Temperature dependent phosphorylation of [3H]thymidine and its incorporation into DNA by KB cells

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Experimental Cell Research 104 (1977) 3 19-324

TEMPERATURE

DEPENDENT

r3H]THYMIDINE

PHOSPHORYLATION

OF

AND ITS INCORPORATION

INTO

DNA BY KB CELLS

T. OOKA and J. DAILLIE Epartement

de Biologie GPn6rale et Appliqke, Universitt Claude Bernard (Lyon-l),

Laboratoire associe au CNRS no 92, 69621 Villeurbanne, France

SUMMARY The incorporation of [aH]TdR into DNA by Kl3 cells cooled to 4°C falls rapidly to about l-2 % of that of controls held at 37°C. The amounts of four enzymes involved in TdR metabolism: TdR kinase, thymidylate kinase, cytoplasmic DNA polymerase, and nuclear DNA polymemse, never fall below 50 % of those in the control cells even after 12 h at 4°C. The activities of these enzymes were measured in vitro at different temperatures and it was found that whereas the two kinases retained appreciable activity at low temperature, the DNA polymemse activities were severely inhibited. Cultures of cells rewarmed to 37°C after 12 h at 4°C immediately re-started incotporation of labelled TdR into DNA, showing that sufficient enzyme activity for normal functioning had been preserved during the cold period.

Cell cultures maintained at low temperature (+4”(J) appear to continue to incorporate labelled TdR into DNA, although at a much reduced rate [l-4]. For HeLa cells at 4°C the amount of labelled TdR incorporated into DNA increases with time, and depends upon the number of cells in S phase in the culture. The DNA produced is undistinguishable by ultracentrifugation analysis from that produced by HeLa cells at 37°C. This suggests that the enzyme systems involved in the phosphorylation of TdR, and its incorporation into DNA can still function, although at a considerably lower rate at temperature as low as 4°C. Scholtissek has shown that, in chick fibroblasts, the process of phosphorylation of labelled deoxynucleosides was less inhibited by low temperature than was the in-

corporation of these phosphorylated precursor into the DNA [5,6]. We have examined the phosphorylation of [3H]TdR and its incorporation into the DNA of KB cells at low temperature by measuring the activities of TdR kinase, thymidylate kinase and nuclear and cytoplasmic DNA polymerases in suspension cultures of cells cooled for different lengths of time to 4°C. Some cultures were returned to 37°C after a period at low temperature and the recovery of their enzymatic activities relative to the incorporation of [3H]TdR into DNA was studied. MATERIALS

AND METHODS

Cell cultures KR cells were grown in suspension culture at a density of 2x 10” cells/ml Eagle MEM, modified as described Exp

Cell

Res

104 (I 977)

320

Ooka and Daillie ylated products, samples containing lx 10’ cells were incubated with methyl-[3H]TdR (8 &i/ml) for 20 min and washed. Cold 5% TCA was added and insoluble materials removed by centrifugation. The supernatants were twice extracted with an equal volume of cold ether to remove the TCA, then the TCAsoluble phosphorylated products of TdR were examined by chromatography as described below.

-Chromatography -10 -20 0

2

.

6

8

10

12

1;

16

,I

10

21

2.

15

111

10

32

Fig. 1. Abscissa: time from start of culture (hours); ordinare: cell multiplication (%).

Cell multiplication in three cultures. O-O, Control culture maintained constantly at 37°C; A---A, culture incubated at 4°C (placed at 4°C after 2 h of incubation at 37°C); O-O, culture returned at 37°C after 12 h at 4°C. The number of cells was counted with a Coulter counter. Cell number of inoculum is 3.6X 105cells/ml.

by Joklik [7] and supplemented with 5% heat-inactivated horse serum. This culture was maintained by replacement of one-half of the old culture medium with fresh medium every 24 h. Svnchronized cells were obtained bv a double TdR block method as previously described [8]. Cell concentrations were determined with a Coulter counter (Coultronic). The following experimental conditions were used to study the effects of low temperature on DNA synthesis. At 2 h after the inoculation (at a density of 360x 103 cells/ml), the culture was divided into two culture bottles; one was placed immediately at 4°C and kept for 12 h at this temperature and then returned at 37°C. This culture was kept for 18 h. The other one was kept continually at 37°C for 23 h (control culture). Samples were removed from these cultures at the times indicated in figs 1 and 4. For studies on cells in S phase, cultures prepared with a double TdR block method were used. Two aliquots of cell suspension were removed from the synchronous cultures at 2 h intervals during the cell cycle. One aliquot was immediately transferred at 4°C for 50 min and the other one was used as control at 37°C.

Labelling

of cells with tritiated

TdR

Ahquots (3.6~106 cells) were removed from the cultures at the times indicated in BPS 2 and 4 and incubated with methyl[aH]TdR (2 -pCi/ml, spec. act. 18 &i/mM, from the Radiochemical centre, Amersham) for 20 min at 37°C or at 4°C in haemolysis tubes. At the end of the incubation, the cells were washed twice at 0°C with 5 ml of medium containing non radioactive TdR (0.6 mM) and the nucleic acids precipitated with cold trichloroacetic acid (TCA) (5 %). The recipitate was recovered on Whatman GF/C Pdters, washed with further 5% TCA, and counted after drying. For measurement of non-precipitated phosphorExp Cell Res 104 (1977)

Descending chromatography on Whatman DE 81 paper using an aqueous ammonium fonnate solvent system [9] [0.75 ml of 25 % NHIOH, 15.83 ml of 98% (v/v) HCOOH made up to 100 ml with H,O] was used to separate TdR from its phosphorylated products. Chromatography on Whatman no. 1 paper using the FINK solvent [lo] system was used to separate TdR from its degradation products. A portion (30 ~1) of TCA-soluble material was spotted onto the paper with a marker mixture composed of TdR, dTMP and dTTP, and then eluted with ammonium formate solvent. The strip was scanned in ultraviolet light to locate the reference compounds and then cut into 1 or 2 cm segments, suspended in Scintillator, and counted in a Spectrometer (Packard).

Preparation

of cell extracts

All manipulations during the preparation were carried out at 0-4”C. The cell extracts were nrenared as nreviously described [8]. Briefly, the ceils *were pefieted, washed with TKMB buffer (10 mM KCI,. 1 mM MgCl,, 1 mM /3-mercaptoethanol and 50 mM Tris-HCl, pH 7.5) containing 0.25 M sucrose then centrifuged at 1600 g for 6 min. The cells resuspended at 10x 1Og cells/ml in the TKMB buffer were disrupted in a Dounce homogenizer. The homogenate was centrifuged at 1600 g for a 6 min to sediment nuclei and cell debris, followed by a further centrifugation at 20000 g (Sorvall rotor SS 34) for 10 min to sediment organelles. The supernatant after a further centrifugation (at 105Ooog for 60 min in Spinco rotor SW 50.1) was used as a source of TdR kinase, thymidylate kinase and DNA uolvmerase. The* crude nuclear preparation was suspended in TKMB buffer containing 0.3% Triton N,,,. and allowed to stand for 10 mm at 0-4”C. The nuclei were centrifuged, washed twice in buffer without Triton N,,, resuspended in TKMB buffer containing 0.1 M sucrose and treated with an eaual volume of 4 M NaCl at OXC for 2 h; this was followed by centrifugation at 150000 a for 4 h (rotor SW50.1). The suwmatant was diaiysed for 24 h against TKMB buyer. The precipitate of nucleoprotein was removed by centrifugation at 20000 g for 10 min. This nuclear preparation was tested only for DNA polymerase, since we have not detected significant thymidine and thymidylate kinase activities in nuclei.

Enzyme assays kinases. The rate of phosphorylation of deoxythymidine to the corresponding nucleotide (dTMP) and of dTMP to the nucleotides

TdR and thymidylate

Table 1. Incorporation

of labelled precursors of KB cells grown at 37°C and 4°C”

TdR metabolism

at low temperature

into the TCA-soluble

and -insoluble

cpm in nucleotides

321

material

cpm in total TCAsoluble material

cpm in TCAinsoluble material

Temperature and time of culture

Monophos- Diphosphate phate

Triphosphate

Total

%

DNA

%

37°C [2 h]* 4°C [1 h 301

3 630 9 220

4 590 10 120

183 070 93 150

191 2.90 112 500

100 59

265 990 5 650

100 2.1

4°C [12 37°C [1 hlC h]

920 45 570

8 920 120

241 51 710 140

253 66 830 550

133 35

330 247 3 360

9::;

D Pulse labelling 20 min in the presence of 8 &i * Initial value. c Culture returned to 37°C.

of [3H]TdR/10B cells.

(dTDP+dTTP) were taken as a measure of TdR kinase and of thymidylate kinase activity respectively, and determined by the use of PH]TdR and [3H]TdR monophosphate as substrates. In the course of our experiments, we have found that TdR and thymidylate kinase activities can be separately detected by the different requirements of Mgz+ ion concentration in their reaction mixtures. TdR kinase requires 1 mM Mg*+ to convert only into TMP whereas thymidylate kinase requires 20 mM Mg*+ to convert into TDP+ TTP [ 111. The incubation mixtures contained, in a final volume of 250 ~1, 50 ~1 of enzyme solution; Tris-HCl buffer 25 @moles (pH 8.0 for TdR kinase and pH 7.5 for thymidylate kinase); ATP 2.5 pmoles; pGK (3-phosphoglyceric acid, barium salt) 3 pmoles; MgCl, 0.25 pmoles for TdR kinase and 5 pmoles for thymidylate kinase; 1.25 pmoles non-radioactive TdR containing 0.5 &i of [3H]TdR (spec. act. 20 Ci/mM, Amersham) for TdR kinase and 2.5 &i of rH]TMP (spec. act. 1 Cilmmole, Amersham) for thymidylate kinase. After 15 min of incubation at the different temperatures (4°C and 37”C), the reactions were terminated by heating at 100°Cfor 2 min. For TdR kinase, 50 pl was spotted on 20 mm diameter DEAE 81 paper discs and unphosphorylated TdR was washed from the discs with 1 mM ammonium formate and water. The discs were then dried and counted. For thymidylate kinase, 30 ~1 samples were chromatographed as previously described (see Materials and Methods).

and enzyme solution was preincubated at 37°C for 30 min. The deoxynucleotide triphosphates were then added and the mixture was re-incubated for 30 min. At the end of this time the reaction mixtures were transferred to a Whatman GF/C glass fiber disc. DNA was precipitated with 5% cold TCA and label not incorporated into DNA was removed by repeated washings with 5% TCA containing 0.05 M Na,P,O,. The discs were then dried with ethanol and ether.

RESULTS

Cooling a culture of KB cells from 37°C to 4°C results in little immediate cell death as measured by the trypan blue exclusion test; the number of viable cells remains essentially unchanged over the 12 h period in the cold. When the cooled culture is rewarmed to 37”C, however, there is a rapid mortality of some 10% of the cells during the first 2 h, then the culture begins to multiply only slightly less rapidly than an uncooled control culture (fig. 1). This behavior resembles that which we have previously described DNA polymerases. The DNA dependent DNA polymerase assays as previously described [8], con- for HeLa cells [I]. tained in a total volume of 125 ~I:50 ~1 of enzyme The incorporation of r3H]TdR into TCAsolution; Tris-HCl buffer (pH 7.0 for cytoplasmic polymerase and pH 8.5 for nuclear polymerase) 7.5 insoluble material in cooled KEI cells falls pmoles; MgCl, 0.3 (for cytoplasmic polymerase) or very quickly to a level of l-2 % as compared 0.55 pmoles (for nuclear polymerase); KC1 0.5 pmoles; to control cultures held at 37°C (table 1 and 6.25 pg of native or heat denatured calf thymus DNA; 6.25 nmoles each of thymidine triphosphate (dTTP), fig. 4a). The incorporation into TCAdeoxyguanosine triphosphate (dGTP), deoxycytidine soluble phosphorylated material (TMP, triphosphate (dCTP) and deoxyadenosine triphosphate (dATP) containing 0.5 &i of r3H]TTP (spec. act. 16 TDP and TTP), however, falls less quickly, Ci/mMole, Amersham). Before adding the radioactive some 59% of that is of control cells retracer and deoxynucleotide triphosphates. The DNA

322

Ooka and Daillie

Fig. 2. Abscissa: time from start of culture (hours); ordinate: incorporation of [3H]TdR per culture. O-O, at 37°C (cpmX 10’); O-O, at 4°C (cpmx 103); A---A, no. of cells (Xl@). Incorporation at 37°C and at 4°C of [3H]TdR at different phases of cell cycle following release of Kl3 cells in suspension culture from a double 2 mM TdR block. A culture containing 3.6~105 cells/ml was treated twice with 2 mM TdR. After a second block, the cells were harvested by centrifugation, washed with TdR free medium and resuspended in this same medium. After this treatment ceils entered the S phase. Two aliauots of cells (3.6X lo6 cells each) suspension were removed from the cultures at 2 h intervals. One was incubated immediately with [3H]TdR (2 &i/ml) for 20 min at 37°C. The other one was placed at 4°C for 30 min before adding rH]TdR and then incubated in the presence of [SH]TdR (2 @i/ml) for 20 min at 4°C. DNA synthesis was determined from the incorporation of radioactive label into acid-insoluble material.

maining after 90 min at 4”C, and some 35 % after 12 h (table 1). When such cultures are rewarmed to 37°C the incorporation of [3H]TdR into acid-insoluble material regains the initial value observed before cooling the cells within 1 h, then continues to rise over the next 6 h (table 1, fig. 4~). The incorporation into acid-soluble phosphorylated products rises even more rapidly, 133% of the initial value being obServed after the first hour at 37°C (table 1). Samples taken from synchronized cultures at different points in the cell cycle, cooled to 4”C, then allowed to incorporate Exp Cell Res 104 (1977)

[3H]TdR at that temperature showed that the maximum incorporation into acidinsoluble material occurred some 2 h later than the maximum for the mother culture kept at 37°C (fig. 2). The actual amount of incorporation was at all times less than for the control cultures. We have previously described a similar phenomenon using HeLa cells [ 11. The activities of four enzymes concerned with the metabolism of TdR; TdR kinase, thymidylate kinase, nuclear DNA polymerase, and cytoplasmic DNA polymerase, were studied in vitro at 37”C, 21°C and 4°C. By comparison with a maximum value obtained after 2 h of incubation at 37”C, TdR kinase retained some 70% of its activity at 21”C, and about 10% at 4°C (fig. 3). Thymidylate kinase also retained appreciable activity at these temperatures, but TDP was the major product and TTP was more severely diminished whereas at 37°C only TTP could be detected as a product of the

Fig. 3. Abscissa: incubation time (hours); ordinate: enzyme activity measured k-k, at 0°C; l - - -0, at 4°C; O-O, at 21°C; A---A, at 37°C; A-A, at 40°C. (A) TdR kinase; (B) thymidylate kinase; (C) DNA polymerase (a poiymerase) activities measured in vitro by extracts from exponentially growing KB cells as a function of time of incubation. Enzvme activities are expressed as nmoles rH]TMP fo&ed/lS min/5X 1oJ cells for TdR kinase, nmoles [3H]TTP and [3H]TDP formed/l5 min/SX 1DJcells for thymidylate kinase, and pmoles rH]TTP incorporated/30 min/SX 105 cells for DNA polymerase .

TdR metabolism at low temperature

323

plasmic DNA polymerase returned to the initial level over the first 6 h. Thymidylate kinase showed an upswing during the first 2 h, but technical difficulties presented further measurements on this enzyme or on nuclear DNA polymerase.

DISCUSSION Although the cooling of KEI cells to 4°C results in very little immediate mortality, this procedure would seem to result in some Fig. 4. Abscissn: time (hours); ordinnte: (a) % TdR in- degree of damage as about 10% of the cells colporation relative to that at time 0: (bb)% enzvme die quickly after a return of the culture to * . ’ activity relative to that at time 0. 37°C. Of the remaining 90% of viable cells, (a) DNA synthesis determined from the incorporation of [3H]TdR (2 &i/ml, 18 Ci/mM) for 20 min at some may be unable to undergo division as different temperature into acid-insoluble material (cum the slope of the growth curve for the reat time 0= 13b.600 cpm/20 min/3.6X lo6 cells). *--*, Cells held constantly at 37°C; It-- -Ir, cells placed at warmed cultures is a little flatter than that 4°C; +- * -*, cells rewarmed to 37°C. (6) Enzyme activities in KB cells, held constantly at for a control culture maintained at 37°C. 37°C (control), cooled to 4”C, and rewarmed to 37°C. RR cells at 4°C continue to take up -, Cells at 37°C (control); ---, cells at 4°C; -. -, cells C3H]TdR from the medium, and to produce rewarmed to 37°C. A, TdR kinase [activity at time 0= 0.45 nmoles r$HlTMP formed/l5 min/lOB cellsl: A. TMP, TDP and TTP although at a reduced thymidylate Ginaie [activity at time 0=0.21 nmoles rate. The incorporation of labelled TdR into [3H]TTP formed/l5 min/lOg cells]; 0, cytoplasmic DNA polymerase [activity at time 0=0.76 pmoles acid-insoluble products is much more conrH]TTP incorporated/30 min/lOB cells]; 0, nuclear DNA polymerase [activity at time 0=0.21 pmoles siderably reduced; to l-2% of that ob[3H]TTP incorporated/30 min/lOB cells]. served in control cultures. We have observed a parallel reduction in the in vitro activities for some of the enzymes conreaction of thymidylate kinase on the TMP cerned in the utilisation of TdR; whereas substrate. The DNA polymerases retained the activities of TdR kinase and thymidylate only some 9% of their activity at 21°C and kinase at 4°C remained appreciable, that of I-2 % at 4°C (fig. 3). the DNA polymerases was very markedly Enzymes taken from cells incubated at reduced. The amount of TdR kinase in cooled KB 37°C or at 4°C behaved identically in vitro at the two temperatures. When the cells cells diminishes by about 20% during the were cooled to 4°C the quantity of these first 4 h, then remains stable for the rest of four enzymes per cell fell over the first the 12 h period at 4°C. The amounts of thymidylate kinase and the two DNA poly3-4 h, reaching a level of 50% of the initial amount for thymidylate kinase and the two merases are reduced by 50 % over the same DNA polymerases, but remaining at nearly period. It would seem, however, that the 80% of initial amount for TdR kinase (fig. cells retain sufficient enzyme activity for of 46). When the cells were rewarmed to 37°C DNA replication as incorporation the amounts of TdR kinase and of cyto- rH]TdR into acid-insoluble products reExp Cell Res 104 (1977)

324

Ooka and Daillie

commences almost immediately on rewarming the cultures to 37°C. These observations suggest that the reduction in incorporation of tritiated TdR into DNA by cells at 4°C is not due to a deficiency in the phosphorylation process. The production of DNA-could be inhibited by a rate limiting reduction in the phosphorylation of guanosine, cytidine or adenosine, however, Scholtissek [5] found that these nucleosides were actively phosphorylated in chick embryo fibroblasts held at 4°C. The reduction in activity of the DNA polymerases would appear to be sufficient to explain the lower rate of DNA synthesis at low temperature, but it is quite possible that other factors also intervene. Studies of the incorporation of [3H]TdR into cell cultures synchronized by the double TdR block technique evidently suffer from the disadvantage that the intracellular TdR pool is probably considerably altered by the manipulation. We believe, however, that by comparing samples of the same synchronized culture labelled either at 37°C or at 4°C at different points in the cycle we have minimised the consequences of this effect. The delay of some 2 h in the maximum of incorporation of [3H]TdR into

Exp Cell Res 104 (1977)

DNA in the cooled cells as compared to the mother culture controls maintained at 37°C could be due to a greater inhibition of the initiation of DNA chains, than the elongation of previously initiated molecules. An analogous observation has been described for L cells where the initiation of polypeptide chains is much more inhibited than their elongation at low temperature [ 121. We are grateful to Dr T. Greeland for critical reading of this manuscript. We also acknowledge the skilled technical assistance of Miss A. Larama. This work was performed at Unite de Virologie (U 51), INSERM, Lyon, France.

REFERENCES 1. Ooka, T, Girgis, A & Daillie, J, Exp cell res 81 (1973) 207. 2. Cleaver, J E, Radiat res 30 (1%7) 795. 3. Shapiro, I M & Lubennikova, E I, Exp cell res 49 (1968) 305. 4. Nelson, R J, Kruuv, J, Koch, C J & Frey, H E, Exp cell res 68 (1%7) 247. 5. Scrohissek, C, Biochim biophys acta 145 (1967) 6. 7. 8. 9.

- I’bid 145 (1967) 238. Joklik, W K, GJBCo catalog (1968) 60. Ooka, T & Daillie, J, Biochimie 57 (1975) 235. Ives, D H, Morse, P A, Potter, Jr & Potter, V R, J biol them 238 (1963) 1467. 10. Fink, K, Cline, R E, Henderson, R B & Fink, R M, J biol them 221 (1956) 425. 11. Ooka, T, Biochimie 58 (1976) 1135. 12. Craig, N, Cell 4 (1975) 329. Received August 6, 1976 Accepted August 18, 1976