Phenotypic drug resistance in mammalian cells in vitro
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Somatic Cell Genetics, Vol. 8, No. 3, 1982, pp. 307-317
Phenotypic Drug Resistance in Mammalian Cells in Vitro
P.C.E.M. Verschure and J.W.I.M. Simons
Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Sylvius Laboratories, Wassenaarseweg 72, 2353 AL Leiden, The Netherlands
Received 22 September 1981--Final 23 December 1981
Abstract--When mammalian cells are cultured at low concentrations of toxic drugs, they often become phenotypically resistant. We studied whether this phenotypic resistance is due to selection of preexisting variants. The drugs 8-azaguaine (AG) and 6-thioguanine (TG) were used and, as a parameter for resistance, the incorporation of hypoxanthine was determined. Preexisting variation among clones in the uptake of hypoxanthine was found, and this variation has a hereditary component. This transmission of aberrant incorporation of hypoxanthine does not appear a stable trait, and the aberrant cell lines returned gradually to the original steady state. There are indications that within a cell population cells with altered levels of incorpo- ration of hypoxanthine arise continuously and at a high frequency. Treat- ment with marginally toxic concentrations of AG or TG indicates that, at least for AG, survival is not related to the preexisting variation in hypoxan- thine uptake. The observed phenomena could be of importance for the selection of drugs to be used in cancer chemotherapy.
Until some years ago it was uncertain whether the mutants which are obtained in the mutational assay systems with mammalian cells in vitro are due to alterations in the DNA which codes for the genes involved or whether the mutants arise because of stable phenotypic variation (1-3). This question has largely subsided since it became gradually clear that mutants show alterations in their enzyme kinetics if stringent selection conditions are used (4--8). This is supported by the finding that with the BrdU-light methods mutants can be induced in specific parts of the S phase, indicating that these mutants are induced at the time of DNA replication (9). Moreover the genetic
0098-0366/82/0500-0307503.00/0 9 1982 Plenum Publishing Corporation
308 Verschure and Simons
nature of the mutants became evident in studies with repair-deficient mutants in which the defective repair of DNA damage results in elevated induced mutant frequencies (10, 11). Over the past years it also gradually became clear that a phenotypic drug resistance can be obtained if the selection conditions used are not so stringent and the nonmutant cells do not die shortly after the application of the selective drug. Unstable phenotypic drug resis- tance can be obtained after low concentrations of 8-azaguanine (12), and recently it has been shown that prolonged application of low concentrations of purine analogs may even lead to stable resistance (13). The same phenomenon also has been observed after prolonged application of dexamethasone (14) or other drugs (15). The nature of this stable phenotypic variation is unknown.
The aim of this study was to gain more insight in this phenomenon. We considered phenotypic drug resistance to be an important problem as it could occur in vivo in patients which are treated with cytostatics, leading to resistant tumor cells (16) or to very high frequencies of resistant lymphocytes (17). In particular, the question we wanted to study was whether phenotypic drug resistance was due to selection of cells or to adaptation of cells. Selection of cells could occur if there is a preexisting variation among cells which is continuously arising at a high frequency. For resistance to purine analogs this would be reflected in hypoxanthine uptake. The existence of variation in enzyme activities among clonal populations has been shown to occur (18). Therefore a method was developed to measure the uptake of hypoxanthine in a large number of clones. It was found that in mammalian cells a kind of steady state occurs in the uptake of hypoxanthine which can alter spontaneously. It is probable that this type of change is involved in the occurrence of stable phenotypic drug resistance.
MATERIALS AND METHODS
Cell Cultures. The experiments were performed with BSC-1 cells (Afri- can green monkey) and with human diploid skin fibroblasts. As standard medium Ham's F- 10 was used, modified by the omission of hypoxanthine and supplemented with 15% newborn calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin sulfate.
The cells were grown in plastic petri dishes. For subculturing, the petri dishes were rinsed once with Ca-free and Mg-free Hanks' BSS (Gibco Bio-cult) and incubated at 37~ for 5 min with 1 ml 0.5% trypsin and 0.02% EDTA (Gibco Bio-cult) in Hanks' BSS. The tryptic activity was neutralized by the addition of standard medium. For the experiments, petri dishes 90 mm (from Greiner, P90) and 30 mm in diameter (from Limbro, P30) were used. Clones were obtained within 14-21 days after seeding 150-300 cells per P90
Phenotypic Drug Resistance 309
and changing the medium twice a week. The clones were grown to at least 5-7 mm in diameter before studying their incorporation of hypoxanthine.
Measurement of Uptake of FH]Hypoxanthine in Clones. For this assay the medium from the cultures was replaced by F-10 medium without serum containing [3H]hypoxanthine (570 mCi/mmol, Amersham); 4 ml/P90 or 1 ml/P30. The final radioactivity was 7.2 ~Ci/ml. After 24 h the nonincorpo- rated hypoxanthine was removed from the cells by incubation in 5% cold TCA (Baker). Subsequently the cultures were fixed with methanol (Merck). The amount of incorporation was measured in a Philips liquid scintillator after cutting out the clones and dissolving the piece of plastic with the clone in scintillation fluid (toluene containing 0.4% PPO, Baker).
Determination of Amount of Protein in Clones in Situ. The clones were stained with a saturated solution of naphthol yellow S (NYS, Fluka) in methanol at pH 2.8 for 2 min. The NYS was removed by washing 3 times with methanol. Subsequently the extinction per clone was measured in a flying spot densitometer (Vitatron LTD100) connected with an integrating recorder (Vitatron UR406).
Hypoxanthine-Guanine-Phosphoribosyltransferase Assay. The cells were lysed in 0.01 M Tris HC1, pH 7.4. The HGPRT activity was assayed as described previously (19).
Determination of Variation in Uptake of Hypoxanthine in Clones in Situ. As it was considered important to characterize the hypoxanthine uptake of clones as soon as possible after their origin, the amount of uptake per unit protein was determined in the clones in situ. After the incorporation of labeled hypoxanthine, the clones were stained with NYS and the extinction per clone measured in a densitometer (see Materials and Methods). This measurement is in IU (integrator units). The incorporation per clone was expressed per 100 IU which corresponds with 3.2 #g protein or about 11,000 cells.
The application of NYS for staining and quantitative determination of proteins has been described (20, 21). This staining proved to be suitable for our purpose. The clones stain within 15 sec, and no further increase in staining was observed c~ver a period of 12 h. Extensive washing of the stained clones with methonol did not reduce the stain. The extinction is linearly related with cell density (measured up to 2.5 x 106 cells/P90). Furthermore the stain does not influence the number Of cpm during liquid scintillation counting.
The degree of accuracy of this method was tested by determining the uptake of hypoxanthine in colonies of BSC-1 cells. These colonies were obtained with droplets of 20t~l containing 8000 cells. These droplets were
310 Verschure and Simons
placed in P90 dishes, and the cells were allowed to attach for 6 h. After attachment, medium with [3H]hypoxanthine was given for 24 h after which the colonies were fixed and stained with NYS The mean incorporation per 100 IU in 42 colonies measured was 9652 cpm with a standard deviation of 2202 cpm (Fig. 1A). This standard deviation is 23% of the mean. In two other experiments this percentage was 30% and 26%, respectively. The weighed mean value for the standard deviation amounted to 28%, which can be considered to be the accuracy for each individual measurement. Therefore the 95% confidence limits for the determination will be about 12% for 20 measurements, 10% for 30 measurement, and 8% for 50 measurements.
In order to determine the biological variation among clones in the uptake of hypoxanthine, the incorporation was determined in 53 clones of BSC- 1 cells (Fig. 1B). A mean of 8267 cpm with a standard deviation of 3579 cpm was found. This standard deviation is 43% of the mean. Therefore the standard deviation in this experiment is much larger than the standard deviation obtained for the accuracy of the method (43% instead of 28%). This indicates that there is a biological variation among clones in the uptake of hypoxan- thine. This variation proved not to be correlated with the size of the clones. This uptake of hypoxanthine in clones of BSC-1 cells appeared to be quite reproducible in three experiments (Table 1), which supports the conclusion about the occurrence of a biological variation among clones.
Experiments on Heredity of Differences in Uptake of Hypoxanthine. To determine whether there is a hereditary component in this biological variation
Number of colonies
0 20 40 60 -tin 80 100 120
Number of clones
20 40 60 80 100 120 1/.0 '160
10 2 CPM/IoOI.U
Fig. 1. Histogram of [~H] hypoxanthine uptake in cell populations of BSC-1 cells. (A) Uptake of hypoxanthine in 42 colonies of BSC-1 cells. The colonies were obtained by means of droplets of cell suspension (8000 cells in 20 #1). (B) Uptake of hypoxanthine in 53 clones of BSC-1 cells.
Phenotypic Drug Resistance 311
Table 1. Incorporation of ~H-Labeled Hypoxanthine in Clones of BSC-1 Cells
Mean incorporation Standard deviation Number of of hypoxanthine expressed as
Experiment clones (cpm/100 IU/24 h) percentage No. measured 95% confidence limits of the mean
1 43 8421 _+ 1092 41 2 53 8267 _+ 988 43 3 64 8469 _+ 918 43
1 + 2 + 3 160 8389 _+ 562 43
among clones in the uptake of hypoxanthine, an experiment was performed analogous to the classic experiment of Johannsen on the heredity of bean size (23).
About 60 clones obtained from Bsc - i cells were isolated with a micropipet in such a way that half of the clone was removed and the other half left in the dish. This procedure allowed for a subsequent determinat ion of hypoxanthine uptake in the unremoved part of the clone. In this way it was possible to proceed only with those clones which were character ized by a high or a low uptake of hypoxanthine. Therefore, from the 60 clones, only 5 were propagated to cell lines, and the uptake of hypoxanthine in these cell lines was measured in clones derived from them (Table 2). In order to avoid semantic ditticulties, clones obtained from the original population are called pr imary clones and clones derived from pr imary clones are called secondary clones (Tables 3, 4, and 5); tert iary clones are clones derived from secondary clonal populations (table 4). Table 2 shows that three of the five clones have the same mean incorporation of hypoxanthine as the parental cells in Table 1; two clones are character ized by a significantly lower incorporation, which points to a hereditary factor in the variation in uptake of hypoxanthine. When there is such a hereditary factor, one would expect that the clonal populations would have standard deviations which are lower than the standard deviation of the parental population. According to Table 2 this appears to be the case for three of the clones but not for the two others, which could mean that a new variation is present within the clones. Therefore the stabil ity of the reduction in
Table 2. Incorporation of 3H-Labeled Hypoxanthine in Secondary Clones of BSC- 1 Cells
Number of Mean incorporation Standard deviation secondary of hypoxanthine expressed as
Primary clones clones (cpm/100 IU/24 h) percentage of BSC- 1 cells measured 95% confidence limits of the mean
P-I 42 3669 _+ 470 41 P-2 65 8082 _+ 932 47 P-3 75 8244 _+ 676 36 P-4 37 8626 _+ 978 34 P-5 57 6073 _+ 563 35
312 Verschure and Simons
Table 3. Incorporation of 3H-Labeled Hypoxanthine in Secondary Clones of BSC-1 Cells Obtained from Primary Clones at Different Passages
Number of Mean incorporation Standard deviation secondary of hypoxanthine expressed as
Primary Passage clones (cpm/100 IU/24 h) percentage clone number measured 95% confidence limits of the mean
P-5 4 57 6073 _+ 563 35 6 77 8602 _+ 961 49 7 57 8880 _+ 1006 43 9 53 8724_+ 731 30
P-I 4 42 3669 _+ 470 41 6 42 4415 _+ 1048 76 7 79 5605 _+ 549 43
16 72 8700 _+ 1141 42
hypoxanthine uptake was determined (Table 3). The latter table shows that the amount of incorporation returns to that demonstrated by the parental line and that therefore the trait is not stable. To check whether this restoration occurs in all the cells concomitantly, some clones derived from passage 4 were isolated, grown to cell lines, and again tested (Table 4). It turned out that, while two of the clones showed reversion to parental values, one clone still demonstrated a reduced incorporation and one other had a significantly higher incorporation. This indicates that within the clonal cell populations new variations are arising continuously and at a high frequency.
An explanation for this could be chromosomal variation as BSC-1 cells are aneuploid and differences between clones in chromosomal content are bound to occur. This point was investigated by performing experiments with normal human skin fibroblasts, which are character ized by a stable diploid karyotype (Table 5). This table shows that three of the seven clones are character ized by a significantly lower uptake of hypoxanthine. Therefore this phenomenon is also present in diploid cell strains. Also in this case no reduction in standard deviation is apparent, which points to instabil ity in these clones also.
Table 4. Incorporation of 3H-Labeled Hypoxanthine in Tertiary Clones of BSC-1 Cells Indicates Heterogeneity in Uptake of Hypoxanthine within Clonal Populations
Number of Mean incorporation Standard deviation tertiary of hypoxanthine expressed as
Primary Secundary clones (cpm/100 IU/24 h) percentage clone clone measured 95% confidence limits of the mean
P.1 P-I-i 22 5970 _+ 1056 40 P-l-2 11 8457 _+ 1987 35
P-5 P-5-1 3! 8215 2360 79 P-5.2 25 13162 1866 34
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Table 5. Incorporation of all-Labeled Hypoxanthine in Primary and Secondary Clones of Human Diploid Skin Fibroblasts ~
Mean incorporation Standard deviation Clones Number of of hypoxanthine expressed as derived clones (cpm/100 IU/24 h) percentage from measured 95% confidence limits of the mean
FS-2 50 4892 761 55 FS-2 29 4524 726 42 FS-8 40 4283 827 61 FS-8 39 4491 878 66 Clone P-ll 64 3780 470 50 Clone P-12 21 3230 794 54 Clone P-13 70 3950 339 36 Clone P-14 17 2500 737 58 Clone P-15 41 1370 e 208 48 Clone P-16 23 2660 548 48 Clone P-17 33 5410 1108 58
~The clones P1 I-P/7 were derived from FS-8.
Determination of HGPRT Activity in Clones Differing in Uptake of Hypoxanthine. The HGPRT activity was determined in the cell extracts from 21 BSC-1 clones which differed, at the time of isolation, in uptake of hypoxanthine from 1327 to 18,088 cpm/100 IU. No significant differences in HGPRT activity were found. Also the cell extracts from the two cell lines C25 and C26 with a reduced uptake (Table 2) showed the same activity as the parental line, which indicates that the amount of HGPRT is not responsible for the variation in uptake of hypoxanthine.
Mutation and Adaptation of BSC-1 Cells to 6-Thioguanine. No sponta- neous mutants resistant to 5 #g 6-thioguanine (6TG) could be recovered from 7.5 106 BSC-1 cells, probably due to the aneuploidy of the cells. The presence in these cells of more than one intact HGPRT gene will considerably reduce mutant frequencies. After treatment with EMS (3 10 -2 M, 2 h) mutants were obtained with a frequency of 0.9 10-5. Seven of these mutants tested for HGPRT activity had 0.5% or less HGPRT activity, and they did not incorporate hypoxanthine.
Adaptation to 6TG appeared possible by culturing the cells at first at low concentrations of 6TG and gradually increasing the amount of TG. Starting at a concentration of 0.1 ~tg/TG/ml, the cells became resistant to 10 #g/ml in 6 months and to 20 ~tg/ml in 2 more months. This trait was stable for the period tested (14 generations). Cell extracts showed 100% HGPRT activity, but the incorporation of hypoxanthine was reduced to 37% (3140 _+ 287).
Effect of Purine Analogs on Uptake of Hypoxanthine. In order to check whether low concentrations of purine analogs select for cells which are characterized by a low uptake of hypoxanthine, BSC-1 cells were seeded in
314 Verschure and Simons
Table 6. Incorporation of ~H-Labeled Hypoxanthine in Clones of BSC-1 Cells Formed in the Presence of Purine Analogs
Mean Standard incorporation deviation
Number of hypoxanthine expressed Relative of (cpm/100 IU/24 h) as
Purine Concentration cloning clones 95% confidence percentage analog 0tg/ml) efficiency measured limits of mean
100.0 64 8469 918 43 TG 1.0 8.2 40 5663 +_ 529 29 TG 1.5 1.2 52 4643 758 59 AG 5.0 4.7 22 7654 _+ 1308 39
medium containing 1.0 lzg TG/ml , 1.5 ~tg TG/ml , and 5 ~tg AG/ml. After formation of the clones this medium was removed and the uptake of hypoxanthine measured (Table 6). The clones grown in the presence of AG do not show an appreciable reduction in uptake, although the cloning efficiency is drastically reduced. In contrast the clones grown in the presence of TG do show a reduction in uptake. However, this reduction appears not as strong as could be expected by removal of clones with the highest uptake. The incorporation in the nine clones from the control group with the lowest uptake, which represent 15% survival, is lower than the incorporation actually found for clones from the TG groups with 1.2% and 8.2% survival. The data indicate that AG did not selectively remove clones with high uptake of hypoxanthine.
The purpose of this study was to obtain information on whether pheno- typic drug resistance is due to selection of spontaneously occurring variation in the incorporation of drugs or whether adaptional processes within the cells play a role. The data suggest that there is a large biological variation in clones in the uptake of hypoxanthine. Pooling of experiments gives a mean incorpora- tion of 8389 cpm for BSC-1 cells and a standard deviation of 3589. For human diploid skin fibroblasts these figures are 4571 and 2619. As the standard deviation for the accuracy of the determination is known, it is possible to calculate the standard deviation which is only caused by biological variation. This results in 8389 ___ 2714 for BSC-1 cells and 4571 ___ 2285 for the skin fibroblasts. These figures mean that there is a wide variation in uptake. If the distribution is normal, it means that there exist clones which show, over a period of 24 h, hardly any uptake in hypoxanthine.
Diploid fibroblasts are characterized by a higher variation than the aneuploid BSC-1 cells, indicating that the aneuploidy is not a main factor in the generation of this variation. Because of the large number of determina-
Phenotypic Drug Resistance 315
tions, the mean incorporation per clone can be measured quite accurately. The 95% confidence limit for the mean incorporation is 8389 cpm _+ 562 for BSC-1 cells and 4571 cpm _+ 410 for the fibroblasts. The ratio of these numbers corresponds roughly with the ratio in HGPRT activity in these cell lines, which is, respectively, 184 (19) and 120 nmol/h/mg protein. Therefore the amount of HGPRT could reflect the number of gene copies per cell (24, 25) which could indicate that the BSC-1 cells have, on average, two HGPRT genes per cell. This indication is reinforced by the finding that spontaneous mutants deficient in HGPRT were not observed and that the induced mutant frequency after 2 h treatment with 3 x 10 -2 M EMS was only 0.9 x 10 -5, which is much lower than the frequencies found for Chinese hamster cells (26) and mouse lymphoma cells (27).
The data obtained with daughter clones from isolated clones showed that a part of the variation in the use of hypoxanthine is transmitted to daughter cells over several generations. The same phenomenon was observed for both the aneuploid BSC-1 cells and the diploid human fibroblasts, indicating that this hereditary variation is not due to karyotypic differences between clones. Therefore the source of this variation could be called epigenetic. It appeared that this hereditary variation is not stable but that aberrant clones returned to parental values of incorporation. Therefore this reproducible amount of incorporation observed in the parental cells and in a number of the clonal populations points to a kind of steady state in substrate utilization. Sponta- neous alterations in this steady state, resulting in decreased or increased substrate utilization, do occur. These alterations will be subject to selection if nonstringent selection conditions are used, but this variation probably is not the only factor responsible for the phenotypic drug resistance, as the aberrant clones remain sensitive for purine analogs. However, the cell line which was obtained by culturing the cells in increasing concentrations of TG became ultimately insensitive to TG, despite its ability still to utilize hypoxanthine. That this latter cell line did not originate from a mutation is indicated by the rarity of mutants because of the supposed multiplicity of HGPRT genes and by the different nature of the mutants which were selected after EMS treatment.
The purine analogs AG and TG appeared to differ with respect to selection of clones with reduced uptake of hypoxanthine. While clones arising in low concentrations of TG were characterized by a reduced uptake of hypoxanthine clones arising in a low concentration of AG appeared normal in their uptake. This may be a consequence of the fact that the incorporation of TG occurs in the DNA while AG is mainly incorporated in the RNA (28, 29).
Our data indicate that steady states occur in the uptake of hypoxanthine and that stable and unstable alterations in these steady states do occur. A
316 Verschure and Simons
further characterization of these steady states is needed. While it was shown that HGPRT is not involved factors such as membrane transport, PRPP availability, relative activity of the salvage pathway to the activity of the de novo pathway, and also ATP availability might be involved.
Understanding the phenomenon of alterations of substrate utilization in steady states will be of importance in the development of cytostatic drugs to be used in cancer chemotherapy. Furthermore information on the phenomenon as such could be of interest for understanding the functioning of mammal ian cells, and it is possible that this will give insight into the still largely ill-understood processes of cellular differentiation and malignant transforma- tion.
This research was sponsored jointly by the Foundation for Basic Medical Research (Fungo), Euratom (contract No. 102-722-BIAN), and the J.A. Cohen Institute for Radiopathology and Radiation Protection. The valuable technical assistance of Miss S. de Vogel is gratefully acknowledged.
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