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w.- TOIiACqO INDUSTRY RESEARCH COMMITTEE 150 EAST FORTY SECOND STREET NEW YORK 17, N. Y. Application For Research Grant Date: October 6 9 1959 1. Name of Investigator: Christopher M® Martin, M. D.# ~a'33 2. Title: Assistant Professor of Medicine 9 and Director, 9 Division of Infectious Diseases 3. Institution & Address: Seton Hall College of Medicine 24 Baldwin Avenue Jersey City 49 New Jersey 4. Project or Subject: INTERAC T I ONS OF V IRUSES ,CARC INO GENS' AND NUC LEIC AC IDS Specific Aims: As a major corollary to virological studies described in USPHS Research Grant Application E-3257, "Transferrin-Virus-Metal-Carcinogen-Interactions "* it is proposed to explore the several fundamental implications in the field of o carcinogenesis arising from the "transferrin effec.t" on viral syn- thesis, as recently described by Martin and Jandl*.` Specifically, it is proposed to study (1) the binding of carcinogens and ca•rcinogen- me al com e es t n c ei acid and (2 carcinogen-binding by viruses an~ its r~a~~ on ~o ~n~u~ ep~o ~asa~ 5. Detailed Plan o roce ure (Use-reverse-s~ 0..(V, #ona~°s e A. Carcinogen-metal complexes and nucleic acids: Selected mutagenic organic carcinogens of varying degrees of metal affinity will be studied by equilibrium-dialysis and differential centrifugation techniques, and their rate of binding to human DNA, RNA, cell nuclei, other cell fractions, and plasma proteins compared, in the presence and absence of chelating agents. Binding affinities in the presence of various trace metals will be studied, particularly those metals shown to be both nucleic-acid associated and carcinogenic (nickel, chromium). B. Carcinogen-bindina by Viruses: The uptake of fluorescent organic carcinogens by viruses and by viral nucleic acid will be studied by the techniques noted above and a/variety of tissue culture and.anii4al systems explored with a view to demonstrating the posi?ible r.ole:o~ wiruses or viral nucleic acids as > vdetors for the-delivery of.car~c-inogens to susceptible intracellular loci. Depending upon, the_ results .o.f,.i v.itro affinity studies, various virus carcinogen pairs wil,l' ,be adminis.t;er~c~ to_`appropriate animal hosts e_ djsditlhe;~a6~;~ro~iu~ing zciericy of~virusplus carcinogen separately, vi~us-carcinogen complex~vi~us al.one, >and carcino~en--al,one will be com- pared with suitab1e controls. :Particular.attention,will_;be g'iv~n to resp~rs.tox~y: t~a'ct.,~rirtases` (influenza; adenoviruses ~ herna'dsorption ~r.iruses).-•-Tissueculttire systems will include human embryonic kidney, lung, and tracheo-bronchial epithelium. For the present, animal studies will be limited to mice and hamst.ers, over.• #Copies of Curriculum Vitae attathed, *Copies attached.

Hurnan-embryonic tissues derived fram:°s.pontaneous-1y_ .abokte,4 fetixses up ~o 1+ months: gest_atiorial age' 'suitable~ both' fo,rf tissue r.. ~ c-ttlture -and for cell f'ractionatiori studies. ` wIl1 be available through the Ob-stet_r`zcs ;and4:Gynecology Departxnent: o~ the Niar;garet' Hagiae Mater~i;ty -HOspital at the . Jerspy C i'ty -Me.dZCa3 GEntar c; . . . _. ., _ ._ . _ D. Metal~ determination tec : See ericlo`sed:-USPHS E I v .

3 . 6. Budget Plan: 7. Anticipated Duration of Work: Salaries Other - Overhead ~ ra- djo Cp ) ~ 3 0 q P p Pp Permanent E ui ment 1 st Year S 6'000 Ex endable Su lies D % 000 (l) 2nd Year ~ 7'000 2,000 1 000 1,350 T o t a l $ 1 0 Total $11X0 2 years : January 1, 1960 - December 31, 1961 8. Facilities and Staff Available: January 1, ~60 - May 1, 1960: Temporary laboratory in Department of Microbiology' Seton Hall College of Medicine, fully equipped to conduct virus research (freezer, refrigerator, incubator, storage facilities; spacious animal quarters shared by pre-clinical depart- ments; adequate space for ultracehtrifugation studies with Spinco Model L and for equilibrium dialysis studies; inadequate space for metals analysis studies). Permanent laboratory facilities (estimated cost $39,000) under ~onstx°uction at this time.~- June 1. 1960 - December 31 , 1961 : Permanent facilities in the Jersey City Medical Center completed' consisting of 2 virology- bacteriology laboratories; 1 chemistry laboratory with 12-foot fume hood' extensive bench space' centrifuges, spectrophotometer automatic precision balance, plus equipment applied for in USPHS E325J. Personnel: January 1, 1959 - June 30, 1959: Investigator, 2 tech- nicians, secretary9 glassware diener. July 1 1 9- December 31, 1960: Investigator; 2 Research Fellows Dr. Clyde Wu - present address: Boston City Hospital, Boston 18, Mass.; Dr..Re.gina McCormack - present address: University of Virginia Hospital, Charlottesville, Va.); 2 technicians, secretary.'glassware diener. (Bulk of support for this group to come from USPHS E3257 and 2 USPHS post-doctoral fellowships.) 9. Additional Requirements: (Explanation of Budget Plan) 1) Personnel d$13 000 - major portion of salaries of 1 technician and secretary for 2 years. inclizding 2) Permanent Eguipment: $5,000 -/portion ($3,000) of cost of Spinco Model L Ultracentrifuge and of Zeiss Spectrophotometer ($2'000). 10. Additional Information (including relation of work to other projects and other sources of supply): A. Background of Studies: The proposed studies of virus-carcinogen-nucleic acid inter- actiona -_represent a _ --?og - ~- 1c-a 1 _ evten-^,~~-.^.n- of- --increasi--"A=.r~ •dence- -th~, "~~--. ng~ SMEx~MICXxxxxxxxxxxxxxxxxxxxxx xxxxxx ~t~t~x~tf~eXt XXXXXXXXXxXxxxxxxxxxxxxXxxxxXX

(1) Viral nucleic acid is an extremely reactive substance, chemically; and (2) Viral infections can be viewed as repeated assaults on the basic cellular genetic apparatus by mutagenic substances. The long-term implications of such repeated interactions of cell and virus nucleic acid, interactions.which occur primarily in youth - the period of rapid cellular growth - have barely been studied. B. Previous Work Done on This Pro 'ect: See IISPHS E3 7, pages 8-9. C. Results Obtained bY Others: The large body of evidence that viral nucleic acid is the component which endows viruses with cell- (and nuclei-) invasive properties has been reviewed by Colter (1). The most striking recent demonstration that the nucleic acid of common human enteroviruses possesses such properties which the protein components merely modify, has been reported by Schalfer and Mattern (2). ' ~ Following the fundamental studies of'Zinder and Lederberg (3) on transduction in Salmonella, many workers, particularly in the field.of bacteriophage, have demonstrated the mutagenic properties of viral nucleic acid; these data have been reviewed by Luria (}+). The indirect evidence gathered by Lederberg (5) supporting the concept of such phenomena occurring in animal cells has been extended by Benzer (6) and received striking support from the studies of Ebert (7) who has demonstrated the ability of Rous sarcoma virus to act as a vector in carrying exogenous, genetically active tissue DNA into chick embryo cells. The intimate association of trace metals with nucleic acids has recently been quantitated and partially defined by Wacker and Vallee (8); studies by several investigators on the effect of trace metals and of chelating agents on the integrity of nucleic acid chains and protein-nucleic acid bonds in nucleoproteins have been summarized by Kirby (9). /,_- °1 The mutagenic properties of chemical carcinogens, first demonstrated by Tatum in Neurospora (10), have since been amply con- firmed; progress in.relating carcinogen action to alterations in specific gene loci has been reviewed and extended by Barratt and Tatum (11). Scattered evidence for metal-carcinogen interaction has been summarized by Furst (12). Although suggestive evidence of interaction and synergism between viruses and chemical carcinogens was adduced as early as 19~4 by Rous and Friedewald (13) and in 1952 by Duran-Reynals .(14)' there have been no published systematic studies of such inter- actions in biochemical terms. Stanley (15) has speculated that mutagenic earc-inoger~s- might--induc-e-neoplas ia=stimulating properties i.n- otherw3.se innocuous viruses or proviruses present in cells in the carrier state.

5 REFERENCES 1. Colter, J.S. Nucleic acid as the carrier of viral activity. Chapter in Pro~ress in Medical Virolozy, Vol. I, Ed. by Berger, E. and Melnick, J.L. Hafner, New York, 1958, pp. 1-35• 2. Schaffer, F.L. and Mattern, C.F.T. Infectivity and physiochemical studies on RNA preparations from hi hly purified poliomyelitis and Coxsackie viruses. Fed. Proc. 16: 317' 1959. - Zinder, N.D., and Lederberg, J. Genetic exchange in Salmonella. J. Bact. 64: 679~ 1952. Luria, S.E~.. Mutations of viruses in relation to normal and abnormal cell functions. Ann. N.Y. Acad. Sci. 71: 1085-1091 ' 1958. Lederberg, J. Cell genetics and hereditary symbiosis. Physiol. Rev. 3?: 403-430, 1952. Benzer, S. The Elementary Units of Heredity. Chapter in The Chemical Basis of Heredit_y, Ed. McElroy, W.D. and Glass, B., Johns Hopkins Univ. Press, 1957, .pp. 70-93. Ebert,.J.D. (Carnegie Institute, Baltimore) Studies on Rous sarcoma virus. Paper presented at seminar, Harvard Medical School9 Dept. of Bacteriology and Immunology. February, 1959. 8. Wacker, W.E.C., and Vallee, B.L. Chromium, manganese, nickel, and other metals in RNA. Fed. Proc. 18: 345, 1959. 9. Kirby, K.S. A new method for the isolation of desoxyribonucleic acids: Evidence on the nature of the bonds between desoxyribo- nucleic acid and protein. Biochem. J. 66: 4959 1957. 10. Tatum, E.L. Chemically induced mutations and their bearing on carcinogenesis. Ann. N.Y. Acad. Sci. 4_9: 87-97, 1947. 11. Barratt, R.W. and Tatum, E.L. Carcinogenic mutagens. Ann. N.Y. Acad. Sci. 71: 1072-1084, 1958. 12. Furst, A. Chelation and cancer - a speculative review. Symposium on Metal-Binding in Medicine. Philadelphia, May 6-8, 1959• To be published (J.B. Lippincott). 13. Rous, P. and Friedewald, W.F. The effect of chemical carcinogens on virus-induced rabbit papillomas. J. Exp. Med. 7,,9: 511, 1944. 14. Duran-Reynals,F. Studies on the combined effects of fowl pox virus and methylcholanthrene in chickens. Ann. N.Y. Acad. Sci. 5-4: 977, 1952. 15. Stanley, W.M. Relationships, established and prospective, between viruses andcancer. Ann. N.Y. Acad. Sci. Z1: 1100-11139 1958. D. Discussion: SiRnificance of this Research: Persistent failure by many investigators.in many systems to isolate a human "cancer virus" necessarily raises the question as to whether such a virus exists at all. On the other hand, there is abun- dant evidence, both in animals and in man, that a large number of chemical carcinogens will induce neoplasia. At the same time, however, efforts to demonstrate the etiologic role of proven environmental carcinogen in induction of neoplasia in man are in virtually all -ins-tances -~G.I.-tract-'--- lung-' Yiver)-op-en-to-t'zie-objectlori=txialt-while experimentally exceedingly large doses of carcinogens are required to induce cancers, each of the suspect environments (artificial food dyes, industrial fumes, gasoline fumes, cigarette smoke, etc.) contains exceedingly minute quantities of carcinogen.

The present study proposes to explore a relatively plausible set of conditions, susceptible to controlled experimental analysis'which might square the negative virologic data and the dis- crepant carcinogen data; i.e~' that minute amounts of carcinogens - not ordinarily of mutagenic significance - may,induce neoplasia if bound to any of several common viruses - not ordinarily carcinogenic - and thereby delivered to the chromosomes of cells the virus invades but does not destroy. The evidence that such interactions can occur ia summarized above. If true and widespread, these interactions would carry the implication that neoplasia so induced is preventable (through virus vaccines) and that minute quantities of environmental carcinogens are relatively tolerable. S igna tur e~~.~ ,-`"r'.J Director of Project Charles L.Brown, M.D. Dean Seton Hall College of Medicine

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