Hum. Genet. 51,315--318 (1979) © by Springer-Verlag 1979
Short Communications
Deficiency of Arginine and Lysine Causes Increase in the Frequency of Sister Chromatid Exchanges W. Schempp* and W. Krone Abteilung Humangenetik, Universit~t Ulm, Postfach 4066, D-7900 Ulm, Federal Republic of Germany
Summary. An increase in the rate of sister chromatid exchanges (SCE) was found when V79 Chinese hamster cells were exposed to increasingly severe degrees of arginine and lysine deficiency. The data suggest a possible function of chromosomal proteins, and of histones in particular, in the maintenance of the low normal rate of SCE. The multifunctional alkylating agent trenimon (2,3,5-trisethyleneiminobenzoquinone) belongs to the large group of mutagenic substances which cause a dosedependent increase of the frequency of sister chromatid exchanges (SCE) in a variety of systems (Hayashi and Schmid, 1975; Beek and Obe, 1975; Vogel and Bauknecht, 1976). Also, this drug has been shown by Raydt et al. (1977) to bring about a pronounced decrease of the concentration of functional histone mRNA in Ehrlich ascites tumor cells. This finding is related to the earlier observation by Riches and Harrap (1973) that the histone content of chromatin from Yoshida ascites sarcoma cells decreases upon treatment with the alkylating agent chlorambucil at doses that do not inhibit DNA synthesis simultaneously. These data call into question the idea that enhancement of the SCE rate by trenimon, and perhaps by other alkylating substances as well, is due solely to their interaction with the DNA. Structural changes in chromatin a n d / o r slowdown of DNA synthesis due to a shortage in the supply of histones might be at least partly involved. We have therefore examined the influence of suboptimal concentrations of arginine (Arg) and lysine (Lys) on the SCE rate in the V-79 Chinese hamster cell line. Exponentially growing cultures from which the experimental subcultures were derived were washed twice in Hank's solution before trypsinization. The cells were resuspended in basal Eagle's medium (BME) containing bromodeoxyuridine (BrdU, 20 gg/ml), fluorodeoxyuridine (FdU, 1.0 ~tg/ml), and 10% dialyzed fetal * To whom offprint requests should be sent
0340-6717/79/0051/0315/$ 01.00
316
cO cO O
W. Schempp and W. Krone
30
/ /
[3_ HA
o9
/
20
/ / /
0
1
2
3
-I°log of relative [Arg+Lys]
Fig. 1. Dependence of SCE rate on the relative concentrations of Arg and Lys. Bars represent SEMs. Points one to four are means of 100 mitoses each; point five is the mean of six mitoses. Statistical evaluation of the data reveals significant differences (P < 0.001) in the three intervals between points one through four. The difference in the last interval is not significant (0.20 > P> 0.15)
calf s e r u m ( F C S ) , b u t lacking A r g a n d Lys. Large L e i g h t o n tubes with m i c r o scopic slides each received a b o u t 105 cells a n d a p p r o p r i a t e aliquots of a stock s o l u t i o n o f A r g a n d Lys to achieve final c o n c e n t r a t i o n s o f I, 0.1,0.02, 0.01, 0.002, a n d 0.001 o f the n o r m a l c o n c e n t r a t i o n s o f these a m i n o acids in B M E with 10% d i a l y z e d F C S . The s t a n d a r d c o n c e n t r a t i o n s in this m e d i u m are 0.09 m M for A r g a n d 0.18 m M f o r I, ys. Some cultures r e m a i n e d in the A r g - L y s - f r e e m e d i u m . A 20-h i n c u b a t i o n p e r i o d was used to achieve a single-cycle s u b s t i t u t i o n b y BrdU. Thereafter, the cultures were washed twice in H a n k ' s s o l u t i o n a n d a l l o w e d to g r o w for 21 h in the a p p r o p r i a t e m e d i a in the absence o f B r d U a n d F d U . In situ p r e p a r a t i o n s o f mitoses were m a d e after a l - h e x p o s u r e to vinblastine. The m e t h o d o f E p p l e n et al. (1975) was used to o b t a i n differential c h r o m a t i d staining. C h r o m o s o m e n u m b e r s r a n g e d f r o m 18 to 23 at all c o n c e n t r a t i o n s of A r g a n d Lys a n d 5 0 - - 6 4 % o f the m e t a p h a s e s h a d the m o d a l c h r o m o s o m e n u m b e r o f 21. T e t r a p l o i d cells were d e l i b e r a t e l y e x c l u d e d f r o m the analysis. The cultures were regularly m o n i t o r e d for m y c o p l a s m a a n d f o u n d to be u n c o n t a m i n a t e d .
Deficiency of Arginine and Lysine in Sister Chromatid Exchanges
317
Figure I gives the cumulative results of three experiments performed according to the schedule outlined above. Each point, with the exception of the one at the relative Arg + Lys concentration of 0.002, represents the mean of 100 mitoses. There is a linear relationship between the SCE rate per mitosis and the negative logarithm of the relative concentration of Arg + Lys. Because of the low number of mitoses that could be analyzed at the relative Arg + Lys concentration of 0.002, it cannot be determined whether the curve tends to acquire a sharper slope or to level off under more extreme conditions of Arg + Lys starvation. No differentially stained mitosis was detectable at the relative Arg + Lys concentration of 0.001. Complete absence of these amino acids prevented the appearance of mitoses. Almost complete inhibition of proliferation was found at the relative Arg + Lys concentration of 0.01. However, measurable loss of cells did not seem to occur within the experimental period at lower levels of Arg and Lys or in the complete absence of these amino acids as judged based on protein determinations (Lowry). With decreasing concentration of Arg + Lys there was a steady increase in the number of chromosomal aberrations such as gaps, breaks, and exchange configurations. At the lower levels of Arg + Lys an increasing number of mitoses showed partial or complete "chromosome pulverization." Freed and Schatz (1969) reported on the induction of the whole range of chromosomal anomalies, including complete pulverization, in a different line of Chinese hamster cells after starvation for any one of the essential amino acids. The most severe effects were not found in Arg or Lys starvation, which was investigated separately by these authors, but in deprivation of cystine, isoleucine, methionine, and valine. Whether the limitation of the supply of these amino acids also causes an increased rate of SCE remains to be investigated. Freed and Schatz (1969), however, caution against far-reaching speculations concerning the role of a slowdown of histone synthesis in the causation of an enhanced SCE rate. It is conceivable that interference with the synthesis of a variety of chromosomal proteins--histones as well as nonhistone proteins--might cause an increase in the SCE rate . This might arise from an intensified exposure of D N A to the enzyme system that catalyzes recombination between sister strands. It is well known that nucleosomic histones segregate conservatively during replication (Leffak et al., 1977; Felsenfeld, 1978). Inhibition of protein synthesis by cycloheximide during replication causes one daughter strand of the replication fork to emerge as protein-free unbeaded D N A (Riley and Weintraub, 1979). Hence, there is exposure of D N A upon inhibition of histone synthesis. To what extent histone synthesis is inhibited by the various degree of Arg + Lys starvation in V79 cells remains to be investigated. As expected, limitation of the supply of Arg and Lys caused a decrease of the rate of D N A synthesis in V79 cells, as measured by 3H-thymidine pulse labelling (data not shown). In Bloom's syndrome there is an interesting relationship between the increased SCE rate and a retarded rate of elongation of replicating D N A (Hand and German, 1975). The observation of an elevated frequency of SCE caused by suboptimal supply of Arg and Lys suggests that histones (and perhaps nonhistone proteins as well) may be involved in the maintenance of the normal low rate of SCE. This hypothesis may stimulate speculations about the underlying defect in Bloom's syndrome and about the nature of the corrective factors supplied by normal cells (Bartram et al., 1979).
318
W. Schempp and W. Krone
Acknowledgements. The authors are grateful to Mrs. S. Z6rlein for her excellent technical assistance.
References Bartram, C. R., Rt~diger, H. W., Passarge, E.: Frequency of sister chromatid exchanges in Bloom's syndrome fibroblasts reduced by cocultivation with normal cells. Hum. Genet. 46, 331--334 (1979) Beek, B., Obe, G.: The human leukocyte test system. VI. The use of sister chromatid exchanges as possible indicators for mutagenic activities. Humangenetik 29, 127--134 (1975) Epplen, J. T., Siebers, J.-W., Vogel, W.: DNA replication patterns of chromosomes from fibroblasts and amniotic fluid cells revealed by a Giemsa staining technique. Cytogenet. Cell Genet. 15, 177--185 (1975) Felsenfeld, G.: Chromatin. Nature (Lond.) 271, 115--122 (1978) Freed, J. J., Schatz, S. A.: Chromosome aberrations in cultured cells deprived of single essential amino acids. Exp. Cell Res. 55, 393--409 (1969) Hand, R., German, J.: A retarded rate of DNA chain growth in Bloom's syndrome. Proc. Natl. Acad. Sci. USA 72, 758--762 (1975) Hayashi, K., Schmid, W.: The rate of sister chromatid exchanges parallel to spontaneous chromosome breakage in Fanconi's anemia and trenimon induced in human lymphocytes and fibroblasts. Humangenetik 29, 201--206 (1975) Leffak, J. M., Grainger, R., Weintraub, H.: Conservative assembly and segregation of nucleosomal histones. Cell 12, 837--845 (1977) Raydt, G., Holzweber, F., Puschendorf, B., Grunicke, H.: Depression of the intracellular histone messenger RNA content by the alkylating agent 2,3,5-trisethyleneiminobenzochinone. FEBS Letters 74, 111--114 (1977) Riches, P. G., Harrap, K. R.: Some effects of chlorambucil on the chromatin of Yoshida ascites carcinoma cells. Cancer Res. 33,389---393 (1973) Riley, D., Weintraub, H.: Conservative segregation of parental histones during replication in the presence of cycloheximide. Proc. Natl. Acad. Sci. USA 76, 328--332 (1979) Vogel, W., Bauknecht, T.: Differential chromatid staining by in vivo treatment as a mutagenicity test system. Nature (Lond.) 260,448--449 (1976) Received June 8, 1979
Short Communications
Deficiency of Arginine and Lysine Causes Increase in the Frequency of Sister Chromatid Exchanges W. Schempp* and W. Krone Abteilung Humangenetik, Universit~t Ulm, Postfach 4066, D-7900 Ulm, Federal Republic of Germany
Summary. An increase in the rate of sister chromatid exchanges (SCE) was found when V79 Chinese hamster cells were exposed to increasingly severe degrees of arginine and lysine deficiency. The data suggest a possible function of chromosomal proteins, and of histones in particular, in the maintenance of the low normal rate of SCE. The multifunctional alkylating agent trenimon (2,3,5-trisethyleneiminobenzoquinone) belongs to the large group of mutagenic substances which cause a dosedependent increase of the frequency of sister chromatid exchanges (SCE) in a variety of systems (Hayashi and Schmid, 1975; Beek and Obe, 1975; Vogel and Bauknecht, 1976). Also, this drug has been shown by Raydt et al. (1977) to bring about a pronounced decrease of the concentration of functional histone mRNA in Ehrlich ascites tumor cells. This finding is related to the earlier observation by Riches and Harrap (1973) that the histone content of chromatin from Yoshida ascites sarcoma cells decreases upon treatment with the alkylating agent chlorambucil at doses that do not inhibit DNA synthesis simultaneously. These data call into question the idea that enhancement of the SCE rate by trenimon, and perhaps by other alkylating substances as well, is due solely to their interaction with the DNA. Structural changes in chromatin a n d / o r slowdown of DNA synthesis due to a shortage in the supply of histones might be at least partly involved. We have therefore examined the influence of suboptimal concentrations of arginine (Arg) and lysine (Lys) on the SCE rate in the V-79 Chinese hamster cell line. Exponentially growing cultures from which the experimental subcultures were derived were washed twice in Hank's solution before trypsinization. The cells were resuspended in basal Eagle's medium (BME) containing bromodeoxyuridine (BrdU, 20 gg/ml), fluorodeoxyuridine (FdU, 1.0 ~tg/ml), and 10% dialyzed fetal * To whom offprint requests should be sent
0340-6717/79/0051/0315/$ 01.00
316
cO cO O
W. Schempp and W. Krone
30
/ /
[3_ HA
o9
/
20
/ / /
0
1
2
3
-I°log of relative [Arg+Lys]
Fig. 1. Dependence of SCE rate on the relative concentrations of Arg and Lys. Bars represent SEMs. Points one to four are means of 100 mitoses each; point five is the mean of six mitoses. Statistical evaluation of the data reveals significant differences (P < 0.001) in the three intervals between points one through four. The difference in the last interval is not significant (0.20 > P> 0.15)
calf s e r u m ( F C S ) , b u t lacking A r g a n d Lys. Large L e i g h t o n tubes with m i c r o scopic slides each received a b o u t 105 cells a n d a p p r o p r i a t e aliquots of a stock s o l u t i o n o f A r g a n d Lys to achieve final c o n c e n t r a t i o n s o f I, 0.1,0.02, 0.01, 0.002, a n d 0.001 o f the n o r m a l c o n c e n t r a t i o n s o f these a m i n o acids in B M E with 10% d i a l y z e d F C S . The s t a n d a r d c o n c e n t r a t i o n s in this m e d i u m are 0.09 m M for A r g a n d 0.18 m M f o r I, ys. Some cultures r e m a i n e d in the A r g - L y s - f r e e m e d i u m . A 20-h i n c u b a t i o n p e r i o d was used to achieve a single-cycle s u b s t i t u t i o n b y BrdU. Thereafter, the cultures were washed twice in H a n k ' s s o l u t i o n a n d a l l o w e d to g r o w for 21 h in the a p p r o p r i a t e m e d i a in the absence o f B r d U a n d F d U . In situ p r e p a r a t i o n s o f mitoses were m a d e after a l - h e x p o s u r e to vinblastine. The m e t h o d o f E p p l e n et al. (1975) was used to o b t a i n differential c h r o m a t i d staining. C h r o m o s o m e n u m b e r s r a n g e d f r o m 18 to 23 at all c o n c e n t r a t i o n s of A r g a n d Lys a n d 5 0 - - 6 4 % o f the m e t a p h a s e s h a d the m o d a l c h r o m o s o m e n u m b e r o f 21. T e t r a p l o i d cells were d e l i b e r a t e l y e x c l u d e d f r o m the analysis. The cultures were regularly m o n i t o r e d for m y c o p l a s m a a n d f o u n d to be u n c o n t a m i n a t e d .
Deficiency of Arginine and Lysine in Sister Chromatid Exchanges
317
Figure I gives the cumulative results of three experiments performed according to the schedule outlined above. Each point, with the exception of the one at the relative Arg + Lys concentration of 0.002, represents the mean of 100 mitoses. There is a linear relationship between the SCE rate per mitosis and the negative logarithm of the relative concentration of Arg + Lys. Because of the low number of mitoses that could be analyzed at the relative Arg + Lys concentration of 0.002, it cannot be determined whether the curve tends to acquire a sharper slope or to level off under more extreme conditions of Arg + Lys starvation. No differentially stained mitosis was detectable at the relative Arg + Lys concentration of 0.001. Complete absence of these amino acids prevented the appearance of mitoses. Almost complete inhibition of proliferation was found at the relative Arg + Lys concentration of 0.01. However, measurable loss of cells did not seem to occur within the experimental period at lower levels of Arg and Lys or in the complete absence of these amino acids as judged based on protein determinations (Lowry). With decreasing concentration of Arg + Lys there was a steady increase in the number of chromosomal aberrations such as gaps, breaks, and exchange configurations. At the lower levels of Arg + Lys an increasing number of mitoses showed partial or complete "chromosome pulverization." Freed and Schatz (1969) reported on the induction of the whole range of chromosomal anomalies, including complete pulverization, in a different line of Chinese hamster cells after starvation for any one of the essential amino acids. The most severe effects were not found in Arg or Lys starvation, which was investigated separately by these authors, but in deprivation of cystine, isoleucine, methionine, and valine. Whether the limitation of the supply of these amino acids also causes an increased rate of SCE remains to be investigated. Freed and Schatz (1969), however, caution against far-reaching speculations concerning the role of a slowdown of histone synthesis in the causation of an enhanced SCE rate. It is conceivable that interference with the synthesis of a variety of chromosomal proteins--histones as well as nonhistone proteins--might cause an increase in the SCE rate . This might arise from an intensified exposure of D N A to the enzyme system that catalyzes recombination between sister strands. It is well known that nucleosomic histones segregate conservatively during replication (Leffak et al., 1977; Felsenfeld, 1978). Inhibition of protein synthesis by cycloheximide during replication causes one daughter strand of the replication fork to emerge as protein-free unbeaded D N A (Riley and Weintraub, 1979). Hence, there is exposure of D N A upon inhibition of histone synthesis. To what extent histone synthesis is inhibited by the various degree of Arg + Lys starvation in V79 cells remains to be investigated. As expected, limitation of the supply of Arg and Lys caused a decrease of the rate of D N A synthesis in V79 cells, as measured by 3H-thymidine pulse labelling (data not shown). In Bloom's syndrome there is an interesting relationship between the increased SCE rate and a retarded rate of elongation of replicating D N A (Hand and German, 1975). The observation of an elevated frequency of SCE caused by suboptimal supply of Arg and Lys suggests that histones (and perhaps nonhistone proteins as well) may be involved in the maintenance of the normal low rate of SCE. This hypothesis may stimulate speculations about the underlying defect in Bloom's syndrome and about the nature of the corrective factors supplied by normal cells (Bartram et al., 1979).
318
W. Schempp and W. Krone
Acknowledgements. The authors are grateful to Mrs. S. Z6rlein for her excellent technical assistance.
References Bartram, C. R., Rt~diger, H. W., Passarge, E.: Frequency of sister chromatid exchanges in Bloom's syndrome fibroblasts reduced by cocultivation with normal cells. Hum. Genet. 46, 331--334 (1979) Beek, B., Obe, G.: The human leukocyte test system. VI. The use of sister chromatid exchanges as possible indicators for mutagenic activities. Humangenetik 29, 127--134 (1975) Epplen, J. T., Siebers, J.-W., Vogel, W.: DNA replication patterns of chromosomes from fibroblasts and amniotic fluid cells revealed by a Giemsa staining technique. Cytogenet. Cell Genet. 15, 177--185 (1975) Felsenfeld, G.: Chromatin. Nature (Lond.) 271, 115--122 (1978) Freed, J. J., Schatz, S. A.: Chromosome aberrations in cultured cells deprived of single essential amino acids. Exp. Cell Res. 55, 393--409 (1969) Hand, R., German, J.: A retarded rate of DNA chain growth in Bloom's syndrome. Proc. Natl. Acad. Sci. USA 72, 758--762 (1975) Hayashi, K., Schmid, W.: The rate of sister chromatid exchanges parallel to spontaneous chromosome breakage in Fanconi's anemia and trenimon induced in human lymphocytes and fibroblasts. Humangenetik 29, 201--206 (1975) Leffak, J. M., Grainger, R., Weintraub, H.: Conservative assembly and segregation of nucleosomal histones. Cell 12, 837--845 (1977) Raydt, G., Holzweber, F., Puschendorf, B., Grunicke, H.: Depression of the intracellular histone messenger RNA content by the alkylating agent 2,3,5-trisethyleneiminobenzochinone. FEBS Letters 74, 111--114 (1977) Riches, P. G., Harrap, K. R.: Some effects of chlorambucil on the chromatin of Yoshida ascites carcinoma cells. Cancer Res. 33,389---393 (1973) Riley, D., Weintraub, H.: Conservative segregation of parental histones during replication in the presence of cycloheximide. Proc. Natl. Acad. Sci. USA 76, 328--332 (1979) Vogel, W., Bauknecht, T.: Differential chromatid staining by in vivo treatment as a mutagenicity test system. Nature (Lond.) 260,448--449 (1976) Received June 8, 1979
