ) with 0 . 5 % triton x100 , and 25 μg / ml each leupeptin and aprotinin ). cells were homogenized and protein was partially purified as previously described ( hallahan et al ., 1990b ). protein extract ( 25 μl ) was added to 25 μl of tem , 5 μl of phospholipid ( 2 . 8 mg / ml phosphatidyl serine and 10 mm phorbol ester in triton x100 mixed micelles , gibco ) ( yasuda et al ., 1990 ) and 10 μl of 32 p - atp / substrate containing 5 × 10 7 cpm / ml of 32 p - atp ( new england nuclear ), 100 μm atp , 250 μm synthetic peptide gln - lys - arg - pro - ser ( 8 )- gln - arg - ser - lys - tyr - leu ( seq id no : 1 ), 5 mm cacl , 100 mm mgcl 2 ( gibco ) ( yasuda et al ., 1990 ) in 20 mm tris hcl , ph 7 . 5 . following incubation for 5 min at 30 ° c ., samples were dried on phosphocellulose and washed in 1 % h 3 po 4 twice for 5 minutes followed by washing in h 2 o twice for 5 min . scintillation counts of each sample and 10 μl of unwashed 32 p - atp / substrate were performed . to calculate the rate of 32 p incorporation into the peptide substrate , 100 μm of synthetic pkc specific inhibitor peptide ( arg - phe - ala - arg - lys - gly - ala leu - arg - gln - lys - asn - val - his - glu - val - lys - asn ( seq id no : 2 )) ( gibco ) ( house et al ., 1989 ) in 20 mm tris 7 . 5 was added to pkc assays prior to 32p - atp / substrate and samples were incubated , washed and counted as described above . background 32 p incorporation was subtracted from that of assays without inhibitor and the rate of 32 p incorporated into the peptide substrate was calculated ( pmol / min ) as previously described ( hallahan et al ., 1990 b , yasuda et al ., 1990 ). phosphorylation rates were normalized to 10 6 cells per assay . to determine the effects of pla2 inhibitors on radiation - mediated pkc activation , mepacrine , bpb , dexamethasone or pentoxifylline were added to hl - 60 cell cultures 1 hr prior to γ - irradiated with 10 . 8 gy using a cobalt - 60 source ( gammacell 220 ) at a dose rate of 2 . 7 gy / second . cells were placed on ice and lysis buffer was added at 60 seconds following irradiation ( hallahan et al ., 1991b ). phosphotransferase activity was assayed as previously described above . phosphorylation rates were normalized to 10 6 cells per assay . cells were grown to a density of 10 6 / ml and exposed to 10 gy ( ge maxitron ™ x - ray generator ) as previously described ( hallahan et al ., 1989 ). rna was extracted using the single step guanidinium thiocyanate - phenol / chloroform method ( chomczynski et al ., 1987 ) at 1 hour following irradiation . control rna from nonirradiated cells treated with otherwise identical conditions and rna from irradiated cells was size fractionated by 1 % agarose formaldehyde electrophoresis . ethidium bromide staining of the rna demonstrated equal loading of each lane . rna gels were then transferred to a nylon membrane ( genescreen plus ™, new england nuclear ). northern blots were hybridized to the 32 p labeled tnf cdna probe ( spriggs et al ., 1990 ) followed by autoradiography for 3 days at − 85 ° c . with intensifying screens . 7s rna hybridization was used to demonstrate equal loading of lanes . mepacrine , dexamethasone , bpb , or pentoxifylline were added to hl - 60 cell cultures 1 hr prior to irradiation with 10 gy ( 1 gy / min ) using a ge maxitron generator . 1 . the effects of the phospholipase a2 inhibitors on radiation - induced fatty acid hydrolysis arachidonic acid production was quantified in irradiated hl - 60 cells which have served as a model for the study of radiation - mediated tnf gene induction and pkc - dependent signal transduction ( hallahan et al ., 1991b ). hl - 60 cells were incubated with [ 3 h ] arachidonic acid for 3 hours , washed , irradiated with 10 gy at 1 gy / min . the hl - 60 cells in logarithmic growth were incubated for 16 hours in rpmi 640 medium supplemented with 1 . 0 mg / ml fatty acid free bovine serum albumin and 0 . 2 μci / ml [ 3 h ] arachidonic acid as described ( spriggs et al ., 1990 , godfrey et al ., 1987 ). mepacrine 20 um , bpb 10 gm , dexamethasone 1 μm , or pentoxifylline ( 3 , 7 - dimethyl - 1 -( 5 - oxo - hexyl )- xanthine , hoffman - la roche , basel , switzerland ) 1 mm were added to labeled cells 1 hr prior to irradiation with 10 gy at 1 gy / min . at the indicated times , a 1 ml aliquot of supernatant was assayed by the addition of hydrofluor and scintillation counting . fatty acid release into the medium was significantly increased following irradiation . previous work has also shown that arachidonic acid release is increased following treatment with h 2 o 2 ( gustafson et al ., 1991 , shasby et al ., 1988 ), which served as a positive control . to confirm that arachidonate was produced , the inventors performed gas chromatographic analysis of lipids extracted from irradiated hl - 60 cells . using this approach , an increase in arachidonate was detectable at 30 minutes following irradiation . conversely , diacylglycerol levels did not change following irradiation as determined by the dag kinase assay . the effects of the phospholipase a2 inhibitors mepacrine , bromphenylbromide ( bpb ) dexamethasone , and pentoxifylline on radiation - induced fatty acid hydrolysis were studied . each attenuated arachidonic acid release into the medium of cells treated with x - rays or h 2 o 2 . since pkc has been shown to activate phospholipase - mediated hydrolysis of membrane phospholipids ( godson et al ., 1990 , sporn et al ., 1990 ), the pkc inhibitor h7 was added to determine whether pkc activation contributes to lipid hydrolysis following irradiation . h7 pretreatment had no detectable effect on arachidonic acid release following irradiation of hl - 60 cells . this is consistent with the finding that pkc inhibition produced no reduction in arachidonic acid release following h 2 o 2 treatment ( sporn et al ., 1990 ). 2 . the effects of the phospholipase a2 inhibitors on radiation - induced protein kinase c activation pkc phosphotransferase activity is increased following the addition of arachidonate ( peters - golden et al ., 1991 , mcphail et al ., 1984 ). taken together with the findings that pkc is activated rapidly and transiently following ionizing radiation exposure and that pkc activity is required for radiation - induced tnf gene induction in hl - 60 cells ( hallahan et al ., 1991b ), these data suggested that arachidonate activation of pkc might be the signalling pathway which confers tnf induction . to determine whether radiation - induced arachidonate production is associated with pkc activation , the phosphotransferase activity of pkc was quantified in irradiated hl - 60 cells pretreated with phospholipase a2 inhibitors . mepacrine μm , bpb , dexamethasone or pentoxifylline were added to hl - 60 cell cultures 1 hr prior to γ - irradiated with 10 . 8 gy using a cobalt - 60 source ( gammacell 220 ) at a dose rate of 2 . 7 gy / second . cells were placed on ice and lysis buffer was added at 60 seconds following irradiation as described ( 9 ). phosphotransferase activity was assayed as previously described ( hallahan et al . 1991b ) using 32 p - atp / substrate containing 5 × 10 7 cpm / ml of 32 p - atp ( new england nuclear ) and synthetic peptide gln - lys - arg - pro - ser ( 8 )- gln - arg - ser - lys - tyr - leu ( seq id no : 1 ). phosphorylation rates were normalized to 10 6 cells per assay . protein was extracted at 60 seconds following irradiation , and phosphotransferase activity was quantified in vitro . the pkc specific peptide substrate from myelin basic protein ( yasuda et al ., 1990 ) and the pkc inhibitor peptide from the pkc regulatory domain ( house et al ., 1987 ) were employed to quantify pkc activity following irradiation . a 3 - fold increase in phosphotransferase activity was found at 45 seconds following irradiation as compared to untreated control cells . mepacrine , bpb , pentoxifylline and dexamethasone , added 1 hr prior to irradiation , reduced the x - ray induced increase in pkc phosphotransferase activity . these data are in concordance with a previously undescribed x - ray - induced signalling pathway following x - ray exposure , whereby oxidized membrane phospholipids are hydrolyzed to arachidonate which in turn activates pkc . 3 . the effects of the phospholipase a2 inhibitors on radiation - induced tnf gene expression although transcription of certain radiation - inducible genes occurs through both pkc - dependent and independent signalling pathways ( datta et al ., 1992 ), tnf induction is dependent upon pkc activation ( hallahan et al ., 1991b ) and thus represents a radiation - mediated gene which can be studied to determine the significance of phospholipase inhibition on radiation - mediated gene induction . mepacrine 20 μm , dexamethasone , bpb , or pentoxifylline were added to hl - 60 cell cultures 1 hr prior to irradiation with 10 gy ( 1 gy / min ) using a ge maxitron generator . rna was extracted at 1 hour after irradiation as previously described ( hallahan et al ., 1989 ). control rna from nonirradiated cells treated under otherwise identical conditions and rna from irradiated cells was size fractionated by 1 % agarose formaldehyde electrophoresis and hybridized to a 32 p labeled tnf cdna probe ( sherman et al ., 1991 ). 7s rna hybridization was used to demonstrate equal loading of lanes . rna was isolated 1 hr following irradiation at the time of peak tnf expression . the finding that each of these agents each blocked radiation - induced tnf gene expression indicated that radiation - induced tnf expression is dependent on signaling through phospholipase - a2 . moreover , attenuation of radiation - mediated gene induction by these phospholipase a2 inhibitors suggests that signal transduction activated by ionizing radiation is in part initiated through hydrolysis of oxidized membrane lipids . second messengers such as diacylglycerol ( dag ), arachidonic acid and calcium participate in pkc activation in response to a number of external stimuli ( nishizuka 1992 ). the inventors have investigated the mechanism of pkc activation following irradiation by analyzing the second messengers diacylglycerol , arachidonic acid and calcium which participate in pkc activation in response to a number of external stimuli ( nishizuka 1992 ). it was found that diacylglycerol levels were not increased following irradiation as determined by the dag kinase assay . furthermore , intracellular calcium flux did not occur as determined by quantifying uv absorption in fura - 2 treated cells during irradiation with 90 sr ( 60 cgy / sec ) ( hallahan et al ., 1994 ). taken together , these data support the finding that phosphoinositol - specific phospholipase c is not activated during irradiation since the coincident increase in inositol triphosphate would mobilize intracellular ca ++ . the acute effects of ionizing radiation on the lung has been shown to be associated with endothelial leakage ( ward et al ., 1993a ). corticosteroids prevent the acute effect when given to animals at the tie of irradiation , but this treatment did not affect lung fibrosis ( ward et al ., 1993b ) indicating that steroids prevent the inflammatory component of radiation injury but not the fibrotic component . in the present study , arachidonic acid release was reduced when irradiated cells were pretreated with pentoxifylline , dexamethasone or bpb . these findings are significant in that the reduction in radiation - mediated phospholipase a2 activity in turn diminished pkc activation and tnf induction . lipid oxidation occurs in the cell membrane of irradiated cells ( yatvin et al ., 1979 ). indeed , the probable mechanism of arachidonic acid release following irradiation is phospholipase a 2 - mediated hydrolysis of oxidized membrane lipids . in support of this hypothesis , whole body irradiation of animal models results in increased arachidonic acid metabolites ( eldor et al ., 1979 ). oxidative injury following h 2 o 2 treatment results in phospholipase a 2 - mediated arachidonic acid release in epithelial and endothelial cells ( gustafson et al ., 1991 , au et al ., 1987 , sevanian et al ., 1983 ). phosphatidylcholine hydrolysis to arachidonic acid is reduced by pentoxifylline in platelets stimulated with thrombin ( rossignol et al ., 1988 ). in concert with these findings and the demonstration that arachidonic acid activates pkc both in vitro ( 34 , 35 , 36 ) and in vivo ( sporn et al ., 1990 , khan et al ., 1991 , fan et al ., 1990 , lester et al ., 1991 ) the inventors have found that inhibition of phospholipase a2 attenuates radiation - induced pkc activation . these results demonstrate for the first time that fatty acid hydrolysis an early step in a signalling pathway activated by ionizing radiation which may be independent of dna damage . on the basis of these results , it is surmised that evolution of phospholipase a2 - dependent signaling pathways provides a mechanism for higher eukaryotes to respond to reactive oxygen intermediates with cytokine production . in support of this consideration , neta et al have shown that tnf protects hematopoietic cells from killing by ionizing radiation ( neta et al ., 1991 ). a practical application of these findings relates to reduction of radiation sequelae during the treatment of cancer . although the effects of ionizing radiation on proliferating cell renewal systems are theorized to be due to the direct killing effects of radiation on stem cells within the injured organ , other work has suggested that tnf induction plays a role in the acute effects of radiation therapy ( reviewed in weichselbaum et al ., 1993 ). for example , elevated tnf serum levels in patients receiving total body irradiation prior to bone marrow transplantation is associated with a greater incidence of complications such as mucositis , pneumonitis , hepatitis and nephritis than in patients with relatively lower tnf serum levels ( holler et al ., 1992 ). because tnf induction is associated with acute and subacute complications of therapeutic radiation , inhibition of phospholipase a2 represents a novel means of abating these sequelae . indeed , pharmacologic agents used to ameliorate the acute and subacute sequelae of radiotherapy include glucocorticoids and pentoxifylline ( phillips et al ., 1975 , gross , 1980 , bianco et al ., 1991 ). for example , acute effects of radiation , such as pneumonitis and the central nervous system syndrome , have been abated by these drugs ( phillips et al ., 1975 ). the identification of a signal transduction pathway responsible for radiation - mediated arachidonic acid production , pkc activation and tnf induction may allow for rational design of radioprotective drugs that do not adversely affect tumor cure rates and avoid the serious side effects of glucocorticoids . such strategies of radioprotection offer new avenues to enhance the therapeutic ratio in clinical oncology . all of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the composition , methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . au , a ., chan , p ., & amp ; fishman , r . 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