- Email: [email protected]
Altered protein synthesis rate in ovaries of D. melanogaster caused by new antitumour alkylating agents
Inr. J. Eiochem. Vol. 23, No. II, pp. 1251-1254, Printed in Great Britain. All rights reserved
1991 Copyright
0
0020-71 IX/91 $3.00 + 0.00 1991 Pergamon Press plc
ALTERED PROTEIN SYNTHESIS RATE IN OVARIES OF D. MELANUGASTER CAUSED BY NEW ANTITUMOUR ALKYLATING AGENTS G. STRPHANOU,’N. A. DEMOPOULOS’and P. CATSOULACOS* ‘Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, Patras and *Laboratory of Pharmaceutical Chemistry, University of Patras, Patras, Greece (Received 27 November 1990) Abstract-l.
The effect of two homo-aza-steroidal esters with antineoplastic activity, namely 3/l-hydroxy13a-amino-13,17-seco-5a-androstan-17-oic-13,17-lactam-p-bis(2-ch1oroethy1)aminophenoxyacetate (NSC 294859) and 3~-hydroxy-13a-amino-13,17-seco-5a-androstan-17-oic-13,17-lactam-~-bis(2-ch~oroethyl)aminophenylacetate (ASE) on protein synthesis rate was studied in ovaries of Drosophila melanogaster females. 2. Two different concentrations for each compound were examined. 3. Both esters containing the same alkylating agent have been shown to decrease protein synthesis in relation to control.
INTRODUCTION Alkylating agents are commonly used as cytotoxic drugs for the treatment of some neoplastic diseases. Modified steroids, such as lactams, are used as bio-
logical carriers for transporting alkylating compounds to target tumours in a rather specific manner (Catsoulacos et al., 1976). This has been used after the discovery that steroidal hormones, as carriers of cytotoxic agents, reduce systemic toxicity and improve cancer therapy. Compounds, such as esters, in which the alkylating agent is linked to the steroid by an easily cleaved bond produce satisfactory activity (Wall et al., 1969). NSC 294859 and ASE are two molecules belonging to this category. NSC 294859 is a new homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenoxy acetic acid. The compound 3fl-hydroxy-13a-amino-13,17-seco-5aandrostan- 17-oic- 13,17-lactam-p-bis(2-chloroethyl)aminophenoxyacetate was prepared by heating 3/?-hydroxy-13a-amino-13,17-seco-5a-androstan-l7oic-I 3,17-lactam with p-[N,N-bis(2-chloroethyl)amino] phenoxyacetylchloride in benzene. The synthesis and the chemical properties of this compound have been reported previously (Catsoulacos et al., 1979). ASE is a homo-aza-steroidal ester of p-bis(2chloroethyl)aminophenyl acetic acid and consists of a nitrogen mustard that is bound to a modified steroid (a lactam, characterized by the group -CO-NH) via a phenyl acetic acid bond (Catsoulacos and Wampler, 1982) and has been synthesized in pure form (Wampler and Catsoulacos, 1977). Both compounds have been shown to have antineoplastic activity. ASE gives a 50% increased lifespan over controls in the treatment of L1210 leukemia in mice and a 150% increased lifespan in the treatment of P388 leukemia (Wampler and Catsoulacos, 1977). NSC 294859 seems to be more
drastic giving 100 and 383% correspondingly. ASE is also active against B,, melanoma on CS7b, mice and T, Guerin tumour on rats (Catsoulacos and Wampler, 1982). As to NSC 294859 the treatment of colon 26 tumour and Br6 melanoma gave positive antineoplastic activity, but the drug was not active in a melphalan resistant P383 line (Catsoulacos, 1983). In addition, both compounds also show mutagenic activity. ASE was found to induce sister chromatid exchanges in CHO cells (Athanasiou et al., 1983), and to be positive in strains TA1535 and TAlOO and in the newer strain TA102 with and without metabolic activation in the Salmonella/microsome system (Athanasiou and Arzimanoglou, 1986). This drug also induces sister chromatid exchanges and cell cycle delay in cultured human lymphocytes (Mourelatos et al., 1987). On the other hand NSC 294859 was found to be effective in causing statistically significant increases in sister chromatid exchange rates and cell division delays (Tselepi et al., 1989) and to induce chromosomal aberrations in human lymphocyte cultures (Tselepi et al., 1985). According to Maier and Schawalder (1986) most of the chemicals which exert mutagenic, carcinogenic or cytotoxic properties affect DNA synthesis, disturb the cell cycle and influence protein synthesis in individual cells. So it would be interesting to study the effect of ASE and NSC 294859, both of them being SCE inducers and mutagens, on protein synthesis rate. For this purpose the incorporation of 35S-methionine in newly synthesized proteins was measured in ovaries of D. melunoguster. MATERIALS AND METHODS
Test compounds NSC 294859 was dissolved in DMSO and two concentrations were tested, 10 and 2Opg/ml. ASE was also dissolved in DMSO and the concentrations used were 50 and
1251
G. STEPHANOU et al.
1252
(a) (Cl CH,CH,)s N
(b) (Cl CH,CH2)1 N
Fig. 1. Chemical structures of (a) 3~-hydroxy-13a-amino-13,17-seco-5a-androstan-l7-oic-l3,17-lactam-~bis(2-chloroethyl)aminophenylacetate (ASE). (b) 3~-hydroxy-13a-amino-l3,17-seco-5a-androstan-l7-oic13,17-lactam-p-bis(2-chloroethyl)aminophenoxyacetate (NSC 294859). 100 pg/ml. The chemical formulas of these compounds are given in Fig. 1. Lubelling
ofproteins
The strain C-S (Lindsley and Grell, 1968) of Drosophila melanogasfer was used in this study, and it is retained under standard conditions of temperature (25°C) and humidity (43 k 4% r.h.) in a standard cornmeal food. Ovaries from adult females 3-5 days old were excised and placed in depression slides in Robb’s saline (Robb, 1969). The solution contained 550 pCi/ml 3SS-methionine (Amersham, specific activity 1000 Ci/ml). Preparations were covered with coverslips and incubated at 25°C for 1 hr. The experimental preparations also contained the drugs, NSC 294859 at final concentration of IO and 20 pg/ml and ASE at final concentration of 50 and lOO~g/ml. There were also control preparations that either contained DMSO (4%) or none of the drugs were in the incubation medium. Protein synthesis
The rate of protein synthesis was measured by precipitation in 5 ~1 aliquots of homogenized ovaries with 10% trichloroacetic acid on Whatman 3MM filters as described by Mans and Novelli (1961). The radioactivity was counted in an LKB 1215 RACKBETA liquid scintillation counter. RESULTS
AND DISCUSSION
Several experiments were carried out in order to study the probable effect of NSC 294859 and ASE on protein synthesis. This was achieved by measuring the incorporation of “S-methionine into the total proteins of treated ovaries in Drosophila mefunogaster, Canton-S strain. Experiments were scheduled as follows: (1) Ovaries of C-S treated with 10 or 20 pg/ml of NSC 294859 for 1 hr and labelled simulTable
I. Incorporation
of “S-methionine treatment
taneously with ?S-methionine (550 pCi/ml) at 25°C. (2) Ovaries of C-S treated with 50 or 100 pg/ml of ASE for 1 hr and labelled simultaneously with “S-methionine (550 pCi/ml) at 25°C. (3) Ovaries of C-S treated with DMSO 4% for 1 hr and labelled simultaneously with 35S-methionine (550pCi/ml) at 25°C. DMSO is tested because the compounds are dissolved in DMSO 4%. (4) Ovaries of C-S that were not treated at all but were labelled with 35S-methionine at 25°C for 1 hr. The variability in ovary size among flies could be considered as a source of variation in those experiments, but this variation is not significant because: (1) Care was taken to select explanted ovaries of similar size (ovaries of somewhat reduced size were not used). (2) Six females were chosen randomly in each experiment and in the case where ovaries of reduced size were observed another female was used. (3) Each couple of ovaries was diversed and the first ovary was used in experimental sample and the other in control sample. (4) Our results are obtained from at least three or more experiments for each treatment. As it is shown in Table 1 NSC 294859 decreases the protein synthesis rate in both concentrations. We can see that at 10 pg/ml concentration the protein synthesis is reduced to 70.16% in relation to control preparation. This reduction seems to be higher at 20 pg/ml concentration where protein synthesis is observed to be at 68.81% of the control rate. The same seems to be true for the ASE although this compound seems to be less active not only because the concentrations tested were much higher than
in total ovarian proteins with NSC 294559
of D. melanogasrer
after
hLR/rnl)
(cumlovarv)
no treatment
% Incorporation in relation to DMSO treatment
NSC NSC NONE
IO 20 -
21135.24 20727.00 30122.40
70.16 68.81 100.00
74.34 72.75 -
DMSO
4%
28488.60
94.57
100.00
Concentration Comoound
% Incorporation relation to
Mean
Mean numbers have been extracted from
in
three or more different experiments.
New antitumour agents that alter protein synthesis rate those of NSC 294859 but also less reduction of protein synthesis rate was observed in relation to control samples. Table 2 shows that at SOpg/ml the protein synthesis rate is reduced to 78.87% while at 100 pg/ml it is reduced to 77.53%. DMSO seems also to slightly reduce the protein synthesis rate. Preparations treated with 4% DMSO show 94.57 (Table 1) and 97.21% (Table 2) of the control samples protein synthesis rate, for the experiments with NSC 294859 and ASE respectively. In addition, if the mean cpm/ovary of the drugtreated samples is compared with the samples treated with DMSO only, a profound decrease of protein synthesis is observed. For the NSC 294859 (Table 1) it is decreased to 74.34 and 72.75% for the 10 pg/ml and 20 pg/ml concentrations respectively and for the ASE (Table 2) it is reduced to 8 1.13 and 79.75% for the 50 and 100 pg/ml concentrations respectively. The effect of the two compounds under study on protein synthesis in isolated ovaries of D. melanogaster is also shown in Fig. 2. Most mutagens that have been studied show an interaction with proteins of individual cells. Excised root tips of Vicia faba exposed to 0.1% caffeine resulted in inhibition of protein synthesis to about 60% of the control rate (Zuk and Swietlinska, 1973). Cultured, freshly isolated rat fibroblasts that were exposed in vitro to vincristine sulphate, amethopterin, bleomycin and benomyl have shown a variable interaction with cellular proteins (Maier and Schawalder, 1986). Amethopterin and bleomycin resulted in increased protein content while vincristine sulphate did not significantly alter the protein content of individual cells. Stephanou and Demopoulos (1987) have shown that bleomycin treatment of preincubated mycelia of Aspergillus nidulans during heat shock enhances the incorporation of 35S-methionine into heat-shock bands. On the other hand it is known that proteins represent one of the biological molecules that react with alkylating agents (Vogel and Natarayan, 1981). Boffa et al. (1987) present data suggesting that qualitative and quantitative correlations between specific targets and alkylating agents apply not only to nucleic acids but also to DNAassociated nuclear proteins. In addition, it is not only the covalent modification of biomacromolecules such as DNA which causes genetic alterations. More subtle effects of the solvation coat, the tertiary structure and aggregation behaviour of proteins can also lead to important genetic changes (Zimmerman et al., 1985). Our results are in accordance with the above observation from the point of view that both alkylating agents tested, which are potent mutagens, also affect protein synthesis. This effect may be the result Table 2. Incorporation
5
SS-
A NSC 294659
a
60-
i !s 294959-DMSo . ASE-DMSO
55 -
501 ’ 0 10
’
20
Compound
’
’
30
40
’
so
Cancentrotiin
’
60
Mean @pm/ovary) 22381.68 22008.00 28384.44 27594.00
’
90
’
so
’
loo
of the interaction of the drugs with the DNA preventing the expression of some genes with protein products. However this hypothesis does not exclude the possibility that NSC 294859 and ASE act at different levels of translation. Acknowledgement-This study was supported by a grant from the Greek General Secretary of Research and Technology to N.A.D. REFERENCES
Athanasiou K. and Arzimanoglou J. (1986) Mutagenicity and clastogenicity of the antineoplastic agents homoaza-steroidal ester of p-bis(Zchloroethy1) aminophenyl acetic acid and chlorambucil. Mutat. Res. 175, 165-169. Athanasiou K., Demopoulos N. A. and Catsoulacos P. (1983) Chromosome damage and SCE induced by the cytostatic factor homo-aza-steroidal ester of p-bis(2chloroethyl) aminophenyl acetic acid in CHO cells in culture. Environ. Mutagen. 5, 219-283. Boffa L. C., Bolognesi C. and Mariani M. R. (1987) Specific targets of alkylating agents in nuclear proteins of cultured hepatocytes. Mutat. Rex 190, 119-123. Catsoulacos P. (1983) Activity of 3/?-hydroxy-13a-amino13,17-seco-5a-androstan-17-oic-13,17-lac~am-p-bis(2chloroethyl)aminophenoxyacetate (NSC 294859) on experimental tumor and leukemia systems. Oncology 40, 290-292. Catsoulacos P. and Wampler G. (1982) Activity of 3phydroxy- 13a-amino-13,17-seco-5a-androsanI ‘I-oic13,17-lactam-p[bis(2-chloroethyl)amino]phenyl acetate (NSC 290905) in murine solid tumors. Oncology 39, 109-l 12. Catsoulacos P., Boutis L. and Dimitropoulos K. (1976) Antitumor activity of steroidal lactone esters of N,N-bis (2-chloroethyl) aminophenyl acetic acid. Eur. J. Med. Chem. Chim. Therap. 11, 189-191. Catsoulacos P., Politis D. and Wampler G. (1979) A new steroidal alkylating agent with improved activity
of “S-methionine in total ovarian proteins of D. melanogaster after treatment with ASE
Concentration WW 50 loo 4%
’
70
(pg/ml)
Fig. 2. Reduction of protein synthesis rate in ovaries of D. melanogaster by the antitumour agents NSC 294859 and ASE.
% Incorporation in
ASE ASE NONE DMSO
1253
relation to no treatment
% Incorporation in relation to DMSO treatment
78.87 77.53 100.00 97.21
81.13 79.7s 100.00
Mean numbers have been extracted from three or more different experiments.
1254
G. STEPHANOUet al.
in advanced murine leukemias. Cancer Chemother. Pharmacol. 3, 61-10. Lindlsey D. L. and Grell E. H. (1968) Genetic variations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627, 431. Maier P. and Schawalder H. P. (1986) A two-parameter flow cytometry protocol for the detection and characterization of the clastogenic, cytostatic and cytotoxic activities of chemicals. Mutat. Res. 164, 369-379. Mans R. J. and Novelli G. D. (1961) Measurements of the incorporation of radioactive amino acids into proteins, a filter-paper disc method. Arch. Biochem. Eiophys. 14, 48-53. Mourelatos D., Petrou C., Boutis L., Papageorgiou A., Catsoulacos P. and Dozi-Vassiliades J. (1987) Induction of cytogenetic damage by modified steroidal derivatives of p-bis(2-chloroethyl)aminophenylacetic acid in human lymphocytes. Mutat: Res. l!%, 2055210. Robb J. A. (1969) Maintenance of imaainal discs of Drosophila melanigaster in chemically defined media. J. Cell Biol. 41, 876-888. Stephanou G. and Demopoulos N. A. (1987) Heat shock phenomena in Aspergillus nidulans. II. Combined effect of heat and Bleomycin to heat shock protein synthesis, survival rate and induction of mutations. Curr. Gener. 12, 443448. Tselepi R., Zacharopoulou A. and Demopoulos N. A. (1985) Genetic activity of 3/?-hydroxy-13a-amino-13, 17-seco-5a-androstan-17-oic-13,17-lactam-p-bis(2-chloroethyl)aminophenoxyacetate (NSC 294859) on human
lymphocyte cultures in vitro. Abstracts of 4th International Conference on Environmental Mutagens, p. 243. Stockholm, Sweden. Tselepi R., Demopoulos N. A. and Catsoulacos P. (1989) Induction of sister chromatid exchanges and cell division delays in human lymphocytes by the antitumour agent homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenoxy acetic acid. Mutagenesis 4(5), 361-364. Vogel E. and Natarayan A. (1981) The relation between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eucaryotic systems: interspecies comparisons. In Chemical Mutagens, Principles and Methods for their Detection (Edited by Hollander A. and De Serres F. J.), Vol 7, pp. 295-336. Plenum Press, New York. Wall M. E., Abernathy G. S. Jr., Carol1 F. J. and Taylor D. J. (1969) The effects of some steroidal alkylating agents on experimental animal mammary tumor and leukemia systems. J. Medicin. Chem. 12, 810-818. Wampler G. L. and Catsoulacos P. (1977) Antileukemic effects of homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenylacetic acid. Cancer Treat. Rep. 61, 3741. Zimmermann F. K., Groschel-Stewart U., Scheel I. and Resnick M. A. (1985) Genetic change may be caused by interference with protein-protein interactions, Mufat. Res. 150, 203-210. Zuk J. and Swietlinska Z. (1973) Effect of caffeine on nucleic acid and protein synthesis in Vicia faba. Mulat. Res. 17, 207-212.
1991 Copyright
0
0020-71 IX/91 $3.00 + 0.00 1991 Pergamon Press plc
ALTERED PROTEIN SYNTHESIS RATE IN OVARIES OF D. MELANUGASTER CAUSED BY NEW ANTITUMOUR ALKYLATING AGENTS G. STRPHANOU,’N. A. DEMOPOULOS’and P. CATSOULACOS* ‘Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, Patras and *Laboratory of Pharmaceutical Chemistry, University of Patras, Patras, Greece (Received 27 November 1990) Abstract-l.
The effect of two homo-aza-steroidal esters with antineoplastic activity, namely 3/l-hydroxy13a-amino-13,17-seco-5a-androstan-17-oic-13,17-lactam-p-bis(2-ch1oroethy1)aminophenoxyacetate (NSC 294859) and 3~-hydroxy-13a-amino-13,17-seco-5a-androstan-17-oic-13,17-lactam-~-bis(2-ch~oroethyl)aminophenylacetate (ASE) on protein synthesis rate was studied in ovaries of Drosophila melanogaster females. 2. Two different concentrations for each compound were examined. 3. Both esters containing the same alkylating agent have been shown to decrease protein synthesis in relation to control.
INTRODUCTION Alkylating agents are commonly used as cytotoxic drugs for the treatment of some neoplastic diseases. Modified steroids, such as lactams, are used as bio-
logical carriers for transporting alkylating compounds to target tumours in a rather specific manner (Catsoulacos et al., 1976). This has been used after the discovery that steroidal hormones, as carriers of cytotoxic agents, reduce systemic toxicity and improve cancer therapy. Compounds, such as esters, in which the alkylating agent is linked to the steroid by an easily cleaved bond produce satisfactory activity (Wall et al., 1969). NSC 294859 and ASE are two molecules belonging to this category. NSC 294859 is a new homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenoxy acetic acid. The compound 3fl-hydroxy-13a-amino-13,17-seco-5aandrostan- 17-oic- 13,17-lactam-p-bis(2-chloroethyl)aminophenoxyacetate was prepared by heating 3/?-hydroxy-13a-amino-13,17-seco-5a-androstan-l7oic-I 3,17-lactam with p-[N,N-bis(2-chloroethyl)amino] phenoxyacetylchloride in benzene. The synthesis and the chemical properties of this compound have been reported previously (Catsoulacos et al., 1979). ASE is a homo-aza-steroidal ester of p-bis(2chloroethyl)aminophenyl acetic acid and consists of a nitrogen mustard that is bound to a modified steroid (a lactam, characterized by the group -CO-NH) via a phenyl acetic acid bond (Catsoulacos and Wampler, 1982) and has been synthesized in pure form (Wampler and Catsoulacos, 1977). Both compounds have been shown to have antineoplastic activity. ASE gives a 50% increased lifespan over controls in the treatment of L1210 leukemia in mice and a 150% increased lifespan in the treatment of P388 leukemia (Wampler and Catsoulacos, 1977). NSC 294859 seems to be more
drastic giving 100 and 383% correspondingly. ASE is also active against B,, melanoma on CS7b, mice and T, Guerin tumour on rats (Catsoulacos and Wampler, 1982). As to NSC 294859 the treatment of colon 26 tumour and Br6 melanoma gave positive antineoplastic activity, but the drug was not active in a melphalan resistant P383 line (Catsoulacos, 1983). In addition, both compounds also show mutagenic activity. ASE was found to induce sister chromatid exchanges in CHO cells (Athanasiou et al., 1983), and to be positive in strains TA1535 and TAlOO and in the newer strain TA102 with and without metabolic activation in the Salmonella/microsome system (Athanasiou and Arzimanoglou, 1986). This drug also induces sister chromatid exchanges and cell cycle delay in cultured human lymphocytes (Mourelatos et al., 1987). On the other hand NSC 294859 was found to be effective in causing statistically significant increases in sister chromatid exchange rates and cell division delays (Tselepi et al., 1989) and to induce chromosomal aberrations in human lymphocyte cultures (Tselepi et al., 1985). According to Maier and Schawalder (1986) most of the chemicals which exert mutagenic, carcinogenic or cytotoxic properties affect DNA synthesis, disturb the cell cycle and influence protein synthesis in individual cells. So it would be interesting to study the effect of ASE and NSC 294859, both of them being SCE inducers and mutagens, on protein synthesis rate. For this purpose the incorporation of 35S-methionine in newly synthesized proteins was measured in ovaries of D. melunoguster. MATERIALS AND METHODS
Test compounds NSC 294859 was dissolved in DMSO and two concentrations were tested, 10 and 2Opg/ml. ASE was also dissolved in DMSO and the concentrations used were 50 and
1251
G. STEPHANOU et al.
1252
(a) (Cl CH,CH,)s N
(b) (Cl CH,CH2)1 N
Fig. 1. Chemical structures of (a) 3~-hydroxy-13a-amino-13,17-seco-5a-androstan-l7-oic-l3,17-lactam-~bis(2-chloroethyl)aminophenylacetate (ASE). (b) 3~-hydroxy-13a-amino-l3,17-seco-5a-androstan-l7-oic13,17-lactam-p-bis(2-chloroethyl)aminophenoxyacetate (NSC 294859). 100 pg/ml. The chemical formulas of these compounds are given in Fig. 1. Lubelling
ofproteins
The strain C-S (Lindsley and Grell, 1968) of Drosophila melanogasfer was used in this study, and it is retained under standard conditions of temperature (25°C) and humidity (43 k 4% r.h.) in a standard cornmeal food. Ovaries from adult females 3-5 days old were excised and placed in depression slides in Robb’s saline (Robb, 1969). The solution contained 550 pCi/ml 3SS-methionine (Amersham, specific activity 1000 Ci/ml). Preparations were covered with coverslips and incubated at 25°C for 1 hr. The experimental preparations also contained the drugs, NSC 294859 at final concentration of IO and 20 pg/ml and ASE at final concentration of 50 and lOO~g/ml. There were also control preparations that either contained DMSO (4%) or none of the drugs were in the incubation medium. Protein synthesis
The rate of protein synthesis was measured by precipitation in 5 ~1 aliquots of homogenized ovaries with 10% trichloroacetic acid on Whatman 3MM filters as described by Mans and Novelli (1961). The radioactivity was counted in an LKB 1215 RACKBETA liquid scintillation counter. RESULTS
AND DISCUSSION
Several experiments were carried out in order to study the probable effect of NSC 294859 and ASE on protein synthesis. This was achieved by measuring the incorporation of “S-methionine into the total proteins of treated ovaries in Drosophila mefunogaster, Canton-S strain. Experiments were scheduled as follows: (1) Ovaries of C-S treated with 10 or 20 pg/ml of NSC 294859 for 1 hr and labelled simulTable
I. Incorporation
of “S-methionine treatment
taneously with ?S-methionine (550 pCi/ml) at 25°C. (2) Ovaries of C-S treated with 50 or 100 pg/ml of ASE for 1 hr and labelled simultaneously with “S-methionine (550 pCi/ml) at 25°C. (3) Ovaries of C-S treated with DMSO 4% for 1 hr and labelled simultaneously with 35S-methionine (550pCi/ml) at 25°C. DMSO is tested because the compounds are dissolved in DMSO 4%. (4) Ovaries of C-S that were not treated at all but were labelled with 35S-methionine at 25°C for 1 hr. The variability in ovary size among flies could be considered as a source of variation in those experiments, but this variation is not significant because: (1) Care was taken to select explanted ovaries of similar size (ovaries of somewhat reduced size were not used). (2) Six females were chosen randomly in each experiment and in the case where ovaries of reduced size were observed another female was used. (3) Each couple of ovaries was diversed and the first ovary was used in experimental sample and the other in control sample. (4) Our results are obtained from at least three or more experiments for each treatment. As it is shown in Table 1 NSC 294859 decreases the protein synthesis rate in both concentrations. We can see that at 10 pg/ml concentration the protein synthesis is reduced to 70.16% in relation to control preparation. This reduction seems to be higher at 20 pg/ml concentration where protein synthesis is observed to be at 68.81% of the control rate. The same seems to be true for the ASE although this compound seems to be less active not only because the concentrations tested were much higher than
in total ovarian proteins with NSC 294559
of D. melanogasrer
after
hLR/rnl)
(cumlovarv)
no treatment
% Incorporation in relation to DMSO treatment
NSC NSC NONE
IO 20 -
21135.24 20727.00 30122.40
70.16 68.81 100.00
74.34 72.75 -
DMSO
4%
28488.60
94.57
100.00
Concentration Comoound
% Incorporation relation to
Mean
Mean numbers have been extracted from
in
three or more different experiments.
New antitumour agents that alter protein synthesis rate those of NSC 294859 but also less reduction of protein synthesis rate was observed in relation to control samples. Table 2 shows that at SOpg/ml the protein synthesis rate is reduced to 78.87% while at 100 pg/ml it is reduced to 77.53%. DMSO seems also to slightly reduce the protein synthesis rate. Preparations treated with 4% DMSO show 94.57 (Table 1) and 97.21% (Table 2) of the control samples protein synthesis rate, for the experiments with NSC 294859 and ASE respectively. In addition, if the mean cpm/ovary of the drugtreated samples is compared with the samples treated with DMSO only, a profound decrease of protein synthesis is observed. For the NSC 294859 (Table 1) it is decreased to 74.34 and 72.75% for the 10 pg/ml and 20 pg/ml concentrations respectively and for the ASE (Table 2) it is reduced to 8 1.13 and 79.75% for the 50 and 100 pg/ml concentrations respectively. The effect of the two compounds under study on protein synthesis in isolated ovaries of D. melanogaster is also shown in Fig. 2. Most mutagens that have been studied show an interaction with proteins of individual cells. Excised root tips of Vicia faba exposed to 0.1% caffeine resulted in inhibition of protein synthesis to about 60% of the control rate (Zuk and Swietlinska, 1973). Cultured, freshly isolated rat fibroblasts that were exposed in vitro to vincristine sulphate, amethopterin, bleomycin and benomyl have shown a variable interaction with cellular proteins (Maier and Schawalder, 1986). Amethopterin and bleomycin resulted in increased protein content while vincristine sulphate did not significantly alter the protein content of individual cells. Stephanou and Demopoulos (1987) have shown that bleomycin treatment of preincubated mycelia of Aspergillus nidulans during heat shock enhances the incorporation of 35S-methionine into heat-shock bands. On the other hand it is known that proteins represent one of the biological molecules that react with alkylating agents (Vogel and Natarayan, 1981). Boffa et al. (1987) present data suggesting that qualitative and quantitative correlations between specific targets and alkylating agents apply not only to nucleic acids but also to DNAassociated nuclear proteins. In addition, it is not only the covalent modification of biomacromolecules such as DNA which causes genetic alterations. More subtle effects of the solvation coat, the tertiary structure and aggregation behaviour of proteins can also lead to important genetic changes (Zimmerman et al., 1985). Our results are in accordance with the above observation from the point of view that both alkylating agents tested, which are potent mutagens, also affect protein synthesis. This effect may be the result Table 2. Incorporation
5
SS-
A NSC 294659
a
60-
i !s 294959-DMSo . ASE-DMSO
55 -
501 ’ 0 10
’
20
Compound
’
’
30
40
’
so
Cancentrotiin
’
60
Mean @pm/ovary) 22381.68 22008.00 28384.44 27594.00
’
90
’
so
’
loo
of the interaction of the drugs with the DNA preventing the expression of some genes with protein products. However this hypothesis does not exclude the possibility that NSC 294859 and ASE act at different levels of translation. Acknowledgement-This study was supported by a grant from the Greek General Secretary of Research and Technology to N.A.D. REFERENCES
Athanasiou K. and Arzimanoglou J. (1986) Mutagenicity and clastogenicity of the antineoplastic agents homoaza-steroidal ester of p-bis(Zchloroethy1) aminophenyl acetic acid and chlorambucil. Mutat. Res. 175, 165-169. Athanasiou K., Demopoulos N. A. and Catsoulacos P. (1983) Chromosome damage and SCE induced by the cytostatic factor homo-aza-steroidal ester of p-bis(2chloroethyl) aminophenyl acetic acid in CHO cells in culture. Environ. Mutagen. 5, 219-283. Boffa L. C., Bolognesi C. and Mariani M. R. (1987) Specific targets of alkylating agents in nuclear proteins of cultured hepatocytes. Mutat. Rex 190, 119-123. Catsoulacos P. (1983) Activity of 3/?-hydroxy-13a-amino13,17-seco-5a-androstan-17-oic-13,17-lac~am-p-bis(2chloroethyl)aminophenoxyacetate (NSC 294859) on experimental tumor and leukemia systems. Oncology 40, 290-292. Catsoulacos P. and Wampler G. (1982) Activity of 3phydroxy- 13a-amino-13,17-seco-5a-androsanI ‘I-oic13,17-lactam-p[bis(2-chloroethyl)amino]phenyl acetate (NSC 290905) in murine solid tumors. Oncology 39, 109-l 12. Catsoulacos P., Boutis L. and Dimitropoulos K. (1976) Antitumor activity of steroidal lactone esters of N,N-bis (2-chloroethyl) aminophenyl acetic acid. Eur. J. Med. Chem. Chim. Therap. 11, 189-191. Catsoulacos P., Politis D. and Wampler G. (1979) A new steroidal alkylating agent with improved activity
of “S-methionine in total ovarian proteins of D. melanogaster after treatment with ASE
Concentration WW 50 loo 4%
’
70
(pg/ml)
Fig. 2. Reduction of protein synthesis rate in ovaries of D. melanogaster by the antitumour agents NSC 294859 and ASE.
% Incorporation in
ASE ASE NONE DMSO
1253
relation to no treatment
% Incorporation in relation to DMSO treatment
78.87 77.53 100.00 97.21
81.13 79.7s 100.00
Mean numbers have been extracted from three or more different experiments.
1254
G. STEPHANOUet al.
in advanced murine leukemias. Cancer Chemother. Pharmacol. 3, 61-10. Lindlsey D. L. and Grell E. H. (1968) Genetic variations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627, 431. Maier P. and Schawalder H. P. (1986) A two-parameter flow cytometry protocol for the detection and characterization of the clastogenic, cytostatic and cytotoxic activities of chemicals. Mutat. Res. 164, 369-379. Mans R. J. and Novelli G. D. (1961) Measurements of the incorporation of radioactive amino acids into proteins, a filter-paper disc method. Arch. Biochem. Eiophys. 14, 48-53. Mourelatos D., Petrou C., Boutis L., Papageorgiou A., Catsoulacos P. and Dozi-Vassiliades J. (1987) Induction of cytogenetic damage by modified steroidal derivatives of p-bis(2-chloroethyl)aminophenylacetic acid in human lymphocytes. Mutat: Res. l!%, 2055210. Robb J. A. (1969) Maintenance of imaainal discs of Drosophila melanigaster in chemically defined media. J. Cell Biol. 41, 876-888. Stephanou G. and Demopoulos N. A. (1987) Heat shock phenomena in Aspergillus nidulans. II. Combined effect of heat and Bleomycin to heat shock protein synthesis, survival rate and induction of mutations. Curr. Gener. 12, 443448. Tselepi R., Zacharopoulou A. and Demopoulos N. A. (1985) Genetic activity of 3/?-hydroxy-13a-amino-13, 17-seco-5a-androstan-17-oic-13,17-lactam-p-bis(2-chloroethyl)aminophenoxyacetate (NSC 294859) on human
lymphocyte cultures in vitro. Abstracts of 4th International Conference on Environmental Mutagens, p. 243. Stockholm, Sweden. Tselepi R., Demopoulos N. A. and Catsoulacos P. (1989) Induction of sister chromatid exchanges and cell division delays in human lymphocytes by the antitumour agent homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenoxy acetic acid. Mutagenesis 4(5), 361-364. Vogel E. and Natarayan A. (1981) The relation between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eucaryotic systems: interspecies comparisons. In Chemical Mutagens, Principles and Methods for their Detection (Edited by Hollander A. and De Serres F. J.), Vol 7, pp. 295-336. Plenum Press, New York. Wall M. E., Abernathy G. S. Jr., Carol1 F. J. and Taylor D. J. (1969) The effects of some steroidal alkylating agents on experimental animal mammary tumor and leukemia systems. J. Medicin. Chem. 12, 810-818. Wampler G. L. and Catsoulacos P. (1977) Antileukemic effects of homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenylacetic acid. Cancer Treat. Rep. 61, 3741. Zimmermann F. K., Groschel-Stewart U., Scheel I. and Resnick M. A. (1985) Genetic change may be caused by interference with protein-protein interactions, Mufat. Res. 150, 203-210. Zuk J. and Swietlinska Z. (1973) Effect of caffeine on nucleic acid and protein synthesis in Vicia faba. Mulat. Res. 17, 207-212.