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Induction of thioredoxin in human lymphocytes with low-dose ionizing radiation
Biochimica et Biophysica Acta 1359 Ž1997. 65–70
Induction of thioredoxin in human lymphocytes with low-dose ionizing radiation Yuko Hoshi a
a,),1
, Hiroshi Tanooka
b,c
, Kunihisa Miyazaki
d,2
, Hiro Wakasugi
d
Nuclear Energy Systems Department, Central Research Institute of Electric Power Industry, Iwado-kita, Komae-shi, Tokyo 201, Japan b Research AdÕisor, Central Research Institute of Electric Power Industry, Iwado-kita, Komae-shi, Tokyo 201, Japan c Genetics DiÕision, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104, Japan d Section of Study of Host Immune Response, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104, Japan Received 17 March 1997; accepted 27 May 1997
Abstract Induction of the expression of the thioredoxin ŽTRX. gene, producing a key protein in regulating cellular functions through redox reaction as well as being a radioprotector, was followed after ionizing irradiation of lymphocytes from human donors. The TRX mRNA level increased to a peak, 5.7-fold higher than the control at maximum, 6 h after irradiation, and then decreased. The optimum radiation dose for enhancement of induction of the TRX mRNA was 0.25 Gy. The TRX protein also increased to a peak, a 3-fold increase at maximum, with the same timing as that for TRX mRNA. q 1997 Elsevier Science B.V. Keywords: Thioredoxin; Induction of gene expression; Ionizing radiation; Low dose
1. Introduction Biological effects of low-level ionizing radiation are now becoming increasingly important from the viewpoint of human health in the environment w1x. At the molecular level, ionizing radiation modulates expression of a number of mammalian genes. These include plasminogen activator gene w2x, proto-
)
Corresponding author. Bio-Science Department Komae Branch, Central Research Institute of Electric Power Industry, 2-11-1, Iwado-kita, Komae-shi, Tokyo 201, Japan. Fax: q81 3 3480 3539; E-mail: [email protected] 1 Present address: Bio-Science Department Komae Branch, Central Research Institute of Electric Power Industry, Iwado-kita, Komae-shi, Tokyo 201, Japan. 2 Present address: Department of Surgery, Jichi Medical School Omiya Medical Center, Omiya, Saitama-ken 330, Japan.
oncogenes w3–5x, and genes controlling cell growth such as cyclin B w6x and p53 w7x, and apoptosis w8x. Involvement of a more numerous variety of radiation-inducible genes controlling cellular functions was shown from analysis of radiation-inducible transcripts w9x. The presence of the responsive element to ionizing radiation w10x and the alteration of transcription factor binding after radiation w11x indicate that ionizing radiation modulates gene expression at the transcription level. The molecular mechanism of these ionizing radiation-inducible events is thought to be related to such radiation-inducible biological phenomena as adaptive response w12x and induced radioresistance w13x, although the molecular data have been obtained with a relatively high radiation dose. On the other hand, the redox status of a number of gene products also regulates various cellular func-
0167-4889r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 8 9 Ž 9 7 . 0 0 0 8 5 - 2
66
Y. Hoshi et al.r Biochimica et Biophysica Acta 1359 (1997) 65–70
tions, such as the DNA binding activity of Jun and Fos and the HAP-1rRef-1 apurinic endonucleaseDNA repair activity w14,15x and the p53 function w16x. Recently, the redoxrDNA repair protein, Ref-1, was shown to be essential for early embryonic development in mice w17x. Thioredoxin ŽTRX. is a key protein which controls cellular redox status. TRX was originally purified from Escherichia coli w18x and shown to be an oxidoreductase enzyme consisting of 104 amino acids w19,20x. The nucleotide sequence of the human TRX gene has been determined w21x and was shown to be identical to that of adult T cell leukemia-derived factor w22,23x, having cytokine activity w24x. Interestingly, TRX is a surface-associated sulfhydryl protein w25x and a radio-protector w26x. TRX reduces hydrogen peroxide and scavenges free radicals w27,28x. Therefore, TRX gene expression potentially can play an important role in cellular functions responding to radiation. It would be interesting to test whether ionizing radiation can induce TRX. In this study, we observed the cellular TRX mRNA and protein levels after ionizing irradiation of human lymphocytes and found that TRX mRNA as well as TRX protein is indeed induced.
2. Materials and methods
Lymphocytes were suspended at 10 6 cellsrml in the medium and kept at 378C for 1 h prior to irradiation. The human B-lymphoblastoid cell line 1G8 w29x, which was immortalized after infection with Epstein-Barr virus Ž EBV., the Jurkat cell line, and lymphocytes from a human donor stimulated for growth of T-cells by interleukin 2 Ž IL-2. and CD3, provided by Dr. Sekine, National Cancer Center, were also used as TRX expression controls. They were incubated in the same medium as described. 2.2. Radiation and chemical treatment Lymphocytes suspended in the culture medium at 10 6 cellsrml were exposed at room temperature to various single doses of g rays generated by a 60 Co source ŽToshiba, Tokyo, Japan. at a dose-rate of 0.72 Gyrmin, as calibrated by a dosimeter ŽFarmer 2570r1B, NE Technology, Reading, UK. , or to Xrays generated by a 150 Kvp generator Ž Model MBR1505R2, Hitachi, Tokyo, Japan. with filters of 0.2 mm Cu and 1.0 mm Al at a dose rate of 0.25 Gyrmin. Irradiated cells were incubated at 378C in humidified 5% CO 2r95% air for various periods of time. Aliquots, containing 10 7 cells each, were used for extraction of RNA and protein. As a positive control, cells suspended at 10 6 cellsrml in the culture medium were treated with 1 ng of 12-O-tetradecanoilphorbol-13-acetate ŽTPA. per ml for various periods of time under the culture conditions.
2.1. Cells 2.3. Measurements of TRX mRNA and TRX protein Fresh human venous blood was obtained from one of the authors, HT Ž donor A. , and was used immediately for experiments after sampling. Other blood samples were obtained from healthy male donors ŽB–E. supplied by the Japan Red Cross Blood Center, Tokyo, and were kept at 48C overnight after separation of serum. Lymphocytes were isolated by centrifugation at 1000 rpm for 30 min in a layer of LYMPHOSEPAL Ž Nippon Gene, Toyama, Japan. . Cells were washed once in phosphate-buffered saline and hemolyzed with hypotonic solution. They were then washed with the cell culture medium, RPMI 1640 ŽNissui, Tokyo, Japan. supplemented with 10% heat-treated fetal calf serum ŽGIBCO BRL; Gaithersburg, MD, USA. , 2 mmol of L-glutamine, 0.2 mmol of sodium pyruvate and 100 units of penicillinrml.
Total cellular RNA was extracted according to protocols of the manufacturer of the ISOGEN kit ŽNippon Gene, Toyama, Japan. , and analyzed according to the method described by Sambrook et al. w30x. Briefly, 10 7 cells for each aliquot were collected by centrifugation Ž 1500 rpm, 10 min., suspended in ISOGEN, containing 10 mM Tris-HCl, pH 7.6, 250 mM NaCl, 0.5% NP-40, and 1 mM phenylmethylsulfonyl fluoride, and then sonicated. RNA was isolated after precipitation with isopropanol. RNA Ž5 m g. was denatured with formaldehyde for 15 min at 658C, and subjected to electrophoresis Ž5 Vrcm. in 1% agarose gel containing 6.6% of formaldehyde. RNA was then transferred to a nitrocellulose membrane ŽNITROPLUS; Micron Separations, Westboro,
Y. Hoshi et al.r Biochimica et Biophysica Acta 1359 91997) 65–70
MA, USA., which was then prehybridized with salmon sperm DNA for 4 h, followed by hybridization for 18–20 h at 458C with a 32 P-labeled probe of the TRX cDNA Ž600 bp. in the hybridization solution. The Northern blots were taken by autoradiography on an X-ray film ŽType AR, KODAK; Rochester, NY, USA. . To quantify TRX mRNA amounts, the blots were taken on an imaging plate and the density was measured by an analyzer Ž Model BAS2000, FUJIX PICTOGRAM; Fuji Film, Tokyo, Japan. . The exposure time was usually overnight for X-ray film and 1 h for the imaging plate. The linear intensity response portion was used to obtain relative TRX mRNA levels. As controls, the 18S ribosomal RNA Ž1900 bp. bands were taken by rehybridization on the same membrane. To extract total proteins, irradiated cells were washed with phosphate-buffered saline and suspended in lysis solution containing 10 mM Tris-HCl, pH 7.6; 250 mM NaCl; 0.5% NP-40 and 1 mM phenylmethylsulfonyl fluoride. Proteins were extracted by freeze-thawing five times followed by sonication. Amounts of TRX proteins in these extracts were measured by the ELISA method w31x, using anti-TRX antibody developed by the National Cancer Center and Immuno-Biological Laboratories ŽFujioka, Gumma-ken, Japan. . Recombinant human TRX ŽIMO, Stockholm, Sweden. was used to obtain the calibration curve.
3. Results Expression of TRX mRNA in human lymphocytes was induced after irradiation of lymphocytes in the culture medium with single radiation doses. Fig. 1 shows Northern blots of TRX mRNA of lymphocytes separated from fresh human blood Ždonor A. following irradiation with 0.25 Gy. The TRX mRNA level increased with time and reached a 5.7-fold higher level after 6 h. Fig. 1 also includes the TRX mRNA in the immortalized human B cell line 1G8, which showed a level 11-fold higher than untreated lymphocytes. As loading controls, the 18S ribosomal RNA was detected in the same membrane filter as that used for the TRX mRNA, which remained unchanged during post-irradiation incubation Ž Fig. 1.. Fig. 2 shows TRX mRNA levels expressed in
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Fig. 1. Northern blots of thioredoxin ŽTRX. mRNA of human lymphocytes incubated in the culture medium for various times after irradiation with 0.25 Gy. Upper lanes: TRX mRNA Ž600 nucleotides.; lower lanes: 18S ribosomal RNA Ž1900 nucleotides.. Lane 1–4: RNA Žtotal loading: 5 m g. from fresh human lymphocytes at 0, 2, 4 and 6 h after irradiation. Lane 5: RNA Žtotal loading: 10 m g. from the human lymphocyte cell line, 1G8, as a positive control. Detection of 18S ribosomal RNA was undertaken to check for gel loading errors.
various preparations of human lymphocytes. Lymphocytes prepared from fresh venous blood Ž donor A. and the Jurkat cells showed the lowest TRX mRNA level. Lymphocytes stimulated for growth with IL-2 and CD3 exhibited a 3.6-fold higher TRX mRNA level. The highest TRX mRNA expression so far measured was exhibited in the immortalized cell line 1G8. Fig. 3 shows quantitative data of the post-irradiation time course of TRX mRNA and TRX protein levels in lymphocytes taken from different donors. Lymphocytes freshly prepared from venous blood showed the highest response ŽFig. 3A.. The peak of the TRX mRNA expression appeared 6 h after irradiation, with a decline afterwards. Red Cross blood samples ŽFig. 3B–E. also showed the increase of TRX mRNA level with a peak at 6–8 h after irradia-
Fig. 2. TRX mRNA levels in human lymphocytes of various preparations and cell lines. a, lymphocytes from fresh blood of donor A. b, Jurkat cells. c, lymphocytes from fresh blood, stimulated for growth with IL-2 and CD3. d. 1G8 cells. Bar: SEM.
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Y. Hoshi et al.r Biochimica et Biophysica Acta 1359 (1997) 65–70
Fig. 3. Time course of the induction of TRX mRNA Župper column. and TRX protein Žlower column. in human lymphocytes from different donors after irradiation with 0.25 Gy. Age of donors Žall males.: A, 63; B, 43; C, 41; D, 62; E, 30. TRX mRNA levels after treatment with TPA Ž'. and without radiation Ž`. are shown. Bars: SEM.
tion, but the increased level was lower than that of sample from donor A. Lymphocytes treated by a tumor promoter TPA also showed the elevated TRX mRNA level with a peak at 2 h. ŽFig. 3A.. The TRX mRNA level in non-irradiated lymphocytes remained unchanged during incubation Ž Fig. 3A. . The TRX protein level was also tracked after irradiation of the lymphocytes with 0.25 Gy ŽFig. 3, lower columns.. The peak expression was found 6 h after irradiation, just as for mRNA, although its increased level was not so high as the mRNA increase. The TRX protein level in non-irradiated sample A lymphocytes was 85 pgrcell or 20.3 ngrmg of total cellular protein. To see the dose response of the TRX mRNA and
protein induction, lymphocytes were irradiated with various radiation doses and incubated in the culture medium for 6 h ŽFig. 4.. The optimum dose for the TRX mRNA increase was found to be 0.25 Gy most clearly for lymphocytes freshly prepared from donor A. The optimum dose for TRX mRNA and protein appeared to be 0.25–0.5 Gy for the other samples also.
4. Discussion The present study showed that the expression of the TRX gene in human lymphocytes increased with a low dose of ionizing radiation. This increase was
Fig. 4. Dose response of the induction of TRX mRNA Župper column. and TRX protein Žlower column. in human lymphocytes 6 h after irradiation. Donors are the same as shown in Fig. 3. Dotted line: donor E. Bars: SEM.
Y. Hoshi et al.r Biochimica et Biophysica Acta 1359 91997) 65–70
most clearly observed in lymphocytes prepared from fresh blood and immediately used. Other samples kept overnight after separation of serum also showed an increase of TRX mRNA and protein after ionizing irradiation, although the expression level and timing varied. Therefore, this increase is thought to occur commonly in human lymphocytes. The optimum radiation dose for TRX induction was 0.25 Gy, which is much lower than doses used for molecular studies reviewed in Section 1. Interestingly, the elevated level of p53 protein is observed in tissue specimens of various organs of mice, with the same optimum dose of 0.25 Gy as that in the present study w32x. The biological consequence of the TRX induction by ionizing radiation is unknown at the time of the present study. However, TRX appears to be a ubiquitous source of reducing potential in mammalian cells and it is now becoming clear from various evidences as stated in Section 1 that the cellular redox status may play an important regulatory role in transcription factor activity and cell cycle control. In this consequence, the TRX induction by ionizing radiation must have some biological effects. This problem will be pursued in a future study.
Acknowledgements We thank Dr. S. Hattori and Mr. K. Ishida, Central Research Institute for Electric Power Industry, and Dr. M. Terada, Director of the National Cancer Research Institute, for their support of this research project, Dr. M.S. Sasaki, Kyoto University, for valuable information, and Dr. D.A. Boothman, University of Wisconsin Medical School, for critical reading of the manuscripts. This work was supported by a 2ndterm Grant-in-Aid for a Comprehensive 10-year Strategy for Cancer Control from the Ministry of Health and Welfare and a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.
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w2x D.A. Boothman, M. Wang, S.W. Lee, Cancer Res. 51 Ž1991. 5587–5595. w3x M.L. Sherman, R. Datta, D.E. Hallahan, R.R. Weichselbaum, D.W. Kufe, Proc. Natl. Acad. Sci. USA 87 Ž1990. 5663–5666. w4x D.E. Hallahan, V.P. Sukhatme, M.L. Sherman, S. Virudachalan, D. Kufe, R.R. Weichselbaum, Proc. Natl. Acad. Sci. USA 88 Ž1991. 2156–2160. w5x A. Anderson, G.E. Woloschak, Radiat. Res. 130 Ž1992. 340–344. w6x R.J. Mushel, H.B. Zhang, G. Iliakis, W.G. McKenna, Cancer Res. 51 Ž1991. 5113–5117. w7x M.B. Kastan, O. Onyekwere, D. Sidransky, B. Volgelstein, R.W. Craig, Cancer Res. 51 Ž1991. 6304–6311. w8x F.M. Uckun, L. Tuel-Ahlgren, C.W. Song, K. Waddick, D.E. Myers, J. Kirihara, J.A. Ledbetter, G.L. Schieven, Proc. Natl. Acad. Sci. USA 89 Ž1992. 9005–9009. w9x D.A. Boothman, M. Meyers, N. Fukunaga, S.W. Lee, Proc. Natl. Acad. Sci. USA 90 Ž1993. 7200–7204. w10x R. Datta, E. Rubin, V. Sukhatme, S. Qureshi, D. Hallahan, R.R. Weichselbaum, D.W. Kufe, Proc. Natl. Acad. Sci. USA 89 Ž1992. 10149–10153. w11x W.M. Sahijdak, C.-R. Yang, J.S. Zuckerman, M. Meyers, D.A. Boothman, Radiat. Res. 138 Ž1994. S47–S51. w12x G. Olivieri, J. Bodicote, S. Wolff, Science 223 Ž1984. 594–597. w13x M.C. Joiner, Int. J. Radiat. Biol. 65 Ž1994. 79–84. w14x S. Xanthoudakis, G. Miao, F. Wang, Y.-C.E. Pan, T. Curran, EMBO J. 11 Ž1992. 3323–3335. w15x L.J. Walker, C.N. Robson, E. Black, D. Gillespie, I.D. Hickson, Mol. Cell. Biol. 13 Ž1993. 5370–5376. w16x R. Rainwater, D. Parks, M.E. Anderson, P. Tegtmeyer, K. Mann, Mol. Cell. Biol. 15 Ž1995. 3892–3903. w17x S. Xanthoudakis, R.J. Smeyne, J.D. Wallace, T. Curran, Proc. Natl. Acad. Sci. USA 93 Ž1996. 8919–8923. w18x T.C. Laurent, E.C. Moore, P. Reichard, J. Biol. Chem. 269 Ž1964. 3436–3444. w19x A. Holmgren, Annu. Rev. Biochem. 54 Ž1985. 237–271. w20x E.E. Wollman, L. D’Auriol, L. Rimsky, A. Shaw, J.-P. Jacquot, P. Wingfield, P. Graber, F. Dessarps, P. Robin, F. Galibert, J. Bertoglio, D. Fradelizi, J. Biol. Chem. 263 Ž1988. 15506–15512. w21x K.F. Tonissen, J.R. Wells, Gene 102 Ž1991. 221–228. w22x H. Wakasugi, L. Rimsky, Y. Mahey, A.M. Kamel, D. Fradelizi, T. Tursz, J. Bertoglio, Proc. Natl. Acad. Sci. USA 84 Ž1987. 804–808. w23x Y. Tagaya, Y. Maeda, A. Matsui, N. Kondo, J. Matsui, N. Brown, K. Arai, T. Yokota, H. Wakasugi, J. Yodoi, EMBO J. 8 Ž1989. 757–764. w24x C. Biguet, N. Wakasugi, Z. Mishal, A. Holmgren, S. Chouaib, T. Tursz, H. Wakazugi, J. Biol. Chem. 269 Ž1994. 28865–28870. w25x M.F. Dean, H. Martin, P.A. Sansom, Biochem. J. 304 Ž1994. 861–867. w26x C.A. Lunn, V.P. Pigiet, Int. J. Radiat. Biol. 51 Ž1987. 29–38.
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w27x K.U. Schallreuter, J.M. Wood, Biochem. Biophys. Res. Commun. 136 Ž1986. 630–637. w28x A. Spector, G.Z. Yan, R.R. Huang, M.J. McDermott, P.R. Gascoyne, V. Pigiet, J. Biol. Chem. 263 Ž1988. 4984–4990. w29x N. Wakasugi, Y. Tagaya, H. Wakasugi, A. Matsui, M. Maeda, J. Yodoi, T. Tursz, Proc. Natl. Acad. Sci. USA 87 Ž1990. 8282–8286. w30x J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning,
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, 1989. w31x K. Miyazaki, M. Miyata, T. Sugimura, M. Terada, H. Wakasugi, Proc. Jpn. Cancer Association 54th Annu. Meeting, 1995, p. 545. w32x H. Matsumoto, X. Wang, T. Ohnishi, Cancer Lett. 92 Ž1995. 127–133.
Induction of thioredoxin in human lymphocytes with low-dose ionizing radiation Yuko Hoshi a
a,),1
, Hiroshi Tanooka
b,c
, Kunihisa Miyazaki
d,2
, Hiro Wakasugi
d
Nuclear Energy Systems Department, Central Research Institute of Electric Power Industry, Iwado-kita, Komae-shi, Tokyo 201, Japan b Research AdÕisor, Central Research Institute of Electric Power Industry, Iwado-kita, Komae-shi, Tokyo 201, Japan c Genetics DiÕision, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104, Japan d Section of Study of Host Immune Response, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104, Japan Received 17 March 1997; accepted 27 May 1997
Abstract Induction of the expression of the thioredoxin ŽTRX. gene, producing a key protein in regulating cellular functions through redox reaction as well as being a radioprotector, was followed after ionizing irradiation of lymphocytes from human donors. The TRX mRNA level increased to a peak, 5.7-fold higher than the control at maximum, 6 h after irradiation, and then decreased. The optimum radiation dose for enhancement of induction of the TRX mRNA was 0.25 Gy. The TRX protein also increased to a peak, a 3-fold increase at maximum, with the same timing as that for TRX mRNA. q 1997 Elsevier Science B.V. Keywords: Thioredoxin; Induction of gene expression; Ionizing radiation; Low dose
1. Introduction Biological effects of low-level ionizing radiation are now becoming increasingly important from the viewpoint of human health in the environment w1x. At the molecular level, ionizing radiation modulates expression of a number of mammalian genes. These include plasminogen activator gene w2x, proto-
)
Corresponding author. Bio-Science Department Komae Branch, Central Research Institute of Electric Power Industry, 2-11-1, Iwado-kita, Komae-shi, Tokyo 201, Japan. Fax: q81 3 3480 3539; E-mail: [email protected] 1 Present address: Bio-Science Department Komae Branch, Central Research Institute of Electric Power Industry, Iwado-kita, Komae-shi, Tokyo 201, Japan. 2 Present address: Department of Surgery, Jichi Medical School Omiya Medical Center, Omiya, Saitama-ken 330, Japan.
oncogenes w3–5x, and genes controlling cell growth such as cyclin B w6x and p53 w7x, and apoptosis w8x. Involvement of a more numerous variety of radiation-inducible genes controlling cellular functions was shown from analysis of radiation-inducible transcripts w9x. The presence of the responsive element to ionizing radiation w10x and the alteration of transcription factor binding after radiation w11x indicate that ionizing radiation modulates gene expression at the transcription level. The molecular mechanism of these ionizing radiation-inducible events is thought to be related to such radiation-inducible biological phenomena as adaptive response w12x and induced radioresistance w13x, although the molecular data have been obtained with a relatively high radiation dose. On the other hand, the redox status of a number of gene products also regulates various cellular func-
0167-4889r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 8 9 Ž 9 7 . 0 0 0 8 5 - 2
66
Y. Hoshi et al.r Biochimica et Biophysica Acta 1359 (1997) 65–70
tions, such as the DNA binding activity of Jun and Fos and the HAP-1rRef-1 apurinic endonucleaseDNA repair activity w14,15x and the p53 function w16x. Recently, the redoxrDNA repair protein, Ref-1, was shown to be essential for early embryonic development in mice w17x. Thioredoxin ŽTRX. is a key protein which controls cellular redox status. TRX was originally purified from Escherichia coli w18x and shown to be an oxidoreductase enzyme consisting of 104 amino acids w19,20x. The nucleotide sequence of the human TRX gene has been determined w21x and was shown to be identical to that of adult T cell leukemia-derived factor w22,23x, having cytokine activity w24x. Interestingly, TRX is a surface-associated sulfhydryl protein w25x and a radio-protector w26x. TRX reduces hydrogen peroxide and scavenges free radicals w27,28x. Therefore, TRX gene expression potentially can play an important role in cellular functions responding to radiation. It would be interesting to test whether ionizing radiation can induce TRX. In this study, we observed the cellular TRX mRNA and protein levels after ionizing irradiation of human lymphocytes and found that TRX mRNA as well as TRX protein is indeed induced.
2. Materials and methods
Lymphocytes were suspended at 10 6 cellsrml in the medium and kept at 378C for 1 h prior to irradiation. The human B-lymphoblastoid cell line 1G8 w29x, which was immortalized after infection with Epstein-Barr virus Ž EBV., the Jurkat cell line, and lymphocytes from a human donor stimulated for growth of T-cells by interleukin 2 Ž IL-2. and CD3, provided by Dr. Sekine, National Cancer Center, were also used as TRX expression controls. They were incubated in the same medium as described. 2.2. Radiation and chemical treatment Lymphocytes suspended in the culture medium at 10 6 cellsrml were exposed at room temperature to various single doses of g rays generated by a 60 Co source ŽToshiba, Tokyo, Japan. at a dose-rate of 0.72 Gyrmin, as calibrated by a dosimeter ŽFarmer 2570r1B, NE Technology, Reading, UK. , or to Xrays generated by a 150 Kvp generator Ž Model MBR1505R2, Hitachi, Tokyo, Japan. with filters of 0.2 mm Cu and 1.0 mm Al at a dose rate of 0.25 Gyrmin. Irradiated cells were incubated at 378C in humidified 5% CO 2r95% air for various periods of time. Aliquots, containing 10 7 cells each, were used for extraction of RNA and protein. As a positive control, cells suspended at 10 6 cellsrml in the culture medium were treated with 1 ng of 12-O-tetradecanoilphorbol-13-acetate ŽTPA. per ml for various periods of time under the culture conditions.
2.1. Cells 2.3. Measurements of TRX mRNA and TRX protein Fresh human venous blood was obtained from one of the authors, HT Ž donor A. , and was used immediately for experiments after sampling. Other blood samples were obtained from healthy male donors ŽB–E. supplied by the Japan Red Cross Blood Center, Tokyo, and were kept at 48C overnight after separation of serum. Lymphocytes were isolated by centrifugation at 1000 rpm for 30 min in a layer of LYMPHOSEPAL Ž Nippon Gene, Toyama, Japan. . Cells were washed once in phosphate-buffered saline and hemolyzed with hypotonic solution. They were then washed with the cell culture medium, RPMI 1640 ŽNissui, Tokyo, Japan. supplemented with 10% heat-treated fetal calf serum ŽGIBCO BRL; Gaithersburg, MD, USA. , 2 mmol of L-glutamine, 0.2 mmol of sodium pyruvate and 100 units of penicillinrml.
Total cellular RNA was extracted according to protocols of the manufacturer of the ISOGEN kit ŽNippon Gene, Toyama, Japan. , and analyzed according to the method described by Sambrook et al. w30x. Briefly, 10 7 cells for each aliquot were collected by centrifugation Ž 1500 rpm, 10 min., suspended in ISOGEN, containing 10 mM Tris-HCl, pH 7.6, 250 mM NaCl, 0.5% NP-40, and 1 mM phenylmethylsulfonyl fluoride, and then sonicated. RNA was isolated after precipitation with isopropanol. RNA Ž5 m g. was denatured with formaldehyde for 15 min at 658C, and subjected to electrophoresis Ž5 Vrcm. in 1% agarose gel containing 6.6% of formaldehyde. RNA was then transferred to a nitrocellulose membrane ŽNITROPLUS; Micron Separations, Westboro,
Y. Hoshi et al.r Biochimica et Biophysica Acta 1359 91997) 65–70
MA, USA., which was then prehybridized with salmon sperm DNA for 4 h, followed by hybridization for 18–20 h at 458C with a 32 P-labeled probe of the TRX cDNA Ž600 bp. in the hybridization solution. The Northern blots were taken by autoradiography on an X-ray film ŽType AR, KODAK; Rochester, NY, USA. . To quantify TRX mRNA amounts, the blots were taken on an imaging plate and the density was measured by an analyzer Ž Model BAS2000, FUJIX PICTOGRAM; Fuji Film, Tokyo, Japan. . The exposure time was usually overnight for X-ray film and 1 h for the imaging plate. The linear intensity response portion was used to obtain relative TRX mRNA levels. As controls, the 18S ribosomal RNA Ž1900 bp. bands were taken by rehybridization on the same membrane. To extract total proteins, irradiated cells were washed with phosphate-buffered saline and suspended in lysis solution containing 10 mM Tris-HCl, pH 7.6; 250 mM NaCl; 0.5% NP-40 and 1 mM phenylmethylsulfonyl fluoride. Proteins were extracted by freeze-thawing five times followed by sonication. Amounts of TRX proteins in these extracts were measured by the ELISA method w31x, using anti-TRX antibody developed by the National Cancer Center and Immuno-Biological Laboratories ŽFujioka, Gumma-ken, Japan. . Recombinant human TRX ŽIMO, Stockholm, Sweden. was used to obtain the calibration curve.
3. Results Expression of TRX mRNA in human lymphocytes was induced after irradiation of lymphocytes in the culture medium with single radiation doses. Fig. 1 shows Northern blots of TRX mRNA of lymphocytes separated from fresh human blood Ždonor A. following irradiation with 0.25 Gy. The TRX mRNA level increased with time and reached a 5.7-fold higher level after 6 h. Fig. 1 also includes the TRX mRNA in the immortalized human B cell line 1G8, which showed a level 11-fold higher than untreated lymphocytes. As loading controls, the 18S ribosomal RNA was detected in the same membrane filter as that used for the TRX mRNA, which remained unchanged during post-irradiation incubation Ž Fig. 1.. Fig. 2 shows TRX mRNA levels expressed in
67
Fig. 1. Northern blots of thioredoxin ŽTRX. mRNA of human lymphocytes incubated in the culture medium for various times after irradiation with 0.25 Gy. Upper lanes: TRX mRNA Ž600 nucleotides.; lower lanes: 18S ribosomal RNA Ž1900 nucleotides.. Lane 1–4: RNA Žtotal loading: 5 m g. from fresh human lymphocytes at 0, 2, 4 and 6 h after irradiation. Lane 5: RNA Žtotal loading: 10 m g. from the human lymphocyte cell line, 1G8, as a positive control. Detection of 18S ribosomal RNA was undertaken to check for gel loading errors.
various preparations of human lymphocytes. Lymphocytes prepared from fresh venous blood Ž donor A. and the Jurkat cells showed the lowest TRX mRNA level. Lymphocytes stimulated for growth with IL-2 and CD3 exhibited a 3.6-fold higher TRX mRNA level. The highest TRX mRNA expression so far measured was exhibited in the immortalized cell line 1G8. Fig. 3 shows quantitative data of the post-irradiation time course of TRX mRNA and TRX protein levels in lymphocytes taken from different donors. Lymphocytes freshly prepared from venous blood showed the highest response ŽFig. 3A.. The peak of the TRX mRNA expression appeared 6 h after irradiation, with a decline afterwards. Red Cross blood samples ŽFig. 3B–E. also showed the increase of TRX mRNA level with a peak at 6–8 h after irradia-
Fig. 2. TRX mRNA levels in human lymphocytes of various preparations and cell lines. a, lymphocytes from fresh blood of donor A. b, Jurkat cells. c, lymphocytes from fresh blood, stimulated for growth with IL-2 and CD3. d. 1G8 cells. Bar: SEM.
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Fig. 3. Time course of the induction of TRX mRNA Župper column. and TRX protein Žlower column. in human lymphocytes from different donors after irradiation with 0.25 Gy. Age of donors Žall males.: A, 63; B, 43; C, 41; D, 62; E, 30. TRX mRNA levels after treatment with TPA Ž'. and without radiation Ž`. are shown. Bars: SEM.
tion, but the increased level was lower than that of sample from donor A. Lymphocytes treated by a tumor promoter TPA also showed the elevated TRX mRNA level with a peak at 2 h. ŽFig. 3A.. The TRX mRNA level in non-irradiated lymphocytes remained unchanged during incubation Ž Fig. 3A. . The TRX protein level was also tracked after irradiation of the lymphocytes with 0.25 Gy ŽFig. 3, lower columns.. The peak expression was found 6 h after irradiation, just as for mRNA, although its increased level was not so high as the mRNA increase. The TRX protein level in non-irradiated sample A lymphocytes was 85 pgrcell or 20.3 ngrmg of total cellular protein. To see the dose response of the TRX mRNA and
protein induction, lymphocytes were irradiated with various radiation doses and incubated in the culture medium for 6 h ŽFig. 4.. The optimum dose for the TRX mRNA increase was found to be 0.25 Gy most clearly for lymphocytes freshly prepared from donor A. The optimum dose for TRX mRNA and protein appeared to be 0.25–0.5 Gy for the other samples also.
4. Discussion The present study showed that the expression of the TRX gene in human lymphocytes increased with a low dose of ionizing radiation. This increase was
Fig. 4. Dose response of the induction of TRX mRNA Župper column. and TRX protein Žlower column. in human lymphocytes 6 h after irradiation. Donors are the same as shown in Fig. 3. Dotted line: donor E. Bars: SEM.
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most clearly observed in lymphocytes prepared from fresh blood and immediately used. Other samples kept overnight after separation of serum also showed an increase of TRX mRNA and protein after ionizing irradiation, although the expression level and timing varied. Therefore, this increase is thought to occur commonly in human lymphocytes. The optimum radiation dose for TRX induction was 0.25 Gy, which is much lower than doses used for molecular studies reviewed in Section 1. Interestingly, the elevated level of p53 protein is observed in tissue specimens of various organs of mice, with the same optimum dose of 0.25 Gy as that in the present study w32x. The biological consequence of the TRX induction by ionizing radiation is unknown at the time of the present study. However, TRX appears to be a ubiquitous source of reducing potential in mammalian cells and it is now becoming clear from various evidences as stated in Section 1 that the cellular redox status may play an important regulatory role in transcription factor activity and cell cycle control. In this consequence, the TRX induction by ionizing radiation must have some biological effects. This problem will be pursued in a future study.
Acknowledgements We thank Dr. S. Hattori and Mr. K. Ishida, Central Research Institute for Electric Power Industry, and Dr. M. Terada, Director of the National Cancer Research Institute, for their support of this research project, Dr. M.S. Sasaki, Kyoto University, for valuable information, and Dr. D.A. Boothman, University of Wisconsin Medical School, for critical reading of the manuscripts. This work was supported by a 2ndterm Grant-in-Aid for a Comprehensive 10-year Strategy for Cancer Control from the Ministry of Health and Welfare and a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.
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