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~ BIqASSAY, CAROINUGl;Nk;615 and T1S5Uh• CULT'UHE: NO. 567R1 Activated: 10 1/6b : Txr, Couxcu, roR ToBicco RicsmRcu - U~S.A. ' ~ ---- ' T - • 633l1atD AvEXUE . . . . COl4.ffTTEE: . NEW 7ocu:: rr: r. 20017 Dr. Little, Chm. • Sommers )?" ;;' Dr. R0im8[1n - App(ication For Renewal of Research Grant -D L 'r.oosli . _ _ F•sst 9 Second [] : Datrr, 19th July, 1967 1. NameafInrestigator(:)o(includetitleandde9rees) PTOf". I)uei0 Severi, iZ`.D. Principal Investigator; '.Cesare Biancifiori, M.D. Go-Principal Investigator; ~ RqdQlro Ribacchi, a.D. Assistant; Emilio Buceiarelli, rii.D. Assistant; `~ 2 Huhtutian :Gaetano Giraldo, M.D•. Assistant ~;"'r`'Adtlress k ;z; -- ~~ .M:-. .-. . C-':'~'`:i•£... ; Division of Cancer Research, University of Perugia 0. Box 327 ' :r;•. 'FERUGIA ZTAT~Y Fn ~1 U~:x~::;iC%"ti~~~c~ ~ ,~ r3. ShortTitleof Proiect: -, :5"6n Approach to the Study of Internal Factors in Dung Carcinogenesis. `-Influence'oi.Hormones. d. ProposedRen.wafStadingDates(Anniversaryorother) OetOber 1St, 1907 3 - . s" s. Discuuseny,important Changes or Additioni to objectlre: or Specific Atms: There have been no basic changes ~W"';.made to the ob3ectives proposed•in the Application of 2nd August, 1966 and the Semiannual Progreas Report no. 1 forwarded in April, 1967 to The +c: Council for Tobacco Research. ' .~. cta '? The observation of adranallimage in carcinogenesis b ;oreal dy hydrazine/isonicotinic acid' hydi•azide (IP)H) seems a profitable line of .; invest3gation.:":~In lurg cancer patients changes in endocrine glands have been described. ''.This is still the starting point for study on ~~~ ~yrhether internal factor exists in iungcaneer. -~ an1' Y .3'~*~~The aims of this ;research' ther•efor•e remain chiefly, o study whether the adrenals are involved in spontaneous .i and INH; and hydrazine sulphate (h.s.)..induced pulman.arg _ .tunours in mice and To study whether the thyroid•is involvedin-spontaneous .• 'and INH and h.a. induced pulmonary tumours in mice. '~q. . Q

7. Changes or Additions to Experimental Design and Procedures: (Attach Separate Pages) (SEE ATTAC_TiED PAGES) 8. Additional Requirements: Nil 9. Changes in Personnel with Biographical Sketches of new Personnel (append), New personnel: Dr. Gaetano Giraldo (See biographical sketch attached) •10. Publications or Papers in Press resulting from the Project or closely related work (SEE ATTACIiED LIST )

Changes or Additions to Experimental' Design and Procedures: A. Spontaneous and 1NFI and h.s. induced pulmonary tumours in the mouse in relation to adrenal hormones. Incidence of spontaneous and induced pulmonary tumours : in susceptible and resistant mice. .There have been no changes made to these experiments, which have been described in the Application and in Progress Report no. 1. Incidence of spontaneous pul.nonary tumours in susceptible - ' adrenalectomized mice into which the adrena7.s of resistant ' mice have been grafted, and vice versa. - At the beginning of this-dxperiment (see Application and Progress the C57/Cb/Se the donors. None of the technical procedures used was effective in preventing the death of the test mice. Por this reason, days; the same thing happened when A/Lac/Se nice were the tests and C57BZ,/Cb/Se mice treated with A/Lac/Se lymphocytes lived beyond a few .:. The administration (intravenous or intraperitoneal) of lyrzphocytes between these two substrains, however, is badly tolerated: none of the .treat the test mice at birth with lymphocytes of the donor mouse. and resistant to lung tumour induetion, were used. To assure that the adrenals take in the host it is necessary to Report no. 1) A/Lae/Se and C57BL/Cb/Se mice, respectively susceptible the C57BL/Cb/Se mice were replaced by CBA/Cb/Se. The tolerance ~ ., 11 betweeri CBA/Cb/Se and A/Lao/Se mice has proved good, provided the test the C57BL/Cb/Se. considered resistant to lung tumour induction and therefore may replatee mice are treated within a few hours of birth. CBA/Cb/Se mice are and have the adrenals of the lymphocyte donor grafted into them; they will be examined at natural death. This has already been d'escribed in the Application and in Progress Rcport no. 1. . .. Several newborn mice of the two substrains have already been `treated, and survival is good; at 8 weeks they will be adrenalectomized, c. Pulnronary tu~~origene sis by IlNH and h. s. in suscept ible adrenalect-ouized. mice into which the,adrenals of resistant mice have beengrafted, and vice versa. These experiments will be carried out as described in the Application, with CBA/Cb/5e mice replacing the G57Bh/Cb/Se. 100354G'756

incidence and the susceptibility to lung tumour induction are different. If these characteristics in the adrenalectomized hybrid test mice are the same as in the parent donor of the adrenals, the influence of these glands in lung carcinogenesis could be hypothesized. 1 . Incidence of spontane ous pulraonary tumours in intact and . ~:. in,adrenalectomized (C5?BZ/Cb/Se x A/Lac/Se)Fl hybrid mice. No:change has been made to the experiment as described in the :,.:Application but, as mentioned in Progress Report no. 1, : fi t ~ ;(BALB/c/Cb/Se• x C57BL/Cb/Se)F' hybrid' mice will also be used. ''Incidenc.e of sp ont ane ous pulmonary tumours in adrenale ct o- "; `;': ;: • niized (C57BL/Cb/Se x A/Lac/Se)F mice into which the e n grafted. : adrenals of the parents have be • No changes: the experiments will be carried out as described e x ~e y w s u . _.Q, ; in the Application. In these experiments as well (BALB/c/Cb/Se ' ill al o be sed C57BL/Cb/~ )F h brid mic Pu]:monary tumorigenesis by INH and h.s. in intact and in ! adrenalectomized (C57Bh/Cb/Se x A/Lac%Se)F1 hybrid mice. (as shown in Progress Report no. 1). (BALB/c/Cb~Se x C57B1,/Cb/Se)Fi hybrid mice willl be used Also in these experiments, in addition to the (C57BL/Cb/Se x A/Lac/Se)F hybrid nice (as shown in the Applicat'ion), g. Pulnonary tuinorigenesis by INR, and h.s. in adrenalectomized 05?BL/Cb/Se x A/hao/ae)Fi hybrid mice into which the adrenals of the parents have been grafted. - These•experiments will be carried out as previously described in the Application; (BALB/c/Cb/Se x.C57BL/Cb/Se)Fl hybrid e m.ice will be added. If it seems of use, (CBA/Cb/Se x A/hac/Se)F1 hybrid mice will be ".'added to the experiments described'in paragraphs d,e,f,g. These different types of hybrids will be used because they are the product of substrains of mice in each of which the spontaneoug B•. Spontaneous and J21H and h.s. induced pulmonary tun¢ours in the mouse in relation to thyroid hormones. No changes made to the experiments, as described in a,b,e,d- of the Application. 1003546'7S7

0. Additional experiments. In addition to the original experimental design, investigations ;` are be irig carried out, on: Mitotic cell activity in the mouse lung and Immunologzcal {:. .approaches of INH and h. s. induced lung tumours in mice. . a. Preliminary studies on the mitotic cell turnover in the mouse lung. This experiment is being carried out tirith special fixation and staining techniques in lung of lung.tumour induction susceptible (BALB/c/Cb/Se) and~ resistant (CBA/Cb/Se and' C5?BL/Cb/Se) mice, treated and non treated and in different hormonal states. This should allow us to observe the early cell changes and the cells•chiefly involved in the regenerative process. Differences between the three groups of mice - treated, non-treaiied and in different hormonal states - could be of significant interest. b. T.mmunological approaches to chemically induced lung tuzaours in nice. (i) Attempts to show whether lung tumours obtained with INH and h.s. are merely accelerated spontaneous tumours or whether they are indtzced. -:It has been shown that chemically induced tumours develop specific antigens, whereas spontaneous tumours do not. Moreover, whereas virus in.duced tumours usually carry antigens specific to the virus and therefore cor_L-aon to every tumour induced by that virus, the tumour specific antigens of chemically induced tumours differ from tumour to tumour. The application of this approach to INFi and h,.s. induced lung tumours could proceed along the following lines: part of a large lung adenoma could be transplanted into mice of the same strain; when it grew out, a part of it could be excised an•d fragments iMplanted into other mice, and removed when they started to grow out. The ability of further fragments of the same tunour, or of measured numbers of cells from it, to be rejected by the immunized mice would • de:aonstrat e the ant igeni city of the se tumours. 100354r.7sg

R: REDACTED MATERIAL This experiment will have to be repeated several tiMes. shoul.d, however, be confirmed also by other experiments, with the ,a,iA of showing that the participation. of a virus in the development .of the pulmonary tumour can be excluded, but this will not show that : a hormonal factor is not involved. The latter is•an internal component _ which probably does not act by itself but, in favourable conditions, might allow the action of 7Iv'H, or h.s. or of any other careinogen. , (ii) Attempts to influence the growth of urethane induced - primary lung tumours in BAhB/c mice by stimulation of imTaunocompetent cells of the host. Previous work done in this laboratoxy (1965, 1966) has shownn that (the depression of in~.'nunoco-apetent. cells obtained by) neonatal : y thynmectomy poten.tiates the oneogenic effect of a single •dose of ~ urethane in BALB/c mice. In fact, in thym-ectomized mice lung• tursours develop earlier, are more nu.*ierous, grow to"a.-greater size, metastasize more frequently than in controls treated' with the sane dose of urethane, and they only rarely show foci of necrosis and/or fibrosis. Theoretically, a stimulation of immunocompetent cells in mice treated with a single dose of urethane could reduce the oncogenic effect of the chemical compound. Following this approach, BAhB/c mice inoculated with urethane will be treated with incomplete and complete Friend's adjuvant, with urethane-induced secondary BAZB/c lung tum•our grafts, and with a cell-free homogenate of the same tu.*~our. - _ 8. (See printed form) I 9. Biographical Sketch of Dr. Gaetan~o Giraldo. Title: Assistant scientist, Division of Cancer Research, -Universi:ty of Peru gia a _ Place of Birth: Naples, Italy Pre-sent Citizenship: Italian Sex; Male Education and Experience: Dr. Giraldo graduated M.I?. from the University of Naples in. 1962. H:e is a staff member of the Institute of Pathological Anatomy. and Division of Cancer Research, University of Perug•ia, from 19b3- Dr. Girald6 has:published 17 papers on-various subjects. ~ _ 1oo3i4s'7s9 -

-Publications or Papers in Pre.ss resulting.from the Project or closely related work. Ribaechi, R. and Girald•o, Cr.: Sui fattori intrinseci della cancerogenesi polraonare del topo: effetti della tinectomia '; ~ .r.r ' neonatale. hav. Anat. Pat. Peruga a 26, 127-136, 1966. Severi, Z. and Biancifiori, C.: Hepatic carcinogenesis in CBA/Cb/Se mice and Cb/Se rats by hydraza.ne sulphate. "~ 29th-30th October, 1966. (in:press) International Conference on Hepatic Hegeneration, Montecatini., 3. Severi, Z. and Biancifiori, C.: Hepatic carcinogenesis in CBE1 mice and Cb rats by isonicotinic acid hyd-rGzide and hydrazine sulphate. Symposium on Hepatomas, Philadelphia, 19th-20th May, 1967. (The paper will be forwarded to the Journal of the National Cancer Institute for publication) 0

R: REDACTED MATERIAL -- 'c,....-. . .. • ~ R Consumable Supplies (list by categories). ? Animal food and sawdust ~~,Chem1cal drnizs 5taining nateriala ,•_,:Sttrgical instruraents 01,000 500 . 250 150 100 Record oards, registers, labels :Glassware C. Olher Expenses (itemize) 300 -1,700 1,700 15:000 L it is understood•that the applicant and institutional officers in appljring for a grant have read and found accepttble the Counc3l's "Statement of;Policy Containing Conditions and:Terms Under Which Project Grants Are Made." Sub-Tatal 300 Tatal $11,600 Signature Ls-,+% ofrsctar ot h.ieet Signature~ ~+4nps Ollibi at'Iha ImGwltow Telephone `

Oth.r sourca of Financial Support ! iF . Ulttinanciat,upport for ret.arch from ali .ourc.s, Including own InNttution, for this and/or r.iat.d rtswrch pro ~ ?! Hormonal i'actor(s)'in pulmonary oaroi.ndgeneeis'. (by hydrazine). (Severi, L. and Bianoifiori, 0.) Lung tumorigenesis by Isoniazid (INH), its' i netabolite hvdrazine su].nhate and derivativesi of hydrazine. (8iancifiori, 0.) , • V . The Counoil for Tbbacoo ;. i4i - Research, 633 Third Avenue,t~ ' New York N.Y. 10017