Effect of chloramphenicol on the growth and viability of exponentially growing mouse leukemic cells (L5178Y)

Copyright All rights

0 1973 by Academic Press, Inc. in any form restwed

of reproduction

Experimental Cell Research 77 (1973) 346-350

EFFECT

OF CHLORAMPHENICOL

OF EXPONENTIALLY

ON THE

GROWING

MOUSE

D. F. LIBERMANi

GROWTH LEUKEMIC

AND

VIABILITY

CELLS

(L5178Y)

and J. L. ROT1 ROTF

Department of Radiation Biology and Biophysics, University of Rochester, Rochester, N. Y. 14623, USA

SUMMARY Two effects of chloramphenicol on mouse leukemic cells (L5178Y) are described. The drug induces a prolongation of the cell cycle (reversible effect). The degree of prolongation is directly proportional to the concentration of the drug. The effect is observed only in the presence of chloramphenicol and cells return to normal cell-growth kinetics when the drug is removed from the culture medium. Chloramphenicol also kills a portion of the cell population immediately (irreversible effect). Data are presented which suggest that chloramphenicol is toxic to cells in the G2 phase of the cell cycle.

Recently, Lenk & Penman [I] reported that when HeLa cells are grown in suspension culture in the presence of ethidium bromide or chloramphenicol, i.e., specific inhibitors of mitochondrial RNA and/or protein synthesis [2], the population doubling time increased from 24 to more than 48 h [l]. The drug-treated cells grew for two generations after which growth ceased and the cells began to disintegrate. Since our laboratory has been particularly interested in the role of cytoplasmic and mitochondrial synthetic events in the regulation of the transition steps necessary for progression through the cell cycle, we were intrigued by the reported effects of chloramphenicol on the cell growth cycle. In this paper we describe some of our experiments exploring the effects of chloramphenico1 on the cell growth cycle of L5178Y cells. 1 Present address: Department of Microbiology, University of Rochester, Rochester, N.Y. 14620, USA. 2 Present address: Department of Biochemistry, University of Florida, Gainesville, Fla 32601, USA. ExptI Cell Res 77 (1973)

MATERIALS

AND METHODS

Cell line Mouse leukemic cells (L5178Y) were used throughout this study. They were grown in suspension culture in Fischer medium (GIBCo, Grand Island, N.Y.) and supplemented with 10 % horse serum and antibiotics penicillin “G” potassium (225 pg/ml) and dihydrostreptomycin sulfate (Nutritional Biochemical Co., Cleveland, Ohio) (45 yg/ml). Normally, the culture medium was adjusted to pH 7.5 with 1 N NaOH. The population doubling time under standard conditions was approx. 9.5 h.

Preparation of exponential cultures To insure that all experiments were started with cultures in log phase growth, cells were subcultured to a maximum concentration of 1 x lo5 cells/ml 12 h prior to their use.

Chloramphenicol treatment Cells were suspended in fresh Fischer medium and in media containing the various concentrations of chloramphenicol (CalBiochem Corp., Los Angeles, Calif.). Cells were dispersed in 5 ml aliauots and cell number was determined at various time intervals thereafter. The cell concentration was determined with a Coulter counter (Coulter Electronics, Hialeah, Fla). All manipulations were performed aseptically.

Effect of chloramphenicol

_’

,l

I. Abscissa: (a) time after addition of chloramphenicol (hours); (b) concentration of chloramphenicol @g/ml); ordinate: (a) cell number; (b) doubling time (hours). (a) Growth of L5178Y cells in various concentrations of chloramphenicol. Cells were grown in the presence of 0, 0.0 pg/ml, control; 0, 25 pg/ml; 0, 50 pg/ml; A, 100 pg/ml; l , 200 pg/ml. The data are means from 2 expts; (b) Population doubling time in various concentrations of chloramphenicol. These points were derived from the slopes of the growth curves in (a). Fig.

Eosin staining The effect of chloramphenicol on viability was determined by the eosin dye exclusion procedure [3]. Living cells exclude the dye whereas dead cells absorb it [4]. A 1 % eosin Y solution (w/v with saline) was prepared and a drop of dye was added to a drop of cell suspension on a glass slide. A coverslip was placed on- the mixture \;hich was then allowed to equilibrate for 2 min before counting IS, 61. Cells were counted for 5 min after the equilibration period under a phase contrast microscope at x 600 magnification. The ratio of stained cells (dead cells) to the total number of cells is henceforth referred to as the necrotic index.

on cell growth

and Gahility

347

various concentrations of chloramphenicol (fig. 1 a). The cells continued to grow exponentially at all the concentrations tested: however, the rate of growth was reduced and the cell population doubling time was proportional to the drug concentration (fig. I b). At 50 ,ug/ml, the drug concentration we chose for all our subsequent studies, the population doubling time was 15 h, an increase of 5b h over the control value. If cells are incubated for 9 or 18 h in chloramphenicol and then released into fresh warm medium, they continue to grow but do so at the control rate (fig. 2). Since there is no apparent lag, the growth block appears to be fully and immediately reversible. Since the cell growth cycle is composed of four phases 191,it was possible that the change in growth rate was due to the prolongation of one or all of these phases. In order to determine the duration of the cell cycle stages for cells which had been growing in the continuous presence of chloramphenicol for more than one generation as well as for the control cells, the following experiment was performed.

Cell cycle measurements The graphic method of Okada [7] was used to determine the time duration of each phase of the cell cycle of exponentially growing cells in the presence and absence of chloramphenicol. This method requires four pieces of information: (a) population doubling time (obtained from growth curve); (b) mitotic index determined microscopically; (c) the percent of S-stage cells pulse-labeled with 3H-thymidine [7]; (d) the duration of G2 period by the method of Puck & Steffen [8].

RESULTS In order to evaluate the effect of chloramphenicol on L5178Y cell growth, cells were grown exponentially in the presence of

2. Abscissa: time (hours); ordinate: cell number. Reversibility of the chloramphenicol block. Cells were grown l , in absence of chloramphenicol, control; 0, in presence of chloramphenicol(50 jhg/ml). Arrows indicate when chloramphenicol was removed. Release after a, 9; [1, 18 h. These data are the means of 2 expts. Fig.

Exptl

Cell

Res 77 (1973)

348 D. F. Liberman

& J. L. Roti Roti

The percentage of S phase cells was determined by pulse-labeling the cells for 10 min with 3H-thymidine (3H-TdR), autoradiographs were prepared and the percentage of labeled cells calculated [lo]. The percentage of mitotic cells was also determined on the same slides. The time of G2 stage was then measured for both the treated and untreated cells by the method of Puck & Steffen [8]. This method involves pulse-labeling cells with 3H-thymidine and treating them simultaneously with Colcemid. Aliquots of cells are fixed at hourly intervals following the addition of chloramphenicol and, then, prepared for autoradiography. The number of labeled and unlabeled mitotic figures are then recorded as a function of time (fig. 3a, c). The results are presented in table 1. Using the graphic method of Okada [7], the remaining cell cycle parameters were determined [lo] and these are also presented in table 1. The times of both Gl and S are increased while the G2 and M phases are essentially unchanged in the presence of 50 pug/ml of chloramphenicol after one generation. In order to measure any immediate effect on G2, chloramphenicol (50 ,ug/ml) and Colcemid (0.027 pg/ml) were added to the cells immediately following the 10 min 3H-TdR pulse. The result is shown in fig. 3b. In comparing fig. 3a-c, it is evident that following the immediate addition of chloramphenicol (fig. 3b) the cells that enter mitosis are labeled cells, whereas in the control and the one generation treatment (fig. 3a, c, respectively), labeled cells enter mitosis about l-14 h later. This would suggest that there was an immediate effect on the G2 cells. Either they are delayed and thus enter mitosis at a later time (i.e., after labeled cells have appeared), or they may have been killed by-the drug and thus are permanently lost fro$the population. To test this latter possibility, we incubated cells with chloramphenicol and, at Exptl

Cell Res 77 (1973)

;!j&/fy

a,

0123456789

Abscissa: time (hours); ordinate: log (1 + M/N). mitotic; q , labeled mitotic cells [8]. Effect of chloramphenicol (50 ,ug/ml) on the duration of the G2 phase of the cell growth cycle. Determination of the time of G2: (a) for normal cells, i.e. control; (b) following immediate addition of chloramphenicol; (c) for cells growing in media containing chloramphenicol for one generation. These data are the means of 2 separate expts. Fig. 3. 0,

various times thereafter, aliquots of cells were assayed for chloramphenicol toxicity by a vital staining technique. Eosin exclusion has been widely used as a viability index [3, 41. Healthy cells will exclude it, while dead or sick cells stain with this dye. The results of the time course experiment are reported in fig. 4. There was an increase in the necrotic index (the ratio of stained cells to total cells). The index reached a maximum between 15 and 20 min and by 40 min had returned to the base line. DISCUSSION There are two kinds of effects of chloramphenicol on L5 178Y cells, irreversible and immediately reversible. The irreversible effect is an apparent killing of G2 cells as evidenced by the fact that the first cells which enter mitosis following chloramphenicol addition

Effect of chloramphenicol Table 1. Cell cycle parameters normal medium % in stage Stage

Control

for cells growing

in the presence of chloramphenicol

Time in stage (hours) CAP

on cell growth and viability

Control

CAP

Time ratio CAP/Control

Gl S

26 591t5

33 59+6

1.7 5.5

3.9 9.2

2.2 1.6

M G2

4.110.9 Ly0.F

1.86;20.9”

0.6 1.3a

0.5 l.@

iii

349 and in

--

a Measured parameter.

are labeled cells (fig. 3b) and the fact that the necrotic index increased upon the immediate addition of D-chloramphenicol (fig. 4). This increase in necrotic index (eosin uptake) indicates that a certain fraction of the population has been injured by the presence of chloramphenicol. The fact that the first cells to reach mitosis after the pulse-labeling procedure in the presenceof chloramphenicol and Colcemid are labeled cells and that the labeled cells mitotic index increasesin parallel with the total population mitotic index (fig. 36) indicates that the cells which were in G2 at the time when the pulse with 3H-thymidine was performed (these G2 cells would not incorporate tritiated thymidine since they are post-S phase cells) have been blocked and do not proceed directly into mitosis. The reversible change is the effect of D-chloramphenicol on the growth rate of L5178Y cells (fig. 1). This effect exists only as long as the drug is present. When the drug is removed, the cells return to their normal growth kinetics. There is no noticeable lag so we feel that this growth is fully reversible. Recently, Fettes et al. [I l] reported that spinner flask cultures of mouse L cells likewise show full recovery of growth when D-chloramphenicol or Tevenel (the sulfamoyl analogue of Dchloramphenicol) are removed from the culture. They studied only one concentration of these drugs (100 pg/ml). They found that

there was no lag between the releaseand the return to normal growth kinetics. It is of interest to note that these authors studied cell viability using the eosin Y exclusion method. However, they were interested in the proportion of viable cells some 70 h after growth in media containing D-chloramphenicol or its analogue. They found that in the presence of Tevenel, the necrotic index was on the order of 1 ‘%, whereas in D-chloramphenicol the necrotic index was 5 16. It is unfortunate that they did not study cell viability following the immediate addition of these drugs. In the introduction we stated that Lenk & Penman [l] found that when HeLa cells were cultured in media containing chloramphenico1(100 lug/ml) they grew for two generations and subsequently began to lyse. Our growth data agree with theirs with respect to the fact that we observe a decrease in growth rate in the presence of chloramphenicol. L5 I78Y cells, however, continue to grow in the presence of chloramphenicol. They grow exponentially at a rate which is proportional to the drug concentration (over the range studied: O-200 pg/ml) and eventually reach stationary phase (data not presented). There are several features to the eosin-staining experiment which require comment. First, the fact that the necrotic index curve does not continue to increase proportionally to the Iate of progresExptl

Cell Res 77 (1973)

350

D. F. Liberman

& J. L. Roti Roti somehow becomes resistant to this lethal action of chloramphenicol. If lymphoma cells can adapt to the presence of chloramphenicol in this fashion and HeLa cells cannot, then one would expect HeLa cell cultures to disintegrate as Lenk & Penman have reported [l]. We are presently investigating this possibility with a suspension culture of HeLa cells.

Al----

” 0 IO 20 30 40 Fig. 4. Abscissa: time (min); ordinate: necrotic index (X). Change in the necrotic index following chloramphenicol (50 pg/ml) addition. Open and closed circles denote 2 separate expts. (See Materials and Methods sections and text for details.)

sion from S+G2 suggests that not all the cells are equally sensitive to this effect of chloramphenicol. Otherwise, we could expect the necrotic index eventually to reach 100 % (fig. 4). Second, the fact that the eosin curve peaks at 16 % and then falls back to the pre.exposure level rather than leveling off indicates that these dead cells must leave the population by lysing. The 16 % level is approximately the percentage we would expect if the G2 cells (in an exponential culture approx. 15-20 % of the cells are in G2) and .only the G2 cells are sensitive to this effect of chloramphenicol. The remainder of the population then

~Exptl Cell Res 77 (1973)

The authors would like to acknowledge the fact that this research was performed in the laboratory of Dr C. S. Lange under contract with the US AEC in the Department of Experimental Radiology (contract no. AT(30-l&4284) and at the University of Rochester Atomic Energy Project and has been assigned Report No. UR-3490-92. The authors would also like to thank Drs Helen Eberle, S. Okada, L. H. Hempelmann and C. S. Lange for helpful discussions, MS Lorraine Sheck for assistance with some of the experiments and MS Madeline Phillips for preparation of the manuscript.

REFERENCES 1. Lenk, R & Penman, S, J cell biol 49 (1971) 541. 2. Zylber, E, Vesco, C & Penman, S, J mol biol 44 (1969) 195. 3. Eaton, M D, Scala, A R & Jewel& M, Cancer res 19 (1958) 945. 4. Watanabe, I & Okada, S, Nature 216 (1967) 380. 5. - J cell biol 35 (1967) 285. 6. - Ibid 32 (1967) 309. 7. Okada, S, J cell biol 34 (1967) 915. 8. Puck, E T & Steffen, J, Biophys j 3 (1963) 379. 9. Howard, A & Pelt, S, Heredity 6, suppl. 1 (1953) 261. 10. Roti Roti, J L, Ph D Thesis. Univ of Rochester (1972). 11. Fettes, I M, Haldar, D & Freeman, K B, Can j biochem 50 (1972) 200. Received April 7, 1972 Revised version received September 14, 1972