Detection of Reverse Transcriptase Activity in Human Cells1

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[CANCER RESEARCH 39, 2062-2069, June 1979] 0008-5472/79/0030-0000$02.00 Detection of Reverse Transcriptase Activity in Human Cells1

Ann A. Kiessling2 and Mehran Goulian3

Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093

ABSTRACT human malignant cells. Reconstituted models prepared by adding known quantities of MuLV4 to human nonleukemic or Samples of three nonmalignant and seven leukemic human leukemic cells prior to cell disruption and enzyme assay were cells were examined for DNA polymerase activity that could be used to determine sensitivity for detection of virus in human identified as RNA tumor virus reverse transcriptase. Expeni cells. Control cells used for comparison with malignant cells ments on virus-infected model animal cells provided the basis were from normal human placenta and spleen, a cultured for cell fractionation procedures, and reconstituted systems of human lymphocyte cell line (8866), and HeLa, a line that has known virus, added to human cells, established a threshold of been found to be negative for reverse transcniptase (1 1, 46). virus detection by enzyme assay at 1 to 10 particles/cell. DNA polymemaseactivity with some properties similar to a reverse MATERIALS AND METHODS transcniptase was detected in some of the human leukemic cells. However, parallel analyses of nonmalignant cells showed Viruses and Virus-infected Animal Cells sufficient similarities to raise serious questions about the spec ificity of the criteria. Reverse transcniptase activity has been AMV and AMV-infected chick myeloblasts were gifts of Dr. reported to be present in white blood cells from a proportion of George Beaudreau, Oregon State University. MuLV was grown cases of leukemia; however, it is concluded from the present and harvested from infected 3T3 cells (clone 1), a gift originally study that the usual enzymatic criteria using synthetic template from Dr. Hung Fan, Salk Institute. Details of purification and primers, which were used in most of the studies reported, are virus titration were as previously described (23). Virus particle not sufficient to identity a DNA polymerase activity as viral counts were determined by electron microscopy by Dr. Peter reverse transcniptase. Kiessling, University of California, San Diego. Human Cells and Human Tissues INTRODUCTION Human leukemia cells were obtained from peripheral blood The unique properties of RNA tumor virus DNA polymerase with sodium citrate or hepanin as anticoagulant and harvested (â€˜â€˜reverse transcniptase' â€˜)(47), especially its ability to tran by gravity sedimentation after addition of 1% dextran. The scribe natural and synthetic RNA's, have suggested the pos buffy coat was washed twice with 5 to 10 volumes of minimum sibility that this enzyme activity would provide a sensitive assay essential medium, pelleted at 800 x g for 10 mm, and me for RNA tumor viruses. For this reason, much effort has been washed with homogenization buffer (Buffer H), which contained directed toward detecting human tumor viruses by assaying 0.05 M Tnis-HCI (pH 7.4), 0.1 M KCI, 0.25 M sucrose, 0.005 malignant cells for reverse transcniptase activity (1, 3, 5, 12â€” M MgCI2, 0.001 M EDTA, and 0.002 M DTT. The cells were 21, 25, 26, 28, 29, 32, 36, 37, 48). However, the difficulties homogenized either fresh, or following storage as packed cell inherent in detecting a viral enzyme expected to be present in pellets at â€”70Â°orin liquid nitrogen banks. HeLa and nonleu low concentrations in malignant cells have been compounded kemic human lymphoblasts (8866 cells) were propagated as by the presence of cellular DNA polymemasesthat can also suspension cultures in minimum essential medium with 10% respond to certain assays that were earlier thought to be fetal calf serum. The cells were harvested and washed as specific for viral reverse transcriptase (4, 8, 11, 27, 39, 40, described for the human leukemic cells. Human placental tis 46). A variety of criteria have been proposed to minimize sues were obtained as aborted early products of conception, confusion of viral enzyme with normal cellular enzymes, includ or from cesameansections. Human spleens were obtained as a ing specific responses by viral DNA polymerase to particular fresh surgery specimen from a patient with nonmalignant dis templates or divalent metal ions (13, 16, 18â€”20)andantigenic ease (hereditary sphemocytosis)or as fresh autopsy specimens similarity of virus enzyme to primate virus reverse transcnip from patients with no known cancers. The solid tissues were tases (9, 12, 48). rendered single-cell suspensions by removing fascia, suspend This communication describes studies to determine the use ing in 2 volumes of Buffer H, and homogenizing in 15-sec fulness of some of these criteria in detection of viral reverse bursts in a Sorvall Omnimix 4 to 6 times, followed by filtration transcriptase in human leukemic cells. Animal cells infected through 3 to 4 layers of gauze to remove connective tissue with tumor viruses provided the positive controls necessary to fibers. The resulting cell suspension was homogenized in the establish guidelines for cell fractionation and assay of the same manner as for the other cells.

@ This research was supported in part by USPHS Research Grants NIH-CA 4 The abbreviations used are: MuLV, Moloney murine virus: AMy, avian 11705 and American Cancer Society Grant NP-i 02. myeloblastosis virus; OTT, dithiothreitol; poly(mC). oligo(dG), poly(2'-o-methyl 2Recipientof USPHSAward(Institutional)HL-07107.Presentaddress:De cytidylate). oligodeoxyguanylate; poly(dA). oligo(dT), polydeoxyadenylate .oh partment of Anatomy, School of Medicine, University of Oregon Health Science godeoxythymidylate; poly(C). ohigo(dG), polycytidylate .ohigodeoxyguanylate; @ Center, 3181 S. W. Sam Jackson Park Road, Portland, Oreg. 97201. poly(A). oligo(dT), polyadenylate ohigodeoxythymidylate.dNTP, deoxyribonucle 3To whom requestsfor reprintsshouldbe addressed,at Departmentof oside triphosphate; CML, chronic myelogenous leukemia; SiSV, simian sarcoma Medicine, M-Oi 3, University of California, San Diego, La Jolla, Calif. 92093. virus; AMML, acute myelomonocytic leukemia; AML, acute myelogenous leuke Received November 13, 1978; accepted February 23, 1979. mia.

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Cell Fractionation Procedures used for assay of endogenous reactions, with [3H]dTTP (2 @sM) in all cases. Where indicated, actinomycin 0 was added at 100 Cells were suspended in 3 to 5 volumes of Buffer H and @g/mlandpancreatic RNase was added at 20 @g/ml. homogenized in a tight-fitting Dounce homogenizer until less Other Template Primers. Activities with poly(C). oligo(dG) than 10% whole cells remained (50 to 60 strokes for normal and poly(mC).oligo(dG) were assayed in System 2 minus KCI, cells, 20 to 40 strokes for malignant cells). substituting dCTP for dATP and [3H]dGTP for [3H]dTTP. These Nuclei were removed by 2 centnifugations at 800 x g for 10 assays were incubated at 37Â°for 20 mm. In the experiments mm. Mitochondnia were removed by 2 centrifugations at described in Charts 1 and 2, using poly(C).oligo(dG), MgCI2 10,000 x g for 10 mm. For some experiments, the postmito (5 mM) was substituted for MnCI2. Activity with poly(dA). chondnial supennatant was layered directly over 10 to 70% oligo(dT) was assayed in System 2 substituting 5 m@MgCl2 for sucrose density gradients in Buffer A [0.01 MTmis-HCI(pH7.4), 0.5 mM MnCl2. 0.001 M EDTA, and 0.001 M DTT] containing 0.1 M KCI, and Products were precipitated by direct absorption of assay centrifuged to equilibrium. The gradients were fractionated, mixtures into glass fiber papers and in situ precipitation in 10% and each fraction was assayed in System 1 or 2 (see below). trichlomoacetic acid as before (23), or by addition of 50 sg In other experiments, particularly with large initial volumes such carrier DNA and 5 assay volumes 10% tnichloroacetic acid, 0.1 as the spleen or placenta preparations, membrane fractions M KH2PO4, and 0.01 M EDTA for 1 0 mm at 0Â°, followed by were obtained from postmitochondnial supemnatantsby centrif collection onto Whatman GF/C glass fiber filters (Whatman, ugation at 30,000 rpm in the SW4O rotor (Beckman, Palo Alto, Co., Clifton N. J.) under vacuum. Filters containing precipitated Calif.) for 3 hm,or 40,000 rpm in the SW5O motorfor 2 hr DNA products from both precipitation techniques were washed through 20% (w/v) sucrose in Buffer A. The membrane fraction 3 to 5 times with cold 0.1 M HCL-0.01 M KH2P04, followed by thus obtained was resuspended by gentle sonication in Buffer 3 to 5 washes with 0.1 M HCI containing no phosphates. DNA A containing 5% sucrose, layered over a continuous 10 to 70% products were quantified on dried filters by liquid scintillation sucrose density gradient as above, or over a discontinuous 20 counting in a PPO-POPOP-toluene system. to 40% sucrose gradient in Buffer A, centrifuged to equilibrium All template primer-directed activities reported here have as described in the text, fractionated, and assayed in System been corrected for acid-insoluble radioactivity in the absence 1 or 2 (see below). of template primer. Tests for terminal deoxynucleotidyltmans Enzyme Assays ferase, by inclusion of primer without template and/or by substitution of noncomplementany dNTP, were consistently DNA was prepared from normal human spleen according to negative. the method of Manmur (34) and maximally â€˜â€˜activated'â€˜by treatment with pancreatic DNase (17 to 19% solubility) (23, Thin-Layer Chromatography 24). Poly(mC).oligo(dG)1218 (1:1, w/w) and poly(dA). One- to 2-psIaliquots of completed assay mixtures were oligo(dT)1218(1:1, w/w) were obtained from P-L Biochemicals chromatogmaphed on polyethyleneimine thin-layer plates (EM (Waltham, Mass.). PoIy(C).oligo(dG)1218 was either from P-L Reagents, Dammstadt, Germany) for 1 hr in 1.0 M LiCI to Biochemicals (1:1, w/w) or from Collaborative Research separate dNTP's from mono- and diphosphates. (Milwaukee, Wis) (2:1 , w/w) as was poly(A).oligo(dT)1215 (2: 1, w/w). Nonradioactive dNTP's and OTT were obtained from RESULTS P-L Biochemicals. [3H]dGTP (20 Ci/mmol) was from New Eng land Nuclear Corp. (Boston, Mass.), [3HJdTTPwas prepared Animal Model Systems from [3H]thymidine (60 Ci/mmol) as previously described (23). Radioactive dNTP's were used without dilution of the specific Cell homogenates were prepared from AMV-infected mye activity unless otherwise stated. loblasts and MuLV-infected 3T3 cells to establish guidelines Enzyme aliquots were adjusted to 0.25% Nonidet P-40 (Shell for fractionation and enzyme assay. The 2 systems allowed Oil Co., Houston, Texas) and 5 mp,i OTT and kept at 0Â°for at comparisons to be made between avian and mammalian viruses least 10 mm prior to addition of assay mixtures, which resulted as well as between circulating leukemic cells and tissue culture in a 5-fold dilution of enzyme-detergent. fibroblasts. The postmitochondnial supemnatant fractions ob System 1. DNA-dependent polymerase was assayed by in tamed from either the AMy- or the MuLV-infected cells yielded cubation for 20 mm at 37Â°ofa mixture (50 to 150 @l)containing the same profiles of enzyme activity when centrifuged to equi 30 mM Tnis-HCI(pH 8.3), 20 m@KCI, 5 mp,iMgCI2, 2 mti OTT, librium in isopycnic sucrose gradients (Chart 1). The same 0.2 mMconcentrations of each nonmadioactivedNTP, activated results were obtained with fresh cells, with cells subjected to DNA (500 @.tg/ml)(31), bovine plasma albumin (1 mg/mI), and 2 to 3 freeze-thaw cycles, or with cells stored at â€”20Â°for 3 [3H]dTTP (2 LM),except as in Table 1 (reaction with activated years. The virus enzyme activity in both the myeloblast and DNA for AMV myeloblasts, HeLa, and soluble fraction of AML 3T3 cell homogenates was equivalent to 1O@to 1O@virus 2 for which [3H]dGTP (5 jIM) was used). particles/cell (23), a concentration of virus particles easily System 2. Poly(A). oligo(dT)-stimulated DNA polymerase ac Visualized in electron micrographs. However, even in this ex tivity was assessed by incubation for 20 mm at 30Â°ina mixture ample of active viral proliferation, the virus enzyme accounted (50 to 150 s1)containing 30 mM Tris-HCI (pH 8.3), 80 mM KCI, for less than 10% of total myeloblast DNA polymerase activity. 0.5 mM MnCI2,2 mM OTT, 0.2 mMdATP, poly(A).oligo(dT) (40 No more than 5% of the viral reverse tmanscmiptase,as @sg/ml),bovineplasma albumin (1 mg/mI), and 2 @.LM[3H]dTTP. defined here as activity with poly(C).oligo(dG), was found in Endogenous Reactions. System 1 minus activated DNA was the â€˜â€˜soluble'â€˜cytoplasmicmaterial (Chart 1, Fractions 16 to

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A peak of enzyme activity responding to the poly(C). oligo(dG) template was also seen in the CML cells with no added virus (Chart 2A). The activity appeared in the gradient in the density region of RNA tumor viruses (1 .16 g/ml) although slightly less dense than the MuLV particles (Chart 2B). How ever, the poly(C).oligo(dG)-dependent enzyme activity in the 0 membrane fraction of the CML cells with no added virus was 90% inhibited by actinomycin 0, whereas, the poly(C). 0. oligo(dG)-dependent activity of virus enzyme was resistant to actinomycin 0 even in the cell lysates. Similar procedures following addition of purified MuLV at the same 2 concentrations to cultured lymphocytes (8866 cells) resulted in 10% recovery of MuLV reverse transcniptase (data not shown). As was the case with the CML cells, none of the

FractionNumber viral enzyme appeared as solubilized activity at the top of the Chart 1. lsopycnic sucrose gradients of postmitochondrial cytoplasmic su gradient, as viral comes or with the nuclei or mitochondria, pernatant fractions from AMV-infected chick myeloblasts (A) and MuLV-infected fractions. Aliquots of the sucrose gradient fractions containing mouse 3T3 cells (B). A 200-id packed-cell volume of washed myeloblasts and a 100-id packed-cell volume of washed 3T3 cells were processed as described in the virus-lymphocyte membrane fractions were slightly more â€œMaterialsandMethods,' â€˜andthe postmitochondrial supernatant fractions were inhibitory (15 to 20%) to virus enzyme in the presence of layered over 10 to 70% sucrose gradients in Buffer A, centrifuged to equilibrium poly(C).oligo(dG) than were the CML cell fractions, but they at 25,000 rpm in a Beckman SW 40 rotor for 16 hr at 4Â°,and fractionated from the bottom into 0.6-mI fractions. Aliquots of 5 @ilwereassayed with DNA template did not account for the loss of the viral enzyme activity. (â€¢)and[3H)dTTP(1500 cpm/pmol),andwith poly(C).oligo(dg)(0)and @ [3H)dGTP(2100 cpm/pmol), as described in â€˜â€˜MaterialsandMethods. Sensitivity of Virus Detection by Reverse Transcriptase Assay 20), and none of the viral enzyme appeared at the buoyant The reported threshold of detection of virus by enzyme assay density ofviral cores (1.19 to 1.21 g/ml). These results indicate varies from 2 x 1o@particles (30) to 2 x 108 particles (43). that virus is either assembled on shiooth membranes or readily associates with membranes having a density of 1.15 to 1.19 We were able to detect 1 to 10 x 10@virus particles by DNA polymerase assay (23), in close agreement with Allaudeen et g/ml. The possibility of inhibitors preventing detection of virus enzymes in the nonmembrane fractions was excluded by me a!. (2); i.e. , with poly(A).oligo(dT) at optimal assay conditions, covery of virtually complete activity when aliquots of the gra 106 particles of AMy, MuLV, feline leukemia virus, and SiSV dient fractions were combined with known quantities of virus each contain sufficient DNA polymerase to incorporate 5 fmol enzyme in assays containing poly(C).oligo(dG). of dTMP (1 50 cpm from [3H]dTTP, 60 Ci/mmol, at 20% effi Actinomycin 0 also did not inhibit the reaction of the intra ciency) into product in 20 mm. We have previously reported cellular virus enzymes with poly(C).oligo(dG). This assay con that poly(C).oligo(dG), the more specific template primer, is dition was applied to test fidelity of the characteristics of intracellular virus enzyme. The reaction of RNA tumor virus enzyme (purified or in detergent-disrupted vinions)with poly(C). oligo(dG) is less than 5% depressed at concentrations of b actinomycin 0 up to 100 @sg/ml,conditionsthat inhibit 95% of 0. the reaction with DNA templates.5 U Reconstruction Experiments. Purified MuLV particles were added to CML cells at proportions of 10 particles/cell and 1 particle/cell. The cell-virus mixtures were homogenized, and the cytoplasmic membrane fractions (see â€˜â€˜Materialsand Meth ods' â€˜)werecentrifuged to equilibrium in isopycnic sucrose gradients along with a similar sample from CML cells with no added virus. The recovery of virus reverse transcriptase added b to the cells (appearing as a distinct peak at a density of 1.18 0. g/ml) was 30 to 50% from both cell-virus mixtures (Chart 2). U None of the virus enzyme was found at the buoyant density of viral comes(1.19 to 1.21 g/ml) or at the top of the gradient in the form of solubilized enzyme. Aliquots of the gradient frac tions did not inhibit reverse transcriptase when added to stan dard virus enzyme assays. If inactivation of the virus enzyme Chart 2. lsopycnic sucrose gradients of postmitochondrial cytoplasmic frac accounted for the loss of 50 to 70% of the added reverse tions from 2 x 1o@CMLcells (A), 1 x 1O@CMLcells plus 1 x 10@MuLVparticles (B). and 1 x iO@CML cells plus 1 x 10Â° MuLV particles (0). C contains 1 x transcriptase activity, the inactivation must require exposure to 1O'Â°MuLVparticlesaloneas a control.Viruswasaddedto the cells prior to @ other cell fractions or exposure times that were longer than the disruption and separation of the cytoplasmic fraction as described in Materials @@ length of incubation for enzyme activities. and Methods. Centrifugation was for 4 hr at 35,000 rpm in a Beckman SW 40 rotor; each gradient was separated into 0.35-mi fractions from the bottom, and ahiquotsof 10 il were assayed with poly(C).ohigo(dG)and [3H)dGTP(3000 cpm/ @ 5A. A. Kiesshing,unpublishedobservations. pmol) (see Materials and Methods â€˜).

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Reverse Transcriptase in Human Cells twice as active with AMV DNA polymemase as is poly(A). magnitude lower than when poly(A).oligo(dT) is included (23). oligo(dT), but only one-tenth as active with the MuLV and feline This is partly due to the fact that the virus enzyme is not leukemia virus enzymes, and at least 4 orders of magnitude saturated with templates in the virions, but at saturation with less active with SiSV (23). added viral RNA or other hetenopolymemRNA, with or without The reconstruction experiments indicated that a single assay oligodeoxythymidylate, the reaction mateisstill at least an order could detect intracellular virus enzyme in as few as 1 million of magnitude below the mateachieved with poly(A).oligo(dT) leukemic cells containing on the average 1 particle/cell. Based (22, 23). Thus, detection of a cellular reverse transcniptase by on our data from whole leukemic cells (Table 1) and isolated an endogenous reaction, or a reaction with hetemopolymerRNA particles (23), at 1 particle/cell, virus enzyme would constitute as template, would require on the order of 1O@to 1O@virus approximately 0.02% of total cellular DNA-dependent DNA particle equivalents of enzyme activity. polymemaseactivity. However, assays at this level of sensitivity The â€˜â€˜simultaneousdetection'â€˜ofDNA synthesis on a high will also detect weak reactions of other DNA polymerases, and molecular-weight RNA template (5, 21) has been offered as assay specificity for reverse transcriptases becomes a critical evidence that some DNA polymerases in human tumors are problem. reverse transcniptases associated with intracellular viral RNA. The Endogenous Reaction. The most specific enzymatic However, this technique was not included here because, in our property of reverse transcniptase is the ability to transcribe hands, only a small percentage of early DNA product synthe heteropolymer RNA's. This is the basis for the â€˜â€˜endogenoussized by detergent-disrupted AMV or MuLV particles was as reaction' â€˜describedfor virus particles, i.e. , DNA synthesis in sociated with high-molecular-weight RNA, and the results were detergent-disrupted vinions using endogenous viral RNA as not reproducible with or without NaF present to inhibit the viral template. In agreement with previous reports (22), the endog RNase H. enous reactions of MuLV and AMV were found to be approxi mately 80% sensitive to RNase and 60 to 70% resistant to Screening Procedure for Human Cells actinomycin 0 (data not shown). The cell fractions containing the virus enzymes from the The data from the experiments with the animal model systems animal model systems were 40 to 60% resistant to actinomycin and the virus recovery experiments from human cells indicated 0 but only 30 to 40% sensitive to RNase (Table 1). The limited reverse transcriptase in human malignant cells would most sensitivity to RNase may reflect the presence of alternative probably be associated with the smooth membrane fraction templates, such as contaminating DNA fragments. (1 .15 to 1.I 8 g/ml) isolated from cytoplasm by isopyknic The amount of product formed in the endogenous reaction sucrose gradients. This is also the cell fraction in which reverse of detergent-disrupted vinions is approximately 2 orders of transcriptase was reported to be present in human leukemic

Table 1 cellsSource DNApolymeraseactivitiesin fractionsfromleukemicandcontrol minb)Endogenous (pmol/5 x 1O@cells/20

primerStandard reaction Exogenous template

as DMembrane of enzyme8IncorporationsayPoIy(C).oligo(dG)+ANase + Actinomycin D Activated DNA Poly(A). oligo(dT) â€”Actinomycin 0 + Actinomycin fraction AMV myeloblasts 22 792 (1O)c 1402 1236 1106 MuLV-3T3 19 13 12 144 (8) 1010 460 464 HeLa 11 10 4 125 (3) 75 0.4 0.1 8866 3 3 1 52 (2) ii 0.1 0.1 Placenta 4 5 1 11 (1) 2 0.1 0.1 0.1CML-iaHumanspleen51 1331 17 9 21 (2) 2 0.1

3 147 (4) 37 0.1 0.1 CML-ib 21 19 12 221 (4) 123 ii O.i CML-2 22 17 12 185 (4) 42 3 0.1 CML-3 9 9 4 98 (4) 76 1 0.1 Acute lymphocytic 4 3 2 120 (4) 22 2 0.1 leukemia AML-i 3 2 1 17 (0.3) 18 0.1 0.1 AML-2 2 2 0.4 72 (3) 18 0.1 0.2 3SolubleAMML8 99 14 8 232 (2) 45 8

fraction 8866 4 2167 (97) 1 9 1 Human spleen 0.1 NT NT 249 (15) 25 0.1 0.1 AML-2 0.1 NT NT 257 (6) 25 0.1 0.1 CML-ia37.4 0.8NTâ€• NT 0.1 4205(97) 5 17 2

a Membrane and soluble fractions, 1 . 1 5- to 1 .20- and 1 .07- to 1 .09-g/mh fractions, respectively, from isopycnic sucrose gradient centrifugations (â€˜â€˜Materials and Methods' â€˜).Leukemiccells are designated by diagnosis and patient number; a and b, separate samples from the same patient. b Incorporation of radiolabeled deoxyribonucleoside monophosphate into product, normalized to standard assay (50 @d)containing enzyme from 5 x 1o@ cells incubated at 37Â°for 20 mm. Actual assay conditions varied, e.g., in specific activities of labeled dNTP's (see â€˜MaterialsandMethodsâ€•). C Numbers in parentheses, percentage of total cytoplasmic (postnuclear) DNA-dependent DNA polymerase represented by that fraction. ci NT. not tested.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. A. A. Kiess!ing and M. Goulian cells by Baxt et a!. (5) â€˜andGalloet a!. (13, 14). An additional the infected model cells fulfilled the expectation, with the result advantage of initially isolating the membrane fraction was that that with Mg2@poly(dA).oligo(dT) was only 10 to 20% as active only a small proportion (5 to 10%) of cellular DNA-dependent as was poly(A).oligo(dT). However, the opposite results were DNA polymemaseremained associated with this fraction. obtained with the human enzyme samples, in that poly(dA). The assay in which poly(A).oligo(dT) with Mn2@was used oligo(dT)was a more effective template primer than was poly(A). was selected to provide maximum sensitivity for initial detec oligo(dT) in the presence of [email protected] one exception to this tion. Although this system will also detect cellular DNA polym was the AMML sample, for which poly(dA).oligo(dT) and erases fi and y, it is the most stimulatory template primer for poly(A). oligo(dT) were equally effective in the presence of the mammalian virus enzymes (23). DNA polymerase activity [email protected] results seemed to indicate that the poly(A). detected by poiy(A).oligo(dT) in the membrane fraction was oligo(dT)-Mn2@assay system was detecting DNA polymenase further characterized by: (a) response to poly(C).oligo(dG) in â€˜yinmost of the membrane fraction. However, this interpretation the presence and absence of actinomycin 0; (b) the effect of is now open to question in light of more recent reports (27, 44) actinomycin 0 and RNase on the endogenous reaction; (C)the [in contrast to the earlier report (16)] that DNA polymerase y activity with poly(A).oligo(dT) and poly(dA).oligo(dT) in the from human lymphocytes also prefers poly(A).oligo(dT) to presence of Mg2@;and(d) a test for inhibitors by measurement poly(dA).oligo(dT) in the presence of Mg2t of the activity of MuLV or AMV virus enzymes when mixed with Enzyme activity with poly(C).oligo(dG) was found in the the human leukemic cell fractions. membrane fractions from some cells (malignant and nonmalig Characteristics of the Enzymes in Human Leukemic and nant) but, in contrast to the virus-infected cells, this activity Nonleukemic Cells. Total cellular DNA-dependent DNA polym was sensitive to actinomycin 0 in all cases but one (Table 1). erase was greater in the malignant cells than in the tissue The one exception was the AMML sample, for which the activity culture cells; the normal tissues, placenta or spleen, contained with poly(C).oligo(dG) was 38% resistant to actinomycin 0. even less DNA-dependent DNA polymerase than either malig Experiments in which various cell fractions were added to viral nant or tissue culture cells (Table 1). All membrane-associated DNA polymemasewith poly(C).oligo(dG) did not reveal signifi enzyme activities (indicated as a percentage of total cellular cant inhibitors, and the poly(C).oligo(dG) stimulation of the DNA-dependent DNA polymerase in Table 1), were stimulated viral enzymes was not depressed by actinomycin 0 in the at least 50% by the addition of Nonidet P-40, but this was not presence of aliquots of the leukemic cell fractions. In addition, the case for enzyme in the soluble, cytoplasmic fractions. [3H]polycytidylate, with or without oligodeoxyguanylate primer, An endogenous reaction, as measured by synthesis of DNA was degraded less than 10% by enzyme fractions from the without added template primers, was detected in all membrane leukemic cells, and at least 70% of the initial [3H]dGTP[as well fractions examined. The endogenous reaction of membrane as [3HJdTTPfrom assays with poly(A). oIigo(dT)] was recovered fractions from acute lymphocytic leukemia, AMML, and the 4 at the termination of the assay. Thus, degradation of neither samples of CML were at least 38% resistant to the presence of template primer (13, 16) nor substrates (19) was a significant actinomycin 0, but the same was true for normal human spleen problem with any of the postmitochondrial supemnatant cell (Table 1). In none of these samples, i.e. , neither malignant nor fractions obtained by these procedures. nonmalignant, was the endogenous reaction greater than 20 Of the samples (placenta, spleen, AML-1 , AML-2, and to 30% sensitive to RNase treatment; in fact, most were actually AMML) that were also tested with poly(mC).oligo(dG), only one stimulated by RNase. The endogenous reactions most sensitive had activity greater than 1% of the DNA-dependent activity. In to RNase were with membrane fractions from acute lympho this sample, which again was the AMML referred to above, the cytic leukemia, AML-1 , and CML-2. activity was the same as with poly(C).oligo(dG) (Table 1). Activity with poly(A).oligo(dT) and Mn2@was found in the The principal cytoplasmic DNA polymerase from one sample isolated cytoplasmic membranes of all cells examined. Greater of CML cells, which was sensitive to N-ethylmaleimide and had than 80% of the total cellular activity stimulated by poly(A). chromatographic properties of DNA polymerase a, was found oligo(dT), from either malignant or nonmalignant cells, was to be stimulated by either poly(C).oligo(dG) (Chart 3) or membrane bound and remained so in spite of repeated frac poly(A). oligo(dT) (data not shown) at a level of 1% of the tionation by isopyknic sucrose gradient centnifugation (data not stimulation by activated DNA. These 3 activities remained shown). The values for poly(A)- oligo(dT) stimulation reported associated at the same ratio through DEAE-cellulose and DNA in Table 1 are with Mn2@for this template primer. In general, agamosechromatography, eluting at salt concentrations differ the leukemic cells contained more poiy(A).oligo(dT)-stimulated ent from the oncornavimalenzymes. Thus, it would appear that, activity than did the control cells, but the leukemic cells also at detection sensitivities of 1% or less of reactions with DNA contained more DNA-stimulated DNA polymerase, so that the template, it may be possible to detect weak reactions of the poly(A).oligo(dT)-stimulated activity was 10 to 30% of the cellular enzymes with the synthetic template primers, as has DNA-stimulated activity in membrane fractions from all cells been indicated previously (8). studied except 4, i.e. , HeLa (60%), AML-1 (100%), CML-3 The 4 cell samples containing enzyme that satisfied the most (75%), and CML-1 b (50%). criteria for a reverse transcniptase (see Table 1) were leukemic It has been reported that, in the presence of Mg2@,DNA cells, i.e. , AML-1 , CML-1b, CML-3, and AMML, with HeLa cells polymemasey prefers poly(dA). oligo(dT) to poly(A). oligo(dT), being not too dissimilar. Only the AMML cells contained a whereas the reverse is true for the virus enzymes (16). For this poly(C). oligo(dG)-stimulated enzyme that was resistant to ac reason, some cell fractions (AMV myeloblasts, MuLV-3T3, tinomycin D. The AML and CML samples contained a high ratio 8866, placenta, spleen, AML-1 , AML-2, and AMML) were of poly(A).oligo(dT) to DNA-stimulated activity but exhibited no assayed with poly(dA). oligo(dT) as well as poly(A). oligo(dT), reaction with poly(C). oligo(dG) that was resistant to actino each with Mg2@instead of [email protected] animal virus enzyme in mycin 0. However, the properties of SiSV DNA polymenase

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Reverse Transcriptase in Human Cells ate in the presence of Mn2'@,have also been proposed (44); but the former has subsequently been shown to be the same for cellular enzymes as for viral enzymes (27, 44), and the latter will not distinguish small amounts of viral enzyme in the presence of equal or greater amounts of cellular enzymes. Poly(C). oligo(dG) has been shown to be a template primer for cellular enzymes also (27, 46); in addition to this problem of lack of specificity, it is not a consistently effective template primer with all virus enzymes, particularly SiSV (23). The meth ylated derivative, poly(mC).oligo(dG), has been used as a specific template primer for virus reverse transcniptase (18â€” 20), but it is much less active with virus enzymes than is poly(C). oligo(dG) (23). Moreover, it has recently been reported to stimulate some cellular enzymes at a level of 0.2% of their activity with DNA (44). HeteropolymemRNA remains the most specific template for virus reverse transcriptase, but enzyme activity is much less with natural RNA than with the synthetic template primers, e.g., IflCub0tiOfl , mm by 2 orders of magnitude for a munine viral enzyme (23). In Chart 3. Kinetics of response of DNA polymerase from CML cells with no addition, it must be shown in each case that the natural RNA added template primer (i@@)orin the presence of DNA (â€¢),andpoly(C).oligo(dG) (0). Soluble cytoplasmic DNA polymerase from CML cells was separated from is, in fact, serving as template for synthesis of the product, nucleic acids by passing through DEAE-cellulose equilibrated with 0.3 M KCI in which has been difficult to demonstrate clearly (6, 13, 16, 37, @ Buffer B (Buffer A plus 10% glycerol; see Materials and Methods' â€˜).The 41 , 42, 45). Because of the limited amounts of clinical material passthrough solution containing enzyme was dialyzed against 0.05 M KCI in Buffer B and adsorbed to a DEAE-cellulose column equilibrated with the same ordinarily available for assay, principal reliance has been buffer. Enzyme was eluted with a linear gradient of 0.05 to 0.5 M KCI in Buffer B, placed upon reactions with synthetic template primers for and the peak fraction was further chromatographed twice on DNA-agarose (42) detection and identification of virus-related reverse transcnip with a linear gradient of 0.03 to 0.8 M KCI in Buffer B. The peak fraction eluting at 0.2 M KCI was added to assay mixtures containing [32P]dGTP(100,000 cpm/ tase in human cells (1, 3, 5, 12â€”21,25, 26, 28, 29, 32, 36, pmol) and the indicated templates as described in â€˜â€˜MaterialsandMethods.â€• 37, 48). Our results indicate that this approach suffers seriously from lack of specificity. (23) indicate that all RNA tumor virus enzymes might not be We were able to detect virus enzyme in the membrane stimulated by this template primer. fraction of the cell-virus mixtures at a level as low as 1 virus particle/cell in the reconstruction experiments. This is 2 orders DISCUSSION of magnitude more sensitive than a previous report of a recon stituted system (2). The parallel sucrose gradient from the cell Our objective was to test for the presence of reverse tran fraction containing no added virus also showed a peak of scriptase in human leukemia by the most sensitive and specific enzyme activity with poly(C).oligo(dG). Although this cellular enzymatic procedures available and to compare with the results enzyme activity was inhibited by actinomycin 0, whereas the in nonmalignant cells. This required an appraisal of the sensi virus enzymes were not, the presence of this cellular enzyme tivity and specificity of currently available assay procedures for made it difficult to interpret low levels of virus-like enzyme viral reverse transcriptase in cell homogenates. activity. A simple cell fractionation procedure was used to enrich for Enzyme activities from malignant cells with any of the prop viral enzyme. Our results with the animal model cells, indicating erties of viral transcriptase were compared with similar frac that viral enzyme remains associated with a particulate mem tions from nonmalignant cells. Distinct qualitative differences brane cell fraction, are in agreement with previous reports from could not be demonstrated, however, because none of the other laboratories with similar animal model systems (13, 21). leukemic cells examined in this study contained enzyme activ The reports of detection of reverse transcriptase in human ities that were not also associated with control cells; nonwere leukemia cells have also emphasized the particulate or mem there any leukemic cell enzymes that satisfied all of the criteria brane-bound nature of the enzyme (5, 14). Therefore, the for a reverse transcriptase. In general, the differences between detection procedure used here focused on the membrane malignant and control cells were quantitative, i.e. , the leukemic fraction (1.15 to 1.20 g/ml) obtained by isopyknic sucrose cells contained more poly(A). oligo(dT)-stimulated activity than gradient centnifugation. did the control cells, and the leukemic enzymes that responded The choice of an assay system specific for virus reverse to poly(C).oligo(dG) exhibited higher levels of this activity than transcniptase was less straightforward. Each of the synthetic did the control cells. The fact that the malignant cells also template primer/metal ion combinations previously thought to contained a higher concentration of DNA-dependent DNA po be specific for virus reverse transcniptase activity has subse lymenase than did any of the control cells used created an quently been reported to be utilized to some extent by cellular additional problem in interpretation of the quantitative differ enzymes that are not virus reverse transcriptases. Ratios of ences observed, due to the increased risk of detecting minor enzyme activity with one template primer relative to another, activities of cellular enzymes. e.g. , poly(A).oligo(dT) to poly(dA).oligo(dT) in the presence of Although it is possible that one or more of the enzymes Mg2@(16), or the most recently reported polydeoxythymidylate. detected in this sample of leukemic cells may be virus-related oligoniboadenylate to polyniboadenylate .oligodeoxythymidyl reverse transcniptase, the assay techniques were incapable of

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clearly distinguishing between enzyme activities found in nor leukemic cells; implications to leukemogenesis. Cold Spring Harbor Symp. mal and malignant cells. Enzymes with the properties of viral Quant. Biol., 34: 933-973, 1974. 14. Gallo, A. C., Miller, N. A., Saxinger, W. W., and Gillespie, 0. H. DNA related reverse transcniptase have been found in normal monkey and to a primate ANA tumor virus genome which is synthesized endogenously human placentas (35, 38). With the use of physical and enzy by reverse transcriptase in virus-like particles from human acute leukemia matic properties, as here, it is also possible to confuse the DNA cells. Proc. NatI. Acad. Sci. U. S. A., 70: 3219â€”3224,1973. 15. Gallo,R. C., Sarin,P. S., Allen,P. T., Newton,W.A., Priori,E. S., Bowen, polymemaseactivities of viral particles with those of mitochon J. M., and Dmochowski, L. Reverse transcriptase in Type C virus particles dna (7, 33, 42). It is apparent that none of the human leukemic of human origin. Nat. New Biol., 232:1 40â€”142,1971. 16. Gallo,A.C.,Sarin,P.5., Smith,A.G.,Bobrow,S.N.,Sarngadharan,M.G., cells examined contains virus reverse transcniptase approach Aeitz, M. S., Jr., and Abrell, J. W. ANA-directed and primed DNApolymerase ing the level of the animal model systems. Even if all the poly(A). activities in tumor viruses and human lymphocytes. In: A. Wells and A. Inman oligo(dT)-stimulated activity in the leukemic cells is assumed to (eds.), Proceedings of the Second Annual Harry Steenbock Symposia on DNA Synthesis in Vitro, pp. 251â€”256.Baltimore: University Park Press, be virus enzyme, which seems unlikely, the human leukemic 1973. cells contain the equivalent of less than 10 particles/cell. 17. Gallo, A. C., Yang, S., and Ting, A. ANA-dependent DNA polymerase of In a much larger series of leukemic patients than the one we human acute leukemic cells. Nature (London) 228: 927â€”929,1970. 18. Gerard, G. F. Poly(2'-o-methylcytidylate).oligodeoxyguanylate, a template report here, Gallo eta!. (13) have reported that, by their criteria, primer specific for reverse transcriptase is not utilized by HeLa cell DNA approximately 10% of the human leukemic cells examined polymerase. Biochem. Biophys. Res. Commun., 63: 706â€”7,1975. were positive for reverse transcniptase activity, and most of 19. Gerard, G. F., Lowenstein, P. M., and Green, M. Detection of reverse transcriptase in human breast tumors with poly(Cm). oligo(dG). Nature these were of myelogenous origin. The fact that 90% of human (Lond.), 256: 140â€”143,1975. leukemias may be negative for virus reverse transcmiptase 20. Gerard, G. F., Aottman, A., and Green, M. Poly(2'-o-methylcytidylate). ohigodeoxyguanylateas a template for the directed DNA polymerase in RNA indicates several possibilities, including: (a) most human leu tumor virus particles and a specific probe for the ANA directed enzyme in kemias are transformed, nonproducer cells; (b) most human transformed murine cells. Biochemistry, 13: 1632â€”i641, 1974. leukemias are not associated with tumor virus infection; or (c) 21. Gulati, S., Axel, A., and Spiegelman, S. Detection of RNA instructed DNA poly-merase and high molecular weight RNA in malignant tissue. Proc. NatI. occasional tumor virus infection occurs; however it is not Acad. Sci. U. S. A., 69: 2020â€”2024,1972. etiological, but secondary on even unrelated to the leukemia. 22. Kiessling, A. A., Deeney, A. O'C.. and Beaudreau, G. S. DNA and ANA from At the least, the difficulty in detecting RNA tumor viruses in AMV as templates for viral DNA polymerase. FEBS Left., 20: 57â€”60,1972. 23. Kiessling, A. A., and Goulian, M. A comparison of the enzymatic responses humans, the absence of epidemiological clustering typical of of the DNA polymerases from four ANA tumor viruses. Biochem. Biophys. infectious agents, and the clonal nature of the human leukemias Res. Commun., 71: 1069â€”77,1976. 24. Kiesshing,A. A., and Nieman, P. ANA tumor virus DNA polymerase: Activity and lymphomas (10) indicate some differences between animal with exogenous primers. Biochim. Biophys. Acts, 272: 145â€”147,1972. models and the human diseases. 25. Kiesshing,A. A., Weber, G., Deeney, A. O'C., Possehl, E., and Beaudreau, G. DNA polymerase activity associated with a plasma particulate fraction from patients with CLL. J. Virol., 7: 221â€”226,1971. REFERENCES 26. Kotler, M., Weinberg, E., Haspel, 0. T., Olshevsky, U., and Becker, Y. Particles released from arginine-deprived human leukemic cells. Nat. New 1. Ackerman, W., Murphy, W., and Miller, B. 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Ann A. Kiessling and Mehran Goulian

Cancer Res 1979;39:2062-2069.

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