EXPERIMENTALCELLRESEARCH
187,177-179
(1990)
SHORT NOTE Glutathione Content and Growth in A549 Human Lung Carcinoma Cells Yu-JIAN Department
KANG AND M. DUANE ENGER’
of Zoology, Iowa State University,
Ames, Iowa 50011
growth-related change in cellular glutathione levels suggests the possibility that glutathione may be involved in the regulation of cell growth. In the present work, the effect of L-buthionine-SRsulfoximine (BSO), a specific inhibitor of y-glutamylcysteine synthetase [ll], on the growth of A549 human lung carcinoma cells and the relationship between initial cellular levels of glutathione and cell growth have been studied. The results obtained demonstrate that subcultured A549 cells with lower initial glutathione levels grow with a longer lag phase, and that decreasing cellular glutathione levels with BSO inhibits cell growth.
The relationship between glutathione content and cell growth was investigated in A549 human lung carcinoma cells. A decreased cellular glutathione content was achieved by exposing the cells to L-buthionine-SRsulfoximine (BSO). It also occurred in these cells as they approached their plateau phase of growth. During exponential growth, a lower initial glutathione content correlated with a longer lag phase in subcultured cells. Further, depletion of cellular glutathione by BSO inhibited cell growth. This inhibition became apparent 36 h after the addition of BSO. These observations raise the possibility that a critical concentration of GSH may be required for optimal growth of A549 human lung carcinoma cells. 0 1990 Academic Press, Inc.
MATERIALS
INTRODUCTION Glutathione (GSH)’ is known to play a significant role in a number of important cellular processes including metabolism, transport, enzyme activity, structural integrity, synthesis of macromolecules, and protection [l31. There is as yet, however, no definition of the role or function of glutathione in regulating or modulating cell growth. Cellular levels of glutathione differ dramatically among cells of differing origins [l, 41. Even in the same type of cells, glutathione levels change as a function of cell culture conditions and cell growth stages [5-lo]. Studies on human lung carcinoma-derived A549 cells showed that cellular levels of glutathione change as a function of growth phase. The highest level of glutathione was observed to occur during the early portion of the exponential phase of growth and the lowest level was found to exist in plateau phase [5], a phenomenon similar to that reported to occur in other cells [4,7-lo]. This i To whom correspondence and reprint requests should be addressed at Department of Zoology, Iowa State University, 339 Science II, Ames, IA 50011. s Abbreviations used: BSO, L-buthionine-SR-sulfoximine; GSH, glutathione; GSSG, glutathione disulfide; PBS, phosphate-buffered saline; DTNB, 5,5’-dithiobis(2-nitrobenzoate).
AND
METHODS
Materials. Glutathione, GSSG reductase, NADPH, L-buthionineSR-sulfoximine, and 5,5’-dithiobis(2-nitrobenzoate) (DTNB) were obtained from Sigma Chemical Co. (St. Louis, MO). McCoy’s 5A medium was purchased from GIBCO (Grand Island, NY), and fetal calf serum from HyClone (Logan, UT). Cell culture. Human lung carcinoma-derived line A549 cells were routinely grown in McCoy’s 5A medium supplemented with 10% fetal calf serum, 2.2 g/liter of sodium bicarbonate, and antibiotics at 37°C and pH 7.0-7.2 in a humidified atmosphere of 95% air and 5% CO,. Stock cultures were passaged at 4-day intervals. Tests for mycoplasma were negative. Cells were removed from monolayer stock cultures with trypsin-EDTA, counted with a Coulter counter, and plated in 60-mm round cell culture dishes at 0.5 X 10s cells/dish or in 35-mm dishes at 0.5 X lo5 cells/dish. Cell growth determination. Cell growth was followed by determining the total number of cells in each culture dish at 12-h intervals. At the end of each interval, the monolayer culture was washed with warm PBS, trypsinized, and diluted with an exact volume of medium. The resulting cell suspension was further diluted with Isoton II saline solution prior to determination of cell number in a Coulter counter. Determination of total cellular GSH levekr. The DTNB-GSSG reductase recycling assay [ 121 was used to determine total cellular glutathione content. Briefly, the cells were trypsinized, rinsed with cold PBS, centrifuged at 4”C, resuspended in cold 0.6% sulfosalicylic acid, mixed vigorously with a “vortex” apparatus, and recentrifuged. The supernatant was then assayed for total glutathione by measuring the color change of DTNB at 412 nm in the presence of GSSG reductase and NADPH.
RESULTS
AND
DISCUSSION
Cellular glutathione content was decreased to 20% of that of control when A549 cells were exposed to 10 mM 177
0014-4827/90
$3.09
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
178
SHORT
NOTE
TABLE
1
Relationship between Initial GSH Content and Rates of Cell Growth for Subcultures” Hours in culture 24 48 12
96
GSH (nmol/106
cells) *
35.4 + 8.6d 30.7 + 4.6
19.4 f 0.6 15.2 + 0.8
Cell number (X105)’ 1.33 f 0.04 0.97 iz 0.06 0.70 + 0.05 0.60 f 0.02
a Cells, cultured for different times (24, 48, 72, and 96 h), were reseeded in 35mm dishes at 0.5 X lo5 cells/dish and subcultured for 36 h. * Initial GSH content at subculture time 0. ’ Cell numbers at the end of 36-h subculture. d Data are mean + SD from triplicate dishes.
2 1
,
0
12
24
/
36
I
40 60 72 Tm ( Hours )
64
96
106
120
FIG. 1. Effects of BSO on the growth of A549-T27 cells subcultured from a 36-h culture. Ten millimolar BSO was added 0 (A), 12 (O), or 24 (0) h after subculture. The inserted panel shows the effect of BSO (A) on the growth of cells from a 96-h culture to which BSO was added at 0 time. Open circles (0) represent untreated, control cell numbers and the solid circles (0) represent control as well as treated cell numbers during the period of no differential growth in control and treated samples.
BSO for 24 h. A further decrease to 5% or less of control GSH levels was achieved after 36 h. As shown in Fig. 1, cells that were exposed to 10 mM BSO at different times following subculture exhibited growth inhibition within 36 h. These results are consistent with the possibility that reduced GSH content inhibits cell growth. However, it is also possible that BSO exerts a toxic effect that results in reduced growth. Although BSO at low concentrations is reportedly a specific inhibitor of GSH synthesis, concentrations comparable to that used in these experiments inhibit cystine uptake in A549 cells [13] and transport of y-glutamyl amino acids in mouse kidneys [ 141, suggesting that cellular functions other than those affecting GSH status may be affected by BSO. When exponential-phase A549 cells were subcultured at 24-h intervals, the subcultures showed differential rates of growth that correlate with the initial glutathione levels of these cells (Table l), i.e., cells with higher levels of glutathione showed a more rapid resumption of growth as indicated by the number of cells existing 36 h after subculture. This result accords well with the report of Murata and Kimura [ 151 that yeast mutants deficient in glutathione synthesis have an extremely long generation time. To further define the differences in cell growth patterns that occur when cultures are established at different initial glutathione levels, the growth of subcultures established from early (36-h) or late (96-h) log-phase cells were monitored at 12-h intervals for 120 h. As shown in Fig. 2, these subcultures differed primarily in
the length of their lag phase. Cells with higher levels of glutathione showed a shorter lag phase and vice versa. This correlation is consistent with the possibility that a certain level of GSH is required for cell growth. There are of course numerous other changes that occur during logarithmic growth that may affect the length of the lag period in subcultures. Exploring possible mechanisms by which [GSH] may affect growth will be important in determining whether a causal relationship indeed exists. It is well known that ribonucleotide reductase is required in all cells to produce deoxyribonucleotides. This essential enzyme for DNA synthesis depends on thioredoxin or glutaredoxin as hydrogen donors [ 16,171. Some mutants of Escherichia coli depend on the glutaredoxinglutathione system as a hydrogen donor for ribonucleotide reduction [la-201. Should this be the case in A549 cells, inhibition of glutathione synthesis could result in corresponding inhibition of DNA synthesis. The result would be much the same as that which results from the 30
Time
( Hours )
FIG. 2. Comparison of cell growth in cells subcultured from 36-h (0) and from 96-h (0) cultures. Cell growth is expressed as the ratio of the number of growing cells in the subculture to those seeded. Data are presented as means + SDS.
SHORT
treatment of cells with hydroxyurea which, by inhibiting ribonucleotide reductase, inhibits DNA synthesis and arrests cells in the early S phase of cell cycle [21]. As shown in Fig. 1, there is about a 36-h delay after BSO addition for growth inhibition to become apparent. This length of time corresponds roughly to that necessary to deplete cellular glutathione to its lowest level. One would thus expect that this level is not adequate for efficient DNA synthesis if indeed the cells depend on the glutaredoxin system for DNA synthesis. Such may be the case in murine mammary carcinoma cells [22], in which depletion of glutathione by BSO inhibits DNA synthesis and arrests cell growth. In addition to possibly being requisite for DNA synthesis, an adequate glutathione level is necessary for efficient protein synthesis in a variety of cells [23-261. This may provide another explanation for the results obtained in this study. Further studies are required to delineate the mechanism(s) involved in growth inhibition mediated by BSO and to determine whether the observed correlation between GSH content and cell growth reflects a causal relationship. This work was supported by NIEHS/PHS Grant ES03863-02 and by Iowa State University. We thank Mary Nims for manuscript preparation.
REFERENCES 1. 2. 3. 4.
Kosower, N. S., and Kosower, E. M. (1978) Znt. Reu. Cytol. 54, 109. Meister, A., and Anderson, M. E. (1983) Annu. Reu. Biochem. 52,711. Ketterer, B. (1982) Drug Metub. Reu. 13, 161. Allalunis-Turner, M. J., Lee, F. Y. F., and Siemann, D. W. (1988) Cancer Res. 48,3657.
Received August 15,1989 Revised version received November
13,1989
179
NOTE 5.
Post, G. B., Keller, D. A., Connor, K. A., and Menzel, D. B. (1983) Biochem. Biophys. Rex Commun. 114,737.
6.
Meredith,
7.
Harris, J. W., and Teng, S. S. (1973) J. Cell. Physiol. 81,91.
8.
Harris, J. W., Painter, R. B., and Hahn, G. M. (1969) Znt. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 15,289.
M. J. (1986) Cell Biol. Toxicol. 2,495.
9. Harris, J. W., and Patt, H. M. (1969) Exp. Cell Res. 56, 134. 10.
Ohara, H., and Terasima,
11.
Griffith,
12.
Tietze, F. (1969) Anal. Biochem. 27,502.
13.
Brodie, A. E., and Reed, D. J. (1985) Tonicol. Appl. Pharmacol. 77,381.
14.
Griffith, 0. W., Bridges, R. J., and Meister, Acad. Sci. USA 76,6319.
T. (1969) Exp. Cell Res. 58, 182.
0. W., and Meister,
A. (1979) J. Biol. Chem. 254, 7558.
A. (1979) Proc. Natl.
15.
Murata,
16.
Holmgren,
A. (1988) Biochem. Sot. Trans. 16,95.
17.
Holmgren,
A. (1985) Annu. Reu. Biochem. 54,237.
18.
Holmgren,
A. (1976) Proc. N&l. Acad. Sci. USA 73,2275.
19.
Mark, D. F., and Richardson, USA 73,780.
20.
Holmgren, A., Ohlsson, I., and Grankvist, Chem. 253,430.
21.
Meyn, R. E., Hewitt, R. R., and Humphrey, R. M. (1975) in Methods in Cell Biology (Prescott, D. M., Ed.), Vol. IX, Academic Press, New York.
22.
Dethlefsen, L. A., Biaglow, J. E., Peck, V. M., and Ridinger, (1986) Znt. J. Radiat. Oncol. Biol. Phys. 12,1157.
D. N.
23.
Wax, R., Rosenberg, E., Kosower, (1970) J. Bacterial. 101, 1092.
E. M.
24.
Rossman, T., Norris, C., and Troll, W. (1974) J. Biol. Chem. 249, 3412.
25.
Kosower, N. S., Kosower, J. Biochem. 77,529.
26.
Hibberd, K. A., Berget, P. B., Werner, (1978) J. Bucteriol. 133, 1150.
K., and Kimura,
A. (1986) Agric. Biol. Chem. 50, 1055.
C. C. (1976) Proc. N&l. Acad. Sci. M.-L.
(1978) J. Biol.
N. S., and Kosower,
E. M., and Koppel,
R. L. (1977) Eur.
H. R., and Tuchs, J. A.
187,177-179
(1990)
SHORT NOTE Glutathione Content and Growth in A549 Human Lung Carcinoma Cells Yu-JIAN Department
KANG AND M. DUANE ENGER’
of Zoology, Iowa State University,
Ames, Iowa 50011
growth-related change in cellular glutathione levels suggests the possibility that glutathione may be involved in the regulation of cell growth. In the present work, the effect of L-buthionine-SRsulfoximine (BSO), a specific inhibitor of y-glutamylcysteine synthetase [ll], on the growth of A549 human lung carcinoma cells and the relationship between initial cellular levels of glutathione and cell growth have been studied. The results obtained demonstrate that subcultured A549 cells with lower initial glutathione levels grow with a longer lag phase, and that decreasing cellular glutathione levels with BSO inhibits cell growth.
The relationship between glutathione content and cell growth was investigated in A549 human lung carcinoma cells. A decreased cellular glutathione content was achieved by exposing the cells to L-buthionine-SRsulfoximine (BSO). It also occurred in these cells as they approached their plateau phase of growth. During exponential growth, a lower initial glutathione content correlated with a longer lag phase in subcultured cells. Further, depletion of cellular glutathione by BSO inhibited cell growth. This inhibition became apparent 36 h after the addition of BSO. These observations raise the possibility that a critical concentration of GSH may be required for optimal growth of A549 human lung carcinoma cells. 0 1990 Academic Press, Inc.
MATERIALS
INTRODUCTION Glutathione (GSH)’ is known to play a significant role in a number of important cellular processes including metabolism, transport, enzyme activity, structural integrity, synthesis of macromolecules, and protection [l31. There is as yet, however, no definition of the role or function of glutathione in regulating or modulating cell growth. Cellular levels of glutathione differ dramatically among cells of differing origins [l, 41. Even in the same type of cells, glutathione levels change as a function of cell culture conditions and cell growth stages [5-lo]. Studies on human lung carcinoma-derived A549 cells showed that cellular levels of glutathione change as a function of growth phase. The highest level of glutathione was observed to occur during the early portion of the exponential phase of growth and the lowest level was found to exist in plateau phase [5], a phenomenon similar to that reported to occur in other cells [4,7-lo]. This i To whom correspondence and reprint requests should be addressed at Department of Zoology, Iowa State University, 339 Science II, Ames, IA 50011. s Abbreviations used: BSO, L-buthionine-SR-sulfoximine; GSH, glutathione; GSSG, glutathione disulfide; PBS, phosphate-buffered saline; DTNB, 5,5’-dithiobis(2-nitrobenzoate).
AND
METHODS
Materials. Glutathione, GSSG reductase, NADPH, L-buthionineSR-sulfoximine, and 5,5’-dithiobis(2-nitrobenzoate) (DTNB) were obtained from Sigma Chemical Co. (St. Louis, MO). McCoy’s 5A medium was purchased from GIBCO (Grand Island, NY), and fetal calf serum from HyClone (Logan, UT). Cell culture. Human lung carcinoma-derived line A549 cells were routinely grown in McCoy’s 5A medium supplemented with 10% fetal calf serum, 2.2 g/liter of sodium bicarbonate, and antibiotics at 37°C and pH 7.0-7.2 in a humidified atmosphere of 95% air and 5% CO,. Stock cultures were passaged at 4-day intervals. Tests for mycoplasma were negative. Cells were removed from monolayer stock cultures with trypsin-EDTA, counted with a Coulter counter, and plated in 60-mm round cell culture dishes at 0.5 X 10s cells/dish or in 35-mm dishes at 0.5 X lo5 cells/dish. Cell growth determination. Cell growth was followed by determining the total number of cells in each culture dish at 12-h intervals. At the end of each interval, the monolayer culture was washed with warm PBS, trypsinized, and diluted with an exact volume of medium. The resulting cell suspension was further diluted with Isoton II saline solution prior to determination of cell number in a Coulter counter. Determination of total cellular GSH levekr. The DTNB-GSSG reductase recycling assay [ 121 was used to determine total cellular glutathione content. Briefly, the cells were trypsinized, rinsed with cold PBS, centrifuged at 4”C, resuspended in cold 0.6% sulfosalicylic acid, mixed vigorously with a “vortex” apparatus, and recentrifuged. The supernatant was then assayed for total glutathione by measuring the color change of DTNB at 412 nm in the presence of GSSG reductase and NADPH.
RESULTS
AND
DISCUSSION
Cellular glutathione content was decreased to 20% of that of control when A549 cells were exposed to 10 mM 177
0014-4827/90
$3.09
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
178
SHORT
NOTE
TABLE
1
Relationship between Initial GSH Content and Rates of Cell Growth for Subcultures” Hours in culture 24 48 12
96
GSH (nmol/106
cells) *
35.4 + 8.6d 30.7 + 4.6
19.4 f 0.6 15.2 + 0.8
Cell number (X105)’ 1.33 f 0.04 0.97 iz 0.06 0.70 + 0.05 0.60 f 0.02
a Cells, cultured for different times (24, 48, 72, and 96 h), were reseeded in 35mm dishes at 0.5 X lo5 cells/dish and subcultured for 36 h. * Initial GSH content at subculture time 0. ’ Cell numbers at the end of 36-h subculture. d Data are mean + SD from triplicate dishes.
2 1
,
0
12
24
/
36
I
40 60 72 Tm ( Hours )
64
96
106
120
FIG. 1. Effects of BSO on the growth of A549-T27 cells subcultured from a 36-h culture. Ten millimolar BSO was added 0 (A), 12 (O), or 24 (0) h after subculture. The inserted panel shows the effect of BSO (A) on the growth of cells from a 96-h culture to which BSO was added at 0 time. Open circles (0) represent untreated, control cell numbers and the solid circles (0) represent control as well as treated cell numbers during the period of no differential growth in control and treated samples.
BSO for 24 h. A further decrease to 5% or less of control GSH levels was achieved after 36 h. As shown in Fig. 1, cells that were exposed to 10 mM BSO at different times following subculture exhibited growth inhibition within 36 h. These results are consistent with the possibility that reduced GSH content inhibits cell growth. However, it is also possible that BSO exerts a toxic effect that results in reduced growth. Although BSO at low concentrations is reportedly a specific inhibitor of GSH synthesis, concentrations comparable to that used in these experiments inhibit cystine uptake in A549 cells [13] and transport of y-glutamyl amino acids in mouse kidneys [ 141, suggesting that cellular functions other than those affecting GSH status may be affected by BSO. When exponential-phase A549 cells were subcultured at 24-h intervals, the subcultures showed differential rates of growth that correlate with the initial glutathione levels of these cells (Table l), i.e., cells with higher levels of glutathione showed a more rapid resumption of growth as indicated by the number of cells existing 36 h after subculture. This result accords well with the report of Murata and Kimura [ 151 that yeast mutants deficient in glutathione synthesis have an extremely long generation time. To further define the differences in cell growth patterns that occur when cultures are established at different initial glutathione levels, the growth of subcultures established from early (36-h) or late (96-h) log-phase cells were monitored at 12-h intervals for 120 h. As shown in Fig. 2, these subcultures differed primarily in
the length of their lag phase. Cells with higher levels of glutathione showed a shorter lag phase and vice versa. This correlation is consistent with the possibility that a certain level of GSH is required for cell growth. There are of course numerous other changes that occur during logarithmic growth that may affect the length of the lag period in subcultures. Exploring possible mechanisms by which [GSH] may affect growth will be important in determining whether a causal relationship indeed exists. It is well known that ribonucleotide reductase is required in all cells to produce deoxyribonucleotides. This essential enzyme for DNA synthesis depends on thioredoxin or glutaredoxin as hydrogen donors [ 16,171. Some mutants of Escherichia coli depend on the glutaredoxinglutathione system as a hydrogen donor for ribonucleotide reduction [la-201. Should this be the case in A549 cells, inhibition of glutathione synthesis could result in corresponding inhibition of DNA synthesis. The result would be much the same as that which results from the 30
Time
( Hours )
FIG. 2. Comparison of cell growth in cells subcultured from 36-h (0) and from 96-h (0) cultures. Cell growth is expressed as the ratio of the number of growing cells in the subculture to those seeded. Data are presented as means + SDS.
SHORT
treatment of cells with hydroxyurea which, by inhibiting ribonucleotide reductase, inhibits DNA synthesis and arrests cells in the early S phase of cell cycle [21]. As shown in Fig. 1, there is about a 36-h delay after BSO addition for growth inhibition to become apparent. This length of time corresponds roughly to that necessary to deplete cellular glutathione to its lowest level. One would thus expect that this level is not adequate for efficient DNA synthesis if indeed the cells depend on the glutaredoxin system for DNA synthesis. Such may be the case in murine mammary carcinoma cells [22], in which depletion of glutathione by BSO inhibits DNA synthesis and arrests cell growth. In addition to possibly being requisite for DNA synthesis, an adequate glutathione level is necessary for efficient protein synthesis in a variety of cells [23-261. This may provide another explanation for the results obtained in this study. Further studies are required to delineate the mechanism(s) involved in growth inhibition mediated by BSO and to determine whether the observed correlation between GSH content and cell growth reflects a causal relationship. This work was supported by NIEHS/PHS Grant ES03863-02 and by Iowa State University. We thank Mary Nims for manuscript preparation.
REFERENCES 1. 2. 3. 4.
Kosower, N. S., and Kosower, E. M. (1978) Znt. Reu. Cytol. 54, 109. Meister, A., and Anderson, M. E. (1983) Annu. Reu. Biochem. 52,711. Ketterer, B. (1982) Drug Metub. Reu. 13, 161. Allalunis-Turner, M. J., Lee, F. Y. F., and Siemann, D. W. (1988) Cancer Res. 48,3657.
Received August 15,1989 Revised version received November
13,1989
179
NOTE 5.
Post, G. B., Keller, D. A., Connor, K. A., and Menzel, D. B. (1983) Biochem. Biophys. Rex Commun. 114,737.
6.
Meredith,
7.
Harris, J. W., and Teng, S. S. (1973) J. Cell. Physiol. 81,91.
8.
Harris, J. W., Painter, R. B., and Hahn, G. M. (1969) Znt. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 15,289.
M. J. (1986) Cell Biol. Toxicol. 2,495.
9. Harris, J. W., and Patt, H. M. (1969) Exp. Cell Res. 56, 134. 10.
Ohara, H., and Terasima,
11.
Griffith,
12.
Tietze, F. (1969) Anal. Biochem. 27,502.
13.
Brodie, A. E., and Reed, D. J. (1985) Tonicol. Appl. Pharmacol. 77,381.
14.
Griffith, 0. W., Bridges, R. J., and Meister, Acad. Sci. USA 76,6319.
T. (1969) Exp. Cell Res. 58, 182.
0. W., and Meister,
A. (1979) J. Biol. Chem. 254, 7558.
A. (1979) Proc. Natl.
15.
Murata,
16.
Holmgren,
A. (1988) Biochem. Sot. Trans. 16,95.
17.
Holmgren,
A. (1985) Annu. Reu. Biochem. 54,237.
18.
Holmgren,
A. (1976) Proc. N&l. Acad. Sci. USA 73,2275.
19.
Mark, D. F., and Richardson, USA 73,780.
20.
Holmgren, A., Ohlsson, I., and Grankvist, Chem. 253,430.
21.
Meyn, R. E., Hewitt, R. R., and Humphrey, R. M. (1975) in Methods in Cell Biology (Prescott, D. M., Ed.), Vol. IX, Academic Press, New York.
22.
Dethlefsen, L. A., Biaglow, J. E., Peck, V. M., and Ridinger, (1986) Znt. J. Radiat. Oncol. Biol. Phys. 12,1157.
D. N.
23.
Wax, R., Rosenberg, E., Kosower, (1970) J. Bacterial. 101, 1092.
E. M.
24.
Rossman, T., Norris, C., and Troll, W. (1974) J. Biol. Chem. 249, 3412.
25.
Kosower, N. S., Kosower, J. Biochem. 77,529.
26.
Hibberd, K. A., Berget, P. B., Werner, (1978) J. Bucteriol. 133, 1150.
K., and Kimura,
A. (1986) Agric. Biol. Chem. 50, 1055.
C. C. (1976) Proc. N&l. Acad. Sci. M.-L.
(1978) J. Biol.
N. S., and Kosower,
E. M., and Koppel,
R. L. (1977) Eur.
H. R., and Tuchs, J. A.
