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inefficient selel:tion, when transformed cells were near. Examination of four suchplanu revealed that tlireecontained the expected DNA sequences' homolo- gous with pMI0N2DCl, indicating that loss of foreign gene expression may account, for' many of the apparent escapes. Thee problem could be managed!ea'sily byfint screening for'planderrooting in thepres- ent:e of 100 µg of kanamycin, per mI ter and then applying the nopaline' and leatcallus assays to assure stablrexpres- sion in plants for further analysis and testing of 'progeny. Unexpected'changesin expressionI of' 'f-DNA genes in plant cells and frequent loss of expression of opine synthase genes have been reported (T4). Induction of genes for opine synthase upon transfer of explants to culture medium has also been observed (15). The predictable in- duction of these genes in I certain 1 of our transformants and the transmission of this property, to progeny may be due to influences from surrounding DNA or chromatin I strueture at the site of inser- tion of' the foreign genes into a plant chromosome. Such position effects have been observed frequently in other eu- karyotic systems, such as transgenic mice (16) and Drosophila (17). The leaf disk transformation systemm should be applicable to many of the plant species that'are susceptible to rl . tumefa- ciens and can be regenerated from leaf explants. By integrating the transforma• tion, selection, and regeneration pro- cesses into a simple and efficient proce• dure; the production of transformed plants could I become routine for studies of gene expression and of the physiology or biochemistry of plants, even in labora- tornes with little expertise in tissue cul-, ture methods. R. B. HioRScH J. E. FRY N. L. HOFFMANN D. , E'ICHHOLTZ S.,G: ROGERS R. T: FRALElf Biological'Sciences; Monsanto Company; St. Louis, Missouri 63167 RererRSas e.d Nep. 1. R. B. Hbeuls er ol.'. S'cienrr221. 496 (09E4); M. DeBlbck: t:. Hernera-Estrnlla. M. Van Monta- ~tt J. ScheO. EMBO J: 3. 116l1 (1914)j . 2. R. Fraley er al., in preparation. 3. J: F. Shep~ ; D. Bidiuy, E. Shahin. Science 2t1~. 17 (1t+d0). 4. P. Tamtnyski n a1.. E11lI8t9,J: 2. 2143 (19q): K. Barton rt al:. Cell 32, 1033 (1983). 5. M. DeCkene er al., Bot: Rev: 42. 3!9 Y1976). 6. 1. Vasil ei al:, Adv. Grnet: 29.127 (1979). 7, R. Fraky et ad:,,Proc. NorL Aced. Sei. (J.S,A. M. a1103 t19tt3):, a. S. Rosen er al.. in preparation. 9. I.eat disks were incutsated! on nurse culture plt<tes with medium i(pH adjusted to 3,7) icontain. 1114 MS salis (Gibco). B5 vnamins. sucrose (30aJ ritet). benzy{adettine (1.0 µyml), naphthtlette acetic scid! (0.1 vyml), and 0.8 percent saar: I MARCH 1983' S~tenc~ Sboots were tvoted!on!similarmtidium lacking 7lien F1 h~' fe' 10. tuniawas produced by ctossina rines V23 p R31. The same media were,used forpetunia as for tot»cco (9). I l. B. 7liosna and D. Pratt. TAeor. App1. Genet. !9, 215 (19l1I). The nursecunuts medium con- aioed oneaenth the stattdard'' amount of MS' salts (Gibco). BJ vitamins. sucrose (30 yliten). 2,4+D 1 (2 tnVliter): and 2iP' (1 mS/liter). Leaf dislus were then I transferred to medium conuio- ina MS salts. BS'vitamins, suctose (30 yliterl, and zeatin (2'tttS/liter). etIrbenieiWin (30) uymqd kaoamycio 1300 4mU: Shoots were rooted land transpIanted to sotl as,for petunia and Itobacco. 12. E. F: GeosSe and P. D. Sherr6natom Plant Proparation by Tiurt.e Cirltare (ESegetics. En- etand 19a4hpp. 311-32; A. Binns. H! Wood. A. Brwn„Ddireasiarion,19, 99'(1%1): U. S. McCormiek er at.. in perp~fr~tion: 14. K. Barton er a1., Cell 32.1033 (1BE31: G. WuM letm. L. Mokndijk, G. ooms, R. Schilperoort:, ibid. 24: 719 (0989 )t A: Hepbutst. L. Ciarke. L. Pi:arsou. J. Whi(e, J: Mol, Appf. Gener. 2.315 (1983). ISi A: Bituss. personal communication. 16. R: Psliniter, H. Chen. R. Brinster;,CeU'29. 701 1191121: T. A. S'tewart, E. F. Wapror. B. Mints: Science 217, 2046 (19l2): E. Lacy rt a1.. Ce1134, 343 (i19E3). 17. T. Hatehia; _R. Levis. ~h~' Cell 3If,,469 (19lN); D. deCicco. A. S ibid. 31, 45 (19aN): I I. We thank ahe Department of Genetics. Universi; 9 ofiAmsteniam for the F, hybnd petuniaaad' . Ptan for the L2 tomato plants. 1(7etober 1914; aaccepted 19 November 1984 NNOTICE Phagocytes as Carcinogensc Malignant TransformathonTh'b matp'tl'alilnay be 'cl c.;:j,:~,;ht pratrcted Produced byHuman! Nkutrophils law (ritla 11 U,S- Code). Abstract. In a'ttudy of the relation between chronic inflammation and carcinogen-, esis, C3Rf mouseJibroblasts of'the 10T 1J3 clone8'line (!0'T 112 cells) were exposed to human neturophils stimulated'to synthesize reactive oxygen intermediates or to a cell-free enrymatic system'generating'superoxide (;ranthine oxidase plus hypoxan* th'inI After exposure, the JOT 112 cells were either placed in tissue culture or immediately ithiected' into atJtymic nude mice. BbtJt malighant and' benign tumors developed in the mice ir'(jecred with treated cells, but not in those injected with control celLs; in one instance cells grown from one of the benign tumors subsequently developed'a malignant phenotype. Malignant transformation was also oi+served in treated cells in'the experiments in vitro: We showed previously thar human' groups concur with these' observations phagocytes can produce mutations in, (J-10). We conducted the present study bacteria and mutations and sister chro- to address this question more'directly. matid exchanges' im cultured hamster The cells used were C3H mouse fibro- ovary cells, and that reactive oxygen blasts of the lOT 1'l2'clone 8 line (10T 112 metabolites were important for the pro- cells). At passages l0'through 1'2, lOT 112 duction, of' these genetic lesions (1-4)- cells were grown in monolayer culture The results suggested that the toxic in- and treated as shown in Table Il After termediates or by-products of oxygen the treatment the cells were washed; metabolism generated by inflammatory removed from the petri dishes, and plat- phagocytes may play a role in the carci- ed according to standard' methods for nogenic' process. The findings of other ttansformationa'ssa'ys (1h-13). After 6 to Table 1. Transformation of C3N{' 10'r 112 cells in vitro. The C3H 10'r 112 cell line andl tranaformation assay methods were described previously Bri'efly, plataau.phase (-4 x l0` cells per square centimeter) cells; Qown in Eagl!'s basal medium containing 10' percent'fttal calf serum: were treated while they were attached to plastic 100-mm petri dishes. Neutrophils were prepared from healthy human volunteem as described (20). In jeneral, tar`et 10T'1T2 cells were incubated for 60 minutes at3TC with neutrophils and with or without TPA (Consolidated Midland; 1.0 t."). In some experiments; xaathine oltidase (13' yyml) and hypoxanthine (7' (almi), both from Siama, were Iayerttd onto the 10T !r3' cells insteaI neutrophs7s. Attheend ofthe incubation period the 104' I/Z monolayers were washed, removed from the dishes, and plated into 1Mmm dishes at densities such that less thao 2000 surviving cells per,dish were expected (on the basis of plating efficiency determinations).,Medium wass changed twice weekly until confluence was reachedl then weekly thereafter until fixation and staining were performed at 6 to S weeks, Number of cells Trrcatntent survivi Total number f' Dishes with type !l]'foci lW N ng per dish o dishes Total Percentage None (control) 1265 210 7 3.3 ~ Hypoxartthine plus xanthine oxidase 1335 201 3• 13.01 1+Ieutrophils (110s) 1 1380 20 2 10.0 >,Ieutrophils ds x 10*) 1200 20 10- 30.0 r Neutrophils plus TPA 1320 20 7' 35.0 ]+leutnophilt (s' x 110') plus TPA 480 20 3' 15.0 TPA (I Wtnl) 1003 20 I 3:0 -Siptihcandy peater (P <.03) trsmsfonnation frequency than untreued control I(211. 123 t Vo 1. a Z-7 66 ql)

Table 2: Tumor development in athymic nude mice inoculated with IOT 11,t: cells treated as described in Table 1. Thrcells were washed. removed from the dishes, aspirated into syrinses, and iqjected subcutaneously (8 X' tl!>"to 106 cells per mouse). The aaimals were then observed! tesular(y for the appearance of tumors at the injection site. Treatment Maliptant ttutton Benign lesions Tota1! number of mice Number of mice with tumor Nooe 0 0 32 0 TPA 0 0 2'1 0 Hypoxanthine plus xantbine oxidase 1 2 10 3 Neutrophils (S X 1B`) plus TPA 3 2 22 5 Neuutsphils (107) PMN'plus TPA 1 2 111 3 8 weeks in culture, the cultures were stained and examined for the presence of transformed, type III, foci (YA When foci with type III morphology are im- planted in genetically approptiate mice, malignant tumors develop in 80 to 90 percent of the recipients (11, 12). As shown in Table 1, humanw neutruphils were able to induce significantly in- creased numbers of'transformants. This transforming effect occurred whether or not the neutrophils were exposed to 12- tetradecanoylphorbol-13'-acetzte CI'PAY during the 1-hour incubation period with the lOT lil2 cells. The TPA had been added'to aetivate the oxidative metatio- fism of neutrophils (d. 7. 8). but we subsequently observedi that the l0T 1J2' • ~ .li, 41J ~ ~ ~~ " ~ ~~ ~`" ~ A. ~I ~.\~. Fig. 1. (A) t.ow-power (x100) photomicro- gtaph of histologic section of'sarcoma from nude mouse, showing dense ceilularity,. (B) . Hisli power (x6D0) view of same tumor. dem- onstrating hyperchromatic nuclei and irregu+ lar cell size and shape. cells stimulated the t elease of superoxide by the neutrophils of ' two of seven healthy volunteers (including those used in these experiments) directly, without the addition of TPA or other activators. Maximum transformation wasi ob- served when the 1OT' li/2 cells had been incubated with, either 10° neutrophils plus TPA or with 5 x l(Is neutrophils alone. To determine whether reduced' oxygen, metabolites generated by the phagocytes could be contributing to the transforming activity, we performed ex- periments,in which the enzymatic sys• tem generating the superoxide: (xanthinr oxidase plus hypoxanthine) replacedlthe neutrophils in incubations with the I(YT 1/2 cells. As shown in Table I, the enzy- matic system also induced transforma- tion, confirming a role for reduced oxy - gen ~ species in the process. Zimmerman and Cerutti recently reported similar findings with xanthine oxidhsr plus xan+ thine (3): In a parallel series of'expenments we determined whether 10T 1/2 cells im+ planted into athymic nude mice immedi+ ately after, treatment in, vitro could un- dengo transformation in vivo and devel+ op into tumors. [Blair, er a!. (14) found that freshly transfected NIH 3T3 cells gave rise to tumors in nude mice in S to 9 weeks.J As, shown in Table 2, tumors developed in nude mice injected with treated' cells, but not in. 53' control ani- mals (32 injected with untreated' lOT 1/2 cells and 21 injected with lOT' 1/2 cells tteated! with, TPA alone). The tumors appeared' 13 to 22 weeks, after injection and were excise&and examined histolog- ically; five of the tumors were malignant and six were benign. Four of the malig- nant lesions were poorly differentiated sarcomas (Fig. 1)i and one was not clas- siflable. The benign lesions were more heterogeneous. One was an angioma with a completely benign histologic ap- pearance (Fig; 2A), one was a fibrous mass that appeared at 18 ' weeks, grew, and then regressed ito about half its maxi- mum size prior to exeision, and the re- maining four were multiloculated cystic structures of uncertain type (Fig. 2B). Cells from three of the malignant: tumors and the benign angioma were successful- ly placed!into tissue culture, where they all grew with typical transformed mor- phology: Arearly passages (3 through S). cells derived from the tFuee fibrosarco- tttas and from the benign angioma were injected back into nude mice (1!0¢ cells per mouse). The cell lines derived: from the three sarcomas produced identical tumors in 2 to 4 weeks in four out of four, two out of two, and'one out of five mice, respectively; the cell Une deriired from the benign angioma gave rise to maliQ- nant sarcomas in 4 toS'weeks.in four out of four mice (Fig. 2C). Cytogenetic stud- ~.-a •~..~r. -~~--~ •w. /r''y~i ~~ 2 Q '~ ~ . .~a _ ~ Fig. 2. L.ow-power (x 100) photomicroQaphs of (A) the benign angioma [~see ie+ui iBit one ~ of the multiloculated cystic struceures ansine, ~ at the site of'injcctionof'lIOTI':'cellsf and iCl ifA of'the sarcoma that arose from celhs grown ini ~ O vitro from the tumor shown in tAi and in)ect•, ed back into a nude mouse. 1233' St:IEvC'E. V,OL. :-•'

ibs confirmed that the sarcoma-derived eell !ines were of murine cell origin. These experiments demonstrate tfiatt inflammatory phagocycic cells can in, duce malignant transformation. The rea. sons for the absence of an obvious dose: response effecr are not clear, but such an absence has been observed previously (1, 2, 4). Some possible explanations are as follows. (i) While at low doses the cells can repair the' multiple types of cellular lesions that may be produced by phagoeytes: as the dose increases there may be a narrow range within whichI the cells undergo transformation, with death resulting',from any dose above this range. (ii) Some clumping of eiellfr may occur and resulrin differenrJevels of exposure. (iii) Dead or nonproducing cells may quench or buffer the toxic products. (iv) Cells may vary in their susceptibility too transformation according to their stage in the cell cyele. It is interesting that S x 10e' neutrophil's plus TPA induced, fewertransformed foci than6' x 10° neu- trophils alone (Table 1). This suggests that explanations (i) or (iii)' are more likely than the others to account for the lack of a clear-cur dose response. The finding of benign lesions in some: of the animals is alko of interest. While in some settings transformation in vitro catt, appear to occur by way of a single step (JS), in others a multistage modell seems more applicable (.7, 16). The benign angi- oma observed here, and' its subsequentt evolution into a malignant eell6ne, sug- gests that a process involving,more than one step occurred in our experAmentail system, despite the single brief exposure tolthe transforming agent. Although phagocytes stimul4ted by aa variety of agents (bacteria„ TPA, opsa nized zymosan, exposure to lOT 1/2 cells) to, produce oxygen metabolites cause DNA and chromosomal damage and transformation (1-4, 6-.8), and al- though a, cell-free oxidizing system can have similar effects (3, 3; 9), the specific molecular species ultimately responsible for these events are unknI In addi- tion to producing,the superoxide anion, hydrogen peroxide, and the hydroxyl radical, phagocytes produce strongly ox+ idizing halogenated amines and hypohal; ous acids (17-19). Furthermore, the in• teraction of these' strong oxidants with membrane constituents of both phago- e}nes and the target cells can generate multiple biologically active or toxic in, termediates (for exatnple, peroxides and aldehydes). While earlier work suggests an iinportant role for the hydroxyl radi- cal in the overall process, this compound! is so reactive that when it is generated! extracellularly it probably cannot reach, the target nucleus. However, its reac- tions might generate other species that can. The ultimate carcinogen remains to be de5ned. SIGMUND A. WEITZMAN ALAN B. WEITBERG Hematology-Oncology Unit, Massachusetts General lafospital Cancer Center, and!Departntent of Medicine, Harvard Medical School, Bioston, Massaclursetts 02114 EDWARD P. CLAR[ Department of Radiation Medicine, Massachusetts General Isfospital Cancer Center, and 1 ATarvard' Medical 'School TstoMAs P. STOSSEL fllematolopy-Oncology Unit, Massachusetts General Fitospital ' Cancer Center, and Department'of Medicine, Harvard Medical School' R.fen~ W New 1. S. A. Weiuman snd'T. P. Stossel. Science 212. S16'(19l1): 2. .,J: lniawnol. 12/, 2770 (i19t2). 3. A. B. Weabeq., S. A. Weitraun. M. Des- erempes. S. A.,Lan. T. P. Stossel. N. Eesl. J: Med. 3M. 26 (i1913): 4. S! A. Weitstrun and T. P. Stossel. Cancer Lett. 22. 317 (19ta). 5. R. Irmeutmun and P. Csnmi. Proc. Nert. Atad.' Sci. U.S.A. ah 2013 (1964). 6. D. E. Levin. M. Hollstein. M. F. Cliristman: E. A'. SichwieR„B. N. Ames. ibid. 79; 7445 (1912)1 A. M. Fuhoa, S. E. Lmreku, G. H. Heppner, Cancer Rtr 44.4309 (19i4). 7. H! C. Bitttsoim. in Radioproterrors and Anti- caroinoraua 0. Nypard and M. Sianc. Eds. (Academic Ptess, New York. 19R3). pp. 539- ss6. a. B. D. Goldstein. G. Witz. M. Amorsuo. D. S. Suoee. W. Troll. Cancer Lert. 11. 257 (1991). 9. S. A. Lesko. RI 1. Lotsntzea, P. , O. P. Ts'o: BiorAereiurry 19J 3023 (19t0): 10. "Nationall Institutes of Health workshop re• pott:" edited by E: S. Copeland. in ~CaecerRet. 43. 3631 0963). 11. C. A. ResnikoQ: J. S. Bertram. D. W, Brankow. C. Heidelberyer: ibid. 33. 3239 (1973). 12. C. HeidelbetZer et af.. Ilfarat. Rer: I!1MJ,2l3 (19i3); 13. E. P. Clark. G. M. Hahe.li B. , Little. Radiar. Res. asJ 619 (0961): 14. D. G: BWr, C. S. Cooper. M: K. OYktwson. L. A. Eader. G. F. Vande Woude, Scieece 21t. 1122 (19[2). 13: R: E. Letts4ten. Natrre fJ.oadonl 2;3. 246 nseo), 16'. S. Moedal. D. W. Brankow, C. Heidebersen Cancer Rcs, ,36, 2234 (1976). 17: S: I: Bearman. G: A. Schwertieq. E. Hl Ko- (bMy. B. M. Babior, J. lab. Clie. Med. 9ti,1193 tf: S~B,1Mi'). Weist, R. Klien, A. Sfivka, M. Weii J. Ctie: Inre:t, 76. 5% (19E2). 19. E! L. Thomas: M. Gnsham, M: M! JeQerson. ibid. 72. M P(19R0): S. J. Weits. M. B. Lampert. S. T. Test. Science 2II2, 615 (19E3): 20: L. A. Boxer and iT. P: StwsN. J. CW. brvest. $3. 1374(1974). 21. F. E: Cnoxton. EJe+eeatary Starirric. (Dover: New York. 1959). pp. 246-283: 22. We thank S. Lan for perfoeminti'the cytot;eeetic ttudies, M. Belmontefo, technical!u:istaoce. and L. Anderson. Ni Hartis, T. Mayer, tmd iT. Hartist' for reriew of patAalopcal, meterial. Funding support was Orovided by the,Amenean Cancer Society (Fant CI1190). an American C<,mer Society 7uoiorFaculty Fcllowship. NIH VSm CA 00962, the Council for Tobacco Rx. sevch (Srant IeOU); Edwin Hiam. and the Edrrie Webster Foundatim E. Campos provided secl retarial auistance. D. Harmon prowid.d help with the statistia. 24 ' September 1964; 'aaccepted 13 December 11164 Monoclonal Antibodies Against the Aster Yellows Agent Abstract. Hybridoma clones secreting, specific monoclonal antibodies against the aster yellows agent, a mycoplasma-like organistn, were produced by using patriallyy pur'{y'ted salivary gland preparations from irJ,rctedleafhopper vectors as tJt'e imrnuno- gen. After 39I7'hybridontas from 20 ind 'ependent ftrsions were screened for specific antibody against the aster yellows agent, two table clones were obtained. With theree tnonoclonal antibodies the aster yellows agent in diseased'lettuce, periwinkles, and inoculative insects was specifically identified by enzyme-linked im.nunosorbentt assay. Thraster yellows agenrwas serologjeally diferentiatedJrom the mycoplas- ma-like organisms associated'with ash yellows, loofah witehes'-broom, paulownia witch'es'-broom, sweet potato witch'es'-brootn, peanut rosette, maize bushy stunt, and elm pltloem necrosis. Aster, yellows (AY), an economically important disease affecting many erops. (1), is caused by amycoplasma-like orga- nism (MLO). Although the AY' a:gentt was believed for more than 50 years to be a virus (2), in 1967'mycoplasmas were implicated as the causal agent of AY and other plant diseases with similar symp- toms (d, 4). Since then, more than 200 plant diseases have been ascribed to MLO's. The AY agenr has been eonsidl ered to be the most prominent member of the group because it has been impli+ cated l in the induction of important dis- eases of' many hosts. During the time in which the agent was thought of as a virus, many unsuccessful lattempts were made to purify it (5). Even since the discovery that the causative a;ents:were wall-lessprokaryotes, attempts to isolate and cultivate these microorganisms havee been unsuccessfull To our knowledge, none of the MLO's associated! with yel- lows diseases of plants have been suc- cessfully grown in vitro. Thus, in, thee absence of cultivation of'the causative agents, the numerous yellows diseases t:ould!be differentiated'only by indefinite biological properties such as host range, symptomatology, and insect vector rela- tions. The AY agent, like otlter MLO"s, in-, habits only the sieve tube elements of the. phloem tissues of its plant hosts. Since. i MARCH I9W 1233