Podophyllotoxin resistance: A codominant selection system for quantitative mutagenesis studies in mammalian cells

261

Mutation Research, 83 (1981) 261--270 Elsevier/North-Holland Biomedical Press

P O D O P H Y L L O T O X I N RESISTANCE: A CODOMINANT SELECTION SYSTEM F O R QUANTITATIVE MUTAGENESIS STUDIES IN MAMMALIAN CELLS

RADHEY S. GUPTA

Department of Biochemistry, McMaster University, Hamilton, Ont. L8N 3Z5 (Canada) (Received 28 November 1981) (Revision received 1 April 1981) (Accepted 21 April 1981)

Summary Mutants resistant to the microtubule inhibitor podophyllotoxin (Poda), a codominant marker, can be readily selected in various mammalian cell lines such as, CHO, HeLa, mouse L cells, Syrian hamster cells (BHK2~) and a mouse teratocarcinoma cell line OC15. In CHO cells, the recovery of Pod a mutants is n o t affected by cell density (up to 1 × 106 cells per 100-mm diameter dish), and after treatment with the mutagen ethyl methanesulfonate maximum mutagenic effect is achieved after a relatively short expression time (40--48 h). The frequency of Pod a mutants in various cell lines increased in a dose-dependent manner in response to treatment with the mutagens ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine. The Pod a selection system thus provides a new genetic marker which should prove useful in studies of quantitative mutagenesis in mammalian cells.

In recent years, mutants of mammalian cells resistance to a variety of cytotoxic drugs have been selected in established cell lines, such as the Chinese hamster ovary (CHO) line [2--5,12--22,27,32]. These selection systems have provided valuable tools in biochemical [14--18,20--22,25,27], genetic [7--9,11] and quantitative mutagenesis studies. Such systems also form the basis of developing short-term assay systems for mutagens/carcinogens detection in mammalian cells [1,5--7,12,19,26,35]. However, the large majority of mutants that have been obtained in CHO line are of recessive t y p e [7,32], a fact that is accounted for by a high degree of functional hemizygosity in this line [4,23,24, 32], and hence such mutants cannot be readily selected in other types of cells [4,8,18]. In contrast to recessive mutants, selection systems based on codominant mutations should be applicable to different types of cells. However, there is a paucity of good selection systems of this type, at present. 002%5107/81/0000--0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press

262 We have recently reported that mutants resistant to the microtubule inhibitor podophyllotoxin (Pod R) can be readily selected in CHO cells [10]. In cell hybrids formed between resistant and sensitive cells the Pod a mutation behaved codominantly as indicated by the intermediate level of resistance of hybrid cells to the drug [10]. This latter characteristic of the Pod R mutants suggested that this selection system should also be applicable to other types of cells. In the present paper we report the selection of Pod R mutants in a variety of other established cell lines such as, mouse L cells, baby hamster kidney cell (BHK 21), HeLa cells and a mouse teratocarcinoma cell line. Several characteristics of the Pod s selection system in CHO cells, e.g. optimal expression time, effect of cell density and selective drug concentration, and effect of treatment with various concentrations of mutagens on the frequency of mutants, which should prove useful in its application as a probe for quantitative mutagenesis studies in mammalian cells have also been investigated. Materials and methods Cell lines and culture conditions

The different cell lines employed in these studies are listed in Table 1. All the cell lines except CHO and its derivatives, which were frequently grown in suspension cultures, were routinely cultivated as monolayer cultures in MEM alpha medium (Grand Island Biological Company) supplemented with 5% fetal

TABLE CELL

1 LINES

Cell lines

Species of origin

Phenotype

Ref.

CHO(Pro-)

Chinese h a m s t e r

P r o - P o d S, a u x o t r o P h i c for proline

(13--20), 28

PodR 5

Chinese h a m s t e r

Pro- Pod R _ derived f r o m CliO Pro"

10

LtK-

Mouse

P o d S t K - ( t h y m i d i n e kinase deficient)

29

LtK PodR2 H e L a $3

Mouse Human

Pod~tK" Pod

p r e s e n t studies 31

H e L a P o d R 2 2 and HeLa PodR23

Human

p o d o p h y l l o toxin-resistant derivatives o f HeLa ceUs

p r e s e n t studies

BHK 21-13

Syrian h a m s t e r

Pod S

34

B H K P o d R 2 and PodR3

Syrian h a m s t e r

podophyllotoxin-resistant m u t a n t s o f B H K 2 1 - 1 3 cells

present studies

Mouse (teratocarcinoma)

A s u b e l o n e o f cell line O C 1 5 , k i n d l y provided by Dr. M,W. M e B u r n e y , O t t a w a

30, 33

Pod R derivatives o f O C 1 5 S l cell line

present studies

OC15S1

O C P o d R 4 and PodRl3

Mouse (teratocarcinoma)

263 calf serum (FCS). The cell counts measurements were made using a Coulter Electronic Counter. Podophyllotoxin was purchased from Polysciences, Inc., Warrington, Philadelphia. Plating effeciencies. Plating efficiencies (same as cloning efficiency) of the cells were determined by plating a known number of cells (based on coulter cell counter measurement) of a single suspension of the culture 5n tissue culture dishes containing the growth medium. The dishes were incubated for 7--10 days at 37°C, after which they were stained with 0.5% methylene blue in 50% methanol and aggregates of 50 or more cells were counted as colonies. The relative plating efficiencies were determined as the ratios of the number of colonies at a certain drug concentration to that obtained in the absence of the drug. The absolute plating efficiencies of various cell lines were in the range of 30--60% in different experiments.

Semi-quantitative measurement of resistance to a drug The degree of resistance of mutant cells towards podophyllotoxin was determined by a semi-quantitative procedure [10]. The procedure involved seeding separately a b o u t 200 and 500 cells of the sensitive and resistant cell lines (in 0.5 ml volume of growth medium) into the wells of 24-well tissue culture dishes (Costar), containing 0.5 ml of the various dilutions of the drugs (at twice the desired final concentrations) in growth m~tium. The dishes were incubated for 6--7 days at 37°C after which they were stained by the usual procedure [10]. Mutagenesis and mutant selection protocols A typical mutagenesis experiment involved adding the mutagen to exponentially growing cells at the desired final concentration. To a parallel culture which served as control, an equivalent amount of solvent was added. After the appropriate treatment period, mutagen was removed, cells were washed and then resuspended in fresh growth medium. A known number (usually 250-300) of mutagen-treated and untreated cells were plated in nonselective medium to determine the fraction of cells surviving mutagen treatment. The remaining cells were grown in the nonselective medium for the indicated periods to allow time for mutation expression. Mutant selection was carried o u t by plating 5 × l 0 s cells/100-mm dish, on several dishes in medium containing the appropriate concentration of podophyUotoxin. The plating efficiency of the cells at the time of plating was again determined by plating a known number of cells in nonselective medium. In all of the experiments described mutation frequencies have been corrected for the plating efficiency of the cells [19]. Results

Fig. 1 shows the survival curves of several mammalian cell lines towards podophyllotoxin. The cell lines which were examined include, (i) the proline requiring Chinese hamster ovary (CHO) cell line, (ii) the mouse L cells (LtK-), (iii) the Syrian hamster kidney line, BHK 21-13, (iv) the mouse teratocarcinoma cell line, OC15S1 and (v) the HeLa cells. The cell lines showed significant differences in their sensitivity towards podophyllotoxin, and among these,

264

1°-'t-t

~i~,, ! ~

I, I

'i

~

)Z

1 0 -=

,

;

!

i i

5

!

i',

i Z t.-

1 0 -3

i

i

i

MJ >

10-"

lO-S

10-* 0

10

20

PODOPHYLLOTOXIN

30

(ng/ml)

Fig. I. Dose--response curves of variois cell lines towards podophyllotoxin o m o u s e Ltk- cells; t, HeLa; 21-13 cells; ~, C H O (Pro-) line; o0 O C 1 5 S 1 (mouse teratocarcinoma) cell line. The plating efficiency of cells was less than 10 -6 at drug concentrations to which survival curves are extrapolated by dotted lines. T h e arrows (?) indicate the drug concentrations at which clones were picked and examined for their relative resistance.

A BHK

mouse L t K - cells were clearly more sensitive to podophyllotoxin. The concentrations of podophyllotoxin which resulted in 10% survival (Dl0 values) of different cell lines were as follows: CHO, 8.3 ng/ml; BHK 21-13, 7 ng/ml; OC15S1, 9.7 ng/ml; L t K - , 3.3 ng/ml; HeLa, 6 ng/ml. At concentrations of the drug which were slightly higher than the D10 values, a sharp decrease in the plating efficiency of various cell lines was noted. However, upon plating larger number of cells at somewhat higher concentrations of the drug, discrete clones of growing cells were observed. This is indicated by the plateau regions in survival curves in the frequency range of 10 -s to 10 -6 (Fig. 1). The concentration ranges of podophyllotoxin over which discrete clones were observed differed for various cell lines and were relatively narrow as compared to some of the other selective systems such as resistance to ouabain, emetine, thioguanine or diphtheria toxin [19]. The decline in the plateau regions observed with increasing concentrations of podophyllotoxin suggests the clones exhibiting different degree of resistance are present in cultures, and at higher concentrations only the more resistant types are selected. A few of the clones of various cell lines which were seen growing in the pres-

265

TABLE

2

DEGREE

OF

SENSITIVITY

LINES TOWARDS

OF

Concentrations

CeH ~ n e s

VARIOUS

PARENTAL

AND

Pod R MUTANT

MAMMALIAN

CELL

PODOPHYLLOTOXIN of podophyllotoxin 5

7.5

(ng/ml)

0

2

10

15

20

CHO(Pro-)

+++

+++

++

CHO PodR2

+++

+++

+++

+

=

.

+++

+++

+++

HeLa

+++

+++

+

+-

.

HeLa-PodR22

+++

+++

+++

++

++

+

±

HeLa PodR23

+++

+++

+++

+++

++

+

+-

BHK 21-13 BHK PodR2

+++ +++

+++ +++

++ +++

+ +++

± +++

. +÷

BHK PodR3

+++

+++

+++

+++

++

+

.

.

+++

+++

÷++

++

-+

.

++÷ +++

+++ +++

+++ ÷++

+++ +++

+++ +++

+÷+ +÷

LtK LtK-PodR2

+++ ++÷

++ +++

. +++

. ++

--

--

_

_

--

--

--

.

.

. ±

--

--

--

--

--

+ --

± --

.

.

.

++ ± .

.

m

__

.

±

.

50

.

+

.

40

.

.

+++ +

. .

-+

. +

.

. +

35

.

.

OC PodR4 OC PodR13

30

++ .

OC15S1

.

25

. .

~_

The degree of resistance of various cell lines towards podophyllotoxin w a s d e t e r m i n e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e g r o w t h o f c e l l s as d e t e r m i n e d b y t h e i r a p p r o x i m a t e p l a t i n g e f f i c i e n c i e s w a s r e c o r d e d as f o l l o w s : +++, normal growth similar to that in 0 concentration

control.

÷+, slightly reduced growth, i.e. between 50--90% of the control. *, greatly reduced growth, between 5--50% of the control cells. ±, n e g l i g i b l e ( ~ 5 % ) g r o w t h . --, no growth.

ence of podophyUotoxin (at drug concentrations indicated by arrows in Fig. 1) were picked, grown in non-selective medium and their degree of resistance towards podophyllotoxin was examined by a semi-quantitative procedure. Results of these tests for some representative mutants are shown in Table 2. As TABLE

3

RECONSTRUCTION EXPERIMENT SITY EFFECT IN SELECTION a Number

of Pod S

ceils plated

Number

WITH

Pod R CELLS

of

Pod R cells

TO SHOW THE

Average number

of

colonies observed

ABSENCE

OF CELL

DEN-

Relative b plating efficiency

-1

200 ×

106

--

118 2

100 --

1 × 105

200

117

100

2 × 105 5 × 105

200 200

121 118

102 99

1 × 106

200

122

101

a Cells were plated in 100-mm dishes containing 25 ng/ml of podophyllotoxin. C H O ( P r o - ) a n d P o d R 5 (a P o d R d e r i v a t i v e o f C H O cells) w e r e e m p l o y e d as t h e s e n s i t i v e a n d r e s i s t a n t cells. T h e n u m b e r of ceils p l a t e d is b a s e d u p o n c e l l - c o u n t m e a s u r e m e n t cells in non-selective medium was about 75%.

with

b Relative plating efficiencies have been corrected nating from sensitive ceils.

a coulter counter.

for the approximate

The plating efficiency number

of Pod R

of Pod R colonies origi-

266 NUMBER 0

OF CELL D I V I S I O N S

2 i

4 i

100 .

6 i e~--.......~

g lo

I HOURS AFTER MUTAGENESIS

Fig. 2. E x p r e s s i o n t i m e for the Pod R m u t a t i o n a f t e r m u t a g e n t r e a t m e n t . E x p o n e n t i a l l y g r o w i n g P r o C H O cells w e r e t r e a t e d w i t h t h e m u t a g e n EMS ( 3 5 0 # g / m l ) at 0 t i m e . A f t e r 16 h, t h e m u t a g e n w a s r e m o v e d a n d t h e cells were g r o w n in fresh m e d i u m . A t v a r i o u s t i m e intervals, p o r t i o n s of the m u t a g e n t r e a t e d c u l t u r e w e r e p l a t e d in m e d i u m c o n t a i n i n g 25 n g / m l p o d o p h y l l o t o x i n t o d e t e r m i n e t h e f r e q u e n c y of Pod R m u t a n t s . T h e d o u b l i n g t i m e of P r o - C H O cells u n d e r t h e s e c o n d i t i o n s was a p p r o x . 13 h.

may be seen,various Pod a clones were about 2-4-fold more resistant to podophyUotoxin as compared to the parental sensitive cell lines. Some of these clones have been grown in non-selective medium for more than a month, and have found to stably maintain their degree of resistance towards podophyllotoxin. In view of the stability of the mutants obtained at the above drug concentrations, these same concentrations have been employed in subsequent mutagenesis experiments with various cell lines. Before a selection system could be employed for quantitative mutagenesis purposes, knowledge of certain additional characteristics of the system is required. These include (a) recovery of mutants at different cell density (b) optimal expression time after mutagen treatment and (c) dose--response relationship between mutagen concentration and the frequency of mutants. The above characteristics of the Pod a selection system in CHO cells, therefore, were examined. Table 3 shows the results of a reconstruction experiment, where a k n o w n number of Pod a cells were plated together with different numbers (in the range of 1 × l 0 s to 1 × 106) of Pod s cells, in medium containing 25 ng/ml podophyllotoxin. It is apparent from the results of this experiment that recovery of the Pod R m u t a n t cells was not adversely affected by high cell density even when up to 1 × 106 cell were plated in a 100 mm diameter dish. Fig. 2 shows the results of an experiment carried out to obtain information regarding optimal expression time for the Pod a mutation. In this experiment, Pro- CHO cells were treated with the mutagen ethyl methanesulfonate, and at various times before and after mutagenesis portions of the cells were harvested and plated in selective medium containing 25 ng/ml podophyllotoxin. As may be seen (Fig. 2), a marked increase in the frequency of Pod a mutants in culture

267 1000

50

m ,.i

4O

100

o O v-

kz

<

30

uJ IZ < I-.

z

2O

10

d Z

I0

O!

0

A

100

a

200

,

300

ETHYLMETHANE SULFONATE (ug/ml)

I

400

,n 0

1

2

[ M N N G ] (;xg/ml)

Fig. 3. D o s e - - ~ e s p o n s e r e l a t i o n s h i p b e t w e e n t h e c o n c e n t r a t i o n s of m u t a g e n a n d t h e f r e q u e n c y o f P o d R m u t a n t s in C H O cells. P r o - C H O cells w e r e t r e a t e d w i t h 50, 100, 2 0 0 , 3 0 0 a n d 4 0 0 / ~ g / m l o f EMS for 20 h at 3 7 ° C . T h e f r a c t i o n o f cells w h i c h s u r v i v e d t r e a t m e n t w i t h d i f f e r e n t c o n c e n t r a t i o n s o f m u t a g e n s w a s 0 . 9 8 at 50 p g / m l , 0 . 9 5 at 1 0 0 ~ g / m l , 0 . 7 6 a t 2 0 0 ~ g / m l , 0 . 4 6 at 3 0 0 p g / m l a n d 0 . 3 0 at 4 0 0 ~ g / m l . T h e m u t a g e n - t r e a t e d c u l t u r e s w e r e g r o w n for 3 d a y s in n o n - s e l e c t i v e m e d i u m b e f o r e p l a t i n g t h e m in p r e s ence of 25 ng/ml of podophyllotoxin. Fig. 4. E f f e c t o f M N N G t r e a t m e n t o n t h e f r e q u e n c y o f Pod R m u t a n t s in d i f f e r e n t cell lines. E x p o e n e t i a l l y growing eultttres of different cell lines were treated with 0 (control, 1 and 2 $4g/ml of M N N G for 2 h. T h e cell killing was in the range of 20--35% at 1 /~g/ml of M N N G and between 40 and 6 5 % at 2/~g/ml of the mutagcn. The mutagen-treated cells were grown for 3 days in non-selectlve m e d i u m before selecting for podophyllotoxin resistance. T h e selective concentrations of podophyllotoxln for different celllines were, L M T K - (o), 10 ng/ml; B H K 2 I ($), 15 ng/ml; H e L a (4), 15 ng/ml.

was evident at the earliest time examined (16 h). The frequency of Pod R mutants reached a plateau level by 40 h and stayed constant for the next 2--3 days, during which it was examined. The short expression time and the failure to see any apparent lag in expression as seen here for the Pod a marker, are features that appear to be common in various codominant markers [19,20]. To investigate the dose--response reaitionship between mutagen concentration and the frequency of Pod a mutants, Pro- CHO cells were treated with different concentrations of the mutagens ethyl methanesulfonate and after an expression period of 3 days, the frequency of Pod a mutants in different cultures was determined. Results of such studies are presented in Fig. 3. As may be seen, the frequency of Pod R mutants in cultures increased linearly with

268 increasing concentrations of the mutagen over the range of 50--400 pg/ml which was examined. A similar linear dose--response with increasing EMS concentrations, has been observed earlier for a number of other markers in CHO cells [5,19,20,26]. The effect of treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on the frequency of Pod ~ mutants in different cell lines is shown in Fig. 4. As can be seen, treatment with this mutagen cause a marked increase in the frequency of Pod R mutants in various cell lines and a larger effect was observed at the higher concentration of the mutagen. Discussion The results presented in this paper show that mutants resistant to the microtubule inhibitor podophyllotoxin can be readily selected in a variety of mammalian cell lines e.g. HeLa, mouse L t K - , Syrian hamster line BHK 21, mouse teratocarcinoma line OCI5S1 and in CHO cells. In view of the codominant nature of the Pod a lesion as seen in studies with CHO cells [10], the relative ease with which such mutants are obtained at comparable mutation frequency in various cell lines is not surprising. The Pod R mutants of CHO cells which have been examined in earlier studies show a highly specific cross-resistance pattern to various microtubule inhibitors and other drugs, indicating that the lesions in these mutants do not involve a membrane permeability alteration, such as that seen with the colchicine-resistant mutants [3]. In CHO cells, mutants showing higher levels of resistance to podophyllotoxin have also been obtained after a second step selection on the Pod R mutants (Pod RII mutants), and the lesion in some of the Pod RII class of mutants have been shown to affect a microtubule associated protein of approx. 64 000 dalton [10, and unpublished results]. Some of the useful characteristics of the Pod R system that the present study has revealed are (i) stable phenotype (ii) very short expression period (~-2 days), (iii) absence of any affect of cell density on m u t a n t recovery and (iv) a concentration-dependent increase in the frequency of Pod R mutants upon treatment with mutagens in various cell lines. Although, some of the above characteristics have been examined only for the CHO cells, in view of the genetic and biochemical nature of the lesion involved, it is likely that these parameters (e.g. expression time) will also hold true for the other cell lines. One possible limitation of the Pod a selection system that has become apparent from the present studies is the variation in m u t a t i o n frequency with the selective concentration of the drug due to which selection for mutants need be carried out over a narrow selective range. In view of the different sensitivity of various cell lines towards this drug, the selective concentration of podophyllotoxin for any given cell line should be carefully determined before this marker is employed for quantitative mutagenesis purposes. However, once this has been carried out, the Pod a selection system should provide a valuable genetic marker for mutagenesis studies with almost any mammalian cell line. With the inclusion of Pod R marker, a large number of well-characterized genetic markers affecting a variety of functions and loci are now available in CHO cells for studies of quantitative mutagenesis [see 5,19,20]. In such cells,

269 the mutagenic responses after treatment with a given agent can simultaneously be assessed at a large number of genetic end-points. A system such as this, in principle, should not only prove highly sensitive in detecting various types of mutagenic compounds, but should also provide valuable information regarding specificity in the response of various genetic loci to different mutagenic agents. The response of such a system to a variety of mutagens is currently being investigated in our laboratory. Acknowledgements I thank Rajni Gupta and Malcolm Moffat for excellent technical assistance. The work was supported by grants from the Medical Research Council of Canada during the tenure of a Scholarship. References 1 A r l e t t , C . F . , D. T u r n b u l l , S.A. H a r c o u r t , A . R . L e h m a n a n d C.M. Colella, A c o m p a r i s o n o f t h e 8-azag u a n i n e - a n d o u a b a i n - r e s i s t a n t s y s t e m s f o r t h e s e l e c t i o n o f i n d u c e d m u t a n t C h i n e s e h a m s t e r cells, Mutation Res., 33 (1975) 261--278. 2 B a k e r , R . M . . D.M. B r u n e t t e , R. M a n k o w i t z , L . H . T h o m p s o n , G . F . W h i t m o r e 0 L. S i m i n o v i t c h a n d J . E . Till, O u a b a i n - r e s i s t a n t m u t a n t s o f m o u s e a n d h a m s t e r cells in c u l t u r e , Cell, 1 ( 1 9 7 4 ) 9 - - 2 1 . 3 B e e h - H a n s e n , N . T . . J . E . Till a n d V. L i n g , P l e i o t r o p i c p h e n o t y p e o f c o l c h i c i n e r e s i s t a n t C H O cells: C r o s s r e s i s t a n c e a n d c o l l a t e r a l s e n s i t i v i t y , J. Cell. P h y s i o l . , 8 8 ( 1 9 7 6 ) 2 3 - - 3 9 . 4 C a m p b e l l , C.E., a n d R . G . W o r t o n , E v i d e n c e o b t a i n e d b y i n d u c e d m u t a t i o n f r e q u e n c y a n a l y s i s f o r f u n c t i o n a l h e m i z y g o s i t y a t t h e e m t l o c u s in C H O cells, S o m a t i c Cell G e n e t . . 5 ( 1 9 7 9 ) 5 1 - - 6 5 . 5 C a r v e r , J . H . , G . M . A d a i r a n d D . L . W a n d r e s , M u t a g e n i c i t y t e s t i n g in m a m m a l i a n cells, II. V a l i d a t i o n o f multiple drug resistance markers having practical application for screening potential mutagens, Mutat i o n Res., 7 2 ( 1 9 8 0 ) 2 0 7 - - 2 3 0 . 6 Clive, D., K . O . J o h n s o n , J . F . S . S p e c t o r , A . G . B a t s o n a n d M.M.M. B r o w n , V a l i d a t i o n a n d c h a r a c t e r i z a t i o n o f t h e L 5 1 7 8 Y / T K +/- m o u s e l y m p h o m a m u t a g e n a s s a y s y s t e m . M u t a t i o n Res., 59 ( 1 9 7 9 ) 6 1 - 108.

7 F r i e d r i c h , V., a n d P. C o f f i n o , M u t a g e n e s i s in $ 4 9 m o u s e l y m p h o m a cells: I n d u c t i o n o f r e s i s t a n c e t o o u a b a i n . 6 - t h i o g u a n i n e a n d d i b u t y r y l c A M P , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 4 ( 1 9 7 7 ) 6 7 9 - - 6 8 3 . 8 G u p t a , R . S . , R e p e a t e d m u t a g e n e s i s a n d t h e s e l e c t i o n of recessive a n d d o m i n a n t m u t a t i o n s in c u l t u r e d m a m m a l i a n cells, M u t a t i o n Res., 7 4 ( 1 9 8 0 ) 5 0 3 - - 5 0 8 . 9 G u p t a , R . S . S e n e s c e n c e o f c u l t u r e d h u m a n d i p l o i d f i b r o b l a s t s , A r e m u t a t i o n s r e s p o n s i b l e ? J. Cell Physiol.° 1 0 3 ( 1 9 8 0 ) 2 0 9 - - 2 1 6 . 10 Gupta, R.S., Resistance to the microtubule inhibitor podophyllotoxin: Selection and partial charact e r i z a t i o n o f m u t a n t s in C H O cells, S o m a t i c Cell G e n e t . , 7 ( 1 9 8 1 ) 5 9 - - 7 1 . 11 G u p t a ° R . S . , a n d S. G o l d s t e i n , D i p h t h e r i a t o x i n r e s i s t a n c e in h u m a n f i b r o b l a s t cell s t r a i n s f r o m n o r real a n d c a n c e r - p r o n e i n d i v i d u a l s , M u t a t i o n Res.. 7 3 ( 1 9 8 0 ) 3 3 1 - - 3 3 8 . 12 Gupta, R.S., and S. Goldstein, M u t a g e n testing in the h u m a n fibroblast diphtheria toxin resistance system, in: a M o n o g r a p h describing the Results of International Collaborative Studies on the Evaluation of Short-Term Tests for Carcinogenesis, Elsevier/North-Holland, Amsterdam, 1981. 13 Gupta, R.S., and L. Siminovitch, The isolation and preliminary characterization of somatic cell m u tants resistant to the protein synthesis inhibitor emetine, Cell, 9 (1976) 213--219. 14 Gupta, R.S., and L. Siminovltch, T h e molecular basis of emetine resistance in Chinese hamster ovary cells: Alteration in the 4 0 S ribosomal subunit, Cell, 10 (1977) 61--66. 15 Gupta, R.S., and L. Siminovitch, Mutants of C H O cells resistant to the protein synthesis inhibitors cryptopleurine and tylocrebrine: Genetic and biochemical evidence for c o m m o n site of action of emetine, cryptopleurine, tylocrebine and tubulosine, Biochemistry, 16 (1977) 3209--3214. 16 Gupta, R.S., and L. Sirninovitch, Mutants of C H O cells resistant to the protein synthesis inhibitor emetine: Genetic and biochemical characterization of second-step mutants, Somatic Cell Genet., 4 (1978) 77--94. 1"/ Gupta, R.S., and L. Siminovitch, Genetic and biochemical characterization of mutants of C H O cells resistant to the protein synthesis inhibitor trichodcrmin, Somatic Cell Genet., 4 (1978) 355--374. 18 Gupta, R.S., and L. Slminovitch, Genetic and biochemical studies with the adenosine analogs toyocamycin and tubercidin: Mutation at the adenosine kinase locus in Chinese hamster cells,Somatic Cell Genet., 4 (1978) 715--736.

270 19 G u p t a , R . S . , a n d L. S i m i n o v i t c h , G e n e t i c m a r k e r s f o r q u a n t i t a t i v e m u t a g e n e s i s s t u d i e s in C h i n e s e h a m s t e r o v a r y cells: C h a r a c t e r i s t i c s o f s o m e r e c e n t l y d e v e l o p e d selective s y s t e m s , M u t a t i o n Res., 6 9 (1980) 113--126. 2 0 G u p t a , R . S . , a n d L. S i m i n o v i t c h , D R B r e s i s t a n c e in C h i n e s e h a m s t e r a n d h u m a n cells: G e n e t i c a n d b i o c h e m i c a l c h a r a c t e r i s t i c s o f t h e s e l e c t i o n s y s t e m , S o m a t i c Cell G e n e t . , 6 ( 1 9 8 0 ) 1 5 1 - - 1 6 9 . 21 G u p t a , R . S . , a n d L. S i m i n o v i t c h , P a c t a m y c i n r e s i s t a n c e in C H O cells: M o p h o l o g i c a l c h a n g e s i n d u c e d b y t h e d r u g in t h e w i l d - t y p e a n d m u t a n t cells, J. Cell. P h y s i o l . , 1 0 2 ( 1 9 8 0 ) 3 0 5 - - 3 1 6 . 2 2 G u p t a , R.S., a n d L. S i m i n o v i t c h , D i p h t h e r i a t o x i n r e s i s t a n c e in C h i n e s e h a m s t e r cells: G e n e t i c a n d b i o c h e m i c a l c h a r a c t e r i s t i c s o f t h e m u t a n t s a f f e c t e d in p r o t e i n s y n t h e s i s , S o m a t i c Cell G e n e t . , 6 ( 1 9 8 0 ) 361--369. 23 Gupta, R.S., D.H.Y. Chart and L. Siminovitch, Evidence obtained by segregation analysis for functional hemizygosity at the Emt r locus in CHO cells, Cell, 14 (1978) 1007--1013. 24 Gupta, R.S., D.H.Y. Chan and L. Siminovitch, Evidence for variation in the number of functional gene copies at the AreaR locus in Chinese hamster cells, J. Cell Physiol., 97 (1978) 461--467. 25 Gupta, R.S., J.J. Krepinsky and L. Siminovitch, Structural determinants responsible for the biological activity of (--)-emetine, (--)-cryptopleurine and (--)-tylocrebrine: Structure--activity relationship a m o n g r e l a t e d c o m p o u n d s , Mol. P h a r m a c o l . , 1 8 ( 1 9 8 0 ) 1 3 6 - - 1 4 3 . 2 6 Hsie, A.W., P.A. B r i m e r , T . J . Mitchell a n d D . G . Gosalee, T h e d o s e - - r e s p o n s e r e l a t i o n s h i p f o r e t h y l methanesulfonate-induced mutations at the hypoxanthine--guaninephosphoribosyl transferase locus in C h i n e s e h a m s t e r o v a r y cells, S o m a t i c Cell G e n e t . , 1 ( 1 9 7 5 ) 2 4 7 - - 2 6 1 . 27 Ingles, C . J . , B . G . B e a t t y , A. G u i a l i a , M.L. P e a r s o n , M.M. C r e r a r , P.E. L o b b a n , L. S i r n i n o v i t c h , D . G . S o m e r s a n d M. B u c h w a l d , cz-Amanitin r e s i s t a n t m u t a n t s o f m a m m a l i a n cells a n d r e g u l a t i o n o f R N A p o l y m e r a s e II a c t i v i t y , in: R. L o s i c k a n d M. C h a m b e r i i n ( E d s . ) , R N A P o l y m e r a s e , C o l d S p r i n g H a r b o r L a b o r a t o r y Press, N e w Y o r k , 1 9 7 6 . 2 8 K a o , F . T . , a n d T . T . P u c k , G e n e t i c s o f s o m a t i c m a m m a l i a n cells, IV. P r o p e r t i e s o f C h i n e s e h a m s t e r cell mutants with respect to the requirement for proline, Genetics. 55 (1967) 513--524. 2 9 K i t , S., D . R . D u b b s , L . J . P i e k a r s k i a n d T.C. H s u , D e l e t i o n o f t h y m i d i n e k i n a s e a c t i v i t y f r o m L cells r e s i s t a n t t o b r o m o d e o x y u r i d i n e , E x p . Cell Res., 31 ( 1 9 6 3 ) 2 9 7 - - 3 1 2 . 3 0 M c B u r n e y , M.W., C l o n a l lines o f t e r a t o c a r c i n o m a cells in vitro: D i f f e r e n t i a t i o n a n d c y t o g e n e t i c c h a r a c t e r i s t i c s , J. Cell. P h y s i o l . , 8 9 ( 1 9 7 6 ) 4 5 1 - - 4 5 6 . 31 P u c k , T . T . . P.I. M a r c u s a n d S.J. C i e c i u r a , C l o n a l g r o w t h o f m a m m a l i a n cells in vitro: G r o w t h c h a r a c t e r i s t i c s o f c o l o n i e s f r o m single H e L a cells w i t h a n d w i t h o u t f e e d e r l a y e r , J. E x p t l . M e d . , 1 0 3 ( 1 9 5 6 ) 273--284. 3 2 S i m i n o v i t c h , L., O n t h e n a t u r e o f h e r e d i t a b l e v a r i a t i o n in c u l t u r e d s o m a t i c cells, Cell, 1 ( 1 9 7 6 ) 1 - - 1 1 . 3 3 S t e v e n s , L . C . , T h e d e v e l o p m e n t o f t r a n s p l a n t a b l e t e r a t o c a r c i n o m a f r o m i n t r a t e s t i c u l a r g r a f t s o f prea n d p o s t - i m p l a n t a t i o n m o u s e e m b r y o s , D e v e l o p . Biol., 21 ( 1 9 7 0 ) 3 6 4 - - 3 8 2 . 3 4 S t o k e r , M., a n d I. M a c P h e r s o n , S y r i a n h a m s t e r f i b r o b l a s t cell line B H K 21 a n d its d e r i v a t i v e s , N a t u r e (London), 203 (1964) 1355--1357. 3 5 Y o t t i , L . P . , C.C. C h a n g a n d J . E . T r o s k o , E l i m i n a t i o n o f m e t a b o l i c c o o p e r a t i o n in C h i n e s e h a m s t e r cells b y a t u m o r p r o m o t e r , S c i n e c e s , 2 0 6 ( 1 9 7 9 ) 1 0 8 9 - - 1 0 9 1 .