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Biochemical and serological characteristics of reverse transcriptase from human spleen in a case of childhood myelofibrotic syndrome
cancel'Letters, 2 (1.977)291~29~8 © Elsevier/N0rth:E011andScientificPublishersL~[.
29t
B I O C H E M I C A L A N D SEROLOGICAL C~.4~RACrERISTICS OF REVERSE TRANSCRIPTASE FROM.HUMAN SPLEE,N IN A CASE OF CHILDHOOD MYELOFIBROTIC S Y N D R O M E : ,
•
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LINDA K.•STEEL,H. LAUBE and P.CHANDRA* GUstav:Etnbden-zentrurn der Biologist.hen Chemic, .Abtei~ung fur Molc~ularbiologle, Theodor.Stern,l,:ai 7, Frani'~fnrt70 (G.ER.) (Received'3 December1976) SUMMARY A reverse transcriptase isolatedand purified from the spleen of a child with myelofibrotic syndrome has biochemical and immunolog~cal properties similar to that of known type-C viral reverse transcriptases, and, in particular, to the enzymes from two primate type.C viruses (Gibbon Ape leukeroia virus and Simian Sarcoma virus}.
INTROD,C
ON
:
. . . .
:
The discoveryof RNA-directed DNA p01yme:rase (reverse ~ranscript~se in RNA tumor viruses [3~25] ) has led to much investiga~ioR of the possible role of DNA polymerases in neoplastic transformation of human cells. The use of the 'simul~aneous detection test' has led to the identification of reverse transcriptase activity in severedhuman malignancies; however, the enzyme has been purified.inoililya few cases [4,7,28], Human revm.~etranscrip~e, isolated and highly purified from fresh leu;¢emic ceils [23], has been demons~rated to be irnmunolottcaUy related to reverse ~ranscriptases of several primate RNA tumor viruses. Since the first reported cases were publi~hed [10], myetofibrotic syndrome has been found to resull; from a number of etiological facl~ors(review by Hunstein and Hauswald [11 ]). Pa~hologica/inw.~s~igationhas indicated a distinct nature, differing from ~hat of leukemia [8,12,13,t9,27]. The firs~ indication of a virus etiology of thlis disease came from data reported recently from our laboratory [4]. This report described the is,~latio:a,of a DNA-pelymera~ in a post-operative spleen specimen from a 2~Jfi-year-oldgirl having myelofibrotic syndrome. PuriJ'ication ~techniquesand some of its biochemical characteristics were described. This emTme was found to re~emble the reverse transcriptaze of RNA [minor vir~.,,;esin many of its biochemical parameters, including tern*Address correspondence to: P, Chaadra.
292
, ........
plate requirements and io~ic specificity.The purifiedenzyme was completely devoid of terminal transferaseactivity.In thi~:paper, we present fu:L~her physiochemical characteri~.:ation of the purified myelofibrotic enzyme, ~nd itsserologic'Arelatednessto other primate orLcornaviruses. MATERIALS AND METHODS Labelled deoxynucleoside triphosphates we:.~eobtained from NEN-Chemicals GmbH, Germany and unlabelled deoxynudeoside triphosphates from Calbiochem or Boehringer, Mannhei=,= Tutzini~.Anti-reversetranscldptase IgG from Gibbon Ape leukemia virus (GaLV), Avian My=:oblastosi:!~virus (AMV), Simian Sarcoma virus (SiSV) and SiSV reverse transcriptasewere obtained from Litton Bionetics.DEAE-23 celluloseand phosphocellulose P were Se~a products. DEAE-52 cellulosewas obtained from Whatm~m. D N A cellulosewas prepared by the method of Alberts and Herrick [2]. 'Activated' D N A was prepared by the method o:~Sehlabacb et el. [20]. DEAE-52 cellulose and ~,hosphocelluloseP were pretreated with bovine serum albumin by the method of Sedwick et el. [221..Alloperations were performed at 0-,-4°C.Salt concentrations were determined with a conductivity meter. Enzyme assay Isolation of the oncamavi~al-like DNA polymerase enzyme was previously described [4]. Po~ymerases a (I), ~ (II) and 7 (III) were isolated and purified by a modification of the procedures presented by Lewis et el. [14,15] and Gallo et el. ~7]. The D N A polymerases were assayed using poly[rA, dTr~], poly[dA-dT], oligo[dG] •poly[rC] and 'activated'D N A as template primers. The ~i~tandard assay was performed by adding 0.025 ml of enzyme to a volume of 0.225 ml containhug: 0.066 M Tris--HCl (pH 7.8), 0.001 M dithiothreitol(DTT), 0.01 M MgCl: or 0.001 M MnCl2, 0.06 M KCI or 0.125 M KCI, I #Ci [3H]dq['TP (I #Ci [3H]dI3TP for oligo[dG] •poly[rC]primed reactio~ts),0.04 M each of unle:belled dATP, dCTP and dGTP (dTTP for oligo[dG] •poly.[rC]primed reactJions),~md I #g of template p:dmer. Polymerases ~ (II)and reverse transcriptasewere assayed using poly[rA, dTL~] and poly[dA-dT] (0,06 M KCI and 0.00'.'.[ M MnCl: in the reaction mixture); and oligo[dG] •poly[rC] (0.125 M KCI ant:[0.001 M MnCI: or 0.01 M MgCI~.)as template primer. Polymc:rasesa (1) and 7 (Ill)were assayed with 'activated'D N A (containing 0.06 M KCI and 0.01 M MgCI: in the assay system), and poly[rA, dT12] (utilizing0.125 M KCI and 0.001 M MnCl2). Reactions were incubated for 60 rain at 87°C (MgCi2 present) or 30°C (MnCl2 present);and stopped by the addition of 0.36 m g B S A and 3 ml of cold 10% trichloroaceticacid (TCA}. Acid precipitablem.'~terialwas collectedou Whatman glassfiberpaper discs,washed 3 times with 5% TCA, and comlted in a liquid scintillationspectrometer.
293
Molecular weight determination of spleen reverse transcripta:~e Reverse transcriPtase isolated from a myelofibro'~ic spleen specimen [4] was maintained at -70°C in 0:5 ml aliquots. Molecular weight of the enzyme was determineci by glycerol gradient centrifugation. One millilitre of enzyme (140 pg/ml) was layered on to apreforined 9 mi 5--20% glycerol gradient in a buffer Containing: 0.05 M Tris---HC1 (pH 7.5), 0.00! M DT% 0.001 M EDTA and 0.20M NaCI; and subjected to velocity sedimentation for 24 h at 200,000 Xg in a Damon IEC ultracentrifuge. Three external marker proteins, hen egg albumin,:BSA and aldolase, Were similarly centril'uged (1 mg/ml). Gradients were fractlonated using an ISCO density gradient fractionator in 0.3 ml aliquots, and the 280 nm absorbance of each aliquot was determined. Assay of [3H]dGMP incorporation of the aliquots was perfcrmed with 0.04 ml of the test aliquot and oligo[dG] • poly[rC] as template primer (conditions previe~ly described). As a ta~her measure of the molecular weight, the enzyme and same marker proteins were subjected to disc-gel electrophoresis using the procedure of Davis [6] and Ornstein [18]. Immunological procedures Immune and preimmune GaLV IgG and immune AMV IgG were prepared to concentrations of 4, 8, 16 and 32 pg in a buffer described by Chandra et al. [5]. Myelofibrotic spleen reverse transcriptase (3.4 ug) and SiSV reverse transcriptase (32.1 ng), prepared in the same buffer, were separately combined with each respective IgG concentration and preincubated at 4°C for 16 h. Mixtures were assayed as described, using oligo[dG] • poly[rC] and ~:ncreasingthe [~H]dGTP to 2 uCi (total voI., 0.15 ml). Anti-SiSV reverse transcriptase and preimmune IgG at concentrations of 4, 8 and 16 pg in 0.1 M Tris---HC1 (pH 8.0) were combkned with 3.4 pg of spleen reverse ~;ranscriptaseand preincubated as above. MLxtures were assayed with 2 vg poly[rA-dT~2] and 2 vCi [3H]dTTP (total incubation vol., 0.15 ml). Spleen reverse tmnscriptase antisera Seventy micrograms of spleen reverse transcriptase, emulsified in Freunds adjuva~t, was injected intramuscularly into 2 female goats, followed by 3 booster injections of 35 pg given at 2-week intervals. The blood was collected, serum recovered, an IgG fraction obtained by ammonium sulfate precipitation and purification on a Sephadex G-200 column. Normal goat serum IgG frac~on was used as a control in the enzyme neutraJ.izatio:a assays. Myelofibrotic spleen anti-reverse transcriptase IgG and preimmune IgG were prepared to concentrations of 2, 4, 8, 16 and ~2 pg in 0.1 M Trts-HC1 (pH 8.0). ]?olymerases a, ~, ~ and reverse transcripta~e isolated from myelofibrotic spleen, and SiSV reverse transcr~ptase, were challenged for response against immune and preimmune IgG as described in the legend tc Table 2.
294
RESULTS
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Figure !a presents the glycerol gradient analysis,of 3exter~,al proi:,d-ln ...... m~rkers detected by abs0rbance at 280 n m . Assay of'.[?H]dGMP incorporation of enzyme, aliquots off the: gly'ceroi gradient is sh0wn in 'Fig: .].blThe results indicate an estimated:mo!ecui;~ weight of about T0,000 (assumi~tga .~10bulm' protein). This agrees with.the molecular weight of reverse itranscripta~es. isolated ~om other mammalian type:C viruses [1,7;9,1.6,26]. Disc e]ectrophoresis of the DNA. polymerase enzyme and marker ]xo~eins demonstJmted a mobility coEesponding to that of BSA. This also is in agreement with the 70,000 molecular weight ss determined by glycer01gradient analysis. Table 1 shows the data.obt~tined when myelofibrotic spleen, reverse transo~ptase was ~exted ag~ainstvariable concentrations of GaLV, SiSV and AMV antireverse transcriptase IgG. Semian Sarcoma virus reverse transc.~ptase, which has been shown to be strongly' inhibited by anti-GaLV reverse transcriptase IgG [1,16,21], was tested agaJmstconcentrations of uns:pecific and specific GaLV anti-reverse transcriptase IgG. The percenb of [~].=l]dGMP incorporation in the polymerase assay system for both SiSV reverse transc) iptase and human o---00v,~.Ibumln
0.5.
( MW
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~0,4-
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5
10
~ FRACTION
2O NO,
25
30
- 25
:~ 400.
- 210
> - 15
r~ < 200-
- 10
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H 100C Lu
I
5
10
15 FRACTION
20
30
NO.
Fig,. 1. Glycerol gradient analysis of the oncornavind-like DNA polyme=~se isoh~ed ~=om myelofibrotic spleen, la: Presents :the opticaI densities at 2E~0nm of 3 ~ roterin standards, lb: Shows the [~H]dGMP incorporation in 0.3 ml aliquots of the purffivd enzYme fractlonated on a glycerol gradient (5--20%). The experimental detailsare d~,.~cribedin text.
TABLE 1
295
THE EFFECT OF ANTI-GaLV ANTI-AMV A N D ANTI.SiSV R E V E R S E TIq-ANSCRIPTASES O N THE ONCORNAVIRAL.LIKE DNA-POLYMERASE ENZYNIE ISOLATED F R O M H U M A N MYELOFIBROTIC 'SPLEEN A N D SiSV REVERSE:TRANSCR]:PTASEa Source Of reverse transcr.iptase .,.: Specific anti-reveme trm'.,~scrlptase (igG)added •'' '
'
': " . . . .
• •
IgG 0tg/' ' '
.
Nora-immune
Anfi-GaLV
reactjmict;) (control)I~:G :' RT IgG
.......
Anti-AMV
Anti-SiSV
R T IgG
RT IgG
[JH]dGN'P incorporation(% of enzyme activityl t~
SiSV 4 8 16 32
100 100 100 lOC
75.2 64.7 42.9 50.3
-----
-----
4
100
8 16, 32
100 100
90.3 57.7
lO0 96.7
44.2
100
39.6
86.4 93.7
69.9 38.6 24.6
; Human. rnyelofibrotic spleen
--
aAntisera (IgG) were obtained from Dr, R.C, Gallo, National Institute of Hea.q:h, U.S.A. 3,4 ug of spleen enzyme and 0.032 , g of SiSV reverse tr~mscriptase were preincubated for 16 h at 4~C with variable concentrations of non-immune and immune GaLV and immune AMV and SiSV IgG (spleen enzyme only), in a totalvol, of 0.06 ml (assay procedures as described in text). bThe % of [~H]dGMP incorporatlon expresses activity in the presence of variable concentrations of immu,ne IgG c0mpared:to that of nonqmmune IgG. TABLE 2
RESPONSES OF D N A PO[,,YMERASES T O MYELOFIBROTtC SPLEEN ANTI-REVERSE TRANSCRIPTASE igGa s0Uree and ~ype. of D N A polymeease
TemPlate primer
Myelofibroticspleenanti-reve~setranseripruse IgG (t~g) % o[" inhibition b 2
4
'Activated'D N A
NT
NT
Poly[dA-dT]
NT
Poly[rA. dT,~]
spleerLRTaze SiSV reverse transeriptase
Myelofibrotic splee3~ I (~) Myelofibrotic spleen II (i~) ,
Mye][ofibrotic spleen III(~)
8
16
32
0
0
0
NT
0
0
0
NT
NT
0
4.5
O,ligo[dG]-polytrC]
8.8
19.8
35.7
49.0
71.2
Pely[rA. dT,~]
NT
4.2
11.9
30.2
54.0
14.,5
Mydofibrotic
al'rei,nrnuneand immune IgG were d:P,Jted to the respectiveeoncentr~,tionwith 0.1 M Tris-HCl (pH 8.0).Each polymerase was preincebatedwith an equal volume of IgG f~r 40 mln at 4°C (totalprelncubationvol.,0.5 ml). Assays contained I ;~gof the optimum temp~lateprimer (2 ~g with'activated'DNA). 0.01 M MgCl:, I uCi [JH~dTrP, 0.05 Trls-HCl (pI-!7.5),0.001 M DTT, 0.06 M KCI and 0.004 M each of unlabdled dATP, dCTP and dGTP were presentwith 'actiw~ed'DNA. Assays utilizingdA-dT and rA •dT~, cont~dned 0.001 M MnCI~, I ;~Ci[~H]dT'IT,0.05 M Tris-HC! (pH 7.8),0.001 M DTT, 0.125 M KCI and 0.004 M each or;dATP, dCTP and dGTP. dG •rC template-prlmedassays contained 0.001 M MnCl~, 1 j~Ci[~H]dGTP, 0.05 M TrL~---HCI,0.001 M DTT, 0.06 M KC] and 0,004 M each of dATP, dCTP and dTTP. Total incubationvolume was 0.15 ml (other conditionsas describedin Materialsand Methods; NT, not tested). bData expressedas the percentageof inhibitiono:~the polymeraso testedwith immune IgG compared with the same qu~antityof preimmur:e (control)IgG,
296 ....
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pleen revere not fected by o -!mmuneo v. gG; ' '. whereas imIrmne GaLV exhibited strong inhibition against botlh enzyme~. Anti-SiSV reverse transcript~e IgG also showed strong inhibition oil t h e spleen reverse transcriptase.i The anti-AMV reverse:transcriptase IgG minimally inl~ibited polym~ase activity Of the spleen enzyme. These data indicate an.: immunologi.cal relatedness of our purified enzyme to the reverse %ranscriptases of pri~aateR N A tumor viruses. '['able2 ~ummarizes the zesponses of polymerases ~.,~, 7 and reverse tr~nscr.iptaseisolatedfrom myelofibrotic spleen, and SiSV reversetranscriptase, when challenged with varying concentrations of purified myelofibrotic spleen anti.reversetranscriptaseIgG. Myeiofibrotic spleen e and ~ polymerases were not inhibited by ~he spleen anti-reversetranscriptaseIgG; whereas the myelofibrc,ticspleen reverse transcriptaseand SiSV reversetranscriptasewel~einhibited 71.2 and 54.0% respectively,when tested a~ainst 32 ;(g of IgG. My~lofibrotic spleen 7 polymerase was inhibitedat a very low level(14.5% with 32'?.pg of IgG). DISCUSSION ,[~e results presented indicate that myelofibrotic spleen reverse tlmnscrip'lase is similar to reverse transeriptase of leukemic cells in several aspects. Two si:~e classes of the leukemic enzyme (70,000 and 130,000 daltons) have been reported [16], whereas we could detect only one molecular species of molecular weight 70,000, Witkin et al. [28] recently purified a reverse transcriptase from the human spleen of a patient with chronic lymphocytic ieuke:mia, and have also found a singular molecul~ species of 70,000 dattons. Thvs the cellular enzymes may exis~ in monomer and dimer species, whereas th.~ tissue enzymes r.nay contain a single active monomer. q?he data in Table I indicate that anti-SiSV IgG in the same eonc~,ntration range is significantly more inhibitory to our spleen reverse transcriptase than anti-GaLV under the same conditions. One plausible explanation is that the specific titer for anti-SiSV in the igG fraction is higher than ~;hatfor anti-GaLV. The other :indication could be that SiSV reverse transcriptase is mo~e closely rob.ted to the human enzyme than the GaLV reverse transeriptase. The latter indication is supported by 2 facts. Simian Sarcoma virus is the closest candidate (but not identical) to HL23Vq, a human leukemic virus purified from longterm cultm'es of myeloid cells from a leukemic patient [24]. Moreover, Nooter et al. [17] recently described the isolation of a SiSV~related virus from huma.~ leukemic cells. Preliminm'y studies ~ t h the myelofibrotic anti-reverse transcrip~ase ][gG have indicated thai':,the SiSV reverse transcziptase is more sensitive towards t~s IgG than the GaLV reverse transcriptase. More experiments ar(,~'necessm'y to clarify this issue. One major aspect of these studies was to look for the possibility of detecting leukemic cells in human patients, using splenic antireverse transcriptase. These studies are in progress.
297 ACKNOWLEDGEMENT
The authors are grateful to Dr. Robert C. Gallo for the gift of anti-SiSV and anti:GaLV samples. REFERENCES 1 Abrell,J.W. and Gallo,R.C. (1973) Purification,characterizationand comparison of the D N A polymerases from two primate R N A tumor v~uses. J. Virol.,12, 431--439. 2 Alberts,B.M. and Herrick,G: (1971) D N A cellulosechromatography, Methods Eazymol. 21, 198--217. 3 Baltimore,D. (1970) D N A polymerase in virionsof Rous-Sarcoma. Nature (Lond.), 226, 1209-1211. 4 Chandra, P., Steel,L.K, Laube, H. and Kor~huber, B. (1975) Oncornaviral-likeD N A polymerase activityin a case of childhood myelofibrotie syndrome. FEBS Left, 58, 71--75. 5 Chandra, P., Zunino, F., GStz, A., Gericke, D., Thorbeek, R. and Di Marco, A. (1972) Specific inhibitionof D N A polymerases from R N A tumor virusesby some new daunomycln derivatives.FEBS Left.,21,264. 6 Davis,J.B. (1964) Disc electrophoresisH: method and applicationto human serum proteins.Ann. N.Y. Acad. Sci.,121/2, 404--427. 7 Gallo,R.C., Gallagher,R.E., Miller,N.R., Mondal, H., Saxinger,W.C., Mayer, E.J., SmiCA, R.G. and Gillespie,D.H. (1975) Relationshipsbe~tween components in primate ~ N A tumor virusesand in the cytoplasm of humark leukemic cells:implicationsto leukemogenesis. Cold Spring Harbor Syrup. Quant. Biol,,39, 933--961. 8 Gilbert,H.S. (1970) A reappraisalof the "myelo-p;:oUferativedisease"-- concept. Mt. Sinai J. Med., N.Y., 87,426.-806. 9 Grandgenett, D.P., Gerard, G,F. and Green, M. (1972) Ribonuclease H: a ubiquitous activityin virionsof ribonucleicacid tumor viruses.J. Virol.,12, 431--439. 10 Heuek, G. (1879) Zwei F~flleyon LeukSmie mit eigentiimlichenBlur -- resp. Knochenrnarke befund. Vkchows Arch, Pathol. Anat, Physiol.,78, 475--496. 11 Hunstein, W. and Hauswald, Ch. (1974) Die Osteomyelofibrose. Kiln. Wichenschr., 52, 305--317. 12 Jordan, H.E. and Scott,J.K. (1941) A case of osteoscl¢roslswith extensiveextramedu'~. lary hematopoiesis and a leukemic blood reaction.Arch. Pathol.,31, 895. 13 Leonard, B.J.,Israels,M.C. and Wilkinson, J.F. (1957) Myel(~sclerosis:a clinicopathological study. Quart. J. Med., 26,131--148. 14 Lewis, B.J.,Abrell,J.W., Smith, R.G. and Gallo, R.C. (1974) Human D N A polymerase III (R-DNA polymerase): distinction from DNA polymerase I and reverse transcriptase. Science, 183, 867--869. 15 Lewis, B.J, Abrell, J~W., Smith, R.G. and GalIo, R.C. (1974) DNA polymerases in human lymphoblastoid cells infected with Simian Sarcoma virus. Biochim. Biophys. Ac~a., 349, 148~160. 16 Mondal, H., Gallagher, R.E. and GaUo, R.C. {1975} RNA-dkected DNA poiymerase from human leukemic blood cells and from primate type-C virus-producing cells: high and low molecular weight forms with variant biochemical and immunological propertie.~. Proc. Natl. Acad. Sci. USA, 72, 1194--1198. 17 Nooter, K., Aarssen, A.M., Bentvelzen, P., DeGroot, F.G. and Van Pelt, F.G. (1975) Isolation of infectious C-type oneornavirus from human leukemic bone marrow cells. Nature, 2t56, 595--597. 18 Ornstein, L. (1964)Disc-eleetrophoresis I: background and theory. Ann. N.Y. Acad. Sci., ]2112, 321--349.
298 19 Rosenthal, N. and Eft, L.A. (1943) Clinicalobservations on os~e0petresisand myel0fibrosis.Arch. Intern.Med., 71,793--813.. . . 20 Schlabach,.A., Friedlender,B., B01den, A~ and We.~ssbi~ch,A. (1971:)DNA-dependent DNA polylmerases from: HeLa cellnuclei.II. Template and sub'stra~eufilizatign. ' Biochem. Biophys. Res. Commun., 4~, 879--885. ' : '~ ' ' 21 Scolnick, E.M., Parks, W.P. and Todaro, G.J. (1972.)Rev!drset:ranscript;~seof primate viruses as immunological markers. Science,.177, 119---121. . ...:: 22 Sedwick, W., Wang, T. and Kern, D. (1972) The DNA polymeras~s of ]!~BCells.J. Biol. Chem., 247, 5026--503.3.. . ' . ..... : ....... 23 Smith, R.G., Abrell, J.W., Lewis, B,J. and Gallo, RIC. (1975) Serol'.0giczlanalysisof human deoxyribonucleic acid polymerases: preparation and properties of anti.,~erum to deoxyribonucleic acld polymerase I from human lymphoid c_~11s.J. Biol. Chem., 250, 1702~1709. 24 Teich, N.M., Weiss, R.A., Salahuddin, S.Z' Gallagher, R.E., Gillespie,D.H. and Gallo, R.C. (19751) Evidence for type-C virus expression.inhuman leukemia. Nature, :256, 551--555. 25 Temin, H.MI.and Mizutani, S. (~970) DNA polymerase in vkions of Re'as Sarcoma virus. Nature(London), 226, 2211--1213. 26 Tronick, S.R., Scolnick, E.M. and Parks, W.P. (1972) Reversible inactivation of the deoxyribonacleic acid polymerase of Rauscher Leukemia virus.J. Vko]., ! 0, 1136-.1t42. .27 Ward, H.P. and Block, M.H. (1971) The natural history of agnogeniom'.lreloidmetaplasia (AMM) and ~ critical evaluation of its relationship with the myeloprolif~rative ~yndrome. Medicine, 50, 357. 28 WitkLn,S.S., Ohno, T. and Spiegelman~ S. (1975) Purification of B,NAqlastructed DNA polyrnerase from human leukemic spleen. ~oc. Natl. Acad. Sci.USA, 72, 4";33--4136.
29t
B I O C H E M I C A L A N D SEROLOGICAL C~.4~RACrERISTICS OF REVERSE TRANSCRIPTASE FROM.HUMAN SPLEE,N IN A CASE OF CHILDHOOD MYELOFIBROTIC S Y N D R O M E : ,
•
.
,,
'
,,..,
,
.
LINDA K.•STEEL,H. LAUBE and P.CHANDRA* GUstav:Etnbden-zentrurn der Biologist.hen Chemic, .Abtei~ung fur Molc~ularbiologle, Theodor.Stern,l,:ai 7, Frani'~fnrt70 (G.ER.) (Received'3 December1976) SUMMARY A reverse transcriptase isolatedand purified from the spleen of a child with myelofibrotic syndrome has biochemical and immunolog~cal properties similar to that of known type-C viral reverse transcriptases, and, in particular, to the enzymes from two primate type.C viruses (Gibbon Ape leukeroia virus and Simian Sarcoma virus}.
INTROD,C
ON
:
. . . .
:
The discoveryof RNA-directed DNA p01yme:rase (reverse ~ranscript~se in RNA tumor viruses [3~25] ) has led to much investiga~ioR of the possible role of DNA polymerases in neoplastic transformation of human cells. The use of the 'simul~aneous detection test' has led to the identification of reverse transcriptase activity in severedhuman malignancies; however, the enzyme has been purified.inoililya few cases [4,7,28], Human revm.~etranscrip~e, isolated and highly purified from fresh leu;¢emic ceils [23], has been demons~rated to be irnmunolottcaUy related to reverse ~ranscriptases of several primate RNA tumor viruses. Since the first reported cases were publi~hed [10], myetofibrotic syndrome has been found to resull; from a number of etiological facl~ors(review by Hunstein and Hauswald [11 ]). Pa~hologica/inw.~s~igationhas indicated a distinct nature, differing from ~hat of leukemia [8,12,13,t9,27]. The firs~ indication of a virus etiology of thlis disease came from data reported recently from our laboratory [4]. This report described the is,~latio:a,of a DNA-pelymera~ in a post-operative spleen specimen from a 2~Jfi-year-oldgirl having myelofibrotic syndrome. PuriJ'ication ~techniquesand some of its biochemical characteristics were described. This emTme was found to re~emble the reverse transcriptaze of RNA [minor vir~.,,;esin many of its biochemical parameters, including tern*Address correspondence to: P, Chaadra.
292
, ........
plate requirements and io~ic specificity.The purifiedenzyme was completely devoid of terminal transferaseactivity.In thi~:paper, we present fu:L~her physiochemical characteri~.:ation of the purified myelofibrotic enzyme, ~nd itsserologic'Arelatednessto other primate orLcornaviruses. MATERIALS AND METHODS Labelled deoxynucleoside triphosphates we:.~eobtained from NEN-Chemicals GmbH, Germany and unlabelled deoxynudeoside triphosphates from Calbiochem or Boehringer, Mannhei=,= Tutzini~.Anti-reversetranscldptase IgG from Gibbon Ape leukemia virus (GaLV), Avian My=:oblastosi:!~virus (AMV), Simian Sarcoma virus (SiSV) and SiSV reverse transcriptasewere obtained from Litton Bionetics.DEAE-23 celluloseand phosphocellulose P were Se~a products. DEAE-52 cellulosewas obtained from Whatm~m. D N A cellulosewas prepared by the method of Alberts and Herrick [2]. 'Activated' D N A was prepared by the method o:~Sehlabacb et el. [20]. DEAE-52 cellulose and ~,hosphocelluloseP were pretreated with bovine serum albumin by the method of Sedwick et el. [221..Alloperations were performed at 0-,-4°C.Salt concentrations were determined with a conductivity meter. Enzyme assay Isolation of the oncamavi~al-like DNA polymerase enzyme was previously described [4]. Po~ymerases a (I), ~ (II) and 7 (III) were isolated and purified by a modification of the procedures presented by Lewis et el. [14,15] and Gallo et el. ~7]. The D N A polymerases were assayed using poly[rA, dTr~], poly[dA-dT], oligo[dG] •poly[rC] and 'activated'D N A as template primers. The ~i~tandard assay was performed by adding 0.025 ml of enzyme to a volume of 0.225 ml containhug: 0.066 M Tris--HCl (pH 7.8), 0.001 M dithiothreitol(DTT), 0.01 M MgCl: or 0.001 M MnCl2, 0.06 M KCI or 0.125 M KCI, I #Ci [3H]dq['TP (I #Ci [3H]dI3TP for oligo[dG] •poly[rC]primed reactio~ts),0.04 M each of unle:belled dATP, dCTP and dGTP (dTTP for oligo[dG] •poly.[rC]primed reactJions),~md I #g of template p:dmer. Polymerases ~ (II)and reverse transcriptasewere assayed using poly[rA, dTL~] and poly[dA-dT] (0,06 M KCI and 0.00'.'.[ M MnCl: in the reaction mixture); and oligo[dG] •poly[rC] (0.125 M KCI ant:[0.001 M MnCI: or 0.01 M MgCI~.)as template primer. Polymc:rasesa (1) and 7 (Ill)were assayed with 'activated'D N A (containing 0.06 M KCI and 0.01 M MgCI: in the assay system), and poly[rA, dT12] (utilizing0.125 M KCI and 0.001 M MnCl2). Reactions were incubated for 60 rain at 87°C (MgCi2 present) or 30°C (MnCl2 present);and stopped by the addition of 0.36 m g B S A and 3 ml of cold 10% trichloroaceticacid (TCA}. Acid precipitablem.'~terialwas collectedou Whatman glassfiberpaper discs,washed 3 times with 5% TCA, and comlted in a liquid scintillationspectrometer.
293
Molecular weight determination of spleen reverse transcripta:~e Reverse transcriPtase isolated from a myelofibro'~ic spleen specimen [4] was maintained at -70°C in 0:5 ml aliquots. Molecular weight of the enzyme was determineci by glycerol gradient centrifugation. One millilitre of enzyme (140 pg/ml) was layered on to apreforined 9 mi 5--20% glycerol gradient in a buffer Containing: 0.05 M Tris---HC1 (pH 7.5), 0.00! M DT% 0.001 M EDTA and 0.20M NaCI; and subjected to velocity sedimentation for 24 h at 200,000 Xg in a Damon IEC ultracentrifuge. Three external marker proteins, hen egg albumin,:BSA and aldolase, Were similarly centril'uged (1 mg/ml). Gradients were fractlonated using an ISCO density gradient fractionator in 0.3 ml aliquots, and the 280 nm absorbance of each aliquot was determined. Assay of [3H]dGMP incorporation of the aliquots was perfcrmed with 0.04 ml of the test aliquot and oligo[dG] • poly[rC] as template primer (conditions previe~ly described). As a ta~her measure of the molecular weight, the enzyme and same marker proteins were subjected to disc-gel electrophoresis using the procedure of Davis [6] and Ornstein [18]. Immunological procedures Immune and preimmune GaLV IgG and immune AMV IgG were prepared to concentrations of 4, 8, 16 and 32 pg in a buffer described by Chandra et al. [5]. Myelofibrotic spleen reverse transcriptase (3.4 ug) and SiSV reverse transcriptase (32.1 ng), prepared in the same buffer, were separately combined with each respective IgG concentration and preincubated at 4°C for 16 h. Mixtures were assayed as described, using oligo[dG] • poly[rC] and ~:ncreasingthe [~H]dGTP to 2 uCi (total voI., 0.15 ml). Anti-SiSV reverse transcriptase and preimmune IgG at concentrations of 4, 8 and 16 pg in 0.1 M Tris---HC1 (pH 8.0) were combkned with 3.4 pg of spleen reverse ~;ranscriptaseand preincubated as above. MLxtures were assayed with 2 vg poly[rA-dT~2] and 2 vCi [3H]dTTP (total incubation vol., 0.15 ml). Spleen reverse tmnscriptase antisera Seventy micrograms of spleen reverse transcriptase, emulsified in Freunds adjuva~t, was injected intramuscularly into 2 female goats, followed by 3 booster injections of 35 pg given at 2-week intervals. The blood was collected, serum recovered, an IgG fraction obtained by ammonium sulfate precipitation and purification on a Sephadex G-200 column. Normal goat serum IgG frac~on was used as a control in the enzyme neutraJ.izatio:a assays. Myelofibrotic spleen anti-reverse transcriptase IgG and preimmune IgG were prepared to concentrations of 2, 4, 8, 16 and ~2 pg in 0.1 M Trts-HC1 (pH 8.0). ]?olymerases a, ~, ~ and reverse transcripta~e isolated from myelofibrotic spleen, and SiSV reverse transcr~ptase, were challenged for response against immune and preimmune IgG as described in the legend tc Table 2.
294
RESULTS
. .
', .
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,
"
•
'
"
,
'
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. i
Figure !a presents the glycerol gradient analysis,of 3exter~,al proi:,d-ln ...... m~rkers detected by abs0rbance at 280 n m . Assay of'.[?H]dGMP incorporation of enzyme, aliquots off the: gly'ceroi gradient is sh0wn in 'Fig: .].blThe results indicate an estimated:mo!ecui;~ weight of about T0,000 (assumi~tga .~10bulm' protein). This agrees with.the molecular weight of reverse itranscripta~es. isolated ~om other mammalian type:C viruses [1,7;9,1.6,26]. Disc e]ectrophoresis of the DNA. polymerase enzyme and marker ]xo~eins demonstJmted a mobility coEesponding to that of BSA. This also is in agreement with the 70,000 molecular weight ss determined by glycer01gradient analysis. Table 1 shows the data.obt~tined when myelofibrotic spleen, reverse transo~ptase was ~exted ag~ainstvariable concentrations of GaLV, SiSV and AMV antireverse transcriptase IgG. Semian Sarcoma virus reverse transc.~ptase, which has been shown to be strongly' inhibited by anti-GaLV reverse transcriptase IgG [1,16,21], was tested agaJmstconcentrations of uns:pecific and specific GaLV anti-reverse transcriptase IgG. The percenb of [~].=l]dGMP incorporation in the polymerase assay system for both SiSV reverse transc) iptase and human o---00v,~.Ibumln
0.5.
( MW
4s
0oo}
~0,4-
/,u C O,3. (~
co;, x
uJ 0,I.
5
10
~ FRACTION
2O NO,
25
30
- 25
:~ 400.
- 210
> - 15
r~ < 200-
- 10
.f
H 100C Lu
I
5
10
15 FRACTION
20
30
NO.
Fig,. 1. Glycerol gradient analysis of the oncornavind-like DNA polyme=~se isoh~ed ~=om myelofibrotic spleen, la: Presents :the opticaI densities at 2E~0nm of 3 ~ roterin standards, lb: Shows the [~H]dGMP incorporation in 0.3 ml aliquots of the purffivd enzYme fractlonated on a glycerol gradient (5--20%). The experimental detailsare d~,.~cribedin text.
TABLE 1
295
THE EFFECT OF ANTI-GaLV ANTI-AMV A N D ANTI.SiSV R E V E R S E TIq-ANSCRIPTASES O N THE ONCORNAVIRAL.LIKE DNA-POLYMERASE ENZYNIE ISOLATED F R O M H U M A N MYELOFIBROTIC 'SPLEEN A N D SiSV REVERSE:TRANSCR]:PTASEa Source Of reverse transcr.iptase .,.: Specific anti-reveme trm'.,~scrlptase (igG)added •'' '
'
': " . . . .
• •
IgG 0tg/' ' '
.
Nora-immune
Anfi-GaLV
reactjmict;) (control)I~:G :' RT IgG
.......
Anti-AMV
Anti-SiSV
R T IgG
RT IgG
[JH]dGN'P incorporation(% of enzyme activityl t~
SiSV 4 8 16 32
100 100 100 lOC
75.2 64.7 42.9 50.3
-----
-----
4
100
8 16, 32
100 100
90.3 57.7
lO0 96.7
44.2
100
39.6
86.4 93.7
69.9 38.6 24.6
; Human. rnyelofibrotic spleen
--
aAntisera (IgG) were obtained from Dr, R.C, Gallo, National Institute of Hea.q:h, U.S.A. 3,4 ug of spleen enzyme and 0.032 , g of SiSV reverse tr~mscriptase were preincubated for 16 h at 4~C with variable concentrations of non-immune and immune GaLV and immune AMV and SiSV IgG (spleen enzyme only), in a totalvol, of 0.06 ml (assay procedures as described in text). bThe % of [~H]dGMP incorporatlon expresses activity in the presence of variable concentrations of immu,ne IgG c0mpared:to that of nonqmmune IgG. TABLE 2
RESPONSES OF D N A PO[,,YMERASES T O MYELOFIBROTtC SPLEEN ANTI-REVERSE TRANSCRIPTASE igGa s0Uree and ~ype. of D N A polymeease
TemPlate primer
Myelofibroticspleenanti-reve~setranseripruse IgG (t~g) % o[" inhibition b 2
4
'Activated'D N A
NT
NT
Poly[dA-dT]
NT
Poly[rA. dT,~]
spleerLRTaze SiSV reverse transeriptase
Myelofibrotic splee3~ I (~) Myelofibrotic spleen II (i~) ,
Mye][ofibrotic spleen III(~)
8
16
32
0
0
0
NT
0
0
0
NT
NT
0
4.5
O,ligo[dG]-polytrC]
8.8
19.8
35.7
49.0
71.2
Pely[rA. dT,~]
NT
4.2
11.9
30.2
54.0
14.,5
Mydofibrotic
al'rei,nrnuneand immune IgG were d:P,Jted to the respectiveeoncentr~,tionwith 0.1 M Tris-HCl (pH 8.0).Each polymerase was preincebatedwith an equal volume of IgG f~r 40 mln at 4°C (totalprelncubationvol.,0.5 ml). Assays contained I ;~gof the optimum temp~lateprimer (2 ~g with'activated'DNA). 0.01 M MgCl:, I uCi [JH~dTrP, 0.05 Trls-HCl (pI-!7.5),0.001 M DTT, 0.06 M KCI and 0.004 M each of unlabdled dATP, dCTP and dGTP were presentwith 'actiw~ed'DNA. Assays utilizingdA-dT and rA •dT~, cont~dned 0.001 M MnCI~, I ;~Ci[~H]dT'IT,0.05 M Tris-HC! (pH 7.8),0.001 M DTT, 0.125 M KCI and 0.004 M each or;dATP, dCTP and dGTP. dG •rC template-prlmedassays contained 0.001 M MnCl~, 1 j~Ci[~H]dGTP, 0.05 M TrL~---HCI,0.001 M DTT, 0.06 M KC] and 0,004 M each of dATP, dCTP and dTTP. Total incubationvolume was 0.15 ml (other conditionsas describedin Materialsand Methods; NT, not tested). bData expressedas the percentageof inhibitiono:~the polymeraso testedwith immune IgG compared with the same qu~antityof preimmur:e (control)IgG,
296 ....
.
..
,
.
'.
i
;
"
'
"
pleen revere not fected by o -!mmuneo v. gG; ' '. whereas imIrmne GaLV exhibited strong inhibition against botlh enzyme~. Anti-SiSV reverse transcript~e IgG also showed strong inhibition oil t h e spleen reverse transcriptase.i The anti-AMV reverse:transcriptase IgG minimally inl~ibited polym~ase activity Of the spleen enzyme. These data indicate an.: immunologi.cal relatedness of our purified enzyme to the reverse %ranscriptases of pri~aateR N A tumor viruses. '['able2 ~ummarizes the zesponses of polymerases ~.,~, 7 and reverse tr~nscr.iptaseisolatedfrom myelofibrotic spleen, and SiSV reversetranscriptase, when challenged with varying concentrations of purified myelofibrotic spleen anti.reversetranscriptaseIgG. Myeiofibrotic spleen e and ~ polymerases were not inhibited by ~he spleen anti-reversetranscriptaseIgG; whereas the myelofibrc,ticspleen reverse transcriptaseand SiSV reversetranscriptasewel~einhibited 71.2 and 54.0% respectively,when tested a~ainst 32 ;(g of IgG. My~lofibrotic spleen 7 polymerase was inhibitedat a very low level(14.5% with 32'?.pg of IgG). DISCUSSION ,[~e results presented indicate that myelofibrotic spleen reverse tlmnscrip'lase is similar to reverse transeriptase of leukemic cells in several aspects. Two si:~e classes of the leukemic enzyme (70,000 and 130,000 daltons) have been reported [16], whereas we could detect only one molecular species of molecular weight 70,000, Witkin et al. [28] recently purified a reverse transcriptase from the human spleen of a patient with chronic lymphocytic ieuke:mia, and have also found a singular molecul~ species of 70,000 dattons. Thvs the cellular enzymes may exis~ in monomer and dimer species, whereas th.~ tissue enzymes r.nay contain a single active monomer. q?he data in Table I indicate that anti-SiSV IgG in the same eonc~,ntration range is significantly more inhibitory to our spleen reverse transcriptase than anti-GaLV under the same conditions. One plausible explanation is that the specific titer for anti-SiSV in the igG fraction is higher than ~;hatfor anti-GaLV. The other :indication could be that SiSV reverse transcriptase is mo~e closely rob.ted to the human enzyme than the GaLV reverse transeriptase. The latter indication is supported by 2 facts. Simian Sarcoma virus is the closest candidate (but not identical) to HL23Vq, a human leukemic virus purified from longterm cultm'es of myeloid cells from a leukemic patient [24]. Moreover, Nooter et al. [17] recently described the isolation of a SiSV~related virus from huma.~ leukemic cells. Preliminm'y studies ~ t h the myelofibrotic anti-reverse transcrip~ase ][gG have indicated thai':,the SiSV reverse transcziptase is more sensitive towards t~s IgG than the GaLV reverse transcriptase. More experiments ar(,~'necessm'y to clarify this issue. One major aspect of these studies was to look for the possibility of detecting leukemic cells in human patients, using splenic antireverse transcriptase. These studies are in progress.
297 ACKNOWLEDGEMENT
The authors are grateful to Dr. Robert C. Gallo for the gift of anti-SiSV and anti:GaLV samples. REFERENCES 1 Abrell,J.W. and Gallo,R.C. (1973) Purification,characterizationand comparison of the D N A polymerases from two primate R N A tumor v~uses. J. Virol.,12, 431--439. 2 Alberts,B.M. and Herrick,G: (1971) D N A cellulosechromatography, Methods Eazymol. 21, 198--217. 3 Baltimore,D. (1970) D N A polymerase in virionsof Rous-Sarcoma. Nature (Lond.), 226, 1209-1211. 4 Chandra, P., Steel,L.K, Laube, H. and Kor~huber, B. (1975) Oncornaviral-likeD N A polymerase activityin a case of childhood myelofibrotie syndrome. FEBS Left, 58, 71--75. 5 Chandra, P., Zunino, F., GStz, A., Gericke, D., Thorbeek, R. and Di Marco, A. (1972) Specific inhibitionof D N A polymerases from R N A tumor virusesby some new daunomycln derivatives.FEBS Left.,21,264. 6 Davis,J.B. (1964) Disc electrophoresisH: method and applicationto human serum proteins.Ann. N.Y. Acad. Sci.,121/2, 404--427. 7 Gallo,R.C., Gallagher,R.E., Miller,N.R., Mondal, H., Saxinger,W.C., Mayer, E.J., SmiCA, R.G. and Gillespie,D.H. (1975) Relationshipsbe~tween components in primate ~ N A tumor virusesand in the cytoplasm of humark leukemic cells:implicationsto leukemogenesis. Cold Spring Harbor Syrup. Quant. Biol,,39, 933--961. 8 Gilbert,H.S. (1970) A reappraisalof the "myelo-p;:oUferativedisease"-- concept. Mt. Sinai J. Med., N.Y., 87,426.-806. 9 Grandgenett, D.P., Gerard, G,F. and Green, M. (1972) Ribonuclease H: a ubiquitous activityin virionsof ribonucleicacid tumor viruses.J. Virol.,12, 431--439. 10 Heuek, G. (1879) Zwei F~flleyon LeukSmie mit eigentiimlichenBlur -- resp. Knochenrnarke befund. Vkchows Arch, Pathol. Anat, Physiol.,78, 475--496. 11 Hunstein, W. and Hauswald, Ch. (1974) Die Osteomyelofibrose. Kiln. Wichenschr., 52, 305--317. 12 Jordan, H.E. and Scott,J.K. (1941) A case of osteoscl¢roslswith extensiveextramedu'~. lary hematopoiesis and a leukemic blood reaction.Arch. Pathol.,31, 895. 13 Leonard, B.J.,Israels,M.C. and Wilkinson, J.F. (1957) Myel(~sclerosis:a clinicopathological study. Quart. J. Med., 26,131--148. 14 Lewis, B.J.,Abrell,J.W., Smith, R.G. and Gallo, R.C. (1974) Human D N A polymerase III (R-DNA polymerase): distinction from DNA polymerase I and reverse transcriptase. Science, 183, 867--869. 15 Lewis, B.J, Abrell, J~W., Smith, R.G. and GalIo, R.C. (1974) DNA polymerases in human lymphoblastoid cells infected with Simian Sarcoma virus. Biochim. Biophys. Ac~a., 349, 148~160. 16 Mondal, H., Gallagher, R.E. and GaUo, R.C. {1975} RNA-dkected DNA poiymerase from human leukemic blood cells and from primate type-C virus-producing cells: high and low molecular weight forms with variant biochemical and immunological propertie.~. Proc. Natl. Acad. Sci. USA, 72, 1194--1198. 17 Nooter, K., Aarssen, A.M., Bentvelzen, P., DeGroot, F.G. and Van Pelt, F.G. (1975) Isolation of infectious C-type oneornavirus from human leukemic bone marrow cells. Nature, 2t56, 595--597. 18 Ornstein, L. (1964)Disc-eleetrophoresis I: background and theory. Ann. N.Y. Acad. Sci., ]2112, 321--349.
298 19 Rosenthal, N. and Eft, L.A. (1943) Clinicalobservations on os~e0petresisand myel0fibrosis.Arch. Intern.Med., 71,793--813.. . . 20 Schlabach,.A., Friedlender,B., B01den, A~ and We.~ssbi~ch,A. (1971:)DNA-dependent DNA polylmerases from: HeLa cellnuclei.II. Template and sub'stra~eufilizatign. ' Biochem. Biophys. Res. Commun., 4~, 879--885. ' : '~ ' ' 21 Scolnick, E.M., Parks, W.P. and Todaro, G.J. (1972.)Rev!drset:ranscript;~seof primate viruses as immunological markers. Science,.177, 119---121. . ...:: 22 Sedwick, W., Wang, T. and Kern, D. (1972) The DNA polymeras~s of ]!~BCells.J. Biol. Chem., 247, 5026--503.3.. . ' . ..... : ....... 23 Smith, R.G., Abrell, J.W., Lewis, B,J. and Gallo, RIC. (1975) Serol'.0giczlanalysisof human deoxyribonucleic acid polymerases: preparation and properties of anti.,~erum to deoxyribonucleic acld polymerase I from human lymphoid c_~11s.J. Biol. Chem., 250, 1702~1709. 24 Teich, N.M., Weiss, R.A., Salahuddin, S.Z' Gallagher, R.E., Gillespie,D.H. and Gallo, R.C. (19751) Evidence for type-C virus expression.inhuman leukemia. Nature, :256, 551--555. 25 Temin, H.MI.and Mizutani, S. (~970) DNA polymerase in vkions of Re'as Sarcoma virus. Nature(London), 226, 2211--1213. 26 Tronick, S.R., Scolnick, E.M. and Parks, W.P. (1972) Reversible inactivation of the deoxyribonacleic acid polymerase of Rauscher Leukemia virus.J. Vko]., ! 0, 1136-.1t42. .27 Ward, H.P. and Block, M.H. (1971) The natural history of agnogeniom'.lreloidmetaplasia (AMM) and ~ critical evaluation of its relationship with the myeloprolif~rative ~yndrome. Medicine, 50, 357. 28 WitkLn,S.S., Ohno, T. and Spiegelman~ S. (1975) Purification of B,NAqlastructed DNA polyrnerase from human leukemic spleen. ~oc. Natl. Acad. Sci.USA, 72, 4";33--4136.