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Y /R everenees and lotcs 1. F. Duran-Reynals, Ann. N.r. Acod. Scl. 68, 4140 (1957). 2. N. P. 7•tazurenko. Prohlenn o/ Oncology ~, No. 6. 873 (1960). 3. The histological diapnoscs were eonffirmed by Dr. Richard Sicgler, assistant patholoFrst. Chil- dren's Hospital. Philadelphia. Pa., and vrsninl: invcsligator. Institute of Microbiology. Rutgcrs University. 4. This work wav suDportcd by grants from thc Nauonal Institutes of Health anJ i.om she American Cancer Sociny. i• Present address: Instituu of Microbiology, Rutgers University, New Brunswick. N..t. 10 July 1961 Common Human Viruses as Carcinogen Vectors Abstract. Single doses of pairs of v%ruses and organic carcinogens (in amounts too small in themselves to induce tumors) were administered to male Swiss mice frec of polyoma virus. Malignant tumors devel.• oped in groups of mice injected with five of the carcinogen-virus pairs. Prior im- munization against the virus of a pair pre- vented tumor formation by that pair. Car- cinogen binding by poliovirus 2 was dem- onstrated in vitro. It has been a continuing paradox in the field of experimental neoplasia that carcinogens strongly implicated in hu- man tumorigenesis, though present in the human environment in only trace amounts, will ordinarily induce neo- plasia in animals only when admin- istered in relatively large amounts, or when given together with various physi- cal or chemical "cocarcinogens" (1). Sieculation on possible natural cocar- cinogens led us to consider the role of common, nontumor viruses. Viruses are ubiquitous, often occur in family or household patterns, are most easily spread in urban environments, and with relative ease penetrate suscept.blc, non- immune cells, and commonly, cell nu- clei. This report (2) presents evidence of in vitro and in vivo interactions be- tween common human viruses and chemical carcinogens: the results suggest a hypothesis that viruses may serve as natural vectors for the transport of otherwise innocuous amounts of envi- ronmental carcinogens (mutagens) to susceptible intranuclear chronlosomal ` loci. Studies were performed in vivo on male Swiss white ntice (Webster strain ), which were obtained from a colonv proved free of polyoma virus (3) and which were reported to have a low in- cidence of de nr,ro tumors (thyrlloma and lymphoblastic leukemia) (4). The viruses used (vaccinia, ECHO 9, Cox- sackie B., and poliovirus 2) were har- t ^- lW • ILA vested fluids if lul1% Inlccic.l tissue cul- tures of mor.6c> kitincy: by the routes given, thcv cvo}.:.: ri: aiiiblc mortality or morbidil.. 7irc r•rr:inogens injected and thcir r-pt'cuor .toscs. judged to be too small to inducc tumors (I), 9,10-dir ~r rh• 'h: ~a:rnlllraccnc-1? were: (DRS°A). _. anannfluolenc ( A1= ). 1/ A. I ,2,5.6-dihcnzan• thracenc (1)l;/V ). : * 5 : •"t. Each anin,al recrivrtl a single dose, at the same tian ;uld in the same site, of two suh.l wn, c:,+ r. rr us suspension, or lrozcn-l:,..t..-J .., ..S.r? kidney tissuc culture cal:• .- Ilat: „•lI mediunl: . . .. ....anogen or car- cinogen solvent (acctt.nc or propylene glycol). Randomized groups of 6 to 12 animals. 20 tcl '", .1 •.. old, were in- jcctcd 56uhcut.,ncuu,ly, intiraperitoncally, or intranac:llh• an.l ''•^trccd in mul- tiric cnLr-: ........, n.rt survived for 12 months were killed. Cannibalism prior to 3 months of age was hcavy, presumably because of overcrowded cages. Lymphonlas. nlvclc•id Icilkenlias, a re- ticulum cell s:tr:.om:r. and a subcutanc- ous fihrosarcoinla-malignant tumors other than thoi,c reported to arise de vol•o in this strain (4)-oc'curred in five groups of mice that received carcinogen- virus Oairs, and in no other groups (Tables I and 2). The calculated prob- abilities (5) that the tumor incidences in these groups could have occurred by chance arc: DMBA and vaccinia, 2.0 perccnt;. D.Nf BA and poliovirus 2, 0.7 pcrccnt: D%1BA and Coxs6ckie B., 62 pcrccnt: AF and Coxsackic B,, 16.5 percento and AF and ECHO 9, 20 per- cent. \t1 hen the chi-square test with Yates's correction is applied to all the data (Table 1), the probability that tumor incidence associated with the following conditions is due to chance sia^c is: sir.,s, with and without car- cinogetl, .05 > p > .01: carcinogen, with and without virus, .05 > p > .01; virus plus carcinogen, p < .01. Four local- izeJ thvmomas were found in the 161 mice aliNe after 3 months. Multiple pul- rnonary adenonlas occurred in five mice that received DMBA intranasally, with or withoul virus. Half of a group of 108 mice were immunized against vaccinia virus and half against frozen-thawed monkey kid- ney cells. Each was given a single simultaneous intraperitoneal or sub- cutaneous injection, as described above, of either vaccinia virus or frozen-thawed monkey kidney cells plus either DMBA or propylene glycol. The cages were not Table 1. Results ohri,,.c.i hN .nl;,-ctrng mice with carcnnogcn-virus pairs. Data from two experiments arc included. M,.e mre•cted u-ith earemnogen DMOA• AF DBA No. aith mal,8. oant tumors No, ahar ar 3 mo ho. \a. wilh m. mahR- j..rcd nanr tumors, No. alive at 3 mo. Mice injected with carcinogen solvent No No. No- uith No' No. wirh m. mal.8- alive in- mal.g. leercd nant 3 mo. j~ted nant . tumnrs tumors No. al i.ro at 3 mo. No. in• jected 11',rh rorvrmio .,.u: 5 9 16 0 11 16 0 8 t2 tt,r1, £CNO 9 .r•n. 0 11 _ 7 12 0• 6 10 0 12 23 H'r,n C-3arRlt S. ,.rus If 1~ .. - 6 12 0 4 10 0 14 23 5 7 r: o* 6 12 O1 6 11 n'„h ,~ceue carrorr' r,•Ih 0 s r~ O 8 12 0 7 12 0 r. - , . n 6 10 0 6 lo • Prdmnnar. ac,n.•n.+~ in UNIBA gr~nups: ECHO 9. 1; poho 2. 2; tissue culture cells. I: tissue culture media. 1. ? One thyn.nm., l ablc 2 Rource. tumor.. and laty nt periods after injecuon of various ca rci nogen- virus pairs in mice. Abbrc.tal,nn• ~ 1• rntr;,rcritonral; s.c., subcutancous: i.n. intranasal. No. of mice Latent period Carcrnogen..nu% pan Rnine Tumor with tumors (days) DMHA and •n,.m.a rp; L.-mphpma 3 132; 239; 344 uMRA and vaccmia t.p. Mvctad Ieukemia l 202 DN7BA and vacc,n,o s.c. F .broserrcoma 1 168 DMBA and t+nho: i.n. Lvmphoma 4 249; 307; 307:307 DMBA and t•nhn 2 r.n. M)cloid leukemia 1 241 DMBA and eo.sacAre B. rr. lirticulum cell sarcoma 1 307 AF and Cntsa,tic B, tp,. Lymphoma 2 151.202 Ar and ECHO 9 1.p. Lymphoma 2 298; 298 1985 15 DECEMBER 1961 PUBLICATIONa 0024478

overcrowded. After S months, the only tumors observed have been four lymph- omas in a group of eight mice that were not immune to vaccinia viri.ls ar•-~! ,(; tr given vacciniat and D:+1f3A Intrapcri- tonmally. The probability th'at this inci- dence could have occurred by chance- in view of a group of nine in)mune mice that were similarly injected and had no tumors-is 2.9 percent. Purified suspensions of poliovirus 2 (10" to 1'0" nlonke)• kidney TCIDze) and vaccinia ( 10" TCID,°) and of their respective nucleic acids were prepared (6), incubated at 37°C with solutions in aeetone of DMBA-9-C" (7) conaaiil- ing 2.7 X 10' to 5.4 X 10-' milti- micromole of DMBA per milliliter, and ultracentrifuged. Measurements vete made with a gas-flow counter with a sensitivity of 431 to 437 counts per minute per millimicrocurie and a back- ground of 26 to 27 counts per minute; a precision of 0.9 to 1.5 petecent was obtained with prolonged counting times. Binding of DA1BA by whole poliovirus 2 significantly in excess of that by similarly purified suspensions of frozen-thawed monkey kidney cells was demonstrated. Three separate measure- ments of uptake, in molecules pet' TCIDs,, were: 17,000 ~:t 5000 (p < .0l ) : 20,000 -- 12,000 (.05 > p > .01 ) ; and 3100 -!- 900 (p < .01 ). Sigriifi- cant binding of DMBA by vaccinia or by virus nucleic acids was not demon=• strated. The results obtained affirm in vivo interactions of viruses and carcinogens first described hv Rous and Friedewald (8) and by F. Duran-Reynais (9) and since described further by M. L- Duran- Rcynals (10). The results are also con- sistent with the report by 'Wisely er ol. (11) of enhanced chemicsl carcino- genesis in mice repe(ttedly exposed to ~ respiratory viruses. Although the results suggest that common viruses may serve as carcin'o- n gen vectors, other interpretations of these interactions can be made. If such interactions occur in nature, it may prove possible to reduce ncoplasia cur- rently ascribcd to chemical carcinogens by immunir:uion against a virus ( 12). CNRISTOI'11ER M. Mn]iTIN SIGmuNnuR N1nG'a'ssn`° PHIt IP J. ( iosc1EN51:1'` GEReRU F. HaNsrN: Division of 1u/e('fi.nrs Di+rn+rs, Depur)»renr uJ 1lrrlir-i)re. SP)on Hnll Cr,llr'-('r of ilrthc-i»r, Jenev Circ. Nru- Jrr srr t9Nr. Rderrnce+ and Nores 1. P. $hubik anat or Compounds \\ ' ] C>rcd for - `rrrAAc Heotr)r Ca,-- .. . . • .~-.. ,. • . , . ,Ivc7). 2. TLb~rcrwrr u... *tut•-- drqaipbn.rr) R.•,.•r r',thc American 1•ted,cal Arcn: • 1.-,... 2y June 1961. 3. New Animal li-n..- L...+n,, F;vmti. German- town. N-Y. K,ndt+ u•h•,amujlcd by- \V. P. ko+• e. 4. C. Friend pl n/ 1'..., Aan Aaaoc. Cancer nP<va,..l. 9, ^- r l~r•1 t~ C 1 r,end, personal cummunraati.•n 5. R. A Fn§er. .Sr_.,.-n - tr-. .. /n. Resr.••rh ..Jmbwbh, Scot- 17r,.:..J r,,. Iv_.. , v~ 6. J. J. Hollarrd, ei al . J- F.xprl. Med. 110, 65 (1959). 7. NuFle•at•Chicago CnrPnrat,on. st+eeific activity 2 6c m:nnle 8 1'. i ;,nd W. t-. rrledea:,ld, !. Expll. Med. 79. SI1 (1y4J). 9. F. Duran-Rcynal,• rnn \'.) Acad. Sel. 54, 977 (1952). 70. 11 L. Duran-Rcynal.• personal communica. rron 11. 1) V Wisely er 61 A.a. Assoc. Cancer fir+irarrh 3. 278 f 1961 ~ 12. Tlrc•e studics .vere euppnnr.t by U.S. Public Healrh Scrvicc rc•r..rr', rram, E-1695 (Cl) :md. E-3:57 and uJ;.l h. , 4.rtnt from the Tobacco ]nJu~er. R.h ('ommince The as+,•uncr nf ^. (,. Gra.l. and Mary Lu Jacobs is Fratci.•tr+ ,.t....... 'cdceJ • Fellina of the Sur••'•r, for Medical ReseJrrh FnunJ."n•r- i Nat.onal tnsrirut<+ ut Hc.,ith scudent summer rese.irch felloe. 19 June 1961 Estradiol Stimulittion of Glycine Incorporation by Human Endometrittm in Tissue Culture Ahsrrar). The incorporation of C"- labeled glycine intp human endometrium grown in tissue r:utturc is a.celerated by the addition of cslrz.iiol-173 to the cul- ture medium und.'r" ccr7 ,in c.perimental conditions Thi, i. :rccompanied by an increased rol. .•l Jts-.^prarance of the glrcinc from thr medium. and its demonstr.,lion is dc.rrndcnt t:ron the age of the culture at.d thr frcquencV with which the mediurn is rencwrJ E'strogens at~t~~:'r to influence the n7etabollsm and c)1.•mical composition of "target orban>" I+v increasing the rates of endergoluc svnthOic reactions. An enzOme system -.% hich is dependent upon minute amc7unt% of estrogens has been isolated frvrn scver.rl of these target organs. Wh:n .rcii.,,led by estro- Fen, it catal}'zrs th.- Irarr.ic/ of hydro- gen frtsm rcduc:.1 Ir•;•t•.• ..rhupvriJinc nucleou.le tr dipl,,'.ph..hvr-Jine nucico- tidc, which n7as rc%ult in increaseJ energy to accrler.-: th, synthetic activities of the ccl! (I). TimuC culture '::'1~'1 tlr c• sCenled to otler a uceful s~ ~tcn, !or further in- t'l'stig,ttions of c•trvc~n .rii•on Hurl7an cndumetrium, }lril<in-L' tnc,rhhol,.,;rc rcsil.'n,., tt, cstn+ecn,. PUBL ICRTIONS can he propagated in vitro in a variety of media (2, 3). Estradiol, when added to slices of endometrium in vitro, in- creases the rates of oxygen consump- tion and conversion of g!ucose and pyruvate to carbon dioxide. Human endometrium from different phases of the ovulatory cycle shows two peaks of oxygen consumption in vitro. These two peaks, at 6 to 10 and 22 to 24 days, correlate well with the known peak of concentration of endogenous estrogens in blood (4). Treatment of castrated rats with estradiol increases the subsequent rate of incorporation of radioactive glycine and formate into the protein, lipid, adenine, and gua- nine of surviving uteri (5). The estra- diol-sensitive enzyme system has been identified in human endometrium (6). This paper is a preliminary report of the eflects of estradiol on human en- dometriun] in tissue culture. The methods and media used were those devised by Peebles (7). Sterile specimens of endometrium were ob- tained by curettage, and sterile tech- nique was used in all subsequent pro- cedures. The tissue was washed once with Hanks' salt solution, then 20 ml of 0.1-percent trypsin solution was added to each specimen and the mix- ture was stirred rapidly for 15 min- utes. The supernatant fluid was saved, and the remaining tissue fragments were treated twice more in a similar manner. The combined supernatan: ftuids were centrifuged at 600 rev/min for 10 minutes at 12°C. The sediment was resuspended in sufficient medium to give a final •:oncentration of about 750.000 cells per milliliter. Each tube received I ml of this suspension and was incubated in a horizontal position without agitation at 35°C for the de- sired length of time. The medium was composed of 5.5 parts of medium 199, 1.5 parts of beef embryo extract, 3 parts of calf serum, plus penicillin and streptomycin. Gly- cine-2-C"` and estradiol-17Q were added to give final concentrations of 6-6 x 10`16f and 5 X 10'M, respec- tivcly. At the end of the growth periorrt the medium was poured ofC, leaving the ccllc adherent to the tube wall. An equal volurne of 10-percent metaphos- phoric acid was added to each speci- men of inedium. The resulting precipi- tate was removed by centrifugation, and 0.1 nil of the supernatant fluid was pilsetted onto stainless steel planchets. dried tinder an infrared light, and SCIENCE. VOt. 1a4 024479