phenotypic drug resistance in mammalian cells in vitro

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 incorporation 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 hypoxanthine uptake. The observed phenomena could be of importance for the selection of drugs to be used in cancer chemotherapy.

INTRODUCTION

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

307

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 resistance 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).

RESULTS

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


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 hypoxanthine. 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

1A

Number of colonies

15

lO

5

0 20 40 60 -tin 80 100 120

1B

Number of clones

20 40 60 80 100 120 1/.0 '160

10 2 CPM/IoOI.U

180 200

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.


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 up take of hypoxanthine , an exper iment was per formed analogous to the classic exper iment of Johannsen on the heredi ty of bean size (23).

Abou t 60 clones obta ined from B s c - i cells were isolated with a micropipe t in such a way that half of the clone was removed and the o ther ha l f left in the dish. This procedure al lowed for a subsequent de te rmina t ion of hypoxanth ine uptake in the unremoved par t of the clone. In this way it was possible to proceed only with those clones which were charac te r i zed by a high or a low uptake of hypoxanthine. Therefore , f rom the 60 clones, only 5 were p ropaga ted to cell lines, and the up take of hypoxanth ine in these cell lines was measured in clones derived from them (Tab le 2). In order to avoid semant ic ditt iculties, clones obta ined from the original popula t ion are cal led p r imary clones and clones derived from pr imary clones are cal led secondary clones (Tables 3, 4, and 5); t e r t i a ry clones are clones derived from secondary clonal populat ions ( table 4). Tab le 2 shows tha t three of the five clones have the same mean incorporat ion of hypoxanth ine as the pa ren ta l cells in Tab le 1; two clones are charac te r ized by a signif icantly lower incorporat ion, which points to a hered i ta ry factor in the var ia t ion in up take of hypoxanthine . W h e n there is such a he red i t a ry factor, one would expect tha t the clonal populat ions would have s t andard deviat ions which are lower than the s t andard deviat ion of the paren ta l populat ion. Accord ing to Tab le 2 this appears to be the case for three of the clones but not for the two others, which could mean tha t a new var ia t ion is present within the clones. Therefore the s tabi l i ty of the reduct ion 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


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 up take was de te rmined (Table 3). The la t te r table shows that the amount of incorporat ion re turns to tha t demons t ra ted by the paren ta l line and tha t therefore the t ra i t is not s table. To check whether this res torat ion occurs in all the cells concomitant ly , some clones der ived from passage 4 were isolated, grown to cell lines, and again tested (Table 4). I t tu rned out that , while two of the clones showed reversion to paren ta l values, one clone still demons t ra ted a reduced incorporat ion and one other had a significantly higher incorporat ion. This indicates tha t within the clonal cell populat ions new variat ions are ar is ing cont inuously and at a high frequency.

An explanat ion for this could be chromosomal var ia t ion as BSC-1 cells a re aneuploid and differences between clones in chromosomal content are bound to occur. This point was invest igated by per forming exper iments with normal human skin fibroblasts, which are charac te r i zed by a s table diploid karyo type (Table 5). This tab le shows tha t three of the seven clones are charac te r ized by a signif icantly lower up take of hypoxanthine . Therefore this phenomenon is also present in diploid cell strains. Also in this case no reduct ion in s t andard deviat ion is apparen t , which points to instabi l i ty 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


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 H G P R T 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 c p m / 1 0 0 IU. No significant differences in H G P R T activity were found. Also the cell extracts f rom 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 H G P R T is not responsible for the variation in uptake of hypoxanthine.

Mutation and Adaptation of BSC-1 Cells to 6-Thioguanine. No spontaneous 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 t reatment with E M S (3 • 10 -2 M, 2 h) mutants were obtained with a frequency of 0.9 • 10-5. Seven of these mutants tested for H G P R T activity had 0.5% or less H G P R T activity, and they did not incorporate hypoxanthine.

Adaptat ion to 6TG appeared possible by culturing the cells at first at low concentrations of 6TG and gradually increasing the amount of TG. Start ing at a concentration of 0.1 ~tg/TG/ml , the cells became resistant to 10 # g / m l 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% H G P R T 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


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/ m l , 1.5 ~tg TG/ m l , 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.

DISCUSSION

The purpose of this study was to obtain information on whether phenotypic 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 incorporation 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. I f 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-


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 H G P R T activity in these cell lines, which is, respectively, 184 (19) and 120 nmol /h /mg protein. Therefore the amount of HGP R T could reflect the number of gene copies per cell (24, 25) which could indicate that the BSC-1 cells have, on average, two H G P R T 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 H G P R T 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


further characterization of these steady states is needed. While it was shown that HGP R T 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 A T P availabil i ty might be involved.

Unders tanding 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. Fur thermore information on the phenomenon as such could be of interest for unders tanding the funct ioning of mammal i an cells, and it is possible that this will give insight into the still largely ill-understood processes of cellular differentiation and mal ignant t ransforma- tion.

ACKNOWLEDGMENTS

This research was sponsored jointly by the Foundation for Basic Medical Research (Fungo), Euratom (contract No. 102-722-BIAN), and the J.A. Cohen Inst i tute for Radiopathology and Radiat ion Protection. The valuable technical assistance of Miss S. de Vogel is gratefully acknowledged.

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phenotypic drug resistance in mammalian cells in vitro

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