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*'• - .. - .. ~ - `C :; t~~-;BICASSAY, CARCINOGENESIS and TISSUE CULTURE 1i~''~. . ,. ...-... . _._.__..~ .. _ . . -T= CoUlvca FOR T©sAccG Rrsr.nRCS . U.S.A. Ess TIIIFtD 19EAFUE NEH' IoIiH. N. T. 10017 - Dr. Loos13, Chm6 '_ Di. Sommers Apprcation For Researeh Grant -DY : •'Reimann Dr. Little 1. Nameoflnvestipator(s)d(i,ncfudeTitleandDeyrees): Mary Stearns PARSNTE3C, Ph.D. . Assistant Professor of Anatosy in Obstetrics & Gynecolo gy 4 Institur~on ~ Columbia University Address: College of Physicians & Surgeons 630 West 168th Street Nev York, N. Y. 10032 3. 5horf Title of Projech EFFECT OF CONSTITIIENIS OF TOBACCO SMOKE ON! NORMAL AND MAIZGNANT HUMAN RESPIRATORY EPITHELIDM IN VITRO. 1. Proposed Starting Date Novetnber 1, 1967 S.Anticupated-DuranonofthisSpecificStudy: Although some meaningful results should be obtained by the end of the first year, it is expected that an additional period; will be required. C• 6. Brief Descriplon of O6jectives or5pecific Aims: There is an,increasing emphasis on tobaeco smoke as a contributory factor to the development of lung cancer. Tobacco products, the polycyclie aromatic hydro- " carbons in particular, have been shown to induce or enhance neoplastic transfor- taat~oA in laboratory animals (4,9,15). On the other hand soma constituents, namely the oibnoearboxylic acids, have been. reported to have an anti-tumor effect (12);. There is little precise knovledge of the qualitative and quantitative effects on norma1'respiratory epithelium of the nearly 300 organic and inorganic compounds rpported as constituents of tobacco smoke (4,8). These include netal~s, hydro- carbons, alkaloids chieflynicotine, nitrogen products, and volatile elements. Either similar factors.found in polluted air or still other factors must be respon- -r----sib3e-forthe•-occurrence of lung.eancer in non-smokers, and the higher incidence of this disease among male smokers. A great deal of study has been devoted; to the biological activity of tobacco tars or condensates with. emphasis on the pol,ycyclic hydrocarbons which are present only in traces (1,2,5,6,9,15). Not only has it been shown. that these hydrocarbons are not specifically ea'rc•iftogcnia for epithelium (6,9), but the fact that fractions of smoke condensate not containing hydrocarbons also K7~vpToduae neoplastic changes in Iung•epithelium has been demonstrated (6,9). Most animal experiments deal with the carcinogenic effect on skin of smoke condensates and their fractions (4,7,8615). Studies in which animals actually inhaled smoke gave inconsistent results (7). The skin is quite different from the mucus-coated respiratory epithelium and apparently more sensitive to the polycyclic hydrocarbons. Although nicotine content has been considered to be detrimental to the cleansing activity of the respiratory cilia (3), very little is established as to the local biological effect of nicotine (4). Littlo attention has been paid to,the volatile / substances such as carbon monoxide, hydrogen~cyanid'e, and'nitrogen.oxides, present `!• in-many times higher concentration- than that considered to be "safes' in industry (8;1f+). It is possible that neoplastia changes in normal respiratory epithelium 7. G'rveaBriefStatemento.ryourWorkingHypothcsis: (cont. on p.la)- If the development.of malignancy in normal human•respiratory epitheliun is the result of an interference with, the oxygen supply and normal respiration, a simulation , (cont. on p. 1a) G= ~ `_

~ may be caused by an interference on the part of some of these volatile constituents of tobacco and polluted air with the oxygen supply to these cells necessary for aerobic respiration. This anoxia could result in a mutation toward anaerobic glycolysis as a source of energy characteristic of bron,hogenic carcinoma (1k). `,'z e:F!. ~ ~.'::i::;_ ~ The technique of tissue culture makes possible the direct observation of human ,:;...tissues under high magnifications. The biological effect of whole tobacco smoke ;f ; as well as some of its volatile constituents on these cells can be studied microsco_` pically, and the metabolic byproducts of the cells under different conditions can be analyzed. Cell lines provide a source of uniform. test material. In addition test : materials can. be added in a concentration not possible in the intact animal and under .conditions of prolonged contact permitting the equilibrium of poorly soluble or slowly diffusing substances between the medium and the cells. A number of materials possibly related to a neoplastic activity of either tobacco smoke or polluted air have been studied by this method-. Hyperplasia and metaplasia considered preneoplastic - in cultures of human foetal hl.ng treated with polycyclic hydrocarbons and tobacco smoke condensate and its fractions was reported by I,aznitski (5,6). Crocker and coworkers havecarried on similar studies with rat and hamster trachea in tissue culture (1,2,9). These workers reported a nonspecific effect of these carcinogens, and also that analogs of these hydrocarbons not known to be carcinogenic produced similar abnormalities in cultures of respiratory epithelium,. Toxic effects of organic constituents of tobacco smoke and polluted air, acetaldehyde, nitrogen dioxide, , and monocarboxylie acids, on lines of human and animal cells in tissue culture were observed by Pace and his coworkers. (10,11,12). Rounds also observed that nitrogen dioxide introduced as sodium nitrite into the medium of cultures of trypsinized and - suspended cells from the lungs of mice and rats caused nuclear and cytoplasmic changes • • which accompanied a reversible inhibition of respiratory activity (13). See continuation sheet lb for Pertinent Literature Ref, cited above, ~ . . .. . . . .. ... •;::..'.•.;';`n~~:"1' . . It is proposed to study the effect of some of the gaseous constituents of tobacco smoke and polluted air on established lines of normal and malignant human respira_ tory epithelium from. the pharynx, larynx, and lung and to compare their charaeteris- ties as evidenced in tissue culture under these experimental conditions. Effect on rate of proliferation, cell movement and contact relationship, nuclear and cyto- plasmic structure and chromosome content will be correlated with differences in metabolic activity and related to neoplastic capability. A clear understanding of the morphological and metabolic differences between- normal and malignant human respiratory "` epithelium should contribute to an understanding of the neoplastic process itself., 7.,cont. of these conditions in tissue cultures of these cells by a manipulation of the gaseous atmosphere of the culture. should demonstrate this. Similar experimentation with the gaseous phase over respiratory cancer cells in tissue culture might lead to changes in the morphologic and metabolic characteristics of these altered cells which could give a clue to the causes of carcinogenesis. These studies have a direct bearing on neoplastic development and the biological and biochemical mechanisms responsible. ' 1003547058

continuation sheet lb cont. Pertinent Literature References :"l, Crocker, T. T., Nielson, B,:I., and Laznits.ki, I, Carcinogenic hydrocarbons , Arch. of Environ• Health, 10:2:240. 1965 ~ ~ . ..~. . .,.;.; :~~~~:;. ~:: ~• • . . ... • . . . .: . .,. . .- . .. .., . . ,. ,, ., .... . ...,-~ . • ,. . : . .,;~ :,.. '2, --------------, and Nielson, B. I. Effects of carcinogenic and non-carcinogen drocarbons on respiratory epithelium in orhan culture. Tissue Culture Assn. Proc,' Miami June 1965 56 , . r p• • i: ..} . . ' .L 3,Dawson, F. W. Effects of nicotine and influenza on human ciliary activity•in vitro• Fed. Am, Soc, Exp. Biol. and Med. Proc., 24: part I, #260, 1965. 4. Kensler, C. J. The pharmacology of tobacco smoke effects of chronic exposure,. in "Tobacco and Healthil, ed. by G. James and T. Rosenthal, Chap. 1, Charles Thomas;?" Springfield, I11. 1962, p, 5. 5•Iaznitski, I. Effect of 3,4-Benzpyrene on human foetal lung grown in vitro. Brit. J. Cancer, 10:510, 1956, 6, ---------- Observations on the effects of condensates from cigarette smoke on human foetal lung in vitro. Brit. J. Cancer, 12:547. 1958. 7. Leuchtenberger, C., and Leuchtenberger, R. A correlated histological, cyto-"~ logical, and cytochemical study of the major bronchi from mice exposed to cigarette .; smoke. in "Tobacco and Health", ed, by G. James and T. Rosenthal, Chap, 8, Charles Thomas, Springfield, Ill. 1962, p. 81. •- 8. Lindsey, A. J. Some observations upon the chemistry of tobacco smoke. "Tobacco and Health11, ed. by G. James and T. Rosenthal, Chap. 2, Charles Thomas, -~.Springfield, I11., 1962, p. 21. 9, Nielson, B. I., and Crocker, T. T. Epithelial and mesenchymal interactions', :+ in hamster respiratory mucosa exposed. to polycyclic hydrocarbons in organ culture,F Tissue Culture Assn. Proc., Philadelphia, June, 1967, p, 15. • 10. Pace, D. M., and Elliott, A. Studies on the effect of acetaldehyde on R`"r tissue cells cultivated in vitro. Cancer Res,, 20:868, 1960. 11, -----------, Thompson, J. R., Aftonomos, B., and Holck, G, 0. Effects of N02 : and salts of NOZ on established cell lines. Canad, J. Biochem,"and Phys., 39:1247, 1961:- :,.., . 12, -----------, Aftonomos, B., Elliott, A., and Sommer, S. ObserQations on some effects of the sodium salts of certain monocarboxylic acids on established cell lines• Canad. J. Biochem., 45:81, 1967. 13. Rounds, D. E„ and Bils, R. F. Effects of air pollution on cells in culture. Arch. Environ. Health., 10:251, 1965. 14. Stevens, K. M. Lung cancer, an evolutionary approach. Austral. J. Exp. Biol, and Med. Sci,, 43,:421, 1965. 15, Wynder, E, L„ Hoffmann, D., and Auerbach, 0. The role of particulate and volatile components in tobacco carcinogenesis. Am. Assn. Cancer Res., 6:69, 1965. 1003547059

t . Details of Experimental Design and Procedures: (Attach Separate Pages) . It is planned to maintain the following lines of normal and mali•mant human•ies '" ~ ` . piratory epithelium as monolayers of, cells on glass in milk dilution bottles in mix_ ~"'tures of serum (human placental,•horse, and calf) in synthetic media, Parker's :'° Medium 199 or Eagle's minimal essential medium (r1Er1) under an atmosphere of 5% COI~3 i air. :.-. ..a. Line KB, human epidermoid carcinoma of the pharynx ' Line Fd.Ep.42 human epidermoid carcinoma of the larynx carried in our ]aborato- , , i132l h fl l ne L-, normaumanetaung epithelium ,:.We also intend to set up•other lines of freshly explanted normal and malignant "` of Dr. Sheldon C. Sommers, Professor of Pathology at Delafield Hospital. 'These lines`'~ iuman epithelium from biopsy material which will be obtained through the cooperation ~ tissue as opposed to the older lines. will be used to compare the effect of the experimental conditions on freshly explanted' ExpEriments to determine the effect of tobacco smoke and some of its volatile : components Will be carried out as follows. At intervals•series of tubes with and without coverslips will be seeded with a known number of cells. After the cells have '-`s"`r -become confluent the medium will be renewed and the cultures will be gassed by means ` of an apparatus used routinely in our laboratory for adjusting the gaseous phase of the culture with mixtures of 0 and N02 for short periods. In other experi- CO N2 2, . 2, , 9:'i:~y3i2ai Facilties Available (Where Other than Administering Organization Indicate Geographical Location)• (eont, on p.'~2a large well-equipped tissue culture laboratory with glass enclosed sterile room, incubators, refrigerators, deep-freezes, large and small centrifuges, Mettler micro- analytical balances, torsion balances, Leitz Ortholux research microscope with equip- `:1-V~44 ~ ment for bright light and phase contrast microsco py, Zeiss inverted researeh- microscope with, some light and phase contrast equipment, Leitz binocular microscope with Leitz : •th ',, ... • 10I'i~2l~iRiaUi~qSid~6T#t3~ent for photography with lightmeter, camera back, and other equipment f ~.,~ for microphotography and microscopy, sterilizers, stills, special glassware and equip.. „r~;v ment for Carrel flask, Ylaximow slide, Leighton tube, and bottle culture, equipment for histolo ical and histochemi a2 studies g , „• c electrophorator. Dr. Mandl is Asst. Professor of Biochemistry in Obstetrics & Gynecology, phoretic equipment, Spinco I2 ultracentrifuge with swinging bucket, and Elphor FF separation, including cold room, deep freezes, refrigerators, freezer-dryers, analytical balances, homogenizers, two high speed ultracentrifuges, chromatographic and electro- Mandl who has several rooms in Delafield Hospital equipped for biochemical analysis an The chemical investigation will be carried out in collaboration with Dr. Ines r •11. Biographical sketches of ali principal and professional personnel (append) See attached curriculum vitae. ~ 12. List of publications: (Five most recent as pertinent) (append) See attached continuation sheet 2b.

continuation sheet 2a `:. ments the salts, NaSCN; and NaN02, will be added directly to the cult~ure medium or to balanced salt solution in serial dilut2on and allowed to remain in cont t a . c with the cells for.short periods of time. Ci.iltures of freshly explanted normal ';:'~ hu:nan respiratory epithelium will be exposed directly to diluted fresh tobacco ,~~~ •~ ~ smoke by the same apparatus. .,. . .. .... ' . ~ F_S'11y/•I. ia'4 . .. .. , . . . . . , _ .. ~ i• . .. ... , .. .,S.r:.`.~: ~ ~. ~=H.i••~",i iy-.••The effect of these substances on the cells will be determined by the followin 1. Growth rate studies. Effect on rate of growth will be evaluated by dye' exclh bld usion cell counts wit trypanue, an mitotic c OuntS. 2. Mor hp ological Studies. Studies of differences in cell morphology and irC'ty ;number and c aracter of chromosomes will be made as well as histochemical prepara-~`' ..tions to demonstrate different enzyme content. Parallel cultures on coverslips in : Leighton tubes will be fixed and stained w3,th°H. and E., Giemsa, Feulgen, Methyl r Green and Pyronin•Y to observe effect on mitosis and nucleic acid content. Enzyme'coit :tent 'of the cells 'will be observed from preparations in which=the cells have been " reaeted; for sites of alkaline phosphatases, beta_glucuronidase, succinic dehydro-• 4' genase, and leucyl beta-napthylamidase activity. For chromosome studies, cultures will be treated with colchicine to halt mitosis in the metaphase, fixed, spread, stained with acetic-orcein and observed under the oil immersion objective of the :~ microscope and photographed. r 3. Biochemical Studies. The cells and culture medium will be analyzed to evaluate the differences in the metabolic by-products of the eell lines, both treated and-untreated, • a. The effect on cellular respiration and g1YcolYsis will be observed usin g standard l larbur earance and laetic acid ' techni ues In lucose disa articular ! p pp tr.y; r g q g production in the used culture medium will be estimated by the methods of Trevelyan _,~j and Harrison (Biochem. J., 50:299, 1952) and Hullin and Noble (Biochem. J., 55.289. 1953)• . .•.° ._ . . ... ~ •: . b. The cells will be fractionated in a Spinco IZ ultracentrifuge and the mitochondrial fraction. extracted and studied, in the Warburg apparatus by the method of Umbreit (Manometric Techniques and Tissue Metabolism, Chap. I) and the --ratios of oxygen uptake to phosphorus utilization will be estimated and compared. ~~ c.Differences in protein patterns of the metabolic products of the cells will be established by acrylamide gel electrophoresis. .• n ~ d. The enzymatic profiles of both cells and utilized culture medium will be .studied with special reference to proteolytic enzyme activity using chromagenic : substrates and modifications of the Bratton-I:arshall method (Goldberg, Pineda, and Rutenburg, Am. J. Clin. Path., 32:571, 1959; Blackiaood and riandl, Anal. Biochem6, 2: • 3go, 1961). 0 :,;

continuation sheet 2b A ~~ Supporting Data: i . , ~ • .. . , . ... • _ -- • ~ - . .. • .• ~ ' .. . ... , S • A. Previous work done in this or related fields. The principal investigator .'" ~.'.....has carrie on e enszve s u ies of t e e avior of normal and malignant cells in •,rabbit's ear chambers and in tissue cultures, In col.laborationn with H. S. Simms :.,,;j several hundred biological substances and organic and inorganic chemicals were . More recentlv Prowth stimulatine and erowth inhibitin~ materials have been extracted'; .tested for their effect on growth of normal epithelium and connective tissue cells,"-~ ;~Trom normal adult connective tissue and, tested for effect on growtn•ol numan tpmor :~ biopsies, Rous sarcoma, and many lines of normal and malignant human and animal ;~ materials were also tested against animal tumors in vivo, Sarcoma-180 in the mouse cells initiated in this laboratory and maintained in conrinuous cultivation. These:a and Flexner-Jobling carcinoma in the rat and against heterologously transplanted :t9? ~ human ovarian carcinoma in eonditioned rats. The inhibitory material reduced the on Sephadex-G200 and Biogel-300. At the present time biochemical analysis is in specific growth stimulating and growth inhibiting materials have been separated from crude extracts by ion-exchange DEAE-cellulose chrom3tography and•by gel filtration ,.. . in animals when transplanted heterologously into conditioned weanling rats. Active the same line of highly malignant ovarian carcinoma cells in tissue culture and also' effective against malignant epithelium in another. The latter factors inhibited :,0- for malignant fibroblasts in vitro and; in vivo in one case, and materials primarily ~ growth inhibitory properties. We have obtained substances with specific inhibition ".using different methods of fractionation we have separated materials with different showed that these factors inhibited the growthof animal tumors also. Recently by growth of malignant cells in tissue culture from- ?5-100~, Collateral studies progress to determine the exact chemical nature of these inhibitors. • B. Six most pertinent recent references. papers included, 1959. "Transplantation of Tfissuest', ed. by L. Peer. Williams & Wilkins & Co. Baltimore parshley, M. S. Tissue Culture of Adult Tissue. Chap. 17 in Vol. II of the Simms, H. S., and parshley, M. S. The effect of proteins and other substances on , ;,t the growth of adult tissue in vitro. Chap. 7 in "Protein and Amino Acid Nutrition't,:; ed, by A. Albanese. Academic Press. New York, 1959. parshley, M. S., and Mand1, I. Separation and study of a connective tissue consti- for Cancer Assoc Am cell strains m f t hibit t th th i it t i , . , ..-.rSV u or n v ro o ory e g,row o tuen n Res. Proc., 4:1:50, 1963. • parshley, M. S. Effect of inhibitors from•adult connective tissue on•growth of a series of human tumors in vitro. Cancer Research, 25:387, 1965. Parshley, M. S., and•Mandl, I. Inhibition of malignant cells in vitro by a compo- nent of normal adult connective tissue. Nature, 208':800, 1965. Parshley, Ni. S., Mandl, I., and I;anahan, J. Separation and in vitro study of ~ Q tumor cell inhibitors. Tissue Culture Assn, Proc., San Francisco, June, 1966. . W C!1 ~

R: REDACTED MATERIAL C " Reserirch Assistant 100 Diener Fringe Benefits (10) 50, Sub-Total d. Consumable Supplies (list by cotegories) _ Special gUssware and equipment for tissue culture ?ledia, chemica•ls, and stains Sub-Total C.. Other Expenses (itemize) $ 2,000 Travel to scientific meetings $ 500 Sub-Total D. Permanent Equipment (itemize) $ 500 Zeiss Phototaicroscope $ 5,820 Hark Scholander Respirometer ` 97$ E. Overhead (T59'o of lt+Si• C) Estimated Future Requirements: Salaries Consumable Supp{. OtherExpenses Permanent Equip. Overhead ' Totol C Year 2 {'~ Yeor3 ~ ~ .t 2,000 t 500 t 18)5 $14. 070- $ 2,000 $ 500 $ 1 ~4 ~$14 , 440 ItisunderstaodthattheapplIcantandinstitutionalofficen D(r.dw•otProj.ce / l ~ in applying for a grant have read and found acceptable Telephone. . the Council's "Statement of Policy Containing! Conditions Signature and •Terms Uhd'tr Which Project Grants Are Made.:' tu,Lreu Otltc.r oi rF.'. t~rGlmie~ ylCE•FRnS!DENT II'1 DHAKtit Telephone ~ OF MEDICAL AFFAIRS I 111111100=7 ~;

Currant n . j Olher Sourmof Ffnanctal Supporl PendinB ^ Ust flnancial support for mwreh from afl sourea, tncluding own institutlon, forthii and/or rdoNd raaarch projeeft, ' rille of Projec Study of Naturally Occurring Growth Inhibitors Growth Inhibition of Malignant Cells in vitro and in vivo - Effect of Vinca alkaloids on Human Neoplastic Cells in tissue culture Biology and Chemistry of Cultured Ovarian Cancer Ce'. ~lK aVSCOOi' Sourca National Cancer Institute

R: REDACTED MATERIAL CURRICUI,UM VITAE Mary Stearns Parshley >,,9-,College of physicians and Surgeons, Columbia University -.•s" . Assistant Professor of Anatonjy in Obstetrics and Gynecology 630 West 268th Street, New York, New York 10032 -' .:,~.:., ... . .. .. . . .. . - .Zfarried: Thomas Fredricson parshley, New York City, April 15, 1938 Child: Elisabeth, Stearns Parshley,.born June 5,--1947 University Scholar, University of Pennsylvania, 1935-1936. 1934_1938 (Wilder,. Nicolson, and Fine fellowships). University of Pennsylvania, Ph..D. 1938 (Anatorsy, Medical Sciences Travelling Fellow•from Smith College at the University of Penna University of Pennsylvania, M.A. 1935 (Anatomy, Medical Sciences Smith College, A.B.. with High Honors 1933 (Zoology) Bennett Fellow, University of Pennsylvania, 1936-1937. Professional Experience: 1933-1934. 2. Fellow in Anatomy, University of Pennsylvania, 1938-1939. Finney-Howell postdoctoral research Fellow from the Johns 1. Laboratory assistant, Willard Parker Hospital, New York City, ' vessels, tissue transplantation, atherosclerosis, cancer research. World War II: Office of Scientific Research and Development, 1942_1945. (Certificate, O.S.R.D., bronze plaque, Columbia University). Special Fields of'Interest: Microscopic Anatomy and Cell Physiology. The study of living cells and'tissues: by the Clark-Sandison rabbitIs ear chamber method, and-by a variety of tissue culture techniques. Wound healing, regeneratiomof connective tissue, skin and blood Summer: 1. Smith Scholar, Marine Biological Laboratory, Woods Hole, Mass. 1932- 2. Laboratory Assistant, St. John's Hospital, Brooklyn-, New York, 1934. Columbia University, 196 j- 100354'7065 =~~ 6. AssZstant Professor of Anatony in Obstetrics and Gynecology, 5. Lecturer in Biology, Hunter College Graduate School, 1957-1963.;.~ ..~ 1963-1965. Assistant Professor of Anatomy in Surgery, Columbia University,-- " 4. Research Associate in Surgersr, Columbia University, 1949-1 963. ;~3 Research Associate in pathology, Columbia University, 1945-1949...Y 3. Research Assistant in Pathology, Columbia University, 1940_1945.,-,, Hopkins University, 193$-I939• Societies and Committees: phi Beta Kappa, Sigma Xi, AAAS, Am. Assn. Anat., Physiol, Soc., Phila., 1issue,Culture Assn., Am. Heart Assn., Am. Assn. Study of Arteriosclerosis,Am. Soc. Cell Biol., An. Assn, Cancer Research. Corresponding See., Tissue Culture Assn., 1946-1952, Member Course Cozrmiittee and Registrar Tissue Culture Assn. Summer Course, 1945-1964.

PUBLICATIONS - MARY STEARNS PARSHIEY . Stearns, Mary L. the rabbit's ear. I. Am. J. Anat.Z6:° 133_176, 1940. Parshley, M.S., and Si.mms, H.S. tive,tissue in transparent chambers in-; ' the rabbit's ear. II. Am. d. Anat, 67: 55-97. 1940. and Parshley, M. S. Simms, H. S., Parshley, Nf. S., . . J. Geront. 2:3:205_216, 1947. Growth,and differentiation of con- ~ ~ nective tissue as observed micro-`~ scopically in the living rabbit.`Am.`~ J. Med. Sci: 198:144_145, 1939. Proc~ ;:Phys, Soc., Phila., 14:20_21, 1.938_1939 Growth-and differentiation of con-Y ..,,~ -nective tissue as observed microsco-.'- pically in the living rabbit. Anat, ~ .. Rec. 73:3. 1939 Suppi. 2, p.49 (abstract : .. , ' ... .:."s.r:~Y l1~ Studies on the development of connec-,~zl tive tissue in,transparent chambers iri': Studies on the development of connec-:'M. Conditions favoring the growth of adult skin epithelium in vitro. Anat. Ree. 94:3:486 1946 (abstract) , . '.h`~' y Studies on the fat depositing mechaniste' Proc. Am, Soc. Study Arteriosclerosis;A Am. Heart J. 35:860_861, 1947. Fat deposition in vitro caused by lip' and Pitt, R. B. . fanogens and opposed by antilipfanogen.' 8. Simms, H. S., Parshley, M. S., Pitt, R. B., and Fulton, J. B. Further studies on the fat depositing mechanism in vitro. Am. Heart J. 36: •_ 469, 1948 (abstract). (abstractl. YRhi , . , . . , ,. ., Simms, H. S. sue in vitro. Anat. Rec. 103_453, 1949 ~ 9. Fulton J Parshley B . M Growth of blood vessels from adult tis. ;k- ; S 11. Parshley, M,. S'., and Deterling, Cultivation in vitro of fresh and;frozen R. A., Jr., and Coleman, C. C.,Jr.segments of the abdominal aorta of the ': dog. Anat. Rec. 106:64, 1950, (abstract).- 10. Parshley, Nr. S.., and Simms, H, S. Cultivation of adult skin,epithelial cells, chicken, and human, in vitro. Am. J. Anat. 86:163-190, 1950.