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C. TaE CovxcjLFox ToB..cco RESi- A xc$-II.S.A.. Lvc. r 110 EIST tS9TS BTRE~ {o r~ ~! R7fj 1! ^-t NEW TORS. N. T. 100 IY _. . ' ~j' Short title of study: aY MALIGNANT 2i2ANSFORMATION, MUPAGFI`IESIS AND FIMINOLYSIlN PRODUCTION Di' " CIGARETTE SMOKE CONDENSATE FRACTIONS FE B 1 0 1975 Application for Research Gdaru' t;Pate= - - (Use extra pages as nee de~ ) ` 1. Principal Investigator (give title and degrees): Wil.liam F. Ben.edict, M.D., Assistant Professor U.S.C. School of Medicine 2. Institution & address: Division of Hematology-Oncology Childrens Hospital of Los Angeles r 465o Sunset atvd. Los Angeles, California 90027 3. Department(s) where research will be done or collaboration provided: Division of Hematology-0ncology and Medical Genetics Childrens Hospital of Los Angeles 1 ( C 7 . • (214) 421-e8e0 "d. Proposed starting dates June l, 1975 6. Estimated time to complete= 3 years 7.* Brief description of specific research aimn k/p(/!'r. EXHIBIT NO.1.~_ 50114485 c u~~ anN CTTe ~~~ 04367 ~

~ 7. BRIEF DESCRIPTION OF SPECIFIC RESEARCH AIMS: a. To study the transforming ability of various fractions of cigarette smoke condensate from both high and low nicotine cigarettes and to see whether or not equivalent fractions from the different types of cigarettes have similar transforming ability. b. To study the mutagenic activity of the same fractions of cigarette smoke condensate to determine where there Is a correlation between the mutagenic activity of a given fraction and tts ability to transform cells In culture. c. To study the cell cycle specificity of transformation using the various fractions of cigarette smoke condensate. d. To study the transforming abillty and mutagenic activity of the subfractions which have been obtained from other CTR programs derived from those fractions which appear to have potentially tTie highest oncogenlc activlty. a. To study the fibrinolysin activity In normal mouse lung, metaplasttc (bronchial-alveolar) lesions and invasive squamous carcinoma to see if 'sign-ificant differences In fibrino)ysln activity can be found between normal lung'.tissue and early malignant or premalignant changes. This will be done ~ jn.collaboration with Dr. Richard Kouri at Microbiological Associates who will '•provide the lung tissue samples. f.. To study the Increase In fibrinolysln In the mouse celt line IOTICl8 " following treatment with cigarette smoke condensate fractions which produce malignant transformation In these cells. BRIEF STATEMENT OF WORKING HYPOTHESIS: .' We have recently found that certain fractions of the lAl low nicotine : reference cigarette malignantly transforms the mouse cell line IOTf (1). ,These were the Bib and WA1 fractions as well as the starting material and reconstituted condensate. These same fractions have been shown to have sig- nificant mutagenic activity utilizing the salmonella histidine strains developed in the laboratory of Dr. Bruce Ames(V..3). Finally, these exact fractions have been shown to have significant co-carcinogenic activity in vivo when added subcutaneously along with methylcholanthrene as well as also inducing AHH activity (i}).. it is unknown whether the same correlation will be found using other types of cigarette smoke condensate fractions such as that derived from 1R1 high nicotine or hand-suckered cigarettes. The transformation assays have been done on asynchronous cells and it Is unknown whether other fractions will also produce malignant transformation If one studies these fractions during the cell cycle. There Is a good possibility that other fractions will be found to produce malignant transformation ~ In the lOT}C18 mouse cells since it has been shown that certain chemical car- `, . cinogens produce malignant transformation (n this system only when the cell ML cycle assay Is used (5). 50114486 { I C T ~' #~N 4~~• ~~~~~`~~

L. ( transformation rather than selection. -1 The lOT}C18 ceil.iine appears to be an excellent model for the study of chemical carcinogenesis In cell culture for several reasons. This cell line can be transformed by various polycyclic hydrocarbons (6) as wall as various other agents (7) and has significant AHH activtty. It also has a remarkably low level of spontaneous transformation and thus transformed focl obtained In this system are apparently a direct result of the inductlon of Thus, It should be possible to screen the various tobacco smoke condensate fractions from several cigarette sources not only for their transforming ability but also for their mutagenic activitY In the Satmonella system using the new as well as old tester strains (3,8,9). These results can be correlated with J,,,lr vivo data on co-carcinogenic activity which will be determined as part of the '.~-TR program at Microbiological Associates, Bethesda, Maryland. Once the most .active fractions have been identified, further subfractionation can be done and •these subfractions also tested for their transforming ability, mutagenic activity and co-carcinogenic activity. ' It has also been shown by several investigators that malignant cells in that several'of•the growth properties thought to be characteristic of transformed cells require the presence of plasminogen in the culture (12). We have extended mation of chicken cells using a variety of oncogenic viruses (10,11). F1brlnolysl.s Js produced by the activation of serum plasminogen following the secretiori of a serine protease produced'by malignant cells. It is pertinent : general produce a specific proteolytic enzyme In contrast to non-transformed cells. Very recently•Dr. Edward Reich and his colleagues at the Rockefeller University have shown fibrinolysis is associated with the malignant'transfor- ~ thes4 observations showing that there Is a correlation between fibrinolysis and' 1 ' chemical'ly•produced malignant transformation In cell culture (13). Particularly •:.:relevant to thi,s proposal Is the fact that the mouse cell line IOTfC18 shows `• little fibrinolysis wKereas tlie chemfcally transformed cell lines have high fibrinolytic activity. Thus the cell line can be used as a model system to study how early In the trensformation process an increase tn fibrinolytic + actlvity occurs following treatment with cigarette smoke condensate fractions. intracellular and extracellular fibrinolytic activity of cells (Jones et al, paper in preparation). Consequently it will be important to study whether . or'not there are significant differences in fibrinolytic activity between normal mouse lung tissue and bronchial-alveolar lesions end squamous carcinoma In co'llaboration with the CTR program at Microbiological Associates Inc., Bethesda, Md. ~r . .. . . , . • . ......_.. .._-... ..-...... ......_...... _. . . . .-.. . .... . ~ ... .. .. T 6 ` E ' L §' Nc . MEN A QE 9. EXP RI 1 a. Transformation with Fractions of Ciaarette Smoke Condensate Various fractions of 1R1 high nicotine and hand-suckered cigarette smoke condensate:will be studied for their ability to transform the mouse cell line IOTjC18 during the flrst year of the study. An asynchronous ieal_'S. wi'li~be~`silized initially and the results compared with the data obtained using the IA1 cigarette smoke condensate fractions (1). Later In the year we shall begin to study : 50114487 CTR ~~N 0~~~ ~~, Recently we have found that there can be a marked difference between the

• Chapters in Books 1. B'enedict, W.F., Harris, N., and Karon, M., Kinetics of l-A-D-Arat,inofuranosyl- 2. cytosine-Induced Chromosome Breaks. •Experimental Control of Mitosis: II, MSS Information Corporat.ion, New York, p. 44-60, 1972. Nebert, D.W., Benedict, W.F., and Kouri, R., 'Aromatic Hydrocarbon-Produced* Tumorigenesis and the Genetic Differences in Ary•1 Hydrocarbon Induction. Chemical Carcinoaenesis, Edited by Paul Ts'o and Joseph D-iPaolo, Marcel Dekker, Inc., New York, Chapter 12', p. 271-288, 1974. 3• Karon, M., Momparler, R., Benedict, W.F.: Relevance of Molecular Mechanism of Drug Action to Clinical•Trials in Man: Combination Therapy Model using ara-C and aza-C. 27th Annual Symposium on Fundamental Cancer.Research, M.D. 'Anderson Hospital and Tumor Institute, 1974.(in press). . .. , , 50114497 C TR JIN '..;4,387,115-

Lr .';t ~ 1,l0_-12r1:RS Atlu Al;s I knc.fs ( 1. Benedict, W•F•, B.rrn•in, C.S. and Porter, I,I1.,: Hnrkcr Chromosomes in • itDlignancy with Particu.lar Reference to a Long Acrocenl'ric Chromosome. . Amer. Soc. Iluman Genot., December 1967. : 2. Porter,_ I.H., Drown, C.D., and•Benedict, W.F.: Diagnosis of Malignancy in Effusions by Chromosome Analysis. Amer. Soc. Human Gene:., December 1967. ( 3. Benedict, W.F., Brown, C.D.•and Porter, I•H.:,Chromosomes in Effusions.- Lancet I, 1146, 1967• _ 4• Berlcdict, W.F., Portor, I.H., Drown, Malignancy. Lancet I, 922, 1968. 5. ( C ( 6. C.O. and.Doyle,. G.J.,: Chromosomes in Benedict, W.F., Rosen, yl.C., Brown', C.D, and'Porter, I,H.,: Chromosomal Aberratiorls in an Ovarian Cystadenoma. Lancct II, 640, 1969. Benedict, W.F., and Karon, M.,: Mechanism of-Cytosino Arabinoside Induced Call Lethality. IOih Internntional Canccr ConcIress, 1970. Karon,.H• and Benedict, W.F•: Arabinosylcytosine Induced Chromosome Breakage and the Ce1l'Cycle. Clinical Research,.1970. ' Karon, M,-and Benedict, W.F.:•Locus of Action of DNA Inhibitors in the Cell Cycle as Assessed by Chromatid Breakage. Olodd 36, 825m 1970. Benedict, 41.F., Ncbcrt, D.W. and Thomr'srin, E.O.: Expression of Aryl Hydro- carbon Ilydroxylase and Tyrosine a-Ketoglularate Transmninasc Activities in Mouse-Rat Ilybrids. * Fed. Proc. Apri I, 1972. 10. Bcnedict, W.F., Giclcn, J.E-. and Ncbort, D.W.: lrtd~rcibIc Aryr I,ytIrrrr,~rbon HydroxylS se and 7,12-I)imethyIbcnz(a)~nthracenc-produccd Skin l,umurigenesis in the House. Proc. AACR May 1972. ll. Karon, M• and Benedict, W.F.: Comparative Pha nnacology•nf Chromatid Breakage: The Effect of Various ONA Inhibitors: Proc. AACR 11Ay 1972. '• t , furanosylcytosinc (ara-C) Transformatinn and Chromosomai Changes in Hams*ter 1' Fetal Cells. Gcnetics, 74:195-205, 1973. 4. 12. Benedict, W.F• and Y.ouri, R,E.: Thc Rclationsliip Octwacn 1-B-D-arabirio- Evaluatron of Chcmulhcrapy• iroc. AACR, 15: 138, 1974• 15. Dcncdict, W.F., Jones, P.A., Baker, M.S.. and Durtrnm, J.S.: Ccll Cycle SpcCificity* of I-/tl-D-ar,ibinofuranasylcytosinc(ara-C) Transfunna.tion•in C3H/IOTj Calls, Xlth ! Intcrnational Cancer Cuncires j 1974. ' • 1 • ~ 13. Benedict, W'.F• and Kouri, R.E.: Ara-C Produced Transformation in Hamster Fetal i Cells. Proc. Al1CR• 14:94, 1973. 14. Benedict, W.F., Rucknr, N., and Karon, M,: A C,lonal Ilamslcr Ccll Line for the i 16. Benedict, W.F. and Kouri, R.B., Celi Cycle Specificity of 1 f-D-ar.abino- ~ ~ Furanosylcytosine (a.ra-C) Transformation in Hamster Fetal Cclls,. Presented at the • I r • Symposium on the Cell Cycle in Malignancy and Immunity, Jattelle, Washington, ` t Oct. l-2, 1973. I 50114498 " I C T F Z I I N ~~4 3 " G

17. Laug, W.E•., Jones, P.A., and Benedict, W.F.: Relationship Between Fibrinolysis and Malignancy. Clinical Research, 1975. • 18. Jones, P.A., and Benedict, W.F.: Chemotherapeutic Agents Cause Malignant Transformation. Clinical Research, 1975. • ~r 1 .i ~ , , 50114499 r CTR HN 04387. 1-1"'

~~. . . PAPERS PRESENTED SEPTEMBER '73 - OCTOBER '74. ( ( 1. The Interna;ional Genetic Congres.s.• Berkeley, California, September, 1973 2. The Symposium on the Cell Cycle in Malignancy and Immunity. Batelle, Washington, October I and 2, 1973. 3. The American Association for Cancer Research. Houston, Texas, MaPch,'1974. 4. Xlth International Cancer Congress, October 1974. LECTURES GIVEN International 1. Chemical Carcinogenesis: What's Going On? Imperial Cancer Fund, London,.England, 1974. 2. Malignant Transformation in Cell Culture Dopt. of Toxicology and Pharmacology. Liege University, Liege Belgium, 1974. Local 1. Carcinogenesis'and the Epidemiology of Human Cancer, Hematology Review Course, May 1974. December, 1974. 2. Fibrinolysin and Malignancy, Dept. of Pathology, 3. Chromosomes in Ma'lignancy, Dept. of Medical Genetics, USC School of Medicine.l974. 50114500 . r ri..x f f"6 f f N 043876

synchronous populations of calls using both the confluent monolayer and isoleucine deprivation techniques (5). These studies will be done to determine whether there are other fractions of cigarette smoke condensate that produce transformation which the asynchronous studies did not detect. In subsequent years we shall test the subfractions of the various fractions found to •be potentially the most oncogenic for their transformation ability in the 10Ty' cell in order to attempt to (dentify the chemicals which are most likely Involved. b. Mutaoenesis Studies We shall study the same fractions of cigarette smoke condensate that will be analyzed for their transforming ability for their mutagenic activity using the•S mone histldine mutant strains developed In the laboratory of Dr. Bruce Ames 2. We shall not only utilize the strains which have prevlously found to • be mutated by various fractions of cigarette smoke condensate (2), but also the new strains' TA98 and TA100 which have a marked increase in sensitivity, especially with such agdn.ts as polycyciic hydrocarbons (`.g). The mutagenesis studies will be done•-bot;h with and without utilizing an S-9 liver homogenate fraction to activafe the c•igarette smoke condensate to their proximal carcinogenic form (2). •c': 'F16i;1ndlvsln Assav We,sha.11 attempt to determine how early in the transformation process that inrr•eased~Jf•i6r.(nolysin'oc~ys following exposure of cigarette smoke condensate fracttons•uSing both the ~1-fibrinogen assay as well as the new fibrin-overlay tettinique developed In our laboratory which allows the fibrinolysis of single eells'to•be studied.. The lOT}C18 line will be used In this study. We shall also 1'oak at.the Inte.rnal fibrinolytic activity of lung tissue ja vtvo during the process! ' of inductlon of squamous cell carcinoma to determine whether or not significant. di.f.ferences between normal lung tissue and bronchial-alveolar lesions or squamous ' cell 'carcinoma can be found. The tissue for these studies will be provided by Dr.'Richard Kouri at the Microbiological Associates Inc., Bethesda, Md. in collaboration with their CTR program. ' 10. SPACES FOR FACILITIES AVAILABLE: The principal investigator has a well equipped, modern tissue culture laboratory to carry out the various studies mentioned, as well as a biochemistry laboratory to do the fibrinolysin assays. The Division of Hematology-Oncology also has over 800 sq. feet of animal facilities available. • 50114488 I C T R V I N ~".0 4 3' 879.w

r r REFERENCES. Benedict; W.F., Rucker, N., Faust; J., Kouri, R.E:: Malignant Transformation o,f Mouse Celis by Cigarette Smoke''Condensate. Cancer Res., (in press) - paper enclosed. :.. .. .. .... .... 2. Kier, L.D., Yamasaki, E., and Ames, B.N.: Detection of Mutagenic Activity in Cigarette Smoke Condensate, Proc. Nat. Acad. Sel., 71:4159-4163, 1974. 3.:. Detection.. Prcc. Nat1. Aced. Sc,.. U.S., 70:2281-2285, 1973. Kouri, R., Whitmire,..C., and Benedict, W.: In Vivo and I•n Vitro Effects of Ames, B.N., Durstont W.E., Yamasakl, E.: Carcinogens are Mutagens: A. Simpl'e Test System Combining Liver Homogenates"for Activation and Bacteria 'er Cell Cyc1e Dependency of Oncogenic Bertram,J.S.nd Heideib ge~, ~ .: . • A ' ~ ` ~ .Cigarette Smoke Condensate Fractt.ons, AACR (1975).- abstract enclosed. . ' Trans.formationation lnduced by N-Methyl-N~-nitro-N.-nitroguanidine in Cul•ture. Cancer Res.; 34:526-537, 1974. 6.; Reznikoff'; C.A,; Bertram, J.S., Bran.kow, D.W.; and HeTdelberger, C.: Quentitit•lye'and Qualttative Studies of Chemical.Transformatiori of Cloned (i)H• Mouse„Finbryo Cells Sensitive to'Postconfluerice InFiibition of*Cell Divis'ion. Cancer Res., 33:3239-3249, 1973. Jones,•P.A., and Benedict, W.F.: Chemotherapeutic Agents Cause Mal.ignant -- Transformation. Clinical Reslaryh, 1975. - abstract enclosed. ( . , . ... ... . . . 8: Ames., F.D.,. and Durston, W.E.: An Improved Bacterial Test System for'Zhe'Detectiori and Class•ification of Mutagens and Carcinogens. •Pro;~~-. Nat. Acad. Sc1 70:782-786, 1973. __ 9. . McCann, J., Springarn, N.E.,*Kobori, J., and Ames, B.N.; The Detection ~ e C ( Reich, E.: An Enzymatic Function Associated with Transformatlon of Fibroblasts by Oncogenic Viruses*. I. thick Embryo Fibroblast Cultures Transformed by Avian RNA Tumor Vlruses. J. Exe. Med., 137:85-111, 1973. li. Ossowski, L., Urikeless, J.C., Tobla, A.," Quigiey, J:P., Rifkin; D.*B., and 10. Unkeless, J.C., Tobla, A., Ossowski, L., Quigley, P.J., Rifkin, D.B., and Proc. Nat. Acad. Sci., (in press - paper enclosed). of Carcinogens as.Mutagens: Bacterial Tester Strains with R-factor Plasmlds. Reich, E.: An Enzymatic Function Associated with Transformation of Fibroblasts by Oncogenic Viruses. II. Mammalian Fibroblasts Cultures Transformed by DNA and RNA Tumor Viruses. J. Exo. Med., 137:112-126, 1973. 12. Ossowskl, L., Quigley, J.P., Kellerman, G.M., and Reich, E.': Fibrlnolysis Associated with Oncogenlc Transformation. Requirement of Plasminogen for Dorrelated Changes in Cellular•Morphology, Colony Formation in Agar and Cell Migration. J. Exp. Med., 138:1056-1064, 1973. 13. Laug, W.E., Jones, P.A., and Benedict, W.F.: Studies on the Relationship Between Fibrinolysis of Cultured Cells and Malignancy. J. Natl. Cancer Inst. (Jan. 1975) - paper enclosed. 50114489 CTR f fN 04.,__R88O

, 4. 14. First year budget : • A. 5alaries (give names or state "to be recruited") ' Professional (give % time of investigator(s) even if no salary requested) % time Amount Willlam.F. Benedict, M.D.. 20 7,200 Peter A. Jones, Ph.D. 20 3,000 .Walter E. Laug, M.D. 35 5,000 TecFinical Carol Smith. Mary Baker 50 7.912 40 6,125 B. Consuma6le suppliei (by major categories) Se~um*and Medium. 3,000 Ti'ssue'Culture Dtshes and Pipettes 2,000 Radioactive Compounds ' 500 Antilymphocyte Serum 800 Animals and Care ' , 1,000 ' C. Other expenses (itemize) Travel: To cohsult with Dr. Richard Kouri, Microbiological Sub-Total for 8 meeting for Pro ect Director (1,000) 'Publication Costs: (3001 " Associates, Bethesda, Md. on.this project and one Sub-Totalfor C . D. Permanent equipment (itemize) None 7,300 1,300 Running Total of A + B-F C' 37.857 • E. Indirect costs (15% of A+B+C) Sub-Total for D E 15. Estimated future requirements: Total request Salaries Consumable Suppi. Other Expenses Permanent Equip. Year 2 32,160 8030 1300 . 0•. Year3 35,376 8833 _ 1 300 0 T ~~_.. 50114490 Indirect Costs Total 6224 47,714 5-2,335 J 1 CTR MN 043861