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Requirements for competence and commitment of quiescent 3T3-cells to initiate DNA synthesis after growth stimulation in low serum concentration
Copyright 0 1981 by Academic Press. Inc. All nght5 of reproduction in any form reserved 0014.4827/8110901 IS-1 I$(,2 00/O
Experimentd Cell Research 135 (1981) 115-125
REQUIREMENTS OF QUIESCENT AFTER
FOR COMPETENCE 3T3-CELLS
GROWTH
AND COMMITMENT
TO INITIATE
STIMULATION
DNA
SYNTHESIS
IN LOW SERUM
CONCENTRATION
SUMMARY Quiescent serum-starved 3T3 cells in sparse cultures can be stimulated to initiate DNA synthesis and undergo one cell division in low serum concentration after brief exposure to alkaline pH [ 191. This method of mitogenic stimulation was used to investigate the requirements of low molecular weight components during the first cell cycle after onset of stimulation. It was shown that each of the low molecular constituents in Dulbecco’s Modified Eagle Medium (DMEM) could be excluded (one at a time) without influencing the stimulatory response to brief alkaline treatment, with the exception of glutamine, calcium and phosphate ions. The temporal requirements of these factors were studied from onset of sttmulation to initiation of DNA synthesis. It was found that phosphate ions were required immediately after the alkaline treatment. but glutamine was not until 6 h after onset of alkaline stimulation.
Several substances exert a growth-stimuand insulin [IS] that had been prepared so latory influence on cells in vitro, e.g. serum that they were unable to enter the cell in[I], epidermal growth factor (EGF) [2], terior (by attachment of plastic beads to the fibroblast growth factor (FGF) [3], myo- trypsin and insulin molecules) were as efblast factor [4], insulin [5], somatomedins ficient in promoting cell proliferation as [6], proteolytic enzymes in low concentra- were free insulin or trypsin. These findings tion [7, 81, calcium pyrophosphate [9], the suggest that the site of their activity was at calcium ionophone A23187 [lo], ZrP, Ca2+, the cell surface. Also calcium pyrophosand Mg*+ ions [ 1I], or lectins [ 121. phate complexes, which induce proliferaThese substances have been proposed to tion in quiescent cells, have been shown to exert their primary action at the cell sur- precipitate at the cell surface and are not face, from which the mitogenic message is taken up into the cell [9]. mediated intracellularly via some second The mitogenic message can be mediated messenger(s) [ 131. This proposal was sup- from the cell surface into the cell via at ported by experimental evidence that three least two principally different mechanisms. of the substances (trypsin, insulin and cal- First, the mitogenic substance can alter the cium pyrophosphate) that promote cell pro- transmembrane permeability properties for liferation, act at the cell surface but are low molecular weight substances. This not taken up into the cell. Trypsin [14] could result in increased influx or efflux of
116
W. Engstriim
some growth-regulatory substance(s), e.g. amino acids, glucose, or ions. Second, the mitogenic substance(s) could activate (or inhibit) some growth-regulatory enzyme(s) that are associated with the cell membrane. Some support for the first suggestion was given by the findings that mitogenic stimulation of quiescent 3T3 cells resulted in a rapid cellular uptake of low molecular substances, e.g. deoxyglucose [S], uridine [16]. non-metabolizable amino acids [ 171 and phosphate ions [ 181. This study was intended to investigate whether or not the mitogenic message is mediated into the cell via increased cellular uptake of some low molecular weight constituent(s). Swiss 3T3 cells in sparse cultures, starved to quiescence in media with low serum concentration (0.5 c/c), were used for this purpose. These cells are normally dependent on the addition of a high concentration serum (5-10s) to the medium to leave the quiescent state and enter the cell cycle. It was recently shown, however, that quiescent serum-starved 3T3 cells can be stimulated to initiate DNA synthesis and undergo one cell division in medium with low serum concentration (0. l0.5%) after a 5-10 min long treatment in alkaline medium (pH 8.5-10) [ 191. Furthermore, by using this method it was possible to stimulate quiescent 3T3 cells to initiate DNA synthesis in a medium with very low serum concentration (0.0001%). As the serum requirement for mitogenic stimulation was substantially reduced, only minor amounts of low molecular weight substances could be supplied to the culture medium via the presence of serum. Hence it became possible to study the effects on proliferation of the withdrawal of specific low molecular weight components from an almost chemically defined medium. By applying this approach it could be Erp Cell Rrs 135 f/981)
.
shown that each of the low molecular constituents in the growth medium could be excluded (one at a time) without influencing the stimulatory response to brief alkaline treatment, with the exception of glutamine, calcium, or phosphate ions. MATERIAL
AND METHODS
Mouse Swiss 3T3 embryo fibroblast\ (Flow Laboratories Inc.) were maintained in monolayer cultures in Nunc’s plastic tissue culture bottles. The stock cultures were grown in a humified 6% CO,-air mixture in Dulbecco’s modified Eagle medium supplemented with IO% fetal calf serum (FCS) [20], SO units of penicillin per ml and SO pg of streptomycin per ml. The cells were removed from the dish for transfer by treatment with 0.25% trypsin in Tris-buffered saline containing 0.5 mM EDTA. The line wax maintained by seeding 3000 cells/cmZ culture bottle area and transferring them every 3rd day. The cells were never allowed to reach confluency. Cells used for experimental purpose were grown in IO ml plastic tissue culture bottles or in Petri dishes, which contained a glass coverslip on the bottom. Ouiescent cells were obtained bv brieflv washing (2x‘lO set) proliferating cells in Eaile’s balanced sai solution (BSS) and thereafter incubating them in medium supplemented with 0.5% serum for 24 h.
Quiescent. serum-starved cells were treated with an alkaline (pH 9.5) medium for IO min [19]. The cells were then exposed to media with low serum concentration (containing 0.0001 or 0.57r dialysed serum) with different concentrations of low-molecular constituents (cf vdbks l-4 and figure captions) at normal pH (7.3) during a 24-h assay period. These media were produced in our laboratory by adding lowmolecular compounds (ions. amino acids. energy sources. vitamins) either to distilled water or to Earle’s balanced salt solution. The final concentration of each added compound corresponded to that in Dulbecco’s modified Eagle’s medium r201. During the first part of this study. these media, witi different compositions. were present in the cell cultures during the whole 24 h period. In the second part of this investigation. when the temporal requirements for serum, glutamine and phosphate ions were investigated, media deficient with respect to these compounds were present during limited intervals of the assay period. A complete DMEM was present during the other parts of the 24 h assay period.
Autorudiography The (0.5
cell cultures were labelled with [“Hlthymidine pCi/ml culture medium: New England Nuclear
Reyrri,-rmt~,lt.v,fi)r compt~tt~nw trnd c~ommitment to initirrte DNA synthesis
00001
0.001
0.01
0.1
1
10
FY,q. I. Stimulation of DNA synthesis by alkaline treatment in presence of different serum concentrations in culture medium. Ouiescent serum-starved 3T3 cells were exposed for lb min to alkaline (pH 9.5) medium, which was thereafter reolaced bv DMEM with different serum concentrations for 54 h at normal pH (7.3). Two different serum hatches (0. I; n , 2) were tested. Cells continuously exposed to DMEM at normal pH (7.3) with different serum concentration were used as controls (0. I; q . 2). The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure during the 24 h assay period to 0.S @Ii [“H]thymidine/ml culture medium.
2.5 mCi/mmol) for a period of 24 h before fixation. The proportion of cells that had initiated DNA synthesis was determined bv means of autoradioeranhv [2l]. The percentage of *[“H]thymidine-labellei cells was determined by counting at least 1000 cells/slide in the light microscope.
Statistics The Student’s r-test was used to analyse the statistical significance of the differences between the variables obtained in this study. The level of significance was set at P=O.O>.
RESULTS Fig. 1 shows the proportion of cells that initiated DNA synthesis after brief alkaline treatment as a function of the serum concentration (the serum was obtained from two different batches) in the culture medium. An almost complete stimulatory response (-80% labelled cells) could be obtained when the serum concentration was 0.5% or higher (for both batches). However, when the serum concentration was reduced below 0.5 5%the stimulatory response
117
in alkaline-treated cells decreased with decreasing serum concentration. If the concentration of serum was lowered to O.OOOl%, the percentage of labelled cells was below 40% (for both batches). This, however, is still a significantly higher labelling index than that for the corresponding quiescent cells. In the whole interval 0.0001-l c/r serum (for both batches) the alkaline treatment exerted a significant stimulatory effect (P
118
W. Engstriim
Table 1. The qffrct of medium composition on DNA synthesis .fbllort,ing rrlktrline stimulation of quiescent, serum-starved 3T3 cells Quiescent 3T3 cells were exposed to alkaline DMEM (pH 9.5) for IO min and thereafter exposed to media at pH 7.3 with differing composition for 24 h. These media were supplemented either with 0.0001 % or with 0.5% dialysed calf serum. Cells continuously exposed to DMEM at pH 7.3 with different serum concentrations were used as controls. The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure for 24 h to 0.5 &i [:‘H]thymidine/ml medium x , present: -. absent Medium
content
Energy sources
Vitamins
X X X X -
X X X
% Labelled Essential amino acids
0.0001 c/r serum
cells (5% inhibitory effect)
0.5% serum
(c/ inhibitory effect)
77k8.9 64+4.2 67+7.2 33+2.5 37+4. I 40+3.4 63k2.1 39+7. I
(0) (17) (13) (57) (52) (4X) (18) (49)
BSS X X X X X X X X
X X X -
(control)
X -
X -
26t4.6 25f5.1 23k2.6 14k4.1 15k4.9 15t2.5 23k2.0 12+2. I
(0) (4) (12)
(46) (42) (42) (12) (54)
Controls 0.0001% serum+complete DMEM 0. I Cr, serum+complete DMEM 0.5% serum+complete DMEM IO% serum+complete DMEM
treatment. A medium which contained only BSS and essential amino acids and was supplemented with a small amount of dialysed serum, was found to promote a stimulatory response to the same extent as did complete DMEM. It therefore became of interest to elucidate whether withdrawal of any single amino acid or ion resulted in inhibition of the stimulatory response to alkaline treatment. The effect of withdrawal of amino acids from the medium-one at a time-on DNA synthesis in alkaline-treated cells is shown in table 2. Exclusion of glutamine from the medium resulted in a marked and significant (P~0.05) inhibition of the stimulatory response (46-57 % inhibitory effect). Any one of the other amino acids could be excluded without causing any inhibition of the stimulatory response to alkaline treatment. Exp Cell Res 135 (1981)
2+ I .4 6k2.3 IO&S. I 99kO.6
Hence, the withdrawal of glutamine accounted for the entire inhibitory effect that was seen after deprivation of all amino acids from the medium. The impact of glutamine on DNA synthesis in alkaline-treated cells is shown in table 3. It was found that a medium containing only BSS and glutamine-stimulated, alkaline-treated cells initiated DNA synthesis to nearly the same extent (O-23% inhibitory effect) as did complete DMEM. Table 4 illustrates the effect on DNA synthesis of excluding ions from the medium in alkaline-treated cells. When calcium was withdrawn alone or together with magnesium, the cells failed to respond to alkaline treatment. The same result was observed when phosphate was eliminated. In these cases the cells appeared to be in a viable state, judging by microscopic ex-
Quiescent 3T3 cells were exposed to alkaline DMEM (pH 9.5) for IO min and thereafter exposed to a DMEM (pH 7.3) for 24 h from which one amino acid was withdrawn, whereas all other amino acids were held in normal concentration. These media were supplemented with 0.0001 % or 0.5% dialysed calf serum. Cells continuously exposed to DMEM at pH 7.3 with different serum concentrations were used as controls. The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure for 24 h to 0.5 #Zi [“H]thymidine/ml medium. The figures represent means 21 S.D. of four different experiments. The levels within parentheses represent ‘ii inhibitory effect as compared with the controls (30 or 779) Eliminated
amino
acid
% Labelled Addition of 0.000 I dialysed serum
Control Amino acids
0.0001 0. I % 0.5% IO%
None (complete Arginine Cysteine Glutamine Histidine lsoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Tyrosine Valine Glycine
% serum+DMEM serum+ DMEM serum+DMEM serum+DMEM
DMEM)
3Ok4.3 3Ok6.2 29+7.2 14k4.3 2hk7.2 2X&X. I 28k7.7 27-t6. I 26+6.7 3Ok5.3 29k9.4 26kX.h 26L6.5
25k5.6 28k6.2
(% inhibitory effect)
(0) (0) (3) (53) (13) (7) (7) (IO) (13) (0) (3) (131 (13) (17) (7)
cells Addition of0.5% dialysed 77+6.3 7Ok4.2 6726.0 29k5.4 72+ IO.8 70+ 12.3 68i-9.7 7Ok9.8 7Ok6.5 66+5.9 67k8.3 66i: 12.2 7Ok8.6 7226.3 702 IO.5
serum
(% inhibitory effect) (0) (9) (13) (62) (6) (9) (12) (9) (9) (4) (13) (14) (9)
(6) (9)
221.3 61-2.7 lOk3.9 99-to.7
amination. Withdrawal of magnesium (alone), sodium, potassium, chloride or bicarbonate ions from the medium resulted in cellular detachment and cell death. We then studied how long after the alkaline treatment the cells remained competent to initiate their proliferogenic programme, ultimately leading to DNA synthesis and cell division. This was done by treating the cells in alkaline medium for 10 min and then exposing them to a medium from which one constituent had been excluded. The lacking compound was added to the medium at various times after the alkaline treatment
and the % [“H]TdR-labelled cells were determined after 24 h. Fig. 2 shows the competence of alkalinetreated 3T3 cells to respond to readdition of 0.5% serum, glutamine or phosphate ions. after increasing periods in media deficient of either serum, glutamine, or phosphate. It was found that the cells remained competent to synthesize DNA (-75%) if serum or glutamine was added within 6 h after the alkaline treatment. When serum or glutamine was subsequently added, the proportion of cells that entered S phase gradually decreased with time. When serum or gluta-
120
W. Engstriim
Table 3. The effect of’ medium lotion
of quiescent,
serum-stowed
composition 3T3 cells
on DNA
synthrsis
fbllon~ing
olktrlinr
stimrr-
Quiescent 3T3 cells were exposed to alkaline DMEM (pH 9.5) for IO min and thereafter exposed to media at pH 7.3 with different composition for 24 h. These media were either supplemented with 0.0001% or with 0.5 % dialysed calf serum. Cells continuously exposed to DMEM at pH 7.3 with different serum concentrations were used as controls. The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure for 24 h to 0.S &i [“H]thymidine/ml medium X1 Present: ~, absent Medium content c/ Labelled
BSS x X X
*
cells
Glutamine
Other essential amino acids”
Energy sources
Vitamins
0.0001% serum
(% inhibitory effect)
0,s c/r serum
(% inhibitory effect)
x
X
X
X
X
x
26+4.6 26t4.0 1412.1 1222. I
(0) (0) (46) (S4)
77+x.9 59k5.5 3329.5 39?7. I
(0) (23) (57) (49)
X
-
-
X
-
-
-
~‘ot2trol.\ 0.0001 (Z serum-ccomplete DMEM 0.1 % serum+complete DMEM 0.5 % serum+complete DMEM 10%’ serum+complete DMEM ’ Arginine, cysteine, histidine. tryptophan and valine.
‘kl.3 6+2.3 iot5.1 99kO.6 isoleucine,
leucine.
lysine,
mine was added 12 h after alkaline treatment, only a small proportion of cells (-25 5%)responded to addition of the lacking compound. Alkaline-treated cells were able to initiate DNA synthesis C-70%), if phosphate was added immediately after the alkaline treatment. When phosphate was added 3 (or more) h after alkaline treatment, the cells totally failed to respond to the alkaline stimulation. Next we determined for how long after the alkaline treatment the cells became ultimately committed to DNA synthesis, (i.e. how long serum, glutamine or PO:- ions had to be present for irreversible commitment), by removing each compound at various times following alkaline treatment. The commitment to DNA synthesis of alkaline-treated cells after withdrawal of serum, glutamine or phosphate from the cul-
methionine,
3 Hours
phenylalanine.
6
9
in deficient
12
threonine.
tyrosine.
15
media
F;,q, 2. The competence of alkaline-treated 3T3 cells to respond to readdition of glutamine, serum or phosphate, after increasing time in media deficient of either glutamine. serum or phosphate. Quiescent serumstarved 3T3 cells were exposed at time 0 for IO min to alkaline DMEM, which was thereafter replaced by a 0, serum-deficient; n , glutamine-deficient; or A, phosphate-deficient medium at normal pH (7.3). The glutamine- and phosphate-deficient media were supplemented with 0.5 % dialysed serum. The deficient component was thereafter added to the cultures (O-0, 0.5% serum; W--m, 4 mM glutamine, or A-A. 0.9 mM phosphate at various times after onset of alkaline stimulation, as indicated, and was present throughout the experiment. All cell cultures
Table 4. The qffkct of’ depri\wtiotl latiotl
of ions on DNA c!f’yr{ie.sc~c~nt. .sCI.IItIl-.~t~t~l’C(i 3T3 crlls
s~~tlthesis, ,fdlon~ing
uIXtrlhc~ stittlu-
Quiescent 3T3 cells were exposed to alkaline DMEM (pH 9.5) for IO min and thereafter washed briefly with 0.5 mM EDTA (2x5 set). The cells were then exposed to DMEM (pH 7.3) for 24 h from which one ion at a time was excluded. These media were supplemented with 0.0001 % or 0.5% dialysed calf serum. Cells continuously exposed to DMEM at pH 7.3 with different serum concentrations were used as controls. The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure for 24 h to 0.5 FCi [.‘H]thymidine/ml medium. The figures represent means ? I S.D. of four different experiments. The levels within parentheses represent % inhibitory effect as compared with the controls (30 or 77%) X. Cell death Eliminated
ion
“r Labelled Addition of 0.0001 % dialysed serum
Control
None
Ion%
Ca“ ions MgZ* ions Ca” and Mg” Na’ ions KU ions CT ions PO;- ions
(complete
DMEM)
ions
3oi4.3 6i2. I x 2i0.6 x x x 2kl.3
cells Addition of 0.5% dialysed
(V inhibitory effect) (0) (80) (93) 193)
serum
(“4 inhibitory effect)
77k6.3
(0)
II 1-4.4 x 6-cj.3 x x x I I k6.3
X6 92 -
corrrrol.\ 0.0001 V serum+DMEM 0. I I% serum+ DMEM 0.5% serum+DMEM 10% serum+DMEM
2kl.3 622.3 1ot3.4, 99+0.7
ture medium is shown in fig. 3. The cells failed to respond to alkaline stimulation when serum or glutamine was withdrawn within 9 h after onset of stimulation. When serum or glutamine were withdrawn more than 12 h after alkaline stimulation, the cells had been irreversibly committed to DNA synthesis. When phosphate ions were withdrawn within 12 h after alkaline treatment, the cells failed to respond to the alkaline stimulation, whereas when if phosphate was withdrawn after 18 h, the cells had become irreversibly committed to DNA synthesis. were fixed 24 h after onset of alkaline stimulation. The proportion of cells that had initiated DNA synthesis during that 24 h experimental period was determined by autoradiography after continuous exposure to 0.5 &i [“Hlthymidinelml culture medium.
6 Hours
12
18
in deficient
24 media
Fix:. 3. Commitment to DNA synthesis of alkalinetreated cells after withdrawal of serum, glutamine or phosphate from the culture medium. Quiescent serumstarved 3T3 cells were exposed for IO min to alkaline (pH 9.5) DMEM, which was thereafter replaced by DMEM at normal pH (7.3) supplemented with 0.5 % serum, The curves represent time after alkaline treatment at which 0, serum; W, glutamine or A. phosphate was withdrawn from the medium. All cell cultures were fixed 24 h after onset of alkaline stimulation. The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure for 24 h after alkaline treatment by 0.S &i [:‘H]thymidine/ml culture medium.
122
W. Engstriim
DISCUSSION The major aim of this work was to investigate whether the stimulatory effects of mitogenic substances involve increased influx of any low molecular weight constituents of the culture medium. This was studied by investigating the early effects on the first cell cycle after mitogenic stimulation of quiescent cells in media with altered low molecular composition. Quiescent serum-starved 3T3 cells were stimulated to initiate DNA synthesis and undergo one cell division by brief alkaline treatment [19]. In this way it was possible to stimulate quiescent cells to initiate DNA synthesis in a virtually serum-free medium. Since the serum requirement for mitogenic stimulation was substantially reduced, only minor (and probably negligible) quantities of low molecular weight substances were supplied to the culture medium via their presence in serum. It therefore became possible to study the effects on proliferation of the withdrawal of specific low molecular weight components from an almost chemically defined medium. It was found that if all 13 essential amino acids were withdrawn from the medium, the stimulatory response to alkaline treatment was significantly inhibited. A similar inhibition was observed when only glutamine was excluded from the medium. However, the other 12 essential amino acids (except glutamine) could be excluded from the medium without inhibiting the stimulatory response to alkaline treatment on the initiation of DNA synthesis. Nor was the stimulatory effect by alkaline treatment inhibited by withdrawal of vitamins or energy sources (glucose and pyrurate) from the medium. It was even found that cells were almost as capable of responding to brief alkaline
treatment when they were incubated in a medium consisting of only BSS and glutamine, as when they were incubated in complete DMEM. These results indicate that of all low molecular weight components in the culture medium only glutamine can exert a growth regulatory influence. It is possible that increased influx of glutamine into the cell is an important step in the control of cell proliferation. Mitogenic substances that have been shown to act at the cell surface could thus possibly act via altered transmembrane transport of glutamine. It could, however, be firmly concluded from these results that mitogenic signals cannot be mediated intracellularly by increased influx of any of the other lowmolecular substances (i.e. the 12 amino acids except glutamine, energy sources or vitamins), since these factors could be excluded from the medium without inhibiting the stimulatory response to alkaline treatment. This conclusion is further supported by the finding that alkaline-treated cells also initiate DNA synthesis in a medium containing only BSS and glutamine to the same degree as in complete DMEM. The conclusions are in line with earlier findings that glutamine is required during the first cell cycle after stimulation of quiescent cells by 10% serum [22]. When glutamine was withdrawn from the medium, the stimulatory response was about half of that seen in cell stimulated by 10%~serum and a complete DMEM [22]. The removal of glutamine from medium (containing 10% dialysed serum) supporting exponentially growing 3T3 cells resulted in inhibition of growth, which was observable as early as 24 h after medium change [22]. This indicates that glutamine depletion inhibits cell proliferation in the first cell cycle after medium change. The suggested role for glutamine as a critical growth signal was fur-
ther emphasized by the finding that quiescent serum-starved cells could be stimulated to initiate DNA synthesis by exposure to a serum-depleted medium containing a relative glutamine excess [22]. Since it was found that only glutamine and BSS were required for alkaline-treated cells to initiate DNA synthesis it became of interest to analyse whether the removal of any of the ionic components in BSS would inhibit the stimulatory response to alkaline treatment. Two principally different results could be expected after withdrawal of different ions from the medium. Either the cells would remain viable and specifically inhibited in a state of quiescence, or the withdrawal of ions would result in non-specific toxic effects. It was found that when calcium or phosphate ions were excluded from the medium, the cells failed to respond to alkaline stimulation. However, as judged by microscopic examination the cells appeared to be viable. The [‘lH]TdR labelling percentages were similar to those observed when cells were starved to quiescence in low serum concentration. Since exclusion of calcium or phosphate ions from the medium inhibited mitogenic stimulation of quiescent cells without exerting any observable toxic effects, it is possible that these ions are basically involved in the growth control. A mitogenic signal could then possibly be mediated intracellularly via increased influx of calcium or phosphate ions into the cell. The calcium requirement for a stimulatory response of quiescent cells to alkaline treatment was similar to that observed in quiescent cells stimulated by 10% serum. In both stimulatory situations the cells required at least 1O-z M calcium in the external medium to initiate DNA synthesis [23, 241. Calcium ions are also required for
exponential proliferation of 3T3 cells [25]. If the calcium concentration in the medium supporting growing cells is decreased below IO-” M the cells become growth-inhibited. Phosphate ions have also been suggested to play an important role in the control of cell proliferation. Serum stimulation of quiescent 3T3 cells is followed by rapid increase in cellular uptake of phosphate ions [IS]. Furthermore, it was shown that exclusion of phosphate ions from media supporting growing 3T3 cells leads to growth inhibition of the cells [26]. The cells became quiescent in GO/Cl. The elimination of other ions in BSS (Na+, K+, Mg’+, Cl or HCO,?) from the medium resulted in cellular detachment and cell death. The withdrawal of these ions most probably caused unspecific toxic effects, but it cannot be excluded at this stage that they might be involved in the growth control system. It is probable, however, that Na’ and K’ ions are not mediating growth signals intracellularly, since the intracellular concentration of these ions can be varied 5-fold without any observable inhibitory effects on DNA synthesis [15]. These ions seem to be required for other purposes, e.g. cell survival. Magnesium has, however, been suggested to be a central mediator of growth stimulation [15, 251. In analogy with the reports by Rubin & Sanui [l5], we found that when magnesium ions alone were excluded from the medium, the cells detached and died. However, when calcium and magnesium together were excluded from the medium, the cells remained quiescent, but viable. These results indicate that there is an interplay between calcium and magnesium ions within the cell. A normal concentration of calcium ions is toxic to the cells when magnesium is excluded. whereas when both ions are excluded the cells re-
124
W. Engstriim
main viable. Hence it is even possible that calcium regulates cell proliferation by altering the intracellular Mg’+ ion concentration resulting from their competition for divalent ion-binding sites, as has been previously suggested [ 1S]. It was found that a small amount of serum (0.5%) was required for a full stimulatory response to alkaline treatment, but a significantly increased portion of the alkalinetreated cells underwent DNA synthesis in an almost serum-free (0.0001%) medium. The serum concentration did not influence the inhibitory effect of the removal of low molecular compounds from the medium on DNA synthesis in alkaline-treated cells. When either of the two serum concentrations (0.0001 or 0.5%) was used, the withdrawal of glutamine, calcium, or phosphate led to significant inhibition of the stimulatory response to alkaline treatment. The inhibitory effects of the withdrawal of glutamine, calcium or phosphate from the medium were similar in the two serum concentrations tested. It seems thus as if an altered serum concentration in the interval 0.0001-0.5 % neither enhances nor counteracts the inhibitory effects on DNA synthesis by glutamine or ionic depletion. The requirements for serum. glutamine, calcium and phosphate were then analysed with respect to cellular competence and commitment to DNA synthesis [27]. The term competence refers to initial changes in quiescent cells, preparing them to respond to critical growth signals. If these signals do not follow after competence formation. the cells do not enter S phase. The cells remain in this state of competence for only a limited period of time. If the required growth signal is not added to the cell cultures within this time interval, the cells fail to initiate DNA synthesis and return to the state of quiescence. However, if the
required signal(s) is (are) added to competent cells, it (they) must be present for a certain time interval to induce ultimate commitment to DNA synthesis. When the cells have been ultimately committed to DNA synthesis, the critical substance(s) can be withdrawn without affecting the cellular entrance into S phase. This point of ultimate commitment is usually believed to be located a few hours prior to S phase, i.e. in late Gl [27. 281. It was found that phosphate ions were required immediately after alkaline treatment, whereas calcium [24], glutamine and 0.5% serum were not required until 6 h after onset of alkaline stimulation. Thus it seems as if the alkaline treatment renders the cells competent and that this competence formation is dependent on the presence of phosphate ions. Small amounts of serum (OS%), calcium [24] and glutamine are not required for competence formation. but seem necessary to commit the cells irreversibly to DNA synthesis. It appears as if serum contains two sets of growth factors. One set is required for competence formation during serum stimulation, since a high concentration of serum (10%) has to be present for 6-X h after addition. Either the concentration of this type of growth factor in serum is very low. or the cells need large quantities. since a high concentration of serum is required for competence formation during serum stimulation. Another set of growth factors ib needed for ultimate commitment to DNA synthesis in alkaline-treated competent cells. Either the concentration of this type of factor is very high in serum or the cells only require small quantities of these factors for commitment to DNA synthesis. since only a small amount (0.5%) is required for a maximum response to alkaline stimulation. These results and conclusions
are in line with other reports. Pledger et al. found that serum contains two sets of growth factors-one set is a heat-stable ( 100°C) platelet-derived growth factor (PDGF) that is released into serum during the clotting process [37]. This set is responsible for competence formation. A second set of components found in defibrinogenated platelet-poor plasma is subsequently required for ultimate commitment to DNA synthesis. REFERENCES I. Halley. 2. 3. 4. 5. 6. 7. x. 9.
R W. Control of cell proliferation in animal cells (ed B Clarkson 6t R Baserga) p. 13. Cold Spring Harbor lab ( 1974). Cohen, S & Taylor. J M, Recent prog horm res 30. part I (1974) S33. Gospodarowicz. D, J biol them 250 (1975) 25 15. Gosoodarowicz. D. Weseman. J & Moran. J. Naturk268 (1977) 188. Vaheri. A. Ruoslahti. E & Hovi. T. Control ofcell proliferation in animal cells (ed B Clarkson & R Baserrral D. 305. Cold &ring Harbor lab (1974). Van %‘yk, J J, Underwood. L E. Hintz. R I.. Glemmons. D R. Voina, S &Weaver. R P. Recent prog horm res 30 (1974) 259. Burger, M M. Nature 227 (1970) 170. Sefton, B M &. Rubin. H, Nature 227 (1970) 843. Rubin. H & Sanui. H. Proc natl acad sci US 74 (1977) 5026.
Printed
in Sueden
IO. Andersson. R G G & Norrby, K. Virchows arch h cell pathol 23 (1977) 185. II. Rubin. H, J cell physiol 82 (1973) 231. 12. Nicolson, G L, Int rev cytol 39 (1974) 8Y. A. Personal communication. 13. Zetterberg. K D & Burger. M M. Exp cell res 80 14. Noonan. (1973) 405. and cell culIS. Rubin, A H B Sanui. H. Hormones ture. pp. 741-7.50. Cold Spring Harbor conf cell prolif (19791. M J & Rubin. H. J cell physiol 77 (1971) 16. Weber. 157. 17. Foster, D 0 & Pardee. A B. J biol them 244 (I9691 2675. D D & Pardee. A B. Proc natl acad 18. Cunningham, sci US 69 (19691 104Y. A & Engstr6m. W. Proc natl acad xi 19. Zetterberg. US (1981). In press. R. Proc natl acad sci US 46 20. Vogt. M & Dulbecco. (19601 365. A & Killander. 0, Exp cell re\ 40 21. Zetterberg. (1965) 1. A & Engstriim, W, J cell physioi 22. Zetterberg, (1981). In press. 23. Boynton. A L. Whitfields. J F & Isaacs. R J. In vitro I2 (1976) 120. W, Cell biol int rep 5 (1981) SOY. 24. Engstriim. M B Sanui. H. Proc nati 2s. Rubin. A H. Terasaki, acad sci US 76 (1979) 3917. R W & Kiernan. J. Proc natl acad sci IJS 26. Halley, 71 (1974) 2943. 27. Pledger. W J, Stiles. C D. Antoniades, H N & Scher, C D. Proc natl acad sci US 75 (19781 2x3’). 28. Pardee, A. Proc natl acad xi US 71 (1974, 1286.
Received Accepted
March 31. IYXI April 6, IYXi
Experimentd Cell Research 135 (1981) 115-125
REQUIREMENTS OF QUIESCENT AFTER
FOR COMPETENCE 3T3-CELLS
GROWTH
AND COMMITMENT
TO INITIATE
STIMULATION
DNA
SYNTHESIS
IN LOW SERUM
CONCENTRATION
SUMMARY Quiescent serum-starved 3T3 cells in sparse cultures can be stimulated to initiate DNA synthesis and undergo one cell division in low serum concentration after brief exposure to alkaline pH [ 191. This method of mitogenic stimulation was used to investigate the requirements of low molecular weight components during the first cell cycle after onset of stimulation. It was shown that each of the low molecular constituents in Dulbecco’s Modified Eagle Medium (DMEM) could be excluded (one at a time) without influencing the stimulatory response to brief alkaline treatment, with the exception of glutamine, calcium and phosphate ions. The temporal requirements of these factors were studied from onset of sttmulation to initiation of DNA synthesis. It was found that phosphate ions were required immediately after the alkaline treatment. but glutamine was not until 6 h after onset of alkaline stimulation.
Several substances exert a growth-stimuand insulin [IS] that had been prepared so latory influence on cells in vitro, e.g. serum that they were unable to enter the cell in[I], epidermal growth factor (EGF) [2], terior (by attachment of plastic beads to the fibroblast growth factor (FGF) [3], myo- trypsin and insulin molecules) were as efblast factor [4], insulin [5], somatomedins ficient in promoting cell proliferation as [6], proteolytic enzymes in low concentra- were free insulin or trypsin. These findings tion [7, 81, calcium pyrophosphate [9], the suggest that the site of their activity was at calcium ionophone A23187 [lo], ZrP, Ca2+, the cell surface. Also calcium pyrophosand Mg*+ ions [ 1I], or lectins [ 121. phate complexes, which induce proliferaThese substances have been proposed to tion in quiescent cells, have been shown to exert their primary action at the cell sur- precipitate at the cell surface and are not face, from which the mitogenic message is taken up into the cell [9]. mediated intracellularly via some second The mitogenic message can be mediated messenger(s) [ 131. This proposal was sup- from the cell surface into the cell via at ported by experimental evidence that three least two principally different mechanisms. of the substances (trypsin, insulin and cal- First, the mitogenic substance can alter the cium pyrophosphate) that promote cell pro- transmembrane permeability properties for liferation, act at the cell surface but are low molecular weight substances. This not taken up into the cell. Trypsin [14] could result in increased influx or efflux of
116
W. Engstriim
some growth-regulatory substance(s), e.g. amino acids, glucose, or ions. Second, the mitogenic substance(s) could activate (or inhibit) some growth-regulatory enzyme(s) that are associated with the cell membrane. Some support for the first suggestion was given by the findings that mitogenic stimulation of quiescent 3T3 cells resulted in a rapid cellular uptake of low molecular substances, e.g. deoxyglucose [S], uridine [16]. non-metabolizable amino acids [ 171 and phosphate ions [ 181. This study was intended to investigate whether or not the mitogenic message is mediated into the cell via increased cellular uptake of some low molecular weight constituent(s). Swiss 3T3 cells in sparse cultures, starved to quiescence in media with low serum concentration (0.5 c/c), were used for this purpose. These cells are normally dependent on the addition of a high concentration serum (5-10s) to the medium to leave the quiescent state and enter the cell cycle. It was recently shown, however, that quiescent serum-starved 3T3 cells can be stimulated to initiate DNA synthesis and undergo one cell division in medium with low serum concentration (0. l0.5%) after a 5-10 min long treatment in alkaline medium (pH 8.5-10) [ 191. Furthermore, by using this method it was possible to stimulate quiescent 3T3 cells to initiate DNA synthesis in a medium with very low serum concentration (0.0001%). As the serum requirement for mitogenic stimulation was substantially reduced, only minor amounts of low molecular weight substances could be supplied to the culture medium via the presence of serum. Hence it became possible to study the effects on proliferation of the withdrawal of specific low molecular weight components from an almost chemically defined medium. By applying this approach it could be Erp Cell Rrs 135 f/981)
.
shown that each of the low molecular constituents in the growth medium could be excluded (one at a time) without influencing the stimulatory response to brief alkaline treatment, with the exception of glutamine, calcium, or phosphate ions. MATERIAL
AND METHODS
Mouse Swiss 3T3 embryo fibroblast\ (Flow Laboratories Inc.) were maintained in monolayer cultures in Nunc’s plastic tissue culture bottles. The stock cultures were grown in a humified 6% CO,-air mixture in Dulbecco’s modified Eagle medium supplemented with IO% fetal calf serum (FCS) [20], SO units of penicillin per ml and SO pg of streptomycin per ml. The cells were removed from the dish for transfer by treatment with 0.25% trypsin in Tris-buffered saline containing 0.5 mM EDTA. The line wax maintained by seeding 3000 cells/cmZ culture bottle area and transferring them every 3rd day. The cells were never allowed to reach confluency. Cells used for experimental purpose were grown in IO ml plastic tissue culture bottles or in Petri dishes, which contained a glass coverslip on the bottom. Ouiescent cells were obtained bv brieflv washing (2x‘lO set) proliferating cells in Eaile’s balanced sai solution (BSS) and thereafter incubating them in medium supplemented with 0.5% serum for 24 h.
Quiescent. serum-starved cells were treated with an alkaline (pH 9.5) medium for IO min [19]. The cells were then exposed to media with low serum concentration (containing 0.0001 or 0.57r dialysed serum) with different concentrations of low-molecular constituents (cf vdbks l-4 and figure captions) at normal pH (7.3) during a 24-h assay period. These media were produced in our laboratory by adding lowmolecular compounds (ions. amino acids. energy sources. vitamins) either to distilled water or to Earle’s balanced salt solution. The final concentration of each added compound corresponded to that in Dulbecco’s modified Eagle’s medium r201. During the first part of this study. these media, witi different compositions. were present in the cell cultures during the whole 24 h period. In the second part of this investigation. when the temporal requirements for serum, glutamine and phosphate ions were investigated, media deficient with respect to these compounds were present during limited intervals of the assay period. A complete DMEM was present during the other parts of the 24 h assay period.
Autorudiography The (0.5
cell cultures were labelled with [“Hlthymidine pCi/ml culture medium: New England Nuclear
Reyrri,-rmt~,lt.v,fi)r compt~tt~nw trnd c~ommitment to initirrte DNA synthesis
00001
0.001
0.01
0.1
1
10
FY,q. I. Stimulation of DNA synthesis by alkaline treatment in presence of different serum concentrations in culture medium. Ouiescent serum-starved 3T3 cells were exposed for lb min to alkaline (pH 9.5) medium, which was thereafter reolaced bv DMEM with different serum concentrations for 54 h at normal pH (7.3). Two different serum hatches (0. I; n , 2) were tested. Cells continuously exposed to DMEM at normal pH (7.3) with different serum concentration were used as controls (0. I; q . 2). The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure during the 24 h assay period to 0.S @Ii [“H]thymidine/ml culture medium.
2.5 mCi/mmol) for a period of 24 h before fixation. The proportion of cells that had initiated DNA synthesis was determined bv means of autoradioeranhv [2l]. The percentage of *[“H]thymidine-labellei cells was determined by counting at least 1000 cells/slide in the light microscope.
Statistics The Student’s r-test was used to analyse the statistical significance of the differences between the variables obtained in this study. The level of significance was set at P=O.O>.
RESULTS Fig. 1 shows the proportion of cells that initiated DNA synthesis after brief alkaline treatment as a function of the serum concentration (the serum was obtained from two different batches) in the culture medium. An almost complete stimulatory response (-80% labelled cells) could be obtained when the serum concentration was 0.5% or higher (for both batches). However, when the serum concentration was reduced below 0.5 5%the stimulatory response
117
in alkaline-treated cells decreased with decreasing serum concentration. If the concentration of serum was lowered to O.OOOl%, the percentage of labelled cells was below 40% (for both batches). This, however, is still a significantly higher labelling index than that for the corresponding quiescent cells. In the whole interval 0.0001-l c/r serum (for both batches) the alkaline treatment exerted a significant stimulatory effect (P
118
W. Engstriim
Table 1. The qffrct of medium composition on DNA synthesis .fbllort,ing rrlktrline stimulation of quiescent, serum-starved 3T3 cells Quiescent 3T3 cells were exposed to alkaline DMEM (pH 9.5) for IO min and thereafter exposed to media at pH 7.3 with differing composition for 24 h. These media were supplemented either with 0.0001 % or with 0.5% dialysed calf serum. Cells continuously exposed to DMEM at pH 7.3 with different serum concentrations were used as controls. The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure for 24 h to 0.5 &i [:‘H]thymidine/ml medium x , present: -. absent Medium
content
Energy sources
Vitamins
X X X X -
X X X
% Labelled Essential amino acids
0.0001 c/r serum
cells (5% inhibitory effect)
0.5% serum
(c/ inhibitory effect)
77k8.9 64+4.2 67+7.2 33+2.5 37+4. I 40+3.4 63k2.1 39+7. I
(0) (17) (13) (57) (52) (4X) (18) (49)
BSS X X X X X X X X
X X X -
(control)
X -
X -
26t4.6 25f5.1 23k2.6 14k4.1 15k4.9 15t2.5 23k2.0 12+2. I
(0) (4) (12)
(46) (42) (42) (12) (54)
Controls 0.0001% serum+complete DMEM 0. I Cr, serum+complete DMEM 0.5% serum+complete DMEM IO% serum+complete DMEM
treatment. A medium which contained only BSS and essential amino acids and was supplemented with a small amount of dialysed serum, was found to promote a stimulatory response to the same extent as did complete DMEM. It therefore became of interest to elucidate whether withdrawal of any single amino acid or ion resulted in inhibition of the stimulatory response to alkaline treatment. The effect of withdrawal of amino acids from the medium-one at a time-on DNA synthesis in alkaline-treated cells is shown in table 2. Exclusion of glutamine from the medium resulted in a marked and significant (P~0.05) inhibition of the stimulatory response (46-57 % inhibitory effect). Any one of the other amino acids could be excluded without causing any inhibition of the stimulatory response to alkaline treatment. Exp Cell Res 135 (1981)
2+ I .4 6k2.3 IO&S. I 99kO.6
Hence, the withdrawal of glutamine accounted for the entire inhibitory effect that was seen after deprivation of all amino acids from the medium. The impact of glutamine on DNA synthesis in alkaline-treated cells is shown in table 3. It was found that a medium containing only BSS and glutamine-stimulated, alkaline-treated cells initiated DNA synthesis to nearly the same extent (O-23% inhibitory effect) as did complete DMEM. Table 4 illustrates the effect on DNA synthesis of excluding ions from the medium in alkaline-treated cells. When calcium was withdrawn alone or together with magnesium, the cells failed to respond to alkaline treatment. The same result was observed when phosphate was eliminated. In these cases the cells appeared to be in a viable state, judging by microscopic ex-
Quiescent 3T3 cells were exposed to alkaline DMEM (pH 9.5) for IO min and thereafter exposed to a DMEM (pH 7.3) for 24 h from which one amino acid was withdrawn, whereas all other amino acids were held in normal concentration. These media were supplemented with 0.0001 % or 0.5% dialysed calf serum. Cells continuously exposed to DMEM at pH 7.3 with different serum concentrations were used as controls. The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure for 24 h to 0.5 #Zi [“H]thymidine/ml medium. The figures represent means 21 S.D. of four different experiments. The levels within parentheses represent ‘ii inhibitory effect as compared with the controls (30 or 779) Eliminated
amino
acid
% Labelled Addition of 0.000 I dialysed serum
Control Amino acids
0.0001 0. I % 0.5% IO%
None (complete Arginine Cysteine Glutamine Histidine lsoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Tyrosine Valine Glycine
% serum+DMEM serum+ DMEM serum+DMEM serum+DMEM
DMEM)
3Ok4.3 3Ok6.2 29+7.2 14k4.3 2hk7.2 2X&X. I 28k7.7 27-t6. I 26+6.7 3Ok5.3 29k9.4 26kX.h 26L6.5
25k5.6 28k6.2
(% inhibitory effect)
(0) (0) (3) (53) (13) (7) (7) (IO) (13) (0) (3) (131 (13) (17) (7)
cells Addition of0.5% dialysed 77+6.3 7Ok4.2 6726.0 29k5.4 72+ IO.8 70+ 12.3 68i-9.7 7Ok9.8 7Ok6.5 66+5.9 67k8.3 66i: 12.2 7Ok8.6 7226.3 702 IO.5
serum
(% inhibitory effect) (0) (9) (13) (62) (6) (9) (12) (9) (9) (4) (13) (14) (9)
(6) (9)
221.3 61-2.7 lOk3.9 99-to.7
amination. Withdrawal of magnesium (alone), sodium, potassium, chloride or bicarbonate ions from the medium resulted in cellular detachment and cell death. We then studied how long after the alkaline treatment the cells remained competent to initiate their proliferogenic programme, ultimately leading to DNA synthesis and cell division. This was done by treating the cells in alkaline medium for 10 min and then exposing them to a medium from which one constituent had been excluded. The lacking compound was added to the medium at various times after the alkaline treatment
and the % [“H]TdR-labelled cells were determined after 24 h. Fig. 2 shows the competence of alkalinetreated 3T3 cells to respond to readdition of 0.5% serum, glutamine or phosphate ions. after increasing periods in media deficient of either serum, glutamine, or phosphate. It was found that the cells remained competent to synthesize DNA (-75%) if serum or glutamine was added within 6 h after the alkaline treatment. When serum or glutamine was subsequently added, the proportion of cells that entered S phase gradually decreased with time. When serum or gluta-
120
W. Engstriim
Table 3. The effect of’ medium lotion
of quiescent,
serum-stowed
composition 3T3 cells
on DNA
synthrsis
fbllon~ing
olktrlinr
stimrr-
Quiescent 3T3 cells were exposed to alkaline DMEM (pH 9.5) for IO min and thereafter exposed to media at pH 7.3 with different composition for 24 h. These media were either supplemented with 0.0001% or with 0.5 % dialysed calf serum. Cells continuously exposed to DMEM at pH 7.3 with different serum concentrations were used as controls. The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure for 24 h to 0.S &i [“H]thymidine/ml medium X1 Present: ~, absent Medium content c/ Labelled
BSS x X X
*
cells
Glutamine
Other essential amino acids”
Energy sources
Vitamins
0.0001% serum
(% inhibitory effect)
0,s c/r serum
(% inhibitory effect)
x
X
X
X
X
x
26+4.6 26t4.0 1412.1 1222. I
(0) (0) (46) (S4)
77+x.9 59k5.5 3329.5 39?7. I
(0) (23) (57) (49)
X
-
-
X
-
-
-
~‘ot2trol.\ 0.0001 (Z serum-ccomplete DMEM 0.1 % serum+complete DMEM 0.5 % serum+complete DMEM 10%’ serum+complete DMEM ’ Arginine, cysteine, histidine. tryptophan and valine.
‘kl.3 6+2.3 iot5.1 99kO.6 isoleucine,
leucine.
lysine,
mine was added 12 h after alkaline treatment, only a small proportion of cells (-25 5%)responded to addition of the lacking compound. Alkaline-treated cells were able to initiate DNA synthesis C-70%), if phosphate was added immediately after the alkaline treatment. When phosphate was added 3 (or more) h after alkaline treatment, the cells totally failed to respond to the alkaline stimulation. Next we determined for how long after the alkaline treatment the cells became ultimately committed to DNA synthesis, (i.e. how long serum, glutamine or PO:- ions had to be present for irreversible commitment), by removing each compound at various times following alkaline treatment. The commitment to DNA synthesis of alkaline-treated cells after withdrawal of serum, glutamine or phosphate from the cul-
methionine,
3 Hours
phenylalanine.
6
9
in deficient
12
threonine.
tyrosine.
15
media
F;,q, 2. The competence of alkaline-treated 3T3 cells to respond to readdition of glutamine, serum or phosphate, after increasing time in media deficient of either glutamine. serum or phosphate. Quiescent serumstarved 3T3 cells were exposed at time 0 for IO min to alkaline DMEM, which was thereafter replaced by a 0, serum-deficient; n , glutamine-deficient; or A, phosphate-deficient medium at normal pH (7.3). The glutamine- and phosphate-deficient media were supplemented with 0.5 % dialysed serum. The deficient component was thereafter added to the cultures (O-0, 0.5% serum; W--m, 4 mM glutamine, or A-A. 0.9 mM phosphate at various times after onset of alkaline stimulation, as indicated, and was present throughout the experiment. All cell cultures
Table 4. The qffkct of’ depri\wtiotl latiotl
of ions on DNA c!f’yr{ie.sc~c~nt. .sCI.IItIl-.~t~t~l’C(i 3T3 crlls
s~~tlthesis, ,fdlon~ing
uIXtrlhc~ stittlu-
Quiescent 3T3 cells were exposed to alkaline DMEM (pH 9.5) for IO min and thereafter washed briefly with 0.5 mM EDTA (2x5 set). The cells were then exposed to DMEM (pH 7.3) for 24 h from which one ion at a time was excluded. These media were supplemented with 0.0001 % or 0.5% dialysed calf serum. Cells continuously exposed to DMEM at pH 7.3 with different serum concentrations were used as controls. The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure for 24 h to 0.5 FCi [.‘H]thymidine/ml medium. The figures represent means ? I S.D. of four different experiments. The levels within parentheses represent % inhibitory effect as compared with the controls (30 or 77%) X. Cell death Eliminated
ion
“r Labelled Addition of 0.0001 % dialysed serum
Control
None
Ion%
Ca“ ions MgZ* ions Ca” and Mg” Na’ ions KU ions CT ions PO;- ions
(complete
DMEM)
ions
3oi4.3 6i2. I x 2i0.6 x x x 2kl.3
cells Addition of 0.5% dialysed
(V inhibitory effect) (0) (80) (93) 193)
serum
(“4 inhibitory effect)
77k6.3
(0)
II 1-4.4 x 6-cj.3 x x x I I k6.3
X6 92 -
corrrrol.\ 0.0001 V serum+DMEM 0. I I% serum+ DMEM 0.5% serum+DMEM 10% serum+DMEM
2kl.3 622.3 1ot3.4, 99+0.7
ture medium is shown in fig. 3. The cells failed to respond to alkaline stimulation when serum or glutamine was withdrawn within 9 h after onset of stimulation. When serum or glutamine were withdrawn more than 12 h after alkaline stimulation, the cells had been irreversibly committed to DNA synthesis. When phosphate ions were withdrawn within 12 h after alkaline treatment, the cells failed to respond to the alkaline stimulation, whereas when if phosphate was withdrawn after 18 h, the cells had become irreversibly committed to DNA synthesis. were fixed 24 h after onset of alkaline stimulation. The proportion of cells that had initiated DNA synthesis during that 24 h experimental period was determined by autoradiography after continuous exposure to 0.5 &i [“Hlthymidinelml culture medium.
6 Hours
12
18
in deficient
24 media
Fix:. 3. Commitment to DNA synthesis of alkalinetreated cells after withdrawal of serum, glutamine or phosphate from the culture medium. Quiescent serumstarved 3T3 cells were exposed for IO min to alkaline (pH 9.5) DMEM, which was thereafter replaced by DMEM at normal pH (7.3) supplemented with 0.5 % serum, The curves represent time after alkaline treatment at which 0, serum; W, glutamine or A. phosphate was withdrawn from the medium. All cell cultures were fixed 24 h after onset of alkaline stimulation. The proportion of cells synthesizing DNA was determined by autoradiography after continuous exposure for 24 h after alkaline treatment by 0.S &i [:‘H]thymidine/ml culture medium.
122
W. Engstriim
DISCUSSION The major aim of this work was to investigate whether the stimulatory effects of mitogenic substances involve increased influx of any low molecular weight constituents of the culture medium. This was studied by investigating the early effects on the first cell cycle after mitogenic stimulation of quiescent cells in media with altered low molecular composition. Quiescent serum-starved 3T3 cells were stimulated to initiate DNA synthesis and undergo one cell division by brief alkaline treatment [19]. In this way it was possible to stimulate quiescent cells to initiate DNA synthesis in a virtually serum-free medium. Since the serum requirement for mitogenic stimulation was substantially reduced, only minor (and probably negligible) quantities of low molecular weight substances were supplied to the culture medium via their presence in serum. It therefore became possible to study the effects on proliferation of the withdrawal of specific low molecular weight components from an almost chemically defined medium. It was found that if all 13 essential amino acids were withdrawn from the medium, the stimulatory response to alkaline treatment was significantly inhibited. A similar inhibition was observed when only glutamine was excluded from the medium. However, the other 12 essential amino acids (except glutamine) could be excluded from the medium without inhibiting the stimulatory response to alkaline treatment on the initiation of DNA synthesis. Nor was the stimulatory effect by alkaline treatment inhibited by withdrawal of vitamins or energy sources (glucose and pyrurate) from the medium. It was even found that cells were almost as capable of responding to brief alkaline
treatment when they were incubated in a medium consisting of only BSS and glutamine, as when they were incubated in complete DMEM. These results indicate that of all low molecular weight components in the culture medium only glutamine can exert a growth regulatory influence. It is possible that increased influx of glutamine into the cell is an important step in the control of cell proliferation. Mitogenic substances that have been shown to act at the cell surface could thus possibly act via altered transmembrane transport of glutamine. It could, however, be firmly concluded from these results that mitogenic signals cannot be mediated intracellularly by increased influx of any of the other lowmolecular substances (i.e. the 12 amino acids except glutamine, energy sources or vitamins), since these factors could be excluded from the medium without inhibiting the stimulatory response to alkaline treatment. This conclusion is further supported by the finding that alkaline-treated cells also initiate DNA synthesis in a medium containing only BSS and glutamine to the same degree as in complete DMEM. The conclusions are in line with earlier findings that glutamine is required during the first cell cycle after stimulation of quiescent cells by 10% serum [22]. When glutamine was withdrawn from the medium, the stimulatory response was about half of that seen in cell stimulated by 10%~serum and a complete DMEM [22]. The removal of glutamine from medium (containing 10% dialysed serum) supporting exponentially growing 3T3 cells resulted in inhibition of growth, which was observable as early as 24 h after medium change [22]. This indicates that glutamine depletion inhibits cell proliferation in the first cell cycle after medium change. The suggested role for glutamine as a critical growth signal was fur-
ther emphasized by the finding that quiescent serum-starved cells could be stimulated to initiate DNA synthesis by exposure to a serum-depleted medium containing a relative glutamine excess [22]. Since it was found that only glutamine and BSS were required for alkaline-treated cells to initiate DNA synthesis it became of interest to analyse whether the removal of any of the ionic components in BSS would inhibit the stimulatory response to alkaline treatment. Two principally different results could be expected after withdrawal of different ions from the medium. Either the cells would remain viable and specifically inhibited in a state of quiescence, or the withdrawal of ions would result in non-specific toxic effects. It was found that when calcium or phosphate ions were excluded from the medium, the cells failed to respond to alkaline stimulation. However, as judged by microscopic examination the cells appeared to be viable. The [‘lH]TdR labelling percentages were similar to those observed when cells were starved to quiescence in low serum concentration. Since exclusion of calcium or phosphate ions from the medium inhibited mitogenic stimulation of quiescent cells without exerting any observable toxic effects, it is possible that these ions are basically involved in the growth control. A mitogenic signal could then possibly be mediated intracellularly via increased influx of calcium or phosphate ions into the cell. The calcium requirement for a stimulatory response of quiescent cells to alkaline treatment was similar to that observed in quiescent cells stimulated by 10% serum. In both stimulatory situations the cells required at least 1O-z M calcium in the external medium to initiate DNA synthesis [23, 241. Calcium ions are also required for
exponential proliferation of 3T3 cells [25]. If the calcium concentration in the medium supporting growing cells is decreased below IO-” M the cells become growth-inhibited. Phosphate ions have also been suggested to play an important role in the control of cell proliferation. Serum stimulation of quiescent 3T3 cells is followed by rapid increase in cellular uptake of phosphate ions [IS]. Furthermore, it was shown that exclusion of phosphate ions from media supporting growing 3T3 cells leads to growth inhibition of the cells [26]. The cells became quiescent in GO/Cl. The elimination of other ions in BSS (Na+, K+, Mg’+, Cl or HCO,?) from the medium resulted in cellular detachment and cell death. The withdrawal of these ions most probably caused unspecific toxic effects, but it cannot be excluded at this stage that they might be involved in the growth control system. It is probable, however, that Na’ and K’ ions are not mediating growth signals intracellularly, since the intracellular concentration of these ions can be varied 5-fold without any observable inhibitory effects on DNA synthesis [15]. These ions seem to be required for other purposes, e.g. cell survival. Magnesium has, however, been suggested to be a central mediator of growth stimulation [15, 251. In analogy with the reports by Rubin & Sanui [l5], we found that when magnesium ions alone were excluded from the medium, the cells detached and died. However, when calcium and magnesium together were excluded from the medium, the cells remained quiescent, but viable. These results indicate that there is an interplay between calcium and magnesium ions within the cell. A normal concentration of calcium ions is toxic to the cells when magnesium is excluded. whereas when both ions are excluded the cells re-
124
W. Engstriim
main viable. Hence it is even possible that calcium regulates cell proliferation by altering the intracellular Mg’+ ion concentration resulting from their competition for divalent ion-binding sites, as has been previously suggested [ 1S]. It was found that a small amount of serum (0.5%) was required for a full stimulatory response to alkaline treatment, but a significantly increased portion of the alkalinetreated cells underwent DNA synthesis in an almost serum-free (0.0001%) medium. The serum concentration did not influence the inhibitory effect of the removal of low molecular compounds from the medium on DNA synthesis in alkaline-treated cells. When either of the two serum concentrations (0.0001 or 0.5%) was used, the withdrawal of glutamine, calcium, or phosphate led to significant inhibition of the stimulatory response to alkaline treatment. The inhibitory effects of the withdrawal of glutamine, calcium or phosphate from the medium were similar in the two serum concentrations tested. It seems thus as if an altered serum concentration in the interval 0.0001-0.5 % neither enhances nor counteracts the inhibitory effects on DNA synthesis by glutamine or ionic depletion. The requirements for serum. glutamine, calcium and phosphate were then analysed with respect to cellular competence and commitment to DNA synthesis [27]. The term competence refers to initial changes in quiescent cells, preparing them to respond to critical growth signals. If these signals do not follow after competence formation. the cells do not enter S phase. The cells remain in this state of competence for only a limited period of time. If the required growth signal is not added to the cell cultures within this time interval, the cells fail to initiate DNA synthesis and return to the state of quiescence. However, if the
required signal(s) is (are) added to competent cells, it (they) must be present for a certain time interval to induce ultimate commitment to DNA synthesis. When the cells have been ultimately committed to DNA synthesis, the critical substance(s) can be withdrawn without affecting the cellular entrance into S phase. This point of ultimate commitment is usually believed to be located a few hours prior to S phase, i.e. in late Gl [27. 281. It was found that phosphate ions were required immediately after alkaline treatment, whereas calcium [24], glutamine and 0.5% serum were not required until 6 h after onset of alkaline stimulation. Thus it seems as if the alkaline treatment renders the cells competent and that this competence formation is dependent on the presence of phosphate ions. Small amounts of serum (OS%), calcium [24] and glutamine are not required for competence formation. but seem necessary to commit the cells irreversibly to DNA synthesis. It appears as if serum contains two sets of growth factors. One set is required for competence formation during serum stimulation, since a high concentration of serum (10%) has to be present for 6-X h after addition. Either the concentration of this type of growth factor in serum is very low. or the cells need large quantities. since a high concentration of serum is required for competence formation during serum stimulation. Another set of growth factors ib needed for ultimate commitment to DNA synthesis in alkaline-treated competent cells. Either the concentration of this type of factor is very high in serum or the cells only require small quantities of these factors for commitment to DNA synthesis. since only a small amount (0.5%) is required for a maximum response to alkaline stimulation. These results and conclusions
are in line with other reports. Pledger et al. found that serum contains two sets of growth factors-one set is a heat-stable ( 100°C) platelet-derived growth factor (PDGF) that is released into serum during the clotting process [37]. This set is responsible for competence formation. A second set of components found in defibrinogenated platelet-poor plasma is subsequently required for ultimate commitment to DNA synthesis. REFERENCES I. Halley. 2. 3. 4. 5. 6. 7. x. 9.
R W. Control of cell proliferation in animal cells (ed B Clarkson 6t R Baserga) p. 13. Cold Spring Harbor lab ( 1974). Cohen, S & Taylor. J M, Recent prog horm res 30. part I (1974) S33. Gospodarowicz. D, J biol them 250 (1975) 25 15. Gosoodarowicz. D. Weseman. J & Moran. J. Naturk268 (1977) 188. Vaheri. A. Ruoslahti. E & Hovi. T. Control ofcell proliferation in animal cells (ed B Clarkson & R Baserrral D. 305. Cold &ring Harbor lab (1974). Van %‘yk, J J, Underwood. L E. Hintz. R I.. Glemmons. D R. Voina, S &Weaver. R P. Recent prog horm res 30 (1974) 259. Burger, M M. Nature 227 (1970) 170. Sefton, B M &. Rubin. H, Nature 227 (1970) 843. Rubin. H & Sanui. H. Proc natl acad sci US 74 (1977) 5026.
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IO. Andersson. R G G & Norrby, K. Virchows arch h cell pathol 23 (1977) 185. II. Rubin. H, J cell physiol 82 (1973) 231. 12. Nicolson, G L, Int rev cytol 39 (1974) 8Y. A. Personal communication. 13. Zetterberg. K D & Burger. M M. Exp cell res 80 14. Noonan. (1973) 405. and cell culIS. Rubin, A H B Sanui. H. Hormones ture. pp. 741-7.50. Cold Spring Harbor conf cell prolif (19791. M J & Rubin. H. J cell physiol 77 (1971) 16. Weber. 157. 17. Foster, D 0 & Pardee. A B. J biol them 244 (I9691 2675. D D & Pardee. A B. Proc natl acad 18. Cunningham, sci US 69 (19691 104Y. A & Engstr6m. W. Proc natl acad xi 19. Zetterberg. US (1981). In press. R. Proc natl acad sci US 46 20. Vogt. M & Dulbecco. (19601 365. A & Killander. 0, Exp cell re\ 40 21. Zetterberg. (1965) 1. A & Engstriim, W, J cell physioi 22. Zetterberg, (1981). In press. 23. Boynton. A L. Whitfields. J F & Isaacs. R J. In vitro I2 (1976) 120. W, Cell biol int rep 5 (1981) SOY. 24. Engstriim. M B Sanui. H. Proc nati 2s. Rubin. A H. Terasaki, acad sci US 76 (1979) 3917. R W & Kiernan. J. Proc natl acad sci IJS 26. Halley, 71 (1974) 2943. 27. Pledger. W J, Stiles. C D. Antoniades, H N & Scher, C D. Proc natl acad sci US 75 (19781 2x3’). 28. Pardee, A. Proc natl acad xi US 71 (1974, 1286.
Received Accepted
March 31. IYXI April 6, IYXi