Primed in Sweden Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in anyform resewed
ISSN00144827
Experimental
THE ISOLATION
Cell Research 104 (1977) 357-367
AND CHARACTERIZATION
ASPARAGINE-REQUIRING
MUTANTS
CHINESE
CELLS
HAMSTER
P. S. G. GOLDFARB, B. CARRITT,
OF OF
M. L. HOOPER and C. SLACK
Cancer Research Campaign Somatic Cell Genetics Group, Institute of Genetics, University of Glasgow, Glasgow, Scotland
SUMMARY Nine asparagine-requiring mutants were isolated in culture from the Don line of Chinese hamster cells. Investigation of the asparagine requirements of the mutants, the effect of asparagine deprivation on macromolecular synthesis, and the rates of reversion to asparagine independence indicated that there were differences between the mutant clones. Biochemical analysis revealed that the defect in the mutants was due to a deficiency of the enzyme asparagine synthetase, and that the enzyme activity in the mutants and Asn+ revertants obtained from them was not influenced by the concentration of asparagine in the growth medium. Complementation analysis by S&&i virusmediated cell fusion indicated that the lesion behaved as a recessive trait, and was probably located in the same gene in all the mutant clones.
The nutritional requirement of certain tu- contain increased levels of asparagine synmour-derived malignant cell lines for L- thetase [6,7,8, 161appear at low frequency asparagine has been well documented [ 141. in the population and are derived from The inability of such cell lines to synthesise mutational events, rather than as a response asparagine [5] has in all cases been shown to the absence of asparagine [17-211. In to be due to the absence of, or low level of, some instances [14,22] the level of enzyme asparagine synthetase (EC6.3.1.1) in cells activity found in the Asn+ cell lines and passaged both in vitro and as tumours in tumours can be increased by removal of vivo [6-8]. The sensitivity of these aspara- asparagine, suggesting that a regulatory gine-requiring (Asn-) cell lines and tumours system may be present. To date there has been no report of the to L-asparaginase (EC3.5.1.1) from either E. coli or guinea pig serum [9, 10, 111has isolation in cell culture of Asn- cells from formed the basis of chemotherapeutic ap- an Asn+ cell line. The possession of such proaches using asparaginase [12-U]. One cell lines would be of interest not only for Gndrance to these approaches has been the use in the biochemical and genetical anaappearance, when both Asn- tumours and lysis of the regulatory processes involved in 4sn- cell lines are treated with aspara- the expression of asparagine synthetase cinase, of cells which no longer require activity, but also to investigate the sugrsparagine (Asn+ cells). These cells which gested [18, 191 correlation between maExp
Cell
Res
104 (1977)
358
Goldfarb
et al.
lignancy and the appearance of an asparagine requirement in cell lines. In this paper we describe the isolation of Asn- cell lines from an Asn+ line of diploid Chinese hamster cells (Don), and the subsequent derivation from the Asn- lines of Asn+ revertants. Biochemical analysis of the Asnlines and their Asn+ revertants suggests that the observed defect in the Asn- lines is due to a deficiency of asparagine synthetase activity. Complementation analysis suggests that the lesion is probably located in the same gene in all the Asn- clones. MATERIALS
AND METHODS
Chemicals and radiochemicals Ethylmethanesulphonate, 5fluorodeoxyuridine (FUdR), Na*ATP, and L-asparaginase were obtained from Sigma. Aminopterin was purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. Colcemid was supplied by Ciba Laboratories, Horsham, Sussex. UV-inactivated Sendai virus was obtained from Searle Diagnostic Ltd, High Wycombe, Bucks. [6-3H]thymidine 20 Cilmmole, [5 ,6-3H]uridine 40 Ci/ mmole, Lj4,5-3H]leucine 40 Cilmmole, and L#J-“‘Claspartic acid 200 mCi/mmole were obtained from the Radiochemical Centre, Amersham, Bucks.
Cells and cell culture The Don line of Chinese hamster lung fibroblasts was obtained from the American Type Culture Collection, Rockville, Md, as was the Jensen Sarcoma (JS) line of rat cells. The Don TK- derivative a23 was isolated by Dr A. Westerveld and kindly supplied by him. Cells were normally grown in 90 cm* plastic tissue culture flasks (A/S Nunc) in Eagle’s medium (Glasgow modifica~on) contaiining 10% v/v foetal calf serum (GJBCO-Biocult) under an atmosphere of 5% CO, in air, at 37°C. For growth at low cell density (
ISSN00144827
Experimental
THE ISOLATION
Cell Research 104 (1977) 357-367
AND CHARACTERIZATION
ASPARAGINE-REQUIRING
MUTANTS
CHINESE
CELLS
HAMSTER
P. S. G. GOLDFARB, B. CARRITT,
OF OF
M. L. HOOPER and C. SLACK
Cancer Research Campaign Somatic Cell Genetics Group, Institute of Genetics, University of Glasgow, Glasgow, Scotland
SUMMARY Nine asparagine-requiring mutants were isolated in culture from the Don line of Chinese hamster cells. Investigation of the asparagine requirements of the mutants, the effect of asparagine deprivation on macromolecular synthesis, and the rates of reversion to asparagine independence indicated that there were differences between the mutant clones. Biochemical analysis revealed that the defect in the mutants was due to a deficiency of the enzyme asparagine synthetase, and that the enzyme activity in the mutants and Asn+ revertants obtained from them was not influenced by the concentration of asparagine in the growth medium. Complementation analysis by S&&i virusmediated cell fusion indicated that the lesion behaved as a recessive trait, and was probably located in the same gene in all the mutant clones.
The nutritional requirement of certain tu- contain increased levels of asparagine synmour-derived malignant cell lines for L- thetase [6,7,8, 161appear at low frequency asparagine has been well documented [ 141. in the population and are derived from The inability of such cell lines to synthesise mutational events, rather than as a response asparagine [5] has in all cases been shown to the absence of asparagine [17-211. In to be due to the absence of, or low level of, some instances [14,22] the level of enzyme asparagine synthetase (EC6.3.1.1) in cells activity found in the Asn+ cell lines and passaged both in vitro and as tumours in tumours can be increased by removal of vivo [6-8]. The sensitivity of these aspara- asparagine, suggesting that a regulatory gine-requiring (Asn-) cell lines and tumours system may be present. To date there has been no report of the to L-asparaginase (EC3.5.1.1) from either E. coli or guinea pig serum [9, 10, 111has isolation in cell culture of Asn- cells from formed the basis of chemotherapeutic ap- an Asn+ cell line. The possession of such proaches using asparaginase [12-U]. One cell lines would be of interest not only for Gndrance to these approaches has been the use in the biochemical and genetical anaappearance, when both Asn- tumours and lysis of the regulatory processes involved in 4sn- cell lines are treated with aspara- the expression of asparagine synthetase cinase, of cells which no longer require activity, but also to investigate the sugrsparagine (Asn+ cells). These cells which gested [18, 191 correlation between maExp
Cell
Res
104 (1977)
358
Goldfarb
et al.
lignancy and the appearance of an asparagine requirement in cell lines. In this paper we describe the isolation of Asn- cell lines from an Asn+ line of diploid Chinese hamster cells (Don), and the subsequent derivation from the Asn- lines of Asn+ revertants. Biochemical analysis of the Asnlines and their Asn+ revertants suggests that the observed defect in the Asn- lines is due to a deficiency of asparagine synthetase activity. Complementation analysis suggests that the lesion is probably located in the same gene in all the Asn- clones. MATERIALS
AND METHODS
Chemicals and radiochemicals Ethylmethanesulphonate, 5fluorodeoxyuridine (FUdR), Na*ATP, and L-asparaginase were obtained from Sigma. Aminopterin was purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. Colcemid was supplied by Ciba Laboratories, Horsham, Sussex. UV-inactivated Sendai virus was obtained from Searle Diagnostic Ltd, High Wycombe, Bucks. [6-3H]thymidine 20 Cilmmole, [5 ,6-3H]uridine 40 Ci/ mmole, Lj4,5-3H]leucine 40 Cilmmole, and L#J-“‘Claspartic acid 200 mCi/mmole were obtained from the Radiochemical Centre, Amersham, Bucks.
Cells and cell culture The Don line of Chinese hamster lung fibroblasts was obtained from the American Type Culture Collection, Rockville, Md, as was the Jensen Sarcoma (JS) line of rat cells. The Don TK- derivative a23 was isolated by Dr A. Westerveld and kindly supplied by him. Cells were normally grown in 90 cm* plastic tissue culture flasks (A/S Nunc) in Eagle’s medium (Glasgow modifica~on) contaiining 10% v/v foetal calf serum (GJBCO-Biocult) under an atmosphere of 5% CO, in air, at 37°C. For growth at low cell density (
