Serological characterization of a purified reverse transcriptase from osteosarcoma of a child

Cancer Letters, 7 (1979) 189-195 © Elsevier/North-Holland Sclentihc Publishers Ltd.

189

SEROLOGICAL CHARACTERIZATION OF A PURIFIED REVERSE T R A N S C R I P T A S E F R O M OSTEOSAR.COMA O F A CHILD

KARL WELTE, [.~VE EBENER and PRAKASH CHANDRA* Gustav.Embden-Zentrum der Bzologischen ChemJe, A btezlung fur Molekularbiologle, Universit~'t Frankfurt~Main, (F.R.G.)

(Received 12 March 1979) (Accepted 19 April 1979)

SUMMARY Serologic~d analysis o f t h e reverse transcriptase (RTase)., purified from h u m a n osteosm'coma tissue, has s h o w n t h a t it is antigenicaliy related to DNA polymerases from BEV and from RD-114. No cross-reactivity o f t h e osteo. sarcoma RTase was observed with RTases purified f r o m AMV, RLV, SiSV, GaLV mad from h u m a n spleen o f a p a t i e n t with myelofibrosls. INTRODUCTION A m o n g t h e various factors associated to t h e etiology o f malignant bone t u m o r s J~ man, ionizing radiation or r a d m n u c l e o t i d s seem to play an import a n t role (1,10,13,17). Various approaches, in r e c e n t years, indicate the association o f T#~msesto this malignant disease [2,7,9,10,12,20]. This prompted u s [8] to s varch for a v/rus-specific reverse transcriptase in h u m a n osteosarcoma tissue. We have succeeded in purifying a reverse transcriptase from h u m a n osteosarcoma tissue with bmchem~cal properties, resembling those of C-type R N A t u m o r viruses [8]. In t h e present c o m m u n i c a t i o n , evidence is presented t h a t ~he h u m a n osteosarcoma reverse transcnpLase is antlgenicaUy related to D N A polymerases from b a b o o n e n d o g e n o u s virus (BEV) and from r h a b d o m y o s a r c o m a vLrus (RD-114 VLmS). No cross reactivity of t h e osteosarcoma reverse transcript~Lse was f o u n d against antibodies to purffmd reverse transc~ptases from avian myeloblastosis virus (AN[V), Rauscher *Address all correspondence to Professor P. Chandra, Gustav-Embden-Zel~trun~ der Biologlse]hen Chen'ae, Abt. ~'tir Molekulaxbiologie, Khnikum der Johann Wolfgang Goethe-Universit~lJt,Theodor Stern IG~i7, D-6 FPankfurt/Mam 70, F.R.G. Abbreviatzons /UI~IV,Avian myeloblast,c,sis virus, lqEV, Baboon endogenous vtrus; GaLV, Gibbon ape leukemia virus; MFS, Human rnyelofibrotic spleen; RD-114 vtrus, Rhab~omyosarcoma vtru~,,RLV, Rausc[her leukenua wrus; SISV, Simian S,~Lreoma~r~rus.

190 leukemia virus (RLV), Simian sarcoma virus (SiSV), gibbon ape leukemia virus (GaLV), and from human myelofibrotic spleen [5]. Thus, the antigenic character of the osteosarcoma reverse transcnptase ,exhibitsa spectrum of specificity sinzilarto the reverse transcriptase purified from h u m a n melanoma tissue [3]. MATERIALSAND METHODS

Case report A 13-year-old girl complained of pain and sweUmg in the right upper arm and shoulder are~ for about 3 months. The radiographical appearance was characterized by destruction of the proximal humerus with both lytic and osteoblastic features, periostal hone formation with development of spicula and Codman's triangles, and invasion of the tumor into the surrounding soft tissue. Bone scintigraphy showed an enrichment of activity 1D the area of sho~der joint and proximal humerus. The tumor was o, ~tained by en-bloc resection of the right upper extremity. Approximately 100 g tumor tissue was kept at - 8 0 ° C until used. Histologically the tumor tissue revealed the typical pattern of osteosarcoma (osteobiastiLc type with small fibrosarcomatous differentiated areas). SH-Labelled deoxyribonucleosides triphosphates were obtained from NEN. Dreieich, (F.R.G.), and unlabelled deoxyribonucleosides triphosphates were obtained from Boehringer Mannheim. Tutzing. Oligomer-homopolymerkrimer-templates were obtained from Collaborative research, Mdes Laboratories and Boehringer Mannheim. DEAE (DE 23) - cellulose were obtmned mm Serva, Heidelberg, DEAE (DE 52) -- cellulose and Phosphocellulose P 11 from Whatman. Antibodies against reverse Lranscriptase's from SiSV, GaLV, AMV, RLV, BEV and RD-114 vLrus were obtained from Dr. Jack Gruber and Dr Robert C. GaUo (National Cancer Institute, U.S.A.). Ex traction o f DNA polymerases Fifty grams of the osteosarcoma tissue was mined with 500 ml buffer 1 (50 mM Tris--HC1 (pH 7.5), 1 mM DTT (d~thiothreitol), 0.5 mM EDTA, and 5 r~M MgC12) and homogenized under ice-water coohng m a Waring-hlendo c fol 5 min each at ~ow, medium and high speed. The suspeasion wa,~ rehomege~dzed in a Dounce homogenizer and filter,~d through a monolayer of 'nylon stocking. Further purification procedures, outlined in [8], were calTied out by modification ,~f the procedtvres by Lewis et al. [15] and Chandra and Steel [5]. All operations were performed at 0--4°C. Reverse rranscriptase assay The reverse transcriptase was assayed by adding 10 ~1 of the test fractmn to the volume of 40 pl which gave a final concentration of 50 mM Tns--HC1 (pH 8.1), I mM DTT, 60 mM KC1, 1 mM MAC12, 72 ug BSA, 20 pM each c,f the unlabelled deoxynbonucleoside trlpbosphates, 5 ~Ci of [3H]dGTP (sp~,c. act. 776 cpm/pmol) and 1.25 pg of (rC), (dG)12_ is.

191 Reaction mixture was incubated for 60 mm at ~0oC aad ~he reactmn terminated by tlhe addition of 72 #g BSA and 3 ml of cold (4°C) tnchloracetic acid (10%, w/v) containing 20 raM sodium pyrophosphate. Acld precipitable mal;enal was collected on Whatman (GF/C) glass-fiber discs, washed 3 times with 5 ml of 5% (w/,J) tnchloracetic acid containing 20 mM sodium pyrophosphate, dried at 100°C, then suspended m 10 ml of toluene scintillator flukl[ (Quickzmt, Zmsser & Co., Frankfurt) and counted for radioac~lwty m a liquid scintillatlor spectrometer (Mark III Liquid Scintillation System, Searle, Des Plaines, U.S.A.).

SerOlogical procedures Preimmune IgG-fractlons and antibody-fractions agmnst reverse tmnscriptase from v ~ o u s sources (AMV, RLV, S1SV, GaLV, BEV, RD-1 14, human myelohbrotic spleen (MFS)) were prepared at concentrations of 8--32 pg IgG/10 pl of Trls--HCl (pH 7.5) Reverse transcnptase fract~o~ (10 pl) was incubated for 16 h at 4°C with an equal volume of ~he I~;Gfraction. The remaining enzyme acttvlty was measured using ti~e reverse transcriptase assay with (rC) n (dG) L2-is as template-primer in a total volume of 50/11~. All other conditions are described above The ~ercentage of inhibition of Lhe human osteosarcoraa reverse transcriptase activity by ant~bodies against r,~verse transcnptase from various sources was expressed using the same quantity of pre-lmmune (control) IgG (compared with immune IgG) as 100% or actlwty. The enzyme activity of the uninhibited re&-tion was about 1000 pmol/60 mm/mg prgtein. Protein was rL~Leasuredby the method of Lowry et al. [16]. RESULTS AND I~,ISCUSSION The reverse t~anscriptase purified from the osteosarcoma tassue of a child has been characterized bLochemically [8]. As follows from Table 1, the enzyme is located m the mmrosomal pellet. The purified enzyme has a molecular weight of 68,000, and is able to transcribe the heteropolymeric regions of a 70S RNA from R(Mu)LV. Based on these properties, and its specificity towards various template-primers the purifmd enzyme from human osteosarcoma tissue resembles biochemically DNA polymerase~ from RNA tumor v~ruses (retroviruses). Stud,Les on the immunological charactemzation of purif~ted reverse Lranscriptases from human turner biopsies and cells [3--6,11] have led to 2 antigemcally distinl,n~ishable sets of interspecies determinants. Reverse ~ranscriptases purified from human leukemfc cells [ 11 ], spleen of a pstien~t with myelofibrotic syndrome (a preleukemic disorder} 115,6], and from the orbitm tumor (granulocylbic sarcoma) assocmted to A.M.M.L. [4,6] s h ~ e d common antlgeaic deterramants, as shown by their cross-reactivity to DNA polymerases of SiSV and GaLV. On the oth.~r hand, the reverse tran.c;c~ptase purified from human melanoma tissue exblbited a completely different anti-

192 TABLE 1

BIOCHEMICAL PROPERTIES OF REVERSE TRANSCRIPTASE PURIFIED FROM HUMAN OSTEOSARCOMA TISSDE* Property

Results

Cellular location Molecular weight Utlhzatior~ of template-primer

Particulate, fraction 68,000 (rC)n " (dG)~2_, s ~(rCm)n " (dG),2 ; (rA)n • (dT)j 2 ; no transcription of (dA)n " (dT)l o

Transcription of heteropolymeric regmns of viral 70~ RNA Divalent cation preference Terminal deoxynucleotidyl transferase activity

Yes Mn2÷ >> Mg2÷ None

*Ebener, Welte and Chandra [SJ. genic specifmity [3]. The h u m a n m e l a n o m a reverse transcripmse was n o t inhibited by antibodies to D N A polymerases purified f r o m SiSV a n d GaLV. Even m o r e surprising 'was t h e observation t h a t antibodies to reverse transcriptase f r o m h u m a n myelofib~otic spleen failed to inhibit t h e activity o f t h e h m u a n m e l a n o m a reverse transcriptase. Interestingly t h e purified e m T m e from h u m a n m e l a n o m a tissue showed a close antigenic resemblance to D N A polymera~es f r o m BEV a n d RD-114, t h e e n d o g e n o u s virus o f t h e cat. Studies o n animal species have so far indicated t h a t viral DNA polymerases within a given group o f type-C viruses are immunologicaily reJated, or even i d e n t i c a l For example.~ DNA polymera~es f r o m avian type-C viruses (RAVo, AvLVAM v a n d AvSVR) are immunologically closely related []L8,19]. It was therefore o f great inlerest to s t u d y t h e antigenic specificity o f t h e purified reverse transcriptase f r o m h u m a n o s t e o s a r c o m a t,ssue. The effect o f antibodies (IgG) to D N A polymerases from R N A t u m o r on-uses a n d f r o m spleen o f a patient with myelofibrosis on t h e enzymatic activity o f re~,erse transcriptase purified f r o m h u m a n osteosarcoma tissue is s h o w n in Table 2. No inhibition of t h e e n z y m a t i c activity was obtained against antibodies to reverse transcriptases f r o m AMV, RLV, SiSV, GaLV and h u m a n rnyelofibrotic spleen. A similar specl~rum o£ inhibition oi' t h e e n z y m a t i c activity was obtained when antibodies to DNA polymerases from BEV a n d RD-114 were pre~ncubated with t h e e n z y m e . In b o t h cases, a concentration-dependent inhibition o f t h e e n z y m a t i c activity was observed. The spectl~lm o f antigenic specificity o f h u m a n osteosarcoma rew.~rse ~ranscriptase follows s o m e well established facts on t h e n a t u r e of antigenic d e t e r m i n a n t s in reverse transcriptases ~rom endogenous, or exogenous

193 TABLE INHIBITION OF HD.'MANOSTEOSARCOMA REVERSE TRANSCRIPTASE ACTIVITY BY ANTIBODIES (IgG) TO DNA POLYMERASES PURIFIED FROM RNA TUMOR VIRUSES AND FROM A SPLEEN OF A PATIENT WITH MYELOFIBROTIC SYNDROME [5] Source of antl-rew!rse transcriptase

IgG-concentr~tiom~(~g) (A) Non-primate sources. Avian myeloblastosls virus Rauscher leukemm vlrus

Percentage of inh,bition

8 0

16 S

32 12

0

0

0

RD-114 vu us

56

70

74

(B) Primate source ~: Simian sarcoma vh us Gibbon ape leukemia v'.rus Baboon endogeno ~ "~rus Human myeloflbrotic syndrome (spleen)

0

5

3

0 52

0 67

0 71

0

0

4

Antibodies against reverse transcrlp~lse ('ron~various sources were ]prepared at eoncentratmns of 8--32 ~g/10 ~l of Tris--HCl (pH 7.5). Reverse transeripinse fraction (10 #1) was incubated for 16 h at 4°C with an equal volume of the antibody-fraction or p~eimmune IgC The remaining enzyme actim~.y was measured using the reverse transcriptase a~ay w~th (rC)n (dG)~2_~ as temphte-primer in a total volume of 50 ~l. l~eactlon condltim, s are described under Materials and Methods.

class o f viruses [11]. For example, the h u m a n osteosarcoma enzyme crossreacts with the DNA polymerase f r o m BEV, but has no antigenic relationship to DNA polymerases from the e~ogenic primate v~mses, $iSV and GaLV. This is analogous to the fact that BEV-DNA polymerase does n o t cross-react with DNA polymerases from SiSV and GaLV (see ref. 11). The inhibition o f osteosarccma reverse tran,scrip~se by antibodies go DNA polymerase from RD-114 again points to a c o m m o n del~erminan~,. The purffmd polymerase~ f:'om BEV and RD-114 have been shown to be antigenically similar [21]. .In an earlier r e p o ~ , Kufe et ',fl. [14] have shown that human osteo~arcoma contains RNA related to the RNA o f m o u s e leukemia vivJs, Our experiment~ do r~ot exhibit any nnmunological ~,~imflarity between rew~me transcriptases from h u m a n o s t e o ~ r c o m a tissue and RLV or GaLV. Since the hybridization technique involves the formation o~ 'mismatched' hybrids, it is difficult to interpret these rest~lts with respect to viral information. I~ may, however, be useful to repeat the hybridization experiment~ using ~he ~ f i n i t y p a t t e r n ' system reported by Gallo et al. (see ref. 11).

194

Based o n ~he immunological patterson o f t h e purified reverse transcriptase f r o m humvz~ usteosarcoma, ~t is h a r d to tuaders~md t h e role o f leukemic cells o n t h e rele~.,se o f C..type particles b y osteosarcoma cell cultures [7]. In analogy to fine animal situation, it was suggested [7] that; t h e viral information in l e u k e m i c celh~ m a y take over t h e helper f u n c t i o n in t h e release of C-type particles ii'om osteosarcoma cell cultures [7]. If this were t h e case, one w o u l d expect s o m e similarity in t h e viral-related i n f o r m a t i o n between h u m a n osbe~.~arcoma tissue and t h e h u m a n leukemic celts. The present data are noi i]a ~avor o f this possibility. T h u s , it m a y be t h a t t h e h u m a n situation is ,'~otcl~ite analogous i,o the anima~ situation. Further characterization of ~he :~um~,~a osteosarcoma e n z y m e is in prog, ess to establish its i m m u n o logical patt~rn. ACKNOWLE

~)GEMENTS

We wre g~'alt,eful to Professor Dr. H. R l e h m (Universitfits-Kinderl6inik, Berlin-West ~~or providing h u m a n osteosazcoma tissue and t h e radiographic picture. Th/s work was s u p p o r t e d in part by Grant No. 14 0305 of t h e Stlftung Vo~kswagenwerk. REFERENCES 1 Arlen, M.~ Hihinbotharn, N.L., Huvos. A.G., Marcove, R.C. MKler, Th. and Sl~ah, I.C. (1971) R~d~ation-induced sarcoma Lofbone. Cancer, 2-8, 1087--1099. 2 Bowen, J., ~'~Alen,P.T., East, J.L., ~Ivruyama, K. Newton, W.A, Georgiades, J., Priori, S . . ~ J Dmoehowski, L. (1973) Molecular probes m studies of the relationship of virtraes ~o human neopl~da. Am. J. Clin. PathoL, 60, 88--99. 3 Chandra, P.~ Balikcioglu, S. and Mildner, B. (1978) Bioehemica! and immunolo,glcal ¢harac~erzza~lon of a reverse tmnscriptase from h u m a n melanoma tissue.Cancer Letters, 5. 299--310. 4 Chandra, P., Steel,L.K. and Cavdar, A.O. (1979) Ewdence for the presence of an oncornavh~l reverse transcriptasein an orbital tnrnt~rassociated to acute myelom onocytic leukernia in children: Biochemical ~md immunological characterizationof the enzyme. In. Modern Trends in H u m a n Leukemia. Editors. R. Neth and K. Mannweil~r. Sl;~inger-Verlag,Berlin--Heidelberg--New York. 5 Chandra, P. and Steel,L.K. (1977) Purification,bio~l'lerniealcharacterizationand serologics~ analysis of cellulardeoxyribonucleic acid polymerases and a reverse trartseript~ze fxom sl)leenof a patient with rnyalofibroticsyndrome. Bmchern. J., ] 67,

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