Cell, Vol. 11, 447-454,
June
1977,
Copyright
0 1977 by MIT
Selection and Characterization of Cells Resistant to Diphtheria Toxin and Pseudomonas Exotoxin A: Presumptive Translational Mutants T. J. Moehring and J. M. Moehring Department of Medical Microbiology University of Vermont College of Medicine Burlington, Vermont 05401
Summary Two classes of diphtheria toxin-resistant variants were selected from Chinese hamster ovary (CHOKl) cells: permeability variants, in which uptake of toxin was impaired, and a new class of cytoplasmic variants, which were cross-resistant to Pseudomonas exotoxin. EF-2 prepared from the cytoplasmic variants was resistant to ADP-ribosylation by either toxin. The evidence presented suggests that these are translational variants possessing a mutationally altered EF-2 gene product. These studies also confirmed that Pseudomonas toxin ADP-ribosylates EF-2 in toxin-sensitive intact cells, as well as in cell-free systems. Introduction Diphtheria toxin inhibits protein synthesis in sensitive mammalian cells by catalyzing the ADP-ribosylation of elongation factor 2 (EF-2), thereby inactivating it. In this reaction, NAD is an obligatory cofactor, and the ADPR moiety is covalently bound to the enzyme which results in loss of the enzyme’s translocase and GTPase activities (Pappenheimer and Gill, 1973; Collier, 1975). Animals and cultured cells of most mammalian species, with the notable exception of rats and mice, are highly sensitive to the lethal effects of diphtheria toxin. We have previously reported on the selection and characterization of a series of diphtheria toxin-resistant (TOXR) cell strains from toxin-sensitive (TOXS) cultured human and nonhuman cell lines (Moehring and Moehring, 1972, 1976a). All these resistant cell strains, like those cultured from animals which are naturally resistant to the toxin, have belonged to a phenotypic class of resistant cells which we have designated permeability variants. Extracts from these cells, when tested in cell-free, amino acid-incorporating systems, were as sensitive to the action of the toxin as extracts from TOXS parental cells, and EF-2 prepared from these cells was susceptible to ADPribosylation by the toxin. Thus the resistance to toxin of these variants seems to result from an inability of the toxin to become internalized, perhaps because these cells lack either the appropriate surface receptors or a functional toxin-specific transport mechanism. We now wish to report on the selection from Chinese hamster ovary (CHO) cells of a new class of TOXR strains which we have designated transla-
tional variants. These variants possess three important properties which distinguish them from those previously described: first, they are more resistant to diphtheria toxin than any others we have isolated to date; second, their extracts are resistant to toxin in cell-free, amino acid-incorporating systems; and third, EF-2 extracted from these cells is not susceptible to ADP-ribosylation by diphtheria toxin. Furthermore, they are cross-resistant to Pseudomonas aeruginosa exotoxin A which, like diphtheria toxin, has been shown to catalyze the ADP-ribosylation of EF-2 (Iglewski and Kabat, 1975). To our knowledge, these translational variants are the first to be isolated in mammalian cells where the selecting agent interacts with a known specific target protein of the translational machinery. Results Isolation and Initial Characterization of Diphtheria Toxin-Resistant Variants The multiplication of TOXS CHO-Kl cells was progressively inhibited by increasing concentrations of diphtheria toxin from 0.001-10 guinea pig minimal lethal doses (MLD) per ml in complete F12 medium. Figure 1 shows the effect of increasing concentrations of toxin on the plating efficiency of CHO-Kl cells. Virtually all TOXR strains developed from clones of CHO-Kl cells surviving exposure to 0.1 MLD/ml or more for 24 hr showed a stable increased resistance to the toxin even after repeated culture in toxin-free medium. All the TOXR strains discussed here were isolated following exposure of wild-type parental CHO-Kl cells to 1 MLD of toxin per ml. A number of TOXR cell strains were recovered from normal and mutagenized CHO-Kl cells. They fell into two distinct classes based upon their behavior in the whole cell assay for inhibition of protein synthesis by toxins. The first class was made up of the permeability variants previously reported (Moehring and Moehring, 1976a). Permeability variants having many different levels of increased resistance were isolated; some could resist only IO times more toxin, on an MLD per ml basis, than the parental CHO-Kl, and others 100 to more than 10,000 times more. The TOXR permeability variants we discuss here represent the highest level of permeability class resistance recovered from CHOKl cells. This resistance is comparable to that of cells cultured from naturally resistant animals and other selected permeability variants reported previously (Moehring and Moehring, 1976a). The resistance to toxin of all the permeability variants was overcome by exposing them to high concentrations of toxin. The second class of TOXR variant strains that we describe possessed even greater resistance to diphtheria toxin, a resistance which could not be
Cell 440
l-
10-l -
10-Z 6 5 -u ;E iii
10-3 -
F ‘F if Q,
10-q-
Figure 2. The Effect of Increasing Concentrations Toxin on Protein Synthesis in TOXS CHO-Ki Cells iants
10-S -
Cells were exposed to toxin for 24 hr. The average cpm for each set of control cultures for the various strains were: CHO-KI, 10,981; CH-RI, 11,422; CH-REI .12, 12,944; CHI-RI .41, 8467; CHR46.6e, 9688; CH-RE1.22~ 11,523.
.g -0 d
lo-7L .3col
I 001
Concentration
I .Ol
I 0.1
of Diphtheria
Figure 1. The Plating Efficiency the Concentration of Diphtheria (Combined Results from Several
I 1.0
8 l0
, 100
Toxin, MLD/ml
of CHO-Ki Cells as a Function of Toxin in the Culture Medium Experiments)
overcome by increasing concentrations of toxin. In the sections which follow, we demonstrate that this latter group of variants represents a second distinct TOXR phenotype: a class of cytoplasmic variants. Furthermore, we present evidence that these strains are translational variants possessing an altered EF-2 which cannot be ADP-ribosylated. In Figure 2, the relative sensitivity of three independently selected translational variants is compared with that of permeability variants and wildtype parental cells. The concentration of diphtheria toxin which inhibited protein synthesis in CHO-Kl cells by 50% after 24 hr of exposure, referred to as the ID,,(24) value (Moehring and Moehring, 1976a), was 0.0038 MLD per ml. The ID,,(24) values for the two permeability class variant strains, CH-RI and CH-RE1.12, were 260 and 220 MLD per ml, respectively. ID&24) values for the three translational variant strains, CH-RE1.22c, CH-R1.41 and CHR48.6e, could not be computed, since they were not inhibited to a significant extent by any concentration of toxin even up to 10,000 MLD per ml. The frequency of occurrence of permeability variants such as CH-Rl in unmutagenized CHO-Kl populations was 8.9 x lo-‘. The frequency of translational variants such as CH-R1.41 was 1 .O x IO-‘. In CHO-Kl populations treated with the chemical mutagen ethyl methane sulfonate (EMS), the
of Diphtheria and TOXR Var-
frequency of permeability variants was increased to 4.9 x 10-5, an increase of 55 times. The frequency of translational variants was increased to 2.9 x 1Om6, an increase of 29 times. Of the five toxinresistant strains which we discuss, CH-RI, CHR1.41 and CH-R48.6e were isolated from normal parent cell populations, and CH-RE1.12 and CHREl.22~ were isolated from mutagenized populations. Kinetics of Intoxication Figure 3 demonstrates the effect of various concentrations of diphtheria toxin on the toxin-resistant variant strains over a 96 hr period. This type of study shows the effect of the different concentrations of toxin on the growth of cells in culture, since the increased incorporation of amino acids into protein in control, or unintoxicated, cultures parallels the growth and multiplication of the cells. Control cultures of all the strains increased their incorporation over the initial value 11 times or more during the 96 hr period of the experiments. In Figure 3A, a plot of the values for CHO-Kl cells shows that concentrations of toxin of 0.01 MLD per ml and higher depressed incorporation below the initial rate, causing inhibition from which the cells did not recover. The lower concentrations tested inhibited the cells, but still allowed increase above the initial rate and allowed cell growth. In Figure 38, it can be seen that concentrations of lOOO10,000 MLD per ml progressively depressed the incorporation of amino acids into permeability variant CH-RI cells below the initial rate, whereas lOOIO MLD per ml only depressed the rate of growth, allowing an increase over the initial value. Figure 3C shows that concentrations of toxin of 1000 and 10,000 MLD per ml have no effect whatever over
Presumptive 449
Translational
Mutants
time on cultures of the translational RE1.22~. Growth and incorporation treated cultures are exactly as control
- CHO-KI
0
12
24
36
Time
48
60
72
84
96
Hours
in
B. CH-RI
Time
in
I2
24
36
48
Hours
60
72
Time in Hours Figure 3. Kinetics of Inhibition of Protein Synthesis Toxin in TOXS CHO-KI Cells and TOXR Variants The average cpm per culture were: CHO-Ki, 422; CH-RI,
DEAE-Dextran Treatment of Resistant Strains We have previously reported that treatment of permeability variants with the membrane-active substance DEAE-dextran (DEAE-D) increases their sensitivity to diphtheria toxin (Moehring and Moehring, 1976c). Figure 4 shows this effect on CHRl cells. The IDso (24) concentration of the toxin on untreated cells was 260 MLD per ml. When these cells were pretreated with DEAE-D, however, only 5 MLD per ml were necessary to inhibit protein synthesis by 50% in 24 hr. When translational variant strains CH-REI .22c and CH-R48.6e were treated with DEAE-D, no increase in sensitivity to toxin was noted. They reacted exactly as did control cells. Investigating the effects of treating translational variants with DEAE-D accomplished two purposes. First, the results gave further support to the view that they were not permeability variants with a higher level of resistance than previously observed. Second, the results showed that treatment of cells with DEAE-D specifically enhanced the action of diphtheria toxin. DEAE-D was not simply a second toxic agent acting synergistically on toxin-treated cells. Resistance of Cell-Free Extracts Post-mitochrondrial extracts of TOX” and TOX’ Chinese hamster ovary cells were prepared and tested in a cell-free. amino acid-incorporating system. Figure 5 shows that cell-free extracts of the permeability variant. CH-Rl , were equal in sensitivity to extracts of wild-type CHO-Kl cells when titrated against increasing concentrations of diphtheria toxin. Cell-free extracts of the three translational variants, CH-RI .41, CH-REl.22~ and CHR48.6e. were fully resistant, however, and were not significantly inhibited by any concentration of toxin tested. Following these observations. we were able to designate the latter variants as cytoplasmic variants and considered the possibility that their cell extracts contained a diphtheria toxin-resistant EF2.
E ,j , , , , , , I / 0
variant CHof toxincultures.
84
96
by Diphtheria
for the zero time, or initial, 440; CH-REI .22c, 402.
samples
ADP-Ribosylation of EF-2 EF-2 was prepared from each ceil strain and tested for susceptibility to ADP-ribosylation in the presence of excess diphtheria toxin and NAD. as shown in Figure 6. Under our conditions of assay. there was a linear dose response following the addition of increasing amounts of EF-2 prepared from TOX’ wild-type cells and permeability class TOX” variants. The ADPR:EF-2 complex was quantitatively recovered following precipitation with acid and expressed as pmoles of ADPR incorporated. Under
Cell 450
1.o
..I
\
o CH-REl.22~
. CH-REl.22~ A CH-R48.6e A CH-R48,6e q n
0.1
.
*DEAE-D
\
0
+ DEAE-D
CH-RI CH-RI + DEAE-D 1.0
\\
.-
d IO
03
loco
10000
EF- 2 Extract, pg Protein
Cont. of Diphtheria Toxin, MLD/ml Figure theria Strains
4. Effect of Cellular Pretreatment Toxin Activity in Permeability
with DEAE-D on Diphand Translational TOXR
The average cpm for each set of control cultures were: CHREI .22c, 8441; CH-REl.22~ + DEAE-D, 7689: CH-R48.6e, 9688: CH-R48.6e + DEAE-D, 7449: CH-RI, 11,422; CH-RI + DEAE-D, 10,871.
60
Concentration Figure 5. The Effect Labeled Amino Acids Cells
of Diphtheria
of Diphtheria by Cell-Free
Toxin,
MLD/ml
Toxin on Incorporation of ‘%Extracts of TOXS and TOXR CHO
The average cpm for each set of control CHO-Kl, 6334: CH-RI, 6949; CH-R48.6e, CH-REI .22c, 5612.
reaction mixtures 6382: CH-R1.41,
were: 3873;
Figure 8. Diphtheria Toxin-Catalyzed ADP-Ribosylation Extracted from TOXS and TOXR CHO Cells
of EF-2
these conditions, diphtheria toxin did not catalyze the transfer of a measurable amount of ADPR to the EF-2 in extracts prepared from the three cytoplasmic variants, CH-RE1.22c, CH-R48.6e and CHRI .41. The extracts from these cells appeared to be totally resistant to the action of the toxin. The possibility that extracts prepared from these cytoplasmic variants contained a factor which antagonized the toxin-catalyzed ADP-ribosylation reaction was considered and tested. In one type of experiment, we determined the effect of mixing toxin-resistant extracts with extracts from TOX5 cells in the ADP-ribosylation assay. Increasing amounts of EF-2 extract prepared from the cytoplasmic variants CH-RI .41 and CH-REI .22c were incubated with a constant amount of EF-2 extracted from wild-type CHO-Kl cells, in this case 60 pg of protein per reaction mixture, and the transfer of ADPR to EF-2 was measured. Figure 7 shows that the ADP-ribosylation of toxin-sensitive EF-2 from CHO-Kl cells was not altered by the presence in the reaction mixtures of TOX’* extracts at concentrations up to 2.5 times the concentration of the TOXS extract. Post-mitochondrial extracts from cytoplasmic variants were also mixed with extracts from wildtype cells to determine whether that would affect the sensitivity to toxin of the latter in the cell-free, amino acid-incorporating assay. The sensitivity to toxin of the wild-type extract was not altered by extract from cytoplasmic variants. In addition, diphtheria toxin was incubated with post-mitochondrial extracts, prepared as for use in cell-free amino acid assays, and with EF-2 extracts prepared
Presumptive 451
Translational
Mutants
CH-R48.6e 100
&
60
E 8 f!
40
8 C
-$=+=? CH-REL22c
CH-RI,41
TOXs EF-2 Extract @HO-Kl), 6Opg Protein t
g 01’11”‘111”1”” 0
20
40
60
80
100
120
140
160
TOXR EF-2 Extract, pg Protein Figure 7. The Effect on ADP-Ribosylation
of Increasing of a Standard
Amounts Amount
The average ADPR incorporated by control tract was 6.57 pmoles per 60 ~g protein.
of TOXa EF-2 Extract of TOXS EF-2 Extract samples
L
of TOXS ex0 ,sml
’ “,,J1’ ,001
5 1’,‘11’ .Oi
j
““’
0.1
I.0
ul 10
Pseudomonos Exotoxin A , pg/ml from cytoplasmic variants. When the toxin was subsequently tested for activity on intact TOX’ cells, no loss of toxin activity was detected. The results of these experiments strongly suggest that resistance to ADP-ribosylation is an intrinsic property of the enzyme, EF-2. and not due to the presence in extracts or cells of a factor which possesses either antitoxic activity or the ability to modify the susceptibility of EF-2 to ADP-ribosylation by diphtheria toxin. We therefor conclude that they are true translational variants. Cross-Resistance to Pseudomonas Exotoxin A The work of lglewski and Kabat (1975) demonstrating that Pseudomonas aeruginosa exotoxin A, as well t& diphtheria toxin, was capable of ADP-ribosylatlng EF-2 in the presence of NAD+ prompted us to determine whether any of our diphtheria toxinresistant variants were cross-resistant to Pseudomonas toxin. The response of intact CHO cells to Pseudomonas toxin is presented in Figure 8. TOX’ CHO-Kl cells and permeability class TOX” CH-Fil and CH-RE1.12 cells were equally sensitive to the toxin. suggesting that the two toxins either bind to different receptors or enter cells by independent mechanisms. More importantly. however. the translational variants CH-R48.6e. CH-RE1.22~ and CH-RI .41 did show the cross-resistance to Pseudomonas toxin expected if these cells possess an EF-2 which is refractory to the action of diphtheria toxin. Furthermore. as shown in Figure 9. when the ability of Pseudomonas toxin to ADP-ribosylate EF2 was measured directly in the ADP-ribosylation assay, it was found that EF-2 from wild-type CHOKl cells and permeability class TOX” variants was susceptible to ADP-ribosylation, while that from translational variants was totally resistant.
Figure thesis
8. The Effect of Pseudomonas Exotoxin in TOXS CHO-Kl Cells and TOXR Variants
The average cpm for each set of control 8487; CH-RE1.12, 9208; CH-RI, 8657; RE1.22c, 7874; CH-R1.41, 6106.
on Protein
Syn-
cultures were: CHO-Kl, CH-R48.6e, 7590; CH-
Karyotypic Analysis The CHO-Kl cells used in this study had a modal chromosome number of 20, as previously reported (Kao and Puck, 1969). Three of the TOX” strains. CH-REl.12, CH-RI.41 and CH-RE1.22~~ had the same modal chromosome number as the wild-type cells. The permeability variant, CH-RI , had a modal chromosomal number of 19. while that of the translational variant, CH-R48.6e. was 40. All the variants except CH-R48.6e had a growth rate in complete F12 medium which was equal to that of the parent ceil line. The growth rate of CH-R48.6e was approximately one half that of the parent cell. Although marker chromosomes have been identified in some of the variants, we have not as yet been able to associate any specific chromosomal changes with the TOX” phenotypes under study. Discussion The study of the process of diphtheria intoxication at the cellular and molecular level would be facilitated if ceils mutant at different steps in the process of intoxication were available. Such mutants would be useful for elucidating steps in the process of intoxication and for investigating cellular functions which are directly involved in that process. The present communication deals with the selection and biochemical characterization of two phenotypic classes of CHO variants resistant to diphtheria toxin: the permeability variants that we have
Cell 452
10 z E a 5
8-
0.5
1.0
Pseudomonas
5.0
IO
Exotoxin A, pg/Reaction
Mixture
Figure 9. Pseudomonas Exotoxin-Catalyzed ADP-Ribosylation EF-2 Extracted from TOXS and TOXR CHO Cells
of
described previously, and a new class of cytoplasmic variants whose cell-free extracts resist ADPribosylation by diphtheria toxin and Pseudomonas exotoxin. We believe that evidence that these latter variants are translational mutants possessing a mutationally altered EF-2 gene product is compelling. We could find no evidence of a soluble factor that was either antitoxic in nature or able to modify the susceptibility of EF-2 from normal cells to ADPribosylation. The possibility that these variants arose as a result of some epigenetic event seems less probable. The toxin-resistant phenotypes described were both qualitatively and quantitatively diverse; they were stable in the absence of toxin; their low frequencies of appearance in the wild-type population were increased following treatment with the mutagenic agent EMS; and their frequencies of reversion were low. These are properties consistent with a genetic origin. The EF-2 enzyme in the translational variants appears to be altered in its susceptibility to toxin while retaining its normal translocase and GTPase activity. The translational variants CH-R1.41 and CH-REI .22c. for instance, grew at the same rate as wild-type CHO-Kl cells. In addition, their extracts were as active in the cell-free assay for incorporation of amino acids as equal amounts of extracts from wild-type cells. One strong possibility is that
the site on EF-2 where ADP-ribosylation occurs has in some way been modified. Such a modification could result from a point mutation leading to either a conformational change in the molecule or an amino acid substitution for the residue. X, to which the ADPR moiety is covalently bound in the toxincatalyzed inactivation of EF-2 (Robinson, Henrikson and Maxwell, 1974). Similar alterations in gene product have been demonstrated in other systems (Albrecht, Biedler and Hutchison, 1972; Chan, Whitmore and Siminovitch, 1972; Baker et al., 1974; Chasin, 1974). Further studies to characterize the genetic change and the nature of the modification of the enzyme are presently being carried out. When the kinetics of diphtheria intoxication were studied on intact cells, it was found that translational variants were totally resistant to 10.000 MLD of toxin per ml over a period of several days. These results demonstrate that in the CHO system at least. diphtheria toxin does not have a growth inhibitory or cytotoxic action which is alternate or complementary to that of ADP-ribosylation of EF-2. The variants described have proved useful in comparing further the action of Pseudomonas exotoxin with that of diphtheria toxin. Our results demonstrate that the cytotoxic action of Pseudomonas exotoxin in intact cells. like that of diphtheria toxin, is to ADP-ribosylate EF-2. Translational variants were fully cross-resistant to Pseudomonas exotoxin, while permeability variants failed to show any cross-resistance to Pseudomonas exotoxin. It has been observed that cells resistant to one cytotoxic agent may be cross-resistant to other cytotoxic agents having different intracellular targets (Ling and Thompson. 1974; Peterson, O’Neil and Biedler. 1974). indeed. we found that diphtheria toxin-resistant permeability variants of KB cells were cross-resistant to several different viruses (Moehring and Moehring, 1976b). These observations, along with our diphtheria toxin studies, clearly indicated that the resistance of these cells was at the level of the membrane. In the present study. we compared two agents, diphtheria toxin and Pseudomonas exotoxin. having the same molecular basis for cytoxicity and found that they did not share a common mechanism of uptake. These results are similar to those obtained in a comparison of uptake between diphtheria toxin and two other structurally similar (Olsnes, Refsnes and Pihl, 1974) cytotoxic inhibitors of protein synthesis, abrin and ricin (S. Olsnes. J. Moehring and T. Moehring, unpublished data). It is possible that continuing studies will reveal additional TOX” phenotypes as well as heterogeneity within the phenotypes already described. Further development of this experimental system should enable us to seek solutions to biologic
Presumptive 453
Translational
Mutants
problems of wide interest. Translational variants, for instance, should prove useful in studying EF-2, its role in protein synthesis and its regulation. The availability of permeability variants will facilitate further studies on mechanisms of binding and uptake of toxins and other macromolecules. A well characterized system of TOX” biochemical and genetic markers will be of value in studies of mutagenesis in somatic cells. Finally, the results of our studies on the interaction of diphtheria toxin and Pseudomonas exotoxin A with toxin-resistant cells suggest that similar variants could have an important role in elucidating other mechanisms of pathogenesis at the cellular and molecular level. Experimental
Procedures
Ceils, Media and Culture Conditions Chinese hamster ovary cells, line CHO-KI, were obtained from the American Type Culture Collection. Toxin-resistant variants were selected from this line as described below. All cell strains were maintained in Ham’s Nutrient Mixture F12, containing 10% fetal calf serum and 50 pg of gentamicin per ml (which is referred to as “complete” F12) at 37°C in an atmosphere of 5% CO* in air. Toxins Two lots of diphtheria toxin were used in these studies. Columnpurified toxin was a gift from R. John Collier (University of California, Los Angeles). This toxin produced a single band in acrylamide gel electrophoresis in the absence of thiols and had 20,000 guinea pig minimum lethal doses (MLD) per mg of protein. Concentrated and partially purified toxin was obtained from Connaught Medical Research Laboratories (Toronto, Ontario, Canada). This toxin produced a single major band and several trace bands in acrylamide gel electrophoresis, and had 19,000 MLD per mg of protein. Data showing that these two toxin preparations are nearly identical in toxicity when considered on an MLD per ml basis have been published (Moehring and Moehring, 1976a). We report results obtained with the two preparations interchangeably. We are indebted to Stephen H. Leppla (U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland) for providing us with highly purified Pseudomonas aeruginosa exotoxin (Leppla, 1976). This toxin produced a single band in acrylamide gel electrophoresis and had 5000 mouse lethal doses per mg of protein. Selection of Diphtheria Toxin-Resistant Cell Strains Selection of cell strains resistant to diphtheria toxin was carried out following a single exposure of populations of normal and mutagenized CHO-KI cells to a concentration of toxin which would completely, and irreversibly, inhibit protein synthesis in 24 hr-that is, 0.1 MLD per ml or higher (see CHO-KI curve in Figure
2). Cells were mutagenized by exposing them to 300 pg of ethyl methane sulfonate (EMS) (Eastman Kodak) per ml of complete F12 for 18 hr, according to the procedure described by Baker et al. (1974). This treatment caused from 50-60% killing of the cells. Cells were then passaged and maintained in nonselective medium for an expression period of 5 days. To select toxin-resistant strains, normal or mutagenized cells were seeded in 60 mm plastic petri dishes (Falcon Plastics) at 2 x 1 O5 cells per dish. They were allowed to adhere for periods of from 2-18 hr prior to application of toxin in growth medium. After exposure to toxin for 24-48 hr, cultures were washed with Hank’s balanced salts solution and incubated in complete F12 in the usual manner. Cultures were fed with fresh medium on day 4.
After further incubation for 3-4 days, plates were examined for living clones of cells. If these clones were only to be counted, 0.1% 2(p-idophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) (Aldrich Chemical) in complete F12 was added to each plate, and cultures were incubated again for 12-18 hours. Living cells were then stained a deep permanent red. Clones were counted after the plates were drained, washed with distilled water and air-dried. No fixing was required. If living clones were to be selected for further study, they were isolated with porcelain cloning cylinders and transferred to tissue culture multidishes with 16 mm wells (Linbro Chemical) and grown to confluency in toxin-free complete F12. When sufficient cells were generated, the resistance to toxin of the isolated strains was determined using the whole cell assay for inhibition of protein synthesis by toxins, described below. Karyology Exponentially growing monolayer cultures were incubated with 0.05 pg of Velban (Lilly) per ml for 75 min at 37°C. Cells were removed from the substrate using 1 x trypsin-EDTA (Gibco), swelled in 0.06 M KCI for 18 min at room temperature, fixed in three changes of methanol:acetic acid (3:l) and spread on glass slides by blowing. Banded chromosomes were prepared, according to the procedure of Sun, Chu, and Chang (1973), from airdried slides which had aged for 3 days. At least 100 representative chromosome spreads of each variant were counted. Whole Cell Assay for Inhibition of Protein Synthesis by Toxins The assay procedure (Moehring and Moehring, 1976a) was as follows. Cells were seeded at 5 x IO4 cells per culture in 8 ml flint glass vials (I.C.N. Pharmaceuticals, Cleveland, Ohio) and incubated for 48 hr. When cells were pretreated with DEAEdextran (DEAE-D), 500 pg of DEAE-D per ml were applied to cultures immediately prior to application of toxin dilutions, as reported previously (Moehring and Moehring, 197613). Cells were then exposed to appropriate concentrations of toxins in culture medium containing 5% fetal calf serum for 24 hr. At the end of this period, the medium was aspirated, and 0.5 ml of 0.05 M tris(hydroxymethyI)aminomethane-buffered Eagle’s minimum essential medium containing a I:20 dilution of the normal amino acids and 0.4 &I of j4C-labeled amino acid mixture (15 amino acids; 100 &i/ml; New England Nuclear, Boston, Massachusetts) were added. Assay vials were then incubated for 30 min. Labeling medium was then removed, and cells were precipitated on the vial with 4 ml of chilled 5% trichloroacetic acid. The precipitated cell layer was dried following removal of the trichloroacetic acid. 5 ml of Permafluor liquid scintillator in toluene (Packard Instrument) were added to each vial, and the samples were counted directly in a Tri-Carb scintillation spectrometer (Packard lnstrumerit). Preparation of Cell Extracts and Incorporation of Amino Acids by Cell-Free Systems The methods used to prepare cell extracts and to study their incorporation of radioactive amino acids into protein have been described in full elsewhere (Moehring and Moehring, 1968; 1976a). The post-mitochondrial cell extracts used in these studies were a product of 50 million cells per ml of lysing buffer, and a 20 ~1 amount of cell extract was used per standard reaction mixture of 50 ~1. A ‘YXabeled amino acid mixture (NEC-445, New England Nuclear) was used to label peptides, which were collected on Whatman GF/C glass fiber filters and counted by scintillation spectrometry, as were the whole cell samples. Preparation of EF-2 EF-2 was prepared from CHO dure used by Gill and Dinius nates of mammalian tissues. centrifuged at 500 x g for IO cells, was then suspended in
cells by a modification of the proce(1973) to extract EF-2 from homogeCells were harvested, washed and min. The cell pellet, 0.25 ml per lOa 0.25 M sucrose in the ratio of 1 ml of
Cell 454
Table
1. Formation
of ADP-Ribosyl cpm
EF-2 and Poly(ADP-Ribose)
in Acid-Insoluble
in the Presence
and Absence
of 0.18 M Histamine
Material
1.6 PM NAD
3.2 pM NAD
4.6 /.LM NAD
Toxin
Toxin
Toxin
0.18 M Histamine
+
-
A
-t
-
A
+
-
A
-
a829
2745
60a4a
11,596
5353
6242
14,714
a334
6380
+
7068
226
7,497
a37
6660
7,152
364
6788
6842
a The difference in counts (A) following incubation of 115 pg of CHO-Kl EF-2 extract with 1.6 PM NAD in the absence of histamine 6488-1187 = 6304 cpm. Reaction mixtures containing 115 pg of CHO-Kl extract and 250 pg of CH-R48.6e extract were incubated under standard conditions presence of 100 pg of diphtheria toxin and NAD sn the amounts indicated. The specific activity of 14C-NAD was 543 cpm/pmole.
sucrose to lOa cells and homogenized in a tight-fitting Dounce homogenizer. To each 1.25 ml of homogenate were added 0.21 ml of 4M NH&I and 0.33 g of wet, washed, activated charcoal (Norit neutral, Fisher Scientific). The mixture was homogenized intermittently in a Dounce homogenizer with a loose-fitting pestle over a period of 15 min and then centrifuged at 100,000 x g for 1 hr. The clear supernatant was collected and assayed for ADP-ribosylatable EF-2. The extracts were stored at -70°C. Specific activity (pmoles of ADP-ribosylatable EF-2 per ml protein) was not altered by up to five cycles of freeze-thawing. The specific activities of extracts prepared from CHO-KI, CHRl and CH-RI .I2 were 107, 97 and 92 pmoleslmg, respectively, and comparable to a standard preparation of rat liver EF-2 prepared in our laboratory (Moehring and Moehring, 1976c), having a specific activity of 106 pmoles/mg protein. An Average yield of EF2 from CHO-Kl cells was 1825 pmoles per 2 x lOa cells or 5.5 x lo6 molecules per cell. The amount of EF-2 in extracts of the three translational variants, CH-R46.6e, CH-REI .22c, and CH-RI .41, could not be determined since they contained no ADP-ribosylatable EF-2 (Figure 6). ADP-Ribosylation Assay ADP-ribosylation was carried out in 0.25 ml reaction mixtures containing 0.25 M Tris (hydroxymethyl) aminomethane-hydrochloride buffer at pH 6.2, 0.04 M’dithiothreitol, 0.1 M ethylenediamine-tetraacetate and 1.6 PM U-14C-NAD (280 mCi/mmol) Amersham-Searle). The standard amount of diphtheria toxin used was 100 pg per reaction. The amounts of Pseudomonas toxin and EF-2 used are indicated in each experiment. The reaction was initiated by the addition of either toxin or EF-2. Reaction mixtures were incubated at 25°C for 15 min, and the reaction was stopped by the addition of 0.25 ml of 10% trichloroacetic acid. Under these conditions, the reaction went to completion when toxin and NAD were in excess. A complete set of control tubes, without toxin, was included in each experiment, and the amount of ADP-ribosyl EF-2 formed was determined from the difference between counts recovered from incubation mixtures with and without toxin. The concentration of NAD used represented an excess of 25 fold or more over the maximum amount of EF-2 used in each experiment. It was also determined by isotope dilution that this excess was sufficient to overcome completely the dilution effect of the small amount of endogenous NAD which remained in some of the preparations of EF-2 following treatment with charcoal. In the case of EF-2 preparations from translational variants, these latter determinations were made by mixing these extracts with extracts of EF-2 prepared from CHO-KI, as described in Figure 7. Extracts of EF-2 from CHO cells, like those extracted from animal tissues, were found to contain poly(ADP-ribose) transferase activity (Gill and Dinius, 1973). In the presence of sufficient 14C-NAD, this activity leads to the formation of acid-insoluble radioactive poly(ADP-ribose) which interferes with the assay for
was in the
ADP-ribosyl EF-2. At the concentration of NAD used in our experiments, 1.6 PM, a significant amount of poly(ADP-ribose) was formed unless histamine, an inhibitor of transferase activity, was included in the reaction mixture (Table 1). Including histamine in the reaction mixture resulted in a slight but consistent increase in net corn. Acknowledgments This investigation was supported by a USPHS grant from NIH. We wish to acknowledge the capable assistance of D. Danley, C. Rolerson and P. Guigley in carrying out the karyotypic analysis of CHO CELLS. A preliminary report of this study was presented at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7-10, 1976. Received
February
8, 1977;
revised
March
25, 1977
References Albrecht, A. M., Biedler, J. L. and Hutchison, D. (1972). Cancer Res. 32, 1539. Baker, R. M., Brunette, D. M., Mankovitz, R., Thompson, L. H., Whitmore, G. F., Siminovitch, L. and Till, J. E. (1974). Cell 1, 9. Chan, V. L., Whitmore, G. F. and Nat. Acad. Sci. USA 69, 3119.
Siminovitch
I, L. (1972).
Proc.
Chasin, L. A. (1974). Cell 2, 37. Collier, R. J. (1975). Bacterial. Rev. 39, 54. Gill, D. M. and Dinius, L. L. (1973). J. Biol. Chem. 248, 654. Iglewski, B. H. and Kabat, D. (1975). Proc. Nat. Acad. Sci. USA 72, 2284. Kao, F.-T. and Puck, T. T. (1969). J. Cell. Physiol. 74, 245. Leppla, S. H. (1976). Infect Immunol. 14, 1077. Ling, V. and Thompson, L. H. (1974). J. Cell Physiol. 83, 103. Moehring, T. J. and Moehring, J. M. (1968). J. Bacterial. 96, 61. Moehring, T. J. and Moehring, J. M. (1972). Infect. Immunol. 6, 487. Moehring, J. M. and Moehring, T. J. (1976a). Infect. Immunol. 13, 221. Moehring, J. M. and Moehring, T. J. (1976b). Virology69, 786. Moehring, T. J. and Moehring, J. M. (1976c). Infect. Immunol. 73, 1426. Olsnes, Sjur, Refsnes, K. and Pihl. A. (1974). Nature 249, 627. Pappenheimer, A. M., Jr. and Gill, D. M. (1973). Science 782, 353. Peterson, R. H. F., O’Neil, J. A. and Biedler, J. L. (1974). J. Cell Biol. 63, 773. Robinson, E. A., Henrikson, 0. and Maxwell, E. S. (1974). J. Biol. Chem. 249, 5088. Sun, N. C., Chu, E. H. Y. and Chang, C. C. (1973). Mammalian Chromosome Newsletter 74, 26.
June
1977,
Copyright
0 1977 by MIT
Selection and Characterization of Cells Resistant to Diphtheria Toxin and Pseudomonas Exotoxin A: Presumptive Translational Mutants T. J. Moehring and J. M. Moehring Department of Medical Microbiology University of Vermont College of Medicine Burlington, Vermont 05401
Summary Two classes of diphtheria toxin-resistant variants were selected from Chinese hamster ovary (CHOKl) cells: permeability variants, in which uptake of toxin was impaired, and a new class of cytoplasmic variants, which were cross-resistant to Pseudomonas exotoxin. EF-2 prepared from the cytoplasmic variants was resistant to ADP-ribosylation by either toxin. The evidence presented suggests that these are translational variants possessing a mutationally altered EF-2 gene product. These studies also confirmed that Pseudomonas toxin ADP-ribosylates EF-2 in toxin-sensitive intact cells, as well as in cell-free systems. Introduction Diphtheria toxin inhibits protein synthesis in sensitive mammalian cells by catalyzing the ADP-ribosylation of elongation factor 2 (EF-2), thereby inactivating it. In this reaction, NAD is an obligatory cofactor, and the ADPR moiety is covalently bound to the enzyme which results in loss of the enzyme’s translocase and GTPase activities (Pappenheimer and Gill, 1973; Collier, 1975). Animals and cultured cells of most mammalian species, with the notable exception of rats and mice, are highly sensitive to the lethal effects of diphtheria toxin. We have previously reported on the selection and characterization of a series of diphtheria toxin-resistant (TOXR) cell strains from toxin-sensitive (TOXS) cultured human and nonhuman cell lines (Moehring and Moehring, 1972, 1976a). All these resistant cell strains, like those cultured from animals which are naturally resistant to the toxin, have belonged to a phenotypic class of resistant cells which we have designated permeability variants. Extracts from these cells, when tested in cell-free, amino acid-incorporating systems, were as sensitive to the action of the toxin as extracts from TOXS parental cells, and EF-2 prepared from these cells was susceptible to ADPribosylation by the toxin. Thus the resistance to toxin of these variants seems to result from an inability of the toxin to become internalized, perhaps because these cells lack either the appropriate surface receptors or a functional toxin-specific transport mechanism. We now wish to report on the selection from Chinese hamster ovary (CHO) cells of a new class of TOXR strains which we have designated transla-
tional variants. These variants possess three important properties which distinguish them from those previously described: first, they are more resistant to diphtheria toxin than any others we have isolated to date; second, their extracts are resistant to toxin in cell-free, amino acid-incorporating systems; and third, EF-2 extracted from these cells is not susceptible to ADP-ribosylation by diphtheria toxin. Furthermore, they are cross-resistant to Pseudomonas aeruginosa exotoxin A which, like diphtheria toxin, has been shown to catalyze the ADP-ribosylation of EF-2 (Iglewski and Kabat, 1975). To our knowledge, these translational variants are the first to be isolated in mammalian cells where the selecting agent interacts with a known specific target protein of the translational machinery. Results Isolation and Initial Characterization of Diphtheria Toxin-Resistant Variants The multiplication of TOXS CHO-Kl cells was progressively inhibited by increasing concentrations of diphtheria toxin from 0.001-10 guinea pig minimal lethal doses (MLD) per ml in complete F12 medium. Figure 1 shows the effect of increasing concentrations of toxin on the plating efficiency of CHO-Kl cells. Virtually all TOXR strains developed from clones of CHO-Kl cells surviving exposure to 0.1 MLD/ml or more for 24 hr showed a stable increased resistance to the toxin even after repeated culture in toxin-free medium. All the TOXR strains discussed here were isolated following exposure of wild-type parental CHO-Kl cells to 1 MLD of toxin per ml. A number of TOXR cell strains were recovered from normal and mutagenized CHO-Kl cells. They fell into two distinct classes based upon their behavior in the whole cell assay for inhibition of protein synthesis by toxins. The first class was made up of the permeability variants previously reported (Moehring and Moehring, 1976a). Permeability variants having many different levels of increased resistance were isolated; some could resist only IO times more toxin, on an MLD per ml basis, than the parental CHO-Kl, and others 100 to more than 10,000 times more. The TOXR permeability variants we discuss here represent the highest level of permeability class resistance recovered from CHOKl cells. This resistance is comparable to that of cells cultured from naturally resistant animals and other selected permeability variants reported previously (Moehring and Moehring, 1976a). The resistance to toxin of all the permeability variants was overcome by exposing them to high concentrations of toxin. The second class of TOXR variant strains that we describe possessed even greater resistance to diphtheria toxin, a resistance which could not be
Cell 440
l-
10-l -
10-Z 6 5 -u ;E iii
10-3 -
F ‘F if Q,
10-q-
Figure 2. The Effect of Increasing Concentrations Toxin on Protein Synthesis in TOXS CHO-Ki Cells iants
10-S -
Cells were exposed to toxin for 24 hr. The average cpm for each set of control cultures for the various strains were: CHO-KI, 10,981; CH-RI, 11,422; CH-REI .12, 12,944; CHI-RI .41, 8467; CHR46.6e, 9688; CH-RE1.22~ 11,523.
.g -0 d
lo-7L .3col
I 001
Concentration
I .Ol
I 0.1
of Diphtheria
Figure 1. The Plating Efficiency the Concentration of Diphtheria (Combined Results from Several
I 1.0
8 l0
, 100
Toxin, MLD/ml
of CHO-Ki Cells as a Function of Toxin in the Culture Medium Experiments)
overcome by increasing concentrations of toxin. In the sections which follow, we demonstrate that this latter group of variants represents a second distinct TOXR phenotype: a class of cytoplasmic variants. Furthermore, we present evidence that these strains are translational variants possessing an altered EF-2 which cannot be ADP-ribosylated. In Figure 2, the relative sensitivity of three independently selected translational variants is compared with that of permeability variants and wildtype parental cells. The concentration of diphtheria toxin which inhibited protein synthesis in CHO-Kl cells by 50% after 24 hr of exposure, referred to as the ID,,(24) value (Moehring and Moehring, 1976a), was 0.0038 MLD per ml. The ID,,(24) values for the two permeability class variant strains, CH-RI and CH-RE1.12, were 260 and 220 MLD per ml, respectively. ID&24) values for the three translational variant strains, CH-RE1.22c, CH-R1.41 and CHR48.6e, could not be computed, since they were not inhibited to a significant extent by any concentration of toxin even up to 10,000 MLD per ml. The frequency of occurrence of permeability variants such as CH-Rl in unmutagenized CHO-Kl populations was 8.9 x lo-‘. The frequency of translational variants such as CH-R1.41 was 1 .O x IO-‘. In CHO-Kl populations treated with the chemical mutagen ethyl methane sulfonate (EMS), the
of Diphtheria and TOXR Var-
frequency of permeability variants was increased to 4.9 x 10-5, an increase of 55 times. The frequency of translational variants was increased to 2.9 x 1Om6, an increase of 29 times. Of the five toxinresistant strains which we discuss, CH-RI, CHR1.41 and CH-R48.6e were isolated from normal parent cell populations, and CH-RE1.12 and CHREl.22~ were isolated from mutagenized populations. Kinetics of Intoxication Figure 3 demonstrates the effect of various concentrations of diphtheria toxin on the toxin-resistant variant strains over a 96 hr period. This type of study shows the effect of the different concentrations of toxin on the growth of cells in culture, since the increased incorporation of amino acids into protein in control, or unintoxicated, cultures parallels the growth and multiplication of the cells. Control cultures of all the strains increased their incorporation over the initial value 11 times or more during the 96 hr period of the experiments. In Figure 3A, a plot of the values for CHO-Kl cells shows that concentrations of toxin of 0.01 MLD per ml and higher depressed incorporation below the initial rate, causing inhibition from which the cells did not recover. The lower concentrations tested inhibited the cells, but still allowed increase above the initial rate and allowed cell growth. In Figure 38, it can be seen that concentrations of lOOO10,000 MLD per ml progressively depressed the incorporation of amino acids into permeability variant CH-RI cells below the initial rate, whereas lOOIO MLD per ml only depressed the rate of growth, allowing an increase over the initial value. Figure 3C shows that concentrations of toxin of 1000 and 10,000 MLD per ml have no effect whatever over
Presumptive 449
Translational
Mutants
time on cultures of the translational RE1.22~. Growth and incorporation treated cultures are exactly as control
- CHO-KI
0
12
24
36
Time
48
60
72
84
96
Hours
in
B. CH-RI
Time
in
I2
24
36
48
Hours
60
72
Time in Hours Figure 3. Kinetics of Inhibition of Protein Synthesis Toxin in TOXS CHO-KI Cells and TOXR Variants The average cpm per culture were: CHO-Ki, 422; CH-RI,
DEAE-Dextran Treatment of Resistant Strains We have previously reported that treatment of permeability variants with the membrane-active substance DEAE-dextran (DEAE-D) increases their sensitivity to diphtheria toxin (Moehring and Moehring, 1976c). Figure 4 shows this effect on CHRl cells. The IDso (24) concentration of the toxin on untreated cells was 260 MLD per ml. When these cells were pretreated with DEAE-D, however, only 5 MLD per ml were necessary to inhibit protein synthesis by 50% in 24 hr. When translational variant strains CH-REI .22c and CH-R48.6e were treated with DEAE-D, no increase in sensitivity to toxin was noted. They reacted exactly as did control cells. Investigating the effects of treating translational variants with DEAE-D accomplished two purposes. First, the results gave further support to the view that they were not permeability variants with a higher level of resistance than previously observed. Second, the results showed that treatment of cells with DEAE-D specifically enhanced the action of diphtheria toxin. DEAE-D was not simply a second toxic agent acting synergistically on toxin-treated cells. Resistance of Cell-Free Extracts Post-mitochrondrial extracts of TOX” and TOX’ Chinese hamster ovary cells were prepared and tested in a cell-free. amino acid-incorporating system. Figure 5 shows that cell-free extracts of the permeability variant. CH-Rl , were equal in sensitivity to extracts of wild-type CHO-Kl cells when titrated against increasing concentrations of diphtheria toxin. Cell-free extracts of the three translational variants, CH-RI .41, CH-REl.22~ and CHR48.6e. were fully resistant, however, and were not significantly inhibited by any concentration of toxin tested. Following these observations. we were able to designate the latter variants as cytoplasmic variants and considered the possibility that their cell extracts contained a diphtheria toxin-resistant EF2.
E ,j , , , , , , I / 0
variant CHof toxincultures.
84
96
by Diphtheria
for the zero time, or initial, 440; CH-REI .22c, 402.
samples
ADP-Ribosylation of EF-2 EF-2 was prepared from each ceil strain and tested for susceptibility to ADP-ribosylation in the presence of excess diphtheria toxin and NAD. as shown in Figure 6. Under our conditions of assay. there was a linear dose response following the addition of increasing amounts of EF-2 prepared from TOX’ wild-type cells and permeability class TOX” variants. The ADPR:EF-2 complex was quantitatively recovered following precipitation with acid and expressed as pmoles of ADPR incorporated. Under
Cell 450
1.o
..I
\
o CH-REl.22~
. CH-REl.22~ A CH-R48.6e A CH-R48,6e q n
0.1
.
*DEAE-D
\
0
+ DEAE-D
CH-RI CH-RI + DEAE-D 1.0
\\
.-
d IO
03
loco
10000
EF- 2 Extract, pg Protein
Cont. of Diphtheria Toxin, MLD/ml Figure theria Strains
4. Effect of Cellular Pretreatment Toxin Activity in Permeability
with DEAE-D on Diphand Translational TOXR
The average cpm for each set of control cultures were: CHREI .22c, 8441; CH-REl.22~ + DEAE-D, 7689: CH-R48.6e, 9688: CH-R48.6e + DEAE-D, 7449: CH-RI, 11,422; CH-RI + DEAE-D, 10,871.
60
Concentration Figure 5. The Effect Labeled Amino Acids Cells
of Diphtheria
of Diphtheria by Cell-Free
Toxin,
MLD/ml
Toxin on Incorporation of ‘%Extracts of TOXS and TOXR CHO
The average cpm for each set of control CHO-Kl, 6334: CH-RI, 6949; CH-R48.6e, CH-REI .22c, 5612.
reaction mixtures 6382: CH-R1.41,
were: 3873;
Figure 8. Diphtheria Toxin-Catalyzed ADP-Ribosylation Extracted from TOXS and TOXR CHO Cells
of EF-2
these conditions, diphtheria toxin did not catalyze the transfer of a measurable amount of ADPR to the EF-2 in extracts prepared from the three cytoplasmic variants, CH-RE1.22c, CH-R48.6e and CHRI .41. The extracts from these cells appeared to be totally resistant to the action of the toxin. The possibility that extracts prepared from these cytoplasmic variants contained a factor which antagonized the toxin-catalyzed ADP-ribosylation reaction was considered and tested. In one type of experiment, we determined the effect of mixing toxin-resistant extracts with extracts from TOX5 cells in the ADP-ribosylation assay. Increasing amounts of EF-2 extract prepared from the cytoplasmic variants CH-RI .41 and CH-REI .22c were incubated with a constant amount of EF-2 extracted from wild-type CHO-Kl cells, in this case 60 pg of protein per reaction mixture, and the transfer of ADPR to EF-2 was measured. Figure 7 shows that the ADP-ribosylation of toxin-sensitive EF-2 from CHO-Kl cells was not altered by the presence in the reaction mixtures of TOX’* extracts at concentrations up to 2.5 times the concentration of the TOXS extract. Post-mitochondrial extracts from cytoplasmic variants were also mixed with extracts from wildtype cells to determine whether that would affect the sensitivity to toxin of the latter in the cell-free, amino acid-incorporating assay. The sensitivity to toxin of the wild-type extract was not altered by extract from cytoplasmic variants. In addition, diphtheria toxin was incubated with post-mitochondrial extracts, prepared as for use in cell-free amino acid assays, and with EF-2 extracts prepared
Presumptive 451
Translational
Mutants
CH-R48.6e 100
&
60
E 8 f!
40
8 C
-$=+=? CH-REL22c
CH-RI,41
TOXs EF-2 Extract @HO-Kl), 6Opg Protein t
g 01’11”‘111”1”” 0
20
40
60
80
100
120
140
160
TOXR EF-2 Extract, pg Protein Figure 7. The Effect on ADP-Ribosylation
of Increasing of a Standard
Amounts Amount
The average ADPR incorporated by control tract was 6.57 pmoles per 60 ~g protein.
of TOXa EF-2 Extract of TOXS EF-2 Extract samples
L
of TOXS ex0 ,sml
’ “,,J1’ ,001
5 1’,‘11’ .Oi
j
““’
0.1
I.0
ul 10
Pseudomonos Exotoxin A , pg/ml from cytoplasmic variants. When the toxin was subsequently tested for activity on intact TOX’ cells, no loss of toxin activity was detected. The results of these experiments strongly suggest that resistance to ADP-ribosylation is an intrinsic property of the enzyme, EF-2. and not due to the presence in extracts or cells of a factor which possesses either antitoxic activity or the ability to modify the susceptibility of EF-2 to ADP-ribosylation by diphtheria toxin. We therefor conclude that they are true translational variants. Cross-Resistance to Pseudomonas Exotoxin A The work of lglewski and Kabat (1975) demonstrating that Pseudomonas aeruginosa exotoxin A, as well t& diphtheria toxin, was capable of ADP-ribosylatlng EF-2 in the presence of NAD+ prompted us to determine whether any of our diphtheria toxinresistant variants were cross-resistant to Pseudomonas toxin. The response of intact CHO cells to Pseudomonas toxin is presented in Figure 8. TOX’ CHO-Kl cells and permeability class TOX” CH-Fil and CH-RE1.12 cells were equally sensitive to the toxin. suggesting that the two toxins either bind to different receptors or enter cells by independent mechanisms. More importantly. however. the translational variants CH-R48.6e. CH-RE1.22~ and CH-RI .41 did show the cross-resistance to Pseudomonas toxin expected if these cells possess an EF-2 which is refractory to the action of diphtheria toxin. Furthermore. as shown in Figure 9. when the ability of Pseudomonas toxin to ADP-ribosylate EF2 was measured directly in the ADP-ribosylation assay, it was found that EF-2 from wild-type CHOKl cells and permeability class TOX” variants was susceptible to ADP-ribosylation, while that from translational variants was totally resistant.
Figure thesis
8. The Effect of Pseudomonas Exotoxin in TOXS CHO-Kl Cells and TOXR Variants
The average cpm for each set of control 8487; CH-RE1.12, 9208; CH-RI, 8657; RE1.22c, 7874; CH-R1.41, 6106.
on Protein
Syn-
cultures were: CHO-Kl, CH-R48.6e, 7590; CH-
Karyotypic Analysis The CHO-Kl cells used in this study had a modal chromosome number of 20, as previously reported (Kao and Puck, 1969). Three of the TOX” strains. CH-REl.12, CH-RI.41 and CH-RE1.22~~ had the same modal chromosome number as the wild-type cells. The permeability variant, CH-RI , had a modal chromosomal number of 19. while that of the translational variant, CH-R48.6e. was 40. All the variants except CH-R48.6e had a growth rate in complete F12 medium which was equal to that of the parent ceil line. The growth rate of CH-R48.6e was approximately one half that of the parent cell. Although marker chromosomes have been identified in some of the variants, we have not as yet been able to associate any specific chromosomal changes with the TOX” phenotypes under study. Discussion The study of the process of diphtheria intoxication at the cellular and molecular level would be facilitated if ceils mutant at different steps in the process of intoxication were available. Such mutants would be useful for elucidating steps in the process of intoxication and for investigating cellular functions which are directly involved in that process. The present communication deals with the selection and biochemical characterization of two phenotypic classes of CHO variants resistant to diphtheria toxin: the permeability variants that we have
Cell 452
10 z E a 5
8-
0.5
1.0
Pseudomonas
5.0
IO
Exotoxin A, pg/Reaction
Mixture
Figure 9. Pseudomonas Exotoxin-Catalyzed ADP-Ribosylation EF-2 Extracted from TOXS and TOXR CHO Cells
of
described previously, and a new class of cytoplasmic variants whose cell-free extracts resist ADPribosylation by diphtheria toxin and Pseudomonas exotoxin. We believe that evidence that these latter variants are translational mutants possessing a mutationally altered EF-2 gene product is compelling. We could find no evidence of a soluble factor that was either antitoxic in nature or able to modify the susceptibility of EF-2 from normal cells to ADPribosylation. The possibility that these variants arose as a result of some epigenetic event seems less probable. The toxin-resistant phenotypes described were both qualitatively and quantitatively diverse; they were stable in the absence of toxin; their low frequencies of appearance in the wild-type population were increased following treatment with the mutagenic agent EMS; and their frequencies of reversion were low. These are properties consistent with a genetic origin. The EF-2 enzyme in the translational variants appears to be altered in its susceptibility to toxin while retaining its normal translocase and GTPase activity. The translational variants CH-R1.41 and CH-REI .22c. for instance, grew at the same rate as wild-type CHO-Kl cells. In addition, their extracts were as active in the cell-free assay for incorporation of amino acids as equal amounts of extracts from wild-type cells. One strong possibility is that
the site on EF-2 where ADP-ribosylation occurs has in some way been modified. Such a modification could result from a point mutation leading to either a conformational change in the molecule or an amino acid substitution for the residue. X, to which the ADPR moiety is covalently bound in the toxincatalyzed inactivation of EF-2 (Robinson, Henrikson and Maxwell, 1974). Similar alterations in gene product have been demonstrated in other systems (Albrecht, Biedler and Hutchison, 1972; Chan, Whitmore and Siminovitch, 1972; Baker et al., 1974; Chasin, 1974). Further studies to characterize the genetic change and the nature of the modification of the enzyme are presently being carried out. When the kinetics of diphtheria intoxication were studied on intact cells, it was found that translational variants were totally resistant to 10.000 MLD of toxin per ml over a period of several days. These results demonstrate that in the CHO system at least. diphtheria toxin does not have a growth inhibitory or cytotoxic action which is alternate or complementary to that of ADP-ribosylation of EF-2. The variants described have proved useful in comparing further the action of Pseudomonas exotoxin with that of diphtheria toxin. Our results demonstrate that the cytotoxic action of Pseudomonas exotoxin in intact cells. like that of diphtheria toxin, is to ADP-ribosylate EF-2. Translational variants were fully cross-resistant to Pseudomonas exotoxin, while permeability variants failed to show any cross-resistance to Pseudomonas exotoxin. It has been observed that cells resistant to one cytotoxic agent may be cross-resistant to other cytotoxic agents having different intracellular targets (Ling and Thompson. 1974; Peterson, O’Neil and Biedler. 1974). indeed. we found that diphtheria toxin-resistant permeability variants of KB cells were cross-resistant to several different viruses (Moehring and Moehring, 1976b). These observations, along with our diphtheria toxin studies, clearly indicated that the resistance of these cells was at the level of the membrane. In the present study. we compared two agents, diphtheria toxin and Pseudomonas exotoxin. having the same molecular basis for cytoxicity and found that they did not share a common mechanism of uptake. These results are similar to those obtained in a comparison of uptake between diphtheria toxin and two other structurally similar (Olsnes, Refsnes and Pihl, 1974) cytotoxic inhibitors of protein synthesis, abrin and ricin (S. Olsnes. J. Moehring and T. Moehring, unpublished data). It is possible that continuing studies will reveal additional TOX” phenotypes as well as heterogeneity within the phenotypes already described. Further development of this experimental system should enable us to seek solutions to biologic
Presumptive 453
Translational
Mutants
problems of wide interest. Translational variants, for instance, should prove useful in studying EF-2, its role in protein synthesis and its regulation. The availability of permeability variants will facilitate further studies on mechanisms of binding and uptake of toxins and other macromolecules. A well characterized system of TOX” biochemical and genetic markers will be of value in studies of mutagenesis in somatic cells. Finally, the results of our studies on the interaction of diphtheria toxin and Pseudomonas exotoxin A with toxin-resistant cells suggest that similar variants could have an important role in elucidating other mechanisms of pathogenesis at the cellular and molecular level. Experimental
Procedures
Ceils, Media and Culture Conditions Chinese hamster ovary cells, line CHO-KI, were obtained from the American Type Culture Collection. Toxin-resistant variants were selected from this line as described below. All cell strains were maintained in Ham’s Nutrient Mixture F12, containing 10% fetal calf serum and 50 pg of gentamicin per ml (which is referred to as “complete” F12) at 37°C in an atmosphere of 5% CO* in air. Toxins Two lots of diphtheria toxin were used in these studies. Columnpurified toxin was a gift from R. John Collier (University of California, Los Angeles). This toxin produced a single band in acrylamide gel electrophoresis in the absence of thiols and had 20,000 guinea pig minimum lethal doses (MLD) per mg of protein. Concentrated and partially purified toxin was obtained from Connaught Medical Research Laboratories (Toronto, Ontario, Canada). This toxin produced a single major band and several trace bands in acrylamide gel electrophoresis, and had 19,000 MLD per mg of protein. Data showing that these two toxin preparations are nearly identical in toxicity when considered on an MLD per ml basis have been published (Moehring and Moehring, 1976a). We report results obtained with the two preparations interchangeably. We are indebted to Stephen H. Leppla (U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland) for providing us with highly purified Pseudomonas aeruginosa exotoxin (Leppla, 1976). This toxin produced a single band in acrylamide gel electrophoresis and had 5000 mouse lethal doses per mg of protein. Selection of Diphtheria Toxin-Resistant Cell Strains Selection of cell strains resistant to diphtheria toxin was carried out following a single exposure of populations of normal and mutagenized CHO-KI cells to a concentration of toxin which would completely, and irreversibly, inhibit protein synthesis in 24 hr-that is, 0.1 MLD per ml or higher (see CHO-KI curve in Figure
2). Cells were mutagenized by exposing them to 300 pg of ethyl methane sulfonate (EMS) (Eastman Kodak) per ml of complete F12 for 18 hr, according to the procedure described by Baker et al. (1974). This treatment caused from 50-60% killing of the cells. Cells were then passaged and maintained in nonselective medium for an expression period of 5 days. To select toxin-resistant strains, normal or mutagenized cells were seeded in 60 mm plastic petri dishes (Falcon Plastics) at 2 x 1 O5 cells per dish. They were allowed to adhere for periods of from 2-18 hr prior to application of toxin in growth medium. After exposure to toxin for 24-48 hr, cultures were washed with Hank’s balanced salts solution and incubated in complete F12 in the usual manner. Cultures were fed with fresh medium on day 4.
After further incubation for 3-4 days, plates were examined for living clones of cells. If these clones were only to be counted, 0.1% 2(p-idophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) (Aldrich Chemical) in complete F12 was added to each plate, and cultures were incubated again for 12-18 hours. Living cells were then stained a deep permanent red. Clones were counted after the plates were drained, washed with distilled water and air-dried. No fixing was required. If living clones were to be selected for further study, they were isolated with porcelain cloning cylinders and transferred to tissue culture multidishes with 16 mm wells (Linbro Chemical) and grown to confluency in toxin-free complete F12. When sufficient cells were generated, the resistance to toxin of the isolated strains was determined using the whole cell assay for inhibition of protein synthesis by toxins, described below. Karyology Exponentially growing monolayer cultures were incubated with 0.05 pg of Velban (Lilly) per ml for 75 min at 37°C. Cells were removed from the substrate using 1 x trypsin-EDTA (Gibco), swelled in 0.06 M KCI for 18 min at room temperature, fixed in three changes of methanol:acetic acid (3:l) and spread on glass slides by blowing. Banded chromosomes were prepared, according to the procedure of Sun, Chu, and Chang (1973), from airdried slides which had aged for 3 days. At least 100 representative chromosome spreads of each variant were counted. Whole Cell Assay for Inhibition of Protein Synthesis by Toxins The assay procedure (Moehring and Moehring, 1976a) was as follows. Cells were seeded at 5 x IO4 cells per culture in 8 ml flint glass vials (I.C.N. Pharmaceuticals, Cleveland, Ohio) and incubated for 48 hr. When cells were pretreated with DEAEdextran (DEAE-D), 500 pg of DEAE-D per ml were applied to cultures immediately prior to application of toxin dilutions, as reported previously (Moehring and Moehring, 197613). Cells were then exposed to appropriate concentrations of toxins in culture medium containing 5% fetal calf serum for 24 hr. At the end of this period, the medium was aspirated, and 0.5 ml of 0.05 M tris(hydroxymethyI)aminomethane-buffered Eagle’s minimum essential medium containing a I:20 dilution of the normal amino acids and 0.4 &I of j4C-labeled amino acid mixture (15 amino acids; 100 &i/ml; New England Nuclear, Boston, Massachusetts) were added. Assay vials were then incubated for 30 min. Labeling medium was then removed, and cells were precipitated on the vial with 4 ml of chilled 5% trichloroacetic acid. The precipitated cell layer was dried following removal of the trichloroacetic acid. 5 ml of Permafluor liquid scintillator in toluene (Packard Instrument) were added to each vial, and the samples were counted directly in a Tri-Carb scintillation spectrometer (Packard lnstrumerit). Preparation of Cell Extracts and Incorporation of Amino Acids by Cell-Free Systems The methods used to prepare cell extracts and to study their incorporation of radioactive amino acids into protein have been described in full elsewhere (Moehring and Moehring, 1968; 1976a). The post-mitochondrial cell extracts used in these studies were a product of 50 million cells per ml of lysing buffer, and a 20 ~1 amount of cell extract was used per standard reaction mixture of 50 ~1. A ‘YXabeled amino acid mixture (NEC-445, New England Nuclear) was used to label peptides, which were collected on Whatman GF/C glass fiber filters and counted by scintillation spectrometry, as were the whole cell samples. Preparation of EF-2 EF-2 was prepared from CHO dure used by Gill and Dinius nates of mammalian tissues. centrifuged at 500 x g for IO cells, was then suspended in
cells by a modification of the proce(1973) to extract EF-2 from homogeCells were harvested, washed and min. The cell pellet, 0.25 ml per lOa 0.25 M sucrose in the ratio of 1 ml of
Cell 454
Table
1. Formation
of ADP-Ribosyl cpm
EF-2 and Poly(ADP-Ribose)
in Acid-Insoluble
in the Presence
and Absence
of 0.18 M Histamine
Material
1.6 PM NAD
3.2 pM NAD
4.6 /.LM NAD
Toxin
Toxin
Toxin
0.18 M Histamine
+
-
A
-t
-
A
+
-
A
-
a829
2745
60a4a
11,596
5353
6242
14,714
a334
6380
+
7068
226
7,497
a37
6660
7,152
364
6788
6842
a The difference in counts (A) following incubation of 115 pg of CHO-Kl EF-2 extract with 1.6 PM NAD in the absence of histamine 6488-1187 = 6304 cpm. Reaction mixtures containing 115 pg of CHO-Kl extract and 250 pg of CH-R48.6e extract were incubated under standard conditions presence of 100 pg of diphtheria toxin and NAD sn the amounts indicated. The specific activity of 14C-NAD was 543 cpm/pmole.
sucrose to lOa cells and homogenized in a tight-fitting Dounce homogenizer. To each 1.25 ml of homogenate were added 0.21 ml of 4M NH&I and 0.33 g of wet, washed, activated charcoal (Norit neutral, Fisher Scientific). The mixture was homogenized intermittently in a Dounce homogenizer with a loose-fitting pestle over a period of 15 min and then centrifuged at 100,000 x g for 1 hr. The clear supernatant was collected and assayed for ADP-ribosylatable EF-2. The extracts were stored at -70°C. Specific activity (pmoles of ADP-ribosylatable EF-2 per ml protein) was not altered by up to five cycles of freeze-thawing. The specific activities of extracts prepared from CHO-KI, CHRl and CH-RI .I2 were 107, 97 and 92 pmoleslmg, respectively, and comparable to a standard preparation of rat liver EF-2 prepared in our laboratory (Moehring and Moehring, 1976c), having a specific activity of 106 pmoles/mg protein. An Average yield of EF2 from CHO-Kl cells was 1825 pmoles per 2 x lOa cells or 5.5 x lo6 molecules per cell. The amount of EF-2 in extracts of the three translational variants, CH-R46.6e, CH-REI .22c, and CH-RI .41, could not be determined since they contained no ADP-ribosylatable EF-2 (Figure 6). ADP-Ribosylation Assay ADP-ribosylation was carried out in 0.25 ml reaction mixtures containing 0.25 M Tris (hydroxymethyl) aminomethane-hydrochloride buffer at pH 6.2, 0.04 M’dithiothreitol, 0.1 M ethylenediamine-tetraacetate and 1.6 PM U-14C-NAD (280 mCi/mmol) Amersham-Searle). The standard amount of diphtheria toxin used was 100 pg per reaction. The amounts of Pseudomonas toxin and EF-2 used are indicated in each experiment. The reaction was initiated by the addition of either toxin or EF-2. Reaction mixtures were incubated at 25°C for 15 min, and the reaction was stopped by the addition of 0.25 ml of 10% trichloroacetic acid. Under these conditions, the reaction went to completion when toxin and NAD were in excess. A complete set of control tubes, without toxin, was included in each experiment, and the amount of ADP-ribosyl EF-2 formed was determined from the difference between counts recovered from incubation mixtures with and without toxin. The concentration of NAD used represented an excess of 25 fold or more over the maximum amount of EF-2 used in each experiment. It was also determined by isotope dilution that this excess was sufficient to overcome completely the dilution effect of the small amount of endogenous NAD which remained in some of the preparations of EF-2 following treatment with charcoal. In the case of EF-2 preparations from translational variants, these latter determinations were made by mixing these extracts with extracts of EF-2 prepared from CHO-KI, as described in Figure 7. Extracts of EF-2 from CHO cells, like those extracted from animal tissues, were found to contain poly(ADP-ribose) transferase activity (Gill and Dinius, 1973). In the presence of sufficient 14C-NAD, this activity leads to the formation of acid-insoluble radioactive poly(ADP-ribose) which interferes with the assay for
was in the
ADP-ribosyl EF-2. At the concentration of NAD used in our experiments, 1.6 PM, a significant amount of poly(ADP-ribose) was formed unless histamine, an inhibitor of transferase activity, was included in the reaction mixture (Table 1). Including histamine in the reaction mixture resulted in a slight but consistent increase in net corn. Acknowledgments This investigation was supported by a USPHS grant from NIH. We wish to acknowledge the capable assistance of D. Danley, C. Rolerson and P. Guigley in carrying out the karyotypic analysis of CHO CELLS. A preliminary report of this study was presented at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7-10, 1976. Received
February
8, 1977;
revised
March
25, 1977
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I, L. (1972).
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Chasin, L. A. (1974). Cell 2, 37. Collier, R. J. (1975). Bacterial. Rev. 39, 54. Gill, D. M. and Dinius, L. L. (1973). J. Biol. Chem. 248, 654. Iglewski, B. H. and Kabat, D. (1975). Proc. Nat. Acad. Sci. USA 72, 2284. Kao, F.-T. and Puck, T. T. (1969). J. Cell. Physiol. 74, 245. Leppla, S. H. (1976). Infect Immunol. 14, 1077. Ling, V. and Thompson, L. H. (1974). J. Cell Physiol. 83, 103. Moehring, T. J. and Moehring, J. M. (1968). J. Bacterial. 96, 61. Moehring, T. J. and Moehring, J. M. (1972). Infect. Immunol. 6, 487. Moehring, J. M. and Moehring, T. J. (1976a). Infect. Immunol. 13, 221. Moehring, J. M. and Moehring, T. J. (1976b). Virology69, 786. Moehring, T. J. and Moehring, J. M. (1976c). Infect. Immunol. 73, 1426. Olsnes, Sjur, Refsnes, K. and Pihl. A. (1974). Nature 249, 627. Pappenheimer, A. M., Jr. and Gill, D. M. (1973). Science 782, 353. Peterson, R. H. F., O’Neil, J. A. and Biedler, J. L. (1974). J. Cell Biol. 63, 773. Robinson, E. A., Henrikson, 0. and Maxwell, E. S. (1974). J. Biol. Chem. 249, 5088. Sun, N. C., Chu, E. H. Y. and Chang, C. C. (1973). Mammalian Chromosome Newsletter 74, 26.
