Erythromycin resistance in mouse L cells

Somatic Cell Genetics, Vol. 5, No. 5, 1979, pp. 585-595

Erythromycin Resistance in Mouse L Cells

Peter L. Moiloy I and Jerome M. Eisenstadt

Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut

Received 21 February 1979-Final 30 April 1979

Abstract--The sensitivity of mouse cell lines in culture to the macrolide antibiotic, erythromycin stearate, was investigated. Both resistant and sensi- tive lines were found. Experiments indicated that in sensitive cells erythro- mycin stearate inhibits mitochondrial protein synthesis. Mutants resistant to erythromycin stearate were selected from the line LM(TK-), and these are also less sensitive to other macrolide antibiotics such as carbomycin and spiramycin. Attempts to transfer the erythromycin resistance of either the mutants or naturally resistant lines by fusion of cytoplasts with sensitive cells were unsuccessful, and it is concluded that resistance to erythromycin stearate is controlled by nuclear genetic factors.

INTRODUCTION

The assembly of functional mitochondrial respiratory oxidative phospho- rylation and protein synthetic systems requires the synthesis of gene products coded within both the nuclear and mitochondrial genomes. In lower euka- ryotes the use of mutants in all of these systems has contributed greatly to understanding their synthesis and assembly and has enabled the mapping of the genes for several products and investigation of the characteristics of the mitochondrial genetic system (1-3).

Mammalian cell mutants resistant to chloramphenicol and carbomycin, inhibitors of mitochondrial protein synthesis, have been isolated (4-6). Resistance to chloramphenicol is cytoplasmically transferred (for review of system see ref. 7), while resistance to carbomycin is not transferred by enucleated cytoplasts. Recently the isolation of mutants resistant to the inhibitor of oxidative phosphorylation, rutamycin, has been reported (8). Other mutants with defects in enzymes of the respiratory chain have been

1Present address: Department of Biochemistry, University of Adelaide, South Australia.

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586 Molloy and Eisenstadt

isolated from Chinese hamster cells and these are probably coded in the nucleus (9). It would be of particular interest to obtain further mutants of these systems, especially if they are encoded in the mitochondrial genome.

Mutants resistant to the macrolide antibiotic, erythromycin, have been isolated from a number of lower eukaryotes, but the degree of sensitivity of mammalian mitochondrial protein synthesis to erythromycin has been the subject of some disagreement. It is generally agreed that intact mitochondria from rat liver are insensitive to erythromycin. In some cases this has been ascribed to a permeability barrier (10), while others have found that in disrupted mitochondria no significant inhibition was observed, although binding of erythromycin to the ribosomes could be demonstrated (11). Isolated mammalian mitochondrial ribosomes are sensitive to macrolide antibiotics including erythromycin, although the concentrations required for inhibition are considerably higher than those which affect protein synthesis in bacterial or yeast mitochondrial systems (12, 13).

In this report the sensitivity of mitochondrial protein synthesis in vivo and the inhibition of growth of mouse LM(TK-) ceils by erythromycin stearate is described. Mutants resistant to erythromycin stearate have been isolated but neither this resistance nor the naturally occurring resistant phenotype of other mouse lines is transferred by fusion of cytoplasts with erythromycin-sensitive nucleated cells.

MATERIALS AND METHODS

Cell Lines. All lines used were derived from mouse L cells: LM(TK-) lacks thymidine kinase (TK: EC 2.7.1.75) and hence is resistant to bromo- deoxyuridine (BrdU); A9 lacks hypoxanthine phosphoribosyltransferase (HPRT: EC 2.4.2.8) and is resistant to 8-azaguanine (AG) and 6-thioguan- ine (TG); 501-I is a chloramphenicol (CAP)-resistant line derived from A9.

Media. Cells were grown either in suspension in MEM-S or attached to plastic in MEM-E medium (Flow Laboratories) supplemented with 5% (v/v) fetal calf serum (Flow Laboratories), 100 units/ml of penicillin and 100 #g/ml of streptomycin.

Antibiotics. Stock solutions of erythromycin stearate (Sehwarz-Mann) and spiramycin (SPI) were prepared in ethanol and added to the medium in varying concentrations as described in Results. The maximum concentration of ethanol in the medium was 1% (v/v), and no effect on cell growth was observed at this level. Aqueous stock solutions of other antibiotics were prepared and added to the medium to the following final concentrations: BrdU, 30 ~g/ml; AG, 3 ug/ml; TG, 8.4 #g/ml; CAP, 50 ~g/ml. Selective medium containing hypoxanthine-aminopterin-thymidine (HAT) was used as described previously (14).

Erythromycin Resistance 587

Enucleation. Enucleation was carried out as described by Wiglet and Weinstein (15), except that the cells were layered on prepared gradients in 10% Ficoll (Pharmacia) solutions. Cytoplast preparations were examined for cell and karyoplast contamination following staining with lactopropionic orcein (16).

Cell Fusion. Cell-cell and cell-cytoplast fusions were performed as described previously (4) using 500 hemagglutinating units of inactivated Sendal virus. Immediately after fusion cells were inoculated at various dilutions into selective media as described in the text.

Mitochondrial Protein Synthesis in Vivo. Incorporation of [35S]me- thionine into whole cells was carried out essentially as described by Jeffreys and Craig (17). Flasks (25 c m 2) w e r e inoculated with 10 6 cells which were allowed to attach to the surface overnight. The attached cells were rinsed twice with saline and then 5 ml of MEM-E, lacking methionine, supple- mented with 5% dialyzed fetal calf serum were added to each flask. After a period of 30 min in the presence of the indicated antibiotics of 4 h in the case of erythromycin stearate, [35S]methionine (New England Nuclear, Boston, Massachusetts) was added and the incubation continued for 2 h. Cells were then washed three times with cold saline containing methionine, 50 #g/ml, detached from the surface and lysed by the addition of 2 ml of 1 M NaOH for 10 min at 37 ~ This was neutralized by 5 M HCI and protein precipitated with 5 ml of 10% trichloracetic acid containing 50 ug/ml of methionine. Precipi- tates were collected on glass fiber filters (Whatman GF/A) , washed three times with 5% trichloracetic acid, once with 70% ethanol, dried, and radioac- tivity was determined.

RESULTS

Sensitivity o f Cells to Inhibition by Erythromycin Stearate. In a survey of the effects of a number of potential inhibitors of mitochondrial function on mouse L cells, erythromycin stearate (ERY) was found to cause almost complete inhibition of the proliferation of L M ( T K - ) cells at a concentration of 20 ug/ml (Fig. 1). By contrast erythromycin base was only partially effective at a concentration of 100 #g/ml. This probably reflects a difference in the ability of the two derivatives to permeate the cell.

Two approaches were taken to investigate whether this growth inhibition resulted from a specific inhibition of mitochondrial function. First, we had noted that inhibition by a number of inhibitors of mitochondrial functions is enhanced when cells are grown in medium in which the normal 0.1% glucose is replaced by pyruvate or ethanol as the primary energy source and when the only glucose present in the medium is that from the fetal calf serum (about one tenth of the normal level). In Table 1 the effects of a number of

588 Molloy and Eisenstadt

500 -

b

W m '5

Z

_J _J hl (J

I 0 0

5 0

IO

0 I I I I I I 2 4 6 8

D A Y S

Fig. 1. 25-cm / flasks were inoculated with 4 x 10 4 cells. Duplicate flasks were counted after 1, 2, 4, 6, and 8 days. o, LM(TK-) control; U], LM(TK ) ERY, 30 izg/ml; e, ERLI.1 control; I , ERLI.1 ERY, 30 ~zg/ml; A, ERLI.I, 50 ug/ml.

antibiotics on cell growth in normal and "low glucose" media are compared. The effective concent ra t ions of mi tochondr ia l inhibitors C A P and carbomycin (CAR) on protein synthesis, antimycin A on respiration, and rutamycin on oxidative phosphorylation are reduced five- to tenfold in the "low glucose" medium. A similar differential is seen for ERY. By contrast, AG, which does not specifically affect mitochondria, is equally effective in both media. This indicates that the inhibition of growth is the result of an inhibition of mitochondrial function.

The effect in vivo of E R Y on mitochondrial protein synthesis was studied

Erythromycin Resistance 589

Table 1. Inhibitory Effect of Erythromycin Stearate

Cell number (• 10 -4)

Antibiotic Concentration MEME,

(ug/ml) M E M E low glucose

None

8-Azaguanine

Chloramphenicol

Carbomycin

Antimycin A Rutamycin

Erythromycin stearate

104 53

0.3 5 7 3 1 1

5 46 9 10 31 2 50 11

2 15 1 5 3 1

0.1 6 1 0.5 4 0.6

5 34 2 10 8 1 50 3 1

~ ) cells were inoculated at l04 cells/25-cm z flask; cells were harvested and counted after seven days. Medium was M E M E or M E M E in which the 0.1% glucose was substituted for by 0.1% ethanol, each supplemented with 5% fetal calf serum.

by incubating LM(TK ) cells in the presence of [35S]methionine and 10 #g/ml of emetine, an inhibitor of cytoplasmic protein synthesis. Under these conditions the remaining amino acid incorporation into protein is largely inhibited by CAP (50-70% in different cell lines). Residual incorporation occurs mainly in a heterogeneous range of small peptides (17). Emetine- insensitive amino acid incorporation by LM(TK ) is inhibited by 50-60% in the presence of the specific inhibitors of mitochondrial protein synthesis, CAP, SPI, and CAR (Table 2, first column). A similar degree of inhibition by ERY was observed, although it was necessary to preincubate the cells in its presence for 4 h (with emetine present for only the final half hour) to see this effect. Together with the known mode of action of erythromycin on mitochondrial protein synthesis in other systems, it seems reasonable to conclude from these experiments that ERY is inhibiting mitochondrial protein synthesis.

Isolation of Mutants Resistant to Erythromycin Stearate. LM(TK-) cells were treated for 24 h with 300 ug/ml of ethylmethane sulfonate, conditions yielding a cell viability of about 2%. After growth in nonselective media for six days, 6 • 108 cells in a l-liter spinner culture were exposed to 50 #g/ml of ERY. After five days cells were inoculated into flasks and allowed to attach. Twenty-eight clones were picked after 17 days and subclones of primary isolates were analyzed. It was observed that another mouse L cell

590 MoUoy and Eisenstadt

Table 2. Mitochondrial Protein Synthesis in Vivo a

Percentage of control incorporation

Antibiotic LM(TK-) A9 ERLI.1 ERL4.2

Emetine, 10 #g/ml 100 100 100 100 Emetine, plus

Chloramphenicol, 50 ~tg/ml 46 58 55 56 Spiramycin, 50/~g/ml 49 67 63

100 ug/ml 39 58 61 84 Carbomycin, 5 #g/ml 49 70 52

10/~g/ml 44 49 Chloramphenicol, 50 #g/ml b 47 58 56 47 Erythromycin stearate, 50 gg/ml b 82 88 100 100

1 O0 #g/ml b 63 95 1 O0 1 O0 200 #g/ml b 43 83 100 87

"Control incorporation (in the presence of emetine) was 3450, 3816, 355, and 1519 cpm respectively for LM(TK-), A9, ERLI.1, and ERL4.2.

bCells were preincubated in the presence of the antibiotic for 4 h prior to the addition of [3SS]methionine. Incorporation in such cells in the absence of emetine was not significantly different from that in cells not preincubated in the presence of CAP or ERY.

line, A9, is cons iderably more res is tant to E R Y than L M ( T K - ) as a re o ther unre la ted mouse lines, e.g., R A G . The charac ter i s t ics of the na tura l ly res is tant line, A9, are, therefore, considered in para l le l with the mutants .

In Fig. 1 the growth of the mutant , ERL1.1 , in the presence and absence of E R Y is compared with that of L M ( T K - ) . Growth of L M ( T K - ) is to ta l ly inhibi ted by 3 0 / z g / m l of ERY. In nonselective media E R L I . 1 grows with a s l ight ly increased doubling t ime compared to L M ( T K - ) , but continues to grow in 50 g g / m l of E R Y at only a s l ightly decreased ra te compared to

growth in the absence of the drug. The growth charac ter i s t ics of o ther mutan t s a re s imilar to E R L I . 1 , and da ta for some of these are summar ized in Table 3. A9 shows a degree of resis tance s imilar to the mutan t s and grows as well as L M ( T K - ) in nonselective media. Both the mutan t s and A9 clone efficiently when inocula ted at low cell densi ty in concentra t ions of E R Y up to 50 # g / m l . The resis tance of the mutan t s is not affected by growth in the absence of selection for a t least 50 doublings.

The mutan t s were surveyed for resis tance to a number of other inhibi tors of mi tochondr ia l function. Al l mutan t s as well as A9 exhibi t a significant increase in resis tance to C A R and SPI under both cloning and mass cul ture

condit ions when compared to the sensitive line, L M ( T K - ) (Table 3). Cross- res is tance to these other macro l ide ant ibiot ics is not unexpected because in bac te r i a l sys tems they share the same r ibosome binding site (18). Mi tochondr ia l mutan t s of yeast having a number of different pa t te rns of cross-resis tance to these and other ant ibiot ics have been descr ibed (19, 20). Cross-res is tance to ol igomycin, an t imycin A, mikamycin , or C A P was not observed.

Erythromycin Resistance 591

Table 3. Antibiotic Sensitivity of Wild-Type and Mutant Cells Lines"

Antibiotic

Percentage of control

Concen- Growth Cloning efficiency tration

(t~g/ml) LM(TK ) ERLI.I ERL4.2 A9 LM(TK-) ERLI.I ERL4.2 A9

Erythromycin 20 10 0.3 100 100 92 stearate 30 1 92 82 72 <0.1 90 90 92

50 l 48 70 58 87 85 88 Carbomycin 2.5 15 45 82 28 <0.1 74 77 18

5 2 16 20 16 <0.1 0.2 <0.1 l0 1 3 6 3

Spiramycin 25 14 67 60 44 6.6 100 100 100 50 3 42 37 36 0.1 56 75 100

aSensitivity of growth in mass culture was determined by inoculation of 5 • 104 cells into duplicate 25-cm 2 flasks with antibiotic additions as shown. Cells were harvested and counted after six days. Cloning efficiencies were determined by inoculation of 10z-104 cells into duplicate flasks; colonies were stained with methylene blue and counted after 9-20 days.

The growth characteristics in vivo of the resistant lines are reflected in the effects of ERY, SPI, and C A R on the incorporation in vivo of amino acids by the mitochondrial protein synthesizing system (Table 2). E R L I . 1 , ERL4.2, and A9 all show almost complete insensitivity to ERY, much enhanced resistance to SPI, and, in the case of A9, increased resistance to C A R . To determine whether the resistance results from an altered mitochondrial component or some other mechanism, e.g., detoxification or decreased cell permeability, it will be necessary to examine amino acid incorporation by isolated mitochondria or mitochondrial ribosomes.

Inheritance of A9 Phenotype. In mammal ian cells it is possible to demonstrate the cytoplasmic location of a genetic determinant by fusing cytoplasts of the cell carrying the determinant (formed by enucleation using cytochalasin B) with a nucleated cell line lacking the determinant (4). A9 cells were, therefore, enucleated and the cytoplasts fused with the E R Y - sensitive line, L M ( T K ). Selection was made for resistance to both BrdU, the nuclear marker of L M ( T K - ) , and 50 # g / m l of ERY, the putative cytoplasmic marker of A9. Only two clones grew under these conditions. Both showed the morphology and TG resistance of A9, indicating that they were variants of the nucleated parent cell. This lack of transfer suggests that E R Y resistance is not cytoplasmically determined.

To test further the validity of this conclusion, a similar experiment was performed using as the cytoplast donor the line 501-1, a mutant of A9 carrying the cytoplasmically inherited determinant for C A P resistance. The results of the fusion of 501-1 cytoplasts with L M ( T K - ) cells are shown in Table 4. While the frequency of transfer of the determinant for C A P resistance was about 1 per 103 L M ( T K - ) cells, no transfer of E R Y resistance

592 Molloy and Eisenstadt

Table 4. Enucleation of A9 and 501.1 and Fusion with LM(TK- )

Enucleated Sensitive Colonies per 106 line ~ recipient Selection conditions recipient cells

A9 LM(TK-) BrdU, ERY, 50 itg/ml 2 b

501.1 LM(TK ) BrdU, ERY, 30 lzg/ml c BrdU, ERY, 50 izg/ml 0 BrdU, CAP, 50 itg/ml 1010

501.1 BrdU, ERY, 30 #g/ml 0 BrdU, CAP, 50 lzg/ml 0

LM(TK-) BrdU, ERY, 30 izg/ml c BrdU, ERY, 50 ~g/ml 0 BrdU, CAP, 50/zg/ml 8

aThe cytoplast preparation from A9 contained 86% cytoplasts; 2 • 10 6 cytoplasts were fused with 2 • 106 cells; the fusion mixture was inoculated at 105 or 2 • 105 total particles per flask. The cytoplast preparation from 501.1 contained 98.5% cytoplasts; 1.5 • 107 cytoplasts were fused with 3 • 106 cells; the mixture was inoculated at 2 or 5 • 104 recipient cells per flask.

bThese cells had the morphology of A9 and were resistant to AG and TG. Clndividual flasks contained either no colonies or had patches of cells rather than clones arising

from single cells. Of the fusion flasks, patches of cells grew in 7 of 16 flasks incoulated with 5 • 104 recipient cells while in the control LM(TK-) flasks patches of cells grew in 8 of 10 flasks inoculated with 105 cells.

was observed when selection was made in medium containing 50 ug/ml of ERY. In the minimally selective concentration of 30 ~g/ml of ERY, less growth was seen in the experimental than in the control L M ( T K - ) flasks. Analysis of a number of independent CAP-resistant clones from this fusion in ERY-containing medium showed no difference in resistance from LM(TK-) .

The expression of ERY resistance was also examined in hybrid cells formed by fusion of nucleated A9 cells with LM(TK ). All hybrids tested were resistant to 30 /~g/ml. This partial resistance suggests that ERY resistance is a codominant nuclear-determined trait.

Inheritance of Phenotype of ERL Mutants. As LM(TK-) , the parental line of the ERL mutants, was the only stock line sensitive to ERY, a direct enucleation of mutants and fusion of cytoplasts with a sensitive line to test for transfer of resistance was not practicable. Two different approaches were taken, therefore, to examine the inheritance of the ERY resistance of the ERL mutants.

A hybrid line, ALE, was made by fusion of ERLI.1 with A9 followed by selection in HAT medium. As expected, this line is highly resistant to ERY. The hybrid, which should have maintained a cytoplasmic complement from both parents (21) was then enucleated and the cytoplasts were fused with LM(TK-) . Selection was made in medium containing BrdU and ERY (50 #g/ml), but no transfer of resistance was seen up to a total of 4.5 x 106

recipient cells (Table 5).

E r y t h r o m y e i n R e s i s t a n c e 5 9 3

T a b l e 5. Attempted Transmission of ERL Phenotype via Cytoplasts ~

Recipient Colonies per 106 Enucleated parent line Selection conditions recipient cells

ALE LM(TK ) BrdU, ERY, 50 #g/ml 0 ALE - - BrdU, ERY, 50 #g/ml 0 - - LM(TK-) BrdU, ERY, 50 #g/ml 0

ERL1.1 TAL AG,TG,ERY, 40 #g/ml 5 AG,TG,ERY, 50 #g/ml 0

ERL4.2 TAL AG,TG,ERY, 50 #g/ml 0 AG,TG,ERY, 50 #g/ml 0

ERL12.3 TAL AG,TG,ERY, 40 #g/ml 5 AG,TG,ERY, 50 #g/ml 0

ERL29.3 TAL AG,TG,ERY, 40 #g/ml 2 AG,TG,ERY, 50 #g/ml 0

ERLI.1, ERL4.2, - - AG,TG,ERY, 40 tag/ml 0 ERLI2.3 or ERL29.3 - - TAL AG,TG,ERY, 40 #g/ml b

AG,TG,ERY, 50 #g/ml 1

~Cytoplast preparations of ALE, ERLI.I, ERL4.2, ERL12.3, and ERL29.3 contained respec- tively 98%, 96%, 97%, 99%, and 99% cytoplasts. Equal numbers of cells and cytoplasts were fused in all cases; 3 x 106 ALE cells and 2 x 106 of each parent were used in the ERL fusions. Fusion mixtures were inoculated at 2.5 or 5 x 104 recipient cells per flask.

bSmall patches of cells grew in a number of these control flasks.

As a more direct approach, an ERY-sens i t ive recipient line was made by fusing A9 with L M ( T K - ) and subsequent ly selecting for a HPRT-def ic i en t segregant from the hybr id cells in med ium conta ining A G and TG. This line, T A L , is pa r t i a l ly res is tant to ERY, growing on 30 # g / m l but not 40 t~g/ml. Four different E R L lines were enuclea ted and their cytoplasts fused with T A L , selection being made in media containing A G , T G and E R Y (ei ther 40 or 50 t~g/ml). A small number of colonies grew on 40 # g / m l of E R Y and one on 50 ~ g / m l (Table 5). When the growth of these colonies was tested on ERY-con ta in ing media , all proved indis t inguishable from T A L . W e conclude tha t they were sensitive cells which had escaped selection. These two approaches indicate tha t the E R Y resis tance of the E R L mutan t s is encoded in the nuclear genome, as it also appears to be in A9.

D I S C U S S I O N

The exper iments repor ted in this paper s t rongly suggest tha t differences in sensi t ivi ty to ERY, both selected and na tura l ly occurr ing, a re control led by nuclear genet ic factors. A t t e m p t s to t ransfer E R Y resis tance by c y t o p l a s t - cell fusion have consis tent ly failed, a l though in one exper iment C A P resis- tance was eff icient ly t ransfer red . Ce l l - ce l l hybr ids between A9 and L M ( T K ) show an in te rmedia te res is tance to ERY. I f this were the resul t of a mixing of cy toplasmic resis tance and de te rminan t s of sensit ivity, a s imi lar

594 Molloy and Eisenstadt

increase would be expected in cytoplast-cell fusion products. Resistance to ERY appears similar to the previously reported resistance to CAR (6), and indeed the ERY-resistant lines show considerable resistance to CAR and SPI. The CAR-resistant mutant is also partially resistant to ERY and SPI (22). For the CAR-resistant mutant, resistance of amino acid incorporation by isolated mitochondria to inhibition by CAR has been demonstrated. Resis- tance to ERY could be determined at the level of cell permeability, detoxifi- cation, mitochondrial permeability, or the mitochondrial ribosomes. Further experiments are needed to examine this question.

The marked difference in sensitivity of the closely related mouse L-cell lines A9 and LM(TK- ) is interesting in view of the differing reports in the literature on the sensitivity of rat liver mitochondrial protein synthesis to inhibition by erythromycin (10, 11). The possibility is raised that, within a single species, individuals may be either sensitive or resistant to erythromycin. It will be necessary to conduct careful studies in vitro of mitochondrial protein synthesis in different strains under the same laboratory conditions to evaluate this possibility.

ACKNOWLEDGMENTS

The excellent technical assistance of Donna Poirier was greatly appre- ciated. (Grant DRG-68F of the Damon Runyon-Walter Winchell Cancer Fund to P.L.M. and GM21873 and GM22142 to J.M.E.)

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