MUTATIONS OF CHINESE HAMSTER SOMATIC CELLS FROM 2-DEOXYGALACTOSE SENSITIVITY TO RESISTANCE JEAN-MICHEL CLAVERIE*, ARISTIDE COMLAN DE SOUZA* JEAN-PAUL THIRIONf
AND
Dbpartment de Microbiologie, Centre Hospitalier Uniuersitaire, Faculte de Mkdedm, Uniuersitk de Sherbrooke, Sherbrooke, Qukbec, Canada JIH 5N4 Manuscript received November I, 1978 Revised copy received January 23,1979 ABSTRACT
In Chinese hamster somatic cells, the spontaneous change of phcnotype from 2-deoxygalactose sensitivity to resistance was studied using fluctuation test experiments ri la LURIAand DELBRUCK(1943) for four Chinese hamster cell strains derived from V79. The results are consistent with true mutational events. The mutation rates are in the range of 1 to 3.5 x 10-5 per cell per generation. The relationship between the 2-deoxygalactose resistance and the galactokinase markers is discussed.
HE nature of the heritable variations of solmatic cells in culture is not well Tdocumented. There is, however, increasing evidence that they result from mutational events (i.e.,involving heritable nucleotide base changes, deletions or rearrangements in the primary structure of the DNA) instead of epigenetic events (see review by SIMINOVITCH 1976). Thus, the potential usefulness of a new heritable variation as a genetic marker will depend on its genetic characterization and the clear demonstration of its genetic and spontaneous random origin. THIRION, BANVILLEand NOEL (1976) and BANVILLEand THIRION (in preparation) have shown that 2-deoxygalactose (Dga) can be used with Chinese hamster somatic cells as a selective agent to obtain mutants that are resistant to its toxic action. These variants have either no, o r reduced, galactokinase activity (E.C.2.7.1.6) when compared to the wild type. THIRION, BANVILLEand NOEL (1976) and BANVILLE and THIRION (in preparation) have reported evidence that suggests a genetic origin for these variants; namely, the frequency of appearance of the resistant clmes increases after treatment with the mutagen ethyl methanesulfonate. The Dga-resistant phenotype is stable after many generations in the absence of Dga. Resistance behaves as a recessive trait. In this paper, we describe the results of several fluctuation tests ci Za LURIA and DELBRUCK (1943) performed with four Chinese hamster cell lines that we have used in order to isolate DgaR variants. A Kolmogorov-Smirnov statistical analysis of the results demonstrates clearly the spontaneous random appearance of the resistant phenotype in populations of wild-type cells and rules out any * The order of the first two authors was determined by the toss of a coin.
+ To whom reprint request should be addressed. Genetics 92: 563-572 June, 1979.
5 64
J.-M. CLAVERIE, A . C. D E SOUZA A N D J.-P. T H I R I O N
other induction process. This, plus the previous results, clearly demonstrates that the DgaRcharacter is the result of a genetic event and is therefolre a useful marker for the genetic analysis of Chinese hamster cells in culture. MATERIALS A N D M E T H O D S
Cells, media and culture conditions: Chinese hamster cells V79 were cloned three times in succession. V6, one of the clones, was used. V6 cells were grown for five years and recloned several times. Cells from frozen stocks of V6 and of the mutants were grown in air-CO, incubators a t 37" in petri dishes, glass bottles or roller bottles in minimal essential medium (MEM) with 8% fetal calf serum (FCS). A pedigree of the different cell lines used in this study is shown in Figure 1. A line Tyk6 resistant to BrdUrd and a line Hpt13 resistant to 8-azaguanine were isolated as and TALBOT (1978). Previously these lines were called Bu6 and A13. described by THIRION 2-deoxygalactose resistance test: The dose responses of wild-type and mutant cells were BANVILLE and NOEL (1976). The 2-deoxygalactose redetermined as described by THIRION, sponse of a strain was defined as the level of 2-deoxygalactose required for 50% inhibition of clone formation after ten to 16 days and designated as R50. Sensitive, partially resistant and fully resistant strains were defined as strains whose R50 values were in the ranges 0 to 0.6 mM, 0.6 mM to 6 mM and more than 6 mM, respectively. Enzyme assay and protein determination: The galactokinase specific activity of the different strains was determined as described by THIRION, BANVILLE and NOEL(1976). Incorporation of tritium from SH-galactose: About 2 x lo5 cell were seeded in a 3.5 cm petri dish with 2 ml of MEP4 plus 8% FCS and about 0.5 mCi of 0.01 miv 3H-D-galactose. When the cell layer had reached confluency (about two days later), the radioactive medium was removed and the cells washed twice with 2 ml of phosphate buffered saline (pH 7.2). After trypsinization, the cells were collected in 1 ml of phosphate buffered saline, lysed in 0.25 N NaOH and precipitated with 4 ml of 10% trichloroacetic acid solution. They were then collected in 2.4 cm diameter 1958) V79 (FORDand YERGANIAN cloning
mutagenesis
mutagenesis
Hptl3
Tyk6
1 mutagenesis
I
DR2
FIGURE 1.-Pedigree of the different strains used for mutagenesis and fluctuation analysis. Every mutagenesis was carried out with ethyl methanesulfonate as described previously (THIRIONand TALBOT 1978). Hptl3 and Tyk6 were previously called A13 and Bu6, respectively. The V6 cell line was grown for several years, and different culture isolates of V6 were made during that time.
565
MUTATIONS O F CHINESE IJAMSTER CELLS
glass-fiber filters (Whatman GF/A). The filters were washed with about 10 ml of a 5% trichloroacetic acid solution containing 0.01 mM D-galactose, and then with 5 ml of ethanol, dried BANVILLE and NOEL (1976). and the radioactivity counted as described by THIRION, Fluctuation test experiments: Fluctuations tests were performed for each of the four cell lines V6, Hptl3, Tyk6 and DR2 as follows: For each “experiment”, independent replicate cultures were initiated by seeding about 500 cells into each of 35 to 40 three-cm petri dishes containing 2 ml MEM with 8% of FCS. The cells were grown for seven days at 37” to a cell density of about 2 iz 0.2 x IO4 cells per dish. Each one of the replicate cultures was then trypsinized and seeded into a 10-cm petri dish with 48 miv Dga in MEM with 8% FCS for the selection to occur. For the “control,” about 500 cells were seeded as described above, but the cells were grown to a final density of about 4 iz 0.4 x 105 cells per petri dish. After this step, the culture was distributed equally into 20 samples in 10-cm petri dishes with 48 mM Dga in MEM with 8% FCS for the selection t o occur. After three days, for both the “experiment” and the “control,” the medium was removed and replaced by fresh selection medium with the same composition as before. Twelve days later, the resistant clones were stained and counted. RESULTS
Fluctuation tests: In order to understand the mechanism of the change in cell phenotype froni Dga sensitivity to resistance, we have performed fluctuation tests with four different cell strains; V6, Hptl3, Tyk6 and DR2, which were all derived from V79 (Figure 1) . V6, Hptl3 and Tyk6 strains are sensitive to Dga, while DR2 strain is partially resistant. Tables 1 to 4 show the results for each and DELBRUCK (1943), the spontaneous mutational cell line. According to LURIA appearance of the DgaRphenotype implies a large variance distribution f o r the TABLE 1
Fluctuation test with V6 cells Experiment
No. replicate cultures No. samplings per culture No. of replicates with N DgaR colonies: N = 0 colonies 1 2 3-4 5-8 9-1 6 >2000* No. of DgaR colonies per replicate: Range Mean Ratio, variance/mean Mutation rate (F, calculation)
Replicate-sample control
1
37 1
U)
27 1 0 3 2 2 2
12 2 3 3 0 0 0
0-12 1.37 7.1 1.1 i 0.2 x 10-5
0-3 0.85 1.6
* Although such high values are consistent with the expected fluctuations i n a test c i la LURIA and DELBRUCK (1944), they were not used for the calculation of the range, mean and variance in order to make the statistical test more sensitive (see text).
566
J.-M. C L A V E R I E , A. C. DE SOUZA A N D J.-P. THIRION
TABLE 2
Fluctuation test with H p t l 3 cells Experiment
1 20
26 1 1 1 3 3
14 1 3
0-14 1.74 7.2 1.0 i 0.2 x 1 0 - 5
0-3 0.65 1.7
1 2 3-4 5-8 9-1 6
No.of DgaR colonies per replicate: Range Mean Ratio, variance/mean Mutation rate (F, calculation)
Replicate-sample control
35 1
No. replicate cultures No. samplings per culture No. of replicates with N DgaR colonies: N = 0 colonies
e
0 0
number of resistant clones per petri dish for the “experiment” case (independent replicate cultures), whereas that number must follow a Poisson’s distribution (variance = mean) for the “control” (sampling of the same culture). In contrast, if the phenotypic change is induced by Dga (epigenetic event), the distribution for the number of resistant clones per petri dish must follow a Poisson’s law in both “experiment” and “control” cases. Our data are clearly in agreement with the mutational hypothesis since the ratios of the variance to the mean never exceed two for the “controls” but are always greater than six for the “experiments”. TABLE 3
Fluctuation test with Tyk6 cells Experiment
No. replicate cultures No. samplings per culture No. of replicates with N DgaR colonies: N = 0 colonies 1 2 3-4 5-8 9-16 No. of DgaR colonies per replicate: Range Mean Ratio, variance/mean Mutation rate (F, calculation)
Replicate-sample control
36 1
1 20
23
I2 0
6 0 2 3 2 0-13 1.6 6.7 1.5 i 0.3 x 10-5
3
5 0 0 0 4
1.15 1.9
567
MUTATIONS O F CHINESE HAMSTER CELLS
TABLE 4 Fluctuation test with DR2 cells Experiment
No. replicate cultures No. samplings per culture No. of replicates with N DgaR colonies: N = 0 colonies 1 2 3 4 5-8 9-1 6 17-32 33-64 65-1 28 2000* No. of DgaR colonies per replicate: Range Mean Ratio, variance/mean Mutation rate (Focalculation)
Replicate-sample control
36
1
1
U)
13
10
0-65 7.97 22 3.5 2 0.3 x 10-5
0-5 1.5 I.8
* See footnote Table 1.
A Kolmogolrov-Smirnov test was used to check thc compatibility of observed distribution with Poisson’s law, using the calculated mean of each sample as an estimate. We find that all the “controls” are compatible with a Poisson’s distsibution (P>0.05), whereas the “experiments” are not compatible at high significance levels (P
AND
Dbpartment de Microbiologie, Centre Hospitalier Uniuersitaire, Faculte de Mkdedm, Uniuersitk de Sherbrooke, Sherbrooke, Qukbec, Canada JIH 5N4 Manuscript received November I, 1978 Revised copy received January 23,1979 ABSTRACT
In Chinese hamster somatic cells, the spontaneous change of phcnotype from 2-deoxygalactose sensitivity to resistance was studied using fluctuation test experiments ri la LURIAand DELBRUCK(1943) for four Chinese hamster cell strains derived from V79. The results are consistent with true mutational events. The mutation rates are in the range of 1 to 3.5 x 10-5 per cell per generation. The relationship between the 2-deoxygalactose resistance and the galactokinase markers is discussed.
HE nature of the heritable variations of solmatic cells in culture is not well Tdocumented. There is, however, increasing evidence that they result from mutational events (i.e.,involving heritable nucleotide base changes, deletions or rearrangements in the primary structure of the DNA) instead of epigenetic events (see review by SIMINOVITCH 1976). Thus, the potential usefulness of a new heritable variation as a genetic marker will depend on its genetic characterization and the clear demonstration of its genetic and spontaneous random origin. THIRION, BANVILLEand NOEL (1976) and BANVILLEand THIRION (in preparation) have shown that 2-deoxygalactose (Dga) can be used with Chinese hamster somatic cells as a selective agent to obtain mutants that are resistant to its toxic action. These variants have either no, o r reduced, galactokinase activity (E.C.2.7.1.6) when compared to the wild type. THIRION, BANVILLEand NOEL (1976) and BANVILLE and THIRION (in preparation) have reported evidence that suggests a genetic origin for these variants; namely, the frequency of appearance of the resistant clmes increases after treatment with the mutagen ethyl methanesulfonate. The Dga-resistant phenotype is stable after many generations in the absence of Dga. Resistance behaves as a recessive trait. In this paper, we describe the results of several fluctuation tests ci Za LURIA and DELBRUCK (1943) performed with four Chinese hamster cell lines that we have used in order to isolate DgaR variants. A Kolmogorov-Smirnov statistical analysis of the results demonstrates clearly the spontaneous random appearance of the resistant phenotype in populations of wild-type cells and rules out any * The order of the first two authors was determined by the toss of a coin.
+ To whom reprint request should be addressed. Genetics 92: 563-572 June, 1979.
5 64
J.-M. CLAVERIE, A . C. D E SOUZA A N D J.-P. T H I R I O N
other induction process. This, plus the previous results, clearly demonstrates that the DgaRcharacter is the result of a genetic event and is therefolre a useful marker for the genetic analysis of Chinese hamster cells in culture. MATERIALS A N D M E T H O D S
Cells, media and culture conditions: Chinese hamster cells V79 were cloned three times in succession. V6, one of the clones, was used. V6 cells were grown for five years and recloned several times. Cells from frozen stocks of V6 and of the mutants were grown in air-CO, incubators a t 37" in petri dishes, glass bottles or roller bottles in minimal essential medium (MEM) with 8% fetal calf serum (FCS). A pedigree of the different cell lines used in this study is shown in Figure 1. A line Tyk6 resistant to BrdUrd and a line Hpt13 resistant to 8-azaguanine were isolated as and TALBOT (1978). Previously these lines were called Bu6 and A13. described by THIRION 2-deoxygalactose resistance test: The dose responses of wild-type and mutant cells were BANVILLE and NOEL (1976). The 2-deoxygalactose redetermined as described by THIRION, sponse of a strain was defined as the level of 2-deoxygalactose required for 50% inhibition of clone formation after ten to 16 days and designated as R50. Sensitive, partially resistant and fully resistant strains were defined as strains whose R50 values were in the ranges 0 to 0.6 mM, 0.6 mM to 6 mM and more than 6 mM, respectively. Enzyme assay and protein determination: The galactokinase specific activity of the different strains was determined as described by THIRION, BANVILLE and NOEL(1976). Incorporation of tritium from SH-galactose: About 2 x lo5 cell were seeded in a 3.5 cm petri dish with 2 ml of MEP4 plus 8% FCS and about 0.5 mCi of 0.01 miv 3H-D-galactose. When the cell layer had reached confluency (about two days later), the radioactive medium was removed and the cells washed twice with 2 ml of phosphate buffered saline (pH 7.2). After trypsinization, the cells were collected in 1 ml of phosphate buffered saline, lysed in 0.25 N NaOH and precipitated with 4 ml of 10% trichloroacetic acid solution. They were then collected in 2.4 cm diameter 1958) V79 (FORDand YERGANIAN cloning
mutagenesis
mutagenesis
Hptl3
Tyk6
1 mutagenesis
I
DR2
FIGURE 1.-Pedigree of the different strains used for mutagenesis and fluctuation analysis. Every mutagenesis was carried out with ethyl methanesulfonate as described previously (THIRIONand TALBOT 1978). Hptl3 and Tyk6 were previously called A13 and Bu6, respectively. The V6 cell line was grown for several years, and different culture isolates of V6 were made during that time.
565
MUTATIONS O F CHINESE IJAMSTER CELLS
glass-fiber filters (Whatman GF/A). The filters were washed with about 10 ml of a 5% trichloroacetic acid solution containing 0.01 mM D-galactose, and then with 5 ml of ethanol, dried BANVILLE and NOEL (1976). and the radioactivity counted as described by THIRION, Fluctuation test experiments: Fluctuations tests were performed for each of the four cell lines V6, Hptl3, Tyk6 and DR2 as follows: For each “experiment”, independent replicate cultures were initiated by seeding about 500 cells into each of 35 to 40 three-cm petri dishes containing 2 ml MEM with 8% of FCS. The cells were grown for seven days at 37” to a cell density of about 2 iz 0.2 x IO4 cells per dish. Each one of the replicate cultures was then trypsinized and seeded into a 10-cm petri dish with 48 miv Dga in MEM with 8% FCS for the selection to occur. For the “control,” about 500 cells were seeded as described above, but the cells were grown to a final density of about 4 iz 0.4 x 105 cells per petri dish. After this step, the culture was distributed equally into 20 samples in 10-cm petri dishes with 48 mM Dga in MEM with 8% FCS for the selection t o occur. After three days, for both the “experiment” and the “control,” the medium was removed and replaced by fresh selection medium with the same composition as before. Twelve days later, the resistant clones were stained and counted. RESULTS
Fluctuation tests: In order to understand the mechanism of the change in cell phenotype froni Dga sensitivity to resistance, we have performed fluctuation tests with four different cell strains; V6, Hptl3, Tyk6 and DR2, which were all derived from V79 (Figure 1) . V6, Hptl3 and Tyk6 strains are sensitive to Dga, while DR2 strain is partially resistant. Tables 1 to 4 show the results for each and DELBRUCK (1943), the spontaneous mutational cell line. According to LURIA appearance of the DgaRphenotype implies a large variance distribution f o r the TABLE 1
Fluctuation test with V6 cells Experiment
No. replicate cultures No. samplings per culture No. of replicates with N DgaR colonies: N = 0 colonies 1 2 3-4 5-8 9-1 6 >2000* No. of DgaR colonies per replicate: Range Mean Ratio, variance/mean Mutation rate (F, calculation)
Replicate-sample control
1
37 1
U)
27 1 0 3 2 2 2
12 2 3 3 0 0 0
0-12 1.37 7.1 1.1 i 0.2 x 10-5
0-3 0.85 1.6
* Although such high values are consistent with the expected fluctuations i n a test c i la LURIA and DELBRUCK (1944), they were not used for the calculation of the range, mean and variance in order to make the statistical test more sensitive (see text).
566
J.-M. C L A V E R I E , A. C. DE SOUZA A N D J.-P. THIRION
TABLE 2
Fluctuation test with H p t l 3 cells Experiment
1 20
26 1 1 1 3 3
14 1 3
0-14 1.74 7.2 1.0 i 0.2 x 1 0 - 5
0-3 0.65 1.7
1 2 3-4 5-8 9-1 6
No.of DgaR colonies per replicate: Range Mean Ratio, variance/mean Mutation rate (F, calculation)
Replicate-sample control
35 1
No. replicate cultures No. samplings per culture No. of replicates with N DgaR colonies: N = 0 colonies
e
0 0
number of resistant clones per petri dish for the “experiment” case (independent replicate cultures), whereas that number must follow a Poisson’s distribution (variance = mean) for the “control” (sampling of the same culture). In contrast, if the phenotypic change is induced by Dga (epigenetic event), the distribution for the number of resistant clones per petri dish must follow a Poisson’s law in both “experiment” and “control” cases. Our data are clearly in agreement with the mutational hypothesis since the ratios of the variance to the mean never exceed two for the “controls” but are always greater than six for the “experiments”. TABLE 3
Fluctuation test with Tyk6 cells Experiment
No. replicate cultures No. samplings per culture No. of replicates with N DgaR colonies: N = 0 colonies 1 2 3-4 5-8 9-16 No. of DgaR colonies per replicate: Range Mean Ratio, variance/mean Mutation rate (F, calculation)
Replicate-sample control
36 1
1 20
23
I2 0
6 0 2 3 2 0-13 1.6 6.7 1.5 i 0.3 x 10-5
3
5 0 0 0 4
1.15 1.9
567
MUTATIONS O F CHINESE HAMSTER CELLS
TABLE 4 Fluctuation test with DR2 cells Experiment
No. replicate cultures No. samplings per culture No. of replicates with N DgaR colonies: N = 0 colonies 1 2 3 4 5-8 9-1 6 17-32 33-64 65-1 28 2000* No. of DgaR colonies per replicate: Range Mean Ratio, variance/mean Mutation rate (Focalculation)
Replicate-sample control
36
1
1
U)
13
10
0-65 7.97 22 3.5 2 0.3 x 10-5
0-5 1.5 I.8
* See footnote Table 1.
A Kolmogolrov-Smirnov test was used to check thc compatibility of observed distribution with Poisson’s law, using the calculated mean of each sample as an estimate. We find that all the “controls” are compatible with a Poisson’s distsibution (P>0.05), whereas the “experiments” are not compatible at high significance levels (P
