Specific increase in pyrimidine deoxynucleoside transport at the time of deoxyribonucleic acid synthesis in 3T3 mouse cells.

Abstract Initiation of division of density-inhibited 3T3 mouse fibroblasts by fresh serum brought about increased uptake of thymidine and deoxycytidine which coincided closely in time with initiation of DNA synthesis. This increase was specific, for transport of deoxyadenosine, deoxyguanosine and orthophosphate did not change at the time of DNA synthesis. Although similar increases occurred for thymidine and deoxycytidine, kinetic studies indicated that they were taken up by different transport systems. The increased transport of thymidine represented an increase in net uptake, since uptake was stimulated more than efflux. Increased thymidine uptake was not caused by increased rates of DNA synthesis, since inhibition of DNA synthesis did not prevent the transport increase. In addition, it did not require thymidine kinase, since 3T3 cells lacking this enzyme showed the transport increase. Kinetic studies revealed that the increase was accompanied by an increased Vmax with no change in Km, indicating activation and/or increased amount of the same rather than a new transport system. Uptake of thymidine at both the basal and stimulated levels occurred by facilitated diffusion below about 5 µm and by passive diffusion above this concentration. Increased uptake of thymidine was prevented by cycloheximide and further increased by actinomycin D, suggesting that the increase required newly made protein(s) whose synthesis might be controlled at the post-transcriptional level.

This increase was specific, for transport of deoxyadenosine, deoxyguanosine and orthophosphate did not change at the time of DNA synthesis. Although similar increases occurred for thymidine and deoxycytidine, kinetic studies indicated that they were taken up by different transport systems.
The increased transport of thymidine represented an increase in net uptake, since uptake was stimulated more than efllux. Increased thymidine uptake was not caused by increased rates of DNA synthesis, since inhibition of DNA synthesis did not prevent the transport increase.
In addition, it did not require thymidine kinase, since 3T3 cells lacking this enzyme showed the transport increase. Kinetic studies revealed that the increase was accompanied by an increased Y max with no change in K,, indicating activation and/or increased amount of the same rather than a new transport system, Uptake of thymidine at both the basal and stimulated levels occurred by facilitated diffusion below about 5 PM and by passive diffusion above this concentration.
Increased uptake of thymidine was prevented by cycloheximide and further increased by actinomycin D, suggesting that the increase required newly made protein(s) whose synthesis might be controlled at the post-transcriptional level.
Correlations between transport of specific nutrients and growth rates of animal cells in culture have strengthened suggestions t,hat changes in membrane permeability might play an important role in controlling DNA synthesis and cell division (l-4).
For example, when normal cells grow to confluency and form a densityinhibited monolayer, large decreases in transport occur for phosphate (5-7), uridine (6, S), hexoses (9,lO) and thymidine (11,12). These permeability changes appear to be specific, since growth * This work was supported by lJnit.ed States Public Health Service Grant CA-12306. to confluency is accompanied by decreased uptake of some but not all amino acids (13,14).
In addition, when density-inhibited cells are initiated to divide by adding fresh serum, there is a very rapid increase in transport of phosphate and uridine (6) aud also glucose (9). This treatment results in no change of adenosine uptake (6) and rapid decreases in amino acid transport (15), demonstrating specific rather than generalized permeability changes.
The present studies on the relationship between deoxynucleoside t.ransport and DNA synthesis were prompted by several observations relating pool sizes of DNA precursors to the onset of DN4 synthesis.
In addition, synchronization of cells with high levels of thymidine which inhibit DNA synthesis can lead to shortening of the Gr period as measured by collection of cells at the Gl/S boundary (29,30). Finally, pool sizes of deoxynucleotides show considerable fluctuations which appear to correlate with the rate of DNA synthesis (7,26).
Our experiments have shown that uptake of thymidine and deoxycytidine increases at the same time as DNA synthesis in confluent 3T3 cells initiated to divide by addition of fresh serum. In contrast, transport of deoxyadenosine, deoxyguanosine, and orthophosphate does not increase during the period of DNA synthesis.
The increase in thymidine transport is prevented by inhibiting protein synthesis.
It is not a consequence of increased rates of DNA synthesis, and it can take place in 3T3 cells lacking thymidine kinase (3T3TK-cells). The increase is accompanied by an increase in V,,, with no change in K,. These results indicate that the increase in thymidine uptake results from increased synthesis of the same transport system or activation of it by a newly synthesized protein. Growt,h of the cells and all experimental incubations were carried out in an atmosphere of 5y0 CO2 in air so the pH was maintained between 7.3 and 7.5. Care was taken to avoid pH fluctuations which affect transport rates (32, 33). Cells were removed from flasks or dishes with 0.054"c trypsin and counted in a hemacytometer.
Measurelnent oj Substrate Uptake into Acid-soluble Fraction-Cells were incubated at 37" for 10 min with either %labeled deoxynucleoside (2 PC1 per ml, 1.3 Ci per mmole) or [3'P]orthophosphate (100 $Zi per ml, carrier free). Uptake of these compounds was linear during the lo-min incubation period. The cells were then immediately washed four times at O-2" with isotonic phosphate-buffered 0.9% SaCl solution. The washing procedure took less than 1 min. Loss of intracellular isotope during washing was less than 57& The washed cells were subsequently incubated for 15 to 30 min at O-2" with 10% trichloroacetic acid. Radioactivity in cell-free neutralized aliquots was measured in a liquid scint.illation counter. All measurements were made on duplicate plates. Variability between duplicates was generally less than 10%.
Measurement of Rates of DNA Synthesis3T3 cells were incubated at 37" for 10 min with [3H]thymidine (2 PCi per ml, 1.3 Ci per mmole) . They were immediately washed twice at O-2" with isotonic phosphate-buffered 0.9% NaCl solution, and four times at O-2" with 10% trichloroacetic acid. The cells were then dissolved in 0.5 M KOH, and radioactivity in neutralized aliquots was measured in a liquid scintillation counter.
Rates of DNA synthesis in 3T3TK-cells were measured by incubating these cells for 30 min with [3H]deoxyadenosine (7.5 &i per ml, 9 Ci per mmole).
The cells were immediately rinsed twice at O-2" with isotonic phosphate-buffered 0.9% NaCl solution. They were then dissolved in a solution of 1.0% sodium dodecyl sulfate, 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0. An aliquot was diluted 1:20 with water, and pancreatic RNase (heat treated at 90" for 15 min) was added to a final concentration of 10 pg per ml. This mixture was incubated at 37" for 1 hour.
Calf thymus DNA was then added to a final concentration of 10 pg per ml. An equal volume of 10% trichloroacetic acid was added, and the mixture was cooled to O-2" for 30 min. Precipitates were collected on glass filters and washed with 5% trichloroacetic acid. Radioactivity was measured in a liquid scintillation counter.

Disfribution of Radioactivity in Intracellular
Compounds-Following incubation with [3H]thymidine, cells were washed and treated with 10% trichloroacetic acid as described above for measurement of substrate uptake.
Trichloroacetic acid was removed from these extracts by ether extraction.
Carrier thymidine and thymidine nucleotides were added to the extracts which were subjected to paper electrophoresis in 0.05 M citrate buffer, pH 4.6 (34). Whatman No. 3MM paper was used with a gradient of 60 volts per cm. Thymidine-containing compounds were located by ultraviolet light. Radioactivity in these spots and the rest of the electrophoretogram was determined by counting cut segments in a liquid scintillation counter. Protein Determinations-Protein was measured by the Lowry modification of the Folin-Ciocalteau method (35).

Distribution of Radioactivity in Intracellular
Compounds jollowing Incubation with [3H]Thymidine-Transport experiments were carried out on both 3T3 and 3T3TK-cells. We employed the latter cells, because measurements of thymidine transport should not be complicated by extensive metabolic alterations of intracellular thymidine.
The extent to which intracellular thymidine was metabolized by 3T3TK-cells was examined by incubating nonconfluent and confluent cultures of these cells with [3H]thymidine for 10, 60, and 120 min, washing away extracellular [3H]thymidine, and det.ermining the percentage of intracellular radioactivity in thymidine as described under "Experimental Procedure." These experiments demonstrated that over 90% of the intracellular radioactivity was located in thymidine, even after a 120-min incubation period. Similar experiments were carried out on 3T3 cells. As shown in Table I, approximately 10% of the intracellular radioactivity was located in thymidine in these cells regardless of the incubation time.
The percentages of radioactivity in thymidine nucleotides are also shown in the  . These values varied only slightly with growth rate and labeling time. About 2 to 5% of the t,otal intracellular radioactivity was located in other unidentified compounds.
Spec$c Increase in Pyrimidine Deox:ynucleoside Uptake at Time of DNA Synthesis-Previous studies which demonstrated an increase in thymidine transport during the G1 and S periods of t,he cel1 cycIe (24-28) prompted us to measure transport of DSA precursors during these periods and to compare the timing of increased rates with the initiation of DNA synthesis.
These studies \Tere conducted on confluent 3T3 cells initiated to divide brought about a synchronous wave of DNA synthesis which pea,ked at about 22 hours (36).
Transport of DNA precursors during G1 and S is also shown in Fig. 1. As can be seen, uptake of thymidine and deoxycytidine into the acid-soluble fraction of the cells increased about 4-fold during S. This increase appeared to coincide closely with the initiation of DXA synthesis. In contrast, transport of deoxyadenosine, deoxyguanosine, and ort,hophosphate did not increase at the time of DNA synthesis.
Increased transport of thgmidine and deoxycytidine was therefore specific and not a by adding fresh serum. As shown in Fig. 1 Kinetic experiments indicated that thymidine and deoxycytidine were taken up by different transport systems. Although deoxycytidine competitively inhibited thymidine uptake, the Ki was much higher (0.4 X 10d4 M) than the K, for both deoxycytidine (1.1 X lo+ M) and thymidine (0.5 X 10m6 M) uptake.
Efect of Inhibitors of DNA Synthesis on Increased Thymidine Uptake-The increase in thymidine transport during S might be a consequence of increased rates of DNA synthesis which could deplete pools of thymidine or thymidine deoxynucleotides and trigger increased t,ransport. This possibility was checked by inhibiting DNA synthesis during S and measuring transport of thymidine.
Hydroxyurea, cytosine-/3-n-arabinofuranoside, or mitomycin C was added to confluent 3T3 cells 18 hours after initiation with fresh serum. DNA synthesis and thymidine uptake were measured at 20 hours. As shown in Table II, each of these inhibitors markedly reduced the rate of DNA synthesis. In contrast, they had much less effect on thymidine transport. Hydroxyurea and cytosine-P-n-arabinofuranoside did not significantly lower thymidine uptake, while mitomycin C reduced it only about 30 %. These resu1t.s dem0nstrat.e that the increase in thymidine transport during S was not caused by increased rat.es of DS,4 synthesis.
Transport Increase in STSTK-Cells-The increase in thymidine transport might be caused by an increase in thymidine kinase. Levels of this enzyme increase during S (37), and it has been suggested that it plays a roIe in thymidine transport (38). Accordingly, we det,ermined whether the increase in thymidine transport occurred in 3T3TK-cells. As shown in Fig. 2, addition of fresh serum to density-inhibited cultures of these cells brought about initiat,ion of DNA symhesis as in 31'3 cells. This treatment also resulted in increased transport of thymidine at the time of DNA synthesis (Fig. 2), indicating that the increase in thymidine transport did not require thymidine kinase.
We also employed 3'1'3TK-cells to determine whether the   increase in thymidine transport resulted in a net uptake of t,hymidine. Efflux of thymidine was measured by equilibrating these cells for 2 hours with [aH]thymidine (2 PM), and then determining the rate of release of radioactivity at 37" into nonlabeled medium by washed cells. These measurements were conducted on confluent 3T3TK-cells 6 and 22 hours after the addition of fresh serum. As shown in Table III, uptake of thymidine was increased over 2-fold 22 hours after adding fresh serum. Efflux was stimulated less than 2-fold.
Stimulation by serum resulted in a a-fold increase in net uptake of thymidine (Table III) FIG. 2. Thymidine uptake and DNA synthesis by confluent 3T3TK-cells after addition of fresh serum. The experiment. was performed as described in Fig. 1 for 3T3 cells, except that rates of DNA synthesis were measured using [3H]deoxyadenosine as described under "Experimental Procedure." 0, rate of DNA synthesis; l , thymidine uptake.  FIG. 3 (Zejt). Effect of cycloheximide on serum-initiated increase in thymidine transport by confluent 3T3 cells. Cells were plated at a density of 1.5 X lo6 cells per 35.mm dish and grown to confluency over a a-day period. Fresh calf serum was then added to a final concentration of 20'%. At times indicated by the arrows, cyeloheximide was added to a final concentration of 80 /INI. Thymidine uptake into the acid-soluble fraction was measured as described under "Experimental Procedure." l , eontrol cultures; 0, cycloheximidetreated cultures. FIG. 4 (right). Effect of actinomytin D on serum-initiated increase in thymidine transport by confluent 3T3 cells. Cells were plated at a density of 1.5 x lo5 cells per 35-mm dish and grown to confluency over a 3-day period. Fresh calf serum was then added to a final concentration of 20%. At times indicated by the W~DUJS, actinomycin D was added to a final concentration of 6.0 pg per ml. Thymidine uptake into the acid-soluble fraction was measured as described under "Experimental Procedure." 0, control cultures; 0, actinomycin D-treated cultures. and RNA synthesis.
These experiments were carried out on confluent 3T3 cells initiated to divide by adding fresh serum. Preliminary experiments showed t,hat addition of cyclohesimide to a final concentration of 80 PM inhibited incorporation of ['"Clleucine into an acid-insoluble product over 90% within 1 hour. The effect of this concentration of cycloheximide on the seruminitiated increase in thymidine transport is shown in Fig. 3. As can be seen, cycIohesimide inhibited thymidine uptake as well as the transport increase brought about by fresh serum. Cycloheximide (80 pM) inhibited thymidine uptake about 25oj, 1 hour after its addition to nonconfluent 3T3 cells.
Analogous experiments were carried out using actinomycin D to inhibit RNA synthesis.
This inhibitor was added to a final concentration of 6.0 pg per ml, a level which inhibited incorporation of [3H]uridine into an acid-insoluble product over 90% within 1 hour. As shown in Fig. 4, this treat,ment resulted in a stimulation of thymidine uptake. Treatment of mammalian cells with actinomycin D brings about increased activity of a number of enzymes, presumably by modifying translational controls of enzyme synt.hesis (39). The requirement for protein synthesis during the serum-initiated increase in thymidine transport, and the stimulation of transport by actinomycin D suggest that the synthesis of protein(s) required for the transport increase might be controlled at the level of translation.
Kinetic Studies on Thymidine Uptake by ST3 and STSTK-CellsThe requirement for protein synthesis during t,he period of increased uptake of thymidine suggested two possibilities concerning the mechanism of the transport increase. First, increased transport could result from the synthesis of a new transport system which might have a lower K, for thymidine uptake.
Alternatively, increased transport might be a result of increased activity and/or amounts of the same transport system, leading to an increased V,,,, but an unchanged K, for thymidine uptake.
To distinguish between these possibilities, we measured V,, a.nd K, values before and after the seruminitiated transport increase.
The inset in Fig. 5 shows thymidine 5. Inset, effect of t.hymidine concentration on thymidine uptake by confluent 3T3 cells 2 and 22 hours after addition of fresh serum. Cells were plat.ed at a densit.y of 1.5 X lo6 cells per 3.5-mm dish and grown to confluency over a 3-day period. Fresh calf serum was then added to a final concentration of 20%. Thymidine uptake into the acid-soluble fraction of the cells was measured as described under "Experimental Procedure" using a IO-min incubation period. l , 2 hours after serum addition; 0, 22 hours after serum addition.
Xain figure, reciprocal of uptake velocity oersus reciprocal of thymidine concentration. This figure was constructed from data shown in the inset.
uptake by confluent 3T3 cells as a function of tl~ymidine CORcentration 2 and 22 hours aft,er adding fresh serum. These data were used to construct the Lineweaver-Burk plot shown in Fig. 5. As can be seen, uptake followed Michaelis-Menten kinetics at thgmidine concentrations below about 5 FM, indicating that a saturable cell component, was involved in transport. At higher concentrations, uptake was directly proportional to substrate concentration, demonstrating entry by passive diffusion. (Plagemann and Erbe (40) have recently reported Michaelis-Menten kinetics for thymidine uptake below 2 I.~M and simple diffusion above 2 $LM by hepatoma cells growing in SUSpension culture.) V,, and K, values were obtained from the plot of Fig. 5 and are shown in Table IV. As can be seen, the serum-initiated increase in thymidine transport by 3T3 cells was accompanied by an increase in V,,, with practically no change in the K,.
This suggests that increased transport is a result of increased activity and/or amount of the same, rather than a different transport system. Ident,ical experiments were carried out on confluent 3T3TKcells following treatment with fresh serum (Fig. 6 and Table IV). Uptake of thymidine by these cells also followed Michaelis-Menten kinetics below about 5 PM and simple diffusion above this concentration.
These cells had a slightly higher K, and somewhat lower Tr,,, for thymidine uptake than 3T3 cells. However, like 3T3 cells, serum treatment of confluent 3T3TKcells brought about an increase in the V,,, for thymidine uptake with no change in the K,, suggesting activation and/or increase in the transport system that. also takes up thymidine at the basal level.
Co?zcentration of Acid-soluble Tkymidine-containing Compounds in ST3 and STSTK-Cells-To check the possibility that the increased uptake of thymidine might be accompanied by the appearance of an energy coupling system to bring about active transport, we measured the intracellular acid-soluble concentration of thymidine-containing compounds. 3T3 and 3T3TKcells were equilibrated with medium containing [3H]thymidine for 2 hours. This did not significantly lower the isotope concentration in the medium.
The concentration of thymidine-containing compounds in the acid-soluble fraction was measured as a function of thymidine concentration in the medium, as described in Table V. These d&a demonstrated that the intracellular concentration of acid-soluble thymidine-containing compounds was approximately equal to the concentration of thymidine in the medium.
This was true for 3T3 and 3T3TKcells taking up thymidine at both the basal and serum-stimulated levels. Thus, the increase in thymidine uptake was not accompanied by the appearance of an active transport system. DISCUSSION These results demonstrate that initiation of division of densityinhibited 3T3 cells by fresh serum brought about increased uptake of thymidine and deoxycytidine which closely coincided in time with initiation of DNA synthesis. This increased uptake was specific for pyrimidine deoxynucleosides, since transport of deoxyadenosine, deoxyguanosine, and orthophosphate did not change at the time of DNA synthesis.
Although transport of thymidine and deoxycytidine increased in a similar manner, kinetic experiments indicated that they were taken up by different transport systems. In addition, the increased transport of thymidine represented a net increase in uptake by the cells. Measurements on confluent 3T3TK-cells demonstrated that addition of fresh serum stimulated uptake to a greater extent than efflux.
Although initiation of DNA synthesis is accompanied by an increase in thymidine transport, accurate measurements of the rate of DNA synthesis still can be made by following the incorporation of labeled t.hymidine. This conclusion is based on Thus, incubation of cells u-ith labeled thymidine during this period xvould result in similar specific activit,ies of the TTP pool.
Our studies on the mechanism of the increase in thymidine uptake led to the follovGng conclusions.
The increase ITas not a consequence of increased rates of DNA synthesis, since inhibiting DNA synthesis did not prevent the transport increase. r\loreover, it did not require thymidine kinase, since a similar response occurred in 3T3 cells lacking this enzyme.
Kinetic studies demonstrated that the transport increase Ii-as accompanied by an increase in V,,, \Tith no change in R,, indicating increased amounts and/or activity of the same rather than a ne'vv transport system. The transport increase lx-as inhibited by cycloheximide, but further increased by actinomycin D, suggesting that the increase required the synthesis of protein(s) tha.t might be subject to translational control (39). In addition, the increased uptake \vas not associated xvith the appearance of an active system for thymidine transport. Both st'imulated a.nd ba.sal-level cells took up thymidine by facilitated diffusion belolv 5 pM and by passive diffusion above this concent.rat.ion.
Even though 3T3 cells very rapidly phosphorglated thymidine to thymidine nucleotides, this appeared to have 110 large effect on the thymidine transport parameters IT-e examined.
Similar results were obtained \I-ith 3T3TK-cells nhich metabolized intracellular t'llymidine to only a small estent.. Over 9Oc/, of the radioactivity in these cells remained in thymidine even after a Z-hour incubation Jyit.1~ [311]thymidine.
The increase in thymidine and deosycytidine upt.ake at the time of DNA synthesis suggests that increased pool sizes of these compounds or their derivatives might. influence the init.iation of DNA synthesis.
To test this possibility 15-e added varying levels of deoxynucleosides (5 x 10e6 to 2 x lop3 ar) individually and in combination to confluent 3T3 cells, and also to confluent 3T3 cells Lvhich had been initiated \vith 101~ levels of fresh serum. We then measured the percentage of cells making DNA at sarious intervals by autoradiography, and mitotic cells by Giemsa stain. We could detect no significant and reproducible change in either the timing or extent of DNA synthesis or mitosis.' 1~1 addition to these t'ransport increases at the time of DNA synthesis, initiation by fresh serum also brings about some very early changes in uptnke rates of other precursors.
Large increases in uptake of uridine and phosphate (6) and also glucose (9) can be detected Tvithin 15 min. There is no increase in adenosine uptake lvithin 30 min (B), and large decreases in amino acid uptake occur nrithin 1 hour a.ft.er serum addition (15), demonstrat,ing that these early transport changes are also specific. d4ciinowledyment-TYe thank Mr. Tom Ho for capable technical help.