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VIRUS, previous infections N0. 550AM-R2 Activated: 1/1/67 Supp. 9/23/67 THE COUNCIL FOR TOBACCO RESEARCH - U.S.A., INC.Ren: 1/1/68 COMMITTEE: ~ Dr. Huebner, Chin. Dr. Andervont Dr. Loosli Dr. Lynch 110 EAST 59TH STREET NEW YORK, N. Y. 10022 Ren: 1/1/69 Supp. for Con't. 1/1/70 - 3/31/70 NO. 550AM Activated: 4/1/70 Renewed: 4/1/71 Date: Oct. 20, 1971 1. Name of Investigator(s): (include title and degrees) ~~ '' '-" a~ %iu~ y i J~ W ~.. John E. Craighead, M.D.; Professor of Pathology 2. Institution & University of Vermont Address: Department of Pathology Medical Alumni Building Burlington, Vermont 05401 3. Short Title of Project: Epithelial Cell Transformation and Carcinoma Induction by "C" Type RNA 4. Proposed Renewal Starting Date: (Anniversary or other) April 1, 1972' 5. Discuss any Important Changes or Additions to Objectives or Specific Aims: The major objectives of the work supported by this grant are 1) to produce clones of epithelial cells which have been.transformed by "C" type oncornaviruses, and 2) to induce neoplasms having the pro- perties of carcinomas by transplanting these cells into appropriate animals. Strains of mouse sarcoma virus and cells of~renal`and hepatic origin are being employed in these studies. Various tissue culture methods have been developed in an effort to select and grow clones of transformed epithelial cells for transplantation. Since polycyclic hydrocarbons enhance the oncogenic effects of oncorna- viruses in the intact animal and produce epithelial tumors in animals, Viruses these chemicals will be used as cocarcinogens in in vitro transformation experiments. ' The objectives of this study as briefly outlined above, have not changed during the first 18 months of work supported by this grant. The postulates upon which our initial proposal was based appear sound and justify continued pursuit of these objectives. ® 6. Give a Brief Statement of your Working Hypothesis if altered or modified: Our ;-~ ~ wo _ rking hypothesis has no~ eeen ;r~od~~zeu .

2. 0 7. Changes or Additions to Experimental Design and Procedures: (Attach Separate Pages) No changes in the experimental design and procedures are anticipated during the third and final year of support by this grant.' We are directirfg every effort to the selection of clones of transformed epithelial cells and are attempting to culture large numbers of these cells for transplantation experiments in mice. Thusfar, it has been difficult f or us to grow sufficient numbers of cells to carry out conclusive experiments. These efforts will be continued.using roller tube methods, an assortment_of_different.types of inedium-and serum. When and if transplants are established the tumors,will be examined histologically to determine whether or not"they exhibit properties of carcinomas. These lesions will also be compared with tumors formed from transplanted, transformed fibroblasts and those resulting from the injection of animals with cell-free preparations of virus. An evaluation of the infectious process in transformed epithelial cells has begun using standard virological techniques and ultrastruc- tural approaches. This work will be continued during the coming year in an effort to determine whether or not and to what extent mouse sarcoma viruses replicate in transformed and non-transformed-epithelial cells. 8. In our initial application for research grant support we proposed to use polycyclic hydrocarbons as cocarcinogen with mouse sarcoma virus in transplantation experiments. This.work has not yet been initiated but will be undertaken during the coming year. In these experiments non- S toxic or marginally toxic dosages of methylcholanthrene and benzopyrene , will be added to cultures of primary mouse hepatic cells. At appropriate intervals thereafter mouse sarcoma virus,. with and without t1helpertt mouse leukemia virus, will be introduced. t'HelpertT fibroblast cultures will also be used when appropriate in efforts to support the growth of the-epithelial cells. Clones of epithelial cells will be selected and established to test for transformation. 9. Changes in Personnel with Biographical Sketches of new Personnel (append) No changes 0 10. Publications or Papers in Press resulting from the Project or closely related work None

Budget (for coming year) 11. 3. 4/1/72 - 3/31/73 A. Salaries (Personnel by names or category) % time Amount Professional . John E. Craighead, M.D. 10% 2551 Technical M.P. Smith 50% 3838 J.B. Kessler 100% 7676 H.G. West 42.93% 3062 L.G. Pudvah Sub-ToaT % 1258 18,385 - P l e s 14/ 2574 ist by categories) 5uppl e B. Consmable ( Tissue Culture Media 1000 Glassware and disposable plastics 1000 Animals 1000 Photographic supplies 250 Miscellaneous 750 Sub-Total 4000 C. Other Expenses (itemize) None Sub-Total - D. Permanent Equipment (itemize) None E. Overhead (15% of A+ B-hC) It is understood that the applicant and institutional officers in applying for a grant have read and, found acceptable the Council's "Statement of Policy Containing Conditions and Terms Under Which Project Grants Are Made." 3,744 Total 28,703 Business Officer of the I4titution Signature Telephone Telephone

, ~'. Other Sources of Financial Support ' Current List financial support for research from all sources, including own institution, for this and/or related research projects. Title of Project Source Includes indirect Amount Duration Correlated Studies of Pulmonary Disease in PHS 1 P17 HE 14212-01 Proj. 4 $46,202.00 6/1/71 - a Rural Population: 5/31/72 Studies on the Pathogenicity of EMC Virus PHS 5 R01 A109118-04 $32,667.00 10/1/71.- 9/30/72 Pathogenesis of Adult Human Cytomegalovirus PHS 5 R01 A109112-03 $43,450.00 3/1/71 - Infection 2/29/72 terminates 2/29/72 Pending None