Tobacco Documents Online

VIRUS, previous infections 357R1 Activated: 11/1/62 . C ONJNLL'I'TEE • Dr. Jacobson Chairman Dr. Kotin Dr. Lynch TOBACCO INDUSTRY RESEARCH COMMITTEE 150 EAST FORTY SECOND STREET NEW YORK 17, N. Y. Application for Research Grant Date: August 13, 1963 1. Name of Investigator: Morris Pollard 2. Title: Director-of Lobund Laboratory, Professor and Associate Chairman of Biology 3. Institution & Address: University of Notre Dame Notre Dame, Indiana 4. Project or Subject: Carcinogenesis in Germfree Animals 5. Detailed Plan of Procedure (Use reverse side if additional space is required). Studies on carcinogenesis in Lobund Laboratory during the past year have been reviewed in the annual report. Three projects are here outlined for which we solicit support: Relationship of viral, chemical, and physical- factors in the carcinogenic process. - - 1. Inoculations of inethylcholanthrene (MC) and subsequently of viruses into adult mice did not modify the response. Susceptibility of rodents to oncogenic viral action is usually demonstrable only if inoculated at birth, and this shall be done. Enhancing or promoting effect of chemical agents can thus be clearly delineated in animals which have had exposure to a viral initiator. Not all viral inocula are oncogenic and not all mouse strains develop tumors thereof; however it would be interesting to deter- mine -their influence on subsequent exposure to chemical or physical carcinogenic agents. We will inoculate viruses such as polyoma, adeno _ . _ 12, va __ccinia, or influenza viruses into germfree newborn mice of C3H, Swiss-Webster, and ICR strains, and thereafter superimpose inoculations of MC, DMBA, applications of croton oil, or exposure to sublethal doses

• • 2 of x-irradiation. Control groups (germfree and conventional) will receive virus or a drug or x-rays alone. The animals will be maintained either germfree or conventional for the duration of the experiment. When tumors develop, or at an arbitrarily determined 'time, the animals will be exsanguinated and examined by autopsy. Lesions will be examined microscopically and the serums will be tested for specific viral antibody. 2. We know that Swiss-Webster mice inoculated at birth with MC develop pulmonary adenomas within 8 weeks. This occurs in 100% of germfree and 83% of conventional mice. The lungs are inflated with Zenkers solution through~the trachea; and when washed thereafter the surface tumor nodules can be counted with low magnification. In this procedure mice have developed average of 35 tumors per lung at 8 weeks; 45 at 10 weeks; and 169 at 15 weeks; and individual cases have had as many as 320 tumors per lung. We therefore have a useful and uniform medium in germfree mice with which to evaluate modifying influences. We will inoculate new born germfree Swiss-Webster mice subcutaneously with MC and at intervals thereafter with viruses such as influenza, adeno- virus, polyoma and vaccinia. We will then determine if the superimposed viral infection caused the lesion to assume malignant character. We have a system which assures 100% benign pulmonary neoplasia and which clearly permits evaluation of occurrence and of malignancy. Since 100% of germfree Swiss-Webster mice develop lung adenomas, the effect of preventive measures too can be evaluated. Boyland (Chester Beatty Institute) claims that the carcinogenic effect of MC can be inhibited by coincident inoculations of thymidine. This warrants further investigation. 3. Im_mun_ol_ogical_b_asis o_f_c_h_e_mic_al a_n_d_v_i_r_al carcinogenesis. Germfree rodents have a low level of gamma globulin and a relatively inactive reticuloendothelial system as reflected by the histology of the spleen, lymph nodes and cell systems in the intestinal wall. Adult germfree mice and rats will be inoculated with methylcholanthrene or with DMBA. When neoplasms appear, either in the lungs, in the inoc- ulated area or in the breast the animals will be exsanguinated and autopsied. Components of the RES will be examined histologically for evidence of activation, and the serum will be examined electrophoretically for evi- dence of globulin formation. Control tissues will come from germfree animals which had been inoculated with conventional and oncogenic . . . ,_ ~.~ _ ~d~'L'--e~J--nd= ro~ir=.on~~e-^,n a -`~n2~i'Fa;-s==3T.CiicI3 i-eC:ei'veCi.-lv"L"l:,- -D!bTt513,--oT• 0

3 • viruses. Preliminary trials have indicated that viral agents induce tissue reactions which reflect activation, and that the development of chemical tumors in germfree mice is accompanied by similar tissue reactions. This may mean that the neoplastic transformation is the same, whether initiated by viral, chemical, or physical factor; and that the oncogenic process may be related to the status of the RES. If this is the case then (1) specific immunization against chemical carcinogenesis may be envisioned, or (2) the immunological response is unrelated to the chemical carcinogen and reflects the "unmasking" of a virus, or (3) the neoplastic cell represents a mutant which the host reticuloendothelial system recognizes, and/or (4) the response may reflect benign or malig- nant transformation (dependent-autonomous) stages of tumor development. This we will continue to study. From the results accumulated thus far, it is apparent that the neoplastic process does not require a bacterial flora. This means that one of the most important problems in carcinogenesis is still the mecha- nism whereby a"normal" cell is transformed to a neoplastic one, and how the body defenses (including the immunologic mechanism) can function to the advantage of the host. Pertinent References: Kidd, John G. , 1961. Does the host react against his own cancer cells? Cancer Res. , 21:1170-83. Furth, Jacob, 1963. Influence of host factors on the growth of neoplastic cells. Cancer Res. , 23:21-34. Old, L. J., B. Benacerraf, D. A. Clarke, E. A. Carswell, and E. Stockert, 1961. The role of the RES in the host reaction to neoplasia. Cancer Res. , 21:1281-1300. Kelly, Margaret G. and R. W. O'Gara, 1961. Induction of tumors in newborn mice with dibenz(a, h)anthracene and 3-methylcholanthrene. J. Nat. Cancer lnst., 26:651-673. Duran-Reynals, M., 1963. Combined effects of chemical carcino- genic_aj~en_tG_a.nd-vi-rasPs_ p;-`3:i.48-i.85;-Kar•g-er, Basel. 0

• - 4 - Kotin, Paul. Ibid. 186- 215. i Malmgren, R. A. and A. S. Rabson, 1963. Failure of inoculation with polyoma virus to influence chemical carcinogenesis in mice. J. Nat. Cancer Inst. , 30:203-206. Pollard, Morris and J. C. Salomon, 1963. Oncogenic effect of methylcholanthrene in new-born germfree mice. P. S. E. B. &,M., 112;256-259. Pollard, M. , 1963. Induction of tumors in germfree rodents by methylcholanthrene. Fed. Proc. Pollard, M. and B. A. Teah, 1963. Spontaneous tumors in germfree rats. J. Nat. Cancer Inst. , 31:155-158. Pollard, Morris, 1963. Chemical induction of breast tumors in germfree rats. Nature. Submitted for publication. Pollard, Morris, 1963. Response of germfree rodents to chemical carcinogens. In preparation. Addendum We now have evidence of a leukemia virus in germfree C3H mice which was activated by three doses of 150 r x-rays administered whole- body a week apart, started at 1 month of age. 0

- 5 - 6. Budget Plan: a. Salaries $ 9,000 b. Expendable Supplies 6,000 c. Permanent Equipment d. Overhead (15~/0 of a, b, e) 2,304 e. Other (FOAB & Comp. In.s. ) 360 Total $17, 664 • 7. Anticipated Duration of Work: 8. Facilities and Staff Available: One Year See attached sheet. 9. Additional Requirements: None 10. Additional Information (Including relation of work to other projects and other sources of supply): U. S. Public Health Service - Virology of germfree animals U. S. Public Health Serviee - Intracellular viral chemical indicator system Cancer Society of St. Joseph County - Electron microscope and its maintenance .Office of Naval Research - Physiology of immune reaction in germfree animals (Co-principal Investigator with R. Wilson, Ph. D. ) Maintenance of the germfree colony of animals is supported by funds provided by the National Institutes of Health and the. Office of Naval Research. ~ ~ Signature Director of Project Morris Pollard 'siness Officer of h Institution ev. J. J. ilson, C. S. C.

• • 0 - 6 - 8. Facilities and Staff Available: Lobund Laboratory is the most widely diversified operation involving germfree methodology. The staff consists of 14 professional personnel (M. D. , Ph. D. , and D. V. M. ) plus 60 technicians. The facilities and equipment are excellent and the library provides 400 journal titles in the biological sciences. Current projects involve radiation biology, enzymology of wound regeneration, origin of antibody, nutrition, dental caries, thymus function, cytology, viruses and stress, biochemistry of viral replication, viral latency, electron microscopy, and oncology. A team of technicians provides the germfree animals to the investigator and another team cares for them during the experimental period. During the past year over 6, 000 germfree mice and 3, 000 germfree rats have been produced. The Labora- tory occupies approximately 16, 000 square feet of space. The National Science Foundation has approved construction of 20, 000 square feet addi- tional laboratory facilities for Lobund Laboratory. 0