The Effect of Hexose on Chloramphenicol Sensitivity and Resistance in Chinese Hamster Cells MICHAEL L. ZIEGLER AND RICHARD L. DAVIDSON Division of Human Genetics, Children's Hospital Medical Center and Department of Microbiology and Molecular Genetics, Haruard Medical School, Boston, Massachusetts 021 15

ABSTRACT

The toxicity and extent of growth inhibition produced by chloramphenicol (CAP) in CAPSChinese hamster cells (line V79-5) was found to be dependent on the type and concentration of hexose in the medium. In high levels of glucose (6.5 mM), cultures of CAPScells underwent 7-8 population doublings in the presence of 100 pg/ml CAP and viability then dropped rapidly. In contrast, lower concentrations of glucose (1.0 mM) permitted only limited growth (2-3 doublings) in the presence of 100 pg/ml CAP, but the cells remained viable and apparently quiescent for prolonged periods of time. The growth potential of V79-5 cells in CAP appeared specifically dependent on glucose, as mannose and galactose could not substitute for glucose. The toxicity of CAP to these cells seemed to be determined primarily by the number of cell doublings in t h e presence of the drug. A CAPR derivative of V79-5, designated 5-3, was analyzed in order to determine whether the requirement for glucose for cell growth in the presence of CAP also occurred in cells that were isolated as resistant to the drug. In order to rigorously control the hexose in the medium, some experiments were performed with medium containing dialysed, instead of whole, fetal calf serum. It was seen that the growth of the CAPRline in the presence (but not the absence) of 100 pg/ml CAP was dependent on glucose in the medium. Thus, resistance to CAP in these cells appears to be a conditional state, dependent on glucose for expression. Furthermore, the glucose auxotrophy of these cells in the presence of CAP suggests that CAP is still affecting some activities in cells isolated as CAPR.

In eukaryotic cells chloramphenicol (CAP) has been shown to selectively inhibit mitochondrial protein synthesis without affecting cytoplasmic protein synthesis (Galper and Darnell, '711, leading t o a progressive loss of components of the electron transport system (Clark-Walker and Linnane, '67). In mammalian cells, i t has been shown that cytochromes aa3, b, and c, are reduced in cells exposed to CAP (Firkin and Linnane, '68; Lipton and McMurray, '77). In addition, incubation of cells in CAP may lead to alterations in mitochondrial ultrastructure, including disruption of cristae and decrease in matrix density (Lipton and McMurray, '77; Lenk and Penman, '71). These findings suggest that cells in the presence of CAP undergo a progressive decrease in mitochondria1 respiratory ability. J. CELL. PHYSIOL. (1979) 98; 627-636

Recently a group of respiratory deficient mammalian cells has been isolated (Ditta et al., '76) and found to have an absolute dependency on glucose for growth. Since glucose auxotrophy was shown to accompany a decrease in respiratory competence, we have carried out experiments to determine whether incubation of somatic cells in CAP can lead to alterations in cellular sugar requirements, and conversely, whether changes in the sugar source can have an effect on the cells' response to CAP. It was observed that the ability of Chinese hamster cells to grow and to survive in the presence of CAP is altered significantly by changes in the hexose in the medium. In addition, evidence was found to suggest that resistance to CAP in drug resistant variants is Received July 18, '78. Accepted Oct. 9, '78.

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a conditional state which depends upon the type and concentration of exogenous hexose. MATERIALS AND METHODS

Cells and media The CAPScell line used in this study was the Chinese hamster cell line V79-5 (Harris, '74). A CAPR subline, designated 5-3, was isolated by a single step selection (without mutagenesis) in the presence of 100 pg/ml CAP. The frequency of CAPRcolonies was 6.4 X and a single colony was subcultured and propagated as line 5-3. The basic medium was glucose free Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum, 1.0 mM sodium pyruvate, and 0.026 M sodium bicarbonate (LG medium). Since the fetal calf serum contained approximately 10 mM glucose, as determined by a glucose reagent kit (Sigma), LG medium contained about 1.0 mM glucose. LG medium was supplemented with additional hexose to make the following media: HG medium received a supplement of 5.5 mM glucose; GT medium - 5.5 mM galactose (Sigma, glucose free); MA medium - 5.5 mM mannose. HG, GT and MA media thus each contain a total hexose concentration of 6.5 mM. For some experiments dialysed fetal calf serum was used instead of whole serum. Dialysed serum was tested for glucose as indicated above and none could be detected. Media containing dialysed serum was designated with t h e prefix ds (e.g., dsHG). CAP was prepared and stored as a stock solution (10mg/ml) in 70% EtOH. Medium with a final CAP concentration of 100 pg/ml is denoted as 100 CAP, etc. Growth tests In order to obtain uniformity between assays, cells to be tested were cultured for four days in 25 cm2 culture flasks from an inoculum of lo5 cells/flask in 10 ml of HG medium. The cells then were collected and a series of 60-mm culture dishes was inoculated with 1-2 X lo' cells in 5 ml of the various media. At approximately 24-hour intervals, cells from duplicate cultures were harvested with trypsin and counted in a Coulter counter. The cultures were renewed with 5 ml fresh media of the same type on days 3 and 5 and daily thereafter. Assays for viability Plating efficiency tests were performed in order to assay cell viability after exposure of cells to CAP under various conditions. Cells

for these tests were plated a t 100 cellddish in triplicate petri dishes contained 5 ml of HG medium. After seven t o ten days, the colonies were fixed with methanol, stained with Giemsa and counted. RESULTS

Growth potential of CAPscells in CAP Growth tests were performed with V79-5 cells in order to determine whether the concentration or type of sugar in the medium had an effect on the ability of CAPScells to proliferate in the presence of CAP. Growth curves for V79-5 cells in LG medium (1.0 mM glucose) with various supplements are shown in figure 1A. It can be seen t h a t LG medium alone supported rapid growth, with the cells exhibiting a population doubling time of approximately 1 3 hours. The addition of 5.5 mM glucose (in HG medium) or 5.5 mM galactose (in GT medium) to the basal (LG) medium did not affect the growth rate of t h e cells, nor did it affect the maximal cell density attained. These results show that, under the conditions of the present assay, the small amount of glucose contributed by the serum in LG medium did not limit cell multiplication. When V79-5cells were exposed to 100 pg/ml CAP, the growth patterns of the cells in the various media were very different. In HG/100 CAP medium, the growth rate of the cells was initially similar to t h a t of cells in t h e absence of CAP. However, figure 1A demonstrates that population growth gradually slowed and by day 6 the cells began to detach from the substrate and the cell number declined. Even though the cells could not grow indefinitely in CAP, they underwent approximately seven population doublings before growth inhibition became pronounced. In contrast to t h e delayed inhibition of growth in HG/100 CAP medium, cell division was rapidly inhibited when t h e cells were exposed to 100 pg/ml CAP in either LG or GT medium. Essentially complete cessation of growth occurred within 2.5 doublings, after which the cell number remained relatively stable. Thus, with either low levels of glucose alone (LG) or a supplement of a n alternative hexose (GT), cells had a more limited growth potential in the presence of 100 pg/ml CAP than cells in medium containing high glucose (HG) plus CAP. Viability of cells grown in CAP As shown in figure lA, V79-5 cells began to degenerate after six days' growth in HG/100

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TIME (Hours) Fig. 1 Effect of CAP and hexose on growth. The growth curves for V79-5 (part A) and 5-3 (part B) were obtained as described in MATERIALS AND METHODS. The cells were grown in the following media: A , LG; W, GT; 0 , HG; A, LW100 CAP; 0, GT/100 CAP; 0, HG/100 CAP. LG medium contained approximately 1.0 mM glucose. Hexose supplements were added to LG medium to make HG (final glucose concentration 6.5 mM) or GT (5.5 mM galactose) medium.

CAP medium. In contrast, V79-5 cells grown in GT/100 CAP medium did not degenerate after even longer periods of time. At least two explanations could account for the persistence of the cells in GT/100 CAP medium. First, cell death may have occurred but the cells remained attached during the assay period. Second, a plateau phase may have been reached where neither cell death nor multiplication occurred (or in which cell death and multiplication balanced each other). In order to test these possibilities, the viability of V79-5 cells cultured in these media was determined (as the plating efficiency of the cells in HG medium in the absence of CAP) for periods up to ten days. The results of these tests are presented in figure 2. When cultured in HG/100 CAP medium, the cells retained high viability (around 90% plating efficiency) until day 4. After that time, plating efficiency dropped rapidly, falling to approximately 5%by day 7. It appeared as if the cultures could attain a certain maximum cell number and then viability would drop rapidly. A different picture emerged for cells grown in GT/100 CAP medium. In this medium, the

cells maintained relatively high plating efficiencies (around 30%)up to the end of the 10day assay period, including the period during which the cell number was not increasing. Since similar results were found for cells grown in LG/100 CAP medium (plating efficiency after 10 days was 35%),the results observed with GT/100 CAP medium probably are due to the low levels of glucose in the medium, rather than any specific effect of galactose. These data suggest that the plateau in cell number observed in LG/100 CAP or GT/100 CAP medium represents a steady state in which a significant part of the population remains viable for prolonged periods without an increase in cell number.

Limitation of growth of cells i n CAP The long term retention of viability of V795 cells in LG/100 CAP and GT/100 CAP media indicates that the absolute time in the presence of 100 pg/ml CAP is not the critical factor in determining toxicity. Thus other explanations for the disparity in the extent of killing by CAP in the different media had to be

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considered. Since the cells in the presence of 100 pg/ml CAP reached different densities depending on sugar type and concentration (fig. lA), it was conceivable that a certain maximum cell density might be tolerated in the presence of CAP, above which cell death would ensue. If this were true, i.e., if CAP toxicity were determined solely by cell density, cells plated a t a low density in the presence of CAP would be expected to undergo more doublings before losing viability than cells in sister cultures plated at a higher density. In order to test this possibility, V79-5 cells were plated at two different initial inocula (10-fold apart) in HG/100 CAP medium. After five, seven, and ten days the cell number and viability of the cells (in the absence of CAP) were determined. As shown in table 1, both the high and low density cultures maintained high plating efficiencies during approximately 7-8 population doublings. However, beyond that point, cultures at both densities degenerated rapidly (and apparently in parallel). At the point when degeneration began, between

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days 5 and 7, the high density cultures had approximately ten times more cells than the low density cultures, reflecting the difference in the initial cell inocula. These data suggest that V79-5 cells are not limited in proliferation by a fixed maximal cell density, but have a rather fixed and limited number of potential doublings (approximately 7-8) in the presence of CAP (in HG/100 CAP medium), after which viability drops to a low level. If, as suggested above, the total number of doublings in CAP were the determinant for toxicity and could equal but not exceed 7-8, then the cells which had become quiescent after undergoing 2-3 doublings in LG/100 CAP or GT/100 CAP medium could have retained some potential for further growth in the presence of CAP. This prediction is tested in the experiment presented in figure 2. Cultures were inoculated either in GT/100 CAP or HG/ 100 CAP medium and cell number and cell viability were monitored periodically. On days 4 and 6, the medium in a number of GT/100 CAP cultures was replaced with HG/100 CAP

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The effect of hexose on chloramphenicol sensitivity and resistance in Chinese hamster cells.

The Effect of Hexose on Chloramphenicol Sensitivity and Resistance in Chinese Hamster Cells MICHAEL L. ZIEGLER AND RICHARD L. DAVIDSON Division of Hum...
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