dna transformation studies with mammalian cells in culture

DNA Transformation Studies with Mammalian Cells in Culture

T. RAY BRADLEY,' ROBERT A. ROOSA2 AND LLOYD W. LAW3 Laboratory of Biology, National Cancer Institute, Bethesdu, Md., and The Wistar Institute, Philadelphia, Pa.

A series of experiments using mammalian cells was designed analogous to those with bacteria in which resistance to drugs has been induced with desoxyribonucleic acid extracted from populations of cells resistant to the drug (Hotchkiss, '51).

In the present experiments resistance to 8-azaguanine, a purine analog, has been the genetic marker used. Since resistance to 8-azaguanine has been shown to occur in distinct large steps both in vivo (Law, '51) and in vitro (Szybalski and Smith, '59; and Roosa et al., '61) and to be stably inherited the phenomenon of resistance to this drug appeared to be eminently suitable for experiments of this nature. Two types of experiments will be de- scribed. In the first series of experiments parental cells (sensitive to 8-azaguanine), after incubation with DNA preparations, yielded cells which grew to form colonies resistant to the drug. In the second series of experiments using different cultural conditions an artifact, which produces results simulating resistance to 8-azaguanine, has been demonstrated. The rele- vance of these latter observations to the first series of experiments is discussed.

EXPERIMENTAL MATERIALS AND METHODS

Cell line characteristics All experiments were completed i n vitro

using cloned continuous cell lines isolated from P388, a lymphocytic leukemia first established i n vitro by Dawe and Potter ('57). Herzenberg and Roosa ('60) studied conditions for optimal nutrition and the growth of dispersed cell suspensions into discrete colonies. Subsequently Rossa et al. ('62) by selection with 8-azaguanine, isolated and characterized cloned sublines,

which were stable in their resistance to the inhibitor.

Medium and culture conditions Cell lines were maintained in pyrex

Blake bottles in Eagle's basal medium supplemented with L-serine (0.2 mM), sodium pyruvate (1 mM) and whole calf serum (5% ). This will be referred to as complete growth medium. Eagle's basal medium containing L-serine and sodium pyruvate but not calf serum will be referred to as Basal Medium.

Characterization of cell populations with respect to their growth inhibition by 8- azaguanine was made by inoculating cell suspensions into pyrex petri dishes (6 cm diameter), containing various concentrations of 8-azaguanine. The dishes were incubated at 37°C in a humid atmosphere of 5% COz and 95% air for seven days. In these cases a known number of cells, approximately 300-500, was used per dish and the plating efficiency, the per cent of cells growing into colonies in the absence of drug, determined for each experiment. Table 1 shows the behavior of the parental and resistant sublines to 8-azaguanine.

DNA preparations Extraction of DNA from various cell

populations or tissues was made according to the method of Kay, Simmons and

Received July 6, '62. Accepted July 27, '62. 1Part of the investigation was carried out while

serving as Visiting Associate, NCI, and part under a grant from the Anna Fuller Fund. Present address: Department of Physiology, University of Melbourne, Australia - supported by PHS Grant C-5360 and Anti-cancer Council of Victoria.

2Part of the investigation was carried out during the tenure of a post-doctoral fellowship awarded b the NCI. Part was supported by a PHS Researcg Grant C-4134 and by Research Grants E88A and E89D from the American Cancer Society. Present address: The Wistar Institute, Philadelphia, Pennsylvania.

a Laboratory of Biology, NCI, Bethesda, Maryland.

127

128 T. RAY BRADLEY, ROBERT A. ROOSA A N D LLOYD W. LAW

TABLE 1 Percentage of cells from various sublines o f P388 capable o f forming colonies

in the presence of 8-azaguanine (AZG)

Concentration of AZG

P388 Sublines]

Parental AZGr-1 AZGr-2 AZGr-3 No.22 No. 1 R B N0.R-58 No. 55

5 X Molar 1002 1 x Molar 9 0 95 2 X Molar 7 9 3 X Molar 5 9 77 5 X lo-' Molar 1 0 6 9 8 x Molar 0 1 x 10-BMolar 0 64 100 2 x lo-" Molar 21 3 x Molar 9 58 5 X lo-" Molar 0 6 x Molar 8 X lO-"Molar 1 x 10-5 M O ~ S 40 3 x 10-5 M O ~ X 2 5 8 x Molar 0 1 x lo-* Molar 1 X Molar 100 2 x 10-3 M O ~ S 9 2 5 X Molar 24 1 X Molar 0

1Parental line, P388/P, was a cloned line. 388/AZGr-l was selected and cloned after exposure of parental cells to 1 x 10-8 Molar AZG. 388/AZGr-2 after exposure of parental cells to 2 x 10-5 M 8-AZG; 388/AZv-3 after exposure of AZGr-2 cells to 1 X 10-4 M 8-AZG. .All sublines have maintained their resistance following continuous passage in inhibitor-free me&a and following lengthy periods of storage at -70°C.

x 100. 2 Number of cells forming colonies in presence of inhibitor Number of cells forming colonies in absence of inhibitor

Dounce ('52). The solutions were stored either at 6°C or frozen at -20°C.

Estimation of the DNA content of the preparations was made by the method of Stumpf ('47).

Various dye stacking parameters and other information was obtained by Dr. Audrey L. Stone, National Institute of Mental Health, from one DNA preparation used here, no. 1-DNA-R. This preparation was found to be comparable, with respect to nativity, with bacterial preparations known to have transforming activity. No dialyzable ultraviolet absorbing fraction was obtained from this preparation.

Preparation of cultures for seTies 1 experiments

As a standard procedure in preparation of cultures of parental cells for incubation with DNA extracts, 50 ml soda glass pre- scription bottles were inoculated with cells in 5 ml of growth medium. At various times after the cells had become attached to the glass, the growth medium was removed and the cultures were incubated

with the various DNA preparations. The medium used for incubation of these subcultures with DNA extract was either 0.14 M NaC1, Locke's solution or Basal medium. Control cultures were incubated with the same medium and for the same times as those treated with DNA. After the DNA incubation normal growth medium was added and the cells were used for plating experiments at various times.

For plating experiments the growth medium was removed and replaced by 2 ml of fresh growth medium. The cells were scraped into this medium with a rubber policeman, mixed and counted in a hemo- cytometer chamber. Usually 0.1 ml to 0.5 ml of this cell suspension was th,en added to a flask containing 150-300 ml of growth medium, stirred magnetically with a Tef- lon covered rod and dispensed with a Cornwall automatic syringe into petri dishes containing a concentration of 8- azaguanine known to arrest the growth of all but resistant cells.

In this series of experiments large numbers of cells were inoculated per dish,

DNA TRANSFORMATION STUDIES 129

routinely, 10,000 viable cells per dish as determined by lack of nigrosin staining (Kaltenback et al., '58).

Detection and counting of colonies The colonies were stained by the addi-

tion of 2 ml of 0.1% aqueous crystal vio- let directly to the 5 ml of medium in the dishes. After standing for 20 minutes the fluid was poured off, the dish very gently washed with distilled H 2 0 and inverted to drain and dry. The dishes were then examined on a grid with a binocular dis- secting microscope at X 13 magnification.

In series 1 experiments, where usually only a few colonies were found per dish, the colonies were circled with ink while being observed at X 13 magnification and then, on another microscope, the number of cells per colony was counted at X 125 magnification. Since it had been determined, in a preliminary experiment, that cells resistant to 8-azaguanine grew from single cells to a colony of 64 cells (average value) in seven days under these incubation conditions, the criterion was adopted that only those colonies having 64 or more cells were counted.

All results in the series 1 experiments were then calculated as the number of colonies found per 1 X lo6 efficient cells plated. Since plating efficiency varies from culture to culture, this was determined for each culture plated and used in calcula- tions of the results. For example, in an experiment where a total of 200,000 cells were plated and a plating efficiency determination gave a value of 20% then the results are calculated on the basis of 40,000 efficient cells used.

RESULTS OF SERIES 1 EXPERIMENTS

Since these experiments were of an ex- ploratory nature, a variety of conditions were used with respect to the exposure of the cultures to the DNA extracts. These included the stage of growth of the culture, the duration of the incubation with DNA extract, the incubation medium, the amount of DNA extract used, and the duration between the first exposure to the DNA and the plating out of the cells.

Table 2 shows the results obtained in several of the experiments in this series. For each experiment the number of colo-

nies arising from cell samples from the control cultures are compared with those arising after DNA treatment of identically prepared cultures.

In each experiment the DNA treated cultures yielded larger numbers of resistant colonies than the control cultures and this quantitative difference was not re- lated to any differences in the plating efficiencies of cells from the control and DNA treated colonies. This table presents experiments in which a single dosage of DNA was used as well as others in which the cells were exposed to DNA several times. The length of incubation in the presence of DNA and the period in the growth cycle of these cultures after split- ting has been varied. Many of the experiments were also done with several concentrations of 8-azaguanine.

Considering only those experiments in which 1 X M 8-azaguanine was used, the experiments can be divided into two groups on the basis of the type of treatment with DNA. For example, in experiments nos. 1, 3, 5, 6, 7, 9 and 10 the times of incubation with DNA extracts were short and the cultures were plated out 24 hours after the end of the DNA incubation period. In experiments nos. 4 and 8 a small number of cells was used to start the cultures and growth took place in the presence of the DNA extracts which were added on the third and second days of growth respectively. In experiment no. 2 the experimental culture was treated with a relatively large amount of DNA extract on the second day; since this treatment resulted in the death of an appreciable number of the cells, the culture was al- lowed to grow for several days and once more incubated with DNA extract. In comparing the results of the two groups, it is clear that a significantly larger number of DNA induced resistant colonies were produced in those experiments (nos. 2 , 4 and 8) in which the cells were incubated with DNA for a longer period of time.

The results of experiments no. 5, 6, 7, in which increasing dosages of DNA were used suggest that a dose response relationship may be demonstrable although it can- not, at present, be regarded as established.

130 T. RAY BRADLEY, ROBERT A. ROOSA AND LLOYD W. LAW

TABLE 2 Production of resistant cells from P 3 8 8 / P by treatment of cultures with DNA extracts and subsequent

isolation in 8-azaguanine (AZG)

Total t m e of

growth of Expt. cultures

Treatment] No. of colonies2

AZG AZG 1 x 10-6 M 2 xl0-6 M

days

1 13 Control 0 0 No. 1 DNA-R; 40 pg at day 11 for 24 hours 330 0

2 16 Control 190 54

80 p g at day 13 for 48 hours 3435 1249 No. 1 DNA-R; 400 p g at day 2 for 3 hours, then

6

7

8

9

4

Control 204 122 No. 1 DNA-R; 40 pg at day 3 for 3 hours 456 107

Control No. 1 DNA-R; 8 pg at day 2 for 4 days

Control No. 1 DNA-R; 12.5 p,g at day 3 for 3 hours No. 1 DNA-R; 25 pg at day 3 for 3 hours No. 1 DNA-R; 50 pg at day 3 for 3 hours No. 1 DNA-R; 100 pg at day 3 for 3 hours

Control No. 1 DNA-R; 12.5 pg at day 3 for 3 hours No. 1 DNA-R; 25 pg at day 3 for 3 hours No. 1 DNA-R; 50 pg at day 3 for 3 hours No. 1 DNA-R; 100 pg at day 3 for 3 hours

No. 1 DNA-R; 2 p g at day 3 for 3 hours No. 1 DNA-R; 10 pg at day 3 for 3 hours No. 1 DNA-R; 50 pg at day 3 for 3 hours

4 Control

20 Control No. 1 DNA-R; 40 pg at day 2 for 18 days

4 Control No, 2 DNA-P; 50 p g at day 3 for 5 hours No. 3 DNA-R; 50 ,ug at day 3 for 5 hours

75 225 687 366

0 0 0 57 62 0 73 73 76 76

45 a2

- -

115 - 119 - 175 - 36 0 43 0 3 76 0 263 0 2 74 - 1555 - 39 19 28 0 204 68

10 6 Control 71 - No. 2 DNA-P; 25 pg at day 3 for 48 hours 158 - No. 3 DNA-R; 25 p g at day 3 for 48 hours 107 -

1 DNA preparations. No. 1. DNA-R from P388/AZGr-3, approx. 400 f ig DNA/ml. No. 2. DNA-P from P388/P, approx. 200 fig DNA/ml. No. 3. .DNA-R from P388/AZGr-3, approx. 300 fig DNA/ml.

Number of resistant colonies per 1 xlO-6 effxient cells plated (see text).

Generally, very little effect of DNA treatment was found at the slightly higher concentration (2 X lo-' M ) of 8-azaguanine, but, apart from experiment no. 3 and the 25 vg DNA level in experiment no. 5, the incubation of sensitive cells with resistant DNA yielded an increased number of colonies. The results of experiment no. 2 (although this experiment is peculiar in its incubation cycle) certainly demonstrate this.

In experiment no. 9 a comparison was made of the effects of DNA preparations

extracted from parental cells (P388/P) and 8-azaguanine resistant cells (P388/ AZ(Y3). The extracts were prepared simultaneously under identical conditions and were used immediately after the DNA solutions were obtained. In this case there was no response to the treatment with the DNA preparation extracted from the parental cell population, whereas the DNA extracted from the cells resistant to 8-azaguanine produced a result similar to that found in other experiments, that is, an increase in the number of resistant


colonies. However, experiment no. 10 was made with the same DNA preparations only 24 hours later (DNA preparations stored overnight at 6°C) and yielded results which indicate that at least under some conditions a DNA extract from parental cells might give results essentially identical with those obtained with the DNA extracted from resistant cells. It should be noted, however, that although the cells were from the same cloned line they were split from a different culture bottle for experiment no. 10 and were in a different growth stage at the time of this experiment; further the concentration of DNA and the duration of DNA treatment differed from the conditions of experiment no. 9.

Nevertheless the results of experiment no. 10 tend to weaken an interpretation that these data constitute evidence for DNA-mediated transformations such as has been shown in bacteria (Demerec, '45, and Hotchkiss, '51 and '55) . These experiments are representative of many experiments and whereas the incubation of sensitive cells with DNA extracted from resistant cells has consistently yielded an increase in the number of colonies the incubation of sensitive cells with parental DNA has given the variable results as indicated in experiments 9 and 10 (table 2). No consistent pattern has been achieved when parental DNA was used and many more experiments must be con-

ducted before an interpretation can be made.

On obtaining the results of some of the early experiments in this series, some isolated colonies arising from the populations treated with P388/AZG'+ DNA were chosen for further study. These colonies were picked up with a platinum loop and seeded separately into petri dishes in growth medium without 8-azaguanine. As these populations grew, samples from them were tested for their responses to 8-azaguanine at various times after the isolation of the original colonies into growth medium. Thus these experiments tested the stability of the resistant colonies after growth in the absence of the inhibitor. Five populations were successfully grown. Table 3 shows that the five isolated populations did exhibit stability and that the titers for each of the populations were very similar. Also it is to be noted that these titers are indeed similar to those obtained with the population of cells isolated from the parental population (P388/ P ) by selection with 1 X lo-' M 8-azaguanine (see table 1, subline AZG'-').

RESULTS OF SERIES 2 EXPERIMENTS

In this series of experiments the cells were not incubated with DNA prior to plating out. The DNA preparations were pipetted into the petri dishes with the 8-azaguanine immediately prior to the dis- pensing of the cells into the dishes. This was done in order to see whether the

TABLE 3

The effect of 8-azaguanine (AZG) on the growth of various sublines of P388/P isolated after D N A treatment as compared with parental cells and a drug-selected subline

P388 - Sublines

AZG concentration

Sublines isolated after DNA incubation

1 2 3 4 5 19 32 19 32 17 43 46 16 292

1 Parental AZGr-1

1 x 1 0 - 7 ~ 903 95 100 73 88 86 100 93 80 100 100 3 x 1 0 - 7 ~ 59 77 94 65 66 89 58 87 74 100 93 5 x lO-'M 10 69 53 63 56 57 54 66 61 75 79 8 x 10-?M 0 1 x 10+M 64 61 52 58 57 35 63 74 62 60 2 x M 11 11 26 19 12 53 43 3 17 3 x M 21 5 4 12 5 16 34 29 1 3

1 Population isolated from P388/P by selection with AZG, 1 x 10-6 M. SDays after isolation of the resistant colony and tune of growth of the cells on drug free 8 Number of cells forming colonies in presence of drug

Number of cells forming colonies in absence of drug

growth medium. 100 1 _ _ ~ _ _ _ _

132 T. RAY BRADLEY, ROBERT A.

continual presence of DNA extract during the growth period while colonies were being formed, could produce responses similar to those shown in the experiments in Series 1.

Initial results indicated that under these different conditions, antagonism of 8-azaguanine could be demonstrated (Bradley et al., '62) and further details of the nature of this effect are given in the following experiments.

Table 4 shows the effect of the presence of a DNA preparation during the period in which the cells were forming colonies in the presence of various concentrations of 8-azaguanine. At all concentrations of 8-azaguanine the number of colonies obtained in the presence of the DNA was greater than in the control dishes. At a concentration of 8-azaguanine (8 X M ) which completely inhibits the growth in the control dishes, the presence of the DNA preparation allows the growth of approximately two-thirds of the cells used. Decreasing numbers of colonies were found with increasing concentration of 8-azaguanine.

This antagonism of the 8-azaguanine inhibition of growth was demonstrated with DNA preparations extracted from either parental or 8-azaguanine resistant cells (table 5). The effect was a graded one with respect to the amount of the DNA extract used per dish. Thus, low concentrations of DNA extract produced no colonies and increasing the amount of

ROOSA AND LLOYD W. LAW

TABLE 4 Antagonism by a DNA extract of the 8-azaguanine

(AZG) inhibition of growth of P 3 8 8 / P

AZG concentration

- 1 x 10-'M 3 x 10-7 M 5 x 10-7 M 8 X M 1 x 16-6 M 3 x M 5 x M 8 x M 1 x 1 0 - 5 ~ 3 x 10-5 M

DNA treated1 Control

2512(82% ) 3 236(77%)3 228 249 159 243 22 242

0 211 204 126 95 37 31

0 1 No. 4 DNA extract from P388/AZG*-3; 0.1 ml per

ZEach value is the average number of colonies dish = 50 fig DNA per 5 ml medium.

~.. from three dishes.

3 Plating efficiencies; 307 cells inoculated per dish.

DNA extract per dish resulted in an increase in the number of colonies until a plateau value was reached which was not identical for each DNA extract (table 5).

A number of DNA preparations were examined and found to be active by this test. Preparations of DNA were extracted from the following sources: P388/P the parental line of tissue culture cells (2 preparations) ; P388/AZG'-' ( 1 preparation) ; P388/AZG'-3 (4 preparations) ; P388 ascites tumor from (BALB/c X DBA/2)Fl mice; L1210 ascites tumor from (BALB/c X DBA/2)F1 mice; mouse liver from C57BL/Ka strain; rat spleen from Sprague- Dawley stock.

Since it was important to know whether the antagonism of the 8-azaguanine in-

TABLE 5

The effect of increasing the concentration of DNA extracts in the presence of a constant concentration of 8-azaguanine

No. 6 DNA1 No. 7 DNA2

(fig per dish) colonies (fig per dish) colonies

AZG concentration Dose Number of Dose Number of

1 x 10-EM 7.59 0 3 3.72 03 1 x M 15.4 1 7.44 1 1 x M 30.8 21 14.4 35 1 x M 60.5 46 30.0 69 1 x 10-6M 121.0 47 60.0 65

Controls 8 A Z G l x 1 0 - 6 M 0

74(34% )4

1 No 6 DNA extract from P388/P. approx. 1100 &g/ml DNA No: 7 DNA extract from P388/LZGr-:; approx. 120 fig/ml' DNA. Average values of four dishes.

4 215 cells inoculated per dish.


hibition of growth could be obtained with degraded DNA or not, an experiment was made in which the DNA preparation was tested for activity after incubation with desoxyribonuclease. Incubation of the DNA with DNase was done in veronal buffer (pH 7.0) and added Mg++. A control sample of the incubation system prior to addition of the DNase and the two samples after addition of the DNase were taken. These three samples were heated at 60°C for 15 minutes to inactivate the enzyme. The samples were then used for the experiment.

The results shown in table 6 indicate that there was no decrease in activity in the DNA either with incubation at 37”C, or with DNase for 2 or 24 hours. Whether th,ere was any increase in activity was not investigated.

The observation that the activity of the DNA preparation was not lost on diges- tion with desoxyribonuclease suggested that some of the constituents of nucleic acids might also antagonize the 8-azaguanine growth inhibition of P388/P. The simplest possibility, that free purine or pyrimidine bases could block the inhibitory action of 8-azaguanine was tested. Thus table 7 shows the results obtained when adenine, guanine, uracil, thymine or cytosine were added directly to the petri dishes together with the 8-azaguanine and prior to inoculation of the pa-

TABLE 6 Effect of DNase extract wi th respect to antagonism of the growth inhibition of

8-azaguanine on P388/P

AZG Av. no. of concentration Treatment colonies1

- - 276(74% - 30 pg DNA3 271

0 1 x M - 1 x M 30 pg DNA 236 1 x 10+ M 30pg DNA;

incubated4 244 1 X M 30pg DNA;

DNase5 2 hours 244 1 x M 30pg DNA;

DNaseS 24 hours 236

1Average value of four dishes. 9 Plating efficiency; 375 cells inoculated per dish. 3No. 3 DNA extract from P388/AZGr-3; approx.

30; g DNA/ml.

5 DNase (Worthington). Incubation system - 1.6 ml DNA, 0.8 ml veronal bbffer pH 7.0, 0.3 ml MgS04 (0.03M). 0.3 ml DNase (10 &g/ml).

Lcubated at 37” for 24 hours.

TABLE 7 Growth of P388/P cells in the presence of

8-azaguanine antagonized by nucleic acid bases

AZG Av. no. concentration Base3 colonies1

193(56% )2 - - 0 1 x M -

1 X M Adenine 1 X M 80 1 x M 1 x 1 0 - 5 ~ 81 1 x M 1 x 1 0 - 4 ~ 67 1 x M Guanine 1 x M 0 1 x M 1 x 10-5 M 0 1 x M 1 x 1 0 - 4 ~ 0 1 x M Uracil 1 x M 0 1 x 10-GM 1 x 10-5M 0 1 x M 1 x 1 0 - 4 ~ 0 1 x M Thymine 1 x M 0 1 x M 1 x 10-5 M 0 1 x 10-6 M 1 x 10-*M 0 1 X M Cytosine 1 x M 0 1 x 10-6M 1 x 1 0 - 4 ~ 0 1 X 10-6M Mixture 1 X 10-6M 67 1 X 10-6M of above{ 1 X 10-5M 70

1 Average value of three dishes. 2 Plating efficiency; 344 cells inoculated per dish. 3 Adenine Guanine Uracil, Thymine California

Foundation ’for Biochkmical Research, i o s Angeles California. Cytosine, Nutritional Biochemical Corpd ration, Cleveland, Ohio.

rental cells. Of the bases tested, adenine alone was able to antagonize the growth inhibitory activity of 8-azaguanine.

The data in table 8 demonstrate that increasing the concentration of 8-azaguanine overcame the effect of a constant concentration of adenine, and increasing the concentration of adenine in the presence of a constant concentration of 8-azaguanine led to the growth of larger numbers of cells into colonies.

It was important to determine whether the colonies arising in the series 2 experiments are different from those in the series 1 “transformation” experiments in which the heritability of the selected clones was established (see table 4). Three to four hundred P388/P cells were introduced into 5 ml of complete growth medium containing 1 X lo+ molar 8-azaguanine and 1 X lo-‘ molar adenine. In- cubation was carried out in a humid atmosphere of 5% C 0 2 and 95% air for seven to ten days at which times apparently resistant clones were isolated and reintroduced into complete growth medium. Characterization of these cell populations with respect to their growth inhibition by 8-azaguanine was then


determined at various times after isolation and growth in complete medium, without inhibitors. The results are shown in table 9.

The first column presents the average control titer for seven separate clones isolated from the parental population. The second column is the average titer for nine separate clones isolated from the same population after ten days incubation in 1 X lo-’ M 8-azaguanine, then removed to growth medium for an average of 48 days before testing. The titer of these cells, as one can determine by comparison with table 1, lies between the parental and the first step resistant populations. Ten isolated clones are then shown in the

remainder of the table. These were selected after growth in azaguanine plus adenine, maintained in growth medium for the times indicated, and then tested. Each of these, as can be seen, is completely sensitive to 8-azaguanine.

DISCUSSION Several distinct advantages are appar-

ent in the systems employed here for studies of the in vitro demonstration of transformations to drug resistance in mammalian lymphoblasts. The cell population is relatively homogeneous and ex- tremely sensitive to the purine inhibitor, 8-azaguanine. The media support the clonal proliferation of these leukemic lym-

TABLE 8 Antagonism of 8-azaguanine (AZG) growth inhibition on P388/P cells by adenine

AZG concentration

Control av. no. of colonies

Adenine

Concentration

- 118(23% )2 1 x 1 0 - 6 M 112 1 x 1 0 - 7 ~ 117 1 x M 110 3 x 10-7 M 63 1 x M 98 5 x 1 0 - 7 ~ 23 1 x M 89 8 X lo-? M 0 1 x M 87 1 x M 0 1 x 10-6M 91 3 x M 1 x M 44 5 x M 1 x M 17 8 x M 1 x 10-6M 2 1 x 10-5 M 1 x M 2 3 x 10-5 M 1 x M 0

1 x M 0 5 x 10+M 0 1 x 10+M 0 1 x 1 0 - 7 ~ 3 1 x M 0 5 x 1 0 - 7 ~ 13 1 x 10-6M 0 1 x 10-6M 91

1 Average value from three dishes. 2 Plating efficiency; 515 cells inoculated per dish.

TABLE 9 The effect of 8-azaguanine (AZG) on the growth of various sublines of P388/P

Isolated after growth in complete medium plus 1 x 10-6 M 8-azaguanine and 1 x 1 0 - 6 M adenine

1 2 3 4 4 5 6 7 8 9 1 0 B3 AZG

concentration p388’p1

None - 4S4 35 66 67 15 50 38 38 39 39 28 38

1 x 1 0 - 7 ~ 7S2 80 55 100 78 66 48 91 96 71 74 76 100 2 x 1 0 - 7 ~ 53 82 14 72 50 11 16 72 62 75 37 22 92 5 x 10-7 M 15 55 0 10 1 0 - 7 9 4 9 1 1 7 8 X 10-7M 4 34 - - - - 4 0 0 0 0 0 0

2 x 10-6M 0 7 0 0 0 0 0 0 0 0 0 0 0 1 x M 0 17 o o o o o o o o o a o

1 Average titer for nine separate clones. *Percentage of cells capable of forming colonies in the presence of 8-azaguanine. 3B=Average titer for seven separate clones, derived from P388/P and isolated after ten daya

incubation in 1 x 10-6 AZG, then removed to growth media lackjng the inhibitor. 4Days after isolation of colonies and time of growth in inhibitor-free medium.


phoblasts, permitting the isolation of mu- tant sublines. Step-wise mutational selec- tions to resistant sublines requiring at the highest level 4,000 times the concentration of inhibitor for comparable inhibition of the parental line were obtained (Ftoosa et al., '62). First step changes were of the order of a 5 to 10-fold increase thus permitting the use of a level of 8-azaguanine sufficient to inhibit reproduction of sensitive parental cells and allowing iden- tification of first step markers.

The resistant sublines isolated here in the first series of experiments after DNA treatment with DNA from resistant populations resemble in many characteristics the bacterial transformants discussed by Demerec ('45), and Hotchkiss ('51 and '55). Clones selected after DNA treatment have retained their resistance to 8-azaguanine through repeated subcultures in the absence of drug. Not only was there stability of the resistance character but also uniformity in the degree of resistance to inhibitor. Of the sublines tested, all exhibited resistance characteristic of the first step clones isolated after selection with inhibitor, an approximate five to ten-fold increase in resistance although the DNA was derived from genetically stable sublines showing a 4,000-fold increase in resistance. In the limited results reported here there appears to be a direct relationship between the concentration of DNA to which the cells were exposed and the yield of resistant clones.

Yet the variable results obtained after treatment of cells with DNA isolated from sensitive cells strongly suggest that the DNA, under certain conditions, might be acting as an antagonist to 8-azaguanine. This effect, which is demonstrated by the second series of experiments carried out under quite different cultural conditions, could be that of protection of the cell by DNA at the cell surface or inside the cell. Evidence for incorporation of DNA by mammalian cells has been shown by Bensch and King ('61) and by Boren- freund and Bendich ('61).

The suggestion could, of course, be made that the colonies arising after incubation of cells with the DNA plus azaguanine are mutants induced by the azaguanine. A comparison of tables 3 and

9 shows that R-DNA treated colonies after a shorter exposure to azaguanine are more resistant than cells exposed to 8-azaguanine alone (table 9, column 2) and that they certainly are different from the cells isolated after exposure to adenine plus azaguanine (table 9).

In the second series of experiments the inhibition of growth by 8-azaguanine was effectively overcome by the simultaneous addition of any one of a number of DNA preparations to the medium. There was no obvious specificity with respect to the source of the DNA preparation. Further- more, the integrity of the DNA was not essential since prior incubation with DNase did not remove the activity. Thus it seems likely that the antagonism of 8-azaguanine is due either to a contaminant common to the DNA preparations or to a breakdown product of the DNA and/ or contaminant in the preparations re- leased either in the cells or the medium.

Adenine was also demonstrated to antagonize the inhibition of growth produced by 8-azaguanine in a competitive manner similar to that shown with the use of the DNA preparations. This would suggest that the activity of the DNA preparations might be due to the presence of adenine. Considering that concentrations of adenine equimolar with 8-azaguanine are sufficient to block the inhibition of growth by the latter, suggests the possibility that sufficient adenine could be generated by complete breakdown of the DNA and any RNA contamination to account for the protective activity of the nucleic acid preparations. Since one of the DNA preparations used was found not to be degraded (as evidenced by the absence of dialyzable components), it must also be considered that DNA may act as a non- specific inhibitor, in contrast to adenine. This may be the result of its polyanionic character. If so, other highly charged anions such as sulfated or phosphorylated polysaccharides may show the same effect.

At the moment, the main interest in this second series of experiments is whether the antagonism of 8-azaguanine by adenine can explain adequately the results obtained on induction of resistance in the first series of experiments. The problem resolves into a question of


whether sufficient DNA could remain after the incubation and the various dilu- tions and manipulations to account for the results obtained when the cells were finally plated out in the presence of 8-azaguanine.

In the first series of experiments (table 2) amounts of DNA of the order 10 - 100 wg were used in the incubations with the cells prior to pIating them out in the 8-azaguanine medium. The manipulations of the sampling procedure were such that any of the DNA preparation left in the medium would be diluted approximately 10,000-fold at the time the cells were inoculated into the 8-azaguanine medium in petri dishes. Thus the amounts of DNA preparation likely to be inoculated into the dishes with the cells would be of the order of 0.001 - 0.01 wg per 5 mI of medium. Assuming that this amount of DNA were degraded to consti- tuent purine and pyrimidine bases, an amount of adenine smaller than that found to be necessary to antagonize a concentration of 1 X 10-8 M 8-azaguanine would be generated.

The occurrence of resistant colonies in the first series of experiments (parental cells exposed to DNA from resistant cultures) was of the order of 2 to 30 times greater than are found spontaneously. If it were assumed that these colonies arose as the result of protection by adenine against the 8-azaguanine this might indicate a type of selection phenomenon (i.e., some cells being preferentially protected). However, assuming this to be the mecha- nism by which the colonies were produced in the first series of experiments, one wouId not expect that such colonies, when isolated into drug-free growth medium, would remain resistant to the 8-azaguanine for up to 40 cell generations. Col- onies arising after treatment with DNA obtained from populations resistant to 8-azaguanine did show stability, a degree of uniformity in their resistance to 8-azaguanine, and a degree of resistance cor- responding to that of the first known step in the development of resistance in this cell line. At present, this constitutes evidence that resistance has been induced in the cells which gave rise to the colonies.

Further evidence indicating the exist- ence of two different phenomena occur- ring in the two different systems used is to be found in the studies of the apparent resistant clonal sublines isolated in the second series experiments. All ten of those isolated and characterized over a period of time were found to be entirely sensitive populations.

The general applicability of the results concerning adenine antagonism of 8-azaguanine growth inhibition must be further tested since Szybalski and Smith ('59) state that adenine antagonism was not found in their in vitro systems employing D98, a cell line established from human bone marrow.

The possibility of encountering an adenine - or DNA - effect, characteristic of the second series experiments must be kept in mind when designing experiments to study transformations to drug-resistance in mammalian cells. Experiments which involve the addition of large numbers of heat-killed drug resistant cells to a small number of drug sensitive cells either in vitro or in vivo (Blumenthal et al., '61) must, for the present, be considered unsatisfactory. Detection of the induced resistance by treatment with a short course of the relevant inhibitor may lead to false assumptions of induction of resistance in sensitive cells.

The results from the two types of experiments demonstrate the difficulty in proving that transformation to 8-azaguanine resistance can be achieved in mammalian lymphoblasts. Some of the criteria of transformation reactions, as observed in bacteria, have been fulfilled, but these are not sufficient to unequivocally prove transformation.

SUMMARY

Parental P388 cells, cultivated in vitro and sensitive to 8-azaguanine, were incubated with DNA extracted from cells of a subline P388/AZG'-3, highly resistant to 8-azaguanine. Large numbers of the cells were then tested for the presence of resistant cells in a concentration of 8-azaguanine which inhibited the growth of sensitive parental cells.

Control cultures (not treated with DNA) contained either no or very few resistant


cells, whereas the DNA treated cultures always yielded appreciably larger numbers of resistant cells. These resistant cells obtained after DNA treatment were cultivated for several cell generations in the absence of 8-azaguanine and showed stability in their resistance to the drug. The resistance induced corresponded with that found in the first known step of resistance to 8-azaguanine with this cell line. Re- sistance was at a lower level than the resistance of the cell population from which the DNA was extracted.

Interpretation of the results is compli- cated since anomalous effects were obtained with DNA extracted from populations of cells of the parental line P388/P. Also in experiments under different conditions of incubation, it was shown that a number of DNA preparations could antagonize the growth inhibitory activity of 8-azaguanine. This antagonism was not altered by treatment of the DNA with DNase and it was subsequently found that adenine produced an identical antagonism of 8-azaguanine. Apparently resistant coloni,es obtained by incubation in the presence of adenine and 8-azaguanine were shown, however, not to be truly resistant to 8-azaguanine after cultivation in the absence of the drug.

The relationship between the results of the experiments in induction of resistant cells with DNA and those involving the antagonism of 8-azaguanine with adenine is discussed.

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dna transformation studies with mammalian cells in culture

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