DNA Polymerase and DNA Replication During Lymphocyte 1

(CANCERRFSEARCH30, 2514â€”2520,October1970] DNA Polymerase and DNA Replication during Lymphocyte 1

LawrenceA. Loeb,2 Joan L. Ewald,and ShyamS. Agarwal3 The Institute for CancerResearch, Fox Chase,Philadelphia,Pennsylvania 19111

SUMMARY animal systems (4). Exceptions are those of stationary cell cultures infected with oncogenic viruses and those of organ The initiation of DNA synthesis in human peripheral blood cultures (4). The present study deals with the initiation of lymphocytes is coincident with the induction of DNA poly replication in cultures of human lymphocytes by PHA.4 This merase activity. Phytohemagglutinin was added to cultures of system is particularly advantageous for studying the control lymphocytes, and, at sequential times thereafter, incorpora of DNA synthesis since metabolic changes can be affected tion of thymidine into DNA and various enzymatic activities and monitored entirely in vitro. Furthermore, the influence in the DNA synthetic pathway were determined. An increase of genetic factors and various disease states can be investi of 30- to 150-fold in DNA polymerase activity paralleled in gated. time and magnitude the ability of the cells to synthesize Human peripheral blood lymphocytes are thought to be DNA. An increase in DNase activity also paralleled DNA well differentiated cells at the end stage in lymphoid matura synthesis and the rise in polymerase. The activities of tion. They seldom grow or divide in vivo. This quiescent thymidine kinase and thymidine monophosphate kinase behavior is usually maintained when these cells are cultured. multiplied about 2- to 10-fold. In contrast, kinases such as However, the addition of PHA [an extract of the kidney deoxyguanosine monophosphate kinase and guanosine mono bean (Phaseolus vulgaris)] to lymphocyte cultures causes a phosphate kinase, which were present in greater activity, striking transformation: as many as 90% of the cells enlarge, were not increased by phytohemagglutinin. synthesize DNA, divide, and assume a less differentiated The induction of these enzyme activities by phytohemag appearance (21). A similar but less extensive transformation glutinin appears to require RNA synthesis. Actinomycin D in is brought about by a variety of other mitogens as well as sufficient amounts abolished the phytohemagglutinin appropriate immunological stimuli (22). Alterations in RNA mediated increases in RNA and DNA synthesis, enzyme (6), lipid (13), protein (12), nuclear phosphoprotein (16), activities, and lymphocyte transformation. Smaller amounts and histone (24) metabolism have been reported to occur of actinomycin prevented the induction of DNA polymerase, prior to the onset of DNA replication. We have started to thymidine kinase, and thymidine monophosphate kinase, as study in detail the control mechanism for the initiation of well as the increase in RNA and DNA synthesis, but did not DNA synthesis during lymphocyte transformation. This hinder lymphocyte transformation. Accordingly, the morpho report is on the relationship of enzymes governing DNA logical changes characterizing lymphocyte transformation do synthesis (DNA polymerase, thymidine kinase, TMP kinase, not require DNA replication or increases in enzyme activities dGMP kinase) and degradation (DNase) to DNA replication associated with this replication. In contrast, the stimulation during lymphocyte transformation. of DNA synthesis is rigidly dependent on the induction of DNA polymerase. MATERIALS AND METHODS

INTRODUCTION Cell Culture and Thymidine Incorporation The initiation of DNA synthesis in nonproliferating mam Lymphocytes were isolated from the blood of healthy malian cells has been studied almost exclusively in whole volunteers by the method of Bach and Hirschhorn (3). The final preparation was suspended at a concentration of 7.5 X @ cells/ml in Eagle's minimal essential medium (Spinner modification) (Grand Island Biological Co., Grand Island, 1This investigation was supported by NIH Grants CA-i 1524 and CA-0655 1, and by grants to this Institute from NIH (CA-06927 and N. Y.), containing 20% fetal calfserum, 1%L-glutamine, penidil FR.05539), Stanley C. Dordick Foundation, and an appropriation lin (100 units/mi), and streptomycin (100 @.tg/ml).Portions from the Commonwealth of Pennsylvania. 2M@ a member of the Department of Pathology, School of Medicine, University of Pennsylvania,Philadelphia, Pa. 3Research training fellowship awarded by the International Agency for Research on Cancer, World Health Organization. 4The abbreviations used are: PHA, phytohemagglutinin; DEAE, Received April 7, 1970; accepted June 18, 1970. diethylaminoethyl.

2@14 CANCER RESEARCH VOL.30

(2 ml) of the cell suspension were incubated at 37Â°in the cooling in an ice water bath. Then 0.1 ml of native calf

. absence and presence of 0.05 ml of PHA-M (General Bio thymus DNA (2 mg/ml in 0.02 M KC1) and 0.2 ml of 0.5 N chemicals Corp., Chagrin Falls, Ohio) in 16- x 125-mm perchloric acid were added to each tube. After vigorous disposable plastic culture tubes. mixing and cooling for 20 mm in an ice bath, the digests One hr prior to harvesting, 5.0 pCi of methylthymidine-3H were centrifuged at 8000 X g for 20 mm. Thereafter, 250 j.tl (Schwarz BioResearch, Inc., Orangeburg, N. Y.; 11 of the supernatants containing acid-soluble constituents were Ci/mmole) were added to each culture. Incorporation of pipetted into glass vials containing 1.5 ml of 1.5 M Hyamine labeled thymidine was terminated by adding 10 j.imoles of chloride (Packard Instrument Co., Downer's Grove, Ill.) in unlabeled thymidine and placing the cultures in an ice water methanol, followed by 17 ml of a toluene-phosphor mixture, bath. The cells were then centrifuged for 10 mm at 2500 X and radioactivity was determined. g and washed with 2.0 ml of 0.15 M potassium chloride. To Nucleotide Kinase Activity. The rates of phosphorylations @ the final pellet were added 0.5 ml of a solution made up of of 4C-labeled nucleoside monophosphates were determined 20% (w/v) glycerol (Matheson, Coleman and Bell, East by the method of Furlong (9). Unlike the monophosphate Rutherford, N. J., Spectroquality grade), 0.02 M potassium precursor, the nucleoside diphosphates and triphosphates phosphate buffer (pH 7.4), 0.001 M potassium EDTA, and produced by these reactions are resistant to the action of 0.004 M reduced glutathione. After freezing and thawing semen phosphatase and can be tightly adsorbed onto DEAE twice, the disrupted cell preparations were used for enzyme paper. The reaction mixture for determining TMP kinase assays. The amount of radioactivity incorporated into acid activity contained, in a volume of 0.05 ml in 6- x 50-mm insoluble material was determined concurrently with the culture tubes, the following: 2.15 pmoles of Tris-maleate, pH assay of DNA polymerase activity (1). 8.0; 1.2 pmoles MgCl2 ; 1.15 j.smoles ATP; 0.8 j.tmole phosphoenolpyruvate; 2 j.ig pyruvic kinase; 3.0 mjimoles Enzyme Assays TMP-' 4C, 25,000 dpm/mj.tmole; and 0.03 ml of the dis rupted lymphocyte preparation. Following incubation for 1 DNA Polymerase Activity. The assay for DNA polymerase hr at 37Â°,the reaction was stopped by heating at 95Â°for 3 activity measured the incorporation of an appropriately min. After cooling, 40 jzl of 0.4 M sodium acetate buffer labeled deoxynucleoside triphosphate into an acid-insoluble (pH 4.9) and 10 @zlof human semen phosphatase (18) product (26). The reaction mixture, in a total volume of 03 (approximately20units)wereadded,andthemixtureswere ml, consisted of the following: 25 pmoles Tris-maleate incubated an additional S mm at 37Â°. The tubes were buffer, pH 8.0; 3 pmoles magnesium chloride; 1 pmole centrifuged for 10 mm at 4,000 X g, and 50-j.zl aliquots of potassium chloride; 03 @.tmole j3-mercaptoethanol; 25 the clear supernatant were applied to DEAE paper discs, 2.2 m@zmoleseach of dATP, dCTP, and dGTP; 10 m@@tmolesof cm in diameter (H. Reeve Angel & Co., Clifton, N. J.). The dTTP-[a-3 2P] (approximately 5 X l0@ dpm/mpmole) (Inter filters were washed successively in 0.004 M ammonium national Chemical Nuclear Corp., City of Industry, Calif.); formate, water, and 95% ethanol. Radioactivity was deter 266 mpmoles â€œmaximallyactivatedâ€• calf thymus DNA (19); mined by scintifiation counting. Determinations of GMP and 0.05 ml of the lymphocyte preparation. Incubation was kinase and dGMP kinase activities were carried out in a for 1 hr at 37Â°,and the reaction was stopped by adding 0.5 similar manner. The reaction mixtures (0.05 ml) contained ml of cold 1 M perchloric acid containing 0.01 M sodium 2.15 j.zmoles Tris-maleate, pH 8.0; 0.8 j.tmole MgCl2 ; 0.64 pyrophosphate. With each group of reaction mixtures, a pmole ATP; 0.8 pmole phosphoenolpyruvate; 2 @.igpymvic known amount of purified sea urchin nuclear DNA poly kinase; 2 pmoles KC1 ; and 3 mpmoles of either dGMP-' 4C merase (19) was assayed simultaneously. The acid-insoluble or GMP-' 4C. With dGMP-' 4C as a substrate, 0.03 ml of the material was collected on glass fiber filters, and its radio lymphocyte preparation was used as an enzyme source, and activity was determined by standard dual labeling techniques incubation was for 20 mm at 37Â°.With GMP-' 4C, 0.01 ml with liquid scintillation spectroscopy. After corrections for of the lymphocyte preparation was used and incubation was crossover, the radioactivity in the tritium channel represents for 15 mm. the amount of thymidine-3H which the cells had incorpora Nucleoside Kinase Activity. The reaction mixture for assay ted in culture. In control experiments, there was no decrease of thymidine kinase contained, in a volume of 90 j.zl, the in this value during the DNA polymerase reaction. The following: 8 @tmolesof Tris-HC1, pH 8.0; 0.9 @zmoleof @ amount of radioactivity in the 2P channel represents DNA MgCl2 ; 1.0 pmole of AlP; 1.2 @moles of 3-phospho polymerase activity as measured by the incorporation of glycerate; 5 mj.imoles of thymidmne-' 4C; and 50 @.zlof the residues of TMP-[a-32PJ into an acid-insoluble product. lymphocyte preparation. Incubation was for 1 hi; the DNase Activity. DNase activity was assayed by measuring reaction was stopped by heating at 95Â° for 3 mm. After the amount of added DNA-3H rendered acid soluble. The centrifugation, portions of the supernatant were either incubation mixture contained in a volume of 0.08 ml in applied to DEAE paper discs as in assays of nucleotide culture tubes (6 x 50 mm) the following: 20 mpmoles of kinase activity or chromatographed on strips of DEAE paper partially degraded sea urchin DNA-3 H, 3639 dpm/[email protected]; 8 by the method of Ives et al. (111). i.tmoles of glycine-NaOH buffer, pH 8.5 ; 0.66 pmole of Morphological Transfonnation. The extent of blast transfor MgCl2 ; and 0.04 ml of lymphocyte preparation which had mation was determined by adding 0.1 ml of Velban (Eli been cultured in the absence of radioactive precursors. After Lffly and Co., Indianapolis, md.) (0.5 p.g/ml) to designated 2 incubation for 2 hr at 37Â°, the reaction was stopped by ml cultures. After 2 Kr, the cells were fixed in acetic

OCTOBER 1970 2515

acid:methanol (1 :3). Air-dried preparation was made by the DNase Activity. No DNase activity was evident in initial method of Hungerford (10) and stained with Giemsa stain. studies on the induction of DNA polymerase activity in From each culture, a total of 1000 cells were counted and PHA-stimulated lymphocytes (20). The polymerase reaction classified as small lymphocytes, lymphoblasts, and cells in was linear for 2 hr, and the DNA product of the reaction mitosis. Cell viability was determined by the trypan blue dye was not degraded to acid-soluble oligonucleotides when exclusion technique (23). incubation was carried out for as long as 8 hr. However, when a more sensitive assay in the present study, involving RESULTS use of partially degraded radioactive DNA of high specific activity as @substrate, was used, DNase activity was easily Enzyme Activity during Lymphocyte Transformation. To demonstrated (Chart 2). The increase in DNase activity study the relationship of enzyme activity to DNA synthesis, appears to approximate the increase in polymerase. Exonu we directly compared in the same cultures at various times clease activities are associated with purified Escherichia coil after stimulation by PHA the ability of the cells to incor DNA polymerase (26). By analogy, this DNase activity could porate thymidine and the activities of enzymes associated in part reflect an exonuclease function of the lymphoblast with DNA synthesis. DNA synthesis was determined with polymerase. thymidine-3 H and enzyme assays were carried out under @ optimal conditions with â€˜4C-or 2P-labeled substrates. The ,@ onset and magnitude of DNA synthesis varied in detail in 0@? cultures from different individuals, but the results given here are representative. The increase in thymidine incorporation was first detectable 16 to 18 hr after the addition of PHA. 5Q The rate of thymidine incorporation thereafter increased until maximal at 70 to 90 hr. DNA Polymerase Activity. The amount of thymidine incor .. porated and the DNA polymerase activity were determined on the same samples as detailed in â€œMaterialsand Methods.â€• DNose ACTIVITY In nonstimulated cells, there is little DNA polymerase activity or thyrnidine incorporation (Chart 1). Stimulation by PHA results in a 30- to 150-fold increase in DNA polymerase activity (20). The increase occurs at about the 3 4 I same time as the increase in the ability of the cells to INCUBATION (DAYS) incorporate thymidine into DNA. The enhancement in DNA Chart 2 polymerase activity during the course of PHA stimulation is remarkably parallel to the increase in the rate of thymidine Thymidine Kinase and TMP Kinase Activity. Other enzyme incorporation. Further, once DNA synthesis is diminished, activities functioning in the DNA synthesis are also stimu DNA polymerase activity also decreases. lated by PHA. Thymidine kinase, an activity associated with the â€œsalvagepathwayâ€• for DNA synthesis, was stimulated 20 about 3-fold (Chart 3). Similarly, TMP kinase activity >- . 0 increased at about the same time as DNA replication (Chart

/OÂ½4\\ DNA â€˜5â€˜LI 4). In cells not stimulated with PHA, the activities of these â€œJo. Cg) Polymerose 4 enzymes are constant or, if anything, become diminished I @@@Activityâ€¢0 during a similar period of culture. 1*

â€˜5 y @ 4 0D3- Thymidine Kinase a U I I- @ I 2 :a INCUBATION (DAYS) Chart 1 Charts 1 to 7. The relationship of DNA synthesis to enzyme j 3 5 I activities during lymphocyte transformation. Lymphocytes were INCUBATION (DAYS) I- cultured in the presence of PHA for the times indicated. Enzyme activities were determined as detailed in â€œMaterialsandMethodsâ€•on Chart 3 disrupted lymphocytes that had incorporated thymidine into DNA. Thymidine incorporation is given in cpm; the value on the ordinate is Deoxyguanosine and GMP Kinase. The addition of PHA to to be multiplied by i0@. The latter represents incorporation/0.i ml lymphocyte cultures does not appear to stimulate the of the fmal lymphocyte preparation for 1 hr prior to harvesting the activity of these kinases (Charts 5 and 6). This may be cultures. Fumarase activity was determined by measuring the disap related to the high levels of these activities before PHA pea.rance of fumarate by the method of Racker (25). Each point stimulation. The amount of dGMP kinase activity in non represents the averageof a set of triplicate cultures. stimulated lymphocytes is about 5-fold greater than the TMP

2516 CANCER RESEARCH VOL.30

Initiation of DNA Synthesis

>.â€˜-. kinase activity and 50-fold greater than thymidine kinase

o TMP Kinase activity. @ â€˜@ 0.1 /=@@ r5 U Fumarase. In order to delineate the specificity of induction U@ -I @ U)rn @. ._II / r by PHA, we determined the activity of fumarase, an activity @ z@3 tIE not directly involved in DNA synthesis (Chart 7). As shown, @ @k%â€¢..rn,7 â€˜$,@ the amount of fumarase activity elevates gradually and @ o@o5-1I@I@@JI , @@ @- I â€¢T reaches a maximal increase of about 50%. This increment is

IINCUBATION 3 5@: small compared to that of polymerase and is not temporally (DAYS)Chart related or proportional to the DNA synthetic activity of 4>- these cells. .@ Fumarase @@ dGMPActivity Kinose @@@ .01. I@ h L %@ I @ % In, @â€”IUuI @ @!02 / â€˜ â€˜ 3 5 . @d b'rS â€˜ a -L@@.1 , 1 INCUBATION(DAYS) 5INCUBATION 3 (DAYS)I @- Chart 7 Chart 5 Prevention of Enzyme Induction by Actinomycin

.,. Temporal relationships between RNA, protein, and DNA A synthesis, and the morphological changes characterizing GMP U 5@ lymphocyte transformation are directly compared in Chart 8. Replicate cultures were incubated with PHA for the number Ix of days indicated. One hr prior to harvesting the cultures, the appropriate radioactive precursors were added and their Iz .â€”o-1@' 0 incorporations into an acid-insoluble precipitate were deter

3 5 >. mined. As observed by others (5, 12), one of the earliest I lICUBATION (DAYS) biochemical events induced by PHA is an increase in the rate of RNA synthesis. Later, and perhaps programmed by the Chart 6

z 0 T 0 U) a: 0 E 0@ 0. a: U 0- I ,,@.0@@Thymidine 8z N@) @i0 @ w IZ LL V a- wc5 z 0 0 ow U) @-iJ @ Iâ€” Lymphoblast Â°@ @. w 0 a- I 2 3 4 5 P1-tA INCUBATKDN(DAYS)

Chart 8. Alterations in lymphocyte metabolism induced by PHA. Replicate 2-mi portions of lymphocytes were incubated simultaneously with PHA for the number of days indicated. One hr prior to harvesting the cells, 4 @iCiofuridine-3H (specific activity, 20 Ci/mmole) or 4 MCi of L-leucine-14C(specific activity, 180 mQ/mmole) or 5 MCIof thymidine-3H (specific activity, 11 Q/mmole) were added to the indicated cultures. Incorporation into acid-insoluble material was determined after precipitating the lymphocytes and washing by centrifugation with i N perchioric acid. The final pellet was dissolvedin Hyamine hydroxide (Packard) (17). Eachpoint represents the averageincorporation in 3 cultures. In other cultures, the extent of blast transformation was determined as outlined in â€œMaterialsandMethods.â€•

OCTOBER 1970 2517

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1970 American Association for Cancer Research. L. A. Loeb, J. L. Ewald, and S. S. Agarwal newly synthesized RNA, there is an increase in protein The present result indicates that the induction of DNA synthetic activity. The sudden rise in the rate of DNA replication by PHA is accompanied by striking changes in synthesis in these cultures is usually not detected for 18 hr the activities of some of the enzymes responsible for this and may require the prior increase in RNA and protein synthesis. Nonstimulated lymphocytes contain little DNA synthesis. The complete morphological transformation of the polymerase, thymidine kinase, DNase, and TMP kinase small lymphocyte with its compact nucleus and scanty activities. These activities are enhanced by the presence of cytoplasm into a much larger lymphoblast with reticular PHA. This enhancement occurs at the same time as DNA nucleus, abundant cytoplasm, and prominent nucleoli was replication and is sensitive to actinomycin D and cyclohexi not observed until after DNA replication. mide (unpublished results). Since the increase in amino acid In order to measure the dependence of lymphocyte trans incorporation starts at a much earlier time, it is possible that formation on the synthesis of RNA, we investigated the these enzymes were synthesized at this earlier time in effects of actinomycin D. PHA and graded amounts of inactive forms. actinomycin were added at the start of the experiment dGMP kinase is an example of an enzyme activity that does Crable1).After72hr,thecultureswereevaluatedforthe not increase during lymphocyte transformation. It is present indicated metabolic and morphological changes of lympho in greater activity in nonstimulated lymphocytes than TMP cyte transformation . At sufficient concentrations (0.015 kinase, as if there is sufficient dGMP kinase activity to @.tg/mlor greater), the effect of actinomycin D was to satisfy the requirements for subsequent lymphocyte transfor prevent the PHA-mediated induction of thymidine incorpora mation. tion, DNA polymerase, thymidine kinase, TMP kinase Evidence indicates that the pathway by which thymidine is activity, uridine incorporation, and lymphoblast transforma incorporated into DNA is not identical to the pathway by tion. However, actinomycin at a concentration of 0.005 which the cell synthesizes DNA de novo. In the former, @.zg/m1,which abolished the increase in polymerase activity, thymidine is sequentially phosphorylated to TIP, one of the as well as nearly all augmentation of thymidine and uridine immediate substrates for DNA polymerase. In the latter incorporation, did not effect subsequent morphological trans pathway, reduction of ribonucleotides occurs at the diphos formation. phate level (17). The increase in thymidine incorporation during lymphoblast transformation resulting from PHA DISCUSSION occurs at the same time as the increase in thymidme kinase and DNA polymerase activity. The most direct explanation Human peripheral lymphocytes seldom divide in vivo and for this relationship is that both the â€œsalvageâ€•andde novo reportedly live for many years (8). In culture, their nonpro pathways are under coordinate controls. Since thymidine liferative behavior is usually continued. Quantitative cyto kinase is not on the de novo pathway for DNA replication logical measurements indicate that these cells contain a and the activity is not proportional to the increase in diploid complement of DNA; their DNA content must thymidine incorporation, DNA polymerase might stifi limit double prior to mitosis and cell division (6). The addition of DNA replication. PHA to lymphocyte cultures initiates a series of metabolic The increase in DNA polymerase, thymidine kinase, and alterations resulting in DNA synthesis and the transformation TMP kinase activity as well as the replication of DNA by the of these quiescent lymphocytes into actively replicating cells could be abolished, for the most part, by the addition lymphoblasts. of as little as 0.005 pg of actinomycin per ml of culture

Table1

Effect ofactinomycin D on PHA-stimulated human lymphocytes Multiple 2-mi portions of isolated lymphocytes from the same individual were cultured with and without PHA and actinomycin. The amount of actinomycin refers to the final concentration in the cultures. Thymidine and uridine uptake is the amount incorporated for 1 hr prior to harvestingthe cultures (see legend to Chart 1). Results are averagesof triplicate cultures.

polymer activity kinase TMP Uridine- H incorporation (mpmole TM2 PtThymidineactivity kinase activity incorporation CulturelymphoblastsNo conditionsThymidine-3H(cpm/0.1 ml)DNA hr/0.i ml)ase (mpmole/hr/0.1 ml)(mMmole/hr/0.i ml)3 (cpm/0.1 ml)%

0.158<10Occ/'PHA47520.1130.256PHA110.0020.032 0.442319768PHA D0.001+ actinomycin 0.095312770.005ag/nil10700.0100.034 @g/m1 0.032 0.01 ag/nd 62 <0.001 0.022 <0.002 <10 35 0cc.0.020.015@g/ml238 00.005 <0.0010.030 0.0i5106 <1057 @g/ml0<0.OOi0.004 <0.002<100cc.

a@@es containing less than 1% lymphoblasts.

2518 CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1970 American Association for Cancer Research. Initiation ofDNA Synthesis medium. In these experiments, actinomycin was added at the the induction of polymerase was diminished with actinomy same time as PHA. After 72 hr in culture, the amount of clii D. So far, stimulation of DNA synthesis in lymphocytes actinomycin was sufficient to abolish 90% of the PHA by a variety of methods results in a parallel increase in DNA mediated increase in the rate of uridine incorporation. polymerase activity (2). Inhibition of PHA-mediated stimula However, this low concentration of actinomycin did not tion of polymerase by a variety of methods is accompanied hinder morphological transformation. These results confirm by a proportional decrease in DNA synthesis (unpublished the finding of Kay et al. (14) that actinomycin can prevent results). DNA polymerase activity can only be defmed as the the induction of thymidine incorporation by PHA yet allow DNA-directed polymerization of deoxynucleoside triphos blastogenesis to proceed and are compatible with the experi phates as detailed by Kornberg (18); no other mechanism is ments of Salzman et al. (27) in which fluorodeoxyuridine known. We do not yet know if the enzyme responsible for prevented DNA synthesis without inhibiting blastogenesis. this polymerization during lymphocyte transformation is Our results also indicate that the induction of enzymes similar to E. coil DNA polymerase. associated with DNA synthesis is also not a prerequisite for blastogenesis. The degree of proportionality between the ACKNOWLEDGMENTS amount of actinomycin added to the cultures and the decrease in DNA replication, or the activities of enzymes We thank Dr. Sam Sorof for generous counsel and are indebted associated therein, is an indication of exact coordinate to Mrs. Grace Ziegler and MissSylvia S. Bugbee for most cooperative controls operative in regulating DNA synthesis. Since cells assistance. cultured in the presence of actinomycin and PHA are able to transform successfully, the effects of actinomycin observed REFERENCES here cannot be attributed only to cell death. The viability of the cells was further confirmed by their ability to exclude 1. Agarwal, S. S., Blumberg, B. S., Gerstley, B. J., London, W. T., Sutnick, A. I., and Loeb, L A. DNA Polymerase Activity as an the dye, trypan blue (23). Index of Lymphocyte Stimulation: Studies in Down's Syndrome. Experiments with inhibitors of protein and RNA synthesis J.Qin.Invest.,49:161â€”169,i970. suggest that the increase in polymerase activity observed 2. Agarwal, S. S., Bugbee, S., and Loeb, L. A. Mixed Lymphocyte during lymphocyte transformation results from the de novo Cultures: Induction of DNA Polymerase. Nature, 226: 949â€”950, synthesis of the polymerase (unpublished results). The sensi 1970. tivity of this induction to actinomycin is in accord with a 3. Bach, F., and Hirschhorn, K. Lymphocyte Interaction: A Poten requirement for the synthesis of new RNA. In other experi tial Histocompatibility Test in Vitro. Science, 143: 8i3â€”8i4, ments, puromycin, at concentrations sufficient to prevent 1964. 90% of the PHA-mediated enhancement of protein synthesis, 4. Baserga, R. Biochemistry of the Cell Cycle: A Review. Cell prevents the induction of the polymerase. It is possible that Tissue Kinet,, 1: 167â€”191,1968. PHA effects the removal of an inhibitor of the polymerase; 5. Cooper, E. H., and Barkhan, P. Observations on the Proliferation of Human Leucocytes Cultured with Phytohaemagglutinin. Brit. if so, this effect requires RNA and protein synthesis. J.HaematoL,9:101â€”111,1963. However, we find no evidence for the presence of an 6. Cooper, H. L., and Rubin, A. D. RNA Metabolism in Lympho inhibitor in unstimulated cultures (20). It is always con cytes Stimulated by Phytohemagglutinin: Initial Responses to ceivable that the enzyme is present as an inactive form and Phytohemagglutinin. Blood, 25: i014â€”i027, 1965. that activation requires RNA and protein synthesis. This is 7. de lucia, P., and Cairnes, J. Isolation of an E. coli Strain with a unlikely, since this activation is not limited to polymerase. Mutation Affecting DNA Polymerase. Nature, 224: i 164â€”i166, If our results can be generalized, the induction of replica 1969. tion in nondividing cells requires the synthesis or activation 8. Fitzgerald, P. H. The Immunological Role and Long Life-Span of of a number of enzymes functioning in the DNA-synthetic Small Lymphocytes. J. Theoret. Biol., 6: 13â€”25,1964. pathways. Evidence supporting this concept has been 9. Furlong, N. B. A Rapid Assay for Nucleotide Kinases Using C'4- obtained from a number of eukaryotic systems in which or H3-Labeled Nucleotides. Anal. Biochem., 5: 515â€”522,1963. 10. Hungerford, D. A. Leukocytes Cultured from Small Inocula of DNA replication can be induced in vivo (4), as well as the Whole Blood and the Preparation of Metaphase Chromosomesby induction of DNA replication following viral infection (15). Treatment with Hypotomc KQ. Stain Technol., 40: 333â€”338, The induction of replication by PHA in vitro appears to 1965. be particularly suitable for studying the control mechansim 11. Ives, D. H., Morse, P. A., Jr., and Potter, V. R. Feedback for DNA synthesis. The magnitude of change, both as to the Inhibition of Thymidine Kinase by Thymidine Triphosphate. J. number of cells participating (as much as 90%) and the Biol.Qiem.,238:1467â€”1474,1963. increased enzymatic activities associated with DNA synthesis 12. Kay, J. E. Early Effects of Phytohaemagglutinin on Lymphocyte (as much as 150-fold), facilitates detailed studies in this RNA Synthesis. European J. Biochem., 4: 225â€”232,1968. system. 13. Kay, J. E. Phytohaemagglutinin: An Early Effect on Lymphocyte Lipid Metabolism.Nature, 219: 172â€”173,1968. That DNA polymerase is responsible for DNA replication in 14. Kay, J. E., Leventhal, B. G., and Cooper, H. L. Effects of bacteria has been recently been questioned by de Lucia and Inhibition of Ribosom@ilRNA Synthesis on the Stimulation of Cairns (7). On the other hand, the data presented in this Lymphocytes by Phytohaemagglutinin. ExptL Cell Res., 54: communication indicate a high degree of correlation between 94â€”100,1969. DNA polymerase activity and DNA replication in human 15. Keir, H. M. DNA Polymerases from Mammalian Cells. Progr. lymphocytes. This proportionality was further evident when Nucleic Acid Res. Mol. Biol., 4: 81â€”128,1965.

OCTOBER 1970 2519

16. Kleinsmith, L J., Ailfrey, V. G., and Musky, A. E. Phosphoryla mation to Delayed Hypersensitivity in Guinea Pigs and Man. tion of Nuclear Protein Early in the Course of Gene Activation Federation Proc., 27: 21â€”28,1968. in Lymphocytes. Science, 154: 780â€”781,1%6. 23. Pappenheimer, A. M. Experimental Studies upon Lymphocytes. I. 17. Larsson, A., and Reichard, P. Enzymatic Reduction of Ribonu The Reactions of Lymphocytes under Various Experimental cleotides. Progr. Nucleic Acid Res. Mol. BioL, 7: 303â€”347,1967. Conditions. J. Exptl. Med., 25: 633â€”650, 1916. 18. Lehman, I. R., Bessman, M. J., Simms, E. S., and Kornberg, A. 24. Pogo, B. G. 1., Allfrey, V. G., and Mirsky, A. E. RNA Synthesis Enzymatic Synthesis of Deoxyribonucleic Acid. I. Preparation of and Histone Acetylation during the Course of Gene Activation in Substrates and Partial Purification of an Enzyme from Lymphocytes. Proc. NatL Acad. Sd. U. S., 55: 805â€”812,1966. Escherichiacoli. J. Biol. Chem., 233: 163â€”170,1958. 25. Racker, E. Spectrophotometric Measurements of the Enzymatic 19. Loeb, L. A. Purification and Properties of Deoxyribonucleic Acid Formation of Fumaric and Os-Aconitic Acids. Biochim. Biophys. Polymerase from Nuclei of Sea Urchin Embryos. J. BioL (1cm., Acta,4: 211â€”214,1950. 244: 1672â€”1681,1969. 26. Richardson, C. C., Schildkraut, C. L,, Aposhian, H. V., and 20. Loeb, L. A., Agarwal, S. S., and Woodside, A. M. Induction of Kornberg, A. Enzymatic Synthesis of Deoxyribonucleic Acid. DNA Polymerase in Human Lymphocytes by Phytohemag XIV. Further Purification and Properties of Deoxyribonucleic glutinin. Proc. NatI. Acad. 5th. U. S., 61: 827â€”834, 1968. Acid Polymerase of Escherichia coIl. J. Biol. (1cm., 239: 21. NoweIl, P. C. Phytohema@lutinin: An Initiator of Mitosis in 222â€”231,1964. Qiltures of Normal Human Leukocytes. Cancer Res., 20: 27. Salzman, N. P., Pellegrino, M., and Franceschini, P. Biochemical 462â€”466,1960. Changes in Phytohemagglutinin Stimulated Human Lymphocytes. 22. Oppenheim, J. J. Relationship of in Vitro Lymphocyte Transfor Exptl. Cell Res., 44: 73â€”83,1966.

2520 CANCER RESEARCH VOL. 30

Lawrence A. Loeb, Joan L. Ewald and Shyam S. Agarwal

Cancer Res 1970;30:2514-2520.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/30/10/2514

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/30/10/2514. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.