Synthesis of poly(adenosine diphosphate ribose) in synchronized Chinese hamster cells

Prided in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved @X4-4827/78/ll7l-Ol27$02.00/0

Experimental Cell Research 117 (1978) 127-135

SYNTHESIS

OF POLY(ADENOSINE

SYNCHRONIZED NATHAN

DIPHOSPHATE

CHINESE

HAMSTER

RIBOSE)

IN

CELLS

A. BERGER,’ AARON S. KAICHI,’ PALMER G. STEWARD,Z ROBERT R. KLEVECZ: GERALD L. FORREST3 and STEPHEN D. GROSS3

‘HematologylOncology Division, Department of Medicine, Jewish Hospital of St Louis, 3ection Biology, Division of Radiation Oncology, Mallinckrodt Institute of Radiology, 1,2Washington University, School of Medicine, St Louis, MO 63110, and 3Department of Cell Division of Biology, City of Hope National Medical Center, Duarte, CA 91010, USA

of Cancer Biology,

SUMMARY Chinese hamster ovary cells were synchronized by mitotic selection and used to study the relation of poly(adenosine diphosphate ribose) synthesis to DNA synthesis and the different phases of the cell cycle. DNA synthesis was measured in cells rendered permeable to exogenously supplied nucleotides. Poly(ADPR) synthesis was also measured in permeable cells in the presence of both minimum and maximum DNA damage. The maximum DNA damage was produced by treating the cells with saturating concentrations of DNase. As anticipated, the DNA synthesis complex showed its maximum activity during S phase and showed 4-5-fold less activity during the other phases of the cell cycle. The basal level of poly(ADPR) synthesis was elevated during Gl, fell to its lowest level during S phase, then increased during Cl2 and rose to its highest level during Gl. The DNase responsive activity of poly(ADPR) synthesis was relatively constant thru the cell cycle but showed a peak at the end of S phase; then the activity decreased during the subsequent G2-M period.

Using mouse L cells, we have previously shown that the rate of poly(ADPR) synthesis was low in logarithmically growing cells and high in cells that were in the plateau or stationary phase of cell growth [ 1, 21. Since plateau phase cells are arrested in Gl [3,4], these results suggested that poly(ADPR) synthesis might be increased during this phase of the cell cycle. Previous attempts to use synchronized cells to measure fluctuations in poly(ADPR) synthesis [5-73 produced variable results as to which phases of the cell cycle contained the peak activities of poly(ADPR) synthesis. Those studies all made use of different metabolic inhibitors to temporarily arrest the cells in a specific phase of the cell cycle in order to obtain the synchronized populations. Since we have 9-781819

shown that many agents which arrest cell growth have drastic effects on poly(ADPR) synthesis [ 1,2] it seemed possible that some of the differences in the previous studies could have been due to the treatments with the metabolic inhibitors and the subsequent unbalanced cell growth. In contrast to synchronization procedures using metabolic inhibitors, the technique of mitotic selection gives a high degree of synchrony of essentially unperturbed cells [S]. Since the technique of mitotic selection has been adapted to large scale studies for Chinese Hamster Ovary (CHO) cells [9, lo], we chose to use these cells to study the cell cycle changes in poly(ADPR) synthesis. Before doing so, we confirmed that CHO cells showed high levExp

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els of poly(ADPR) synthesis in plateau and caffeine inhibit the reaction and also by phase and low levels of poly(ADPR) syn- demonstrating that venom phosphodiesterthesis in log phase as previously demon- ase but not DNase or RNase degrade the strated for L cells [ 1,2]. We then proceeded polynucleotide product [l, 21. The advanto characterize the rates of poly(ADPR) tages of measuring poly(ADPR) synthesis in synthesis and DNA synthesis as a function permeable cells as opposed to isolated nuof the cell cycle in the CHO cells synchro- clei have been described both by us [ 1, 21 and by Shall and co-workers [13]. In brief, nized by mitotic selection. the mild treatment involved in cell permeThe synthesis of DNA and poly(ADPR) abilization permits the basal level of polywas measured in CHO cells synchronized by mitotic selection and then made perme- (ADPR) synthesis to be measured [ 1,2, 131, able to exogenously supplied nucleotides whereas the process of nuclear isolation [ 1, 2, 111.DNA synthesis in the permeable causes DNA damage and variable stimulacells is measured by incorporation of radio- tion of poly(ADPR) synthesis [13]. We also activity from r3H]dTTP into DNA. This found that when permeable cells were system requires the presence of all four treated with DNase plus 0.05% Triton Xdeoxynucleoside triphosphates, ATP and 100 to allow the DNase to have rapid access Mg2+; it is inhibited by sullhydryl binding to the nuclear DNA, the degradation of the agents such as N-ethylmaleimide [ 111.DNA cell DNA was associated with a rapid insynthesis in the permeable cells is of the crease in rate of poly(ADPR) synthesis [l]. semiconservative replicative type and pro- At saturating concentrations of DNase, the duces high molecular weight DNA inter- DNase responsive activity can be used to measure the maximum attainable rate of mediates [12]. The DNA synthetic activity in the permeable cells fluctuates with the poly(ADPR) synthesis. Thus, these two asreplicative activity of the cells [I 11. Since says, in which the DNA is minimally or the reaction measured in the permeable maximally perturbed, were used to measure cells probably reflects the combined func- the basal and maximally stimulated levels tion of many of the components of the rep- of poly(ADPR) synthesis. lication fork it is subsequently referred to as DNA replicase activity rather than strictly DNA polymerase activity. The measureMATERIALS AND METHODS ment of DNA replicase differs from measurements of r3H]TdR incorporation in that Cell culture and synchronization the latter are influenced by the rates of up- CHO cells were synchronized by mitotic selection roller bottles using the commercially available take and phosphorylation and the pool sizes from automated synchrony system (Talandic Research of thymidine as well as by the activity of the Corp., Pasadena, Calif.) described in detail elsewhere [9’J. Four roller bottles were inoculated with 5x10’ DNA replication complex. cells/roller in 100ml McCoy’s 5a medium/roller. MitoTo measure the basal level of poly- tic selections were performed ltJ4J h after subculPrior to selection, two changes of medium were (ADPR) synthesis, the permeable cells are ture. rinsed through the rollers. This rinse to remove unsupplied with r3H]NAD in the presence of attached cells was followed by rotation of the bottles at 2S&300 rpm in 30-35 ml medium for l-2 min. The Mg”+ and the incorporation of [3H]ADPR first high-speed selection was discarded and after an into poly(ADPR) is followed [ 1,2]. The spe- additional hour of rotation at slow speed (0.5 tpm) a second selection was performed. These and subsecificity of this reaction has been confirmed quent selections were automatically inoculated into 120 ml spinner cultures at a concentration of 24X 1W by showing that both 5-methylnicotinamide Exp Cdl Rrs 117 (1978)

Synthesis of poly(adenosine cells/ml. One selection was plated in a 75 cm2 flask and examined using an inverted microscope and timelapse video tape recorder.

Video tape recording Cell divisions were scored by continuously monitoring a field of the synchronous cultures using a Nikon MS inverted microscope coupled with a Hitachi SV512 or 512A time-lapse video tape recorder and HV16A low light vidicon cameras as described previously [14]. Timing of cell divisions was registeredby writing the imaged time (from an MMS model CL-l timer generator) of occurrence of each anaphase directly onto the video screen over the dividing cells. These times were subsequently tallied and grouped into half-hour classes. Midpoint (time of occurrence of 50% of divisions) was selected as the most representative statistic to determine generation time. The results were similar if modal values were used. From these studies it was determined that mitotically selected CHO cells had a generation time of 12 h.

Thymidine labeling and autoradiographic estimates of S phase Tritiated thymidine (rH]TdR, 6 Cilmmole) was purchased from New England Nuclear and was used within a month of the purchase date. Chamber slides were inoculated with 5x1s CHO cells, and [3H]TdR at 5 &i/ml was added for 60 min at hourly intervals through the cycle. Following exposure to the isotope and in preparation for autoradiography, monolayers were washed twice in cold Hanks’ BSS. Soluble pools were extracted with two 4”c, 10% TCA washes, and the monolayers rinsed in 4”C, 10% potassium acetate in 80 % ethanol. Cells were then washed twice in 95 % ethanol at room temperature. Autoradiographs were prepared using Kodak NTB-2 liquid emulsion. Slides were routinely developed after 7 days exposure. Autoradiographs were developed for 2 min in Kodak D-19 at room temperature, rinsed and fixed in Kodak Fixer. The washed, air-dried slides were then stained with Giemsa, and coverslips were mounted with Permount.

Flow microfluorometry cells were removed from the spinner culture, centrifuged and resuspended in Hanks’ BSS. After a second resusnension of the &let in 5 ml of BSS. an equal volume of 8% glutaraldehyde in pH 7.2 phosphate buffer was added. Cells were allowed to fix overnight in the cold and then centrifuged and washed once in distilled water. The pellet was resuspended in ethanolic Ba(OH), (6 ml room temperature saturated Ba(OHh in 100 ml 70% ethanol) and the RNA hydrolyzed with agitation for 1 h at 37°C [15]. Cells were centrifuged, rinsed once in distilled water and pelleted again. Cell pellets were resuspended in propidium iodide (Calbiochem, 50 mg/l) and allowed to stain for 30 min before analysis in an Ortho-Biophysics Cytofluorograf. Excitation of DNA specific fluorescence was by means of 488 nm coherent light from a 10 mW argon laser. Whenever possible 50000 cells/ sample were assayed as described previously [ 161. 4xlW

diphosphate

ribose)

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Analysis of FMF data A simple computer method was developed for analysis of the Flow-Microfluotimetry (FMF) data to determine the fraction of cells in a sample in each of the Gl. S. or G2 chases. (In this amdvsis mitosis is not recognized as a separate phase; ceRs before division are a part of G2 phase and following division a part pf Gl phase.) The analysis was based on the rationale that the amount of fluorescence was linearly related to the channel number and also that the fluorescence was linearly related to the DNA content per cell. Thus the channel number for the Gl peak will be located at about half the channel number of the G2 peak. We also assumed that the Gl peak and the G2 peak are each distributed normally. In contrast, S phase cells are distributed among a large number of different channels (approx. 50 channels in this case) due to the inherently broad distribution of DNA content. Finally, for two subpopulations of cells, each with an identical DNA content, the relative uncertaintv of their distribution among the channels should be approximately constant; i.e., the normal distributions describing the Gl and G2 peaks should have approximately the same coefficient of variation. A PC 12/7 comuuter with a scone and an analog to digital converter ‘(A/D converterj was used for-the computer assisted analvsis. The FMF data were plotted on the scope with the abscissa corresponding to the channel number and the ordinate corresponding to the number of cells per channel. In addition, two normal distributions were displayed; the amplitudes, coefftcients of variation, and means for each distribution being controlled by rotating three separate potentiometers on the A/D converter. By manipulation of these potentiometers, one of the normal distributions was superimposed upon the Gl peak and the other upon the G2 peak. Since the FMF data were obtained for a synchronous population of cells, samples taken at certain time points demonstrated distorted Gl or G2 peaks due to the large number of cells either entering or completing DNA synthesis. In these situations the guideline for placing the normal distribution was the fact that the mean for the distribution corresponding to the G2 peak should be approximately twice the channel number of the mean for the distribution corresponding to the Gl peak. Furthermore, the coefficients of variation for the two peaks should be approximately the same. In fact, for the samples analyzed, the ratio between the means of the G2 and Gl beak distributions was 1.94f0.05 (mean f standard deviation), and the coefficients of variation for the Gl and G2 peaks were 8.15+0.94% and 8.45+ 1.01%, respectively. This computer-assisted positioning of the normal distributions over the Gl and G2 peaks was the basis of the analytical method. The number of cells in Gl (or G2) phase was taken as the integral of the normal distribution for Gl (or G2) phase. In theory, half of the Gl and the G2 populations, and probably all of the S phase populations, lie between the two channels corresponding to the means of the two normal distributions. Therefore, the number of cells in S phase was computed by summing the data between these two channels and subtracting half of the Gl plus G2 populations. As an indication of the internal consistency of the analytical method, E.xp CrURes

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Table 1. Variation of DNA and poly(ADPR) synthesis in asynchronously growing Chinese hamster ovary cells

Growth phase Loi3 High density

DNA synthesis (dpm/W cells)

Poly(ADPR) synthesis (dpm/ 105cells) Basal

DNase responsive

14 618

294

3 936

3 015

1 761

4 053

Log phase cells were at a density of 3.4X loS/ml, high density cells were at 1.4~ 1OVnl. Cells were permeabilized and suspended at 2.5~ 10’ cells/ml. Assays for synthesis of DNA or poly(ADPR) were performed as described in Methods.

The assay system to measure basal or physiologic activity of poly(ADPR) synthesis was composed of 0.2 ml containing 5x lo6 permeabilized cells in permeabilizing buffer and 0.1 ml of the reaction mix. The reaction system to measure the DNase responsive poly(ADPR) polymerase activity contained, in addition to the components listed above, 0.05% Triton X-100 and 100 pg DNase. All components were combined in an ice water bath. Reactions were started by transferring the tubes to a 30°C water bath where they were incubated for 30 min. They were terminated by precipitation with 10% TCA and 2% Na4PZ07, COIlected on GF/C discs, washed and prepared for scintillation counting as described above.

RESULTS

Table 1 presents the levels of DNA synthesis and poly(ADPR) synthesis at different time points in an asynchronous culture of CHO cells. As previously noted with for each sample the sum of the cells in the three phases L cells [ 1, 21, the transition from log growth was compared with the sum of the data points for that to plateau phase was associated with a desample. Considering all samples, the maximum difference between these two numbers was 8 % and for more crease in DNA replicase and an increase in than half of the samples they were within 2% of one the basal activity of poly(ADPR) synthesis. another. When the cells were treated with DNase to damage the DNA, the maximal or DNase Cell permeabilization and nucleic responsive activity was the same in log and acid synthesis After all mitotic selections were performed, the cells plateau phase cells. from each spinner culture were counted, then collected Fig. 1 presents the levels of DNA synby centrifugation at 1000 g, 10 min, 4°C. The cells were permeabilized as previously described [l, 2, 11, thesis and poly(ADPR) synthesis in CHO 121by a 15 min cold shock in 0.01 M Tris HCI, pH cells synchronized by mitotic selection 7.8, 1 mM EDTA, 4 mM MgCl*, 30 mM 2-mercaptoethanol. The cells were collected again by centrifugafrom roller bottle cultures, then maintained tion and suspended in the same buffer at 2.5~ 10’ cells/ in spinner culture until the time of assay. ml before being dispersed into reaction tubes. The reaction mixture for DNA synthesis contained During this procedure, the cells were con0.1 M Hepes pH 7.8, 0.02 M MgC1,/0.21 M NaClIl5 tinuously in complete medium at 37°C. The mM ATP10.3 mM dATP/0.3 mM dCTP/0.3 mM dGTP/ 2.2 FM [3H]dTTP (spec. act. 45x 106dpmlnmol) (New upper panel shows the results of the autoEngland Nuclear). The standard incubation system was composed of a 50 ~1 aliquot containing 1.25~ lo6 radiographic study to determine the percent permeabilized cells plus 25 ~1 of DNA synthesis mix. of cells labelled during serial 1 h pulses with The components were combined in an ice water bath and the reactions started by transferring the tubes to r3H]TdR. Each time point represents the a 37°C water bath. Reactions were terminated after 30 percent of cells labelled during the precedmin by precipitation with an excess of cold 10% TCA, ing hour’s incubation, i.e., the 12% labelled 2% Na4P20,. Precipitates were collected on GF/C filters (Whatman), washed five times with TCA, cells at 3 h represents the percentage of NapsO,, twice with ethanol, then dried. The discs were treated overnight with 0.5 ml protosol (New Eng- cells incorporating r3H]TdR during a pulse land Nuclear), then toluene, PPO, POPGP scintillation label from hour 2 to hour 3. These data influid was added and radioactivity determined by dicate that S phase began approx. 3 h after scintillation counting [ 11. The reaction mixture for poly(ADPR) synthesis con- mitotic selection, peaked at 6 h after selectained 100 mM Tris HCl pH 7.8/120 mM MgClJl mM tion, showed a second burst of DNA synnicotinamide [2,VH]adenine dinucleotide (rsH]NAD) (spec. act. 20X 10sdpmlnmol) (New England Nuclear). thesis at 9 h after selection and ended by 10 Exp Cdl Res 117 (1978)

Synthesis of poly(adenosine

60 40 i -20

IO 8 6

-0

4 2

:a 5

IO

I5

I

Fig. 1. Abscissa: time after mitotic selection (hours); ordinate: (a) % labelled nuclei (O-O); (b) % cells in fraction. O-O, Gl; A-A, G2+M; A-A, S; (c) (left) DNA synthesis (dpmx 1O-3/1(r cells) (4-e); (right) poly(ADPR) synthesis dpmx 1O-5/1o6 cells) (O-cl, W-W). Variation of DNA synthesis and poly(ADPR) synthesis in synchronized Chinese hamster ovary cells. Cells were svnchronized bv mitotic selection. (a) Autoradiographic analysis of-cells synthesizing DNA: Cells were oulse-labelled with f5Hlthvmidine for 1 h periods. Percent labelfed nuclei ce) in each sample were determined as described in Methods. (b) Microfluorometric analysis. DNA content/cell was examined bv FMF. The oercent of cells in each ohase of the cell cycle was detkmined as described in’Methods. Cells in Gl (O-O). in S (A-A). in G2+M (A-A). (c) Nucleic acid iynthesi‘s in permeable cells. Synchro: nized cells were permeabilized, then used to measure synthesis of DNA and poly(ADPR) as described in Methods. DNA svnthetic activitv (6 - 4 ): results are expressed as nu-hoactivity incorporated.& 105cells. Poly(ADPR) synthetic activity (E-Cl); DNase responsive poly(ADPR) synthesis (D-m); results are expressed as radioactivity incorporated per 10scells.

h after selection. This burst of DNA synthesis before cells leave S has been noted previously [14, 171.The fact that 80 % of the cells were labelled at mid-S demonstrates that a high degree of synchrony was

diphosphate

ribose)

131

achieved. A second round of DNA synthesis began approx. 14-15 h after mitotic selection and demonstrated progression into the second S phase. Fig. 2 illustrates the FMF curves obtained from the CHO cells at hourly intervals after mitotic collection. The computer assisted assignments for cells in Gl, S, and G2, indicated by the different cross-hatched areas, were made according to the principles outlined in Methods. The values obtained from this analysis were used to plot the middle panel in fig. 1. The data from the FMF analysis demonstrate that the cells entered S between 2-4 h after mitotic selection. The percentage of cells in S peaked between &8 h after selection and then decreased between 9-11 h. The maximum percentage of cells in S phase, measured by FMF, was 65% compared with 80% in S as determined by the autoradiography data. This difference has been noted previously; i.e., FMF underestimates the proportion of cells in S phase as determined by autoradiography [9, 161. This is probably due to the fact that some of the cells in early or late S fall under the Gl or G2 peaks respectively on the FMF analysis. The FMF analysis indicates that in the first 2 h after mitotic selection, approx. 60% of the cells showed a Gl DNA content whereas 27-30 % of the cells showed the G2 DNA content. As previously demonstrated [9], it is probable that the cells which appear to be in G2 at these times actually represent Gl doublets which have not yet completely dissociated. Taking these cells into account, approx. 90 % of cells were in Gl during the first 2 h after mitotic selection. When the S phase cells decreased between hours 9-11 there was a concomitant increase of cells in the G2 phase. The percentage of cells in G2-M peaked at 11 h Exp Cell Res 117 (1978)

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I”

595%G,. 13%5,27A%Gz

6H 222%G,, 649%S,129%G~

IAH 536%G, 15515. 30.9IG1

,iH 508%G, ,74%5,3,8%Gz

0

140

0

140

Fig. 2. Abscissa: channel no.; ordinate: cells/channel. CHO cell cycle analysis. Cells were synchronized by mitotic selection and maintained in spinner culture until analyzed. Each successive panel shows the FMF curves obtained at hourly intervals after mitotic selection. The time after mitotic selection is indicated in the upper right comer of each panel. Computer assisted assignment, as outlined in Methods, was used to determine the fraction of cells in each phase of the cell cycle. The results are illustrated by different striped areas under the FMF curves. The Gl area is indicated by vertical stripes, S is indicated by horizontal stripes and G2 is indicated by diagonal stripes. The percentage of cells in each phase of the cell cycle was calculated from the area under the curves. These percentages are indicated in the upper right comer of each panel.

after selection and then began to decrease. Twelve hours after selection the cells began to enter Gl again. Erp Cell Res 117 (1978)

On the basis of the autoradiographic and FMF analysis the cell cycle parameters were established as follows: Cl phase O-3 h, S phase 3-10 h, G2 plus M phases lO12 h, second Gl phase 12-15 h. The generation time or time to complete one cell cycle was 12 h. This is in agreement with the videotape analysis for anaphase frequencies, which also indicated a 12-h generation time. These cell cycle parameters are also in agreement with those obtained previously for CHO cells [9, 143. When nucleic acid synthesis was measured in the synchronized cells, the activity of the DNA replication complex showed approximately a 3-fold increase in activity during S phase. The peak of activity occurred between 5-6 h after mitotic selection and corresponded to the first peak in the autoradiographic data. It is interesting to note that as the cells left S phase, the declining activity of DNA replicase demonstrated a shoulder at 9 h which corresponds to the second burst in DNA synthesis measured by the autoradiographic techniques. The basal level of poly(ADPR) synthesis was elevated when the cells were in Gl. The activity subsequently declined as the cells entered mid-S phase, then increased again as the cells entered G2. The activity continued to increase and showed its highest value after the cells divided and returned to Gl phase. In contrast to the basal activity, the DNase responsive or maximal poly(ADPR) polymerase activity remained relatively constant during the first 7 h following selection. During the next 2 h there was a 1.5-fold increase in activity. During the subsequent hour, as the cells entered G2-M, the DNase responsive activity decreased to its previous level where it remained for the remainder of the cycle. We have previously suggested that the DNase responsive activity measured the total

Synthesis of poly(adenosine diphosphate ribose) available poly(ADPR) polymerase in the cells [l, 21. These studies suggest that the amount of enzyme in the cell is relatively constant throughout most of the cell cycle. The increase in DNase responsive activity from hours 7-9 suggests that the enzyme protein may be synthesized during the end of S and then the enzyme is restored to its usual level as the cells subsequently divide. DISCUSSION We interpret these studies to indicate that synchronous CHO cells demonstrate their highest levels of poly(ADPR) synthesis in Gl and G2 phases with the greatest activity being in Gl. The rate of poly(ADPR) synthesis decreases to a minimum during S phase, at which time the DNA replication complex reaches its maximum activity. In contrast to the fluctuations in the basal rate of poly(ADPR) synthesis, the DNase responsive poly(ADPR) synthetic activity was relatively constant throughout the cell cycle until late S-early G2, when the activity suddenly increased, then returned to the steady level coincidently with cell division. Since the DNase responsive activity appears to represent the maximum measurable activity, we suggested that this represents the total amount of enzyme protein in the cells capable of responding to this DNA degradation [ 1, 21. According to this interpretation, the increased activity of DNase responsive poly(ADPR) synthesis at 9-10 h after selection probably represents synthesis of new enzyme at the end of S phase, just before the cells prepare to divide . Before comparing these results to those previously described, it should be noted that different results could occur with different cell types. It is also important to

133

reiterate that damage to DNA increases the rate of poly(ADPR) synthesis [l, 2, 13, 181. Thus, Shall and co-workers compared the activity of poly(ADPR) polymerase in isolated nuclei and permeable cells and demonstrated that the isolated nuclei showed the highest levels of poly(ADPR) synthesis which correlated with the increased amount of DNA degradation that occurred in the nuclear preparation [13]. On the basis of similar observations [l, 21, we measured poly(ADPR) synthesis under the two conditions described, i.e., using the gentle cell permeabilizing technique to measure physiologic levels of poly(ADPR) synthesis in the absence of DNA damage, and then adding DNase to measure poly(ADPR) synthesis in the presence of maximal DNA dam-

age[l, 21. In the present study, cells were synchronized by mitotic selection. They were maintained in complete medium at 37°C throughout the synchronization and subsequent incubation. Thus our results are not affected by the use of any inhibitors or by the removal of the cells from complete growth medium, either of which can alter the rate of synthesis of poly(ADPR) [ 1,2]. In partial agreement with our studies, Smulson et al. [6] measured poly(ADPR) synthesis in isolated HeLa cell nuclei and found the activity to be highest in Gl and lowest in S phase. Kidwell & Mage used hydroxyurea to synchronize HeLa cells [SJ. Following release of the cells from hydroxyurea they measured poly(ADPR) synthesis in isolated nuclei and found peaks in G2 with low activity at all other points in the cell cycle. It is probable that their measurements of poly(ADPR) synthesis were elevated because of DNA damage incurred during the nuclear isolation procedure. Thus the measurements of poly(ADPR) synthesis would correspond to our DNase Exp Cell Res 117 (1978)

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responsive activity and actually represent measurements of the total or maximal available enzyme rather than of its physiologic activity. They also investigated the synthesis of poly(ADPR) by a more physiologic technique in which a specific antibody was used to follow the incorporation of radioactive adenosine into poly(ADPR) by intact cells [5]. These results demonstrated two peaks of polymer synthesis, one in Gl and another in G2. Thus, their results from the more physiologic approach to poly(ADPR) synthesis agree with our measurements for the basal activity of poly(ADPR) synthesis. Miwa et al. measured poly(ADPR) synthesis in transformed Syrian hamster lung cells after synchronization with 5-benzyl3(I’-anilinoethylidene) pyrrolidine-2,4-dione [7]. At various times in the cell cycle, the cells were lysed and the incorporation of [3H]NAD into poly(ADPR) was studied by autoradiographic techniques. These studies demonstrated peak activities for poly(ADPR) synthesis in Gl and G2 [7]. These workers also isolated nuclei from the cell at one point in each phase of the cell cycle and found that activity was high in all phases, but highest in G2 [7]. Thus the results of the more physiologic autoradiographic assays correspond to our measurements of the physiologic activity of poly(ADPR) synthesis while the measurements in isolated nuclei generally correspond to our measurements of the DNase responsive activity. Our studies demonstrate that at any time during the cell cycle, two different activities can be measured for poly(ADPR) synthesis. The present work also emphasizes the cautionary note indicated by us [ 1, 21 and by Shall et al. [I31 that measurements of physiologic activity of poly(ADPR) synthesis require extremely gentle preparation of E.rp Cd Rrs 117 (1978)

cells so that DNA is not degraded and the enzyme is not stimulated. This stimulation by damaged DNA explains many of the apparent discrepancies noted in previous studies of the activity of poly(ADPR) synthesis in different phases of the cell cycle. Recent investigations of poly(ADPR) synthesis indicate that this enzyme is involved in modifying chromosomal proteins [19, 201 and may be important in regulating the physical and functional status of the chromatin and DNA. The present studies indicate that in CHO cells, poly(ADPR) synthesis is highest in the Gl phase of the cell cycle and lowest in the S phase. This suggests that under normal conditions, this enzyme system has its major function in the non-DNA synthetic portion of the cell cycle. The studies with added DNase show that there is a large reserve of poly(ADPR) polymerase activity in all phases of the cell cycle. Since poly(ADPR) synthesis increases in response to treatment with several different DNA damaging agents [21, 221, it is possible that this enzyme system represents a reserve mechanism involved in the response to and repair of DNA damage throughout the cell cycle. This researchwas supported by grantsto N. A. B. from the National Leukemia Assbc~ation, Inc., Garden City, N.Y. and the Jewish Hospital of St Louis and to R. R. K. from the National Institute on Ageing. N. A. B. is a Leukemia Society of America Scholar.

REFERENCES 1. Berger, N A, Weber, G & Kaichi, A S, Biochim biouhvs acta 519 (1-978)87. 2. Be&e;, N A, We&, G, Kaichi, A S & Petzold, S J, Biochimbiophysacta519 (1978) 105. 3. Nilhausen, K & Green, H, Exp cell res 40 (1965) 166. 4. Macieira-Coelho, A & Berumen, L, Proc sot exp biol med 144( 1973)43. 5. Kidwell, W R & Mage, M G, Biochemistry 1.5 (1976) 1213. 6. Smulson, M, Henriksen, 0 & Rideau, C, Biochem biophys res commun 43 (1971) 1266.

Synthesis of poly(adenosine 7. Miwa, M, Sugimura, T, Inui, N & Takayama, S, Cancer res 33 (1973) 1306. 8. Terasima, T & Tolmach, L J, Exp cell res 30 (1%3) 344. 9. Klevecz, R R, Biotech bioeng 17 (1975) 675. 10. Klevecz, R R & Forrest, G L, Growth nutrition and metabolism of cells in culture vol. 3 (1977) 149. 11. Berger, N A & Johnson, E S, Biochim biophys acta 425 (1976) 1. 12. Berger, N A, Petzold, S J & Johnson, E S, Biochim biophys acta 478 (1977) 44. 13. Halldorsson, H, Gray, D A & Shall, S, FEBS lett 85 (1978) 349. 14. Klevecz, R R, Proc natl acad sci US 73 (1976) 4012. 15. Bruchhaus, H & Geyer, G, Histochem j 6 (1974) 579.

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16. Klevecz, R R, Keniston, B A & Deaven, L L, Cell 5 (1975) 195. 17. Kapp, L N & Painter, R B, Exp cell res 107 (1977) 429. 18. Miller, E G, Biochim biophys acta 395 (1975) 191. 19. (5);:: M G & Stocken, L A, Biochem j 161 (1977) 20. Okayama, H, Ueda, K & Hayaishi, 0, Proc natl acad sci US 75 (1978) 1111. 21. Davies, M J, Shall, S & Skidmore, C J, Biochem sot trans 5 (1977) 949. 22. Berger, N A, Petzold, S J & Skidmore, C J. In preparation. Received April 12, 1978 Revised version received June 7, 1978 Accepted June 12, 1978

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