Isolation of ouabain-resistant human diploid fibroblasts

Cell, Vol. 3, 221-226,

November

1974,

Copyright

0 1974

by MIT

Isolation of Ouabain-Resistant Human Diploid FibroblastP

Seventeen clones resistant to the cytotoxic action of ouabain were isolated in culture by direct selection from 5 independent strains of diploid human fibroblasts. Resistant clones were recovered at frequencies on the order of IO-7 per wild type cell selected from populations treated with the mutagen EMS, but no resistant cells were detected among 108 unmutagenized cells. Most selected clones remained ouabain-resistant following further propagation in the absence of drug. The growth of wild type cells was inhibited by 50% at ouabain concentrations of 2-5 x 10-s M, while resistant clones required 15-180 fold higher drug concentrations to cause equivalent inhibition. Ouabain-resistant clones showed increased resistance of K+ transport function to ouabain inhibition that paralleled their increased resistance to growth inhibition. Initial experiments suggest that under selective conditions the resistant diploid fibroblasts differ significantly from wild type in binding of 3Houabain per unit surface area. The ouabain-resistant cells were similar to wild type in transport properties unrelated to ouabain inhibition, Resistant cells had normal karyotypes and senesced with a lifespan similar to control clones. The ouabain-resistant phenotypes of these diploid human fibroblast isolates apparently reflect point mutations that specifically affect the Na+/K+ transport ATPase with respect to ouabain-binding and/or response to bound ouabain.

see Thompson and Baker, 1973), but genetically marked diploid human cells have been :obtained;for the most part from individuals with hereditary abnormalities or polymorphisms (Krooth and Sell, 1970; Povey et al., 1973). It is importantthat capabilities for selection of particular mutants in culture be extended where possible to include diploid human cell material in order that the karyotypic normality of these cells can be exploited in somatic cell genetic and molecular biological investigations. Isolations ,in Lculture of several kinds of mutant human diploid fibroblast or lymphocytoid cells have now been reported (Albertini and DeMars, 1973; DeMars and Held, 1972; van Zeeland et al., 1972; Sato, Slesinski, and Littlefield, 1972; Pious, Hawley, and Forrest, 1973; Rappaport and DeMars, 1973). The traits selected to date are evidently recessive and recoverable as a consequence of X linkage or heterozygosity in the genomes of the parental cells. We report here the isolation in culture of human diploid fibroblasts carrying an evidently dominant or codominant genetic marker-ouabain resistance. Aneuploid mouse and hamster cell mutants selected for resistance to the cytotoxic effect of the drug ouabain have been described by Baker et al. (1974). Two noteworthy observations were that the frequency of mutant recovery was not dependent upon density of wild type cells selected, and that the drug resistance was codominantly expressed in somatic cell hybrids. Since ouabain-resistant mutants could also be obtained from the heteroploid human HeLa cell line (R. M. Baker, in preparation; Mankovitz, Baker, and Buchwald, 1973), it was clear that ouabain resistance might be an especially useful marker for work with diploid human fibroblasts. We have obtained clones of human diploid fibroblasts with substantially increased resistance to ouabain cytotoxicity in a number of independent single-step selections (Buchwald, Mankovitz, and Baker, 1973; Mankovitz, Buchwald, and Baker, 1973). The isolation and characteristics of these cells are described below. Our results imply that the ouabain-resistant cells are mutant and that they differ from wild type principally in relative resistance of Na+ /K+ transport to ouabain inhibition.

Introduction

Results

In recent years methods have been developed for the selection of a variety of mutants from aneuploid established lines of mammalian cells (for example

Selection of Ouabain-Resistant Diploid Fibroblast Clones Table 1 describes selections for ouabain-resistant human diploid fibroblasts from five independent wild type strains in two different laboratories (Toronto and Kingston). In each experiment, 2-12 x 106 normal skin fibroblasts were exposed to the mutagen ethyl methane sulphonate (EMS) under conditions that resulted in 20-40% survival.

Ft. Mankovitz Department of Pathology Queen’s University Kingston, Ontario M. Buchwald and R. M. Baker+ Research Institute The Hospital for Sick Children, and Departments of Medical Genetics Medical Biophysics University of Toronto Toronto, Ontario, Canada

and

Summary

‘:Address reprint requests to: Dr. M. Buchwald, Genetics Department, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. +Present address: Department of Biology and the Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.

Cell 222

After 2-6 days growth in the absence of EMS, 2-5 x 107 cells were cultured at appropriate inocula in ouabain at 4 x IO-7 M to 1 x IO-6 M. These selective drug concentrations were found in control experiments to result in complete elimination of wild type cells inoculated at 2.5 x 105 and at ~2.5 x 106 cells, respectively, per 100 mm tissue culture dish. The cultures were kept under ouabain selection until viable colonies could be identified, usually at 3-4 weeks. The phenotypically drug-resistant colonies were trypsinized and subsequently cultured in the absence of ouabain. In these experiments a total of 17 colonies were obtained from approximately 2 x IO* mutagenized cells subjected to ouabain selection for a recovery efficiency of approximately 8 x 1 O-8. Since plating efficiencies of the parental cultures in the absence of ouabain were on the order of IO%, we infer a mutant frequency on the order of one ouabainresistant cell per 106 wild type cells in the mutagenized parental populations. No colonies were recovered from a total of about 1 x 108 cells that were subjected to ouabain selection without prior exposure to mutagen. Characterization of Ouabain-Resistant Human Diploid Fibroblasts Ouabain-Resistance of Cell Multiplication Clones selected in ouabain were assayed in comparison to wild type for their ability to proliferate as a function of increasing ouabain concentration in the medium. As described in experimental procedures, cells were seeded in replicate dishes containing medium with various concentrations of ouabain, and after four or more days total cell number was determined for each condition. Figure 1 illustrates the results for two wild type and two ouabainresistant clones derived from the same parental strain. The extent of multiplication of wild type cells was reduced to half that of drug-free controls at 3 x IO-* M ouabain and was almost completely suppressed at 10-7 M. Multiplication of the two mu-

Table

1, Selection

of Ouabain-Resistant

Experiment Number

Number Strain

sex

treated

T-l

130

F

T-2

134

F

tant clones, however, was unaffected by ouabain concentrations up to 3 x 1 O-7 M, and was reduced to half that of drug-free controls only at concentrations in excess of 10-b M. Table 2 summarizes the results of such assays for wild types and six ouabain-resistant clones from four of the experiments listed in Table 1. To compare the various cell types, we have taken the concentration of ouabain necessary to reduce cell multiplication to half that in drug-free controls (Dso) as a parameter descriptive of the biological resistance

1

10-e Ouoboln

i 10-q

10-e concentrot~on,

M

Figure 1. Growth of Ouabain-Resistant 130-A (II) and 130-B (0) and of Wild Type 130-D and 130-E (A, n ) Clones as a Function of Increasing Concentration of Ouabain in the Culture Medium Replicate samples of 104 cells were seeded in 35 mm culture dishes containing ouabain at concentrations ranging from IO-9 M to 1 O-4 M, as well as in control dishes without drug. The plates were incubated for 6-6 days, and then the cells were trypsinized and counted with an electronic cell counter. The average number of cells in duplicate dishes is normalized to the number of cells in the control dishes for plotting.

Cells of cells with EMS

Number of Cells Plated in Ouabain

Number of Colonies Recovered

Concentration EMS &g/ml, 24 hr)

Expression Time (days)

12 x 106

150

2

4.5

x IO’

3

6 x 106

200

3

3.2 x IO’

2

K-3a

MFl

M

7 x 106

120

6

3.5

x 107

3

K-3b

MFI

M

4 x 106

120

4

3.0 x 107

1

K-4

MF2

M

2 x 106

120

3

5.0 x 10’

6

K-5

MF3

M

5 x 106

120

3

2.0

x IO’

2

21.2

x IO’

17

Various

F/M

10.0

x 107

Total Pool

0

Ouabain-Resistant 223

Table

2. Growth

Diploid

of Wild

Fibroblasts

Type

and Ouabain-Resistant

Clones

in Ouabain Molar Ouabain at 50% Growth

Concentration (Ds~)

Increase in Resistance

Strain

Clone

Phenotype

130

E L

WT

3.3

x IO-8

130

A

OUAR

2.0

x IO-6

60x

130

B

OUAR

3.3 x 10-b

100x

MFl

Parent

WT

2.8

MFl

4141b

OUAR

5.0 x 10-b

MFl

4143

OUAR

2.2 x 10-b

134

D*

WT

1.8 x IO-8

134

B

OUAR

2.8 x IO-’

MF2

Parent

WT

5.0 x IO-~

MF2

123

OUAR

5.0 x 10-b

“Not

selected

x 10-S 180x 80 x

15X

100x

in ouabain.

10~’ lO-5 Ouabaln cancentrst~on, M

Figure 2. The Dose-Response Curves for Wild Type MFI (0) and into Ouabain-Resistant as a Function of Ouabain Concentration

10-3

Relative 86Rb Flux into MFl-4141 b (0) Cells

The assay protocol is described in Experimental Procedures. Influxes into the two cell types were essentially the same in the absence of ouabain. The background at high ouabain concentration is a natural “ouabain-insensitive” flux. The error bars represent the standard deviations of the mean uptakes in triplicate samples.

to ouabain. The DsO.s were 1.8-5.0 x 1 O-8 M for wild type cells, while for the six resistant clones the D5,,s ranged from 2.8 x 10-7 M to 5.0 x 10-b M. These selected clones showed increases in resistance of 15 to 180 fold over their wild type control in terms of drug dose. Of four other ouabain-selected clones that were similarly tested, two exhibited wild type drug sensitivity and two showed resistances comparable to those described above. Thus 8 of 10 tested colonies that survived the selection regimens were reproducibly drug resistant. The ouabain-

resistant clones retained their phenotype over several months in culture to senescence. Karyotype The chromosomes of the ouabain-resistant clones 130-A, 134-B and MFl-4141 b were examined in comparison to the wild types using both banding and nonbanding techniques. These resistant clones had a normal diploid karyotype. Na+ /K+ Transport and Ouabain Binding Ouabain specifically inhibits plasma membrane Na+ /K+ transport ATPase (EC 3.6.1.3), (Schwartz, Lindenmayer, and Allen, 1972), and in ouabain-resistant aneuploid cells this transport activity has appropriately reduced sensitivity to the drug (Baker et al., 1974; R. M. Baker, unpublished data). We therefore examined the effect of ouabain upon K + influx into wild type and resistant diploid human fibroblasts in order to ascertain if there were analogous differences in the susceptibility to ouabain of Na+ /K+ transport in these cells. The data in Figure 2 illustrate the effect of increasing ouabain dosage upon the influx of *6Rb (a tracer for K+) into MFl wild type and the MFI4141 b mutant clone. Whereas for wild type cells the Vtb influx declined substantially for ouabain concentrations greater than 3 x 10-E M, the influx into the mutant cells was appreciably reduced only for drug concentrations in excess of 10-S M. In terms of dose there was a more than 200 fold difference in drug sensitivity of the cation influx. Similar results were obtained for the ouabain-resistant 130-A and 130-B clones in comparison to the wild type 130-D clone (see Figure 1). The clear analogy between the relative dose responses for ouabain inhibition of cell multiplication and of K + transport imply that the drug resistance of the mutants derives from an altered response of their Na+ /K-I- ATPase activity to ouabain.

Cell 224

The MFl wild type and 4141 b mutant cells were compared with respect to several other parameters of cation transport. Intracellular Na:K ratios in the absence of ouabain, as assayed from uptakes to equilibrium of *6Rb and 22Na tracers, did not differ significantly for the two cell types. They were also similar with respect to dependence of *6Rb influx upon external K + concentration. Half-maximal influxes were achieved at approximately 1.3 mM and 1.2 mM K+ , respectively, for the MFl wild type and 4141 b cells; maximum influxes, for ~7 mM external K+ , were found to be 1.4 & 0.1 (SD.) and 1.6 + 0.1 nmoles/lOs cells/min, respectively. We examined the binding of 3H-ouabain by wild type and ouabain-resistant fibroblasts under culture conditions comparable to those utilized for the original selection and for the cell multiplication and *6Rb influx assays (see Experimental Procedures). For the MFl-4141 b mutant, the rate of binding and equilibrium amount of ouabain bound per cell were approximately 25% lower than for a wild type MFl control culture (at equilibrium with 3 x 10-J M ouabain, 6 x 105 and 8 x 105 molecules per cell, respectively). Cell size spectra were determined for freshly trypsinized cultures equivalent to those assayed in the above experiments. These measurements indicated that the mean cell volume and standard deviation were more than twice as great for the 4141 b mutant culture (4400 f 2600 ~3) as for the wild type control (2000 i: 1000 ~3). Taken together, the data thus imply that these mutant cells bound less than haff the wild type level of ouabain per unit of apparent surface area-assuming spherical shape for the cells in suspension and equivalent binding capacities for attached and freshly suspended cells (see Boardman, Lamb, and McCall, 1972). The calculated differences in cell surface areas were in qualitative agreement with differences in saturation densities of the cultures. (Also, the mutants may have had a somewhat reduced V,,, for influx per unit surface area in the absence of ouabain.) We are reluctant to present these results as definitive because it was not possible to reconfirm the observations or to further study the relationships between binding, cell size, and influx under various assay conditions prior to exhaustion of the mutants’ growth potential. Nevertheless, in light of the additional controls described in the following paragraph, it seems apparent that these resistant diploid fibroblasts differed appreciably from wild type in Binding ouabain-binding characteristics. differences of comparable magnitude have been observed between wild type and some ouabain-resistant HeLa cell clones (R. M. Baker, in preparation). The differences in cell size noted between different diploid fibroblast cultures are probably attri-

butable to typical enlargement of cell volumes as the strains approach senescence (Simons, 1970), although we do not yet exclude a possible relation to the mutant phenotype. Quantitatively similar differences in cell volume spectra were observed in parallel control experiments where early and late passage cultures from a wild type strain were compared. Moreover, the wild type cells nearing senescerise bound substantially more ouabain per cell than cells of the same strain at an early passage, so that a constant number of binding sites per unit of apparent surface area was maintained. On the other hand, the early and late passage wild type cells did not differ significantly in intracellular Na:K ratios and varied only slightly in the ouabain sensitivity of *6Rb influx. Discussion Clones with substantially increased resistance to ouabain cytotoxicity were obtained in culture by direct single-step selections from a number of independent wild type populations of diploid human fibroblasts. There were no visible abnormalities in the karyotypes of such ouabain-resistant fibroblast clones. Their characteristics lead us to believe that they are somatic cell mutants. First, for most selected clones the ouabain-resistance phenotype was reproducible, and it was stable in the absence of further selection. Second, the resistant clones were considerably more frequent in populations treated with the chemical mutagen EMS (17 per 2.1 X IO* cells selected) than in unmutagenized populations (0 per 1 x IO* cells selected). Third, the several ouabain-resistant clones examined showed a specific functional alteration-increased resistance to ouabain inhibition of K+ transport-that corresponded closely in extent to their increased resistance to ouabain cytotoxicity. A resistant fibroblast clone appeared similar to wild type in transport and growth properties unrelated to ouabain inhibition, but it appeared to bind fewer ouabain molecules per unit surface area under selective conditions. These observations strongly suggest that the ouabain-resistant character in these cells is due to point mutation that specifically affects the binding of ouabain and/or the response to bound ouabain of the Na+ /K+ ATPase transport enzyme. Except that the relevant drug doses are orders of magnitude lower, the properties reported here for the ouabain-resistance trait in diploid human cells seem generally consistent with those previously described for the ouabain resistance character in aneuploid rodent cells (Baker et al., 1974). Ouabain resistance in Chinese hamster ovary (CHO) cells was shown to be codominant or incompletely dominant in cell-cell hybridization experiments.

Ouabain-Resistant 225

Diploid

Fibroblasts

The occurrence of ouabain-resistant diploid human cells that can be recovered in a single step and are presumably singly mutant is compatible with a dominant nature for the character in these cells as well. It does not seem plausible that each of the five different wild type populations, derived from randomly chosen individuals, could have been heterozygous for an undiscovered recessive ouabain-resistance marker. On the other hand, we certainly do not rule out the possibility that the affected gene(s) is functionally hemizygous-for example, X linked-in the diploid cells. Indeed, the dose response pattern for drug resistance of transport function exemplified in Figure 2 suggests that this may be the case. For the mutant diploid cells there is little reduction in transport activity at doses strongly inhibitory for wild type cells. These results contrast to those for single-step ouabain-resistant mutants of heteroploid HeLa cells, where the dose responses for influx follow a more complex pattern that implies continued presence of a ouabain-sensitive component in addition to a ouabain-resistant component of transport activity (R. M. Baker, in preparation). The difference between the heteroploid HeLa and the diploid fibroblast mutants could mean either that the ouabain-resistance character is X-linked or that the two classes of mutants involve different loci or alleles. Experimental

Procedures

Media and Culture Conditions Cells were maintained in DI medium (Stanners, Eliceiri, and Green, 1971) supplemented with 12-15% fetal calf serum (Flow Laboratories) at 37°C in 5-10% C02. Trypsin (Difco Laboratories) at 0.1% or 0.25% in citrate saline (134 mM tri-sodium citrate and 15 mM KCI, pH 7.8) was routinely used for removing cells from their substrate and preparing single cell suspensions. Ouabain was obtained from Sigma Chemical Company. Basic cell culture techniques and procedures employed in our laboratories have been discussed elsewhere (Thompson and Baker, 1973). Cell Cultures Biopsies of skin or foreskin were minced and subsequently treated by either of 2 methods. In one method the pieces were immobilized under 22 mm2 coverslips in several tissue culture dishes containing medium. The medium was replaced 2 or 3 times weekly, and the primary outgrowths were usually subcultured within 2 or 3 weeks, and again about one week later when the primary subcultures had become confluent. A second procedure was employed with a view to maximizing the recovery of cells capable of proliferation from a given biopsy. In this procedure, the minced pieces were stirred in approximately 10 ml of trypsin solution for 30-45 min at room temperature. The trypsin solution containing floating cells was separated from the undigested biopsy pieces by careful pipetting. The solution containing cells was combined with an equal volume of serum-containing medium, centrifuged, resuspended in medium, and temporarily stored in ice. This procedure was repeated with the undigested tissue three times. The second, third, and fourth collections were pooled, inoculated into a 100 mm dish, and incubated at 37°C. A monolayer containing 2.5 x 106 cells could be recovered in as little as 9 days and routinely within 2-3 weeks.

We have recently observed that using this second procedure one can recover 2-5 X 106 cells routinely within 2-4 days from a biopsy if the trypsin solution is replaced by collagenase (Type I, Sigma Chemical Co.) dissolved in phosphate buffered saline (PBS) supplemented with 5.5 mM glucose (G. W. Conrad, personal communication; Conrad and Hart, 1973). When collagenase is used, the digestion is performed at 37”C, and the first cell collection is retained along with the second and third. Cells from the second or third subculture were used for selection experiments or stored in a liquid nitrogen or -100°C freezer. Selection Experiments One hundred mm dishes were inoculated with 4-6 x 105 cells per dish and incubated for l-2 days, at which time ethyl methane sulfonate (EMS) was added to the cultures at concentrations of 120200 pg/ml. Twenty four hr later the medium was removed, the cells were washed twice with serum free medium or PBS, and fresh drug-free medium was added to each culture. The cells were incubated, with subculturing when necessary, for 2-6 days; at this time the cells were subcultured into medium containing ouabain at the appropriate concentration and incubated for 3-4 weeks, usually without medium changes. Ouabain-resistant colonies could be detected microscopically within 2-3 weeks and could be seen by the eye 3-4 weeks after the addition of ouabain. The colonies were isolated and cultured until there were enough cells for assays and freezing. In an attempt to provide age-matched controls, wild type cells were inoculated at low concentrations into drug-free medium to provide colonies that were picked at the time that the experimental dishes contained mutant colonies. For the K series of selections, however, controls consisted of unmutagenized cells frozen at the time that experimental cells were mutagenized. There did not appear to be any significant difference between these two types of controls with respect to ouabain inhibition of cell proliferation or K+ uptake. Cell Proliferation Assays Replicate samples of cells were inoculated into 35 or 60 mm diameter tissue culture dishes containing appropriate concentrations of ouabain. The cells were cultured for 4-21 days, at the end of which time the medium was removed and the cells were trypsinized and counted with an electronic cell counter. At concentrations at which ouabain is inhibitory, the maximum cell yield per culture is depressed even after prolonged incubation, and the concentration that resulted in a cell proliferation half that of controls was not significantly affected by the duration of incubation, provided that it was at least 4 days. Isotope Assays **Rb influx and3H-ouabain-binding assays were performed according to procedures based on those of Vaughan and Cook (1972). For each sample, a glass scintillation vial (Wheaton Glass Co.) was seeded with l-2 X lo5 cells in 2 ml of normal culture medium to yield a uniform but nonconfluent monolayer of cells on the bottom of the vial after overnight incubation. For measurements of initial rates of *Qb uptake (influx), unlabeled ouabain was then introduced to the desired concentration and allowed to bind for 3 hr at 37°C. (At 3 X 10-r M ouabain, 6 mM K+ , specific ouabain binding saturates in about 2 hr.) After this interval, the vials were inoculated with @Rb (New England Nuclear) at approximately 1 pCi/ml (0.1 mM), and transport was allowed to proceed for 15 min at 37OC. The medium was then removed and the vials washed with 4 consecutive aliquots of 20 ml ice cold PBS. Distilled water was added to lyse the cells, and the retained radioactivity was determined by assaying Cerenkov radiation in the aqueous medium on a scintillation counter. Ouabain binding to the cells was examined using an analogous protocol to follow the retention with time of SH-ouabain (New England Nuclear) at l-10 x 10-b M ouabain (about 12 Ci/mM)

Cell 226

in normal culture medium (6 mM K+). After washing, the cells were lysed with 2 ml distilled water, and 8 ml Aquasol (New England Nuclear) was subsequently added for scintillation counting. Influx as a function of K + concentration was determined in similar experiments where the normal culture medium was removed, the cells were washed in PBS minus KCI, and 86Rb was added in medium initially prepared without KCI and then supplemented with 5% FCS and KCI to the appropriate K+ concentrations. In calculating influxes, the effective specific activity of 8*Rb was corrected as appropriate, assuming that the 86Rb+ tracer is indistinguishable from K+ and that the transport of the two ions is identical (Vaughan and Cook, 1972). Internal ratios of Na and K in the cells were evaluated by comparing uptakes at equilibrium (44 hr) of *2Na (Amersham-Searle) and *bRb by confluent monolayers. 2*Na was assayed by gamma counting after extraction of the cells with distilled water. Each data point was assayed in triplicate. Typical standard deviations among the replicate samples were 5 10% of the mean and appeared to be largely attributable to variations in the total number of cells retained per vial through the washing procedures. Cell counts were determined on six replicate vials that had been processed through the washing procedure and then trypsinized. Cell Sizing Cell volume distributions were determined for cells freshly trypsinized from appropriate scintillation vial cultures, using an electronic cell counter (Taylor, 1970) equipped with multichannel analyzer, in the laboratory of Dr. R. Miller of the Ontario Cancer Institute. Acknowledgments We thank Marie Florian, Souren Chekijian, James Kao, lzzat Khapoya, and Teresa Lam for their technical assistance with various experiments, and Mark McCutcheon for his generous aid in measuring the cell size spectra. We are grateful to Louis Siminovitch and Aser Rothstein for their encouragement and comments on the manuscript. A portion of this work was carried out at the Ontario Cancer Institute. The work was supported by the Medical Research Council of Canada and the National Cancer Institutes of Canada and the United States. Received

August

20, 1974

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