Phosphate Uptake and Control of Fibroblast Growth GREGORY S. BARSH, DEBORAH B. GREENBERG AND DENNIS D. CUNNINGHAM Department of Medical Microbiology, College of Medicine, University ofCalifornia, Iruine, Iriine, California 9271 7

ABSTRACT Previous studies have shown that growth t o quiescence of fibroblast-like cells is accompanied by a large decrease in the rate of phosphate uptake. Since 3T3 cells can be arrested in the G , (or Go) phase of the cell cycle by lowering the concentration of phosphate in the medium, we examined the possibility that the decline in phosphate uptake observed during growth to quiescence might be a key event in the inhibition of DNA synthesis and cell division. The experimental approach consisted of controlling the rate of phosphate uptake by varying the phosphate concentration in the medium. Kinetic experiments showed that phosphate uptake in both growing and quiescent cells was partly accounted for by simple diffusion as well as carrier-mediated uptake. In fact, diffusion of phosphate into the growing cells was 2.5-fold greater than in the quiescent cells. When phosphate uptake was measured in 3T3 cells plated a t different initial densities, we found an inverse relationship between phosphate uptake and cell density, showing that phosphate uptake was correlated with growth rate and did not decline simply as a consequence of time in culture. Measurements of phosphate demonstrated that the lowered rate of phosphate uptake by quiescent cells was not due merely to a reduction of phosphate in the medium. To check the possibility that release of a previously described transport inhibitor might account for the decline in phosphate uptake observed as cells grow to quiescence, we removed media from growing and non-growing cultures and tested its ability to support phosphate uptake. We found that the medium from growing cultures supported a higher rate of phosphate uptake than the medium from the quiescent cultures did, indicating that a transport inhibitor was being released. In addition, we found that the amount of inhibitor released was proportional t o the concentration of phosphate in the medium. To directly determine if the decline in phosphate uptake was a key event in the decline in DNA synthesis as cells grew to quiescence, we switched growing cultures to a medium with low phosphate immediately after cell attachment. This lowered the rate of phosphate uptake to a level below that of quiescent cells grown in the usual concentration of phosphate. This was done for 3T3, Polyoma virus-transformed 3T3, human diploid foreskin, and secondary chick embryo cells. Measurements of DNA synthesis and cell number showed that this lowered rate of phosphate uptake had virtually no effect on cell growth, directly demonstrating t h a t the decline in phosphate uptake observed during growth to confluency was not causing the decline in DNA synthesis. In addition, measurements of intracellular phosphate pool size showed that changes in phosphate uptake were not directly paralleled by changes in intracellular phosphate pool size, and that intracellular phosphate pool size was not regulating DNA synthesis or cell division during growth to quiescence. Received Sept. 28, '76. Accepted Dec. 10, '76.

J. CELL. F'HYSIOL.,92: 115-128.

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G. S. BARSH, D B. GREENBERG AND D. D. CUNNINGHAM

The division of fibroblast-like cells in culture is affected by many factors including serum concentration (Todaro et al., '67; Holley and Kiernan, '681, available growth surface area (Todaro and Green, '63; Stoker and Rubin, '67; Kolodny and Gross, '69; Thrash and Cunningham, '75), and the concentration of low molecular weight nutrients present in the medium (Ley and Tobey, '70; Holley and Kiernan, '74). Specifically, deprivation of glucose (Holley and Kiernan, '741, some amino acids (Ley and Tobey, '701, or phosphate (Pi) (Holley and Kiernan, '74) leads to arrest in the Go or GI state )or the restriction point (Pardee, '74) 1. Readdition of the limiting nutrient results in initiation of cell proliferation. The mechanism by which these low molecular weight nutrients control cell division is unclear; i t has been suggested that proliferation of cells both in culture and in vivo might normally be regulated by changes in growth factor or intracellular nutrient levels which, in turn, are mediated by selective alterations in uptake (Pardee, '64; Holley, '72). Several observations have suggested t h a t Pi may play a key role in the regulation of cell division. Pi uptake decreases as 3T3 cells reach a growth-arrested state (Blade et al., '66; Cunningham and Pardee, '69; Weber and Edlin, '71); when fresh serum is added to these quiescent cells to initiate cell proliferation, increases in Pi uptake can be detected within several minutes (Cunningham and Pardee, '69; Jimenez de Asua e t al., '74). Very low levels of Pi in the medium can limit cell division (Holley and Kiernan, '74; Greenberg et al., '77); proliferation can then be initiated by the addition of Pi to normal levels (Holley and Kiernan, '74). Virally transformed 3T3 cells show radically altered transport properties. Pi uptake is 3- to 5-fold higher than in normal 3T3 cells when compared a t confluent or near-confluent densities (Cunningham and Pardee, '69; Hare1 et al., '751, and addition of fresh serum to Polyoma virus-transformed 3T3 (Py3T3) cells has little effect on Pi uptake (Cunningham and Pardee, '69). We have previously shown that the increase in Pi uptake that occurs upon serum stimulation of quiescent cells is not causally related to the subsequent increase in DNA synthesis and cell number (Greenberg et al., '77). We now report studies on 3T3, human diploid foreskin (HF), and secondary chick embryo (CE) cells examining the possible involve-

ment of P, uptake and intracellular P, pool size in the control of DNA synthesis and cell division during growth to quiescence. Our results demonstrate that the decrease in p, uptake that occurs as cells reach quiescence under usual culture conditions is not a causal event in the cessation of DNA synthesis and cell division. In addition we show that intracellular P, pool size is neither causally related to cessation of cell division during growth to quiescence nor necessarily correlated with the changes that occur in P, uptake. MATERIALS AND METHODS

Materials ( 3 H ) - t h y m i d i n e was purchased from Schartz-Mann. 32P1was obtained from ICN. Medium 199, Dulbecco-Vogt modified Eagle's (DV) medium, glutamine, antibiotics, trypsin, vitamin and amino acid stocks were purchased from Gibco. DV medium without Pi was either purchased from Gibco or made up from analytical reagent grade chemicals, vitamin, and amino acid stocks. Calf and chicken sera and dialyzed calf sera were purchased from Irvine Scientific Company. Tryptose phosphate broth was purchased from Difco. Plastic tissue culture dishes were obtained from Falcon Plastics. Dialysis tubing was purchased from Union Carbide. cells Swiss mouse 3T3 cells (clone 42, obtained from Doctor George J. Todaro) were grown in DV medium containing 100 units of penicillin per milliliter, 100 p g of streptomycin per milliliter, and 10%calf serum. Cultures were restarted from frozen stocks of cells a t 6- to 8week intervals and were always maintained a t subconfluent levels. 3T3 cells were judged free from mycoplasma contamination i n checks by autoradiography with (3H)-thymidine (Nardone et al., '65) and by determinations of the ratio of uridine to uracil incorporated into acid-insoluble material (Schneider et al., '74). HF cells (which were used between passages 10 and 25) were supplied by Doctor David T. Kingsbury and grown like 3T3 cells. CE cells were prepared from 9- t o 10-day-old chick embryos according to the method of Rein and Rubin ('68). Primary CE cultures were grown in Medium 199 supplemented with 2.0% chicken serum, 2.0% tryptose phosc Unless otherwise stated, all references to PI uptake refer to uptake into the acid-soluble fraction ds described In MATERIALS AND METHODS

PHOSPHATE UPTAKE AND GROWTH CONTROL

phate broth, 100 units of penicillin per milliliter and 100 F g of streptomycin per milliliter. Growth of all cells and all experimental incubations were carried out in an atmosphere of 5%CO,. Cells to be used in experiments were seeded in 35-mm culture dishes with 2.0 ml of medium. All experimental points were derived from either duplicate or triplicate plates.

Cell counting Cells were detached in 1.0 ml of phosphatebuffered saline (PBS) containing 0.02%EDTA and 0.05% trypsin. They were then diluted with PBS and counted in a Coulter electronic particle counter. Cell counts, routinely done in duplicate, usually varied less than 5%. Protein All measurements of DNA synthesis, Pi uptake, and intracellular Pi pool size were standardized per F g of cell protein. Cells were dissolved in Reagent A of Lowry e t al. ('51). Protein was measured as described by these authors.

DNA synthesis DNA synthesis was measured by determining the amount of (3H)-thymidine incorporated into acid-insoluble material during a 20-minute incubation as previously described (Thrash and Cunningham, '74).

Piuptake Pi uptake into the acid-soluble fraction of cells was determined by a method similar to one previously described (Cunningham and Pardee, '69). Carrier-free 32Piwas added to the growth medium on the cells to a final concentration of 1.0-2.5 pCi/ml. The cultures were then incubated in a 37' water-bath incubator with an atmosphere of 5%CO, in air. At the end of the incubation period, the cultures were removed from the incubator, the medium was immediately aspirated, and the plates with attached cells were dipped successively into a series of four beakers containing 0.15 M NaCl a t 0-2". This rinsing procedure took less than 30 seconds. Loss of intracellular radioactivity during rinsing was less than 5%. Eight-tenths milliliter of 10% trichloroacetic acid was then added to each plate, and acid-soluble radioactivity was extracted from the attached cells during a 45minute interval with periodic gentle shaking. The trichloroacetic acid extract was then

117

transferred into scintillation vials and counted by liquid scintillation in a Triton-X-100-toluene-based scintillation fluid. A zero time blank (determined by adding 32Pito cultures on ice, aspirating the medium immediately, and rinsing as described) was subtracted from each experimental point. This zero time point was always less than 10%of the uptake measurement. Unless otherwise stated, the incubation period for the uptake assay was 20 minutes. We have found that Pi uptake is linear for as long as two hours, in agreement with the data of Weber and Edlin ('71). Thus, a 20-minute timepoint (corrected by subtracting a zero time blank) yields a rate of Pi uptake into the acid-soluble fraction.

Intracellular pool size of orthophosphate Cells attached to 100-mm culture dishes were rinsed twice with 0.15 M NaC1, and the acid-soluble Pi pools were extracted with 2.0 ml of 10%trichloroacetic acid for 45 minutes. Inorganic Pi was immediately precipitated by the following method of Sugino and Miyoshi ('64).One milliliter of the precipitation mixture (4 parts 5.0% ammonium molybdate, 1 part 0.2 M triethylamine hydrochloride, and 2 parts 2.0 N perchloric acid) was added to the trichloroacetic acid extract. After ten minutes, the mixture was centrifuged at 2,000 X g for 20 minutes and the supernatant was discarded. The precipitate was dissolved in 0.5 ml of acetone and the amount of Pi present was determined by a modified method of Baginski e t al. ('67). (Standards were prepared from 1 mM NaH2P0, and 10% trichloroacetic acid which was then precipitated and dissolved in acetone as described above.) One-tenth milliliter of 20% ascorbic acid was added to each sample, and the contents of each tube were thoroughly mixed. One milliliter of molybdate complexing reagent (2.0%sodium citrate dihydrate and 2.0%anhydrous sodium arsenite in 2.0% acetic acid) was added immediately and the contents of the tube were mixed again. After 15 minutes, absorbance was read a t 690 nm. Values for the amount of Pi present in the cells from 100 mm culture dishes ranged from 5-150 nmoles. These values are well within the sensitivity range of the assay. Our values for absolute amount of Pi present were then standardized per p g of cell protein. Since protein is directly proportional to intracellular fluid space (Foster and Pardee, '691, the

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G. S BARSH, D. B. GREENBERG AND D. D. CUNNINGHAM

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Fig. 1 Kinetics of Pi uptake in quiescent and growing 3T3 cells. 3T3 cells were plated in DV medium containing 10% serum a t a density of either 1.1 X lo3 or 1.1 X lo4 per cm2. Three days later, the cultures set up a t the lower density were still growing (0- -- 0) and those set up a t the higher density were quiescent (0-0). To remove residual Pi, the medium was aspirated and the cultures were rinsed with 0.15 M NaCl buffered with 15 mM Hepes ipH 7.4). They were then incubated for two hours at 37°C in dialyzed depleted medium. (Depleted medium was obtained by collecting medium from 3T3 cultures which hadbeen set up a t a density of 1.1 x 1O'per cm2in DV medium containing 10%serum, and grown to quiescence over a 3-day period. This medium was dialyzed for seven days against 0.15 M NaCl which was changed daily, and finally against two changes of Pi-free DV medium without NaHCO,, but containing 1 5 mM Hepes buffer ipH 7.4) 1. We previously determined t h a t after a 2-hour incubation in Pi-free medium, there was no detectable efflux of intracellular Pi. After the 2-hour preincubation, the medium was aspirated, the cells were rinsed again and then incubated for 15 minutes with 3zPiin dialyzed depleted medium containing the indicated concentrations of 4. P, uptake and represent growing protein were determined as described in MATERIALS AND METHODS. Dashed lines (---I cells; solid lines (-) represent quiescent cells. Upper panel: Total uptake with diffusion estimated as described in the text. Middle panel: Carrier mediated uptake calculated by subtracting diffusion from total uptake. Lower panel: Lineweaver-Burk plot of carrier-mediated uptake data.

values of pmoles PJpg protein are direct measures of intracellular concentrations.

Serum dialysis and adjustment of Pi concentrations Serum was dialyzed as previously described (Greenberg et al., '77). Either this serum or

commercially dialyzed calf serum was used in all experiments where a Pi concentration is specified. We found no differences between commercially dialyzed calf serum and our own dialvzed calf serum. either in the level of residuk P, or the ability to support cell growth when supplemented with Pi. No Pi could be

119

PHOSPHATE UPTAKE AND GROWTH CONTROL

detected in either type of serum by the method of Baginski et al., (‘67), indicating that the Pi concentration was less than 0.001 mM. Media with specified Pi concentrations were prepared by supplementing Pi-free DV medium and dialyzed calf serum with a 12.5 mM Pi solution in DV medium. Salt substitutions were not necessary as the ionic strength of the media did not vary significantly. RESULTS

Kinetic analysis of Pi uptake by growing and quiescent 3T3 cells Pi uptake by proliferating nonconfluent 3T3 cultures and serum-stimulated confluent cultures is generally 3- to 5-fold higher than in quiescent confluent cultures (Cunningham and Pardee, ’69). At physiological Pi concentrations (0.8-1.2 mM), uptake could be due to both a carrier-mediated component and a diffusion component. In order to evaluate the relative contributions of these components to total Pi uptake in both proliferating and quiescent cultures, we measured the rate of Pi uptake as a function of Pi concentration in the culture medium. Before measuring Pi uptake, we preincubated the cells in dialyzed, depleted medium, to reduce intracellular pools of Pi. Previous experiments have shown that 3T3 cells remain viable for as long as three days in Pi-free media (Greenberg et al., ’77). Figure 1shows the kinetic analysis of Pi uptake in both proliferating and resting cultures of 3T3 cells. For the range of Pi concentrations tested (0.075-1.5 mM), Pi uptake in the proliferating cultures (open circles, upper panel) was approximately 2-fold greater than in the quiescent cultures (closed circles, upper panel). We found that there was a significant contribution by a diffusion component (greater than 20%) to the total uptake at concentrations above 0.2 mM. As expected, diffusion accounted for a greater percentage of total uptake at higher Pi concentrations. Diffusion was estimated by drawing a line through the origin that was parallel to the total uptake a t regions where uptake appeared to be linear (0.4-1.5 mM) as described by Renner et al. (‘72) and Neame and Richards (‘72). As can be seen in figure 1, the diffusion component for proliferating, nonconfluent cells (dashed line without symbols, upper panel) was approximately 2.5-fold greater than for quiescent, confluent cells (solid line without symbols, upper panel). This large difference in diffusion of Pi between proliferating and quiescent cells

could mean that the proliferating cells have a higher amount of surface membrane area available for Pi uptake per p g of cell protein than the quiescent cells. In both proliferating and quiescent cultures, diffusion at physiological Pi concentrations (0.8-1.2mM) appeared to account for approximately 40 to 60%of the total uptake. Thus, the change in Pi uptake between growing and quiescent cells resulted from a change in both the carrier-mediated and diffusion components. The middle panel in figure 1 shows carriermediated uptake rates calculated by subtracting the estimated diffusion components (upper panel, fig. 1)from the values for total uptake. The lower panel in figure 1 shows a Lineweaver-Burk plot of the carrier-mediated uptake data from the middle panel. As can be seen, the change in uptake data from the middle panel. As can be seen, the change in uptake between proliferating and quiescent

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cells resulted from a change in the Vmax and not the Km. This last result is analogous to results reported earlier concerning the difference between serum-stimulated confluent and resting confluent cells (Jimenez de Asua e t al., '74).

Effectof towered P, concentration on cell growth Figure 2 shows the effect on cell number when 3T3 cells were switched to conditioned media containing 0.005 mM P, a t different times during their growth. As can be seen, the reduced P, concentration had a marked effect on cell number, confirming the results of Holley ('72) and Holley and Kiernan ('74). Furthermore, i t appeared that the same level of PI (0.005 mM) could support different final

cell densities. Thus, if conditioned medium containing low P, was added to cultures a t different densities, a limitation of cell division occurred which was quantitatively dependent upon the cell density immediately prior to the medium change. We have previously shown t h a t if 3T3 cells are switched to fresh medium containing different Pi concentrations a t one initial cell density, a limitation of cell number results which is proportional to Pi concentration (Greenberg et al., '77). Thus, depending on the experimental conditions employed, final cell density may or may not be quantitatively dependent on P, concentration. The limitation of cell number observed in these experiments is consistent with the possibility that depletion of PI in the medium or the consequent decline in P, uptake brings about a

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Fig. 4 Changes in Pi uptake and DNA synthesis during growth of 3T3 cells to confluency from different initial densities. 3T3 cells were set up at the densities indicated below in DV medium containing 10%serum. Cell number ( A --- A ) , protein (A-A), Pi uptake (0-1, and DNA synthesis (0--- 0)were determined at the indicated times as described in MATERIALS AND METHODS. Panel A: plated at 4.4 X 10' per cm2; Panel B: plated at 2.2 X lo3 per cm2; Panel C: plated at 1.1 X 10' per cm'; Panel D: plated at 4.4 X 10' per cm2.

decrease in DNA synthesis during growth to quiescence. Relationship between p, uptake andDNA synthesis during growth to quiescence If DNA synthesis and Pi uptake were causally related during growth to quiescence, a specific temporal relationship should have existed between the two. As shown in figure 3, the decline in Pi uptake (solid lines) appeared to slightly precede the decline in DNA synthesis (dotted lines) in 3T3 cells grown in the presence of either 10 or 25% calf serum. We considered the possibility that Pi uptake is related not to growth rate or cell density, but instead to time in culture. To answer this question, we plated 3T3 cells at various densities in the presence of 10%serum and measured Pi uptake and DNA synthesis. As shown in figure 4, the initial rate of Pi uptake was inversely related to the seeding density. Furthermore, in the cultures plated at the higher densities where the least cell division oc-

curred, there was little or no decline in P, uptake. Thus, P, uptake appeared to correlate with rate of cell division and cell density rather than time in culture. Factors controlling decline of P, uptake As can be seen from figure 3, the decline in P, uptake appeared to precede somewhat the decline in DNA synthesis, suggesting that the decline in DNA synthesis was probably not causing the inhibition of P, uptake. To check this tentative conclusion, we inhibited DNA synthesis in 3T3 cells t o 10%of control values with 1 mM hydroxyurea and measured the subsequent effect on P, uptake We found that a t either one, six, or ten hours after inhibitor addition, P, uptake showed no differences from the control value (data not presented). Thus, the decline in P, uptake appears not to be caused by the decline in DNA synthesis. I t has been reported that at least some of the decline in P, uptake that occurs as cells grow to quiescence is accounted for by the release of a transport inhibitor into the medium

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Hours Fig. 5 Release of PI uptake inhibitor by 3T3 cells. 3T3 cells were plated a t a density of 1.1 X l o 4 per om2 in DV medium containing 10%serum. After attachment, some of the cells were switched to DV medium containing 10%serum and Pi added back to a level of 0.02 mM, 0.2 mM, or 2.0 mM. Pi uptake ( 0 )was determined a t the indicated times as described in MATERIALS AND METHODS. After four days, media taken from the cells a t 28 hours and 96 hours (stored a t 2°C) were switched to confluent 3T3 cells. These were obtained by growing cells plated a t 1.1 X lo4per cm2in DV medium containing 10%serum for four days. After a 20-minute preincubation with these media, Pi uptake was measured as described in MATERIALS AND METHODS. “Amount of inhibitor released’ is defined as: P, uptake in confluent cells with media from 28 hour cultures - 1. 9 uptake in confluent cells with media from 96-hour cultures

and that the decline is not simply due to an exhaustion of serum factors (Pariser and Cunningham, ’71; Hare1 et al., ’75). We examined the possibility that the amount of inhibitor released during growth to confluency might be quantitatively related to the initial rate of P, uptake in dividing cells, and t h a t high initial rates of uptake could actually “induce” large amounts of a n inhibitor of P, uptake to be released. To test this possibility we grew 3T3 cells in either 2.0, 0.2, or 0.02 mM P, (to alter the rate of P, uptake) and noted the effect on inhibitor release. As can be seen in figure 5, the different PI concentrations had a marked effect on P, uptake (left panel), both in dividing and quiescent cultures. Furthermore, the amount of inhibitor released (right panel) appeared to be proportional to the absolute amount that P, uptake declined.

Thus, where P, uptake was initially highest (the cultures grown in 2.0 mM PI),the most inhibitor was released, and the largest absolute decline in PIuptake was observed. Our assay for amount of inhibitor released was based on the ability of the medium from the 28- and 96-hour experimental cultures to support PI uptake in a different set of confluent cultures. This raised the possibility that the differences observed in amount of inhibitor released may have been influenced by the P, concentration in the medium during the assay for inhibitor release rather than actual differences in release of inhibitor. To test this possibility, we supplemented the media taken from the experimental cultures with additional P, so that the P, concentration during the assay for release of inhibitor was 2.0 mM. We found that this treatment had no effect on

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Fig. 6 Effect of lowered Pi uptake on growth to confluency of CE cells. CE cells were plated a t a density of 1.1 X 10' per cm2 in DV medium containing 2%calf serum. After attachment, the medium was changed t o DV containing 2%calf serum and Pi added back to a level of either 0.1 mM ( - - - J or 1.1mM (-1. At the indicated times, cell number (01,DNA synthesis (m1, Pi pool size (A),and Pi uptake (XI,were determined as described in MATERIALS AND METHODS

the results for amount of inhibitor released. Thus, the media taken from cultures originally grown in 0.02 mM P, still showed very little inhibitor release even when tested at a P, concentration of 2.0 mM. These results verified our conclusion that release of a transport inhibitor accounted for a t least some of the decline in P, uptake observed during the growth of 3T3 cells to quiescence.

Effectofreduced P,concentration on cell growth, DNA synthesis, P,uptake, and intracellular P, pool size To determine directly if the decline in P, uptake affected the decline in DNA synthesis

during growth to quiescence, we switched growing cultures to a medium with low P, immediately after attachment. This low concentration of P, reduced the rate of P, uptake to a level below that observed for quiescent control cultures. This was done for CE, 3T3, HF, and Py3T3 cells. With the CE cells (fig. 61, P, uptake (bottom panel) in the cultures switched to low P, (dotted lines) was well below the levels observed for quiescent control cultures (solid lines). However, cell division (top panel) and DNA synthesis (second panel), were not inhibited a t all. In fact, they were not significantly different from control cultures, directly showing t h a t neither the de-

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G. S. BARSH, D. B. GREENBERG AND D. D. CUNNINGHAM

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cline in Pi uptake that occurred when cells were switched t o low Pi, nor the decline in Pi uptake normally observed when the cultures grow to confluency was causing a decline in DNA synthesis and subsequent cessation of cell division. Although the decline in Pi uptake was not causally related t o the decline in DNA synthesis, the possibility remained that intracellular Pi pool sizes might be regulating cell division during growth to quiescence. With the CE cells (third panel, fig. 61, there was only a small change in intracellular Pi pool size during growth to quiescence. I t seems unlikely that this could be causing the sharp decline in DNA synthesis (second panel).

We found that a n 11-fold difference in PI concentrations between the control and low P, cultures, while causing a t least a 5-fold difference in levels of P, uptake, had almost no effect on intracellular P, pools. Thus, the difference between intracellular P, pools for control and low P, cultures was always very small, possibly reflecting a lower rate of P, efflux in the low P, cultures. Similar results were obtained for 3T3 cells (fig. 7). The level of P, uptake in the cultures switched to low P, was always below the levels observed for quiescent control cells. Since there were virtually no differences between the control and low P, cultures with respect to cell number (top panel) and DNA synthesis,

PHOSPHATE UPTAKE AND GROWTH CONTROL

125

Hours Fig. 8 Effect of lowered l'j uptake on growth of HF cells. HF cells were plated at a density of 5.5 X l o 3 per cm2in DV medium containing 5%calf serum. After attachment, the medium was changed to DV medium containing 10%calf serum and l'j added back t o a level of either 0.1 mM ( - - - ) or 1.1mM (-). At the indicated and Pi uptake (XI,were determined as described in times, cell number ( O ) ,DNA synthesis ( W ,Pi pool size (A), MATERIALS AND METHODS.

(second panel) it appeared that a decline in PI uptake was not inhibiting DNA synthesis in these cultures. In contrast to the CE cells, we found that intracellular PI pools in the 3T3 cells (third panel, fig. 7) increased approximately 2-fold during growth to quiescence. As with the CE cells, however, the difference between intracellular P, pools of the control and low P, cells was very low compared to differences in levels of PIuptake. Results for the HF cells are shown in figure 8. Again, there were no significant differences in DNA synthesis and cell number between the control and low P, cells, showing that PI

uptake was not affecting DNA synthesis during growth to quiescence. Intracellular Pi pool size in the HF cells decreased approximately 50% during growth to quiescence (third panel, fig. 8). This decrease, however, could not be causing the decline in DNA synthesis because DNA synthesis was already decreasing by 50 hours whereas intracellular Pi pool size had not changed. Thus, neither intracellular Pi pool size nor Pi uptake appeared to be affecting DNA synthesis in these cultures. Since transformed cells generally have higher rates of Pi uptake than untransformed cells (Cunningham and Pardee, '69; Hare1 et

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G. S. BARSH, D. B. GREENBERG AND D. D. CUNNINGHAM

Hours Fig. 9 Effect of lowered Pi uptake on growth of Py3T3 cells. Py3T3 cells were plated a t a density of 1.1 X lo3 per om2 in DV medium containing 10%calf serum. After attachment the medium was changed to DV meAt the dium containing 10%calf serum and Pi added back to a level of either 0.1 mM ( - - - ) or 1.1 mM (-). indicated times, cell number ( O ) , DNA synthesis (B), Pi pool size (A),and Pi uptake (XI,were determined as described in MATERIALS AND METHODS.

al., '751, we considered the possibility that low rates of Pi uptake might restore densitydependent growth in transformed cells. With Py3T3 cells (fig. 91, when PI uptake (bottom panel) was reduced (dotted lines) to a level approximately 10-fold below that observed for cultures grown in the presence of 1.1mM P, (solid lines), the low P, cultures actually grew a t a faster rate than the control cultures. The final cell density, however, was similar for both sets of cultures. This demonstrated t h a t the high level of PI uptake characteristic of these cells was not responsible for the loss of density-dependent growth. We previously reported that PI uptake in

Py3T3 cells is not significantly affected by cell density (Cunningham and Pardee, '69).Our present results indicate, however, that P, uptake declines a s much as 10-fold during growth through confluency. It seems likely that this phenomenon was not observed earlier because most of the decline occurs a t a very low cell density (below 1.1 X lo4 cells/ cm?. Measurements of intracellular PI pool size in the Py3T3 cells (third panel, fig. 9) showed large differences between the control and low PI cultures a t the later time points. We found t h a t this was primarily due to depletion of PI in the medium. It should be noted, however,

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incubation time of two hours prior to sampling. We measured Pi pools by a direct chemical method since we have found that Pi uptake is nearly linear for as long as two hours. Weber and Edlin ('71) previously presented DISCUSSION data showing that Pi uptake by 3T3 cells into The role t h a t Pi uptake might play in the both the acid-soluble and acid-insoluble fracregulation of growth can be separated into tions is nearly linear for three hours. Thus, two questions. First, is the increase in Pi up- the measurements of Gray et al. ('76) might take that occurs upon serum stimulation of reflect levels of uptake rather than intracelluquiescent cells a key event in subsequent cell lar concentrations. division? Secondly, is the decline in Pi uptake In summary, the present results, along with that occurs as cells grow to quiescence causal- our other recent results (Greenberg et al., ly related to the decline DNA synthesis? We '77), have shown that under usual culture previously dealt with the former question conditions, changes in Pi uptake and in the in(Greenberg et al., '77) and found that neither tracellular pool size of Pi do not control DNA an increase .in Pi uptake nor intracellular synthesis or cell division. In addition, our Pi pool size was necessary for initiation of studies on glucose uptake have shown that DNA synthesis and subsequent cell division. fluctuations in the uptake of this nutrient do The present experiments address the latter not regulate DNA synthesis or cell division in question. 3T3, HF, or CE cells under the usual culture Our results with HF, CE, and 3T3 cells show conditions (Thrash and Cunningham, '74; that a reduction of Pi uptake in growing cells Naiditch and Cunningham, '77). to a level below that observed for quiescent The key approach in these studies involved cells (grown in the presence of 1.1mM Pi) has controlling the rate of uptake by varying the no effect on DNA synthesis or cell division. concentration of Pi or glucose in the growth This directly demonstrates that the decline in medium. This permitted us t o directly deterPi uptake that occurs during growth to quies- mine whether changes in uptake that occur cence does not cause the decline in DNA syn- during growth stimulation or inhibition were thesis under these conditions. These cells rep- causally involved in the control of cell prolifresent several different animal species, as eration. This approach should be useful in furwell as early passage normal cells and a cell ther studies to evaluate the possible role of line used extensively in studies on growth changes in uptake of other nutrients in the control of cell division. control. The suggestion that Pi uptake may be regACKNOWLEDGMENTS ulating cell growth rests on the assumption that changes in Pi uptake are paralleled by This investigation was supported by a changes in intracellular Pi pool size. Our re- USPHS Research Grant from the National sults show that this is not the case. In the 3T3 Cancer Institute CA-12306. D.D.C. is a recipand Py3T3 cells, while Pi uptake decreases, ient of a USPHS Research Career Developintracellular Pi pool sizes show a surprising ment Award from the National Cancer Instiincrease upon growth to confluency. Weber tute CA-00171. We thank Mr. Tom Ho for and Edlin ('71) have reported that intracellu- technical assistance. lar Pi pool size in 3T3 cells shows little or no change when growing cells are compared to LITERATURE CITED confluent cells. Their results for growing cells, Baginski, E. S., P. P. Foa and B. Zak 1967 Determination however, are taken a t a time when the cells of phosphate: Study of labile organic phosphate interference. Clin. Chim. Acta., 25; 155-158. are very near confluency. Recently, a report concerning intracellular Blade, E., L. Hare1 and N. Hananin 1966 Variation du taux dincorporation du phosphore dans les cellules en foncPi pool sizes in 3T3 cells appeared which pretion de leurs concentrations e t inhibition de contact. Exp. sented results different from ours (Gray e t al., Cell Res., 42: 473-482. '76). These investigators reported that the Cunningham, D. D., and A. B. Pardee 1969 Transport changes rapidly initiated by serum addition to "contact pool size of Pi decreased upon growth to quiesinhibited' 3T3 cells. Proc. Nat. Acad. Sci. (U.S.A.), 64: cence, and increased after stimulation of the 1049-1056. cells by serum. They used a n isotope equilibra- Gray, P. N., M. E. Cullum and M. J. Griffin 1976 Population density and regulation of cell division in 3T3 cells. I. tion method t o measure Pi pools, allowing an that as with normal 3T3 cells (third panel, fig. 7), intracellular Pi pool sizes in the control cultures of Py3T3 cells increased during growth to confluency.

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