- Email: [email protected]
Down-regulation of Mn-SOD by the HIV TAT protein
Session 5: Regulation of Gene Expression in Oxidative Stress, Part I 5 ~5
OXIDANT-MODULATED GENE EXPRESSION IN HAMSTER HA-l CELLS Dana R. Crawford, Cheryl A. Edbauer-Nechamen, Gary P. Schools, Sharon L. Salmon and Kelvin J.A. Davies Department of Biochemistry and Molecular Biology, The Albany Medical College, New York, 12208 USA.
DOWN-REGULATION OF Mn-SOD BY THE HIV TAT PROTEIN Sonia C. Flores and Joe M. McCord Webb- Waring Institute for Biomedical Research, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, Colorado, 80262 USA
The expression of a growing number of genes is now known to be modulated by oxidant stress. These include genes for antioxidant enzymes, proto-oncoproteins, DNA repair enzymes, stress proteins, and others. We have attempted to identify oxidantmodulated genes by using an adaptive response model system. Pretreatment of HA-1 hamster fibroblasts with hydrogen peroxide induces a protective response in some of these cells against a later, higher dose of the same agent. We find an approximate 30-fold protection using this protocol. This protective effect is inhibited by both protein and mRNA synthesis inhibitors. This indicates that protective sequences are being synthesized in the cell following hydrogen peroxide pretreatment. We have attempted to identify oxidant-modulated mRNAs m HA-1 cells using differential display technology. Following mRNA isolation from oxidant-pretreated and -untreated cells, first strand cDNA is synthesized and used as a template for polymerase chain reaction (PCR). Using different combinations of primers, segments of the cDNAs are PCR-amplified and their products compared on a sequencing gel. Using this procedure, we have found several significantly modulated sequences. When used as hybridization probes, these sequences detect modulated mRNA species on Northern blots. The same pretreatment protocol also induces GADD 153, GADD 45, and heme oxygenase mRNAs, all of which are known to be induced by oxidant stress. We are currently characteriung the differential display-derived clones toward determining their possible role in oxidant-stress protection.
Using a HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1, we have found that the expression of the regulatory tat protein suppresses the expression of cellular Mn-SOD. This enzyme is one of the cell’s primary defenses against oxygen-derived free radicals and is vital for maintaining a healthy oxidant/antioxidant balance. The parental HeLa cells express nearly equivalent amounts of Cu,Zn- and Mn-SOD isozymes, while the tat-producing cells contained 52% less Mn-SOD than the parental cells, with the Cu,Zn-SOD enzyme essentially unchanged. The steady-state levels of Mn-SODspecific RNAs were also lower in the HeLa- ta t cell line than in the parental line. Again, no difference was seen in the steady-state levels of Cu,Zn-SOD-specific RNAs. In addition to the decreased MI-SOD activity, HeLa- ta t cells showed evidences of increased oxidative stress. Carbonyl proteins were 75% higher and cellular reduced glutathione content was 50% of parental. Moreover, HeLa- ta t cells were more susceptible to heatshock treatment than parental cells. HeLa and HeLatat extracts were incubated with radiolabeled transcripts of Mn-SOD, followed by ultraviolet cross-linking, digestion with ribonuclease A, and electrophoretic analysis. The results suggest a direct interaction between tat and Mn-superoxide dismutase transcripts.
DEFECTIVE GENE EXPRESSION OF MNSOD IN TUMOR CELLS T. Galeotti, S. Borrello, ME. De Leo, G. Piccirillo, F. Ria, G. Pani, M. Landriscina, H. Wohlrab’ Inst. of Gen. Pathology, Catholic Univ., Rome, Italy, 1Dept. of Metabolic Regulation, Boston Biomed. Res. Inst., MA, USA. Reactive oxygen species (ROS) have been frequently implicated in the initiation and promotion phases of carcinogenesis and it is long-standing the observation that antioxidant enzymes, which can antagonize this process, are lowered in a number of malignancies. The enzyme most commonly decreased is the mitochondrial Mn-containing superoxide dismutase (MnSOD) encoded by a nuclear gene mapped on the band 6q25, a region frequently deleted in several human tumors. The close association of del(6q) with diminution of MnSOD activity, immunoreactive protein and mRNA in SV40-transformed human fibroblasts has led to suggest that MnSOD might be a new type of tumorsuppressor gene. This hypothesis is also sustained by the finding that introduction of a human chromosome 6 by microcell fusion or transfection of MnSOD cDNA into human melanoma cell lines suppress the malignant phenotype. There are, however, conflicting observations?hat t&d to ascribe the deficiency of MnSOD activity more to a defect in the expression of the gene than to its deletion. In many transformed cell lines, including some with marked del(6q), there is no change in the dosage of the MnSOD gene and the enzvme is highly - . inducible by various pro-oxidant agents. Mn ion has been found to be highly deficient in human and rodent tumors. Owing to the second messenger function of ROS in activating transcription factors (NF-kB, AP-1) and to the ability of Mn to facilitate the dismutation of 05 to H202, we propose here that in the early stage of carcinogenesis an impairment of the signal transduction machinery, related to the Mn deficiency, might limit the binding to DNA of transcription factors and cause the defect in the MnSOD gene expression. (Supported by CNR-ACRO project N. 92.02164. PF39).
IN AND IN VIVO VITRO EXPRESSION OF METALLOTHIONEIN IN INJURED TYPE II ALVEOLAR EPITHELIAL CELLS S. Horowitz*, B. Piedboeuf’. W. Maniscalco’, S. Hall?, M. Campbell’, R. Watkins’, ‘Dept. of Pediatrics, Univ. of Rochester Sch. of Med., Rochester, NY 14642, *Pulm. and Crit. Care Med., Mail Code UHN-67, Oregon Health Sci. Univ., Portland, OR 972013098, *Pulmonary Research Instit., Winthrop-Univ. Hosp., SUNY at Stony Brook Med. Sch., Mineola, NY 1 I SOI. Metallothioneins (MTs), a family of proteins that possess antioxidant activity and are involved in Cu and Zn metabolism, are induced at the mRNA and protein levels during acute lung injury following hyperoxia and oleic acid-induced lung injury. In situ hybridizations have revealed patterns of increased MT message abundance in injured lung, and show abundant MT mRNA in unidentified cells of the gas exchange region. To dctcrmine if MT is expressed in type II pneumocytes during lung injury, we used duallabel in situ hybridizations with a type II cell-specific marker--7Hlabeled antisense cRNA for the surfactant protein SP-C--together with digoxigenin-labeled cRNA for MT. Results show that MT transcripts accumulate in type II cells during in viva lung injury either by hyperoxia or olcic acid, in rabbits. To begin to explore what drives this increase, type II cells were isolated from adult rabbits and cultured overnight in media containing serum. After 20 h. cells wcrc fed with serum-free media and exposed to varying concentrations of O? for 24 h. Time-matched controls were kept in serum-free media at 21% 02. Cells, on slides, were fixed in IO% formalin and analyzed directly for MT transcripts by in siru hybridization. MT mRNA was present in many type II cells in controls, and per-cell mRNA abundance increased over time in culture. Relative to 21% O,, exposure to 5% 0, for 24 h decreased the abundance of MT transcripts. By contrast, exposure to 95%1 increased MT mRNA levels, on a oer-cell basis. Under all conditions, SP-C mRNA abundance wak relatively unaffected. These experiments suggest that type II cells may respond directly lo 0, Icvels. Perhaps MT, which is highly induced by hyperoxia and has oxy-radical scavenger activity, may help protect type II cells from oxidative Injury.
503
5 :6
OXIDANT-MODULATED GENE EXPRESSION IN HAMSTER HA-l CELLS Dana R. Crawford, Cheryl A. Edbauer-Nechamen, Gary P. Schools, Sharon L. Salmon and Kelvin J.A. Davies Department of Biochemistry and Molecular Biology, The Albany Medical College, New York, 12208 USA.
DOWN-REGULATION OF Mn-SOD BY THE HIV TAT PROTEIN Sonia C. Flores and Joe M. McCord Webb- Waring Institute for Biomedical Research, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, Colorado, 80262 USA
The expression of a growing number of genes is now known to be modulated by oxidant stress. These include genes for antioxidant enzymes, proto-oncoproteins, DNA repair enzymes, stress proteins, and others. We have attempted to identify oxidantmodulated genes by using an adaptive response model system. Pretreatment of HA-1 hamster fibroblasts with hydrogen peroxide induces a protective response in some of these cells against a later, higher dose of the same agent. We find an approximate 30-fold protection using this protocol. This protective effect is inhibited by both protein and mRNA synthesis inhibitors. This indicates that protective sequences are being synthesized in the cell following hydrogen peroxide pretreatment. We have attempted to identify oxidant-modulated mRNAs m HA-1 cells using differential display technology. Following mRNA isolation from oxidant-pretreated and -untreated cells, first strand cDNA is synthesized and used as a template for polymerase chain reaction (PCR). Using different combinations of primers, segments of the cDNAs are PCR-amplified and their products compared on a sequencing gel. Using this procedure, we have found several significantly modulated sequences. When used as hybridization probes, these sequences detect modulated mRNA species on Northern blots. The same pretreatment protocol also induces GADD 153, GADD 45, and heme oxygenase mRNAs, all of which are known to be induced by oxidant stress. We are currently characteriung the differential display-derived clones toward determining their possible role in oxidant-stress protection.
Using a HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1, we have found that the expression of the regulatory tat protein suppresses the expression of cellular Mn-SOD. This enzyme is one of the cell’s primary defenses against oxygen-derived free radicals and is vital for maintaining a healthy oxidant/antioxidant balance. The parental HeLa cells express nearly equivalent amounts of Cu,Zn- and Mn-SOD isozymes, while the tat-producing cells contained 52% less Mn-SOD than the parental cells, with the Cu,Zn-SOD enzyme essentially unchanged. The steady-state levels of Mn-SODspecific RNAs were also lower in the HeLa- ta t cell line than in the parental line. Again, no difference was seen in the steady-state levels of Cu,Zn-SOD-specific RNAs. In addition to the decreased MI-SOD activity, HeLa- ta t cells showed evidences of increased oxidative stress. Carbonyl proteins were 75% higher and cellular reduced glutathione content was 50% of parental. Moreover, HeLa- ta t cells were more susceptible to heatshock treatment than parental cells. HeLa and HeLatat extracts were incubated with radiolabeled transcripts of Mn-SOD, followed by ultraviolet cross-linking, digestion with ribonuclease A, and electrophoretic analysis. The results suggest a direct interaction between tat and Mn-superoxide dismutase transcripts.
DEFECTIVE GENE EXPRESSION OF MNSOD IN TUMOR CELLS T. Galeotti, S. Borrello, ME. De Leo, G. Piccirillo, F. Ria, G. Pani, M. Landriscina, H. Wohlrab’ Inst. of Gen. Pathology, Catholic Univ., Rome, Italy, 1Dept. of Metabolic Regulation, Boston Biomed. Res. Inst., MA, USA. Reactive oxygen species (ROS) have been frequently implicated in the initiation and promotion phases of carcinogenesis and it is long-standing the observation that antioxidant enzymes, which can antagonize this process, are lowered in a number of malignancies. The enzyme most commonly decreased is the mitochondrial Mn-containing superoxide dismutase (MnSOD) encoded by a nuclear gene mapped on the band 6q25, a region frequently deleted in several human tumors. The close association of del(6q) with diminution of MnSOD activity, immunoreactive protein and mRNA in SV40-transformed human fibroblasts has led to suggest that MnSOD might be a new type of tumorsuppressor gene. This hypothesis is also sustained by the finding that introduction of a human chromosome 6 by microcell fusion or transfection of MnSOD cDNA into human melanoma cell lines suppress the malignant phenotype. There are, however, conflicting observations?hat t&d to ascribe the deficiency of MnSOD activity more to a defect in the expression of the gene than to its deletion. In many transformed cell lines, including some with marked del(6q), there is no change in the dosage of the MnSOD gene and the enzvme is highly - . inducible by various pro-oxidant agents. Mn ion has been found to be highly deficient in human and rodent tumors. Owing to the second messenger function of ROS in activating transcription factors (NF-kB, AP-1) and to the ability of Mn to facilitate the dismutation of 05 to H202, we propose here that in the early stage of carcinogenesis an impairment of the signal transduction machinery, related to the Mn deficiency, might limit the binding to DNA of transcription factors and cause the defect in the MnSOD gene expression. (Supported by CNR-ACRO project N. 92.02164. PF39).
IN AND IN VIVO VITRO EXPRESSION OF METALLOTHIONEIN IN INJURED TYPE II ALVEOLAR EPITHELIAL CELLS S. Horowitz*, B. Piedboeuf’. W. Maniscalco’, S. Hall?, M. Campbell’, R. Watkins’, ‘Dept. of Pediatrics, Univ. of Rochester Sch. of Med., Rochester, NY 14642, *Pulm. and Crit. Care Med., Mail Code UHN-67, Oregon Health Sci. Univ., Portland, OR 972013098, *Pulmonary Research Instit., Winthrop-Univ. Hosp., SUNY at Stony Brook Med. Sch., Mineola, NY 1 I SOI. Metallothioneins (MTs), a family of proteins that possess antioxidant activity and are involved in Cu and Zn metabolism, are induced at the mRNA and protein levels during acute lung injury following hyperoxia and oleic acid-induced lung injury. In situ hybridizations have revealed patterns of increased MT message abundance in injured lung, and show abundant MT mRNA in unidentified cells of the gas exchange region. To dctcrmine if MT is expressed in type II pneumocytes during lung injury, we used duallabel in situ hybridizations with a type II cell-specific marker--7Hlabeled antisense cRNA for the surfactant protein SP-C--together with digoxigenin-labeled cRNA for MT. Results show that MT transcripts accumulate in type II cells during in viva lung injury either by hyperoxia or olcic acid, in rabbits. To begin to explore what drives this increase, type II cells were isolated from adult rabbits and cultured overnight in media containing serum. After 20 h. cells wcrc fed with serum-free media and exposed to varying concentrations of O? for 24 h. Time-matched controls were kept in serum-free media at 21% 02. Cells, on slides, were fixed in IO% formalin and analyzed directly for MT transcripts by in siru hybridization. MT mRNA was present in many type II cells in controls, and per-cell mRNA abundance increased over time in culture. Relative to 21% O,, exposure to 5% 0, for 24 h decreased the abundance of MT transcripts. By contrast, exposure to 95%1 increased MT mRNA levels, on a oer-cell basis. Under all conditions, SP-C mRNA abundance wak relatively unaffected. These experiments suggest that type II cells may respond directly lo 0, Icvels. Perhaps MT, which is highly induced by hyperoxia and has oxy-radical scavenger activity, may help protect type II cells from oxidative Injury.
503
5 :6