- Email: [email protected]
Induction of radioresistance by a nitric oxide-mediated bystander effect
International Congress Series 1236 (2002) 295 – 298
Induction of radioresistance by a nitric oxide-mediated bystander effect Hideki Matsumoto a,*, Sachiko Hayashi a, Zhao-Hui Jin a, Masanori Hatashita a, Hiroki Shioura b, Toshio Ohtsubo c, Ryuhei Kitai d, Yoshiya Furusawa e, Osami Yukawa f, Eiichi Kano a a
Department of Exp Radiol and Health Phys, Fukui Med Univ, Fukui, 910-1193, Japan b Department of Radiol, Fukui Med Univ, Fukui, 910-1193, Japan c Department of Otorhinolaryngol, Fukui Med Univ, Fukui, 910-1193, Japan d Department of Neurosurg, Fukui Med Univ, Fukui, 910-1193, Japan e Heavy-Ion Radiobiol Res Gr, Natl Inst Radiol Sci, Chiba, 263-8555, Japan f Division of Biol and Oncol, Natl Inst Radiol Sci, Chiba, 263-8555, Japan
Abstract To elucidate whether nitric oxide excreted from irradiated cells affects cellular radiosensitivity, we examined (a) the accumulation of inducible nitric oxide synthase, p53 and Hsp70, (b) the concentration of nitrite in the medium of cells after irradiation with X-rays, and (c) cellular radiosensitivity using two human glioblastoma cell lines, A-172, having the wild type p53 gene, and transfectant of A-172 cells, A-172/mp53, bearing a mutated p53 gene. Accumulation of inducible nitric oxide synthase was caused by X-ray irradiation in the mutant p53 cells but not in the wild type p53 cells. Accumulation of p53 and Hsp70 was observed in the wild type p53 cells after exposure to the conditioned medium from X-irradiated mutant p53 cells, and the accumulation was abolished by the addition of a specific nitric oxide scavenger to the medium. The radiosensitivity of wild type p53 cells was reduced when the cells were cultured in the conditioned medium from X-irradiated mutant p53 cells, as compared with the conventional fresh growth medium. These findings indicate that one of the possible mechanisms of the radiation-induced bystander effect is an intercellular signal transduction initiated by nitric oxide radicals. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Radioresistance; Nitric oxide; Irradiated cells
1. Background Recently, there have been several reports that unirradiated cells exhibit biological responses when they are cocultivated with irradiated cells [1,2], or exposed to the *
Corresponding author.
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 7 4 5 - 2
296
H. Matsumoto et al. / International Congress Series 1236 (2002) 295–298
conditioned medium (CM) harvested from irradiated cells [3 –5]. This phenomenon has been termed the bystander effect. However, the mechanism of the bystander effect induced by radiation is still unclear. Although it has reported a relationship between nitric oxide (NO) and cellular responses after irradiation in vitro and in vivo [6], the role of NO that is excreted from irradiated cells as part of the stress response caused by exposure to ionizing radiation is still also unclear. We have previously found that the accumulation of stressinduced proteins and thermoresistance in NO-recipient wtp53 cells cocultivated with heatshocked NO-donor mp53 cells, is induced through an intercellular signal transduction pathway initiated by NO [7,8]. In the present study, to elucidate whether NO excreted from irradiated cells affects cellular radiosensitivity, we examined (a) the kinetics of accumulation of inducible nitric oxide synthase (iNOS) after X-irradiation, (b) the kinetics of accumulation of Hsp70 and p53 in non-irradiated NO-recipient cells cocultivated with X-irradiated NO-donor cells, and (c) the modification of radiosensitivity of NO-recipient cells in the CM from X-irradiated NO-donor cells using the human glioblastoma cell lines, A-172 and A-172/mp53.
2. Methods 2.1. Cells Human glioblastoma cell line, A-172, was purchased from the JCRB Cell Bank (Setagaya, Tokyo, Japan). Two human glioblastoma cell lines A-172 and A-172/mp53, which is transfectant of A-172 with the mutant p53Trp248 gene, were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (DMEM-10). 2.2. Irradiation with X-rays Exponentially growing cells were seeded in dishes or in flasks containing DMEM-10 without irradiated feeder cells. Cells were irradiated with X-rays (1.0 –10.0 Gy at 1.0 Gy/ min) in DMEM-10 using High Technical System X-ray (Model HW-150, Hitex, Tokyo, Japan). 2.3. Cocultivation of cells on slide glasses with cells in dishes Exponentially growing cells were seeded in dishes containing DMEM-10 without irradiated feeder cells. At the same time, cells were seeded on slide glasses. The cells in the dishes were irradiated with X-ray. The slide glasses were then transferred into the dishes, and the cocultures were incubated at 37 jC for up to 10 h. 2.4. Western blot analysis Cells were suspended in the RIPA buffer, and then frozen and thawed three times. Aliquots of the supernatants obtained after centrifugation were subjected to Western blotting analysis for iNOS, Hsp70 and p53.
H. Matsumoto et al. / International Congress Series 1236 (2002) 295–298
297
2.5. Measurement of nitrite concentration in medium The nitrite concentration in the medium was measured according to the method of Saltzman [9]. 2.6. Survival curves The surviving cell fraction after irradiation was determined as colony-forming units, and corrected by the plating efficiency of non-treated cells as a control.
3. Results 3.1. Accumulation of iNOS by X-rays After irradiation with X-rays at 2.5 Gy, the accumulation of iNOS was observed in A172/mp53 cells, but not in A-172. In A-172/mp53 cells, the level of iNOS increased gradually after X-irradiation and reached a level about three-fold greater than the control level at 24 h, whereas the level of iNOS in A-172 cells did not increase after X-irradiation. 3.2. Measurement of nitrite concentration in medium The nitrite concentration increased gradually to over 2.0 AM 24 h after X-irradiation in A-172/mp53 cells. The elevation of nitrite concentration in the medium of A-172/mp53 cells was completely suppressed by the addition of 0.1 mM aminoguanidine (AG) as an iNOS inhibitor. In contrast, in the medium of A-172 cells, the nitrite concentration did not change after X-irradiation. 3.3. Accumulation of Hsp70 and p53 in the NO-recipient cells co-cultivated with the NOdonor cells Stress-responsive proteins Hsp70 and p53 were accumulated in non-irradiated A-172 cells cocultivated with X-irradiated A-172/mp53 cells at 5 Gy, and the levels of both proteins reached about two-fold greater than the control level at 10 h. These accumulations were completely abolished by the addition of 0.1 mM AG to the medium. 3.4. Accumulation of Hsp70 and p53 in the NO-recipient cells exposed to CM from the NOdonor cells The CM from mp53 cells was prepared by culturing them for 10 h after irradiation with X-rays at 5.0 Gy with or without c-PTIO. The levels of Hsp70 and p53 in A-172 cells increased to about two-fold greater than the control level at 10 h after exposure to the CM from X-irradiated A-172/mp53 cells without c-PTIO. The accumulation of these proteins was inhibited by the addition of c-PTIO to the CM from X-irradiated A-172/mp53 cells in a dose-dependent manner.
298
H. Matsumoto et al. / International Congress Series 1236 (2002) 295–298
3.5. Radiosensitivity of the NO-recipient cells exposed to the CM of the NO-donor cells A-172 cells in the CM from A-172/mp53 cells cultured for 10 h after X-irradiation at 5.0 Gy (CM-X) were more radioresistant than those in the fresh growth medium (GM), or in the CM from A-172/mp53 cells cultured for 10 h without irradiation (CM-0). In GM, the D0 of the A-172 cells was 1.5 Gy. In CM-0, the radiosensitivity of the A-172 cells was scarcely different: the D0 of the A-172 cells was 1.6 Gy. However, in the CM-X, the D0 of the A-172 cells was 1.9 Gy. The dose modifying factors in D0 were 1.1 and 1.3 for CM-0 and CM-X, respectively. In addition, the values of Dq of A-172 in GM, CM-0 and CM-X were 0.7, 1.0 and 1.4 Gy, respectively. The dose modifying factors in Dq were 1.4 and 2.0 for CM and CM-X, respectively.
4. Conclusions Nitric oxide (NO) excreted from the X-irradiated donor mp53 cells could induce radioresistance in the recipient non-irradiated wtp53 cells. These findings indicate that one of the possible mechanisms of the radiation-induced bystander effect is an intercellular signal transduction initiated by NO radicals [10,11].
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
H. Nagasawa, J.B. Little, Cancer Res. 52 (1992) 6394 – 6396. E.I. Azzam, S.M. de Toledo, T. Gooding, J.B. Little, Radiat. Res. 150 (1998) 497 – 504. C. Mothersill, C. Seymour, Int. J. Radiat. Biol. 71 (1997) 421 – 427. C. Mothersill, C. Seymour, Radiat. Res. 149 (1998) 256 – 262. R. Iyer, B.E. Lehnert, Cancer Res. 60 (2000) 1290 – 1298. W.K. MacNaughton, A.R. Aurora, J. Bhamra, K.A. Sharkey, M.J. Miller, Int. J. Radiat. Biol. 74 (1998) 255 – 264. H. Matsumoto, S. Hayashi, M. Hatashita, H. Shioura, T. Ohtsubo, R. Kitai, T. Ohnishi, E. Kano, Nitric Oxide 3 (1999) 180 – 189. H. Matsumoto, S. Hayashi, M. Hatashita, K. Ohnishi, T. Ohtsubo, R. Kitai, H. Shioura, T. Ohnishi, E. Kano, Cancer Res. 59 (1999) 3239 – 3244. B.E. Saltzman, Anal. Chem. 26 (1954) 1949 – 1955. H. Matsumoto, S. Hayashi, M. Hatashita, K. Ohnishi, T. Ohtsubo, R. Kitai, H. Shioura, T. Ohnishi, Y. Furusawa, O. Yukawa, E. Kano, Int. J. Radiat. Biol. 76 (2000) 1649 – 1657. H. Matsumoto, S. Hayashi, M. Hatashita, T. Ohtsubo, R. Kitai, H. Shioura, T. Ohnishi, E. Kano, Radiat. Res. 155 (2001) 387 – 396.
Induction of radioresistance by a nitric oxide-mediated bystander effect Hideki Matsumoto a,*, Sachiko Hayashi a, Zhao-Hui Jin a, Masanori Hatashita a, Hiroki Shioura b, Toshio Ohtsubo c, Ryuhei Kitai d, Yoshiya Furusawa e, Osami Yukawa f, Eiichi Kano a a
Department of Exp Radiol and Health Phys, Fukui Med Univ, Fukui, 910-1193, Japan b Department of Radiol, Fukui Med Univ, Fukui, 910-1193, Japan c Department of Otorhinolaryngol, Fukui Med Univ, Fukui, 910-1193, Japan d Department of Neurosurg, Fukui Med Univ, Fukui, 910-1193, Japan e Heavy-Ion Radiobiol Res Gr, Natl Inst Radiol Sci, Chiba, 263-8555, Japan f Division of Biol and Oncol, Natl Inst Radiol Sci, Chiba, 263-8555, Japan
Abstract To elucidate whether nitric oxide excreted from irradiated cells affects cellular radiosensitivity, we examined (a) the accumulation of inducible nitric oxide synthase, p53 and Hsp70, (b) the concentration of nitrite in the medium of cells after irradiation with X-rays, and (c) cellular radiosensitivity using two human glioblastoma cell lines, A-172, having the wild type p53 gene, and transfectant of A-172 cells, A-172/mp53, bearing a mutated p53 gene. Accumulation of inducible nitric oxide synthase was caused by X-ray irradiation in the mutant p53 cells but not in the wild type p53 cells. Accumulation of p53 and Hsp70 was observed in the wild type p53 cells after exposure to the conditioned medium from X-irradiated mutant p53 cells, and the accumulation was abolished by the addition of a specific nitric oxide scavenger to the medium. The radiosensitivity of wild type p53 cells was reduced when the cells were cultured in the conditioned medium from X-irradiated mutant p53 cells, as compared with the conventional fresh growth medium. These findings indicate that one of the possible mechanisms of the radiation-induced bystander effect is an intercellular signal transduction initiated by nitric oxide radicals. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Radioresistance; Nitric oxide; Irradiated cells
1. Background Recently, there have been several reports that unirradiated cells exhibit biological responses when they are cocultivated with irradiated cells [1,2], or exposed to the *
Corresponding author.
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 7 4 5 - 2
296
H. Matsumoto et al. / International Congress Series 1236 (2002) 295–298
conditioned medium (CM) harvested from irradiated cells [3 –5]. This phenomenon has been termed the bystander effect. However, the mechanism of the bystander effect induced by radiation is still unclear. Although it has reported a relationship between nitric oxide (NO) and cellular responses after irradiation in vitro and in vivo [6], the role of NO that is excreted from irradiated cells as part of the stress response caused by exposure to ionizing radiation is still also unclear. We have previously found that the accumulation of stressinduced proteins and thermoresistance in NO-recipient wtp53 cells cocultivated with heatshocked NO-donor mp53 cells, is induced through an intercellular signal transduction pathway initiated by NO [7,8]. In the present study, to elucidate whether NO excreted from irradiated cells affects cellular radiosensitivity, we examined (a) the kinetics of accumulation of inducible nitric oxide synthase (iNOS) after X-irradiation, (b) the kinetics of accumulation of Hsp70 and p53 in non-irradiated NO-recipient cells cocultivated with X-irradiated NO-donor cells, and (c) the modification of radiosensitivity of NO-recipient cells in the CM from X-irradiated NO-donor cells using the human glioblastoma cell lines, A-172 and A-172/mp53.
2. Methods 2.1. Cells Human glioblastoma cell line, A-172, was purchased from the JCRB Cell Bank (Setagaya, Tokyo, Japan). Two human glioblastoma cell lines A-172 and A-172/mp53, which is transfectant of A-172 with the mutant p53Trp248 gene, were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (DMEM-10). 2.2. Irradiation with X-rays Exponentially growing cells were seeded in dishes or in flasks containing DMEM-10 without irradiated feeder cells. Cells were irradiated with X-rays (1.0 –10.0 Gy at 1.0 Gy/ min) in DMEM-10 using High Technical System X-ray (Model HW-150, Hitex, Tokyo, Japan). 2.3. Cocultivation of cells on slide glasses with cells in dishes Exponentially growing cells were seeded in dishes containing DMEM-10 without irradiated feeder cells. At the same time, cells were seeded on slide glasses. The cells in the dishes were irradiated with X-ray. The slide glasses were then transferred into the dishes, and the cocultures were incubated at 37 jC for up to 10 h. 2.4. Western blot analysis Cells were suspended in the RIPA buffer, and then frozen and thawed three times. Aliquots of the supernatants obtained after centrifugation were subjected to Western blotting analysis for iNOS, Hsp70 and p53.
H. Matsumoto et al. / International Congress Series 1236 (2002) 295–298
297
2.5. Measurement of nitrite concentration in medium The nitrite concentration in the medium was measured according to the method of Saltzman [9]. 2.6. Survival curves The surviving cell fraction after irradiation was determined as colony-forming units, and corrected by the plating efficiency of non-treated cells as a control.
3. Results 3.1. Accumulation of iNOS by X-rays After irradiation with X-rays at 2.5 Gy, the accumulation of iNOS was observed in A172/mp53 cells, but not in A-172. In A-172/mp53 cells, the level of iNOS increased gradually after X-irradiation and reached a level about three-fold greater than the control level at 24 h, whereas the level of iNOS in A-172 cells did not increase after X-irradiation. 3.2. Measurement of nitrite concentration in medium The nitrite concentration increased gradually to over 2.0 AM 24 h after X-irradiation in A-172/mp53 cells. The elevation of nitrite concentration in the medium of A-172/mp53 cells was completely suppressed by the addition of 0.1 mM aminoguanidine (AG) as an iNOS inhibitor. In contrast, in the medium of A-172 cells, the nitrite concentration did not change after X-irradiation. 3.3. Accumulation of Hsp70 and p53 in the NO-recipient cells co-cultivated with the NOdonor cells Stress-responsive proteins Hsp70 and p53 were accumulated in non-irradiated A-172 cells cocultivated with X-irradiated A-172/mp53 cells at 5 Gy, and the levels of both proteins reached about two-fold greater than the control level at 10 h. These accumulations were completely abolished by the addition of 0.1 mM AG to the medium. 3.4. Accumulation of Hsp70 and p53 in the NO-recipient cells exposed to CM from the NOdonor cells The CM from mp53 cells was prepared by culturing them for 10 h after irradiation with X-rays at 5.0 Gy with or without c-PTIO. The levels of Hsp70 and p53 in A-172 cells increased to about two-fold greater than the control level at 10 h after exposure to the CM from X-irradiated A-172/mp53 cells without c-PTIO. The accumulation of these proteins was inhibited by the addition of c-PTIO to the CM from X-irradiated A-172/mp53 cells in a dose-dependent manner.
298
H. Matsumoto et al. / International Congress Series 1236 (2002) 295–298
3.5. Radiosensitivity of the NO-recipient cells exposed to the CM of the NO-donor cells A-172 cells in the CM from A-172/mp53 cells cultured for 10 h after X-irradiation at 5.0 Gy (CM-X) were more radioresistant than those in the fresh growth medium (GM), or in the CM from A-172/mp53 cells cultured for 10 h without irradiation (CM-0). In GM, the D0 of the A-172 cells was 1.5 Gy. In CM-0, the radiosensitivity of the A-172 cells was scarcely different: the D0 of the A-172 cells was 1.6 Gy. However, in the CM-X, the D0 of the A-172 cells was 1.9 Gy. The dose modifying factors in D0 were 1.1 and 1.3 for CM-0 and CM-X, respectively. In addition, the values of Dq of A-172 in GM, CM-0 and CM-X were 0.7, 1.0 and 1.4 Gy, respectively. The dose modifying factors in Dq were 1.4 and 2.0 for CM and CM-X, respectively.
4. Conclusions Nitric oxide (NO) excreted from the X-irradiated donor mp53 cells could induce radioresistance in the recipient non-irradiated wtp53 cells. These findings indicate that one of the possible mechanisms of the radiation-induced bystander effect is an intercellular signal transduction initiated by NO radicals [10,11].
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
H. Nagasawa, J.B. Little, Cancer Res. 52 (1992) 6394 – 6396. E.I. Azzam, S.M. de Toledo, T. Gooding, J.B. Little, Radiat. Res. 150 (1998) 497 – 504. C. Mothersill, C. Seymour, Int. J. Radiat. Biol. 71 (1997) 421 – 427. C. Mothersill, C. Seymour, Radiat. Res. 149 (1998) 256 – 262. R. Iyer, B.E. Lehnert, Cancer Res. 60 (2000) 1290 – 1298. W.K. MacNaughton, A.R. Aurora, J. Bhamra, K.A. Sharkey, M.J. Miller, Int. J. Radiat. Biol. 74 (1998) 255 – 264. H. Matsumoto, S. Hayashi, M. Hatashita, H. Shioura, T. Ohtsubo, R. Kitai, T. Ohnishi, E. Kano, Nitric Oxide 3 (1999) 180 – 189. H. Matsumoto, S. Hayashi, M. Hatashita, K. Ohnishi, T. Ohtsubo, R. Kitai, H. Shioura, T. Ohnishi, E. Kano, Cancer Res. 59 (1999) 3239 – 3244. B.E. Saltzman, Anal. Chem. 26 (1954) 1949 – 1955. H. Matsumoto, S. Hayashi, M. Hatashita, K. Ohnishi, T. Ohtsubo, R. Kitai, H. Shioura, T. Ohnishi, Y. Furusawa, O. Yukawa, E. Kano, Int. J. Radiat. Biol. 76 (2000) 1649 – 1657. H. Matsumoto, S. Hayashi, M. Hatashita, T. Ohtsubo, R. Kitai, H. Shioura, T. Ohnishi, E. Kano, Radiat. Res. 155 (2001) 387 – 396.