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Council for Tobacco Research

Report of the Council for Tobacco Research-U.S.A., Inc. [St]

Date: 1982
Length: 60 pages
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L•:X'1'1(A COPY REPORT Of THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., Inc. 1982
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1, Organization and Policy The Council for Tobacco Rasearch-U.S.A., Inc. is tbe sponsoring aycucy of a proFaJn of research into questions of tobacco we and hcalth. It is tbe out- growth of ao ortanir..tioo formed early in 1954 by tobacco manufacturcn, jrowera and warehowemen. Research support has been mainly through a pru- yram of lraota-io-aid supplemented by contracu for rescarch with irutitutionm and laborawrie.. TLe Council does oot operate any researcb facility. The ScicntiCc Advisory Board to The Council meets regularly to cvaluaie applicationa for research aupport, judging tbcro solely on the basis of acicotillc mcrit aod rdevanos. The Council awards research grants to independent scientists who are as- sured complete scieatiflc frocdom in conducting their atudies. Grantces alone are reapoou" tor reportinj or publishint their flodinp in the acccptcd scicn- tibc raaoacr - throuslt modiul aqd acicntitk jouroal. and socictica. WlLLIAM D. Noaas Chairman ~ . ~ 198'l RRI'OR'1' p ~ F ~ o d i ~ C TiIE COUNCIL FOR TOBACCO RESEARCH-U.S.A., Inc. TIIE COUNCIL FOR TOItAC/'O IIESF.AIit'fl-U.S..4., Inc. 110 Eut 59th Street, New York, N.Y. 10022
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c Slal•:NTtI•'ll: ADVISUItY 1tl)AIiI) to Thc Council for Tobacco Rcacarch-U.S.A., Inc. as of December 31, 1992 LEON O. JACOBSON, M.D., Chuirutun lusrph Regcwlcirt Pru/c'ssur uJ lliulugic-ul Scirrn rs ( rinrritus ) I'ruJcssur of rlti• Departmcnt u/ Mrdiccinc (rrncritus) University of Chicago Chicago, Illinois RICf IARD J. BING, M.D. Dirrc•tur u/ fs'.rprrirnrnrul Cardiology ruuf St it•nti/ic /).•vrloq„nc'nl Huntington McJical Research Institutc, PasaJcna, California PruJrssur u/ Medicine (rrneriuo') University of Southern California School of Medicine Los Angcles, California ROSWELL K. BOUTWELL, 1'tt.D. Pru/rasor u/ Onculugy McArdlc Laboratory for Cancer Research Univcrsity of Wisconsin Madison, Wisconsin DRUMMOND 11. LIOWDL•N, M.D. l'rolrssur and Ilrad DcPartmcnt of Pathology Unrversity of Manitoba I Ieahh Sciences Center Winnipeg, Canada MICHAEL J. BRENNAN, M.D. PresiJent and Mulical Director Michigan Cancer Foundation Dctroit, Michigan JOSEPH D. FELDMAN, M.D. Mernber, Research lnstitute of Scripps Clinic Scripps Clinic and Research Foundation La JoUa, California WILLIAM U. GARDNER, Ptt.D. E. K. !lunt Pru/essar u/ .1 natuury ( cnrrritus ) Yale University School of Medicine New tlaven, Connecticut PL'rLR M. I LOWLEY, M.D. l.aboratury uf I'athalogy National Canccr lnstitutc Bethesda, Maryland 11ENRY 1'. I.YNCf f, M.D. l'ru/cssur and Chairman L)cpartment of Preventive Medicine and Public Health Crcightun Univcrsity School of Medicine Umaha, Nebraska G. BARRY I'IERCE, M.D. Aiurrirurt Cunrrr Scx4rty Crurrnniul Rr3rurc h NruJrssur llnivcr.ity of Colorado l{eallh Sclcncc\ Cenlcr 1)cnvcr, CuluraJu GORDON lf. SATO. Ptr.D. f'rujr.r.wr u/ hiulugy University of California, San Diego La Jolla, C'afifurni•a SIIL•I.tX)N C. SUMMIiRS Sric•utifir Uirrrlur, Thc C'uuncil fur Tobacco Rcscarch-U.S.A., Inc. ('Gni4 ul l'ru/rs,fur u/ l'ur/wlugy Collcgc of Physicians & Surgcuns of Colurnbia Univcrsity New York, New York ScieutiGc StrfC of '1'lrc• Council SHELDON C. SOMMERS, M.D. Scic'rui/ic Director ROBERT C. HCKKETT, Pu.D. Research Dirrrtur DONALD II. FORD, Ptt.D. VINCENT F. LISANTI, l).M.D. if ssuuutr Kc•srarrh Director Associutc Kcsc•un b Dirrc rur DAVID STONE, 1'tt.D. Associutr Krarun'/r Dircc lur
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t. CONTENTS Introduction . ... . . . . • • • • • 5 Abstracts of Rcports . • . . . • • • • • 7 Canccr-Rclatcd Studics . . - • • • • • • • • • • 7 The Rcspiratory System . . . . • • • • • • • • 23 Heart and Cireulation . • . . . • • • • • • • • 49 Ncuropharmacology and Physiology . . . . . - . . . 60 Pharmacology and Biochemistry . . . . . • • • . . 65 Immunology and Adaptivc Mcchanisms . . . • • • • 78 Epidcmiology . . . . . . . . . . . . . . . . 89 Activc I'rojccts . . . . . • . • • • • • • • • • • • 95 Cornplctcd Projccts . . . . . • • • . • • . • . . . 105 Index of Principal Investigators . . . . . . . . . • . . IlK Indcx of Scuiur Authors . . . ... . . . • • • • . . . 119 Introduction ® ~ H ~ ~ 'I he ('uuncil tor Tobacco Kescarch's program cxprndcd turthcr tTP1982 in rc.ponx tu a gruwing number of solid and ullcn yultc imgin.ruvc grant apphc:Itiun. 111.11 cuntinucd to comc Iruns indepcndent xnnusks. It xcnted clCar Junog the ycar Ihal evcn with the vasl amount ul rc-scarch done tur ncarty thfcc dccadr. nt/o yucslwns rclatcd to smoking .nd hc.rlth, many in the sci- cntr6c cumnnmuy Inhcvc impurlam gaps in knowlcddc r.mam to be lilkd. 'Ilxrctuic, rcll.cung whal appcars 1o be an in.rc.xd tnlcresl in smoking .md hcahh f.x:uch rmung scwnlhls, the Council suhslanually enlrrgcd its an- nu.t1 cumnuUninl. Ilic acltun :J.o rclkctrd the Councd's suptxrrl lor the pl.ns rnd Jc+ir.N ut ih SaanUtic Adviwty Board, which /s alw:ryN on the luokout lur yuwhh.d mvc.lig.tlurs :u vtutly •pectliC jrYas Ih.A are corlvidered irnpurunl. S-u.r 1'fl 1. the ( uuncJ ha% piuvr.lcd 17b.1MM11MM! La iLs Ics.jrch prugram, wlu.h I. IK•Iw•vcJ lu Iw the I.ug.•.1 .md mua ritlcn.rvc ul its ktnd in the wurld. Ifrlay, a..u Ih: oW.a. Ihr piul;r.urr cmph.f.ucv rrscjfih by independenl sei- culnl. In Ihnl; C.ItJN/v.1~/1IJr Ih.ct.c. arxl chrunic pulnwnary atbucrlK. I bc.r luu.l, wce.• lur M_'9 urnginal granh aoJ nurncruus renewals, smce iuany ol the piul:cl. w.fe Iur 1w.f rnd Ihtre yc:us ..r lun6rr. the ('uuncd h.r-, Ihiuugh the y:..n, Iun.l.•d 466 i..e.u:has in 272 nkJt.:rl schouls, husprlah jnd tr.c.urh m.utuuun.. A nu•.r+uic ot Ihc ('ouiw'd•s vuulnhuUUn Io the jdvan:cmerH uf s.iculilic Inlunn.iUon u.ten m the nun1hcr ul rcpurl% puhh.hed by gnntccs Ihal .c- kouwlcdgc ('uuikil supporl. Ihal ligurc was'_,168 w of 1?rccutbcr 11, I9M_'. the CouncJ r.m:nns Jcflicalcd to cuntinurncc of h% resc.rch ctlort.
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Abstracts of Reports Fulkrwing art ahatracts, approved by the authoo, of rcprxts rnr ncw rescarch ackrrowk;dging sulr{xxt from The Council that have appcucJ in xicnti(ic j.wrnals sincc publication of thc 19g1 Rcport. The name of the grant rccrprcnt is in rtalics. Thc abstracts arc grouped under these headings:t. Canccr-Rclatcd StuJics,11. llrc Rcspiratory Systcm, 111. Heut and Circulation. IV. Ncunrpharmaculugy and Physiulogy. V. Pharmacology and Bwrchcmiury, VI. ImmurKrkrgy and Adaptive Mechanisms, VII. Epidemiology. I. Cancer-Related Studies MECI(ANISM OF ACCION OF fiL•NZO/aIPYR(sNf•. AND NICOTINE ON HORMONE PKODUC"CION BY RAT PITUITARY TUMOR CELLS Akhough hrxTnoncs have bccn associated with induction and pwEres+run of tu- nrxs in many cxpcnmcnul systcros. the role of hurrnones in the process uf initiation and prugrcssiun uf carcinugcncsis is rNx clcarly dchned as yet. In thc present attcmpt to wxkrstand tlr- rncchamsm of action of bcnm(a)pyrcnc (fiaP), a cyclic anunauc hydakarbrm. anJ that of nrcwrnc, the tobacco alkaloid, the clfectu of thesc agents on Ixulactin (1'KI.) arrJ gruwth IKxnrrxrc (Gil) synthcsu by rat pquuary IuuNx cells in culture (GI I cclL ) were audicd. Treatment of Gil cclls with mcrxmc (U.1-XK) µl:/ml) ncuhcr affcctcJ the growth nr)r significantly altcred the gcnrral pattern uf humrunc pu><lucurxt in these cells. BaP at concentrations greater than Sµgnnl xrevcrsrbly inlxhitcd the gruwth uf these cclls. The sublethal cunccntrahuns of HaP. which did not al(cct either (I) cell growth, or (2) amino acid transfxwrt rx (3) aaal protein synthcsrs or Jcgradatirxt, did however inhibit spccihcally hurmunc synthc.is by these cells. More interestingly, axrcentrauons of nicotrrre, which did na affect either cell growth or Ixxnwnc syntlresrs, modulated both of thcse cellular prucc»es in thc prcscnce of HaP. A cunccntratHm dcpcndent sonwlauon uf micrownsal iSaP uwnaraygcnax activity was uhscrved in nicotine or BaP ucatcd cells. The cllccts of ::xsc subsunccs on stimulation uf IIaP mrxwuxygcnase activity scems to be additivc. Nicotine alvr cn- hanccd the asxkiauun uf raJioa.uvity (presumably /'H It3aP nkt,tb.ddcowith DNA m )'H/UaP trcatcJ cells. 1t is concludcd that niatxrnc by uself did skw Jcnwmstratc any cyt.wuxic cfl'cct nur inlluence hurrrwne synthesu in GH cclls. IJuwevcr. nlarNlnc atimulatcJ Bal' munrwaygcnax activity anJ the rrueracuun ul 1'H1BaP nktububtcs wrth ccllular UNA anJ alsu modulatcJ BaP irnlurcJ intubitrun u( MxrrwMk synth.su m Gil cells. CTakrabani, S., Hai>LS- S. D. and Bra»wi, l1. K. !!rw'hemrul und 8rupbi•sirul Rrararrh C'r,m.aunrruuuru IOK(.) S'M-(03, IV%:. Frum t)rc I-abxatury of Ph:unr.rculugy, H:warJ SrM.d of D.nt,rl Medicine anJ Dc - partnknt uf 1'harrnaculugy, FluvarJ MrJrcal SaNrul. Busam. 7
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POSITIVE C'ORRELATION BGTWEEN HIGII ARYL IIYI)R<KAR(iON HYDROXYLASE ACTIVITY AND PRIMARY LUNG CANCER AS ANALY'LF3) IN CRYOPRGSERVED LYMPHOCYTES Blood sampks from SI paticnts at the Veterans Aduunisoratiun 1lu.pital, I luur lon, were collected, cakd, and scm tu Microbiological Asx>tiatcs, Bcthcxla, MD. where the lymphocytes werc isolated and cryoprcxrvcd :n - I'JA°C bclure cxuoina- tion. AI the timc of assay, lymphocyte samples were simultaneously thawed, phytolrc- ongglutinin activated, and analyzed (or benz(a)anthracenc-induccd aryl hydrucarbun hydroxylase (AHH) kvels, ('H/thymidinc incor(wration, and reduced mcuunanridc adenirse dinuckotide-dependent cytuchrome b, (cytuchrume c) rcductasc activity. Uc- lennirrations werc made at both 96 and 12U hr in culture, and pcak activittcs wcrc compared among the SI individuals who cxprc»ed such lesions as syuatnous ccll carcirwmas (22`b), adcnocarcinomas (14%), oat ccll carcinomas (64u), chronic ub- srnactivc pulmonary disease (22'b), and other nonmalignant dueascs. O) the 14 high- est AHH/cytuchromc c activities obscrvcd, all were found in paticnu with pruuary lung cancer. Mean AHH/cytochrome c activities were U. M9 fur lung cancer patients (a Iot:J of 21) and 0.47 for noncancer patients (a lotal of 30). No relationship was obscrvcd belwcen AHH/cytochtomc c activity and age of paticnt, number of cibarcttes srrsuked, family history of cancer, location or histological type of twnur, or Icvcl of phytohernagglutinin blastogcnesis (I'HlthymWine cpm/cytuchrunk c). Although the present communication presents data which show a striking correlation between the prcscnce of pulnwrsary carcinomas and high AHII levels in lytnphucytcs tsulatcd (rum patienu, whether the higher AHH kvcls arc the cause or the result of the primary lung cancer still remains to be determined. Kouri. R.E. rr al. (Micrubiologicrrl Associurrs) Concrr Rrsrwcb 42(12):5030-5037, 1982. O[Aei support: American Cancer Society and the Vctcratu Adnunisltatiun Hu.pttal, 1 iuusuia. From the Dtvisiun of Toxieulogy and Orrculugy, Mtcrubiulugical Asx,ciatcs, Ile- thesda, MD, Departmeru of Biological Sciences, Nonh Tcxas State Univcnity, Den- tun. and the Department of Motlicine, Baylor College of Medtcine, and Veterans Hospiul, Houston. ARYL HYDROCARBON INDUCIBILITY IS NOT ALTERED IN BLADDER CANCER PATIENCS OR THEIR PROGENY The ruk of aryl hydrocarbon hydruxylasc (AHH) urJucrbthty in prcdi.pt.inK perwrrs to cancet has been the subject of considerabk cuntruvcny, with suntc reptms shuwing an increased risk of respiratury cancer in perwns with high AHI I irrducibtltty and others showing no such effect. In the present sttrdy, the pu»ible m0ucrke of AHl I on susceptibility to bladder cancer in humans was carefully investtgatcd. AHH nniuct- bility was mcasured in the cultured lymphcuytes of 16 patients who were tktng lul- luwed a(ter successful treatment for blackkr cancer, in 53 progeny of bladder cancer p.t,cnl., atxl in nutdkd cuntrul.. In both thc progcny and paucnt pupulawm., nu evidence was Irwtkl for a diffcrerxe between the dutnbuuun of AHiI tnduitMltty or induced AHiI activtty ctnnpared to the diuribuhon atnong corurul tndtvtduah. Thus. AHI I aruvity or inducibilhy dtd rwl appear to bc a rnajor dctcrmnant of bladder cancer risk in humans. 1'aigcn, B. rr u(. (Puigrn. A. J lnrrrnuriunul Juurnuf ojCuncrr 23:312-315, 1979. A Urbrr support: National Cancer Institute Frum the Dcpartmcnt of Molecular Biology. Ruswell Park Mcnwnal Institulc. ~ Bulfaks. MONOOXYGENASE AND EPOXIDE HYDROLASE REGULATION IN PRIMARY FtiTAL RAT LIVER CELL CULTURE In this rcpun, various lines of evidence arc presented dcrn,mstratmg Ihat a cyto- chrunx: P450 comparahlc to that of the adult rat liver can be found in /etal hcpauicyte culturc. and that nm appeuancc is strictly controlled by nuticords. The liM.vidcncc cited hcre slwws that the level o( the cyn>Lhromc P450 content in primary Ictal liver cell culturc is rcourkably atablc and does n.N sigmAcantly dtllcr Irutu that hwnJ in Ict.rl hvcr uscd fut.•ulturc prcparattun. Aryl hydnrcarb.m hydruxyl.r.c IA1111) arMl epustdc hydrulasc (El l) Ji uvuUC. arc casrly nx~surabk 10 thc.clls and Icnd tu Jc.rcax sltghtly as a IuwK'ttun of tlK culture duration. High conccntrauutr of phcntbarbual (Ptl l urduces both AI111 and 1i11 acttvitics, whereas hcnt (a)unhraccne t1fAI xts prclcrenn.lly and 2.3,7,1i•tctrachlunxldx ntu-p-dwxin selectively on A)1H acttvtty. Tnns-stiltwne ux- kk atwl ethoxyywn behave as selective induccrs of EH. Other evtdcn.e shuws that AIIFI and EII activities in fetal liver cells uc mudthed in a parallel manner by the aWniun uf dcxanwthasunc to the culture medium. This eflect is biphasic as tl>< eruyme acttvtttcs arc lirst inhibited and then induced when the contcotd axnentrauun is prugrcsswcly raised. The cunirutd also rrxxlities the AHII activity .xt a yualitaNve ba.is. In the absence uf the conicuid, the enzyme activity is inhibited rn r•uru by u• napluMrllavonc but nnt by mctyrapnnc. Dexameth:.xme alsu nKKlthes thc induction of AH) I by PB and BA. both on a qualitative and yuarrutative basu. Ovtrall, un the basts ul' Ihis and other evirknce, it xctns yuuc reaxmabk to assumc that prtnary fetal r:u liver cells tn.ulture might cunstitutc un interesting uwxkl lur studying the physruNppr cal regulatory rncchanunu of drug nxtabulizing enzymes. Girlta. J. t.'% rr ul. In: Snyder rr u( (cd.. ): Htulugrrul Rrurrivr lnrnmrdturrs • ll. Purr A, N. w Yurk: 1'knunt Publuhtng Corp.. 1982. pp. g7-97. Other support: Frnx(s National dc la Rcchcrche Screntrliyue. Protn the Lal>,or.uuuc rk Clwntc Mrdtcalc et Jc Tuxtca,lugte, Inuitut .k P.nMdogn, Uutvtt.dc dk Lt.ge. l.t.tigc. kla'll;tuut. It)
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1 t• MULTIPLICITY OF CYTOCHROME P-450 IN PRIMARY FLTAL HEPATOCY7FS IN CULTURE TM pcrirutal period of life is a critical tiasc for thc quanntauvc and qualitauvc dcvebprncnt of micsusurnal morKwaygcrusscs. and earber ubscrvatiuns suggcst th.d fetal hcpatocytcs in culture might constitute an idcal tuul lur studying the pcnn.tal regufatary mechanism of mooooxygenascs. While it has becn known for a while that pnmary fcul rat hcpatocytcs in culture display diffcrent nwrKxrsygcnasc activiuo which can be induced by several chemical inducers, thcse hcpatucytcs wcrc bclievcd uotil now to produce only one singk cytochrorne P-450 spccies, nanicly the cytu- chrorrre P,rIS0(orP-448). Howevu, it now scems possibic to induce other cynxhrume P-450 spccia in thesc hepatocytes, providing that they receive an appropriate hormu- nal trutmcru. ln the worlt rcportod bers, cxamination was made of the effect of duameQtasooc on various morrooaygenases and on the type of cytochromc P-450 wppatting these enzymic activities. Thrce enzymcs, aryl hydnx:arbun hydruxylasc, ettwaycoumaria doetbylasc and aldnn monooaygcnase, wcre measured for this pur- pose. Results of this study show that the prcsence of dcaamcthasunc in the culturc medium pttoducts qualilative and quantitative changcs in the ownuusygenase-supI^xt- wg cytochrorsre(s) Pr150. For low deaameUusone concentrations, a cytochrumc P-450 is formed displaying biochemical and biophysical propenics simdar to thosc induccd by phenobarbrtat in the adult rat liver. At higher concentratiuns, similar qualitative changes arc observed; but a quantituive phenomenon occurs, the (cynuhrome P-450)- dcpcodeat uszymic activities bciag also induced. Dcxanscthasone also has a syncrgis- tic effoct in the induction of cnzymic activity by the mixturc of p(scnobarbital plus bcrwnthraccae. Tbe various biochemical changes induced by dexamcthawnc in the fetal ccll cultures parallel those observed in vivu during the pennatal period of life. Thcrefore, this ccll culture system may constitute an intcresting model fur studying the onwgcnic development of liver rrwrsooxygcnascs. Krcrsxrs, P.. Goujoa, F., Dc Gracvc, J., Van Cantfon, J. and Gir)rn. J. E. Eruopcan Jo+unal ufBioclremisrry 116:67-72, 1981. OiA.r sswrwt: Foods rk Is Rahen:lte Scicntifiquc Mtrlicalc. From the L..bontoirc ck Chimie Mtdica)e, lnstitut de Pat(wlugie. Untvcrsnt dc Lttgc. LJtgc, Belgium. DNA METIiYLAT1ON IN NORMAL AND SV40-TRANSFORML-D IIUMAN FIBROBLASTS The 5-methykytosine base contcnt of DNA in four numal and (rwr SV4U-(rrnS furmed human diploid fibroblast cullurts was measured by high pcrturnrancc liquid chromatography (HPLC). Rcsults show that the percent uf cytusmes methylatcd fur thc four nonrral ceU lines ranged from 2.83 to 3.18, whrlc thc rangc lur the tuur SV4t1• transforracd cells was from 2.90 to 3.03. The mean (ur thc nNal nuuobcr uf HPLC detcrminations was 2.94 ± 0.28(51 dctcrminatwns) fur thc nurm.l cvll ty(x.:nx13 t10 ± 0.28 (53 dcternuna[ions) for the transformed lines. Thus, in axwa..t to whrr 12 rc{wutu) studics .uatparing normal and oncogcnically transf.uarcd cclls, no appareni dtl lcrcncc was ot»crved in thc 5-rnethyky(osinc to cytusinc base ratius in the two ccll types. It is worth cmplusrzrng that the IIPI.C method used here gives an absolute measurc of the 1)NA bases. In addinun, the purity of the DNA is controlled by urrritunng for thc prescnce uf uracil. Other methods using radioactive label may be hinckrcd by. variuus dtflcrcnt artifacts. Diala, E. S.. Plcnt, M. M., Coalsoa, D. W., and IIu,Q'man, R. M. Biuc)rcmiraf and Biophysical Rrsrarch Carunwsicarions 102(4):1379-1384, 1981. Otbar support: National /nstitutes of Hca)th, The United Cancer Courkil, Inc., TTe Cancer Research Coordinating Committee of the University of California, the Aca- dcmic Senate, University of California, San Diego, and the Leukemia Soctety of Anscrrca. From the Deparunent of Pediatrics, Univcrsity of California at San Diego Schoul uf Mcdicinc, La Jolla. CONSTITUTIVE BEHAVIOR OF METHIONYL-tRNA SYNTHETASE COMPARED TO REPRGSSIBLE BEHAVIOR OF METHIONINE ADENOSYLTRANSFI:RASE IN MAMMAUAN CELLS Methioninc, because of its tules in protein synthesis and in mcthylation, is of central unpurtancc to all cells. In the metabolic prucess, mcthtunmc can be used by the cell through twu dtrfcrcm pathways: (I) nscthioninc can be cunverted to S-aJcrw- syhncthtuninc, the ntajur medtyl source fur cellular uansmcthylation rcacuons ard the source of the prrKrylarninc group for pulyaminc biosynthesa, or (2) it can be convened to nscthiunyl-tRNA, an important inecrmcdiate in protein biusynthesis. In thc paper prcscnted hcrc, it is rcpurted that rnethionyl-tRNA synthccase, unlike rnethioninc ademrsyltransferasc, bchaves in a constitutive manner with respect to the concentratron of methioninc in the culture medium. This behavior is seen in Chinc.c hamster uvary cells and in normal diploid and SV4U-tsansformed hunun 6btublasts. Alahough the kinetics of regulatrun of tncthioninc acknosyltranskrase and rncthiunyl-tRNA synthe- tA.e by cxugenuus rncthiurtinc arc ckatly different, the levels of the two cazymes in the human ccll lines arc similar. Rubnitz, J. E., Jacobscn, S. 1. and Huffman. R. At. Birr htmica tr Bwphystra Acra 677:269-273, 1981. Other sapport: Nauonal Insntuics of Nealth. The United Cancer Cuun.'tl, bk , 71k C'anccr Research Courdinaing Committee of the University ul' California. the A.a- dcmic Senate, Univenrty of Califurnu, San Diego, and the Leukemia S.wrcty of Amcrx a. From the Dcprnn><nt of Pcdratrres. University of CalJornu at San Diego SchcKtl of Muhctnc, La Julla. 13
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FOLATE POLYGLUTAMATE AND MON(X;L(1-fAMA'1'I: AC'C UM(1LA'fIC)N IN NORMAL AND SV4(1TKANSFOKMfiU IIUMAN I7BKO1)LA51:S In the present attcmpt to asccrtain thc role of (utatc palyy.luumalcs in cell divi- siun, it sccmul necessary fint w starvc the cclls of tulatcs uuJ cstnn.nc their total (ulatc requirements for growth. AII four ccll lincs aurdtcd hcrc, the nunnal huu,rn dtpluirl foncskin hbrrsblasl BA, the normal human fctal dipluid AF2. and the SV40-uan.- formod lines P5 and PI, showed similar kinctics lur fulatc starvattun. Scpha.ka O-IQ gel filtratwn chrumatogntphy was used to measure the k-cumulauun of h,latc 1>.dyglu- tunate arKf rranoglutamat.e in all of these cell lincs. After the cells had bcen dcpletcd uf folaru, they were provickd with lirruting anwunts of I'Hl-(uhc acid in order that tlk cells wouW accumulue only forms uf fulate necessary litr pruhfcrauun. Buth the nurmal aod the transformed cells accumulatcd nKmugluumatc and ptolygluwauutc ftxrrrs, but by 72 hours of labeling. the ttansfurn><d cclb cuntairkd 3-Il) unks murc polyglutanutc then the normal cells. Thc growth rates for the tMtnnal atxl lranslunncd ccl(s were similar at this limiting folic acid conccmrauun. Thus, il' (olatc pulygluta- nrates are more important for the prolifcrauun of SV4/1-uanstomxd cclls than thc nrrrnal cells, the inhibition of polyglutarnatc formation could possibly be an tmpurtanl potential target for chemotherapy. llujjnrun, R. M. er ul. Journal of Cellular Physiolugy 109:497-505. 19111. Otltar srpporr: National Institutes of Health, The United Cancei ('riurw•,I, hk'., TIh: Cancer Research Caxdinating Comnuttee of the Univenuy of C'aldorrna, the Aca- dcmic Scnate, University of California. San Diego. arxl the Lcukcmia Society of America. From the f1<partment of Pediatrics, Univcnity of Cahlumu at San Docgo Schuul of Medicine, La Ju(la, and the Genetics Unrt, Children's Scrvtce. Massarhuscas Gcncral fiospital, Department of Pediatrics and Center for Hunun Getx:tic., 1(arvard Medical School. Boston. DNA METHYLATION LEVELS IN NORMAL ANI) CHEMICALLY-TRANSFORMED MOUSE 3T3 CELLS -iltis investigation was undertakcn to assess the effcct uf chcmtcal trnn.funnanun on wul gcrwmic DNA rnethylation as mcasured by high Ixrlunnarxc liquid cluumr- wgr'aphy (HPLC). In the study presented hcrc, nunual nmnrx: cmbryu 3T3 cell cultures and prose oacogcnically transformed by the chemical c:rrcinugcns IxniiNa)pyrcrk aud rncthykholanthrene were analyzed by HPLC tu rktcmmrc the S-nkthycytn.uk to cytosine base ratios in their total gcnumic DNA. Rcsults showed that the nKan hu 10 HPLC dcterminations uf the normal 3T3 cells was 2.1/TJ6 of cytusmes nuahylatcd with a standard dcviation of s0.S8, while thal for the bcntt(a)pyrcnc-tran.funucd 31:3 cells and the methykhufanthrene-transformed 3T3 cells was 2.It11'i• s U. 31 113 rktcrmt- nationsl and 2.81'b 20.18 (IS determinatruns), respecUvcly. These rc.ults led tu thc conclusirm that there is no real diffcrcnce in the catcnt of wtal gcnonu. UNA rnw•ohyla- twn between normal and chcmically-uansformcd 3T3 cells whcn mcawrcd by I IPL('. 1)iala, (:. S. and lluf/nwn, R. M. C11z lLiwhrtnicul und llmphysirul Rr.tnurh Cummunirutrun.t 1(ll(4/:(4K9-14'N, 1482. ~ Other support: National Insuwtcs of Ucalth, The Unitcd Cancer Cuuncil, Inc.. lbe T„I (']nlCr Kcxuch C00ndInating Cumnuttcc of the Univcnny u( Cahhnnta. the Aca- rkm,c Scnatc. University of California. San Dicgu, and the Lcukcmia Society o( Anknca. Prum tlw lhpartn>Lnt uf Ncdiatrics. University of Calilurma at San Diego School of Mcdkutc, La Jolla. M6f111UNINE DEPENDENCE IN CANCER CELLS-A REVIEW Mcthir>,unc dcpcrukncc uccun tn a largc numher and wide variety u( canccr cells and dr>LS not xcm w be a random cumpuncnt of the transfurrncd phenotype. Dchnruun ol' methiuninc rkpcr>d<.nce states that it is a defect found in many cancer ccll hncs that inhibits their growth in culture when methionine is replxed by its imrncdiate precur- sur, humr><ystcine, in the culture medium. Normal cultured cell. do nut have thts dcfcrt. Tlris reprxt lists the diverse and large number of anunal and human cancer lines that arc mcthiuninc-dcperwknt, and critically reviews the ccll biology and mclhiomne birxhcnusuy of the phenomenon. 11u,0'mun, R. M. In Vitru 18(5):421-428, 1982. Olher support: National Inatitutcs of Hcalth, The United Cancer Council. Inc.. The Canrer Keacarch Cr>,ndtnaung Crxtunutce of the Univcrsity af C.rltkxnu, the Aca- demic Senate. Univcnity of Caldrunia, San Diego. anrl the (.cukemia Society of America. prruo Uw lkpanuwm of ('cdrautcs. Unrvcrsuy of Cahlurma at San Diego S:huul of Mcd,ctna, la Jolla. IIYI'OMETIIYI.ATION OF 11i:LA CELL DNA AND THE ABSENCF. UF 5 MI~fHYL(.'YTOSINE IN SV4U AND ADENOVIRUS (TYPE 2) DNA: ANALYSIS t)Y 111'LC In thc study presented hcrc, nxthylatiun of the punhcd viri.nt 1)NA uf b.,th SV4U arKl .nlcnrrvirus (typc 2) wa. n>,'asurcd by high pcA,unt.rncc liquid c1u,MU.rtuKr.rphy (IIPI,(') :url crnulwcd to tlkrr Mx.ts. Afrk:rn gr.rn ru,wtl.ry kidney ccllk and IkL-A cclls, rca>lcuvcly. In SV4O DNA, as nwch as 12 na,MHmrks ul cytusmc has brcn n,.a.urr:d without crNrcumit.tnt rkt.ctn,n of mCyt. $V40 iuruarns 27 C'p(i parnm which ra tlk u.u.rl m.thylabuu sttc. II all Cp(i (t.rr% were nrcthylitcd. tho wuuW ytcW a srgn,bcanr 1.3% rr>Lthylatron uf tutal cytustncs; Mrwcvcr, ut the vtnun DNA studt.d here, nuru sccnKd tu bc n><thylatcd. Alsu, as with SV4U, m'C yt was mM present in the 15 14
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ekµioa patscrn of virion adcnovinu (type 2) DNA basea. Ovcrall, essentially no 5- methykytosine was ducctod in cithcr viral DNA. lmplicatiuns (ur viral gcnc rcgula- lion by metAylatioa are discusscd herc. In comparison with normal human ccll DNA mct(rylatioa kvels, liel.a ccll DNA mcthyluion is rc.luccd stgntficantly. Diala, E. S. and !/o,Q'man. R. M. Biochemical and Biophysical Rcsearch Communicutions 107(l):19-26, 1982. Olkar sstipport: Natioaal IrWitutu of Health. From the Dcpuvncat of Pediatrics, Univcrsity of California at San Dicgu School of Medicine. La Jolla. REDUCED AVAILABIUTY OF ENDOGENOUSLY SYNTIif:Sl"LI:D METHIONINE FOR S-ADENOSYIvfEiH1ONINE FORMATION IN METHIONINE-DEPF1dDFNT CANCER CELLS Mcthioninc (Mcl) dcpendcncc-i.a, the inability of culturcd cells to grow when Met is replaccd by its irrunodiate precursor hornocystcme (Met- Hcy' mca(wm)-is a frequent component of the oncogcnically transformcd phcnotype. Normal cclls, on the other hand, grpw in this rnodium. Thcnc have been reports 1Hof(man, R. M. & Erbc. R. W. (1976) Proc. Natl. Acad. Sci. USA 73, 1523-1527; Hoffman. R. M., Jacobsen, S. J. dc Erbc, R. W. (1978)Biochcm. Biophys. Res. Commun. 82, 225-2341 of normal or Aighcs rates of Met biosynthcsis in Met-dcpcrrdem cells and a postulation that Mct- dcpcndent cclls are dcficicnt in utilization of cadogenously synthcsrud Met as opposed to cxogerwusly supplied Met. To rutswcr the critical question of what biochcmical rcauion(s) requires prcfornrod Met in Mcl-depcndcnt cells, we labclcd cells with Mct- troe IS1 i{cy or ('S) Met and dctcnnincd the kvels of Met. S-sdenosyhoetluunine (AdoMU), and S-sdenosyRromocysteine (AdoHcy). We report hcre cxpcrinrents that dcasonsocare that Met-dcpcrsdcal cclls synthesizc a rxxnul arrxwnt of cndugcmwaly syatDcsizod Met and are deficient in utilizing this Met for AduMu synthesis. In cootraat, exogcrsously suppliod Met is utilized normally for AduMct brusynthcsrs. The ratio of AdoMet to AdoHcy is low in Mcldcpcndent cclls growing in Mct'tlcy' nrodium. Wcducrminod that the low AdoMct/AdoHcy ratro probably lirnita growth of Met-0epceduu cells in Mct-Hcy• medium. Coa)son, D. W., Mccham, J. 0.. Stern, P. H., and IloQman, R. M. Proceedings of the National Acadenry of Sciences of the Unired Srutes of America 79:4248-4251, 1982. O(Jssr srryport: Natioasl Institutes of Hca)th, The Univerauy Cancer Cuunctl, Inc.. The Cancer Research Coordinating Cornmrttcc of the Univcnity of California, the Acadcmic Senate. UNvctsrty of CaJtfornrs at San DKgo. and the Lcukemra Society of America. From the Departnsent of Pcdiatncs. University of California uat San Diego Schr«d of Modiciac, La Jolla. VASCULAR INVASION OF CARTILAGE: CORRELATION OF MOKPHOLOGY WITH LYSOZYME, GLYCOSAMINOGLY('ANS, PKOTEASE, AND PROTEASE-INHllI1TORY ACTIVITY DURING IiNDOCIIONURAL BONE DEVELOPMENT Although it is well known that cartilage vasculanzation is a prercquuitc fur txxte dtllcrcntiation, tlrc prcasc mechanisms of this vascularizatwn arc incompletely wxlcr- strxrJ. In the ptcscnt attcmpt to study these changes. dcmincralrzcd bone matrix prc- parul front rat diaphyacs was trsnsplanted subcutaneously into bilateral sites in the thoracic region of 28- to 35-day old malc Long-Evam strain rats; the day of transplan- uuon was dcsignateJ as Day 0. While using the rnatrix-induced endrkhundnl bone ddfcrenuaticrn as a rnoJel systcm, changes in the kvcls of lysozymc, patterns of glycosarnirurglycans, and actrvitics of protcascs and protcase inhibitors were studted during matrix-induced carrilage, bone, and buttc marrow development The morpbo- hrgtcal transitions were corrclatcul with the biochemical parameters. Rcsults showed that thcre was a pcak in lywzynse content on Day 3. during mcsenchymal cell prolifcr- auun, (ulluwed by a decline during cndochorx)rsl bone formation. The lysozymc levels increased again and attained masimal values during hcmatopoicsis on Day 21. Pro- tcasc-inhibitory activity was maximal during chorxLogencsis and drnunishcd during ostcogcncsis. Prutcase activity was maximal on Day 3 during mescnchymal ccll prolrf- «auon and was apparcntly prcscnt as an enzynre-inhibitor complex. Vasculanzatron and bone formation were accompanied by an increase in protcase activity. Chon- druitin-4-sulfate was the prcdonunant glycosaminoglycan detected in the nutrix-in- duccd cartilagc and bone. Reddi, A. 11. and Kueuner, K. E. Developmentul Biology 82:217-223, 1981. Other supporr: National Institutes of Hcalth. From the Laboratory of Biological Structure, National Institute of fkntal Rcsearch, National Institutes of Health. Bcthexla, MD., and the Depanmcnts of Onhopedic Surgery and Ihochetnrstry, Rush-Presbyterian-St. Luhc's Medreal Ccntcr, Chicago. RESISTANCE OF CARTILAGG TO INVASION In this cxtensrve and cucfully workcd-out chaptcr, a simplified conccpt of the invaaivc procc» is intruduccd, and a unique sequence of cvcnts occurring during physiologic (emkxhelial cells) and paehologic (ostcosarconu cclls) invasion is dcscnbuf. The term invasion, as used here, is dchncd as the pa»rng, rntcrpencuatrun, of infiltration of cells into ad)acent tissues. The events that characterize this prrrccss have been studicd in vuro in a combincd organ-ccll culture syucm consisting of marnalian hyalinc cartilage and TE-85 osteosarcoma cells. Cartilage was ab-e to reaist the invasion by uateusarconu cclls in the organ-cell culture system. Ostarsaramu cells forced to grow in direct contact with the cut surfaces of articular hyalrnc cartilage pikd up to sevcral cell layers, but were unable to penetrate the canilagmous nutnx. In vivo. similar reatrictiona of cantlagc to tumot cell invasion havc been rc(xxtcd in ptinury and n><tastalic tumors. Also, cartilage is thought to be resprmatbk for the slow invasion of carcrnuma cells into the walls of the larynx. Spccihc studies reprwred here shuw that hyilrnc canilagc, mddly cxuarted with vatiuus salt auluriuna, is readily ieGltrated by uateuaarcunu cells in the urgan-eell culture system. These lmdmgs kd to 16 17
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th.: conclusion thar canilagc cuntatns caraelahlc matrrs .anqxrunJs that inlnhn inv.r- rc sNm in an e apenmental system. W frcn these Ji((usahlc and c s tra.-tahtc subslarrccs wC further studicd. it was found that the inhibition ul ostcrrsarconra cell pruhfcnuon was caused by nwkcuks with a molecular weight of less than 50.1(X) Jaltotu. From thu anti-invuive factux of the canilage extract, a prutcasc inluhitur was irknNlic.l that has tbe ability tu inhibit nuntalian collagcnast, InCludlnl7 that CIaMnBICJ by UNtCoAarlUnra cells And errdWlwlial cclls. These espcrmwntal data led ar tlrc hylartlresrs that rnv.rsrun of citlx:r wrnrx tx endothchal cells depcnds on pruteulyu.• (<nll.rgenulyuc) cniyme xtivities. Other studies along these hrres havc Ixen in.ntutcJ tu lidluw the rnvasrvc- ncss and proliferation of bladder carkcr. Krurtnar, K. E. And Pauli, fl. U. In: Gilben. ff. A., Wciss, L. and Monscn, D. C G. /eJs•>: Rrrnr Alrtu.rru.rr.r, lsustom: G. K. Hall Medical Publishcrs, 1981, pp. 131-Ib5. Otbsr srrppat: National lnstitutes uf Health. From the [kpartrnents of Orthopedic Surgcry, Pathology and l)io<Ikmutry, Rush- Presbytcnan-St. Lure's Medical Centcr, Chicago. ANTIINVASION FACTOR MEDIATES AVASCULARITY 01: IIYALINE CARTILAGE To test an aruiinvasiun (actor (AIF) hyputlxxsis uf Q>t resutarwc u( canilag. to vascular rnvrsion, a novel in vitro system was surdicd that cmpluycd twvuu ua: ular canilage as a growth surface for normal hcparin-stimulatcd endrxlxbal cells. In (hrs study, cells were tested for their ability to invade the matns uf viable urd Jcvitalv.cJ extracted cartilage as monitored by thin-sectwn ckctron mtcruscupy. The growth bchavior of cells on rkvitalized extracted cartilage was cxamirred in the prcxnce anJ abserrce of cartilage-dcrived, extractabk AIF in the culture medrum. Whereas nor- mally viable anictrlu cartilage is a poor growth surface for crrdutluhal cdls, the eodothelial eells studied here, in contrast, grew as cuntact-inhrbitcd nwnulaycn u( flattcned cells on the surfaces of extracted cartilage. The cells were +cparatcJ Iruur the cartilage matrix by rrbundant basal lamina. lbcrc were a few rnicruvilli at the basrl plasma merrrbrane, but thcrc was no dcgradation or pcrutr.nun u( the cullagcnuus mauix uf extracted cartilage. Huwtver, when endutfxlral cells were sunrulatcd by heparin, they aswmtd a polyhedral shape and perktratcu thc extrxtcJ canilagc wu;r numerous mierovilli and some cytoplasmic processes. Thrs penetration ul tfic tulla- gcnous rrsasna was associated with tissue rarclaction and degradation ol collagen fibcrs. lnrportandy. howevcr, this invasion of hcparrn-stunulatcd a>,kdhclral cells wns abolishe.J when low concentrations of canilagc-deriveJ AIF werc added tu tik culrurc medium. These data provide evidence that the resrsurrKC uf hyahrrc cartilage to cn- duthclial cell invasion is regulated in pan by tissue JenvcJ prutcin+ac rnhrbuurs anJ an arstiproli(erative activity direeted against errduthehal cclls. Krrrrtrrtr. K.E. er d Scnrinurs in Arthritis 6 Rhtrurwpsrrr 11:67-69, 19Z<1. Ot/ur srroPort: National Institutes of lkalth. From Rush Medreal Culkgc. Chicago. 1>S CUAKACTIiKI-LATION OF ADULT BOVINE ARTICULAK CliONUROCYT(iS IN CUL'1-UKE Anrs:ular cartilage sliccs, obtaincJ from 1g-mrr-ulrl brrvrnc metacarprsphalangcal juints, were used as source nutenal (or this rkscnprive articlc ol eMrnduxytes in culture. A(tcr sequential digestion and hlu.tion, .clls wcrc plated in either nssue cuhure Jrshcs or ruller IxNtks. ChurrJrucytes fixed in bulfcrcd glutarakkhyde corrtain- ing 0.1% rullKnlum rcJ, were examined by light And transmr.sn>rr electron mkru- srupy. Cullagcn lype dclerrninalron of 7l-prohnc-labekd pnrtcins iwlated frunt culrures were IvrlurnkJ by clcctropMxctic and CN(3r pcpridc analyso. (Siu.ynnccsis of protcuglycrns was nrcasured by "JO, rncurporatrun into macromulccuks cxtrxtcJ under drssur:iauvc conditions. Eaaminatton showed that iwlatcJ chundrucytc. prior to culture were typically rounded with scanl territorial matrra, which could be removed by mild trypsiniiation. Throughtwt the progressiun of the cultures, phcnutypw alkra- buns were mN observed. Ekctrofluorugraphs of collagen tfut was exxracted Irrxn I f- prulhnc-labclcd cultures after mild pepsin digestion showed I band in the position of the aI chain; an a2 chain could rrot be detected. Cyanogcn bromide pcplidc analysrs confimxd that the majur radioactive peptides eornigrated with unlabeled peptides obtained from type 11 collagen- type I collagcn was nut dctectabk in these . ultures. Prutcoglycan aggrcgatc was extracted from both culture Jishcs And roller bottle cul- tures, under associative conditions. There were dif(ererrres noted in this cxpcnment suggestmg that the roller culture ccll-associatcJ matrix may hase a greater rkgree of txganization than that grown in standard tissue culture drshcs. Thcse samc data also indicate that articular chorwlrocytcs grown in mass rullcrcultures are capabk of synthc- sizing a plremrtypically stable, tiuuc-like matrix in viuo. Kwttrner. K. E. tr ul. Srmrnun in Anhrrus 6 Rhtumurisnr 11:101-103, 198 1. Odur supporr: National Institutes of Health. From Rush Mcdical College and the University of Illinur. Dental S:h.w1, Chrcagu. KL•GUI.ATIUN OF TUMOR INVASION BY CAKTILAGE-t)ERIVED ANTI-INVASIUN FACTOR IN VITRO Mamrnaltan candagc is highly resistant to invasion by tunwr irll. TTrr. reast- arkc was studied hcrc with the usc uf a novel in vurr.:ullure sysrcm. Anrculu:anilagc obtained Irum Iresh nrctacarlwrtralangcal jornts ul prcaJrrlcsarnt twvuks was uxda. a gnrwth surfacc lrrr huuran TE-g5 ustcusarcoma cclls and hrreskrn hbrubla.ts. ('artrlagc drsAs furmeJ t1k bullunn of stainkss-sleel cylrndera, providing cluscJ growth charn- hrrs for these cells. Hnlh invasive ostcosarcuma rclls And normal hbrubla.ts were unable tu pcnruatc vobl., uneatracted canilage during a 2-wecl cuhure pcrMrJ Wlno cartilage was JcvntalucJ by lrcctrng arxd thawrng, the trssue renr.rrncJ resutanl to invasion Cutrlagc, extractcJ with either I or 3 M gu.worJrnc hydnMhkrrrde, was invar)eJ by ost.usarcrtina cells. but nrM by control bbruMasu. Inva.arn by usteosar- cunra cclls rnlu salt catrx'tcJ cartdagc was ahuhslrcJ wlrrn low c.xr.•rnpatnrns u( a cartrlage-dknveJ, anu; rnvasaxr (actor were adrkd to the culture nreJrunr. These data 19 :'3

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