ABSTRACT. DNA synthesis, for example thymidine kinase (6-8), thymidylate kinase (8),

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1 Volume 4 Number 8 August 1977 Nucleic Acids Research Early effects of Phytohemagglutinin on induction of DNA polymerase, thymidine kinase, deoxyribonucleoside triphosphate pools and DNA synthesis in human lymphocytes Gerda Tyrsted and Birgitte Munch-Petersen Department of Biochemistry C, University of Copenhagen, Panum Institute, Blegdamsvej 3, 2200 Copenhagen N, Denmark Received 6 May 1977 ABSTRACT In Phytohemagglutinin stimulated human lymphocytes the time relationship was determined between induction of the parameters mentioned. The results indicate that the induction occurred in a specific sequence. Thus, a simultaneous increase in the activity of DNA polymerase and thymidinekinase occurred after 15 h of incubation with Phytohemagglutinin. Furthermore, this enhancement occurred 2 h before the expansion of the TTP and dctp pogls and 4 h before the expansion of the datp and dgtp pools. The rate of E H] deoxyguanosine incorporation into DNA increased simultaneouslywith the expansion of the TTPand dctp pools. INTRODUCT ION Human lymphocytes are normally quiescent cells arrested in G or G phase of the cell cycle (1). Cultured in vitro the lymphocytescan be transformed by appropriate mitogens such as Phytohemagglutinin (2). The cells will thenenter the S-phaseof the cell cycle with subsequent cell division. It has been shown that the additionof Phytohemagglutinin to human lymphocytes increases enzyme activities necessary for either DNA synthesis, for example DNA polymerase (3-5), or for the formation of the precursors necessary for DNA synthesis, for example thymidine kinase (6-8), thymidylate kinase (8), deoxycytidine kinase and dcmp deaminase (7). Since we are interested in processes involved in the initiation of DNA synthesis, we have previously studied the pool size of the immediate precursorsfor DNA synthesis, i. e. datp, TTP, dgtp and dctp in nonstimulated and in Phytohemagglutinin stimulatedhuman lymphocytes (9, 10). In non-stimulated lymphocytes we found a molar concentration of about x 10 M of dgtp, dctp and datp whereas the molar concentration of TTP was 2-4 fold lower. At maximal transformation, on the other hand, the molar concentrationof dgtp, dctp and datp increased only 6-11 fold whereas an enhancement of fold was observed for thettp pool. This extreme variation in the pool size of TTP might indicate a controlling functionof one or some of the enzymes leading to TTP formation. C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England 2713

2 We therefore found it of interest to carry out early in transforrration an investigation dealing with the relationship betvween the pool size of ceoxyribonucleoside triphosphates and the activities of thyrridine kinase and DNA polymerase. Part of this work has been reported (1 1). MATERIALS AND METHODS Chemicals. Unlabelled deoxyribonucleotides, deoxyriborucleosides ancl calf thymus DNA from Sigma 3~~~~~~~~~ Chemicals Co., St. Louis. [ HI dctp(spec. act Ci/mmole);[ 3H] dgtp (spec. act Ci/mmole) from Radiochemical Center Ltd., U. K.t 3H]TTP (spec. act Ci/mmole).[ H ] datp (spec. act Ci/mmole).[ 8-3H] deoxyguarosine (spec. act. 6.9 Ci/ mmole) and [Me-3H] thymidine (spec. act. 2 Ci/mmole) were purch.ased frorr. New England Nuclear Corporation. Penicillin, streptomycin and Fischer's medium for leukemic cells of mice were obtained from Flow Laboratories, Irvine Scotland; Ficoll from Pharmacia, Uppsala, and Isopaque frcrr. Nygard and Co., Oslo. Phytohemagglutinin from Difco Laboratories, Detroit, Michigan. The large fragment of DNA polymerase from E. Coli (12) which contains polymerase and 3'-5' exonuclease activity was kindly donated by H. Klenow. Isolation and incubatiorn of human lymphocytes. Peripheral blood was obtained from medical students and the lymphocytes were isolated by the Isopaque-Ficoll gradient centrifugation technique (9). The cells were cultured at a cell density of 10 cells per ml in donor's serum and Fischer's tissue culture medium. The medium was supplemented with sodium bicarbonate, g per liter medium. Glutamine, penioillin and streptomycin were added as described previously (9). The lymphocytes were stimulated by addition of Phytohemagglutinin to the cultures in amounts which gave maximal transformation, measured 3 by the rate of [Me- H] thymidine incorporation into DNA as described earlier (5). Preparation of extract for determination of enzyme activities. The celis were centrifuged at 500 x g for 5 min, the centrifuge tubes were drained and the cell pellet was resuspended in a buffer containing: 0.02 M potassium phosphate (ph 7. 4), 20% w/v glycerol, 1 mm dithiothreitol and 1 mm EDTA. The cells were disrupted by sonication with a MSE Ultrasonic disintegrator for 3 x 10 seconds with intervals of one minute at a frequency of 20 khz. The homogenates were centrifuged for 30 min at x g and the supernatant was used as enzyme source. All operations were carried out at 40C For measuring enzyme activities, deoxynucleotide pool size and the rate of DNA synthesis in the same experiment, it was necessary to use cells 2714

3 from two donors to obtain sufficient amounts of lymphocytes. The cellular material from the two donors was combined in the ratio of 1: 1 after cell disruption. Enzyme assays. The thymidine kinase activity was assayed by measuring the phosphorylation of labelled thym'idine to thymidine mono, di and triphosphate. The standard assay mixture contained in a final volume of : pnoles Tris-HCI buffer ph 8. 0 (22 C), p,moles MgCI p,moles ATP, 45 ILmoles [Me-3H] thymidine (spec. act. 500 Ci/mole) and crude extract 6 o corresponding to 3-5 x 10 cells. After preincubation for 5 min at 37 C the enzymatic reaction was started by addition of crude extract to the assay mixture, and a different intervals 40 Il samples were applied to 2 x 2 cm paper squares of DEAE-cellulose 81 (13). The non-phosphorylated thymidine was eluted from DEAE-cellulose by washing 3 times for 5 min in 5 mm ammonium formate (20 ml per paper square) and then in water and ethanol for 5 min, respectively. The radioactivity was measured in a Nuclear Chicago liquid scintillation counter. The counting efficiency of the tritiated compounds was determined in a Packard sample oxidizer. The conditions for the enzyme assay as described above were found to be optimal for the kinase activity in crude extract from human lymphocytes cultured less than 30 h in the presence of Phytohemagglutinin (14). The enzymatic reactior rate was linear with time for at least 30 min based on 5 time samples and also with varying concentrations of crude extract. The enzyme activity is expressed as pmoles of thymidine phosphorylated per hour per,ug of DNA. DNA polymerase activity was determined at the optimal conditio'ns as described earlier (5). The enzyme activity is obtained as the difference between radioactivity incorporated with all four deoxyribonucleoside triphosphates present in the assay mixture and that incorporated with a single deoxynucleoside triphosphate present. The polymerase activity is then expressed as pmoles dtmp incorporated into acid-insoluble product per hour per,ug of DNA. Measurements of deoxyri bonucleoside triphosphates The deoxyribonucleoside triphosphates were extracted from lymphocytes with 60% ethanol. After evaporation of the pooled supernatants the pool size was determined by using synthetic copolymers, i. e. poly (d(a-t)) and poly d(d(l-c)) in a DNA polymerase catalyzed reaction as previously described (9, 10). The determination was performed in either duplicate or triplicate with different volumes of extract added to the assay mixture. Standard curves were per- 2715

4 formed for each experiment, and the pool size is expressed as pmoles deoxyribonucleoside triphosphate per mg DNA. Determination of the incorporation of precursors into DNA. The transormation of the lymphocytes from the two donors was followed by measuring the rate of incorporation of labelled thymidine into DNA. Triplicate 2 ml lymphocyte cultures from each donor were labelled for 1 h at 370C with [ Me-3H ] thym i di ne (0. 5,uC i/m I of ce I I s, f i na I med i um concentra t ion 2. 5 x 10 M). The radioactivity incorporated into DNA was determined by the glass fiber filter technique as described earlier (9). The results are given as the average of triplicate cultures and expressed as cpm incorporated per 10 cells initially present in the cultures. An average of the DNA content and rate of thymidine incorporation in the lymphocytecultures from the two donors was obtained by isolating labelled cellular DNA in the following way: Aliquots (2 x 12 ml) of cells were removed at 370C from each donor's lymphocyte culture and transferred to prewarmed 30 ml medicine bottles. The cells from the two donors were kept separately until inactivation with 0.2 M HCIO4. The cell aliquots were labelled, with [3H] thymidine (1,uCi/ml of cells, final medium concentration 5 x 10 M) or[ H ]deoxyguanosine (8. 3,uCi/ml of cells, final medium concentration 1. 2 x 10 M), respectively. After equilibrating the gas phase with atmospheric air containing 5% C02, the cells were harvested after incubation at 370C for 1 h with the isotopes. Duplicate 5 ml aliquots of cells were transferred to icecold centrifuge tubes. After centrifugation at 40C for 5min (500 x g) the medium was discarded and the cell pellet suspended in 300 4l ice-cold HCIO4. The HCIO4 suspension obtained from cells labelled with thymidine from the one donor was combined with the respective HCIO4 suspension from the other donors cells. The centrifuge tubes were rinsed twice with 300,ul HC104. With regard to the deoxyguanosine labelled cells the same procedure was performed as described above. DNA was then isolated as described earlier (5) and the amount of DNA was determined by the colorimetric method of Burton (15) with calf thymus DNA as standard. The radioactivity was measured by liquid scintillation spectrometry. The incorporation of radioactivity into DNA is expressed as cpm per ig of DNA per hour. The data are obtained as the average of duplicate samples. RESULTS The transformation of lymphocytes from two donors is illustrated in Fig. 1. The lymphocytes were cultured separately. Phytohemagglutinin was added at zero hour and at the indicated times the rate of labelled thy- 2716

5 z 90 o 70.C/ c o 0,-.C E Hours of incubation with PHA Fig. 1. The rate of thymidine incorporatior (cpm x 10 ) into DNA per 106 cells per h. The cells isolated from donor A. - * and from donor B o - o. midine incorporation into acid-insoluble product was determined by the glass fiber filter technique. The results demonstrate that the predominant rate of thymidine incorporation was observed in the cells from donor A since the incorporation rate in these cells at 27 h is almost 5 times higher than in the cells from donor B. In the following experiments the cellular material from the lymphocyte cultures presented in Fig. 1 is combined in the proportion one to one and determination of the pool size of deoxyriborucleoside triphosphates, enzyme activities and DNA synthesis was performed as described in Methods. The data obtained for the investigated parameters mentioned below are compared to an average value found at 12 and 15 h in the presence of PHA. The cells from the two donors were incubated at 37 C and Phytohemagglutinin was added to the cultures at zero h. After 12 h the pool size of the four deoxyribonucleoside triphosphates, enzyme activities and the rate of radioactive deoxyribonucleoside incorporation were measured and then at intervals of 2-3 h until 27 h after stimulation. The variation in the pool size is shown on a semilogarithmic scale in Fig. 2. The pool size of all four deoxyribonucleoside triphosphates re- 2717

6 it < 400 a z a z E, 200 E b E cx~~~~~~~~~~~~~ a za -5- X50 z E -- - a - ic q11 2 CIL_ _ 18 _ _ 27 _ Hours of incubation with PHA Hours of incubation with PHA Fig. 2. The relationship between deoxynucleotide pools early in transformation. Phytohemagglutinin (PHA) was added at zero h. a) pmoles TTP * - 0, pmoles dctp o - o per mg DNA. b) pmoles datp e - 0, pmoles dgtp o - o per mg DNA. mained constant until 17 h after stimulation. The results shown in Fig. 2a demonstrate that the initial increase in the pool size occurred in the TTP and dctp pools, since at 19 h these pools had increased 1. 8 and 1. 5 fold, respectively. An increase in the datp and dgtp pool was observed two hours later, since at 21 h these pools had increased 1. 8 and 1. 9 fold, respectively (Fig. 2b). The increase in all four deoxynucleotides was exponential until 27 h, except for the dctp pool, es the rate for dctp formation was modified at 24 h. The most pronounced enhancement was observed for the TTP pool, since at 27 h this pool had increased 3-6 fold more than the pools of the other deoxyriborucleoside triphosphates. Whereas the smallest increase (2. 6 fold) was observed for the dgtp pool. DNA polymerase and thymidine kinase activities. The synthesis of DNA is dependent among other things on sufficient amounts of deoxyribonucleoside triphosphates. In view of the observation that the smallest pool in the resting lymphocyte is the TTP pool and that the pyrimidine deoxyribonucleoside triphosphates increase in stimulated cells 2-3 h before the purine deoxyribonucleoside triphosphates, we found it of interest to study or the same batch of cells the correlation between the pool sizes and the activities of DNA polymerase and thymidine kinase. The results in Fig. 3, (semilogarithmic scale) demonstrate that the activity of DNA polymerase and thymidine kinase remained constant until 15 h after stimulation, since we in this and two other experiments have found that the difference between enzymactivities at 12 and 15 h was in the range of 0-7%. At 15 h (Fig. 3)preceding the expansion of the pool size, both enzyme activities increased exponentially. Thus at 17 h the DNA polymerase activity had increased 1. 6 fold, and

7 44 :> - 20 Z > U a U E 10 zn 0 a n Ic 0 W E c >% 5~ Hours of incubation with PHA Fig. 3. The activity of DNA polymerase o - o and thymidine kinase * - * (pmoles per h per,ug DNA). Phytohemagglutinin (PHA) was added at zero h 1000 i 0.' z 300 1oo o d6,0' Ef 30-/.o 0_.,S, E C Hours of incubation with PHA Fig. 4. The incorporation rate of[ H]thymidine *-* and[ H] deoxyguanosine o - o into DNA (cpm per,g DNA per h). Phytohemagglutinin (PHA) was added at zero h. fold after 27 h, whereas at the same time the enhancement in thymidine kinase activity was 1. 3 and 3. 3 fold, respectively. On the other hand the kinase activity during the entire experiment is approximately 3-4 fold greater than the DNA polymerase activity. 2719

8 For obtaining informations about the incorporation rate of radioactive deoxynucleosides into DNA an aliquot of the lymphocyte cultures was labelled with radioactive deoxyguanosine and thymidine 1 h prior to harvesting the cultures for determination of pool size and enzyme activities. The incorporation into DNA and the amount of DNA was determined as described in Methods The results are plotted semilogarithmically in Fig. 4. As seen the incorpo- 3 ration of [ H ]deoxyguanosine remained constant until 17 h after stimulation, followed by an exponential increase until 27 h. At that time the incorporation rate had increased 8 fold compared to the average rate from h. However, the incorporation of [ Me-3H ]thymidine showed a biphasic exponential increase. Thus, a slight increase was observed from h, and a more drastic one from h. The DNA content per 10 cells initially placed in culture was 6. 1,ug at 12 h and 6. 3,ug at 27 h. In similar experiments tritium-labelled deoxycytidine and deoxyadenosine have been employed to measure the rate of DNA synthesis. The results obtained (not shown) are similar to those for[ H ]deoxyguanosine. DISCUSSION In lymphocytes stimulated with Phytohemagglutinin the rate of DNA synthesis begins to increase about 20 h (16) after stimulation, whereas a doubling of the DNA content is found at 72 h (17) or between h (18). With our technique and culture conditions we find that the DNA content per cell increases % of the initial value between h. We have determined the incorporation of precursors into DNA using different[ 3H ]deoxyribonucleosides. Thus the incorporation of deoxyguanosine, deoxyadenosine and deoxycytidine was very similar since the incorporation in stimulated cells increased about 17 h, simultaneously or shortly after the expansion of dctp and TTPF pools. However, in four out of five 3 experiments[ H ]thymidine incorporation into DNA was biphasically exponential. For the moment there is no experimental evidence to interpret this observation. It might indicate that either the present thymidinekinase is activated or new kinases are induced. In fact it is tempting for us to relate the latter suggestion to our findings in Phytohemagglutinin stimulated cells of at least two thymidine kinases, one of them with enzymatic properties different from the enzyme in non-stimulated cells (14). It has been reported, on the other hand, that Phytohemagglutinin stimulated human lymphocytes release thymidine labelled DNA into the medium (19, 20) and are able to perform repair DNA synthesis (21,22). Furthermore Fridlender et al. (23) found that stimulated cells had a higher thymidine uptake than non-stimulated cells. 2720

9 The observed increase in DNA polymerase activity is mainly an increase in DNA polymerase a, since the enzyme activity is inhibited 97% at 12 h and 88% at 31 h in the presence of 1 mm N-ethylmaleimide (unpublished results). This observation is in agreement with results reported by Bertazzoni et al. (21), since they observed early in transformation a more pronounced increase in DNA polymerase a activity than for the 0 -enzyme and they also found both enzymes present in non-stimulated lymphocytes. However, Mayer et al. (18) found only DNA polymerase P activity at 24 h after Phytohemagglutinin addition. Our results, showing an increase in thymidine kinase activity at 15 h differ from those obtained by other authors (7, 8) since they found in Phytohemagglutinin treated human lymphocytes that the thymidine kinase activity began to increase about 36 h and was delayed 12 h with respect to labelled thymidine incorporation into DNA. Bello has studied thymidine kinase activity in synchronized KB cells and suggested that the synthesis of the kinase begins in an activation period prior to DNA synthesis (24). There has been several reports on the initial expansion of the deoxynucleotide pool in synchronized cells. Thus Bray et al. (25) found in synchronized HeLa cells that a sharp rise occurred in the pyrimidine deoxynucleotide pools with the onset of or just prior to DNA replication, while the pools of the purine deoxynucleotides began to rise more slowly at this time. A similar pattern was observed in L 929 (26). In synchronized Chinese Hamster cells Skoog et al. (27) found that the pool size of datp and dgtp increased at the same time as DNA synthesis, while dctp and TTP increased slightly after the onset of DNA synthesis. However, Walthers et al. (28) observed in the same cell system that the time of the initial rise in the pools was similar for each of the four deoxynucleotides and occurred h before initiation of DNA synthesis. However, we found in stimulated lymphocytes that the simultaneous expansion of datp and dgtp pools was delayed h (range of 5 experiments) as compared to the expansion of TTP and dctp pools. The findings in synchronized cells differ thus from those observed in stimulated lymphocytes. This might be due to the fact that the lymphocytes are shifted from resting state to active growth. The sequential induction of the deoxyribonucleoside triphosphate pools in lymphocytes might then indicate a sequential induction of ribonucleoside diphosphate reductase (s) or a sequential activation of the reductase (s) by nucleotide effectors when these have reached appropriate concentrations. Work has therefore been initiated to study CDP reductase activity. A preliminary experiment has 2721

10 shown a pronounced increase in CDP reductase activity at maximal transformation whereas the activity is very low early in transformation. In consequence of the low reductase activity and limiting cellular material we have so far not been able to correlate in time the induction of dctp pool and CDP reductase activity. However, addition of hydroxyurea and Phytohemagglutinin at time zero inhibited CDP reductase activity and the early increase in d- CTP pool (I1). The observed sequential induction of the measured parameters is probably due to regulating mechanisms on transcription/translation levels, since actinomycin added together with Phytohemagglutinin prevented the industion of thymidine kinaseand DNA polymerase activity (8). Furthermore cycloheximide inhibited the increase in DNA polymerase activity and in the pool size of datp and. TTP (5, 8). The observed exponentially increase of the measured activities cannot be interpreted at the moment but could be related to the results obtained by Chiu et al. (29). They have measured T4 induced enzymes involved in initiation of DNA synthesis and the authors propose that T4 DNA synthesis should occur in an integrated complex and the exponential phase of the observed enzyme activities should represent assembly of the complexes. ACKNOWLEDGMENTS We wish to thank Dr. Kay Overgaard-Hansen for reading the manuscript. The skilful technical assistance of Mrs Lisbeth Cloos, Mrs Inger Lyhne, Mrs Gunhild Olesen and Franz Frenzel is gratefully acknowledged. We wish to thank Miss Birthe Rasmussen for making the drawings. The work was supported by a grant from Statens lagevidenskabelige Forskningsrad (Gerda Tyrsted) and Landsforeningen til Kreftens Bekaempelse (Birgitte Munch-Petersen). Birgitte Munch-Petersen has a postdoctoral fellowship from the University of Copenhagen. REFERENCES 1. Baserga,R. (1968) Cell Tissue Kinet NoweIl, P. C, (1960) Cancer Res Loeb,L. A, Agarwal,S. S. and Woodside,A.M. (1968) Proc. NatI. Acad. Sci. USA 61, Rabinowitz, Y., McCluskey, I. S., Wong,P. and Wilhite, B. A. (1969) Exptl.Cell Res. 57, Tyrsted, G., Munch-Petersen,B. and Cloos,L. (1973) Exptl. Cell Res. 77, Rabinowitz, Y., Wong, P. and Wilhite, B. A. (1970) Blood 35,

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