Cell Biology and Toxicology, Vol. 7, No. 3, 1991
203
EFFECT OF MAGNESIUM ON THE GROWTH AND CELL CYCLE OF TRANSFORMED AND NON-TRANSFORMED EPITHELIAL RAT LIVER CELLS IN VITRO N E I L A. L I T T L E F I E L D ,
B R U C E S. HASS, L Y N D A J. M C G A R R I T Y , S U Z A N N E M. M O R R I S
AND
U.S. Public Health Service, Food and Drug Administration National Center for Toxicological Research Division of Comparative Toxicology Jefferson, Arkansas
The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epitheliallike rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 × the concentration in the regular RPMI 1640 media (lOOmg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G 1 and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G 1 with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G 1 nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics. INTRODUCTION
Mg has been shown to influence the transformation process, i.e., the process in which a normal cell loses its ability to control normal growth and reproduction and becomes a malignant cell. In studies conducted by Rubin (1981); Rubin et al. (1981); and Rubin (1982),
1. Address all correspondence to: Neil A. Littlefield, Ph.D., HFT-140/DCT, NCTR, Jefferson, AR 72079. Tel: (501) 543-7551. 2. Key words: Magnesium, flow cytometry, transformed epithelial cells. Cell Biology and Toxicology, Vol. 7, No, 3, pp. 203-214 Copyright © 1991 Princeton Scientific Publishing Co., Inc. ISSN: 0742-2091
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Littlefield et al.
transformed fibroblast 3T3 cells that were maintained in media with very low Mg concentrations lost their transformed appearances and characteristics. The cells became serum dependent, resembled non-transformed cells in appearance, and the cell density changed in that they did not reach a stable saturation in low Mg concentrations. Chick embryo fibroblast cells kept at a reduced rate of proliferation for three days by Mg deprivation, were quickly restored to rapid proliferation upon addition of Mg to the growth serum (Rubin and Chu, 1978). These studies demonstrated that cell transformation resulted in a selective loss of the regulatory role of Mg. Rubin (1981) showed that non-transformed cells require approximately ten times more serum in the medium to attain an equivalent rate of multiplication than do transformed cells. After about three days, the cells reverted and become independent. A defect in regulation by Mg plays a major role in the transformation. Loss of the role of Mg occurs in transformed cells (Rubin 1975; Rubin and Koide, 1976). Tumor cells may secrete their own growth factor. When these tumor ceils are deprived of Mg they take on the appearance of non-transformed cells. McKeehan (1984) indicated that there was a reduction in essential specific growth factors, particularly Mg, needed by transformed cells to grow and thrive in normally suboptimal concenlrations of these nutrients, whereas the growth of normal cells was materially dependent on the presence of these nutrients. These reports have shown that proliferation of transformed cells may be independent of specific nutrients. We know very little at the molecular (or structural) level about how the distribution of Mg is regulated within the cell and may have to learn more about this aspect of cell physiology to understand the basis of malignant transformation. The present study is designed to compare the influence of Mg on transformed vs non-transformed epithelial cell lines with respect to growth regulation, adaptability, and manipulation of the cell cycle. Since altered cellular proliferation is a prime characteristic of the transformed cell, we decided to examine the effect of Mg concenlration on basic growth parameters of normal and transformed liver epithelial cells in culture in order to determine if epithelial ceils respond to Mg in ways similar to fibroblast cells. METHODS Cell Culture Two lines of epithelial-like cells were used: TRL-12-15, a non-transformed cell line derived from rat liver (Williams et al., 1971); and TRL-8, a spontaneously transformed derivative of TRL-12-15 (Poirier and Wilson, 1980).
Cells were maintained using complete RPMI-1640 medium in T-25 flasks (Corning Glass Works, Corning, NY) in a 5% CO2, humidified (90%) atmosphere at 37* C. The cells were harvested by aspirating the medium, rinsing with Hank's Balanced Salt Solution (HBSS) that contained 0.05 M EDTA, then freeing the cells from the flask with 1 ml of 0.05%
Cell Biology and Toxicology, Vol. 7o No. 3, 1991
205
trypsin/EDTA (Sigma, St. Louis, MO). After resuspending the cells in RPMI-1640 medium containing the appropriate amount of Mg, cell counts were made with a Coulter ZM Counter (Coulter Electronics, Hialeah, FL). All media used in this study were prepared from a special formulation containing all the ingredients of RPMI-1640 except NaHCO3 and Mg2SO4'7H20 (Sigma, St. Louis, MO). The basic formulation was supplemented with NaHCO3, fetal bovine serum (10%) (Gibco, Grand Island, NY), 1% L-glutamine, and 1% penicillin/streptomycin (Gibco, Grand Island, NY). For test purposes, Mg2SO4.7H20 was supplemented at 100 rag/1 (Ix), 10 mg/l (0.1x), or 0 rag/1 (0x). (100 mg/1 is the concentration of Mg2SO4"7H20 in the standard preparation of RPMI- 1640).
Growth Curves." Cells from each of the two lines were seeded in polystyrene 6-well dishes with a diameter of 35 mm (Coming Glass Works, Coming NY) at a density of 104 cells in 2 ml of medium. Cells were maintained in a 5% CO2, humidified (90%) atmosphere at 37* C. At each respective time interval, the medium was aspirated from the cells, washed with Hank's Balanced Salt solution (HBSS) containing 0.05 M EDTA, and then with 0.05% trypsin/EDTA. The cells were quantitated using a Coulter ZM Counter. Medium was changed every two to four days as prompted by the pH indicator in the media. Two or three wells for each cell line were counted at specific time points. During the first four days, counts were made daily. The counts were made by washing the wells with HBSS that contained 0.05M EDTA, detaching the cells with one ml of trypsin, diluting the cells in an Isoton Coulter Counting solution and making at least three counts on each dilution.
Flow Cytometry." Ceils from each of the two lines were seeded at a density of 1 × 106 ceils/ml in T25 flasks containing 5 ml of RPMI 1640 media with Mg at 0, 0.1, or 1.0 × optimum levels. The cells were maintained as descrilx,,dabove for one to seven days prior to analysis. At harvest, the cell monolayer was rinsed with HBSS after removal of the medium. Approximately 5.0 ml of propidium iodide (PI) staining solution (Vindelov, 1977; Anson et al., 1983) was added to each flask. Culture flasks were incubated on ice for 30 minutes, after which the suspension of nuclei was decanted into a centrifuge tube. An aliquot of cells was removed for quantitation, the cells were pelleted by cenlrifugation, the medium was aspirated, and the cell pellet was gently resuspended. The suspension of nuclei was filtered through 44 ~t nylon mesh into a 5.0 ml sample tube and stored on ice under an aluminum foil light shield until analysis. The position in the cell-cycle of intact (DNA content), stained nuclei was determined with a FACScan (Becton-Dickinson, San Jose, CA) flow cytometer with an Argon ion laser tuned to 488 nm. The cell suspension from each flask was sampled three times, 10,000 nuclei evaluated per sample. Individual data sets were analyzed with the "S-fit" program for DNA analysis
206
Littlefield et al.
(Becton-Dickinson) which constructs a histogram and determines the percentage of nuclei in each of the cell-cycle phases. Individual histograms were inspected to ensure that the coefficient of variation (CV) of the GO +G1 peak was sufficiently small (less than 5%) to ensure analysis (data not shown). Individual data sets were also analyzed according to Bagwell et al. (1979) in a routine which smoothed the data, translocated the peak channel and determined the percentage of nuclei in each of the three cell-cycle phases. Individual data sets were then combined and differences in the percentages of nuclei in each of the three cell-cycle phases determined by a "t-test" for equality of means.
7
10
106 O') ._1 ._1
LU L.) LL o EE LU
10 s
133
22) Z
10 3
--r
1
r 2
T - - T
3
T - - T - - T - - 1
4
5 DAYS
6
7
8
9
FIGURE 1. The Effect of Magnesium Concentration in Growth Media at l(x), 0.5(x), 0.1 (x), or 0(x) Optimum Concentration (100 mg/L of media) Necessary for Optimum Growth of TRL-12-15 Non-Transformed Rat Liver Cells.
Cell Biology and Toxicology, Vol. 7, No. 3, 1991
207
7
10
6
10 or) ._1 ._1 LU
O
IJ,_
O elIJJ 133
5
10
Z 4
10
10
3 l
1
2
T--I----'-'r
3
4
5
1
1
~
6
7
8
l
9
10
"T---
11
DAYS FIGURE 2. The Effect of the Presence or Absence of Magnesium in Growth Media on the Growth of TRL-8 Transformed Rat Liver Cells. RESULTS The results, as shown in Figures 1 and 2, show that the TRL-12-15 cells (non-transformed) exhibited a definite response to the Mg levels, while the transformed TRL 8 cells appeared to be more independent of the Mg levels in the media. The growth curves for the TRL-12-15, as shown in Figure 1, show that at about day 4 the cells in the Mg-restricted medium essentially stopped growing or the rate decreased substantially and the growth remained constant during the remainder of the test period. The ceils in the 0.1 and 1.0 media continued to grow rapidly throughout the study. The TRL-8 cells (Figure 2) in the Mg-restricted medium grew at about
208
Littlefield et al.
the same rate as the other cells, however, there were occasions at about four days that growth rate slowed temporarily, then continued at an increased rate until it matched the rate of the control cells.
7
10
Mg0.1
6
10
0.5 J J iii O ii
I
1.0 5
O
10
cC uJ nn
Z
10
4
3
10 DAYS
F I G U R E 3. The Effect of Magnesium Concentration in Growth Media at l(x), 0.5(x), 0.1 (x), or 0(x) Optimum Concentration (100 mg/L of media) Necessary for Optimum Growth of Pre-Conditioned TRL-12-15 Non-transformed Rat Liver Cells. It was observed in each study that growth rates were similar in all dose groups during the initial four days. Therefore, a subsequent study was done to examine the possibility that cells
Cell Biology and Toxicology, Vol. 7, No. 3, 1991
209
were preconditioned by the medium used to maintain the cell lines. Each ceil may carry an intracellular quantity of Mg which services their needs for a least four days. Four days prior to the start of the study, each respective cell line was inoculated into a T-25 flask containing media with 0, 0.1, 0.5, or 1.0 x the recommended amounts of Mg. At the start of the growth period, each respective dosed flask was used as the stock to seed the various dose groups. Therefore, each group of cells was equilibrated at its respective Mg concentrations for four days prior to start of the study.
+
Mg-
0.1 1
0.5 I
_.1 ..J
U.l 0
0rr
H-
1.0
11
tl.l nn 10 4
-
'1
Z
10a DAYS
F I G U R E 4, The Effect of Magnesium Concentration in Growth Media at l(x), 0.5(x), 0.1(x), or 0(×) Optimum Concentration (100 mg/L of media) Necessary for Optimum Growth of Pre-Conditioned TRL-8 Transformed Rat Liver Cells.
210
Littlefield et al.
These results as shown in Figures 3 and 4, demonstrate that even the transformed cells reach a limit of independence for the Mg. After four days of the study, growth in both cell lines essentially ceased in the Mg-restricted medium. The results from the flow cytometry analysis were similar to the growth curve data. The effects of Mg concentrations on the cell-cycle distributions on the TRL-12-15 and TRL-8 is presented in Tables 1 and 2, respectively. Interestingly, the effect of Mg restrictions on the transit through the cell-cycle was most pronounced in the TRL-12-15 cell line. As early as three days after seeding, those cells cultured under Mg restrictions were accumulating in GO +G1 (Table 1) and fewer cells were moving into S-phase. At day 4, those cells cultured without exogenous Mg were accumulating in S-phase and also in G2 + M. After a medium change on day 5, the cell-cycle distribution of cultures under minimum Mg restriction more closely approximately the distribution of the control population than those under severe Mg restriction. However, after an additional day in culture, the distrubution of cells in the cell-cycle of those cultures grown under Mg restriction approximated each other and were significantly different (p
203
EFFECT OF MAGNESIUM ON THE GROWTH AND CELL CYCLE OF TRANSFORMED AND NON-TRANSFORMED EPITHELIAL RAT LIVER CELLS IN VITRO N E I L A. L I T T L E F I E L D ,
B R U C E S. HASS, L Y N D A J. M C G A R R I T Y , S U Z A N N E M. M O R R I S
AND
U.S. Public Health Service, Food and Drug Administration National Center for Toxicological Research Division of Comparative Toxicology Jefferson, Arkansas
The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epitheliallike rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 × the concentration in the regular RPMI 1640 media (lOOmg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G 1 and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G 1 with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G 1 nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics. INTRODUCTION
Mg has been shown to influence the transformation process, i.e., the process in which a normal cell loses its ability to control normal growth and reproduction and becomes a malignant cell. In studies conducted by Rubin (1981); Rubin et al. (1981); and Rubin (1982),
1. Address all correspondence to: Neil A. Littlefield, Ph.D., HFT-140/DCT, NCTR, Jefferson, AR 72079. Tel: (501) 543-7551. 2. Key words: Magnesium, flow cytometry, transformed epithelial cells. Cell Biology and Toxicology, Vol. 7, No, 3, pp. 203-214 Copyright © 1991 Princeton Scientific Publishing Co., Inc. ISSN: 0742-2091
204
Littlefield et al.
transformed fibroblast 3T3 cells that were maintained in media with very low Mg concentrations lost their transformed appearances and characteristics. The cells became serum dependent, resembled non-transformed cells in appearance, and the cell density changed in that they did not reach a stable saturation in low Mg concentrations. Chick embryo fibroblast cells kept at a reduced rate of proliferation for three days by Mg deprivation, were quickly restored to rapid proliferation upon addition of Mg to the growth serum (Rubin and Chu, 1978). These studies demonstrated that cell transformation resulted in a selective loss of the regulatory role of Mg. Rubin (1981) showed that non-transformed cells require approximately ten times more serum in the medium to attain an equivalent rate of multiplication than do transformed cells. After about three days, the cells reverted and become independent. A defect in regulation by Mg plays a major role in the transformation. Loss of the role of Mg occurs in transformed cells (Rubin 1975; Rubin and Koide, 1976). Tumor cells may secrete their own growth factor. When these tumor ceils are deprived of Mg they take on the appearance of non-transformed cells. McKeehan (1984) indicated that there was a reduction in essential specific growth factors, particularly Mg, needed by transformed cells to grow and thrive in normally suboptimal concenlrations of these nutrients, whereas the growth of normal cells was materially dependent on the presence of these nutrients. These reports have shown that proliferation of transformed cells may be independent of specific nutrients. We know very little at the molecular (or structural) level about how the distribution of Mg is regulated within the cell and may have to learn more about this aspect of cell physiology to understand the basis of malignant transformation. The present study is designed to compare the influence of Mg on transformed vs non-transformed epithelial cell lines with respect to growth regulation, adaptability, and manipulation of the cell cycle. Since altered cellular proliferation is a prime characteristic of the transformed cell, we decided to examine the effect of Mg concenlration on basic growth parameters of normal and transformed liver epithelial cells in culture in order to determine if epithelial ceils respond to Mg in ways similar to fibroblast cells. METHODS Cell Culture Two lines of epithelial-like cells were used: TRL-12-15, a non-transformed cell line derived from rat liver (Williams et al., 1971); and TRL-8, a spontaneously transformed derivative of TRL-12-15 (Poirier and Wilson, 1980).
Cells were maintained using complete RPMI-1640 medium in T-25 flasks (Corning Glass Works, Corning, NY) in a 5% CO2, humidified (90%) atmosphere at 37* C. The cells were harvested by aspirating the medium, rinsing with Hank's Balanced Salt Solution (HBSS) that contained 0.05 M EDTA, then freeing the cells from the flask with 1 ml of 0.05%
Cell Biology and Toxicology, Vol. 7o No. 3, 1991
205
trypsin/EDTA (Sigma, St. Louis, MO). After resuspending the cells in RPMI-1640 medium containing the appropriate amount of Mg, cell counts were made with a Coulter ZM Counter (Coulter Electronics, Hialeah, FL). All media used in this study were prepared from a special formulation containing all the ingredients of RPMI-1640 except NaHCO3 and Mg2SO4'7H20 (Sigma, St. Louis, MO). The basic formulation was supplemented with NaHCO3, fetal bovine serum (10%) (Gibco, Grand Island, NY), 1% L-glutamine, and 1% penicillin/streptomycin (Gibco, Grand Island, NY). For test purposes, Mg2SO4.7H20 was supplemented at 100 rag/1 (Ix), 10 mg/l (0.1x), or 0 rag/1 (0x). (100 mg/1 is the concentration of Mg2SO4"7H20 in the standard preparation of RPMI- 1640).
Growth Curves." Cells from each of the two lines were seeded in polystyrene 6-well dishes with a diameter of 35 mm (Coming Glass Works, Coming NY) at a density of 104 cells in 2 ml of medium. Cells were maintained in a 5% CO2, humidified (90%) atmosphere at 37* C. At each respective time interval, the medium was aspirated from the cells, washed with Hank's Balanced Salt solution (HBSS) containing 0.05 M EDTA, and then with 0.05% trypsin/EDTA. The cells were quantitated using a Coulter ZM Counter. Medium was changed every two to four days as prompted by the pH indicator in the media. Two or three wells for each cell line were counted at specific time points. During the first four days, counts were made daily. The counts were made by washing the wells with HBSS that contained 0.05M EDTA, detaching the cells with one ml of trypsin, diluting the cells in an Isoton Coulter Counting solution and making at least three counts on each dilution.
Flow Cytometry." Ceils from each of the two lines were seeded at a density of 1 × 106 ceils/ml in T25 flasks containing 5 ml of RPMI 1640 media with Mg at 0, 0.1, or 1.0 × optimum levels. The cells were maintained as descrilx,,dabove for one to seven days prior to analysis. At harvest, the cell monolayer was rinsed with HBSS after removal of the medium. Approximately 5.0 ml of propidium iodide (PI) staining solution (Vindelov, 1977; Anson et al., 1983) was added to each flask. Culture flasks were incubated on ice for 30 minutes, after which the suspension of nuclei was decanted into a centrifuge tube. An aliquot of cells was removed for quantitation, the cells were pelleted by cenlrifugation, the medium was aspirated, and the cell pellet was gently resuspended. The suspension of nuclei was filtered through 44 ~t nylon mesh into a 5.0 ml sample tube and stored on ice under an aluminum foil light shield until analysis. The position in the cell-cycle of intact (DNA content), stained nuclei was determined with a FACScan (Becton-Dickinson, San Jose, CA) flow cytometer with an Argon ion laser tuned to 488 nm. The cell suspension from each flask was sampled three times, 10,000 nuclei evaluated per sample. Individual data sets were analyzed with the "S-fit" program for DNA analysis
206
Littlefield et al.
(Becton-Dickinson) which constructs a histogram and determines the percentage of nuclei in each of the cell-cycle phases. Individual histograms were inspected to ensure that the coefficient of variation (CV) of the GO +G1 peak was sufficiently small (less than 5%) to ensure analysis (data not shown). Individual data sets were also analyzed according to Bagwell et al. (1979) in a routine which smoothed the data, translocated the peak channel and determined the percentage of nuclei in each of the three cell-cycle phases. Individual data sets were then combined and differences in the percentages of nuclei in each of the three cell-cycle phases determined by a "t-test" for equality of means.
7
10
106 O') ._1 ._1
LU L.) LL o EE LU
10 s
133
22) Z
10 3
--r
1
r 2
T - - T
3
T - - T - - T - - 1
4
5 DAYS
6
7
8
9
FIGURE 1. The Effect of Magnesium Concentration in Growth Media at l(x), 0.5(x), 0.1 (x), or 0(x) Optimum Concentration (100 mg/L of media) Necessary for Optimum Growth of TRL-12-15 Non-Transformed Rat Liver Cells.
Cell Biology and Toxicology, Vol. 7, No. 3, 1991
207
7
10
6
10 or) ._1 ._1 LU
O
IJ,_
O elIJJ 133
5
10
Z 4
10
10
3 l
1
2
T--I----'-'r
3
4
5
1
1
~
6
7
8
l
9
10
"T---
11
DAYS FIGURE 2. The Effect of the Presence or Absence of Magnesium in Growth Media on the Growth of TRL-8 Transformed Rat Liver Cells. RESULTS The results, as shown in Figures 1 and 2, show that the TRL-12-15 cells (non-transformed) exhibited a definite response to the Mg levels, while the transformed TRL 8 cells appeared to be more independent of the Mg levels in the media. The growth curves for the TRL-12-15, as shown in Figure 1, show that at about day 4 the cells in the Mg-restricted medium essentially stopped growing or the rate decreased substantially and the growth remained constant during the remainder of the test period. The ceils in the 0.1 and 1.0 media continued to grow rapidly throughout the study. The TRL-8 cells (Figure 2) in the Mg-restricted medium grew at about
208
Littlefield et al.
the same rate as the other cells, however, there were occasions at about four days that growth rate slowed temporarily, then continued at an increased rate until it matched the rate of the control cells.
7
10
Mg0.1
6
10
0.5 J J iii O ii
I
1.0 5
O
10
cC uJ nn
Z
10
4
3
10 DAYS
F I G U R E 3. The Effect of Magnesium Concentration in Growth Media at l(x), 0.5(x), 0.1 (x), or 0(x) Optimum Concentration (100 mg/L of media) Necessary for Optimum Growth of Pre-Conditioned TRL-12-15 Non-transformed Rat Liver Cells. It was observed in each study that growth rates were similar in all dose groups during the initial four days. Therefore, a subsequent study was done to examine the possibility that cells
Cell Biology and Toxicology, Vol. 7, No. 3, 1991
209
were preconditioned by the medium used to maintain the cell lines. Each ceil may carry an intracellular quantity of Mg which services their needs for a least four days. Four days prior to the start of the study, each respective cell line was inoculated into a T-25 flask containing media with 0, 0.1, 0.5, or 1.0 x the recommended amounts of Mg. At the start of the growth period, each respective dosed flask was used as the stock to seed the various dose groups. Therefore, each group of cells was equilibrated at its respective Mg concentrations for four days prior to start of the study.
+
Mg-
0.1 1
0.5 I
_.1 ..J
U.l 0
0rr
H-
1.0
11
tl.l nn 10 4
-
'1
Z
10a DAYS
F I G U R E 4, The Effect of Magnesium Concentration in Growth Media at l(x), 0.5(x), 0.1(x), or 0(×) Optimum Concentration (100 mg/L of media) Necessary for Optimum Growth of Pre-Conditioned TRL-8 Transformed Rat Liver Cells.
210
Littlefield et al.
These results as shown in Figures 3 and 4, demonstrate that even the transformed cells reach a limit of independence for the Mg. After four days of the study, growth in both cell lines essentially ceased in the Mg-restricted medium. The results from the flow cytometry analysis were similar to the growth curve data. The effects of Mg concentrations on the cell-cycle distributions on the TRL-12-15 and TRL-8 is presented in Tables 1 and 2, respectively. Interestingly, the effect of Mg restrictions on the transit through the cell-cycle was most pronounced in the TRL-12-15 cell line. As early as three days after seeding, those cells cultured under Mg restrictions were accumulating in GO +G1 (Table 1) and fewer cells were moving into S-phase. At day 4, those cells cultured without exogenous Mg were accumulating in S-phase and also in G2 + M. After a medium change on day 5, the cell-cycle distribution of cultures under minimum Mg restriction more closely approximately the distribution of the control population than those under severe Mg restriction. However, after an additional day in culture, the distrubution of cells in the cell-cycle of those cultures grown under Mg restriction approximated each other and were significantly different (p
