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Human tumor derived cell lines express phospholipid hydroperoxide glutathione peroxidase (PHGPX)
OxidativeStressGenes and Proteins
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• ~N ?d~ DmlVm ~LL LR~S ~ ~ m~0m~so~ olm~a0~ PmSO~SE (Pm~X) R.S. Es~rdry*, M. Malorino**, F.F. Oau*, J.H. Doroshow*, F. Ursini**. •Department of Med. Onc. ard ~era. Research, City of Hope Natl. Ileal.Can., Duarte, CA 91010, USA and the * M ~ t of Biol. ( ~ . , University of Padova, Padova, Italy 35100. E4SPX uses glutathione to retire all titratsble peroxides in ,r,ahranos and lipoproteins including cholesterol and cholesterol ester Irjdroperoxides. khlle demonstrated in several ~mlrml tissues, ~ tissues hax~ not been e ~ r e d . PHGPX was detected in human tumor derived cell lines after growth in selenium sufficient media for 12-14 days. Only 3 to 10% of the total activity was detected in the ER fraction at 20-60% of the cytopl~-q,lc specific activity and alx~t 15% of ~ activity was detected in the nuclear fraction at 50% of the cytmplasmie specific activity. " l h e c y ~ 1 ~ r l e activit2es values w e r e : ~ HL-60 22 rmoles/min/mg. ; HL-60 WE, I<562WE, AI~ K.562, M2F-7 WE, MCF-7H6 - ii-12 rmolas/min/mg; H e ~ -9.6 nmoles/min/mg; MCF-7 - 7 rmoles/min/mg. Activity levels in selenium deficient media were 0.2 -2.5 rmolas/min/mg. Gel filtration demonstrates that the native size of the species is 18 kDa. After lsbeling the cell lines with 75Se-selenious acid 6-7 selenoproteins cen be resolved by SDS-PAGE - Mr: 57-60; 22; 18; 19 kDa. ~he 22 kDa protein is the ~ t of classical glutathione peroxidase (GSHPX). ~ae 18 Id)a s e ~ r o t e l n fractiormtes on Sephadex G-200 as a monomer resolving completely from (~PX and CST activity and is only selenoprotein associated with the ~ activity. association is substantiated in the HCF-7 WE cell line which has no 22 kDa selex~protein or associated GSaWX activity and expresses GST activity at 3-10% the level of the other cell lines.
1.11
ACUTE OXYGEN TOXICITY CAUSES INCREASED LUNG mRNA SYNTHESIS IN RATS ACUTE Phillip Factor, D.O. and J.I. Sznajder, M.D. Section of Pulmonary and Critical Care Medicine, The University of Chicago and Micheal Reese Hospital To examine the responses to hyperoxia at the molecular level we exposed 18 male Sprague-Dawley rats (45 days old, ave. weightf325g) to high levis of oxygen (F102>0.95) for 66 hours. A second group of 7 control an]mals were kept at the same temperature and humidity and provided with the same ad libitum diet as the hyperoxic group. The rats were sacrificed prior to en bloc removal of the lungs. One lung was saved for wet/dry weight determination. The other was immediately frozen for RNA isolation. Using a Proteinase K/phenol/chloroform separation technique followed by oligo-(dT)-cellulose column chromatography, total and poly-A + RN#t,were isolated and quantified. Total RNA %mRNA Wet/Dry Weight Control 5.75, 1.51 4.24, Hyperoxia 3.23, 1.39, 5.31, 5 Days Recovery 9.93, 2.62, 4.72 10 Days Recovery 9.54 2.55 4.67 p<.05 vs Control
mg Total RNA/gm Dry Lung
Wet/dry weight ratios confirmed the presence of lung injury in the hyperoxic group. Animals exposed to high levels of oxygen had a significant increase in mRNA levels when compared to the control animals. This suggests that part of the underlying pathophysiologic process of oxygen toxicity includes increased protein synthesis as indicated by increased expression of total and mRNA. (Supported by a grant from the Chicago Lung Association).
PUkq~ ~ir_~mI~ PEKED~E IS A DI~HNCr
3
1.10
PROIEIN FROM ~ GIIYfb/~ONE Iq~fKII~E R. Steven Esworthy*, Fo~g-Fc~g Ore*, Raymond J. Paxtor~, Steven Akmm~*0 and James H. Doroslx~. *DeparUmnt of Medical Oncology mxl ~erapeutics Research, and **Division of lmmxmlogy City of Hope National Medical Center and the Beclasan Research Institute of the City of Hope, Duarte, CA 91O1O ~ae presence of a glutathione peroxidase activity in p ~ raises the question of whether the enyne is leaked into plasma from red blood ceils or ~ endothelial cells or represents a distinct secretory form of the er~flre. To answer this question human pl~qm glutathione peroxidase ~ms purified to homogeneity and partially sequenced. Overlapping peptide fragments from two endepeptidase digests permitted the determination of one sequence of 32 contiguous anino acids and one sequence of 25 contiguous amino acids. Additional peptide fragments without obvious overlaps were sequenced. ~he 32 amino acid sequence aligns with positions 82-I13 of bavan cytosolic glutathiore peroxidase with 9 mismatches and no gaps or insertions required. 2he 25 anino acid sequence aligns with positions 157-180 with eight m i s r a ~ and an insertion of one resick~e. On the basis of this sequence information we conclude that }luman plasma glutathione perc0ddase is not derived by modification from the preduninmat intracellular enzyme and is most likely secreted into plamra. We could detect no glutathione peroxidase activity in bile indicating that plasma is the principal site of expression. ~he plasma e~Tme does not have activity toward phoscghn_tidylcholine hydroperoxide suggesting that it is dependent on the action of phospholipase Az.
STRESS RESPONSE AND GLUTATHIONE M.L. Freeman, E.L. Saunders, and M.J. Meredith Radiat. Oncol., Vanderbilt Univ., Nashville TN 37232 and Biochem. Oregon Health Sci. Univ., Portland OR 97201 Glutathione (GSH) oxidation and glutathione depletion induce qualitatively different stress responses. Oxidation of glutathione by exposure to diamide enhances the synthesis of the 110, 90, 73/72, and 50 kDa heat shock proteins (HSP). Induction only occurs when thiol oxidation exceeds the rate of reduction. The consequence of enhanced HSP synthesis is the development of resistance to the cytotoxic effects of heat shock (thermotolerance) and protection against thermal inhibition of protein synthesis. Conversely, enhanced HSP synthesis with the subsequent development of thermotolerance induced by a prior heat shock provide protection from the cytotoxic effects of glutathione oxidation produced by exposure to diamide. Enhanced HSP synthesis and the development of thermotolerance did not prevent thiol oxidation as assessed by 2D thiol blotting but apparently provided protection from the consequence of oxidation. Depletion of GSH by exposure to diethyl maleate (DEM) or buthionine sulfoximine (BSO) does not induce HSP synthesis or trigger the development of thermotolerance. Nor does depletion inhibit the heat shock response. However, GSH depletion by treatment with either DEM or BSO enhances the synthesis of a 32 kDa stress protein. The rate of induction of this stress protein is dependent on the rate of GSH depletion and is induced only when depletion exceeds 80%. This protein has been characterized by 2D PAGE and the 32 kDa protein exhibits a pI of 6.4 and has been tentatively identified as heme oxygenase.
1.12
1.g
• ~N ?d~ DmlVm ~LL LR~S ~ ~ m~0m~so~ olm~a0~ PmSO~SE (Pm~X) R.S. Es~rdry*, M. Malorino**, F.F. Oau*, J.H. Doroshow*, F. Ursini**. •Department of Med. Onc. ard ~era. Research, City of Hope Natl. Ileal.Can., Duarte, CA 91010, USA and the * M ~ t of Biol. ( ~ . , University of Padova, Padova, Italy 35100. E4SPX uses glutathione to retire all titratsble peroxides in ,r,ahranos and lipoproteins including cholesterol and cholesterol ester Irjdroperoxides. khlle demonstrated in several ~mlrml tissues, ~ tissues hax~ not been e ~ r e d . PHGPX was detected in human tumor derived cell lines after growth in selenium sufficient media for 12-14 days. Only 3 to 10% of the total activity was detected in the ER fraction at 20-60% of the cytopl~-q,lc specific activity and alx~t 15% of ~ activity was detected in the nuclear fraction at 50% of the cytmplasmie specific activity. " l h e c y ~ 1 ~ r l e activit2es values w e r e : ~ HL-60 22 rmoles/min/mg. ; HL-60 WE, I<562WE, AI~ K.562, M2F-7 WE, MCF-7H6 - ii-12 rmolas/min/mg; H e ~ -9.6 nmoles/min/mg; MCF-7 - 7 rmoles/min/mg. Activity levels in selenium deficient media were 0.2 -2.5 rmolas/min/mg. Gel filtration demonstrates that the native size of the species is 18 kDa. After lsbeling the cell lines with 75Se-selenious acid 6-7 selenoproteins cen be resolved by SDS-PAGE - Mr: 57-60; 22; 18; 19 kDa. ~he 22 kDa protein is the ~ t of classical glutathione peroxidase (GSHPX). ~ae 18 Id)a s e ~ r o t e l n fractiormtes on Sephadex G-200 as a monomer resolving completely from (~PX and CST activity and is only selenoprotein associated with the ~ activity. association is substantiated in the HCF-7 WE cell line which has no 22 kDa selex~protein or associated GSaWX activity and expresses GST activity at 3-10% the level of the other cell lines.
1.11
ACUTE OXYGEN TOXICITY CAUSES INCREASED LUNG mRNA SYNTHESIS IN RATS ACUTE Phillip Factor, D.O. and J.I. Sznajder, M.D. Section of Pulmonary and Critical Care Medicine, The University of Chicago and Micheal Reese Hospital To examine the responses to hyperoxia at the molecular level we exposed 18 male Sprague-Dawley rats (45 days old, ave. weightf325g) to high levis of oxygen (F102>0.95) for 66 hours. A second group of 7 control an]mals were kept at the same temperature and humidity and provided with the same ad libitum diet as the hyperoxic group. The rats were sacrificed prior to en bloc removal of the lungs. One lung was saved for wet/dry weight determination. The other was immediately frozen for RNA isolation. Using a Proteinase K/phenol/chloroform separation technique followed by oligo-(dT)-cellulose column chromatography, total and poly-A + RN#t,were isolated and quantified. Total RNA %mRNA Wet/Dry Weight Control 5.75, 1.51 4.24, Hyperoxia 3.23, 1.39, 5.31, 5 Days Recovery 9.93, 2.62, 4.72 10 Days Recovery 9.54 2.55 4.67 p<.05 vs Control
mg Total RNA/gm Dry Lung
Wet/dry weight ratios confirmed the presence of lung injury in the hyperoxic group. Animals exposed to high levels of oxygen had a significant increase in mRNA levels when compared to the control animals. This suggests that part of the underlying pathophysiologic process of oxygen toxicity includes increased protein synthesis as indicated by increased expression of total and mRNA. (Supported by a grant from the Chicago Lung Association).
PUkq~ ~ir_~mI~ PEKED~E IS A DI~HNCr
3
1.10
PROIEIN FROM ~ GIIYfb/~ONE Iq~fKII~E R. Steven Esworthy*, Fo~g-Fc~g Ore*, Raymond J. Paxtor~, Steven Akmm~*0 and James H. Doroslx~. *DeparUmnt of Medical Oncology mxl ~erapeutics Research, and **Division of lmmxmlogy City of Hope National Medical Center and the Beclasan Research Institute of the City of Hope, Duarte, CA 91O1O ~ae presence of a glutathione peroxidase activity in p ~ raises the question of whether the enyne is leaked into plasma from red blood ceils or ~ endothelial cells or represents a distinct secretory form of the er~flre. To answer this question human pl~qm glutathione peroxidase ~ms purified to homogeneity and partially sequenced. Overlapping peptide fragments from two endepeptidase digests permitted the determination of one sequence of 32 contiguous anino acids and one sequence of 25 contiguous amino acids. Additional peptide fragments without obvious overlaps were sequenced. ~he 32 amino acid sequence aligns with positions 82-I13 of bavan cytosolic glutathiore peroxidase with 9 mismatches and no gaps or insertions required. 2he 25 anino acid sequence aligns with positions 157-180 with eight m i s r a ~ and an insertion of one resick~e. On the basis of this sequence information we conclude that }luman plasma glutathione perc0ddase is not derived by modification from the preduninmat intracellular enzyme and is most likely secreted into plamra. We could detect no glutathione peroxidase activity in bile indicating that plasma is the principal site of expression. ~he plasma e~Tme does not have activity toward phoscghn_tidylcholine hydroperoxide suggesting that it is dependent on the action of phospholipase Az.
STRESS RESPONSE AND GLUTATHIONE M.L. Freeman, E.L. Saunders, and M.J. Meredith Radiat. Oncol., Vanderbilt Univ., Nashville TN 37232 and Biochem. Oregon Health Sci. Univ., Portland OR 97201 Glutathione (GSH) oxidation and glutathione depletion induce qualitatively different stress responses. Oxidation of glutathione by exposure to diamide enhances the synthesis of the 110, 90, 73/72, and 50 kDa heat shock proteins (HSP). Induction only occurs when thiol oxidation exceeds the rate of reduction. The consequence of enhanced HSP synthesis is the development of resistance to the cytotoxic effects of heat shock (thermotolerance) and protection against thermal inhibition of protein synthesis. Conversely, enhanced HSP synthesis with the subsequent development of thermotolerance induced by a prior heat shock provide protection from the cytotoxic effects of glutathione oxidation produced by exposure to diamide. Enhanced HSP synthesis and the development of thermotolerance did not prevent thiol oxidation as assessed by 2D thiol blotting but apparently provided protection from the consequence of oxidation. Depletion of GSH by exposure to diethyl maleate (DEM) or buthionine sulfoximine (BSO) does not induce HSP synthesis or trigger the development of thermotolerance. Nor does depletion inhibit the heat shock response. However, GSH depletion by treatment with either DEM or BSO enhances the synthesis of a 32 kDa stress protein. The rate of induction of this stress protein is dependent on the rate of GSH depletion and is induced only when depletion exceeds 80%. This protein has been characterized by 2D PAGE and the 32 kDa protein exhibits a pI of 6.4 and has been tentatively identified as heme oxygenase.
1.12