EXPERIMENTAL
CELL
RESEARCH
1%?,635-638
(1991)
SHORT NOTE Stimulation by Xanthine Oxidase of 3T3 Swiss Fibroblasts and Human Lymphocytes FIORENZO STIRPE,’ THERESA HIGGINS, PIER LUIGI TAZZARI,’ AND ENRIQUE ROZENGURT Imperial
Cancer Research Fund, P.O. Box 123, Lincolnk
Inn Fields, London WC2A SPX, linited
Kingdom
and occurred only in the presence of insulin or various other stimulating factors with which xanthine oxidase was synergistic. Lymphocytes were also stimulated by xanthine oxidase in the presence of xanthine and phytohemagglutinin.
Xanthine oxidase stimulates [3H]thymidine incorporation by 3T3 cells even in the absence of any added xanthine, but in the presence of, and synergistically with, insulin or various other growth-stimulating factors. Optimal stimulation was obtained with 2-3 mU enzyme/ml and higher concentrations were toxic. Xanthine oxidase also stimulated human peripheral blood lymphocytes in the presence of phytohemagglutinin and ic) 1991 Academic Press, Inc. xanthine.
MATERIALS
AND METHODS
Materials. Xanthine oxidase (from bovine milk, grade III), superoxide dismutase, catalase, xanthine (sodium salt, cell culture tested), allopurinol, insulin, phorbol 12,13-dibutyrate (PDBu), bombesin, vasopressin, epidermal growth factor (EGF), cholera toxin, and isobutylmethylxanthine were obtained from Sigma; forskolin was from Calbiochem and [mc~thyl-3H]thymidine was from Amersham International. Cell cultures and determination of DNA synthesis. Stock cultures of Swiss 3T3 cells (81 and of Balh/c cells were maintained as described in 191. Cells were subcultured in 33.mm Nunc petri dishes (10s cells/ dish) with 2.5 ml of Dulbecco-Vogt modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and were used 668 days after the last change of medium. Cultures of whole mouse embryos were prepared from mice on the 16th day of pregnancy by standard trypsinization technique. Tertiary cultures of these were plated at lo5 tells/33-mm dish in 10% FBS in DMEM. These were switched to 0.5% FBS in DMEM/Waymouth medium after 3 days and used 4 days later when they were quiescent [lo]. For determination of DNA synthesis, cells were washed twice with DMEM to remove residual serum. The determination of DNA synthesis was performed with 2 ml of a 1:l mixture of DMEM and Waymouth medium containing 1 &f [“Hlthymidine (1 pCi/ml) with the appropriate additions. Cells were incubated for 40 h at 37°C in a humidified atmosphere of 10% CO,/90% air, washed sequentially with PBS, 5% trichloroacetic acid, and 70% ethanol, and dissolved with 1 ml of 0.1 M NaOH containing 2% Na,CO, and 1% SDS. The level of acid precipitable radioactivity was measured in 0.5 ml in a scintillation counter [ 111. Lymphocytes were obtained from heparinized peripheral blood from healthy volunteers. Cells were separated on a Ficoll-Hypaque (Lymphoprep) gradient and were washed in phosphate-buffered saline, pH 7.5. The cells were resuspended in RPM1 1640 medium (GIBCO) with 10% fetal calf serum, glutamine, and antibiotics and were seeded in 96well flat-bottom plates (Sterilin) in the presence of PHA (5 pg/ml) and with the appropriate amounts of xanthine and xanthine oxidase as shown in Table 2. Cells were incubated for 24 h before being pulsed with [3H]thymidine (1 wCi/well) for an additional 24 h. They were then harvested with a Titertek cell harvester on glass
INTRODUCTION Mammalian cells can be stimulated to divide by a variety of factors, many of which act synergistically by activating various signal-transduction pathways (review in [ 11). A growing body of evidence suggests that free radicals or free radical-related species are also involved in tumor promotion [a]. Free radicals produced by the xanthineixanthine oxidase system act as weak promoters of transformation in mouse embryo fibroblasts [3], stimulate DNA synthesis in resting Balb/3T3 fibroblasts [4], and enhance proliferation of human fibroblasts [5] and of BHK uninfected and polyoma virus-infected cells [6]. Moreover, a higher yield of colonies was observed in human bone marrow cultures exposed to xanthineixanthine oxidase ([7] and unpublished observations). In the present experiments we studied the effect of xanthine oxidase on DNA synthesis by 3T3 Swiss cells and human lymphocytes. The enzyme at very low concentrations stimulated DNA synthesis by fibroblasts even in the absence of any added substrate. The stimulation was due to free radicals generated by the enzyme 1 To whom correspondence and reprint requests should be addressed at permanent address: Dipartimento di Patologia sperimentale, Universiti di Bologna, Via S. Giacomo 14, I-40126 Bologna, Italy. ’ Permanent address: Laboratorio di Immunogenetics, Istituto Nazionale per la Ricerca sul Cancro, I-16132, Genova, Italy.
635
0014.4827191
$3.00
Copyright 4 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
636
SHORT
NOTE
0.01 mM 4
Xanthine
XANTHINE
OXIDASE
(mU/ml)
FIG. 2. Effect of allopurinol on the stimulation by xanthine oxidase of [3H]thymidine incorporation by 3T3 Swiss cells. Experimental conditions were the same as those in Fig. 1 with no xanthine, in the absence (0) or in the presence (0) of 0.05 mM allopurinol.
XANTHINE
OXIOASE
(mu/ml)
FIG. 1. Effect of xanthine oxidase on [3H]thymidine incorporation by 3T3 Swiss cells. Experimental conditions are described in the text, without xanthine (0) or in the presence of 0.01 mM (0) or 0.1 mM (A) xanthine.
fiber disks and the insoluble radioactivity was determined lation counter. All experiments were repeated at least once.
in a scintil-
RESULTS
Under the conditions described above, 3T3 cells were arrested in the G,/G, phase, as judged by the very low incorporation of [3H]thymidine. Preliminary experiments, aimed at repeating the results of Shibanuma et al. [4], showed that in the absence of insulin the addition of xanthine oxidase (up to 12.5 mu/ml), both with and without xanthine (1 to 50 PM), did not have any effect on DNA synthesis by 3T3 cells. In the presence of insulin (1 Fg/ml), xanthine oxidase stimulated DNA synthesis even in the absence of any added xanthine and became rapidly toxic when xanthine was present at concentrations above 1 PUM.Essentially similar results (not shown) were obtained when the DMEMfWaymouth medium, which contains traces of hypoxanthine, was replaced by DMEM alone. Optimal stimulation of DNA synthesis was obtained with 2-3 mU of enzyme per milliliter, in the presence of insulin. Higher amounts of enzyme were toxic. The addition of 10 or 100 pM xanthine did not significantly affect the stimulation and actually aggravated the toxicity of xanthine oxidase (Fig. 1). No effect was observed if the enzyme was boiled (results not shown). The stimulation by xanthine oxidase was greatly reduced, but not
completely abolished, by allopurinol (Fig. 2) and by superoxide dismutase and catalase (Table 1). When xanthine oxidase was added to cells together with other growth-stimulating factors, its effect was synergistic with that of PDBu, bombesin (at concentrations giving submaximal stimulation), EGF, cholera toxin, and, to a lesser extent, forskolin, but not vasopressin or isobutylmethylxanthine (Fig. 3). The effect of xanthine oxidase was observed also in cells predepleted of protein kinase C by treatment with PDBu [12] (Fig. 4). Xanthine oxidase (1 to 5 mu/ml), both in the presence and in the absence of xanthine, had very little stimulating effect on DNA synthesis by Balb/c fibroblasts and was only toxic to mouse embryo cells (results not shown). Xanthine oxidase also stimulated human lymphocytes, but to a more limited extent than 3T3 cells, and only in the presence of PHA and of xanthine (Table 2). TABLE Effect of Superoxide Stimulation by Xanthine Cells
1
Dismutase and Oxidase of DNA
Catalase Synthesis
Xanthine
on the by 3T3
oxidase
2 mu/ml ( [3H]Thymidine incorporated; cpm)
None Additions None Fetal calf Insulin Insulin + Insulin + Insulin +
serum SOD catalase SOD + catalase
Note. Experimental conditions are averages of duplicate assays.
4,009 481,360 36,186 35,291 34,876 41,728
93,168 89,285 85,946 57,723
are described
in the text. Figures
SHORT
637
NOTE
TABLE 500
1
N
XANTHINE
OXIDASE
2
Effect of Xanthine Oxidase on DNA Synthesis by Human Peripheral Lymphocytes
(2 mu/ml)
Xanthine
Expt no. 1
2 FIG. 3. Synergistic effect of xanthine oxidase and growth factors on [3H]thymidine incorporation by 3T3 Swiss cells. Additions were at the following concentrations: xanthine oxidase, 2 mu/ml; insulin, 1 Kg/ml; PDBu, 100 rig/ml; bombesin, 1 rig/ml; EGF, 5 rig/ml; cholera toxin, 10 rig/ml, in the presence of 50 PM isobutylmethylxanthine; forskolin, 25 PM.
Xanthine oxidase stimulates DNA synthesis by 3T3 Swiss fibroblasts. This effect is exerted by very low amounts of enzyme, even in the absence of any added xanthine, is abolished by boiling the enzyme, and is greatly reduced by allopurinol and by superoxide dis-
500.
+
XANTHINE
OXIDASE
None 1 2 3 None 1 2 None 0.5 1 1.5
PHA added b.dmU None
5
5
in medium
0.005 mM (Thymidine incorporated, cpm)
None
2442 2205 2058 1464 4533 4682 4067 4384 4907 4485 4057
_t 456 + 284 + 84 k 143 f 202 f 304 f 265 f 229 t 361 + 176 +- 124
2433 2357 2233 1655 4251 6039 4634 3757 4405 3862 5595
zk 193 f 283 f 72 I~I 121 + 194 f 29 t 897 f 447 f 13 f 199 t 214
Note. Experimental conditions are described in the text. Figures are averages I SD of triplicate determinations. Lymphocytes were 105/well in experiment 1 and were 2 X lO’/well in experiments 2 and 3. In experiments 2 and 3 incorporation in the absence of PHA was t300 cpm.
DISCUSSION
Oh3
3
Xanthine oxidase added (mu/ml)
(mU/ml)
FIG. 4. Effect of xanthine oxidase on 13H]thymidine incorporation by normal (0) and protein kinase C-depleted (0) 3T3 Swiss cells. Cells were depleted of protein kinase C by 3 days preincubation in DMEM containing 10% conditioned medium and PDBu (400 rig/ml) and were then switched to the medium for DNA synthesis, in the presence of PDBu (400 rig/ml).
mutase plus catalase, but not by either enzyme alone. This indicates that the stimulation is due to free radicals and to hydrogen peroxide produced by the enzyme, presumably utilizing hypoxanthine or another substrate(s) produced by the cells (xanthine oxidase is an unspecific enzyme which acts on a variety of substrates [13]). The residual stimulation in the presence of the substrate-inhibitor allopurinol is probably due to the small amount of free radicals produced when allopurino1 is oxidized to oxypurinol by xanthine oxidase, as pointed out by Spector [14]. Xanthine oxidase exerts its effect only in the presence of insulin, PDBu, or other stimulating factors with which the enzyme appears to act synergistically. Phorbol esters produce free radicals [ 151 and stimulate protein kinase C [ 161. Stimulation by xanthine oxidase occurs in protein kinase C-deprived cells, and is actually greater than that in normal cells. Thus the present results indicate that free radicals may be formed and act after, rather than before, the PDBu-dependent activation of protein kinase C, as suggested by Shibanuma et al. [17]. The reason for the greater stimulation is unknown: possibly the fact that PDBu had already acted upon protein kinase C facilitates the action of xanthine oxidase. Xanthine oxidase has also a mild stimulatory effect on human blood lymphocytes which is consistent with the action of other oxidizing agents [IS]. The addition of xanthine is required, probably because the amount produced by these cells, fewer than fibroblasts, is not sufficient. Stimulation occurs in the presence of, and syner-
638
SHORT
gistically with, PHA. Together and consistently with the effect on 3T3 cells, these results indicate that only cells “primed” to divide are affected by free radicals. The stimulating activity of xanthine oxidase is seen within a very narrow range of enzyme concentration, is very scarce on Balb/c cells, and does not occur at all on mouse embryo cells, to which the enzyme is toxic. This suggests that the stimulation occurs under a critical set of conditions, which may be different for different types of cells. The conversion of xanthine oxidase from its dehydrogenase (D form) into its oxidase (0 form), either irreversibly by limited proteolysis [ 191 or reversibly by oxidation of its sulfhydryl groups [20], was reported to occur in hypoxic or necrotic tissues due to hypoxia and/or proteolysis, leading to production of free radicals which would aggravate tissue damage [21]. In the light of previous [4,5] and present results, one could envisage that xanthine oxidase-generated radicals, besides damaging tissues, could stimulate cell multiplication, the balance between damage and stimulation probably being regulated by the extent of radical production. Thus xanthine oxidase-produced free radicals could have a role in the postnecrotic repair mechanism and also in the pathogenesis of fibrotic conditions, as postulated in the case of Dupuytren’s contracture [22]. REFERENCES 1. 2.
Rozengurt, E. (1989) Brit. Med. Bull. 45, 515-528. Borek, C., and Troll, W. (1983) Proc. NC&. Acud. Sci. USA 81, 1304-1307.
Received April 25, 1990 Revised version received September
4, 1990
NOTE 3. 4. t5 6. 7.
Zimmerman, R., and Cerutti, P. (1984) Proc. N&l. Acad. Sci. USA 81,2085-2087. Shibanuma, M., Kuroki, T., and Nose, K. (1988) Oncogene 3, 17-21. Murrell, G. A. C.. Francis, M. J. O., and Bromley, L. (1990) Biothem. J. 265, 659-665. Burdon, R. H., and Rice-Evans, C. (1989) Free Radicals Res. Commun. 6,345~358. Tazzari, P. L., Battelli, M. G., Abbondanza, A., Dinota, A., Rizzi, S., Gobbi, M., and Stirpe, F. (1989) Transplantation 48, 119122.
8. Rozengurt, E., and Heppel, L. (1975) Proc. Natl. Acad. Sci. USA 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
72,4492-4495. Rozengurt, E., and Sinnett-Smith, J. (1983) Proc. Natl. Acad. Sci. USA 80, 2936-2940. Rozengurt, E., Mierzejewski, K., and Wigglesworth, N. (1978) J. Cell. Physiol. 97, 241-252. Dicker, P., and Rozengurt, E. (1980) Nature (London) 287,607612. Rodriguez-Pena, A., and Rozengurt, E. (1984) Biochem. Biophys. Res. Commun. 120, 1053-1059. Parks, D. A., and Granger, D. N. (1986) Acta Physiol. Sand. Suppl. 548,87-99. Spector, T. (1988) Biochem. Pharmucol. 37,349-352. Copeland, E. S. (1983) Cancer Res. 43, 5631-5637. Fischer, S. M., and Adams, L. M. (1985) Cancer Res. 45, 31303136. Shibanuma, M., Kuroki, T., and Nose, K. (1987) Biochem. Biophys. Kes. Commun. 144, 1317-1323. O’Brien, R. I,., and Parker, J. W. (1976) Cell 7, 13-20. Stirpe, F., and Della Corte, E. (1969) J. Biol. Chem. 244, 38553863. Della Corte, E., and Stirpe, F. (1972) Biochem. J. 126, 739-745. Granger, D. N., Hiillwarth, M. E., and Parks, D. A. (1986) Actu Physiol. Scund. Suppl. 548, 47-63. Murrell, G. A. C., Francis, M. J. O., and Bromley, L. (1987) Brit. Med. J. 295, 1373&1375.
CELL
RESEARCH
1%?,635-638
(1991)
SHORT NOTE Stimulation by Xanthine Oxidase of 3T3 Swiss Fibroblasts and Human Lymphocytes FIORENZO STIRPE,’ THERESA HIGGINS, PIER LUIGI TAZZARI,’ AND ENRIQUE ROZENGURT Imperial
Cancer Research Fund, P.O. Box 123, Lincolnk
Inn Fields, London WC2A SPX, linited
Kingdom
and occurred only in the presence of insulin or various other stimulating factors with which xanthine oxidase was synergistic. Lymphocytes were also stimulated by xanthine oxidase in the presence of xanthine and phytohemagglutinin.
Xanthine oxidase stimulates [3H]thymidine incorporation by 3T3 cells even in the absence of any added xanthine, but in the presence of, and synergistically with, insulin or various other growth-stimulating factors. Optimal stimulation was obtained with 2-3 mU enzyme/ml and higher concentrations were toxic. Xanthine oxidase also stimulated human peripheral blood lymphocytes in the presence of phytohemagglutinin and ic) 1991 Academic Press, Inc. xanthine.
MATERIALS
AND METHODS
Materials. Xanthine oxidase (from bovine milk, grade III), superoxide dismutase, catalase, xanthine (sodium salt, cell culture tested), allopurinol, insulin, phorbol 12,13-dibutyrate (PDBu), bombesin, vasopressin, epidermal growth factor (EGF), cholera toxin, and isobutylmethylxanthine were obtained from Sigma; forskolin was from Calbiochem and [mc~thyl-3H]thymidine was from Amersham International. Cell cultures and determination of DNA synthesis. Stock cultures of Swiss 3T3 cells (81 and of Balh/c cells were maintained as described in 191. Cells were subcultured in 33.mm Nunc petri dishes (10s cells/ dish) with 2.5 ml of Dulbecco-Vogt modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and were used 668 days after the last change of medium. Cultures of whole mouse embryos were prepared from mice on the 16th day of pregnancy by standard trypsinization technique. Tertiary cultures of these were plated at lo5 tells/33-mm dish in 10% FBS in DMEM. These were switched to 0.5% FBS in DMEM/Waymouth medium after 3 days and used 4 days later when they were quiescent [lo]. For determination of DNA synthesis, cells were washed twice with DMEM to remove residual serum. The determination of DNA synthesis was performed with 2 ml of a 1:l mixture of DMEM and Waymouth medium containing 1 &f [“Hlthymidine (1 pCi/ml) with the appropriate additions. Cells were incubated for 40 h at 37°C in a humidified atmosphere of 10% CO,/90% air, washed sequentially with PBS, 5% trichloroacetic acid, and 70% ethanol, and dissolved with 1 ml of 0.1 M NaOH containing 2% Na,CO, and 1% SDS. The level of acid precipitable radioactivity was measured in 0.5 ml in a scintillation counter [ 111. Lymphocytes were obtained from heparinized peripheral blood from healthy volunteers. Cells were separated on a Ficoll-Hypaque (Lymphoprep) gradient and were washed in phosphate-buffered saline, pH 7.5. The cells were resuspended in RPM1 1640 medium (GIBCO) with 10% fetal calf serum, glutamine, and antibiotics and were seeded in 96well flat-bottom plates (Sterilin) in the presence of PHA (5 pg/ml) and with the appropriate amounts of xanthine and xanthine oxidase as shown in Table 2. Cells were incubated for 24 h before being pulsed with [3H]thymidine (1 wCi/well) for an additional 24 h. They were then harvested with a Titertek cell harvester on glass
INTRODUCTION Mammalian cells can be stimulated to divide by a variety of factors, many of which act synergistically by activating various signal-transduction pathways (review in [ 11). A growing body of evidence suggests that free radicals or free radical-related species are also involved in tumor promotion [a]. Free radicals produced by the xanthineixanthine oxidase system act as weak promoters of transformation in mouse embryo fibroblasts [3], stimulate DNA synthesis in resting Balb/3T3 fibroblasts [4], and enhance proliferation of human fibroblasts [5] and of BHK uninfected and polyoma virus-infected cells [6]. Moreover, a higher yield of colonies was observed in human bone marrow cultures exposed to xanthineixanthine oxidase ([7] and unpublished observations). In the present experiments we studied the effect of xanthine oxidase on DNA synthesis by 3T3 Swiss cells and human lymphocytes. The enzyme at very low concentrations stimulated DNA synthesis by fibroblasts even in the absence of any added substrate. The stimulation was due to free radicals generated by the enzyme 1 To whom correspondence and reprint requests should be addressed at permanent address: Dipartimento di Patologia sperimentale, Universiti di Bologna, Via S. Giacomo 14, I-40126 Bologna, Italy. ’ Permanent address: Laboratorio di Immunogenetics, Istituto Nazionale per la Ricerca sul Cancro, I-16132, Genova, Italy.
635
0014.4827191
$3.00
Copyright 4 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
636
SHORT
NOTE
0.01 mM 4
Xanthine
XANTHINE
OXIDASE
(mU/ml)
FIG. 2. Effect of allopurinol on the stimulation by xanthine oxidase of [3H]thymidine incorporation by 3T3 Swiss cells. Experimental conditions were the same as those in Fig. 1 with no xanthine, in the absence (0) or in the presence (0) of 0.05 mM allopurinol.
XANTHINE
OXIOASE
(mu/ml)
FIG. 1. Effect of xanthine oxidase on [3H]thymidine incorporation by 3T3 Swiss cells. Experimental conditions are described in the text, without xanthine (0) or in the presence of 0.01 mM (0) or 0.1 mM (A) xanthine.
fiber disks and the insoluble radioactivity was determined lation counter. All experiments were repeated at least once.
in a scintil-
RESULTS
Under the conditions described above, 3T3 cells were arrested in the G,/G, phase, as judged by the very low incorporation of [3H]thymidine. Preliminary experiments, aimed at repeating the results of Shibanuma et al. [4], showed that in the absence of insulin the addition of xanthine oxidase (up to 12.5 mu/ml), both with and without xanthine (1 to 50 PM), did not have any effect on DNA synthesis by 3T3 cells. In the presence of insulin (1 Fg/ml), xanthine oxidase stimulated DNA synthesis even in the absence of any added xanthine and became rapidly toxic when xanthine was present at concentrations above 1 PUM.Essentially similar results (not shown) were obtained when the DMEMfWaymouth medium, which contains traces of hypoxanthine, was replaced by DMEM alone. Optimal stimulation of DNA synthesis was obtained with 2-3 mU of enzyme per milliliter, in the presence of insulin. Higher amounts of enzyme were toxic. The addition of 10 or 100 pM xanthine did not significantly affect the stimulation and actually aggravated the toxicity of xanthine oxidase (Fig. 1). No effect was observed if the enzyme was boiled (results not shown). The stimulation by xanthine oxidase was greatly reduced, but not
completely abolished, by allopurinol (Fig. 2) and by superoxide dismutase and catalase (Table 1). When xanthine oxidase was added to cells together with other growth-stimulating factors, its effect was synergistic with that of PDBu, bombesin (at concentrations giving submaximal stimulation), EGF, cholera toxin, and, to a lesser extent, forskolin, but not vasopressin or isobutylmethylxanthine (Fig. 3). The effect of xanthine oxidase was observed also in cells predepleted of protein kinase C by treatment with PDBu [12] (Fig. 4). Xanthine oxidase (1 to 5 mu/ml), both in the presence and in the absence of xanthine, had very little stimulating effect on DNA synthesis by Balb/c fibroblasts and was only toxic to mouse embryo cells (results not shown). Xanthine oxidase also stimulated human lymphocytes, but to a more limited extent than 3T3 cells, and only in the presence of PHA and of xanthine (Table 2). TABLE Effect of Superoxide Stimulation by Xanthine Cells
1
Dismutase and Oxidase of DNA
Catalase Synthesis
Xanthine
on the by 3T3
oxidase
2 mu/ml ( [3H]Thymidine incorporated; cpm)
None Additions None Fetal calf Insulin Insulin + Insulin + Insulin +
serum SOD catalase SOD + catalase
Note. Experimental conditions are averages of duplicate assays.
4,009 481,360 36,186 35,291 34,876 41,728
93,168 89,285 85,946 57,723
are described
in the text. Figures
SHORT
637
NOTE
TABLE 500
1
N
XANTHINE
OXIDASE
2
Effect of Xanthine Oxidase on DNA Synthesis by Human Peripheral Lymphocytes
(2 mu/ml)
Xanthine
Expt no. 1
2 FIG. 3. Synergistic effect of xanthine oxidase and growth factors on [3H]thymidine incorporation by 3T3 Swiss cells. Additions were at the following concentrations: xanthine oxidase, 2 mu/ml; insulin, 1 Kg/ml; PDBu, 100 rig/ml; bombesin, 1 rig/ml; EGF, 5 rig/ml; cholera toxin, 10 rig/ml, in the presence of 50 PM isobutylmethylxanthine; forskolin, 25 PM.
Xanthine oxidase stimulates DNA synthesis by 3T3 Swiss fibroblasts. This effect is exerted by very low amounts of enzyme, even in the absence of any added xanthine, is abolished by boiling the enzyme, and is greatly reduced by allopurinol and by superoxide dis-
500.
+
XANTHINE
OXIDASE
None 1 2 3 None 1 2 None 0.5 1 1.5
PHA added b.dmU None
5
5
in medium
0.005 mM (Thymidine incorporated, cpm)
None
2442 2205 2058 1464 4533 4682 4067 4384 4907 4485 4057
_t 456 + 284 + 84 k 143 f 202 f 304 f 265 f 229 t 361 + 176 +- 124
2433 2357 2233 1655 4251 6039 4634 3757 4405 3862 5595
zk 193 f 283 f 72 I~I 121 + 194 f 29 t 897 f 447 f 13 f 199 t 214
Note. Experimental conditions are described in the text. Figures are averages I SD of triplicate determinations. Lymphocytes were 105/well in experiment 1 and were 2 X lO’/well in experiments 2 and 3. In experiments 2 and 3 incorporation in the absence of PHA was t300 cpm.
DISCUSSION
Oh3
3
Xanthine oxidase added (mu/ml)
(mU/ml)
FIG. 4. Effect of xanthine oxidase on 13H]thymidine incorporation by normal (0) and protein kinase C-depleted (0) 3T3 Swiss cells. Cells were depleted of protein kinase C by 3 days preincubation in DMEM containing 10% conditioned medium and PDBu (400 rig/ml) and were then switched to the medium for DNA synthesis, in the presence of PDBu (400 rig/ml).
mutase plus catalase, but not by either enzyme alone. This indicates that the stimulation is due to free radicals and to hydrogen peroxide produced by the enzyme, presumably utilizing hypoxanthine or another substrate(s) produced by the cells (xanthine oxidase is an unspecific enzyme which acts on a variety of substrates [13]). The residual stimulation in the presence of the substrate-inhibitor allopurinol is probably due to the small amount of free radicals produced when allopurino1 is oxidized to oxypurinol by xanthine oxidase, as pointed out by Spector [14]. Xanthine oxidase exerts its effect only in the presence of insulin, PDBu, or other stimulating factors with which the enzyme appears to act synergistically. Phorbol esters produce free radicals [ 151 and stimulate protein kinase C [ 161. Stimulation by xanthine oxidase occurs in protein kinase C-deprived cells, and is actually greater than that in normal cells. Thus the present results indicate that free radicals may be formed and act after, rather than before, the PDBu-dependent activation of protein kinase C, as suggested by Shibanuma et al. [17]. The reason for the greater stimulation is unknown: possibly the fact that PDBu had already acted upon protein kinase C facilitates the action of xanthine oxidase. Xanthine oxidase has also a mild stimulatory effect on human blood lymphocytes which is consistent with the action of other oxidizing agents [IS]. The addition of xanthine is required, probably because the amount produced by these cells, fewer than fibroblasts, is not sufficient. Stimulation occurs in the presence of, and syner-
638
SHORT
gistically with, PHA. Together and consistently with the effect on 3T3 cells, these results indicate that only cells “primed” to divide are affected by free radicals. The stimulating activity of xanthine oxidase is seen within a very narrow range of enzyme concentration, is very scarce on Balb/c cells, and does not occur at all on mouse embryo cells, to which the enzyme is toxic. This suggests that the stimulation occurs under a critical set of conditions, which may be different for different types of cells. The conversion of xanthine oxidase from its dehydrogenase (D form) into its oxidase (0 form), either irreversibly by limited proteolysis [ 191 or reversibly by oxidation of its sulfhydryl groups [20], was reported to occur in hypoxic or necrotic tissues due to hypoxia and/or proteolysis, leading to production of free radicals which would aggravate tissue damage [21]. In the light of previous [4,5] and present results, one could envisage that xanthine oxidase-generated radicals, besides damaging tissues, could stimulate cell multiplication, the balance between damage and stimulation probably being regulated by the extent of radical production. Thus xanthine oxidase-produced free radicals could have a role in the postnecrotic repair mechanism and also in the pathogenesis of fibrotic conditions, as postulated in the case of Dupuytren’s contracture [22]. REFERENCES 1. 2.
Rozengurt, E. (1989) Brit. Med. Bull. 45, 515-528. Borek, C., and Troll, W. (1983) Proc. NC&. Acud. Sci. USA 81, 1304-1307.
Received April 25, 1990 Revised version received September
4, 1990
NOTE 3. 4. t5 6. 7.
Zimmerman, R., and Cerutti, P. (1984) Proc. N&l. Acad. Sci. USA 81,2085-2087. Shibanuma, M., Kuroki, T., and Nose, K. (1988) Oncogene 3, 17-21. Murrell, G. A. C.. Francis, M. J. O., and Bromley, L. (1990) Biothem. J. 265, 659-665. Burdon, R. H., and Rice-Evans, C. (1989) Free Radicals Res. Commun. 6,345~358. Tazzari, P. L., Battelli, M. G., Abbondanza, A., Dinota, A., Rizzi, S., Gobbi, M., and Stirpe, F. (1989) Transplantation 48, 119122.
8. Rozengurt, E., and Heppel, L. (1975) Proc. Natl. Acad. Sci. USA 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
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