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Philip Morris

the Quest for Human Cancer Viruses

Date: 19620000/P
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1005087308-1005087314
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Taylor, G.
Trentin, J.J.
Yabe, Y.
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LEGAL DEPT/CARLSTADT QRSA
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N28
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Stmn/R1-072
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Md Anderson Hospital & Tumor Inst
NIH, Natl Inst of Health
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Bunting
Duranreynolds
Ginsberg
Kibrick
Rowe
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1005087217/1005087364/Dr Stowell Correspondence and Articles 19 56 A11
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Stmn/Produced
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Baylor Univ
Okayama Univ
Science
Univ of Tx
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1005087272/7317

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MARG, MARGINALIA
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05 Jun 1998
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puz28e00

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r d F ! :1a..:.~. Y. 7a l'~f qk'=1 t ti. 'I. -..;• ..- ~t c ?.• h:~ . .. -~_.~.. . fercnce, and the first steps were taken at that conference to clear channels for radio astronomy. The very important' -'band of frequencies near the hydrogen Bne (1400 to 1427 Mcy/sec) was eleared, as a result of almost complete agreementa Various other, frequency bands were given less protection, gen- eraily by, allowing radio astronomy to '<=ahare the ' band with other users. Al- though the results of the 1959 ewnfer- ; ence obviously fall short of alll that radio astronomers hope for, the eon- .'M1t'''~tf~ .~_ t r+ c t ' ~s^s.. ~d ~ ,.., 1 '4 Y. xrS.:" : SCIENCE 137:835,841, 1962 :r. : Many forms of cancer in' many ~?'species of animals are now known to ' be caused by viruses (1). While all t-attempts to isolate causative viruses from human cancer have thus far been unsuccessful, it would appear to be biologically unusual if at least some forms of human cancer were not also :•,eaused by viruses. By far the most r fruitful technique for the demonstration t: of tumor viruses in animals has been ::. the inoculation of extracts of tumors, r• or of nontumorous tissues from tumor- bearing animals, into newborn animals of the same species. This technique is 7obviously' denied to the searcher for " human cancer viruses. Attempts to in- duce cancer in the young of other species by inoculation of human tumor extracts have thus far been negative or inconclusive. The technique perhaps most widely used at present is the attempted propa- gation of human cancer viruses in vitro by inoculation of tissue cultures with human cancer materials. Yet this tissue culture technique has not resulted in the discovery of a single animal tumor 34 aEP1rEMBER 1961 • .... . . ~ F~x ference represents to scientists a very, valuable first step. This article has attempted to show that'further needs for clear frequency bands exist, and it has told' of the work now going on in preparation for the next radio conference. There will be a special ITU conference in 1963 to allocate frequencies for radio com- munications for space research, and it is the hope of radio astronomers that their science and its needs inay . be further considered at that {ime. ",: The Quest for Human ' _ ancer Viruses ` :-A'new approach to an old problem reveals cancer induction in hamsters by human adenovirus. John J. Trentin, Yoshiro Yabe, Grant Taylor .y `-I; ; .:I #f r. . . Referened and N.td . : . . / .~. . f 'L7. V. Jeller and B. P. C. t.ooper. Ree.,Sel. rnter. 32, 166 (t9s11. ' 2 Tbe National Radio Astronolny Obssrvatory 6 operated by' Aewcialed Unh'enlties,. Ine., nder contract withthe Natlonal Science - Poundation. 3. J.. W. Findtar, lroa, r.R.S. (UeuL Radto Snan.) . 46.. 35 (193!).. 4.. W. E. Morrow and D. C- Maciellan, d+bon. . /. sf, 107 (t96q. i.A. E• I:Oley, ibtd. 66, 116 (1961)....-~- 6, rnthia eaamPle we ha+em toaa+ame lae. eCdtWe area of the tremmdttlnyantenna (we 'ha.etakea 10 mr) . and a receiver band width. -- (0 Mcrhee): . and we hars asmned an eleoa ' uoa content o/ tAe' Ibndphed trPleal of a ..y ~ wiMer eooaw at Wi.hYnyton, D.C. . . ,.. .. ~,°..a...,., virus, and only secondarily has it been possible to grow easily some exceptional animal tumor viruses in tissue culture. Because of the severe limitations im- posed on the virologist concerned'with human eanceri we must continue to use the tissue culture screening approach. But the need for new approaches is obvious. One such new approach is described' in this article. It was demonstrated by Duran-Rey- nals (2) that known animal tumor viruses can„under certain conditions of host susceptiNility, produce acute non- cancerous diseases. The production of tumors appeared'to be a late manifesta- tion of the slow growth of virus in a relatively resistant host. It therefore appeared possible that' some human viruses already' known for their acute disease manifestations might, under other conditions of host resistance or in a host surviving the acute disease, producee the late manifestation known T,e enNm, are amiiated with the di+i,wn of as cancer. SUme of the already known aPrN°~snulbioloey ofBaytor UnWenitr Collere er Meduc+ne and wiN the accHUn of eayeriatenW (or unknown) human viruses of aa tlte Pedieuice,. unirenltr. nf Teaae M„ D. Anderwn diseases of childhood might therefore Hospitet'and'rudor tnalate, ltoeaoon. Dr• Yabe h wkaw frud Okeyanu Vd.eeYpi MWkal also be eancerviruses ar,oot, Japs : . :,...:..• . ~. . . . . .. ~ r`:=- . ... . Alla. ~. . w.~ Another large group of already iso- Iated viruses should also be suspect- the human "orphan" viruses. These are viruses that have been isolated from humans but whose disease manifesta- ' tions are unknown=~iruses in search of 'disease.° Cancer may well be the disease that some or many of these/' viruses are at present "orphan" to! With j ese concepts in mindi we initiated a ~ program of testing known human vi- ._ ruses for cancer-inducing effect in new- born animals. The newborn Syrian i hamster was selected as the test animal of choice because of its known relative susceptibility to tumor induction by tumor viruses of other species (3), and j because of its relatively poor defense against the growth of normal-tissue and tumor-tissue transplants from other species, including the human (4). Newborn hamsters are actually more susceptible to tumor induction by poly- oma virus of the mouse than are new- born mice (5). In spite of the many great advantages of inbred mice in other areas of cancer research, mice were excluded as test animals in this study because of the numerous tumor (and -• nontumor) viruses which they' are known to harbor, and because of past experience with interference by such viruses in attempts to isolate human cancer viruses by other methods (6). One of the first groups of human viruses chosen for extensive testing was the adeno group, because of similarities ' to some known animal tumor viruses, including polyoma virus, and because of the latency of adenoviruses in a large percentage of children. The adeno- t ;p4 l tl I ~K~`Sr* ~ . Ar"t u?''a°pk~ '~" rr IF b1y. .a. 0 .:4 s.: :ax`~dt>r,,,g t : { T ~ .... S
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:?4 :r. .-~ 0 a r sor~e , u s .~ " r Virus ~'.. ~ Viam titer• --~ Dose • : ~,~~ type '_~~(MTCIDt»!0'1 ml)~. '~(ml) ~ Y~ .. ,s. . ,.'. Table 1. Tumors resulting from injaetion of huntan tidenoviruses into newborn hamsters. 2 .'; ._. 102 . .0.1lntraperitoneal : 0.01 • Intracraniai 0.1 Inaraperitoneet ~ ~-~0.01: ~ Intrscnnial ;. 7 102 0.05 ' latraputmonary 7. • 10s ' 0.05 ' Intrapulmonary , ! - 10s '- 0.03 . Intrapulmonary 10 -. 10s" , 0.03 Intrapulmonary 11 ::-10s ~ ; 0.05 lntrapulmonary 12 ;.`10sj lntrapulmonary IOs/ '.' OA3 Inuapulmonary ' 14 ~10f' - - 0.03 . Intrapulmonary Tumorous Dead without Tbwa Virus titer hamsten/ Tumor tumon culture (MTCIDto./ , passaBe• 0.1 ml)l ha injmsters ~ected sters~ (days) deatlu . No. Days. y ~ a. Iloute of~ Tumorous hatnsters/ . - Injection - injected'hamstent•, Tumor deaths (itays) , , Alive without tumors No... Days ~ µ ~.• All tlten"repreum . 1:10 dllutlon of the culture suids sent from the American Type Culture. Cos- • lettloe•.. t"Wected Mmsten"'denian hamrten injeaed within 24 houm after birth.and surrivin6 ••.lonaer than. 3 weeks... Most nontumorous hamsters are B8 to 11 months old and eoDunderor} '' servation at the time of writina• S First shipment of type 12 adenovintss tieauel cu7turee auid received "'. from the.Ameriean~Type Culmre. Coaectiun. 9Second sh'~ipmentoL type. 12edeta.iius dasue `"-qtkun- fIWQ recei.ed from the American Iylte. Cultune. Collection about 3 manths after Uieant shlpmmG ~. „ ,~ viruses grow In the nucleus, and their The breeding colony was established nucleic acid is deoxyribonucleic acid. originally with Syrian hamsters from r; The virus particles occur in crystalline the polyoma-virus-free colony of the arrays comparable to those reported by National Institutes of Health (9). Strict '- Bunting in benign human skin papil- isolation procedures were instituted lotnas (7), and by Banfleld er af. (8) from the outset' with respect to all In tissue culture cells infected with other species of animals and to human . polyoma virus. • visitors. This combined hamster, colony and virus laboratory' is referred to here- after as the hamster laboratory. Materials and Methods FL amnion cells and HeLa cells (10) and' lung cells of human embryos (11) An isolated building was designed ' were used. The FL amnion cells were - and constructed for the study, with cultured in medium 199 with 10 percent three separately air-conditioned hamster calf serum. The lung cells of human rooms, a tissue culture laboratory, a embryos were cultured in medium con- sterile room, and a wash room and sisting of either medium 199 or Eagle's sterilizing facility. One of the rooms minimum essential medium with 10 :" was used exclusively for a hamster percent calf serum: The HeLa celCs breeding colony. Newborn litters and were cultured in Eagle's minimum es- their mothers were transferred to one sential medium with 10 percent calf of the other two hamster rooms as serum (12), c needed for inoculation. The other ex- - Human adenovirus types 2, 3, 7, perimcntal hamster room was used for 7a, 9, 10, 11, 12, and 14 were ob- a concomitant study involving inocula- tained from the American Type Cul- ' tion of newborn hamsters with human ture Collection. The virus tissue culture cancer tissue or extracts, or with fluids fluids thus obtained were directly di- from from tissue cultures inoculated with luted to a concentration of 1:10 with human cancer tissue. No animals were the maintenance medium described "; transferred back from the two experi- later, injected into animals, and at the mental rooms to the breeding room. same time titrated in tissue culture tubes Tabled. Tumor induetion by «ll-free filtnte of human type 12 adenovirus tissue culture fluid :.. injected intrapulmonuily (dose, 0.05 ml)- lst 103 7/8 33-91 1 376 ~ 3rd . ~ 10s. 26/27j~~ 33-157. ~ 1 1'!6, tth 101 6/6 ..29-43 Control tutturol None. - 0/7. 2 ~• 321,327 '~ 5 •.374-376 • Thee second shipment of type 12 Wenorirue from American Typa Culture Cnlleetion w.s usedd for tlisue.culture passaae. t"lnjectM hamstert" sianlaes ham.ters tnjectedlat birth and eurvli/nt over3'weeks.. S Four hanr4ers of this group developedlarae tutnars inn the liver, Iw addition too tumon.lu Ne thoraa. . I Noninoculited HeLa eelt tiseue eulture 6uWd eenttitused at 1500 re.oluclons per mdnutee for to minutesm to remove aellt tK ' . - . . . . , .-. " .. f + .±r <'' ~'~ 4}'-a.N€ ~tf of the cells described in the preceding paragraph. Later, however, type 12 virus was propagated in HeLa cells in the tissue culture room of the hamster -laboratory and' tued for further ex- ` periments. , .. ~ Polyoma virus (13) was recultured' in, and stored in, a building separate ` from the hamster-laboratory. No tissue culture work with polyoma virus was done during the period of the experi- . ment under discussion. The polyoma virus hemagglutination inhibition test was carried out according to the procedure of Rowe et a!. (14) on sera collected by exsanguination. At fust Earle's balanced salt solution, ', medium 199, or Eagle's basal medium . with 5 percent horse serum was used as the maintenance medium for the ten- fold serial dilutions of each adenovirus tissue culture fluid. Each dilution (0.1 : m1) was inoculated into two tubes con- taining a monolayer of' either FL am- nion or human embryonic lung cells newly replenished with 1 milliliter of maintenance medium. Inoculated tubes were incubated at 36°C for 5 days, and ; the final reading was made on the 5th day of incubation. Later, however, HeLa cells and Eagle's basal medium with 10 percent horse serum were con- aistently used' for the titradon of type 12 virus, and the procedure was modified as follows. Maintenance medium was used for tenfold'serial dilutions of virus tissue culture fluid; to 0.1 milliliter of each dilution, 1 milliliter of freshly.pre- pared 0.1 percent HeLa cell suspension in maintenance medium was added;,the tubes were incubated at 36°C for 5 days, and the finaL reading was made on the 5th day, The highest dilution " which produced clear cytopathic effecU (CPE) in both tubes< was taken as the minimum 100 percent tissue culture in- fectious dose (MTCID,«): No very significant difference was observed' be- tween the MTCID,ee obtained by the two procedures, but the latter procedure was simpler and the effect was a' little sharpen The 50 percent tissue. culture infectious dose (T,CIDt.) was detetn mined in a similar manner, with four tubes used for each dilution. HeLa cells were infected with type 12 adenovirus. Tissue culture fluid was harvested 2 days. aftet complete cyto- pathic effect was observed. It was cen- trifuged at 1500 to 2000' revolutions , per minute for 10 minutes and filtered through Selas 02 filter cantlles. To test ~ the completeness of filtration, 0.5 milli- liter of fresh Serraria marceacenr broth • culture was mixed in the tissue culture aCIENCE, VOL• 137 V1~ r n a~E TZ
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5 M1 'i17Z?.~±.. © 9 4a H i Y ,. K. 0 i F.~r~~/:.M•:e~,j'a~~'~ a ' k `,°, .~1~~~q- ~'.rY°~~'jy~~~2q y~~ 21Lf W" !er'. ' 4 ~ ~iwl 3 ~ r t? '!k L.. ~42 (I t A~`i m,u~~ ~~t,~°c°" ~ 4~1. f 6e L ~~/'F. G'W . ~~y t'.a.'~~ ~`y,4 ~YCQ -'W .'.3 ~.c..rs ~••~~~~^S ~ a4~~.,~~ z.i 4 y a 0 s m Figs. 1-3. Fig. I (left). Tumors filling the right thoeacic cavity and in the liver of a hamster injected intrapulmonarily on the right side, at birth„with 0.05 milliliter of type 12 adenovirus (10' MTCID,.J0.1 ml) (about X'fs). Fig. 2 (middle). Intnthoraeie sarcoma adherent to the diaphragm at the site of intrapulmonary injection, 45 days previously, of type 12 adenovirus into a newiiorn hamster (about x 17). Fig. 3 (right). Higher magnification ob a portion of the sarcoma shown in Fig. 2 (about X•175). ~ i. ~4.;5.,.~.yY as~'s"., -4..:; ~- .. . . ~,. . .. • . .. .. . .. . . . ` fluid before filtration. About 0.5 milli. with 10 percent'' horse serum was added~ ;.liter of' each of the fluids, before and and the resulting mixtures were incu- after after filtration, was inoculated into bated at 36° to 36.5°C: On the Sth' broth and tested for the growth of Ser- day of incubation a reading was made,, ratia mnrcescau'. and the highest serum dilution which' Newborn hamsters less than 24 hours completely inhibited the cytopathic ef- `" o' were moved with their mothers fect of adenovirus type 12' was taken from the breeder room to the experi- as the neutralizing antibody titer. mental room rand inoculated'with viruses Four human sera with type-12 adeno• intraperitoneally,, intracerebraliy, or in- virus CPE-neutralizing antibody titers trapulmonarily. For intrapulmonary in- of 1:16 or higher (as a result of expo- oculation the virus was injected through sure in nature) and four negative the chest wall with a 30-gauge oeedle. human sera (with titers lower than 1:2) The young were usually observed every aere otherwise randomly selected' from -•• day for the first week and thereafter among 700 sera tested'' at random' from at least three times a week; they were among .1g70 patients admitted to the r, weaned' at 3 to 4 weeks of age. M. D. Anderson Hospital and Tumor Healthy-looking whitish portions of Institute for all causes over a period tumor were taken aseptically and of approximately 6 months. These eight minced with scissors, and about 27 sera were inactivated by heating at cubic millimeters of tumor tissue was 56°C for 30 minutes, mixed with an transplanted subcutaneously or intra- equal volume of virus fluid (10" peritoneally, by trocaq into otherwise TCID,./0.1 ml)„ and kept at room : untreated'' (unconditioned) hamsters of temperature for 30 minutes; then 0.05 '. different ages. milliliter of this mixture was injected Human serum (stored at temperature into newborn hamsters less thari 24 of -70°C) was inactivated by heating hours old. at 56°C for 30 minutes and diluted in Rabbit anti:simian-virus-40 serum was twofold series to a concentration of obtained from the National Institutes 1:64 with Eagles basal medium con- of Health. The neutralizing antibody taining 10 percent horse serum. To 0.1 titer of this serum, as determined by milliliter of each serum dilution, 0.1 B. E. Eddy, was 1: 1'S00' when tested milliliter of virus fluid (10' TCID,J0.1 against 100 TCIf3,. of SV-40 and'read ml)' was added+ and the mixture was in 10 days (73). Control' rabbit serum kept at rooni temperature for 30 min- was obtained from normal rabbits in utes. To each mixture, 1 milliliter of our laboratory. One volume of un- freshly'prepared 0.1' percent HeLa cell diluted serum was mixed with 3 volumes suspension in Eagle's basal medium of' type-12 adenovirus fluid (l0'•' ' 74 SEPTEMBER t%2 . Y. ~ . •. ., ._ .. _ ... . - t+. . , , . i . ., t+mr-,'.~ TCID,.A0.1 ml) and' kept at 4°C over- night. Of this mixture, 0.05 milliliter was inoculated intraperitoneally into newborn hamsters and'-0.1 milliliter was inoculated into I ml of Hela cell tissue culture. One-tenth milliliter of the particular dilution of simian virus 40 (SV-10) fluid (13) which showed cytopathic effect of 3+ to 4+ (vacuolation) on the 7th day of inoculation in green monkey kidney cell (15) was mixed with 0.1' milliliter each of a 1:4 dilu- tion of rabbit anti-SV-40 serum and a 1:4 dilution of control rabbit serum or undiluted patient's serum. The mixture was kept at room temperature for 30 minutes, inoculated into 1 milliliter of green monkey kidney cell culture, and observed for 2 weeks. Eagle's basal medium with 2 percent horse serum' was used as the maintenance medium for green monkey kidney cells. . f, Results Of the first nine types of human adenovirus injected into newborn ham- sters, none except type 12 has shown' tumorigenic effects to date. Of ten' hamsten injected' intrapulmonarily with :. type 12 virus and surviving longer than 3 weeks, eight developed tumors in 33 to 90 days (Table 1). Tumors devel, • oped in the mediastinum, on the internal chest wall, or on the diaphragm. Most adhered to the surrounding organs or arr n. I ! 1 I -Y1 a ['! A x Y rt
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c Y 4 3~ [ a hi d 1 dt t= -:a n ,"T~... ~ .. @ A•- ' r. _ 3 f { .= Tabl~e 3. Troean transplantation of tmnon induced by human typii 12 adenovieus. tissues, and bloody' pleural fluid was Site of Days w present In gross appearance the tumors "' t primary Transplant A~if ' Site of "Tattes°/No. death from , were whitish with red hemorrhagic •~(indueed)IDeneratioo ':. transplant • transplanted tumor ,•.Kas. Inn some hamsters metastases to tumor . _ (days) . . . . . : arowth -r- the mediastinal lymph nodes were ob- I/I, p served. No extrathoracic tumors were tp• 34 observed in these first eight tumorous 14 ` 3. 26-50 / 3 t- 26-36 ' hamsters. ' ; A second shipment of human adeno- ' t' 3s 62 `} "'virus, type 12, obtained from the Ameri- 2et-90 can Type Culture Collection about 5 months after the first shipment induced * 20.24 30 a similar tumor in a hamster intrapul- monarily injected within 24 hours after • ' Tnrwr AI2. No. 15 Thorax <1 Intraperitoneal ~. Thoraa <1 . Subcutaneous : ThoraaTr , 23-25 Intnperitoneal Thorax _ Tt" • 23-23, Sutioutaneous. ~a.. . ' 1 . Trmor A12. No. 141. Thonx '-y- Tt-•' ~.•2a-31 Intmperitoneal -':3/4 ~ Tt - ~ 2i-31~ Subcuuneous~. _ 71mor A12, No: 6 T, •~25 Intraperitoneat -T,."- 25 Subcutaneous Tt , 31-53 - Intnperitoneal Tr - ;~31-93 Subcutaneous Tawor Al2„Fo: 3 ' ~" Thoru ' Tr •` 14 Intraperitoneal -2/2 f=tF~,,' ThOnx ` . Tt .. =4 . .... Subcutaneous -:. 0/1 v, r: .--. - Tatnor A12, No. 20. 1 '.. ;y 7Tlons ' Tt~ ~ 24-25 Intraperitoneal ` -1(2 a; Thorax - f`' , Ta 24-25 Subcutaneous -' 1/2 ~.. Liver 'Tt ` 20-21 ~ Inttaperitoneaf~ 2/3 .'Liver ~Tt ' 20-21 Subcutaneous ;.1/3 : .. = - Taemr A12u -i•Thoru Ti 34,33_ Intraperi(oneal 2/2 ; , Thorax . Tr ' 31 Intraperi(oneal . ~ 3/3 i Thorax 'Ts :. ''t`~23 Intraperitoneal 3/3-, Thoruc. ..T. 30 Intraperitonesl 713 -~. Thorax.. T, :~..27-26 Intraperitoneal ~ ~3/3 ... Thorax. T. ~ 30. Intraperitoneal : 2/3i Thorax . ..To "-30 , -:..Subcutaneous 2/31 -~ Thoru. ' ..Ty ~ .'•29~ Intraperitoneal -. 2/3lt ~~`~ 29 Subcutaneous ~.' 2/3} . .~.Thoras~ Ty , ~ - - `::-.flnnor Al2b . .. .,. _ ~,.Thonx:Tt . '26-27' - . lotnpertioneal r .- . 4/,4 .:Thorax• Tt. _~182 Intrapentoneal 2/3 -. Thonx . , Ts 28 Intraperitoneal . 3/3 Trwtor A12o - Intraperitoneal 3/3' ' 13-14 Thora>t Tt • 26 - Thorax 37 ~ Intraperitoneal •..2/2' , 22, 22 -•Thonx • -'Tt •. :1~34 - Intraperitoneal ~ 3/) 20-26' Andmal i transplanted with tumor witNe 24 hours after birth, rhich surv(ved' loeaer than 7~ daya• `"t This tumor was taken from a hamster which . was found dead. t'tT14 hamster showed a taks, but •.aetually diedof aubmandibular abscea. 1Smaller Inoculum used to prolona host sur.i.al. Thorax Thorax - " 1horax Th Thorax M. of transplantation of malignant tissue culture cells, cell-free filtrates of this second shipment of type 12 adenovirus tissue culture fluid on He4a cells j~Zp were prepared, by the method de- 23-27 . scribed earlier. There was a very'high 14"21 incidence of intrathoracietumors, simi. 22-42 72§ lar to those observed earlier, I to 3 721,731 months after injection of cell-free fi1- trates of tissue cultures infected with type 12 adbnovirus but not after the ~ 17-300 injection of filtrates of control tissue 3f, 91 culture (Table 2)', Thes•e'tumors devel- 16-27 oped in or on the mediastinum, the internal chest wall, the diaphragm, the lung, and' the heart- In many hamsters multiple intrathoracic tumors were found. In four of 26 tumorous ham- aters which had been injected with the virus fluid of the 3rd passage, one to three lar e tumors were found in the 'Table 4. Evideeoe against Invotvement of sinnian virus 40 in the tumor-induaina activity of type 12 g adcnovirustissueeultureauids.- ' liver in addition to the tumors in the thorax (Fig. 1). Acttve propagation of the virus in HeLa cells is indicated by increased tumor-inducing ability and shorter period of latency after eight tissue-culture passages. Histological sections of several of the tumors at the site of injection and in • the liver were seen by'four pathologists, who agreed than the tumors were un- differentiated sarcomas (Figs. 2 and 3). While the liver tumors may be meta- static, the following isolated observation Indicates the possibility that some of them may be primary. Of three ham- sters injected intravenously aCbirth with type 12 adenovirus, one is still alive, one died' aC 60 days with a subcuta. neous tumor at the site of injection„and one died at 40 days with a large liver tumor but no other detectable tumors. All of eight tumors induced in the thorax by type 12 adenovirus and'one ' of the liver tumors, when transplanted t~ intraperitoneally or subcutaneously'into unconditioned hamsters from I to 182 - . Approx. :uter` ' ' ;- (TC)D,e/ '.. . ;, - Treatmenr --~~. 0.1 ml)'~ ..< 81aNaw rfrut 40 laa + - Ioa Hps (60eC, 30 min) + - - - 10/ Rabbitsnti-SV-40serutn - - - - • 14 -+ 107: - ,Rabbit~anti,SV-40serum - - -" . - 14 Hanna adeav.baa iyye 12 10t -'.:'None , - + - • + ...10-s None 14 Subeulturo~ None ~ ae 14~.days ~ ~ lOt Heat (601.30 min) ~ ~ - .- - - ~ - ,~ lot• ~: ~Heat (60°, 30~min)~ 101.3 ~ Rabbitantl+SV-40serum - ~ + - . + 10= Rabbitanti+SV-40serum - . + - ' +. 103.1. 7 Normd rabbit serum - •. + + . - tOs . Normal rabbit serum - . " + + Control tissue sullure at , Cercoplthicas . kidney cells lation Nonr Adeno- V.. ~, Wion 2/) 2/3 2/3 -_2n t 1/2 H6L celb Obser- vation pcriod bcforo aeu dete- Adeno- rfora- type lan CPE (drya) + , 24, 28.31 ~ 63 ::rc To determine whether the tumors r' 117 139 arising in hamsters injected with type 12 adenovirus might possibly be the result '. 31 . 3,6 ,` 39,:a7.:r 45 14 14 ! The same dauuon beforoheat inactivation aave 6/6 tamon In eS days (eea Table 2). anim+l wass atlll aa.a and tumur-free at. 54 d.ya uf sea• . .. •.. ' . ~: .. Tumor induo- tion in newborn hamsters by 54 days (NO. with tumon/ No. in- jected) 3/4t 4/Sf birth. t y 3 j ~. „ ~ ~'s' t~r•. i . L-_. SCIENCn. VO4 137 . ....,.. ~7.... ~?I t? na ~ r X 1 r S r'Rt' ~. f t c I I. fi ~ ..{ ~
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!v~ ;o. . ey;: v S N, . i ~ ., 7 4:1 }1. t . -1= yt .• VR-4 't0-s ~.~..~ ~'~a"~>±A r i .;i Sk 14SEpTEt+tBER 3962 _...,y t.: 3) The tumors induced by type 12 adenovirus do not resemble those in- duced in the hamster by polyoma virus, which produces primarily kidney sar- comas, of which we saw none. The ".type 12 adenovirus tumor is, by con- trast, typically at the site of injectiom ~/ Hamsters have been shown to develop sarcomas at the site of injection, in the newborn animal, of cell extracts of frozen and'ground primary cultures of rhesus monkey kidney (16). The on- cogenic agent involved has more re- cently been shown to be simian virus 40 (SV-40) (17). The occurrence {of sarcomas at the site of injection in the experiments under discussion there- fore suggests involvement of simian `,..virus 40. However, the tumor-inducing capacity of the type 12 adenovirus cul= tures is readily propagable in HeLa cells, whereas simian virus 40 is not. In our experiments tumors began to ' appear by 29 days (the 8th HeLa pas- sage filtrate), and almost all injected animals had tumors by 90 days, whereas tumors induced by simian virus 40 usually do not appear until after 344 months (17). Nevertheless,, experiments were performed as follows with Ccr- copirhecus monkey, kidney cells, simian virus 40, and rabbit anti-SV-40 serum. Tbese experiments were performed in a building separate from the hamster isolation building and tissue culture lab- oratory, since only human viruses and human tissue culture cell types are per- mitted' in the latter. . t days old, grew and killed a high per- centage of the host animals in 13 to 139,days (Table3)!.' -7hus far, 220 and 950 untreated hamsters in the breeder room have been observed over periods of 12 and 6 months, respectively. Of, these, one developed' a spontaneous tumor involv- +~r ing the intestine and mesenteric lymph None• a nodes in the duodenal region at 14 39479 ;• <t a <1.2 t:t months of age- . The rest are non- ~J <ta (<12 1:1 ~ , 40186'<ta -<12 1:1 tumorous. 40146 Not tested <73 -: 1:1 'The following facts indicate that the 40378 <t:1 1:32 1:1 .,~tumors observed are not attributable 39933; <1:1 1:32 1:1 ~' to contamination of animals or cultures 39328 <L1 1:16 1 11 i.. :. . 44523 `- <t a 1:16 . 1:1 by Ixllyoma virus. 1) Sera of 30 untreated hamsters and of 14 hamsters that developed tumors after injection of type 12 adeno- virus were tested for polyoma (hemag- glutination inhibiting) antibodies at di- lutions of 1 to 10 and higher. All were negative. 2) The tumor-inducing activity of the type 12 cultures was readily pro- pagable in HeLa cells, whereas polyoma virus is not and HeLa) are involved, an announce- ment made on 13 April! 1962 amended' the passage history, to include monkey kidney cells, as follows: "Isolated in and passed for an unknown number of passages in human embryonic kidney cells. It then was passed one or more times in monkey kidney cells. After receipt in Huebner's laboratory, it was passed twice in KB cells after 12 pas- sages in KB or HeLa cells." From the results of our experiments however, it appears improbable that SV-40 con- tamination could have been responsible for the tumors induced (Table 4). Simian virus 40 produced' typical vacuolation in Cercopilhecur kidney cell cultures, not in HeLa cells. Tumor- inducing type 12 adenovirus cultures produced typicat adenovirus-type cyto- pathic effect (rounding up and' clump- ing of cells) on both HeLa and Cerco- pithecus cells, but no vacuolation. Simian virus 40 grows poorly if at all on HeLa cells. In order to test the possibility that slight contamination with simian virus 40 was being obscured by the adenovints-type cytopathic effect, type 12 adenovirus cultures were heated to 60'C for 30 minutes. This treatment is known to inactivate adenovirus but not simian virus 40. This treatment did inactivate the adenovirus-type cyto- pathic effeet' of the type 12 adenovirus cultures and did not inactivate the vac- uolating effect of the simian virus 40 cultures, yet it did notl"unmask" a vao- uolating'effect in the type 12 cultures. Likewise, dilution of type 12 [deno- Table 5. Neutmlization of both cytopathic effect and tttator•indudng aaivity of human type 12 adettovirua by.human antisera (convalexent): ,;. Anti-SV-40 Anti- lutioof Haman~ titer -" type-12 se^nn to serum (vacuo- adenovirm. adenovinu donor ~• lation on - titer : type 12 (No.)' ~CaroWthecw(CPEon (IOt.t -;. hidney). -Het.a) . TCIDt.J 0.t ml) • Zlnu. euttum medium wo u.ed imuad of'humae rrom. y. 'Ia retrospect it appears indeed for.. tunate that these tests for simian virus 40 were performed. Whereas the cata- logue of the American Type Culture CoOection states in the passage history , of prototype 12 adenovirus that only, human tissue - culture cell types (KB No. of tumora/ No. hamsters iqiected' ;a Aao at ~ Aae of death of hamster hamster . alit'eand with - tumoa .. tumor ftee - (days) (days) 6/6 : . 29-45 '.,7/7 .' 3$-79 1/I 42 4/4. -: 32-153 , . .. S/7. . 41-i1 170 0/B . . ' 167-173Y• 014 167 " 0!3 166-172 ., - 013 171 t 7wa ~ da.d, urmx-tn., .t 41 aad W day. a v virus to the point where it no longer destroyed the tissue' culture cells by adenovirus-type cytopathic effect (dilu- tionof 1:10,000) failed toseveal vacu- olation even after subculture. Rabbit anti-SV-40 serum inactivated the vacuolating effect of the SV110 cul- , tures but did not inactivate the adeno- virus type cytopathic effect of the type 12 cultures. In vivo testing of the type 12 cultures treated with anti-SV-40 serum has progressed for 54' days at the time of writing. Simian-virus-40' . antiserum did not inactivate the tumor- inducing ability of our type 12 adeno- virus cultures or prolong the period of latency; the first tumor arose in 31 days (Table 4). Granted that the tumor-inducing ao- tivity is not attributable to polyoma virus or to contamination with simian virus 40, is it indeed due to the type 12 ;~ adenovirus rather than to yet a third, ~ unrecognized, contaminant virus? Eleo- „ tron micrographs of HeLa cells infected = with type 12 adenovirus reveal abundant virus particles of a aingle type con- cordant in location, size, arrangement, and morphology with the adenoviruses (Fig. 4). They occur as crystalline arrays in the nucleus, have.a nudeoid surrounded by an outer limiting mem- brane, and have an average particle size of about 56 millimicrons. This finding, together with the other tissue culture characteristics mentioned and the ade- novirus-type cytopathic effect, leaves little doubt that the predominant if not the only virus is an adenovirus. The data of Table S indicate a strong posi- tive association between the adenovirtts- type cytopathic effect and the tttmor- ` inducing activity. Sera of 700 randomly selected pa- tients from the M. D. Anderson Hw- ' pital were tested for neutralizing anti'+ a a: i ~s( ,lftt~ v r ~ : r...a~ . .. N 0 Q ew i 0 .r? r.. • y 4:y'.-n
Page 6: puz28e00
AV , T u r Q -w? I bodies a~inst the adenovirus-type cyto- :'pathic effect of our type 12 cultures. Approximatdy 26 percent showed neu- tralization at a dilution of 1:4 or higher. ; Approximately 6 percent showed neu- l tralization at a dilution of 1:8 or high- "er. (Another 20 percent showed neu- "` 'tralization only at a dilution of 1:2.) ~' Four such positive "convalescent" antisera, each with a titer of 1:16 or higher, and four negative human sera ; were randomly selected. Aliquots of type 12 adenovirus cultures were incu- bated' with each of these eight human ~ sera and with tissue culture medium as ; a control. IC may be seen that the four negative sera (those that did not neul tralize the adenovirus-type cytopathie effect) did not neutralize the tumor- inducing effect, whereas the four posi- tive sera (those that neutralize the ade- novirus-type cytopathic effect) com- pletely neutralized the tumor-inducing effect. None of the four positive sera . ,.. e oY s x sy - -- contained SVrt0 antibodies, These data 3) The incidence of the tumora/wai ry provide strong indication that the high, and the periods of latency were tumor-inducing activity is indeed at- short. tributable to the type 12 adenovirus 4) The tumors grew progressively m c;;; rather than to an unrecognized passen- and killed, a high percentage of the - ger virus. - unconditioned young adult hamsten into which they were transplanted. ti 5) No such tumors occurred in ham- f t piscussion aters injected with control tissue culture • fluid or with the other viruses tested That human type 12 adenovirus is or in control'breeder hamsters. highly tumorigenic for the hamster is 6) The possibility that polyoma virus indicated by the following results. ~ or simian virus 40 (the only other 1) Each of two separate shipments viruses at' present known to cause tu- 'of type 12 adenovirus received 5 months mora when injected into newborn ham . aparf from the American Type Culture sters) might be responsible for the Collection produced fasi-growing ma- tumors observed was specifically ex lignant tumors at the site of intra- eluded'by a variety of tests. pulmonary injection into newborn 7) The possible involvement of still -hamsten. other, as yet unknown, contaminanC 2) Tumor-inducing activity was re- viruses was excluded by a positive asso- tained or increased after at least eight oiation of the tumor-inducing ability tissue culture passages in HeLa cells with the adenovirus content. Of human and cell-free filtration. sera tested, all those, and only those,, r which neutralized the adenovirus-type cytopathic effect also neutralized.the tumor-inducing effect. This latter result indicates that tumor induction is the result of the type 12 adenovirus, and that newly isolated' strains of the same . virus should therefore also have tumor- inducing effect. Whereas most of the tumors arose at the site of intrathoracic injection, four hamsters also developed from one to ' three large tumors of the same histo- . logicali type in the liver. The question of whether these are metastatic or pri- mary is under investigation.. All animal' tumor viruses that are now . known to produce tumors in a heterolo- gous species, and that have been ade- quately tested' in the species of origin, also produce tumors in the species of origin. Simian virus 40, which has re-y eently been found to have a tumorigenic effect in hamsters, has not yet been adequately tested in newborn monkeys. It is yet to be determined what tumon, if any,, may be produced by type 12 adenovirus in humans. While tumor induction studies in subhuman primates and in human cells in vitro (malignant transformation) may, be helpful, the direct approach via tumor induction studies in the species of origin has ob- vious drawbacks in the case of human tumor viruses. If human type 12 adenovirus were ~ injected into newborn infants one might confidently expect, from what is known of animal tumor viruses, that it would' induce tumors. Yet such a result would' by no means prove that in nature this virus is responsible for even a single .~ © ! w N T , aCIENCE:.VinL. 237 i , s i r. ~ ® © © ,. 0 Q TJ : ,
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A f >9r': ~+'~ ~A Jf +~~ .4.. !:-r~ . ,.. N rfi:Jc' r^ j 5 ~•.,.. .r.. . ~~ ~ ~ 1;'v `e Ke A y w culture fluid or with culture fluids of the other viruses testcdj or in control , breedcr hamsters. The possibility that : contamination with polyoma virus and simian virus 40 might be responsible for the tumors induced was specifically - excluded by a variety of tests. The possible involvement of still othcri as ' yet unknown, contaminant viruses was - excluded by a positive association of ' the tumor-inducing ability with the ade- _. that certain other animal tumor viruses, cells in which they multiply, although novirus content. Of eight human sera > such as the mouse mammary tumor they can damage them markedly. These •, tested„only those four which neutralized milk agent, the mouse leukemia virus properties could explain their ability to the adenovirus-type cytopathic effect t; {or vHuses); and the avian leukosis , persist in human tissues for long po- also neutralized the tumor-inducing ef- viruses are responsible for a high inci- riods. Ginsberg goes on to state, "It is fect. Of 700 human sera tested, 26 h dence of "spontaneous"' breast cancer not unreasonable to believe that adeno- percent contained CPE.neutralizing an- ;-ptlnd leukemia in several strains'of mice viruses, like the herpes simplex virus,• . tfbodies for type 12 adenovirus at titers and of diverse "spontaneous" leukemias, could then, by a still-unknown mecha- of 1:4 and higher (23): . ' lymphomas, and sarcomas in chickens, nism, be provoked to cause another .' R*rtream end Notaa Whether type 12 adenovirus will be disease episode, albeit the proof of such y Nart: canre. rn,t. Mo"arrapn Na. 4. O96oq, ' found to resemble polyoma virus or. etiology by our present techniques may z F. Deraa-ReynaL, in The Phe•iepetbotoey, al these other animalitumor viruses in this not be possible. If the hypothesis pre- s; Ba"E'Edar, s' F Stewart, 1RS9D•cYouny, :- regard remains to be determined. The sented were true, this might explain the G. B. Mider, J. Nmt. Concee.ln,r: 2b,-7v 7_-more feasible line of investigation in the major role of adenoviruses as etiological 6 G~E~ Fotey and A. N. Handlcr, Proc. Soc. '' human population would appear to be agents in man,° _ - E=Ptt•,Bto1. Md. 9s.6r1 (19s7). - ~ careful and extensive studies of the The results presented here focus at epidemiology of this and other adeno- tention on the human adenoviruses as =i viruses, with specialireferenceso cancer, potential tumorigenic viruses etiologi- -, Such studies would include (i) attempts cally related to cancer in man. The to isolate this and other adenoviruses possibility that other human orphan from the cancer tissue, body fluids, and viruses, or virusesnf acute diseases„may 11. xcreta of' cancer, patients, and (li) have a delayed tumorigenic manifesta- ;tudy of the occurrence of antibodies against this and other adenoviruses in ; the human population, with special refT erence to cancer and to individuals in young age groups. Type 12 adenovirus was originally. ;. isolated by Kibrick er a1. from the stool of a case of suspected nonparalytic poliomyelitis (19)'. It was classified as .: ;= a new antigenic type of adenovirus by Rowe et af. (20), designated type 12, ' Summary y `- and placed in the American Type Cul. K ture Collection as a prototype, though A new approach to the important but :.its clinical and pathological'significanee difficult task of revealing possible hu- has been obscure. man tumor viruses has been presented A large proportion of adenovirus in- in this article. By systematic testing of ; fections occur in early childhood. In- already known human viruses for onco- fection with types 1, 2, and 5 is preva- genic properties, it was found that in ',lent chiefly in infancy and early child-. ,. hamsters injected intrapulmonarily with : hood (21). Adenovirus respiratory dis- tissue culture fluid of human type 12 ease epidemics in the general population adenovirus within 24 hours after birth have most often yielded types 3 and 7; there was a very high incidence of epidemics among military, recruits have generally yielded types 4 and 7. Adeno- virus vaccines have been effective for the control of' acute respiratory illness caused by the adenoviruses (22), and protective antibodies produced by ade- novirus infections persist for many years (21). This offers real hope of pro- phylactic vaccination against other ill- nesses with which adenoviruses may be ;'spontancous" human tumor. Polyoma virus of the mouse produces a high'in- i~cidcnce of tumors when injected into newborn mice or newborn hamsters. ?-Polyoma virus is enzootic in most labo- :' -ratory and wild' mouse populations (18). Yet al1 of the evidence at present 'indicates that polyoma virus is respon- siblc for vcry;fcw; if any, "spontaneous" tumors in wild or laboratory mice. On the other hand there is strong evidence associated in the human population,,, including perhaps cancer. Ginsberg has pointed out (22) that the propertics of the adenoviruses should render them ideal as potential, latent agents. They are very stable with respect to changes in temperature or pH; the principal site of propagation is in the nucleus; they do not dissociate readily from the cells that they infect; and' they apparently do not' kill the S. S. . E. Stewart,. G n SI. Supp1J, 291 (t961): G S., Neriishl, Y. Yabe,. N. Gda, N. ida,. F. Gonrales, W: . Sutow, . A: Klnchisaum, H. 0. Taylor, l. J. Trentin, J. Naaf. Cencer laar. • 36, 611 (1961): T. H. Bmntinn, Iroe.. Soe: Eapa(._DbAMad. $I, 327 (1953). tl. W. G: Banfield, C. J. Dawe. D: C BrindEey, J: Na!(. Cancer inar. 13,1123 (1959)~!. The hamsters were obtained through the to- . operation ot,Dr. Charles McPhcrson., n,e Ft mn on ceut and xd a' tion in man should be seriousl eon- lo. cdm were y ,J obtained from Mierobiolo`iea) ASSaeisles, Ine. sidered and investigated. The described It• Thid materul war kindly auppued' by Dn• . Koprewski and Hayniek o6Wis1u Instltute. method of testing such already isolated 1s These materials wem obtained fro® Mkro- bioloeical Associates, Inc. and classified 73 The polyoma vlrva the slmlan rirus60 (etraln .,. A426)1 and tM rabbit antl SV•I0:serum were kindlyy supplied by Dr. B. E. Eddy of the ' ' National Institutea of Heatth.. ]I.W. P. Rowe. J. W. Hartley.L W. Law, • 'R_. J. Huebner, J. Eaptl., Med. 11N,. M9 ~IS. Some ot the. CercopOrhera, aehHaps kidney •eett cutture w.as supptied by..Dr., B. E. Eddy ....- cl ,. , of the National Instftutes of Health and sotae was obtained from Microbiobyical~.Associatea,. , '.IC B. E: , Eddy, 0. a: Borman, W. H. Berketer, IC D. Youn`, Irom . Soe., Eap1(. B(ol. Med. 107.191 (1961). • t7: A. A.. Girardf, B. . H. Sweet, V. B. $lotakk, M. R..Hr7leman, Ib)d. 10Q 649 (1962); B. E. Eddy. 0. S. Borman,. G. E:.. Grubba, R. D. .. Youns, F(Iolo;y. 17, 65 (1%2)„ If..W:,P. Rowe. Bauter(of. Rev. 2s, lt (1961). _ 19. S. Kibrlcck, I,. Meltndea, J. F. Enden, Ahn.. - N.Y. Acad: Srl. 67.311 (1957). ~ 20. W P~ :.. Rowe, J.W. Hartley„ R. 3. Huebner, Proc. Soc. E:pa. e(ol. Med. n, 465 (195s), 21..R. l. Huebner, l. A..Belf„W:..P. Rowe, N.Y. Aced. SH. Spec. Pubb. 5, 393 (1957). ,. 22.M. R... Hitteman„Ibid: S„4o3(1957):' ' . 23.Thia lnresti/ation has been supported by re. . aearchSrants from the National Cancer ]n•- sNtote (yrant C-2e6aD, the American Cancer . Society (trants E-27 and E-56)„ and the El Pasa Better Health Foundationand by, a' . SraM (rom Mr. Earl.Hollandsworth.throueh • • tlte. Greatert.on.riew(Teau)'. United Fund. We are grateful to. Drs. B. E. . Eddy, W. P. •'Rowe„ and. R. 1. Huebner for conaulutlon,.. - bDn• W. G. Banaeldi M. Stanton,.l. FunN, and A. . P. Stout for esamination, of hifto- W lo;lul sectiona oftAe btduced'~ tumon, to Mrs.Ayako Yabe, Mlu Yuki Sato, Mr~ Atnelle Dunque; and Misa Bernadeete Bor- ehen for technical easWtance„ and to Dean Stanley. W. Otaoa,. Dr.. Michael E. DeBakey. '. Mra.An Verner, and Mis. Eleuar Mao- • smtHd for admWstratlw eaWtance.. - Mt . - ~ . ,. t .. 1, .. 14 SEPTEMBER. 1%2~ viruses for tumorigenic activity in newborn laboratory animals may prove to be an important tool- in the armamentarium of the cancer researcher. malignanttumors at the site of injection in from I to 3' months. The tumor- inducing activity was not lost by filtra- tion through Selas 02 filters or by tissue culture passages in HeLa cells. Tumors thus induced grew in, and killed, a high percentage of the unconditioned young adult hamsters into which they were transpianted- No such tumors occurred in hamsters injected with control tissue ' ~' .' :. - . ~-.. .. Y ,-..r~.. x. r Y , ~ ..k . . . ..• ..., f~ S ;U r R ia1 V, .( u s°.,wfXr•.: ?s: Ki y 1 vv T". ..+.yj~G..l - ..~. ~L.~Y ~~... ~... :-;1„~: L.~x SSt4ta~t ~: - r . N lf Y

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