MSU LIBRARIES “- RETURNING MATERIALS: P1ace in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped be10w. EFFECTS OF H-RAS ONCOGENE EXPRESSION ON GAP JUNCTION-MEDIATED INTERCELLULAR COMMUNICATION IN MAMMALIAN CELLS BY Mohamed Hashem El-Fouly A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In Genetics College of Natural Science 1988 5 3') C9 99’4"] ABSTRACT EFFECTS OF H-RAS ONCOGENE EXPRESSION ON GAP JUNCTION HEDIATED INTERCELLULAR.COMMUNICATION IN MAMMALIAN CELLS BY Mohamed Hashem El-Fouly One form of intercellular camunication is mediated by the man- branedmannelskrnwnasthegapjmlcticns. 'Ihesechannelsallowthe exchange of ice, mtrients and regulatory molecules among coupled cellsmyimammm. 'Ihisphermnexmofjmmctimal conmmica- tion has been implicated in the regulation of tissue haneostasis, cell growth and differentiation, as well as synchrcx'xization of tissue func- tion and regeneration. Modulators of gap junctions include chanical tunnr pranoters, growth factors, ham, neurotransmitters and pro— ducts of certain oncogenes. Amongagrowingmmberofhmnornogenes,melbersoftherasa1- cogene family are fond expressed in a large mmber of naturally occurring human malignancies. Recent evidence indicates that the nor- maloroncogenicrasproduct, amatbranebanflprotein, mightbeanin- tegral outpatient of the imsitol phosphate transmmbrane signal trans- ductionsystem. 'Ihissystemwas foundtobeactivatedbythepotent timorprcmoter, TPA, ardthesrcmcogeneproductandcorrelateswith the inhibition of junctional cammication. In order to investigate a possible involvement of the ras product in the modulation of gap junctions, the Chinese hamster V79 cells were transfected with the human c—Ha-ras-l oncogene. The wild type Mohamed Hashem El-Fouly cells and those transfected with H-ras were examined for their ability tocammieatemyitm. ‘meeaqcaressimoftherasamcoproteinwas detectedbyindirectimmofluorescenceusingamonoclonal antibody agairst p21. “mo different techniques were utilized to measure gap junction-mediated intercellular ccmtunication. First, a newly devel- oped, rapid and sensitive scrape-loading/dye transfer assay which al- lows the mitoring of the diffusion of fluorescent dye, presumably acrosspatentgapjunctiors, incontiguous cellsjnyim. 'Ihesecond is the metabolic cooperation assay which measures the transfer of toxic metabolites among coupled cells in culture. 'IheresultsobtainedfrantestingH-rastransfectedardmn-trans- fected cells indicate significant correlaticm between the expression of the p21 protein and the blockage of gap junctional canmunication. The data implicate an important potential role of the ras oncogene in the imu'bition of intercellular coupling. 'Ihis functional similarity betweentheeffect ofrasexpressionandthat of certainunnorprano— tersmgapjmactimsuggestsamletherasaucogenemightplayinthe stage of tumor prmotion during the process of carcinogenesis. To my parents, To Ann for their love and support iv Itismypleasuretoackmwledgethemanypecplewhoassistedme, directlyarriirdirectly,cverthecourseofmygraduateworkinthe past few years. First, my gratitude goes to Dr. J.E. 'I'rosko, my mentor, who offered me valuable advice, guidance and a dame to accarplish my goals and ambitions. His role model as a dedicated scientisthasmadeaninpactonmyrmderstandingoftherealityof being a scientist. My thanks also go to Dr. C.C. (hang for his assistarnethmlglmtmysmdiesarriresearduandtoDr.C.Aylsworth for his friendly ermraganent and support. Aclcmledgatent is certainly due to Dr. T. Friedman for spending valuable time reviewing this thesis and offering helpful hints and suggestions. Mydeepestgratitudeiswelldeservedbysanecnewhccauyhtthe tail end of it all, my dear friend and wife, Ann. Her support, assistance, understanding and love nade it all tolerable am mrtlmhile. Also,mywarnesttharflcsgotoaveryspecial,creofa kindfriend, Dr. ToshioMori. Iwill foreverremerrberhiskirriness, knowledge and assistance during the critical stages of this work. Finally, last but not least, my regards, thanks andgratitudego to Darla Conley and Judy Oopanan for their exceptional skills and relentless efforts in helping type this dissertation thrwghwt its mamm- 'I'ABIEOFWI'S WOFWOOOOOOOOOO0.00.00.00.00. m OF Hm. I O O O O O O O OOOOOOOOOOOOOOO II'IERA'IUREREVIEW................. ..... . Cell-to-Cellcmmm1catim GamerzticnStructure .................. GapJunctionsandDevelcpnent. . . . .......... DistrihrtimomeictimalOmmmicatim. . . . . . . . . ModulationofGameactimFmetim. . . . . . . . . . . Assays for Measureuent of Gap Junctional Comicaticn. . Inhibition of Gap Jmcticral Oatmmication and The Oncogene Hypothesis and cancer ........... (mccgenes and Cell-Cell Oamunication .......... 'naeRasamccgel'iesm....... ....... MATERIALSANDW...... .............. Cells...” ..... ............ MediumandoiltureOcnditions .............. Plasmids. . . . . .................... INA'I'ransfectimAssay.. ........ .. ...... c-Ha-ras-lPrcbeIsolaticn. . . . . . . . . . ...... ExtractionofGermicmA........... ..... Estimation of [NA Concentration by Spectrophotometry. . . DetectionoprlOrccgeneProductbyIndirect Inmmcfluorescence ...... ...... IsolaticnofG'IGrOells. ................ 'IheMstabolicOocperatianssay. . . . . . . ...... Scrape-loading/DyeTransferAssay. ..... WOOOO0.00000000000000 ...... ... TransfectimofW90ells................ c-Ha-ras-l Probe Isolation. . ...... . ....... INAAnalysisbySorthernTransfer ............ Detection of c—Ha-ras-l Expression by Indirect 'IheMetabolicOocperatianssay. . . . . . ....... Scrape-loading/We'l‘ransferAssayW vi 28 28 28 29 29 31 32 32 32 33 34 34 39 41 41 41 44 44 44 55 Page Scrape-Inading and Dye Transfer, A Rapid and Simple Technique to surly Gap Juncticnal Intercellular Dieldrin Inhibition of Gap Junctional Intercellular ConnmicaticninRat Glial OellsAsMeasuredbythe Fluorescence Photobleaching and Scrape-Loading/Dye DISCIISSICII ......... 80 (INCLUSICNS .......................... 91 IISI‘OFREFERENCES...................... 93 vii Table 1. LISTOFTABIES (Jo-cultured combinations in the metabolic cooperation assay of the X2 and )6 c-Ha—ras—l transfected v79 $118. 0 O O I I O O C O O ...... O O O O O 0 Control canbinations in the metabolic cooperation assay for the x2 and )6 c—Ha-ras-l trarsfected V79 cells ....................... The metabolic coqaeration assay: Co-mltured carbinatiads of GIGS (W) of H-ras transfected cells with 6‘1Gr of tmtrarsfected (V79R) or H-ras tr'arsfectedanth)cells............... Statistical analysis of the metabolic cooperation assay performed with the x2 and )6 c—I-Ia-ras-l transfectedclones Statistical analysis (t-test) of experiments I and II ofthemetaboliccccperation............. viii Page 36 37 38 50 51 LISTOF FIGJRES Figure l. Diagrmaticr'qoresartatimofpsvn4plaslnid...... 2. Ibrpl'nlogical features of the V79 cells before and after transfection with pSVzneo or pswo4 plasmids. . . 3. Agaresegelelectrrfixoresisarrlethidiumbranide staining of pSVzneo and pswo4 plasmid digests. . . 4. Southernblotanalys1s. 5. Detectim ofthec-I-Ia-ras-lprodust (p21) byindirect immflmrescenceusingnnnoclaaalantibodyin H-mewwlboooooooooooooooo 6. Detectimoprlproductbyindirectimmoflmrescence usirqmonoclonalantibodyinwildtypecellsarri cells transfected with control plasmid pSVzneo . . . . 7. Clo-cultured canbinations in the metabolic cooperation assay of the X2 and )6 c-Ha-ras-l transfected V79cells ......... ........ ...... 8. Co-wlturedcafloimtiorsofflcsandemrcellswith and without c-Ha-ras-l expression in the metabolic cooperationassay(experimentl)........... 9. (Jo-caltured canbinatiors of GIGS and 6'16): cells with and without c—Ha-ras-l expression in the metabolic cooperationassay(experimentII)........... 10. Gapjunctional mediateddyetransferinprimarymman fibroblasts by the scrape-loading/dye transfer my.......OOOOOOOOOOOOOOOOOO 11. Gapjmictionalmediateddyetransferinprinarymman loeratirncytesasdetectedbythescrape—loading/dye 12. GapjtmctionalnediateddyetrarsferinW9cellsas detectedbyscrape-loading/dyetrarsfer........ 13. Inhibited gap junction-mediated dye transfer in cells transfectedwiththec—Ha—ras-loncogene....... 14 . Schanatic represa'rtaticn of tram-are signal transductionsystans . .......... . ..... ix 3O 42 43 45 46 47 48 53 56 57 59 60 84 'Ihepurposeofthisstudywastoinvestigateapotentialrolethe H-ras oncogene product might play in the modulation of gap junctional intercellular comunication in manmalian cells. A suspected resem- blancebetweenthepropertiesofflmerasoncoproteinardthoseofsone chanicnl tumor promoters suggested a possible mechanistic correlation between the function of both factors in undulating cellular coupling. Inordertoconductthisresearch, thetnmanc-Ha—ras—loncogene, de- rived fron the FJ/T4 bladder carcinoma (Shih and Weinberg 1982), was introduced into cammicatim-cmpetent Chinese hanster fibroblasts. These cells were then tested for their ability to comrmicate via their gap ijctiors before and after the introduction, and expression, of the rasoncogene. ‘Iheexpressim oftheras oncoproteinwasverifiedby indirect inmmcfluorescence utilizing monoclonal antibody agairst v-ras product. The intercellular, gap junction-mediated, communication was tested by two different techniques, the metabolic cooperation and the scrape-loading/dye transfer assays. 'Ihisreseardiwastmdertakeninanattarpttoelucidateapoten- tialrole fortherasoncogeneinthenodulationofgapjunctionalcan— mmication following cellular transfection. 'Ihe transformation of a norual cell to tumorigenic and metastasizing one appears to involve a miltistep process of genetic and epigenetic changes (Linder and Gartler 1965, Marchalonis and Nossal 1968, Fialkmr 1974, Nowell 1976, Trosko 1987) . ‘Ihis neoplastic conversion seats to begin in a single 1 2 "initiated" cell (Nowell 1976, Fialkow 1979) . According to the concept of initiation, pronction and progression stages of carcinogenesis (Potter 1982), an initiated cell is one that sustained a nutation causedbyerrorsofmArepairorreplicatim. mringthestageof timer prunotim, the initiated cell because clcnally anplified thus ircreasingflietargetsizeardthereforethednrneforfiuthergenetic damage (Yotti et al 1979, Trosloo and Chang 1984, Potter 1980,1981). 'Ihe initiatedcellstlmprogresstowardnalignancyarribecaue invasive and netastatic. Cancerhasbeenregardedtobethereafltofhaneostaticdistur— bance (Forth 1963, Iversen 1965). It has also been considered as a "stan cell disease" (Till 1982) and a "disease of differentiation" (rm-1m 1968, Pierce 1974, Potter 1978). Most tumor cells lack con- tact inhibition mien grown in mmolayer cilmres (Borek and Sachs 1966, Aberorutbie 1979). This W of contact inhibitim is believed to play an inportant role in the cartrol of cell proliferation (Levine etal 1965). Manyumorcellsstnaedabsenceofgapjmictional coupling (Corsaro and Migeon 1977, Ioewenstein am Kanno 1966, Fentiman et a1 1979, Charo et a1 1987). ‘Ihe phenanenm of gap junction-mediated can- mmication representsan inpcrtantmeansbymidicellscaninteract directlywitheadlothertoeqiilibrateandendiangeions, nutrients and regulatory molecules of approximately 1000-1500 daltcns (loamstein 1981). This gating mechamism has been implicated in the regulation of tissue hmeostasis, cell proliferation and differentia- tion (Ioewerstein 1979, Bennett and Spray 1985, Pitts and Film 1986). 'Ihedisccverythatseveralomorprunoters, imlrrlingdmicalard physical agents, growth factors and hormones inhibit gap junctional 3 coupling, provided substantial evidence for an inportant role gap junc- tions might play in carcinogenesis (reviewed in Madhukar et a1 1988). Recent studies indicate that the products of certain transforming genes, i.e. oncogenes, are themselves growth factors or growth factor receptors (Bradshaw 1986). Sane of these oncogene proteins are local- ized in, or close to, the plaana umbrane (e.g. src, ras, erb and neu). 'Ihe sroamcogenemsreportedtomodulategapjmictional cammication whemexpressed intransformedmanmaliancells (AzarniaandIoewenstein 1984a, 1984b, Chan; et al 1985, Atkinson et al 1986, Azarnia et al 1988). In addition to src, other oncogenic proteirs, e.g. the SV40 little and large T antigens (Steinberg and Defendi 1981) , and nore re- cently, its middle T antigen (Azarnia and Ioewerstein 1987), have been found to correlate with the inhibition of gap junctions. A possible mechanistic linkbetweentheelqaressimofcertainoncogenesaxrlthe inhibition of gap junction during the process of cancer developnart was thus entertained (Trosko and Chang 1986, Trosko et a1 1983, 1987). Anongthegrovingmmberoftrarsforminggenes, madaersoftheras oncogene family are found expressed in a wide variety of naturally oc- curring mnnan malignancies fron different tissues (Willecke and Schafer 1984, Barbacid 1987), as well as in metastatic timers (Egan 1987, Collard et a1 1987). The ras oncogene differ fronthe normed proto- mbyasinglepointnntatimardthenutatedrasproducthasde- creasedGI'Paseactivityascarparedtothewildtype (reviewedin Barbacid 1987). Ras protein is also known to mdulate the adenylate cyclase signaling system in cells expressing p21 (Hiwasa and Sakiyama 1986). Although the function of the normal or oncogenic ras protein is not fully understood, recent evidence indicate that it represents an 4 integral carponent of the phosphatidylinositol 4,5 diphosphate (PIP2) membrane signal transduction systen (Fleishman et al 1986, Wolfman and Macara 1987). Interestingly, this is the same mamrane systan that mightbeactivatedbythesroornogeneprodtct (Changetall985) and by TPA, the potent unnor pronoter, 12-O-tetradecnnoylphorbol-13-acetate (Castagnaetal 1982). The signal transductionthroighthe PIPz systan leads to the activation of a calcium sensitive protein kinase C (PKC) receptor/enzyme (Nishizuka 1986) . PKC activation has been correlated with the inhibition of gap junctional interoellular cornunication (W ard Yamasaki 1985b, Castagna et al 1982). In addition, an observedsyrergisnbetweenTPAardtherasoicogereduringthepmwss of in 11229 transformation might suggest a cannon biological function forbothTPAandthe rasproteinp21(Dotto et al 1985). lIherefore, in order to explore the possible functional correlation (or the lack of it) between the expression of the ras p21 and interoellular communica- tion, the ability of the marmalian Chinese hamster V79 fibroblasts to performgapjtmctioncoiplingwastestedbeforeandafterthe introduc- tion of the ntrtant, oncogenic human c-Ha-ras—l gene. The expression of p21 protein was verified by indirect inmmofluorescence using a nono- clonalantibodyagainstV-rasle, arrithegapjunctiai-mediatedinter- cellular communication assayed by the two different techniques, the metabolic cooperation and the scrape-loading/dye transfer. IITERA‘IUREREVIEN QED-tfin mijSim Interoellular conmmication in nulticellular organisms has been recognizedasaninportantdeterminantintheregulatimofrmeosta- sis, grovth, developnent, cell proliferation and differentiation (Saxen et al 1976, Ioewenstein 1968, Sheridan 1976, Blunberg et al 1982, Wolpert 1978, Bennett et al 1981, Gilula 1980 ard Yamasaki 1984) . Cellular interactions can be fulfilled by on najor mechanisms. In the first, the cells transmit signals across the extracellular space as is the case with grmth factors, hormones, neurotransmitters, and other radiators, where the target cells with appropriate receptors could be locatedinadistanttissueororgan. 'Iheotherformofcellularinter- actions, on which the enphasis of this dissertation will be focused, involves cells in close contact. These cells commnicate via special- ized structures called the "gap junctions" (Ioewenstein 1981) . This form of cellular camunication was first observed as an elec- tricalsympseintheimertebratenervoissystau (mrshpanandPotter 1959, Bennett 1977) . Folloving that initial observation many metazoan tissueswere fonritohavesimilarnodesofmenbranecorductancee.g., in coelenterates (Hand and Gobel 1972, Wood and Kuda 1980), in fish (Robertson 1963) and in manuals (Revel and Karncvsky 1967). The mlian myocardial cells eluded renarkable reliance on junctional comunication in the synchrmizatim of propagating the action poten- tials (Weidnann 1952, Dreifuss et al 1966) . 5 6 Cells coupled via gap junctions form a large cytoplasmic syncytium in which relatively atoll size organic molecules and ions can diffuse freely among contacting cells (Hertzberg et a1 1981, Ioewenstein 1981, Sprayetal 1982). Thesegapjunctionsarefomdinalmostallmeta— zoanorganisnsardtheyseantohavebeonhighlyconservedthrongholt evolution (Ferrachia 1973) . The general structure arnd functional prop- erties of arthropod arnd vertebrate gap junctions (Sinpson et al 1977, FinbovandPitts 1981), aswellasthcseinawiderangeoftissuesof nulticellular organisms are nuch the same (Paracchia 1980). Studies with antibodies derived against liver gap jonction protein have detec- ted hamlogons polypeptides in different tissues (Dermietzel et a1 1984, Hertzberg and Skibbons 1984). In addition, in m staiies indicatethatgapjmnctioncolplingcantakeplacebetweoncellsfron different metazoan organs and organians (Epstein and Gilula 1977, Michalke and Ioewenstein 1971, Gaunt and Subak-Sharpe 1979, Flagg- Newton and Icewenstein 1980) . The models suggested by Sheridan (1976) and Loewenstein (1979) describe the effect of cell coupling on the concentration of hypotheti- cal diffusible molecules that regulate cell division and differentia- tion. Theconcentration ofsudnmolecules couldbedependentontheir rateofsynthesisardonthetotalvolnmeofcytoplasmicsyrcytitmin which they are distributed. A physiological equilibrium level could be disturbed by the interference with the gap junctional camunication. Such inhibition or down regulation of gap junction function could be triggered by nultitude of factors. Uncoupling could oconr after termi- nal differentiation, cell death, physical occlusion, mitogenic signals, altered ionic concentration, odogenms or oucgenos factors or 7 dnanicalstobedescribedinthefollovingsections. Theconsequence of sudn disturbance of this cellular function coild potentially vary fron no noticeable effect to tumor pronction by permitting the prolif- eration of initiated cells; organ dysfunction, e.g. , in the myocardium, causingabrnnnalsigraltrarsductim;ordiseasecoditiosbydiso1rb- ingor'ganortissuehoneostasis (T‘roskoanddnarg1980). W In 1972, Gilula and colleagues demonstrated the presence of a spe- cialized nodarane structure that is responsible for the transfer of small molecules and electrical signals between contacting cells (Gilula et a1 1972) . Extensive biochemical, electron microscopic and x-ray diffractionshndieshavebeoncorhnctedtostudygapjmnctionstnnchne in different tissues fron different organism arnd at different stages of development and differentiation (for review see Rracd‘nia 1980) . Gapjmctionsarespecializedtransmarbranestmcturesthatare conposedofmiltiplednamnelsorcornnennons. Eadnconnoronisconposed of six identical rod-shaped polypeptide submits of abort 7-8 nm long arranged as oligoners that form continuous onamnels betweon two adja- cent cy'tcplasms allowing for mlecule trarnsfer. Each connenron pro- trudesforaborthintheortracellularspacetojoinitscomter— partfronacortignnnscellflmsfominga"gap"seonintenuptirgflne closelyapposedcellmanbrareswhenstainedardoraminedbyelectmn microscopy (Revel arri Karnovsky 1967). The gap junction units ternd to aggregate in plaque-like structures that vary in size, shape arnd dis- tributioninvariois cellsfrondifferenttissues. Theapposedjunc- tions frontwoadjacentcells aligntogetherto formcontirnuwsaqueons 8 channels that provide direct comunication between their cytoplasm. These pattuays allow the free diffusion of relatively small molecules arnd ions of 1000-1500 daltons m (Ioewenstein 1981) between coupled cells (Sinpson et al 1977) . Each cell is able to maintain its individ- ual characteristics by retaining specific non diffusible nnacronoleoiles (Gilula 1985) . 'nnegapjmnctionproteinhasbeenisolatedfronrodentlivercells (Revel et al 1985). A protein of a molecular weight of abort 27 K dal- tons has been identified (Hertzberg arnd Gilula 1979, Henderson et al 1979, Hertzberg et al 1981). Polyclonal antibodies prepared agairnst liver gap junction proteins were able to recognize similar antigenic determinantsinawidevarietyoftisanesfrundifferontqaecies (Hertzberg & Skibbens 1984, Hertzberg 1980, Ziegler arnd Horwitz 1981, T'raub et a1 1982, Willecke et al 1985). 'Ihis similarity suggests an evolutionary conservation of cannonly recognizable antigenic determin- antsofthegapjmnctionproteirsindifferortorganionsardtissues and, therefore, urnderlines the junctional inportance in nulticellular organisms. MW 'nneroleofmerfloranegapjmnctionsdm'ingdevelomenthasbeen ortersively studied (Barnett 1973) and considered to control the dif- fusion of secorl messengers that regulate cell proliferation and dif- ferentiation (Iawrence et al 1978) . Prior to fertilization and durirg the process of oocyte maturation in a variety of organism (e.g., mice, sheep, chickensandfrogs), itwasdiscoveredthatcells oftheomulus grarmlosa (mothers of follicular cell population) do establish direct 9 commicationwithoocytethr'onghpzrocessesthat penetratethelayer of the zona pellucida (reviewed in: Scimltz 1985, Gilula 1978) . Ovulationappearstobetriggeredbyhormonalstimfliwithsubseqnent disruption of this form of cell-cell communication. Following fertili- zation, studies of electrical and dye coupling in noise abryos showed that intercellular commication begins among all adoryonic cells at the eight cell blastomere stage arnd throughout the blastocyst forma- tion. Further developnent arnd differentiation was fond to correlate with conpartmentalization of cell coupling (revised in Gilula 1980, Spray et al 1982, Warner 1983, Schultz 1985). In Drosophila, the pat- ternof intercellularcolplingwassondiedintheodoryonicwingim- aginal disc during its developnent (lo 1985). Several developnental colpartments shoved restricted pattern of cell coupling. Similar ob- servations were made after studying junctional communication in variols insect epidermis (Warner arnd Iawrence 1982, Blennerhassett arnd Caveney 1984). In addition to its potential role in onbryo develcpnent, gap junctional intercellular cotmmication is thoaght to participate in the regulation of cellular proliferation and differentiation in the fully developed organism (Ioewenstein 1979, 1981, Gilula 1985, In 1985, Wald 1985, Neytm ard Tr'autmam 1986, Pitts arri Finbov 1986, Revel et a1 1985, Schultz 1985) . These firrlings strongly point to functional correlation between gap junctional communication and tissue develop- ment, organization arnd differentiation. Direct evidernce of the role of gap junction intercellular columni- cation in developnent arnd differentiation was dononstrated whm anti- bodies agairnst gap junction proteins were microinjected in cells fron developing onbryos (Warner et al 1984, Hertzberg et a1 1985). Gap 10 junctional intercellular communication was blocked or reduced in Xencpus laevis abryo cells after microinjection of antibodies against thegapjunctionprcteins. Morethanhalftheanbryosthatwereal- loved to mature developed congenital patterning anonalies corresponding tothesegnentsthatwereinjectedwith antibodies (Warneretal 1984). The study of the patterns of irntercellular connunioation in intact tissuesarndorganssuggestarole forgapjunctionsinthecortrolof growth arnd differentiation. Gap junctional interoellular communication was fond to have variable distrihntions in intact differentiated or- garns and in different types of epithelial tissue. The results indicated that met epithelial cells are electrically coupled e.g. in pancreas, lacrimal arnd salivary glans (Petersen 1980). In nnamvalian salivary glarris,gap junction communication exists between cells within the sane ascinihrtnotbetweonasciniofthesanegland (Petersen1980). Inthe testis, spermmaturationappearstodependonthepresenceofintact gap jnmctionsbetweenthe LeydigandSertoli cells (Slurpe et al 1981). Similar pattern of communication was detected anang hepatocytes of weanling rats by electric coupling arnd fluorescent dye trarnsfer (Layer et a1 1981) . The extent of this permeability was downregulated follow- ingpartialhepatectonyandthestartoforganregeneration. Microin- jectionof Inciferyellovdye in intact adultmcusesldnshcwedexten- sive dye transfer annong dermal fibroblasts and a limited diffusion in epiderml keratinocytes (Pitts et al 1987). There was no dye trarnsfer acrossthebasementnenbrane. Thedermal fibroblastswerenotconpled totherneighborirng cells ofthe sebaceousglandsorthenuscle fibers 11 (Pittsetal 1987). Inthesanesbndy, thecellsinsebaceousglands were totally coupled among theselves yet conpletely uncoupled with dermal or other contiguous cells. These observatios indicate that intercellular permeability could be selective among cells of the same organ ortissuemostlikelytoneintain furnctional, biochanioal and/or electrical syncytia. Aninportantdnservationlinkirgtnmorpronotirgagentstothe inhibition of gap junctional commication was nnade by Yotti et al (1979) and Murray arnd Fitzgerald (1979). The potent tumor pronoter phorbol ester T‘PA (lz-o-tetradesanoylphorbol-ln—aoetate) was founi to inhibit the netabolic cooperation between cells grom in culture. This observation was further substantiated in varions cell types using dif- ferent tumor pronoters (En'nwto et al 1981, T‘rosko et a1, 1982, Kalimi arnd Sirsat 1984, W and Yanasaki 1985, Yancey et a1 1987, Trosko et a1 1987, Tsushimto et al 1983, Warrgard et a1 1985). Different mummmpjnmctionconmmicationwereutilizedtotestthe effects ofvarious dnonioalconpomdsonthemonbrareonamnelseg. dye transfer by microinjection (Enoncto and YamasaJd 1985b) , electroconp- ling (Ehcmgto et a1 1981), FRAP analysis (Wade et al 1986) arnd scrape- loading/dye transfer (El-Folly et al 1987) . Using freeze—fracture analysis, cells treated with tnmnor pronoters werefonrdtocontainlessgapjtnnctions, whenconparedtocontroljn intro (Yancey et al 1982) and in 1129 (Kalimi and Sirsat 1984). The tamerpronoter, phorbol ester, wasalso fonndtoenhancecellular transformation while blocking intercellular comunication (We and 12 Yamasaki 1985b). TPA is known to activate a monbrane-bound, calcium- sensitive receptor ernzyne protein kinase-C (PKC) (Castagna et a1 1982) . A strong correlation betweern the activation of PKC arnd the inhibition of gap junctions has been therefore inplicated. Recently it was shown that activated PKC phospnorylates gap jonction proteins of rat liver cells inacell-freesystem (T‘akedaetal 1987). PKCisalsokncwnto be activated by an oflogennns second messenger, diacylglycerol (DAG), a byproduct of pinsphatidylinositol 4,5 hiphoqnate (PIP2) (Kislninnoto et al 1980, Nishizuka 1984, We and Yamasaki 1985, Gainer and Mirray 1985). This PIP2 is a nnajor conpoent in a transmmbrane signaling systonthattransmitsexternalsigralsintotlecelltoprovokebio— logical response. AnotherbyproductofPIPZbreakdownistheinositol triplssphate which releases the intracellular calcium fron its stores in the endoplasmic retionlum. This release raises the cytosolic con- centration of ionic calcium (Berridge arnd Irvine 1984) , which case- quently activates HCC (May et a1 1985) arnd inhibits the junctional con- nlmicatim (Rose and W 1977, Rose arnd Rick 1978) . Otherodogenonsfactorsthatarelomntonodulategapjmctional function include the intracellular pH (T‘urni arnd Warner 1977, 1980, Spray et a1 1981) arnd cyclic AMP (Azarlnia et a1 1981, DeMaziere and Sdneuermann 1985, Flagg-Newton et a1 1981, Kanno et a1 1983, Nehta et a1 1986, Saez et al 1986). Increased levels of cAMPappeartoenharee junctional comunicaticn anrnng contiguous cells (Saez et al 1986, Wiener arnd Icaoenstein 1983, Johnson et al 1985). Recently, certain manhrarebonrlproteins, e.g. thesrconcogeneproduct, havebeenshown to be associated with the modulation of interoellular coupling (Azarnia and Ioendostein 1984a,b, Chang et al 1985, Atkirson and Sheridan 1986) . 13 Furthermore, dnargesinthenanbranepotential (Sprayeta11979, Harris et a1 1983) or alteration of the adhesion molecules that assist in mintaining close juxtaposition of contiguous cells (Edelman 1983, abrink 1986) could also modify the cells' ability to corduct interoel- lular cornunication. Extracellular or "exogenous" factors, including tuner pronotirg agents, were fond capable of undulating the junctional gating syston. Alcohols (Johnston et a1 1980), homes (Merk et al 1972, Decker 1976, 1981, Dahl ard Berger 1978, Garfield et a1 1980), neurotransmitters e.g., acetylcholine (Neyton ard T‘rautmann 1986), vitamin A and its derivatives (Elias and Friend 1976, Elias et a1 1981), certain dnngs e.g., pnennbarbital (Jone et al 1985), valiunn (Trosloo arnd Horrobin 1980) , dietary elanents e.g. , unsaturated fatty acids (Aylsworth et a1 1984) ard saccharin (T'rosko et al 1980), environmental pollutants e.g. polybr'oninated bipnenyls (T‘sushineto et a1 1982) , solvents (Chen et a1 1984), metabolites (Malcolm et a1 1985), pnorbol esters (“my and Fitzgerald 1979, Yotti et al 1979, Fitzgerald and array 1980, annoto et al 1981, Trosko et al 1982, Kalimi ard Simat 1984, W ard Yamsaki 1985, Yancey et a1 1987), dieldrin (Trosko et al 1987), an (Tsushinnto et a1 1983, Wangard et al 1985), grovth factors e.g., no and TGF-B (lbdhukar et al 1988) were all shown capable of modulating gap junction furnction. Physical factors e.g., localized cell death or surgical resection in organs as in partial hepatectony, induce cell proliferation during theregenerationardhealingprocessesandareassociatedwiththere— duction of gap junction umber ard, consequently, with the inhibition of cellular coupling (Yee ard Revel 1978, Yancey et al 1979, Meyer et 14 a1 1981). Other gap junction function modifiers include factors like 002 concentration (T'urinardWarner1977) ardchangesintanperature (Ararcia et al 1986) . The nultitude of factors ard different classes of dnonicals and physical agents that share an ability for undulating gap junction foctionmightbeoperatingviadifferentmednanismsyetthrongha few cellular signals or key regulatory elonents e.g. pi, cAMP or Ca'H' concentration, to induce cellular response i.e. proliferation or dif- fererntiation. In addition to the knovn factors that modulate gap junc- tional interoellular cotmmication, one might speculate that at the neleonlar and biochonical level , imitations affecting the gap junction geneoranyof its regulatoryelementsorothergenesregulatinggap junction function, or any alterations of their expression, translation, post translation modification or manlorare translocation, orientation or internalization might, as a consequence, affect the junction-mediated communication. Depending on the nature of the junction modulator, ex- posure tine, stage of cell cycle, state of differentiation and/or pro- liferation, the cellular response could be adaptive or naladaptive (T‘rosko and Chang 1984). In the latter situation the cosequences conldvaryfronmnremarkabletodisruptive ofncrmal furctionardor proliferation (T'rosko ard Chang 1980, Trosko et al 1987) . Given the passive nature of gap junctional perneability, it appears that the control of signal transmission between contiguous cells might depend on different factors. Ana'q these, the nunber of available nan- brarne jurctios, their diameter, the concentration gradient of the 15 diffusible mlecules and their variable friction factors. A number of assayshavebeendevisedtonsasmethejmctioslpenneabilitybeureen cells either directly or indirectly. ‘Ihe following is a brief descrip- tion of those available methods that are being successfully used to suflygapjunctionfmctions. Sonneoftheseassaysarerevienedin more detail elsewhere (e.g., Sccolar and Ioewestein 1978) . Electrical coupling: In this assay, microelectrodes are placed inntracellularly to measure electric (ionic) currents between con- tactirg cells and between cells and the extracellular medium (Azar'nia anti Ioewenstein 1971, Encncto et a1 1981, Yamasaki et a1 1983). It is a sensitive assay that can be applied mm annd jam tocellsofassmalladiameteraleum (Ioevestein 1979). It is not capable however of testing for transfer of nnoleonles larger than the snnnall charged ios it detects. Jinnctional electric corinctance: 'Ihis method allows the qnantita- tion of electrical coupling. It is a conplicated procedure that requires several microelectrodes annd conpares various parameters inclnriing nnonjunnctional nnanbranne resistance or cellular inpnt re- sistance. Its application may be useful in sinple cell systes (Sccolar and Ioewenstein 1978) . Transfer of radiolabelled mlecules: 'n'nis nethod nunitors the transfer of tritiated uridinne nucleotides fronn donor to recipient cells grown in co-onltnnre. 'Ihe recipient cells can be easily identified by prelabeling with fluorescent microspheres before the co-culture (Keijzer et a1 1982). line degree of cell-cell comuni- cation via gap junctions can be estimated by scoring for presence 16 of transferred radiolabelled moleolles (Mir-ray annd Fitzgerald 1979). 'nnisnnethodislegthyandsonswhattedionsanrithere- sultsareobtainedafterabontz-Sweeksfronstartofexperiuent. Metabolic cocperatios assays: These assays rely on the fact that cellsincontactarecapableofenchanginganmberofmetabolites amcngthenselvesacrossgapjmctios (albak-Sharpeetal1966, mrketal 1968). untentcellswithdeficiecyinthepyrimidine crpurinne syntheticpatlmayscansurviveinselectivemedia ifco- ollunred anti contacted with wild type cells ((13): et a1 1970, Pitts 1971, Pitts annd Sinus 1977) . ‘nne transfer of molecules is presum- edtoocolrviagapjnnctions sincestnxiieshaveinriicatedstrog correlation between nnetabolic cooperation annd electrical coupling (Gilula et a1 1972, Azarnia et al 1972). cell coupling can be measured by transfer of radioactive metabolites (Subak-Sharpe et a1 1968), or by assessment of the number of surviving colonies of Wicient nutants when co-cultured with wild type cells in the presece of 6-thioguanine, 6'16 (Fujimoto et a1 1971, Corsam and Migeon 1977). In the latter modification of the assay the wild type cells would netabolizetheS'IGtoatonicpinosphorylatedbyprochtwhidn re— sults in cell death. ‘Ihe nutant PERI-deficient cells are inher- entlyresistanttothelethaleffectofé'msincetheyareincap- ableofnnetabolizing it. Whenbcthcelltypesaregrominco— cultureinthepresenceof GIG, thetoxicmetabolite 6-thioguannine mocphcspnatecantlmsbetransferredbetweencontactingcells with conpetent junctional comunication and is capable of killing thenutanntcells. Onntheotherhannd, ifthegapjunctionsars 17 totally or partially inhibited, the nnutannt cells survive to form colonieswhichcanbeeasily scoredandconparedtotheplating efficiecy of similar nunber of nutant cells. The netabolic cooperation assay allows the testing of dnemi- calsexertinglogtermmcdulationofgapjunctiosatncncyto— toxic doses annd also provides a good means for quantitative analy- sis of dose-response effects of potential modulators of gap junctionconmmication.‘1heuseofthismetlcdhoweverislimited to a few established nanmalian cell systens. 'Ihe cells should have a relatively higln colony forming ability which is not the case in primry cells. 'Ihe assay also requires the utilization of well dnaracterized mntant cells annd it does not allow for testing of short term, or reversible, innlnibition of gap jmction. Fluorescent dye transfer: The inntracellular microinj ection of nnenbranne inpermeable tracer molecules e.g. Incifer yellow (IX) has been sucwssfully utilized to study gap junctional permeability in coupled cells (Rose et a1 1977, Flagg-Newton and Ioewenstein 1979, flag-Natal at al 1979, Friedman and Steinberg, 1982). This method allows tlne direct visualization of intercellular coupling. It also assists in the quantitative evaluation of the degree of junctional conpetecebycomtingthenunbercfsecoriarydyere— cipient cells annd is applicable to a wide variety of cells fron different tissues, organs annd species. It is a sensitive assay end can be utilized to detect the permeability limits of molecular size (Azarnia annd Icewenstein 1976, Rose et a1 1977) . Fluorescece Recovery After motobleadning (FRAP) annalysis: ‘Ihis is a technologically advanced extension of studying gap junction 18 communication using fluorescent tracer molecules (Wade et a1 1986) . The fluorescent dye G-carboxyflucrescein diacetate (a hydrqlhobic nanbrane—permeable nnonnpolar ester) is absorbed by cellsgrownincultnre. Onceinthecytcplasmitbeconesrapidly hydrolyzed by esterases yielding free flucrescein, a molecule with enallecghsizetopermeateacrosscpengapjmctionsbetween coupled cells. 'nne procedure involves laser pnotobleadning of single cells, coupled annd nno'coupled, annd assessing the degree of dye redistribution across gap junctions fronn contiguous non-photo- bleached cells. When contacting cells are pretreated with anemi- cals known to innhibit gap junction comunication, nc dye recovery isobservedinthepctcbleachedcells. 'nnisassayisverysensi- tiveyetrequiresscfinisticatedequipnentandconpnterprocessing toanalyzeanndquantitatethedata. carcinogenesisisaconplenprocessbywhidnanornelstencell undergoes nultiple changes toward transformation. This conversion is ofannultistepnatureasdeterminedbystudies oftuncrdevelopnentand progressionbotlninhnmnans, aswell asinecperimental animals (Foulds 1954, Nowell 1976, Cairns 1975,1981). 'Ihe clonal origin of cancerous tumors has been determined in several studies (Fialkcw 1976, Nowell 1976, Baylin et al 1978). An operational staging assumes three major phases of carcincgenesis namely initiation, pronction annd progression (Boutwell 1974, Pitot et a1 1931, Foulds 1954, cairns 1975, Berenblum and Annuth 1981) . " ‘ Dirinngtheinitiationnnase.astencellsustainsa stableannd 19 irreversible "prenalignant" genetic damage which could be inherited or induced by a given nutagen (Ames et al 1973). Mutation fixation is achieved by an error—prone repair or replication of 111A (Glover et a1 1978, Maher and McCormick 1977, Warren et a1 1981). The majority of nutagenshavebeenshowntoplayaroleininitiationorsonetimesbe- have as "conplete" carcinogens (Trosko et al 1983) . Ann initiator, at a highenoghanndcytotoxicdcse,mightcauseextesivedamagetothe cells following exposure and miglnt lead to cell killing. This killing could provide pronoting conditions by innducing the surviving cells, including those initiated stem cells that escaped death, to proliferate (loch-Caruso and ‘I‘rosko 1985). There is strong evidence to inplicate nutagenesis in onrcincgenesis (Trosko annd Chang 1981, Yuspa annd Dbrgan 1981) , yet carcinogenesis, as a conpliczted prowss, requires more than just nutations (Trosko annd Chang 1978, Trosko et a1 1985) . In the pronotion phase of carcincgenesis, selective proliferation, i.e. clonal annplification, of the initiated cells by pronoters or mito- gens takes place (Trosko annd Chang 1983, Trosko et a1 1983). The ini- tiatedstencellshaveanatnralgrowthadvantageovernm—stencells given their ability for self renewal annd lack of differentiation (Chang et al 1987, Bykorez and Ivashdnenko 1984, Karrio 1983, Steel and Stephens 1983, Till 1982) . The proliferation of the initiated, prena— lignnant cells to reach a "critical mass", increases their chance of sustainingadditional nutationaleventsanndthusenhancingtheproba— bility of tumor formtion (Boutwell 1974). Bring this stage of car- cinogenesis, i.e. the stage of umcr progression, the cells acquire a series of morphologic, biochemical annd genetic changes that would, in most cases, alter the nnormal differentiation pathway (Sachs 1980 a,b), 20 lead to rapid, autononos growth, invasion and metastasis (reviewed in 'Ircsko and Chang 1984a,b) . Intheabseceofhmcrpronnction, theinitiatedcellspresunnably renainquiescentthmghannneostaticcontrol contributedbytheir normalcounterparts. 'Ihisconditionanpearstobeincontrastwith cancercellswhidnarethoghtofascellsthat losthoneostaticcon- trol (Furth 1963, Iversen 1965) annd the capacity for differentiation (Potter 1978). This assunnptionwas stregthenedbythe fact thatnnncst nalignant cells lack contact inhibition (Borek annd Sachs 1966, Corsaro annd Migeon 1977) , and the majority have lost the ability to comunicate with nnornal cells via gap jnn'ctios (Icewenstein 1979, Icewenstein annd Kannc 1964, Fentinan et al 1979, Kanno 1985). 'Ihe correlation between thenecplasticphenotypeanritheabseceofgapjunctional intercellular communication became more significant following the dis- covery that tumor pr'oncters, e.g. [horbol esters, innhibit this inpor- tantmenbranennediatedfunction (Yottietall979,Murrayand Fitzgerald 1979). 11118 linkwas further substantiatedwhen a number of laboratories using different assays to study the effects of known tunncr proncters on gap junction permeability in cells fronn different tissues andorganscametothesamecoclusios (reviewedin'rroskoanddnang 1984a,b, 1987) . Treatment with tnmcr prmcters renarkably decreased the number of gapjunctionsinW9 cellswhenconparedtothemntreatedcontrolas revealedby freeze-fractureandelectronnmicroscopy studies (Yanceyet al 1982). Similar resultswereobtained inphorbol estertreatedmcuse qaidermis cells (Kalimi and Sirsat 1984 a,b) and in regenerating liver cells in partially hepatectonnized animals (Yancey et a1 1979, Yes annd 21 Revel 1979). Substantial evidence indicate that tnmnor pronoters act primarily on cell menbrane while initiators, acting as nnutages, target the INA (reviewed in 'Ircsko annd Chang 1984a,b). 'Ihese observations lexistrengthtothehypothesisthattumorpronctersactbyblocking gapjunctional conplinngbetweenncrmalanriinitiatedstencellsthus disrupting their contact inhibition (Abercronbie 1979, Borek annd Sachs 1966) annd homeostatic regulation of proliferation annd differentiation (Trosko and Chang 1984a,b) . mstunnorcellsarebelievedtohaveolstainedsonegenetic alteratios due to errors in [NA repair or replication (Strog 1977, Cleaver et a1 1975) . Amog the manny theories dealing with the origin ofcancer, theocogenecoceptisgainingwideacceptance (I-Iuebnnerannd 'Ibdarc 1969, 'I‘odaro annd Huebnner 1972, Marshall 1986). Oncogennes are genes capable of inriucing cellular changes that lead to neoplastic transformation. In the viral hypothesis of ocogennesis, it is postu- lated that all vertebrate cells contain genes (called prom-oncogenes) annalogonstothetransformingelenentspresentintnmcrviruses (Huebner and 'Ibdaro 1969, ‘Ibdaro annd Hnnebner 1972, 353th 1983). 'lhese proto-occgeneshavebeenevolutionaryconservedinallmetazoanorgan- isnns. ‘Ihis fact may underline their critical inportance in normal cel- lular function annd proliferation. 'Ihey can acquire ocogenic activi- ties by one or more of for basic mechaniens, nnamely: inappropriate expression during the wrog stage of cell growth or differentiation, annplifiontion, translcontion annd nutatios. ‘Ihe nutational changes can be innduced by radiation, mutages or viruses. Nonmntational genetic 22 nechaniens can also play a role in oncogenic activation e.g. by changes in [NA methylation, annplification or epigenetic alteration of proto- occgene expression (reviewed in Pimentel 1986) . 'Ihe currently identi- fiedonccgenesrepresentasetofhighlycoservedgenesthatpresmn- ably play an inportant role in cell proliferation and differentiation (Lacey 1986, Pimentel 1986) . Oncogenes have been classified according to different criteria, e.g., intracellular localization of ocanotein, biodnenical charac- teristics, or specific structure or function. Two major classes of ocogeneshavebeenrecognizedbasedonthe localization oftheirpro- ducts. 'mesearethennuclearandthecytoplaenicormenbrane-bon'd ocogenes (Weinberg 1985). Several mrts have indicated that these twoclmofoccgenescananddocccperatetotransformncmalpri- narycells inculture e.g.,thenuclearoccgeneproductofc—mycand either of the menbrane-bomd Ha-ras or N-ras (Land et a1 1983): or N- myc with the activated Ha-ras (Yanccpoulcs et a1 1985) . Another clas- sification segregatesnncstoftheknnownocogenesaccordingtotheir bicchennical activity into two najor families (reviewed in Pinentel 1986) . First, the are family whidn includes those occgenes with tyro— sinne kinase activity, e.g., src, abl, fins, yes, erb-B, fes, and ros. Secod, agronpwithnnoknnwnproteinkinaseactivityandthisincludes myc,myb,mcs, sis, andrasocogenes. 'Iherasfamilyisthemcst ubiquitols in malignnant tumors of human origin (Barbacid 1986) . It has three highly honologous merbers, H-ras, K—ras, and N-ras, all of which bind guanine necloetides and have been inplicated, as disonssed below innmoredetails, intranenenbrannesignaltransduction. Certainonco- geneprodnctsstcwedrenarkablehonclogytolmowncellularproteins 23 (reviewed in Pimentel 1986), e.g., sis is hcmnolcgous to m, erb-A to carbonicanhydrase,erb-Btoml=‘recentor,mostomereonrsor, rasto G-proteins, fes/fgrtoactin,myctobetaandgamnacrystallinsandfns to the mocnmclear-phagocyte colony stinmnlating factor ((SF—l). Sonne oftlcseproteinsarelocalizedatornearthecellmenbrane,andwere thoghttohavepotentialfmctionalasscciationwiththepronction phase of carcincgenesis given their ability, oce activated constitu- tively orby induction, to act asmitogen and induce cell proliferation (Trosko et al 1983,1987). ‘Ihis hypothetical association also attempts tolinnktheexpressionofcertainoccgenestotheinterferecewith gap junction communication ('I‘rosko and Onang 1986) . Recently, the alteration of gap junctional communication has been described in cells transfornned with v—sro ocogenne (Chang et a1 1985, Azarnia and Icenestein 1984a, 1984b, Atkinson et al 1986). The ex- pressionofsrcprotein (3)6051“), atyrosinnekinaselinnkedtothe pnoematidylinoeitol 4,5 bipnosphate (pm) transmenbrane signnaling system (Macara et a1 1984, Sugimoto et al 1984), was correlated with increased protein Kinnase C (PKC) activation and inhibition of gap junc- tion intercellular communication (Chang et al 1985). Other viral ocogenic products, e.g. the little T and the large T anntigens of SV40 (Steinnberg and Defendi 1981), and more recently the micflle T of the samevirus (Azarniaandloewenstein 1987) havebeenassociatedwiththe reduction of junctional permeability. These findings might reflect a conmnroleplayedbydnenical pronoters, ocogenesandviral agents in the inhibition of gap junctios during the process of tumor pronotion 24 (‘I‘rosko and Chang 1986,1987, Trosko et al 1984, Madhukar et a1 1988). new aeoftl'ehighlycoservedandubiquitolsnenbrane-boundocopro- teinsisthelepolypeptideofthesmallfamilyofrasocogees (Barbacid 1987). 'Ihesetransforminggeneswere first discovered in the HarveyandKirstenstrainsofratsarconaretroviruses (Phrvey1964, KirstenandMayer1967). 'Ihecellularcounterpartsoftterasgenes (i.e. c-ras) appeartobehighlycoservedandarefonndinorganisns fronnyeaststomanmnals (ShiloandWeinnberg1981). 'Ihismayreflectan inportant role for the ras gees in basic cellular function. I-Inmn cells have three functional ras gees nanely, c-Ha-ras-l, c-K-ras-Z and c-N-ras which map to dnroncsones llp15.l-p15.5, 12p12.1-pter and 1p22- p32 resqcectively. Atthemoleonlarlevel, allrasgeneshave fonrexons (withthe exception of c-K-ras-z which has two alternnative fonrth axons) , a 5' ncnn-codingemandprrmcterregionscontainingG/C ridnboaeswithcut MorCA‘I‘boaes, acharacteristicofpronotersofncusekeepinggees (Barbacid 1987) . Bicchennically, the p21 protein contains fonr donnains: ahighlycoserved, 85aminoacid firstdonnain, asecoddonainwith 80 amincacidswhidnhave851khnmnologyintnemmnanrasgees, athird with 20 amino acids that show rennarkable variability, and last, a con- served stretch of for aminno acids including cysteine at position 186 followed by two aliphatic and oe variable aminno acid (Barbacid 1987) . 'nep21proteinsarefonndatthecytoplaenicsurfaceoftnecellplas- ma neubrane (Willingham et a1 1980, Willunsen et al 1984, Fujiyama and Tamanoi 1986) . 'Ihey bind guanine nucleotides (GI? and GDP) (Scolnick 25 et a1 1979, Shih et a1 1980, Tamanci et al 1984, measles et a1 1985) and have GTPase activity (neneles et al 1985, Sweet et a1 1984, Mannne et al 1985) . Issproteins alsoappeartobeinvolvedinntranennenbranesignal transaction (Willixgham et a1 1980, mirth et a1 1982, Ievinson 1986). 'lheN—tenninaldonainoftherasgeneproduct (p21protein) showshonn- ology to the manmnalian G-proteins (Hurley et a1 1984) which nediate ligand—induced activation of adenylate cyclase and phospnolipase C (Wakenan et al 1986). 'Be former controls the cellular cyclic AMP (cAMP) level (Rodbell 1980) and the latter catalyzes the breakdown of phosphatidylinncsitol 4 , 5 diphcsphate into inncsitol triphosphate and diacylglyoerol (Nishizuka 1986) . In the NDBTB cells, the ras ocogene expression was found to correlate with decreased activity of the adeny- late cyclase menbrane signnaling system and with low levels of cmlP (Hiwasa and Sakiyana 1986). Decreased cAMP levels correlates with the inhibition of junctional interoellular commmicaticnn (Saez et a1 1986, Wieer and Loewensteinn 1983, Jonnson et al 1985) . In addition,emerg- ingevidence indicatesthattleleproteinmaybeaninntegralconpo— nent of the polyphosphcincsitide signnaling systenn (Fleishman et al 1986). 'Be enpression of either the nnornnal orthe occgenic forns of H-ras innanmnnaliancellswas foundto inducealtered levels offlnoqnha- tidylinncsitol 4,5 biphcsphate (PIPZ) and its catabolites (Fleishman et a1 1986) . One of the PIP2 catabolites is diacylglyoerol (DAG) . The elevation of ms levels is kncwn to activate a calcium sensitive, phos- pholipid dependent protein Kinase c (910:) (Kishimcto et a1 1930, Nishizunoa 1984, Ennoncto and Yamasaki 1985) and to inhibit gap junction- a1 coupling in cultured cells (Gainer and Murray 1985). 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Hooe me> odxu onz osu scum AoIHU. moCoHo oooooHom aHEoocou an ooooHocw Amuob coeov moquom Heuucoo one .oeo mo oocomoue one as mHHoo name: no auo>ooou ucoouoe on» ucomoneou much HH< ..HHucoEsuoexo. xommo newuouoeooo oHHobouoE on» as sewmmoudxo H annals—Tu accrue: oco 5H3 2.30 M93 oco noes mo mcoauonhEoo oououHoouou .m ouomwm cm s. c n m seems. nmezcnonmeg o o. % cm was m on A m co A m. co m on 9 V on m. 9 m. on w 00. 55 WW 'Ihisnewlydevelopedassay, derivedfranamethodtointroduce macro—molecules into cells in culture (reference 27 p. 68), was tested with cells of different types and fran different organisns with and without exposure to chemicals known to block intercellular communica- tion (El-Fouly et al 1987). The results were in agreetent with prev- ious studies. 'Ihe loading of madarane inperneable fluorescent dyes intracellularly was adiieved by scraping cells, grown in mlayer C111- turesasdescribedinMaterialsandMethods. Inprimarycell cultures, e.g. human fibroblasts and keratinocytes and in established cell lines lmown for their gap junctional carpetence, the low m Iucifer yellow (LY) dye diffused within seconds into contiguous cells. The extart of dyetransfer variedfranorecelltypetoanotter. Figure 10 representsthedeqreeofdyetrareferinmmanprinaryfibrcblastsani Figure llstnvsprinerytmmankeratinocytes loadedwithbothmarri rhodamine dextran. 'Ihe rhodamine dextran molecules (167 10,000) being larger than permissible size limit for crossing gap junctions retains in the primary loaded cells, thus labeling those initially scrape load- ed. 'Ihe LY, on the other hand, was able to diffuse freely presumably across the merbrane gap junction thus perneatinq the contiguous cells. Aclearmcalcentratimqradientwasmtedbeinqhigherattleedqeof thescrapedcellsarridecreasinginintensityfurtherawayfrunthe line of scraping. 'Ihe LY diffusim into caltacting cells contirued overtinearriwasdilutedortanibecamealmostmrietectableinabwt orehoun Incellstreatedwithdmicalslcumtoblockqapjmlctim- al intercellular cauumication the dye transfer was inhibited. A shady mtheeffectsofdose—responseardtaporalcwrseofcellexposmeto 56 c Figure 10. Gap junctional mediated dye transfer in primary human fibroblasts by the Scrape-Ioadinq/Dye Tramfer assay. 'Ihe extent of jmctiorelcmpeterneisdetemiredbythedegreeofluciferyellowdye trarefer among contiguous cells. a. Phase—contrast micrograph of tnlman fibroblasts in culture. b. Iucifer yellow transfer to contigu- ous cells. c. Rhodamine dextran marker dye labelling the primary loaded cells. a, b and c are photographs of the sane field using dif— ferent light filters. 57 Figure 11. Gap junctional mediated dye transfer in primary htman kera— tinocytes as detected by the Scrape-Ioadinq/Dye Transfer assay. Pri- nary loaded cells are labelled with the high molewlar weight rhodamine dextran dye (red fluorescence). Iucifer yellow (MW 457.2) permeated coupled cells further away frcm the scrape line. 58 certaingapjunction-modulatinq chanicalswastmdertakenandthere- sults indicated the sensitivity of the assay in detecting such changes (Suter et al 1987). In the experiments done here, wildtypeV79 cells including the six randanly selected subclones of wild type cells and those cells transfected with pSVzneo alone showed efficient dye trars- ‘fer (Figure 12). The cells transfected with c-Ha-ras-l (i.e., the )0 and x5) and their 61617 derivatives, in addition to the six pain-trans- fected clones, i.e. 1112331, 3, 5, 6, 7, and 20 andall their 6'IGr sub- clones showed cmplete irriibiticn of the dye transfer following scrape loading (Figure 13) . 59 Figure 12. Gap junction-mediated dye trarefer in V79 cell detected by Scrape—Ioading/Dye Trarsfer. a. Wild type V79 cells. b. Cells transfected with pSVzneo control plasmid. 60 b Figure 13. Inhibited gap jmetim—nediated dye trarefer in cells transfected with the c-Ha—ras-l oncogene. All six clones (i.e. m1, 3, 5, 6, 7 and 20) showed no dye transfer frcm the primary loaded cells tothecontiquousones. MRaslandMRasSarerepresentedinaandb respectively. 61 Experimental Cell Research 168 (1987) 422-430 Scrape-Loading and Dye Transfer A Rapid and Simple Technique to Study Gap Junctions! Intercellular Communication MOHAMED H. EL-FOULY. JAMES E. TROSKO and CHIA-CHENG CHANG Department of Pediatrics and Human Development. College of Human Medicine. Michigan State University. East Lansing. MI 48824. USA Gap junction-mediated intercellular communication has been recognized in cells from difl'erent tissues of various organisms and has been implicated in a variety of cellular functions and dysfunctions. Here we describe a new. direct and rapid technique with which to study this cellular phenomenon. It employs scrape-loading to introduce a low molecular weight (MW) fluorescent dye, Lucifer yellow CH (MW 457.2) into cells in culture and allows the monitoring of its transfer into contiguous cells. In communication- competcnt cells the dye transmission occurred within minutes after loading. The involve- ment of membrane junctions in Lucifer yellow transfer was verified by the concurrent loading of a high MW marker dye conjugate. rhodaminc dextran (MW 10000). Once introduced intracellularly the rhodarninc dcxtran is unable to cross the relatively narrow membrane junctions. Chemicals of variable potency known to block junctional communica- tion were tested in Chinese hamster V79 cells and other mammalian cells. The results showed effective blockage of the dye transfer at non-cytotoxic doses. This new technique can be applied to a wide variety of mammalian (including human) cells. In addition. it has the potential to be utilized as a rapid screening assay to detect chemicals that can modulate intercellular communication and to study their mechanism of action. (9 1987 Academic Press. lac. Gap junction-mediated intercellular communication has been considered as an important determinant for normal cell growth and differentiation [1—3]. Mamma- lian gap junctions permit the exchange of nutrients, ionic signals and regulatory molecules of approx. 1500 D among contacting, communication-competent cells [4]. Modulation of this phenomenon by either natural physiological states or by xenobiotics has been demonstrated in many studies [5-9]. Inhibition of this form of intercellular communication by various chemicals has been postulated to be a factor in the tumor promotion phase of carcinogenesis [10—12], teratogcnesis [13-16], neurotoxicity [l7]. reproductive dysfunction and other chemically-in- duced disease states [8]. Measurement of gap-junctional communication has been achieved by using electrocoupling [18], dye transfer following microinjection [19-21], radioactive metabolite transfer [22], metabolic cooperation of cells with enzyme deficiencies in certain metabolic pathways [23-25] and FRAP analysis (fluorescence recovery after photobleaching) [26]. While each of these techniques has certain advan- Copyright © 1987 by Academic Press. lac. All nuns at any form reserved «114482737 $03.1!) 62 Scrape-loading and dye transfer 423 tages, the complicated procedures, use of highly sophisticated equipment, or lengthy execution of experiments and other limitations prevent widespread meas- urement of this important biological process. In order to develop a rapid and reliable assay to measure gap-junctional communication, we extended an observation by McNeil et a1. [27], whereby certain non-penneable molecules could be introduced into cells via a ‘scrape- loading' technique. Scrape-loading has been effectively utilized to introduce macromolecules into cells in culture by inducing a transient tear in the plasma membrane without affecting cell viability or colony-forming ability [27]. The tracer dye Lucifer yellow (MW 457 .2) is an intensely fluorescent 4-aminophthali- mide with a high quantum yield of about 0.25 [28, 29]. This yield is stable between pH 1 and 10 and allows its detection with epifluorescence microscopy at low, non-cytotoxic concentrations. Lucifer yellow does not diffuse through intact plasma membranes and its low MW permits its transmission from one cell to another, presumably across patent gap junctions [28-30]. Preliminary experi- ments with the high MW dye conjugate, rhodamine dextran (MW 10000), indicat- ed that it can neither diffuse through intact plasma membranes nor cross the junctional channels. Upon excitation, rhodamine dextran emits red fluorescence with a spectrum distinct from that of Lucifer yellow. The concurrent introduction of both Lucifer yellow and rhodamine dextran into cells allows the identification of the primary loaded cells and therefore verifies that Lucifer yellow transfer to contiguous cells occurs through membrane junctions. We report here experiments indicating that this scrape-loading/dye transfer technique can be used to detect gap junctional communication in a wide variety of mammalian cells grown in vitro. MATERIALS AND METHODS To test the efficiency of the technique in detecting intercellular communication. five different lines and three primary cultures of mammalian cells were utilized. Chinese hamster V79 cells. rat glial primary cell culture derived from the cerebral tissue of a 20th day gestation rat fetus [31]. rat glioma cells. WB rat liver cells. human teratocarcinoma [32], and the primary culture of human foreskin fibroblasts (MSU-Z) were grown to confluency on 35 mm plastic plates in modified Eagle’s medium with Earle’s balanced salt solution with a 50% increase in vitamins and essential amino acids. except glutamine. The medium was supplemented with a 100% increase in non-essential amino acids. 1 mM sodium pyruvate and 3-10% fetal calf serum (FCS) depending on the cell type. The cells were incubated at 37°C in humidified air with 5% C02. A primary culture of calf aorta muscle cells obtained from tissue explants [33] was synchronized in defined, serum-free media (1 :1 F12 and Dulbecco supplemented with insulin 10“ M. transferrin 5 rig/l and ascorbic acid 0.5 M) [34]. and incubated at similar conditions. NIH/3T3 mouse fibroblasts were cultured in Dulbecco‘s modified MEM media supplemented with 10% FCS at 37°C in humidified air containing 8% C02. Cells were rinsed with PBS before the addition of the fluorescent dye mixture. Two milliliters of 0.05 % Lucifer yellow and rhodamine dextran (purchased from Molecular Probes. Inc. Eugene. Oreg.) dissolved in PBS were added to the cells and scrape-loaded at room temperature using a rubber policeman or wooden probe. The dye solution was left on the cells for 2 min. then discarded and the plates rinsed with PBS to remove detached cells and background fluorescence. Two milliliters of media were replaced and cells were examined under a Nikon epifluorescene phase microscope illuminated with an Osram H80 200 W lamp. Control plates were set by exposing the cells under similar Exp Cell Res I68 (I987) 63 424 El-Fouly, Trosko and Chang Fig. I. Positive dye transfer in communication- competent mammalian cells. (a-e) Transmis- sion of Lucifer yellow into contiguous cells detected 3-5 min after scrape-loading in Chi- nese hamster V79 cells. rat liver WB cells. NIH/3T3, calf aorta muscle cells (primary cul- ture) and human foreskin fibroblasts. MSU-Z (primary culture). respectively, conditions to the dye mixture but without scraping. The cells were then examined for fluorescence alter rinsing with PBS. To determine the sensitivity of the assay in detecting agents that block intercellular communication. the cells were exposed to a series of known tumor promotors of variable potency at non-cytotoxic doses. The chemicals used included the phorbol ester lZ-O-tetradecanoyl- phorbol-lJ-acetate (TPA). dieldrin. teleocidin. saccharin and mezerine. in addition to a negative control. 4-phorbol-12. l3-didecanoate (4a-PDD). Cells were treated with each chemical for an exposure time predetermined by previous studies [l0. ll. 35—39]. RESULTS In control experiments, cells treated with the dye mixture without scrape- loading did not pick up either dye after an equivalent exposure time. All cells scrape-loaded in the presence of a mixture of Lucifer yellow and rhodamine dextran showed a positive transfer of the Lucifer yellow alone into contiguous cells (fig. 1). This dye transmission occurred shortly after loading. The marker dye rhodamine dextran remained entrapped. thus labelling the primary loaded Exp Cell Res I68 ([987) 64 Scrape-loading and dye transfer 425 Fig. 2. Identification of pri- mary loaded cells. (a) Phase- contrast photomicrograph of human foreskin fibroblasts (MSU-Z) after scrape-load- ing in the presence of both Lucifer yellow and rhod- amine dextran dyes; (b) posi. tive transmission of Lucifer yellow into contacting cells: (c) rhodamine dextran shown confined to the pri- mary loaded cells. (a. b. c) Photomicrographs repre- senting the same scraped area. cells at the edge of the scraped areas (fig. 2). There was also a noticeable Lucifer yellow fluorescence gradient. with its highest intensity in cells at the periphery of the scraped areas. Making the assumption that there was no differential effect of scrape-loading on gap-junction function between cell types. the various cells examined differed in their ability to communicate as measured by the extent of the dye transfer. Chinese hamster V79 and mouse NIH/3T3 cells were the least efficient. whereas rat glial cells. calf aorta muscle cells. and human foreskin fibroblasts (MSU-Z). all primary cultures. showed extensive spread of fluorescence within the same period of time. In most cells. the decrease in Lucifer yellow fluorescence intensi- ty over time was accompanied by its further spread into contiguous cells. In all Exp Cell Res I681l987) 65 426 El-Fouly, Trosko and Chang Fig. 3. Blockage of dye transfer by tumor promoters. Nonocytotoxic doses of the phorbol ester TPA (5 and 10 ng/ml). dieldrin (7 uglml). teleocidin (1 ng/ml). saccharin (5 mg/ml). mezerine (2 ng/ml) and the negative control 4a-PDD (1 ng/ml) were each added separately to cells in culture prior to scrape- 1oading for a period of time that ranged from 15 min to 8 h (data on teleocidin. saccharin. mezerine and 4-PDD not shown). Effective blockage of dye transmission was only observed in cells pretreated with tumor promoters. (a. b) V79 cells mated with TPA (5 nglml) for 15 min; (c. d) rat liver cells (WB) after 3 h of dieldrin (7 rig/ml); (e. f) NIH/3T3 cells pretreated with TPA (5 ng/ml) for 1 h (g, h) calf aorta muscle cells (primary culture) after 30 min exposure to TPA (10 ng/ml): (Li) human foreskin fibroblasts (MSU-Z) following I h in TPA (5 ng/ml). 66 Scrape-loading and dye transfer 427 Fig. 4. Blocking of effective intercellular communication in rat glial primary culture by the neurotoxin dieldrin. (a) Cells viewed with phase- contrast microscopy after scrape—loading; (b) positive cell—cell communication is shown by Lucifer yellow transfer in contiguous cells; (c) inhibition of Lucifer yel- low transmission following pretreatment with dieldrin (7 pglml) for 3 h. plates treated with tumor promoters. including teleocidin. saccharin and mezer- ine (data not shown), the cells appeared to have lost their ability to communicate and the dye transfer was blocked (figs 3. 4). Both Lucifer yellow and rhodamine dextran were limited to a single row of primary loaded cells. The fluorescence remained intense for hours after loading with no indication of further dye transfer of leakage. In cells treated with the negative control 4a-PDD no inhibition of Lucifer yellow transmission was observed (data not shown). Regeneration and proliferation of primary loaded and secondary recipient cells were observed 24 h following scrape-loading, indicating that the procedure has no apparent adverse effect on cell viability. 28-878332 Exp Cell Res I68 ”987) 67 428 El—Fouly, Trosko and Chang DISCUSSION The results obtained using the scrape-loading and dye transfer technique are consistent with previous studies [10. 11, 35—39]. Cells. shown by other techniques to have gap-junctional communication, were also shown here to have a gap junctiomdependent transfer of dye after scrape-loading. Known tumor promoters inhibit this scrape-loading/dye transfer process in a way similar to that shown by other more complicated methods which, themselves, have been shown to be comparable in measuring gap-junctional communication [40]. The observation that different cell types varied in their ability to communicate might be attributed to several possible factors including cell type, tissue of origin. number of pre- existing and functional membrane junctions. mitotic activity, differential reaction to the scrape-loading process, cell volume and state of transformation or differen- tiation. The decrease in fluorescence with time in communication-competent cells is apparently the result of further dye spreading across membrane junctions. This observation was not made in cells blocked by tumor promoters. Among the tested chemicals the scrape-loading technique was used to study the effect of dieldrin. a known tumor promoter and neurotoxin [41. 42] on a primary culture of fetal rat glial cells. The remarkable communication demon- strated by the extent of Lucifer yellow transfer in untreated cells was strongly inhibited following exposure to a non-cytotoxic dose of dieldrin (7 rig/ml) prior to scrape-loading (fig. 4). This is consistent with the observations that dieldrin inhibited gap junction in human teratocarcinoma cells using metabolic c00pera- tion [39] and FRAP [26] techniques. The detection of this chemically induced interference with junctional communication in cells derived from brain tissue may carry important research implications in that it tends to support the observation that many tumor promoters can be neurotoxins [l7] and that cell—cell communi- cation may play an important role in brain development and function [43, 44]. The described technique might allow the study of cell-cell interaction in various cells derived from the brain and its modulation following exposure to drugs or other environmental factors known or suspected to affect brain function. Further investigations are needed to substantiate the close correlation between the pres- ence of gap junctions and the occurrence of electrotonic and dye coupling in the brain tissue of various animals [43, 44]. The described technique provides a direct and low cost approach to study gap junctional intercellular communication in cultured cells with the results obtained in a few minutes. It can be applied to a wide variety of cells grown in monolayer to study potential inhibitors of junctional communication and explore aspects of their mechanism of action. The follow-up of the same cells after reincubation allows the modification of dose. exposure and recovery time, as well as other experimental parameters. The rapidity of the assay ensures a specific and instant testing of cell-cell communication among secondary dye-recipient cells. It thus minimizes physiological alterations or artifacts that may be caused by other lengthy and complicated procedures. The technique offers sensitive and remark- Exp Cell Res I68 (I987) 68 Scrape-loading and dye transfer 429 able qualitative determination of cell-cell communication. For quantitative evalu- ation. the extent of the dye transfer can be estimated by counting the number of fluorescing secondary recipient cells in randomly selected areas on the plate. The possibility exists for automated quantitation by combining this technique with computerized FRAP analysis [26]. Our preliminary studies have demonstrated that dose—response effects can be observed and measured by both counting the fluorescing secondary recipient cells and FRAP analysis (M. G. Evans & M. H. El-Fouly. unpublished data). A further advantage of the method lies within its ability to monitor intercellular communication among as well as between different cell types grown in co-culture. The application of the technique to cells in vitro may prove valuable in testing the hypothesis that the inhibition of intercellular communication is involved in the promotion phase of carcinogenesis. It may also be developed as a rapid assay to screen for suspected chemical modulators of gap junctional communication and to investigate their effects on tissue development and cellular interactions. We thank R. Loch-Caruso for helpful discussion and M. B. Draznin for the calf aorta muscle cells. Research sponsored by the Air Force Office of Scientific Research. Air Force Systems Command. USAF. under Grant Number AFOSR-86-0084. The US Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. REFERENCES . Loewenstein. W R. Biochim biophys acta 560 (1979) 1. . Bennett. M V L & Goodenough. D A. Neuro res prog bull 16 (1978) 373. Hertzberg. E L. Lawrence, T S & Gilula. N B. Ann rev physiol 43 (1981) 479. Loewenstein. W R. Physiol rev 61 (1981) 829. . Gilula. N B. Reeves. 0 R & Steinbach. A. Nature 235 (1972) 262. . Lawrence. T S. Beers. W H & Gilula. N B. Nature 272 (I978) 501. Larsen. W J & Risinger. M A. 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Received May 6. 1986 Revised version received August 12. 1986 Exp Cell Res I68 (I987) 7O FUNDAMENTAL AND APPLIED TOXICOLOGY 9. 785-794 (1987) Dieldrin Inhibition of Gap Junctional lntercellular Communication in Flat Glial Cells as Measured by the Fluorescence Photobleaching and Scrape Loading/ Dye Transfer Assays1 S. Sun-m." J. E. TROSKO,*‘2 M. H. EL—FOULY,‘ L. R. Locrtwooof AND A. KOESTNER‘!‘ ’C enter for Environmental Toxicology and Department of Pediatrics and Human Development and fDepartment of Pathology. Michigan State University. East Lansing. Michigan 48824 Dieldrin Inhibition of Gap J unctional lntercellular Communication in Rat Glial Cells as Mea- sured by the Fluorescence Photobleaching and Scrape Loading/Dye Transfer Assays. SUTER. S.. Taosrto. J. F... EL-FOULY. M. H.. Locrtwooo. L. R.. AND KOESTNER. A. (1987). Fundam. Appl. Toxicol. 9. 785-794. Application of the fluorescence-recovery after photobleaching (FRAP analysis) technique and scrape loading/dye transfer assay was made to measure the pres- ence of gap junctional communication in primary rat glial cells in vitro in the presence and absence of the neurotoxicant and tumor promoter dieldrin. a chlorinated insecticide. Results demonstrate that primary rat glial cells are able to exhibit gap junctional intercellular communi- cation and that dieldrin at noncytotoxic concentrations can modulate gap junctional communi- cation as early as 10 min after exposure to the chemical and that the effect is reversible after 4 hr recovery from the dieldrin exposure. Both the FRAP analysis and the scrape loading/dye transfer assay have validated the observation that dieldrin inhibits gap junctional communica- tion in other cell types using different techniques to measure gap junction function. These results were interpreted as an indication that inhibition of gap junctional communication might con- tribute to the cellular mechanism of dieldrin‘s neurotoxicity. e 1987 Society ofToxicology. Gap junctional-mediated intercellular com- munication has been regarded as an impor- tant determinant for homeostasis in organ- isms composed of functionally specialized cells for normal cell growth and differentia- tion, reproductive. neuroendocrine. and car- diac function. and a whole host of other nor- mal physiological states (Bennett and Goode- nough. 1978: Loewenstein. 1979; Hertzberg et al.. 1981; Pitts. 1980; Bennett et al.. 1981; ' Research was sponsored. in part. by a grant from the College of Human Medicine and by the Air Force Office of Scientific Research. Air Force Systems Command. USAF. under Grant No. AFOSR-86-0084. to J. E. Trosko and by a National Institute of Health Grant (CA32594) to A. Koestner. 2 To whom all correspondence should be addressed at Department of Pediatrics and Human Development. Michigan State University. 8240 Life Science Building. East Lansing. MI 48824. 785 Schultz. 1985: Larsen. 1983). Low-molecu- lar-weight substances ($1500 MW) can be transported from cell to cell via gap junctions on contiguous cells (Loewenstein. 1979). Dis- ruption of gap junctional intercellular com- munication has been postulated to play a role in carcinogenesis (Loewenstein and Kanno. 1966). specifically during the tumor-promo- tion phases (Yotti et al.. 1979: Murray and Fitzgerald, 1979: Trosko et al.. 1983). In addition. many tumor-promoting chemicals (Jone et al.. 1985) and a few oncogenes (Chang et al.. 1985: Azarnia and Loewen- stein. 1984; Atkinson and Sheridan. 1984: Atkinson et al.. 1986: Azamia and Loewen- stein. 1987) have been associated with inhib- ited intercellular communication. Gap junctional intercellular communica- tion has been measured by a variety of tech- niques. including electrocoupling (Furshpan 0272-0590/87 $3.00 Copyright c 1987 by the Society of Toxicology. All rights of reproduction in any form reserved. 71 786 and Potter, 1959); intercellular transfer of in- jected fluorescent dyes (Loewenstein, 1966); use of genetically deficient cells to measure “metabolic cooperation” (Hooper, 1982; Da- vidson et al.. 1985; Gupta et al.. 1985); and autoradiographic detection of the transfer of low-molecular-weight radioactive labeled compounds (Subak-Sharpe et al.. 1969). The ultrastructural analysis of gap junctions is performed by freeze-fracture analysis of cell membranes (Finbow and Yancey, 1981; Larsen, 1983; Larsen and Risinger. 1985). Recently, two new techniques, one using fluorescence-recovery after photobleaching (FRAP analysis), and the other. the scrape loading/dye transfer assay, have been applied to measure gap junctional intercellular com- munication (Wade et al.. 1986; EI-Fouly et al.1987) Dieldrin, belonging to the cyclodiene class of chlorinated insecticides, is a well-docu- mented toxic chemical. It has been found to be carcinogenic in laboratory rodents. spe- cifically it seems to act as a tumor promoter (Ito et al.. 1980; Tennekes et al.. 1982). Sim- ilar to lZ-tetradecanyolphorbol- l 3-acetate (TPA), dieldrin has been shown to be non- mutagenic in most genotoxic assays (Mc- Carin et al.. 1975; Ashwood-Smith, 1981; Purchase et al.. 1978; Probst et al.. 1981; Tong et al.. 1981; ICPEMC. 1984). On the other hand, dieldrin has been shown to in- hibit metabolic cooperation (a form of gap junctional communication) in Chinese ham- ster V79 cells (Trosko et al.. 1987) and hu- man teratocarcinoma cells (Lin et al.. 1986). In addition. dieldrin is known to be a neuro- toxin (Joy, 1982). Since gap junctions are known to exist in neuroectodermal cells, this study was designed to determine if some of the neurotoxic effects of dieldrin might be re- lated to its ability to inhibit gap junctional in- tercellular communication. MATERIALS AND METHODS Cells. Normal rat glial cells were subcultured from pri- marily cultured rat glial cells isolated from cerebral tissue SUTER ET AL. of rat fetuses at the 20th gestation day (K0 et al.. 1980). Cells within 10 passages were grown in modified Eagle's medium (MEM; GIBCO formulas 78-5470: Earle‘s bal- anced salt solution with 50% increase of vitamins and essential amino acids except glutamine), supplemented with nonessential amino acid (100% increase). 1 we so- dium pyruvate. and 10% fetal calf serum. Under the in- cubation condition with 5% CO; in humidified air at 37°C, cells growing in monolayer. contact-inhibited upon confluency. were subcultured every 5 to 7 days. Chemicals. 5 (and 6)-Carboxyfluorescein diacetate and rhodamine lissamine dextran (Lot SB) were obtained from Molecular Probes ( Eugene. OR). Lucifer yellow CH was from Sigma Chemical Co. (St. Louis. MO). Dieldrin [Shell Chemical Co. (purity 99+%)] was a gifi from Dr. B. V. Madhukar of the Pesticide Research Center at Michigan State University. Methods. Experiments were performed with rat glial cells plated in the modified MEM. Dieldrin. dissolved in ethyl alcohol (ETOH). was added to cells for various lengths of time to give a final concentration of 7 jug/ml of medium (0.1% final concentration of ETOH). An identi- cal volume of ethyl alcohol. the solvent carrier. was added to the control cells. Neither the solvent carrier nor the carrier plus dieldrin was cytotoxic to the cells at this concentration nor did the concentration of ETOH inter- fere with intercellular communication. To measure gap junctional communication using the . ‘ FRAP analysis technique, following 24 hr of growth. the cells were washed with PBS containing calcium (0.9 mM) and magnesium (0.5 mM: PBS/Ca/Mg) and stained with 6-carboxyfluorescein diacetate. The dye and labeling conditions do not affect cell viability. and restaining can be performed on the same cells for several days. All mea- surements are performed at room temperature in PBS/ Caz‘IMg” within a l-hr period. A tissue culture plate of labeled cells is placed on a high-speed computer-con- troled two-dimensional stage of the AC AS 470 worksta- tion (Wade et al.. 1986). The Meridian ACAS 470 (An- chored Cell Analysis and Sorting. Meridian Instruments. Okemos. MI) was the standard instrument which was equipped with a 2-W argon ion laser tuned to the 488- nm line. dichroic filter at 510 nm and barrier filter at 520 nm. inverted phase-contrast microscope and 16-bit mi- crocomputer for data acquisition and processing. and micro-stepping stage. The stage moves the cells in a de- fined manner above the objective (40x) of an inverted epifluorescence microscope. The microscope objective serves to focus the argon laser beam (excitation wave length of 488 nm) to a l-um spot size that excites fluo- rescence in individual cells at 1.5-um steps in a two-di- mensional raster pattern. The single-point emission from each excited step is recorded as an intensity by a photo- multiplier tube. The digital signals representative of flu- orescence intensity are stored in the computer with the source x—y location. The emitted intensities are color 72 GAP JUNCTIONAL INTERCELLULAR COMMUNICATION coded and presented on a computer video screen as a pseudo-color image of the fluorescence distribution in the analyzed cell. In order to measure gap junctional communieation by another independent method. the scrape loading/dye transfer assay was utilized. Rat glial cells were subcul- tured using trypsin (0.01%) without EDTA and plated to attain a confluent monolayer ( 1.5-2.0 X 10‘ cells) in 35- mm plastic dishes. The cells were incubated in the modi- fied Eagle's medium with 5% FCS at 37°C in humidified air with 5% CC; for 12-18 hr. Six plates were prepared for each experimental point including untreated controls and controls with solvent (0.1% absolute ethanol final concentration) only. For temporal studies. the cells were treated with a single dose of dieldrin for various exposure . times (6. 10. 15. 20. 30. 50. and 60 min and 24 hr). This predetermined noncytotoxic dose of 7 rig/m1 has been previously shown to induce a complete blockage of gap junctional intercellular communication and dye transfer in rat glial cells (EI-Fouly er al.. 1987). In addition. to test for the reversibility of the dieldrin effect on gap junction conductance following a short-term exposure. the cells were treated with dieldrin (7 rig/ml) for 1 hr then washed with PBS and reincubated after the addition of fresh me- dia for 24 hr. For dose-response experiments. dieldrin was added to each plate at various noncytoroxic concentrations (1. 2. 3. 5. 6. 7. and 10 ug/ml) for a fixed 2-hr exposure time prior to scrape loading. In preparation for scrape loadi ng/dye transfer, the cells were washed with PBS (kept at room temperature). then exposed to a dye mixture containing 0.05% of each of Lucifer yellow (MW 457.2) and rhodamine lissamine dextran (MW 10.000) dissolved in PBS. The dye mole- cules were loaded intracellularly by scraping or cutting the cells using a wooden probe or a sharp knife. The dye solution was left on the cells for 90 see. then discarded. and the plates were carefully rinsed in PBS to minimize the background fluorescence. The cells were next exam- ined for dye transfer under an inverted Nikon epifluoreo scence phase microsc0pe with uv light generated from an Osram HBO ZOO-W bulb. The degree of communieation was assessed by measuring the extent of Lucifer yellow transfer into contiguous cells. Quantitation was esti- mated by counting the number of secondary recipient cells in a fixed surface area selected at random. Ten different fields were examined per plate. six plates per treatment. and an average count is reported as a relative percentage compared to control plates which were con- sidered to have 100% communication. RESULTS The effect of dieldrin on the colony-form- ing ability is shown in Fig. 1. Results show 787 100 90 80 7O 60 50 40 30 20 10 $5 Survwol IITTTTITT O O O -r- p- .— P. P- “F 2 3 4 5 Concentration lug/ml) 0) FIG. I. Effect of dieldrin on the colony-forming ability of primary rat glial cells. The plating efficiency was 87%. Five plates per dose level were counted. that after a 3-day exposure, even at the high- est concentration (7 ug/ml), very little effect was noted in terms of inhibition of the plating efficiency and formation of colonies. It must be noted that the cytotoxicity assay is per- formed at very low cell densities (200 cells/ 60-mm plate). and long exposures to the chemical (3 days). whereas the effect of diel- drin on gap junctional communication is done on high densities of cells for short peri- ods of time. Therefore. these cytotoxicity data would be considered overestimates of the effective cytotoxic levels. In other words. dieldrin at 7 ug/ml (or up to 10 ug/ml) for scrape loading/dye transfer) should not be cy- totoxic under the conditions used to measure its effect on gap junctional communication. In order to ascertain whether FRAP analy- sis could detect gap junctional intercellular communication in primary rat glial cells. an experiment, as illustrated in Fig. 2, was per- formed. The results clearly demonstrate that 18 min after photobleaching of a single un- treated cell. the fluorescence reappeared in coupled cells. but not in isolated cells. This is interpreted as indicating that the carbox- yfluorescence dye was transferred, via gap junctions. to the photobleached cell. The lack of fluorescence in the isolated cell demon- strates that a new source of dye can be re- placed only from gap junctionally coupled 788 73 SUTER ET AL. Cslor Values 74 CAP JUNCTIONAL INTERCELLULAR COMMUNICATION TABLE 1 Resume or FRAP ANALYSIS ON DIELDRIN-TREATED RAT GLIAL CELLS Percentage of cells with Number fluorescence Treatment of cells recovery Control 25 100 24 hr pretreatment with dieldrin (7 ug/ml) 40 0.0 1 hr pretreatment with dieldrin (7 ug/ml) 6 0.0 10 min pretreatment with dieldrin (7 jug/ml) 43 0.0 1 hr pretreatment with dieldrin plus 4 hr post- treatment minus dieldrin 20 100 1 hr pretreatment with dieldrin plus 1 hr post- treatment minus dieldrin 6 83 unphotobleached cells. This figure is typical of 25 other samples in the same dish. To test if dieldrin could inhibit gap junc- tional intercellular communication in rat glial cells, the experiment showed that 24 hr treatment with 7 rig/ml dieldrin prevented gap junction-mediated transfer of the fluo- rescent dye (Table I). Results in Table I also demonstrate that a l-hr treatment of 7 ug/ml dieldrin was suffr- cient to inhibit gap junctional communica- tion as measured by FRAP analysis. This re- sult was typical of six randomly chosen cou- pled cells. To determine if shorter treatment times could inhibit gap junctional communication a series of experiments were performed. The 789 data in Table I illustrate that a lO-min expo- sure to 7 ug/ml dieldrin was sufficient to in- hibit communication. Again, this result was repeated in 43 other randomly chosen cou- pled cells. Since it is important to determine if the _ dieldrin inhibition of intercellular communi- cation is either irreversible or reversible, cells were treated with dieldrin (7 rig/ml) for 1 hr and allowed to “recover" for l and 4 hr after the dieldrin was removed. Cells were washed twice and placed in fresh non-dieldrin con- taining medium. Data in Table l, representa- tive of 20 random samples. show that a 4-hr post-treatment time was sufficient for the re- establishment of gap junctional communica- tion. In addition, results also indicate that 1 hr seems suflicient for these rat glial cells to reestablish gap junctional communication in six randomly chosen cells. Scrape Loading Results The results obtained from scrape loading/ dye transfer assay are shown in Figs. 3 and 4. Quantitative analysis of the dose-response data, as described under Materials and Meth- ods, indicates a direct correlation between the extent of blockage of gap junctional transfer of Lucifer yellow and the dieldrin concentra- tion applied for a fixed period of 2 hr (Figs. 3 and 4A). When the cells were treated for variable exposure times with a fixed dose of dieldrin (7 ug/ml), the extent of junctional communication was inversly correlated with the duration of treatment (Fig. 48: also data not shown). Complete inhibition of dye transfer was observed after approximately 50 min of initiating the treatment (Fig 4B). The FIG. 2. Restoration of fluorescence in photobleached. control primary rat glial cells. By comparing the images generated before photobleaching. when all cells were highly fluorescent as indicated by the false- color image in (A). with images produced 1 min (B) and 18 min (C) after bleaching. the recovery of fluores- cence could be monitored. The image in (C) clearly shows the contacting, but not the isolated. cell regained its image after 18 min postbleaching. Image is x300. 75 790 SUTER ET AL. 76 GAP JUNCTIONAL INTERCELLULAR COMMUNICATION ITIIIj‘I'Y» o r z s s e 7 ro‘ B Dieldrin Concentration (no/ml) ramming.) Percent of Lucifer Yellow Dye-Recipient Cells TlUF-IVIU' O 0 IS 20 3O 50 2“! Exposure Time (minutes) Dieldrin Frag/rel) FIG. 4. (A) Inhibition of dye transfer in rat glial cells by dieldrin as detected by scrape loading/dye transfer assay. The method for quantitation is described under Materi- als and Methods. Dose-response effect of dieldrin on gap junction-mediated Lucifer yellow transfer. The cells were treated with variable concentrations of dieldrin for a fixed period of 2 hr. Panel (A) is a graphic representa- tion of experiments shown in Fig. 3. (B) Time-course of - the efl‘ect of dieldrin on dye transfer. A single treatment dose of dieldrin (7 ug/rnl) was added to the cells for the indicated time periods followed by scrape loading of Lu- cifer yellow. blockage of cell-cell communication by a sin- gle application of dieldrin was sustained for over 24 hr (Fig. 3). The inhibition of dye transfer was reversed when the cells were transiently exposed to dieldrin (7 rig/ml) for 1 hr then released and re-incubated in fresh medium. These cells resumed their control level of communication when examined 24 hr following the removal of dieldrin (data not shown). DISCUSSION There seem to be several conclusions re- sulting from the observations made during 791 this study: (a) FRAP analysis and the scrape loading/dye transfer assay have verified ear- lier conclusions that gap junctional commu- nication exists in rat glial cells (Orkand, 1977; Massa and Mugnaini, 1985); (b) dieldrin ean inhibit gap junctional communication in rat glial cells, as measured by FRAP analysis, supporting previous observations that noncy- totoxic levels of this toxic chemical inhibited gap junctional communication in Chinese hamster V79 and human teratocarcinoma cells as measured by metabolic cooperation and uridine transfer (Trosko er al.. 1987; Lin et al.. 1986); (c) FRAP analysis and scrape loading/dye transfer techniques, by corrobo- rating the aforementioned studies, seem to be validated as a legitimate means to measure gap junctional intercellular communication; and (d) the effect of dieldrin inhibition of gap junctional communication is a reversible phenomenon. Since gap junctional intercellular commu- nication has been postulated to play a major role in the regulation of development, cell proliferation, regeneration, differentiation, homeostasis, and control of differentiated cell functions (Loewenstein, 1979; Pitts, 1980; Hertzberg etal. 1981; Schultz. I985; Larsen. 1983) in multicellular organisms, it seems logical to conclude that exogenous and en- dogenous chemical modulation of gap junc- tion structure and/or function would have adaptive and nonadaptive consequences (Trosko and Chang, 1984). Many chemicals, which are known to be tumor promoters, have been demonstrated to be inhibitors of gap junctional communication (Jone er al.. 1985: Trosko er al.. 1982; Malcolm et al.. 1985). One of those chemicals which is a tumor promoter of rat liver tumors and which inhib- its gap junctional communication is dieldrin. FIG. 3. Dose-response effect of dieldrin on junctional permeability in rat glial cells as measured by the scrape loading/dye transfer technique. The photomicrographs show Lucifer yellow transfer into contiguous cells pretreated with various concentrations of dieldrin. (A) Untreated control cells; (B-H) cells pretreated for 2 hrwith l. 2. 3. 5. 6. 7. and 10 ug dieldrin/ml. respectively. 77 792 What makes the dieldrin effect on the inhibi- tion of gap junctional communication in rat glial cells relevant to these results is that diel- drin is also a known neurotoxin (Joy, 1982). In the former case, it has been postulated that when gap junctional communication is in- hibited in tissues where a single carcinogen- initiated stem cell is repressed by surrounding normal cells, the initiated cell then clonally expands to form a tumor (Yotti et a1., 1979; Trosko et al.. 1983). In the latter case, al- though the role of gap junctional communi- cation in neural cells has not been as well studied as the chemical neurotransmission form of intercellular communication, it is known to exist in brain tissue (Andrew et al.. 1981). In addition, two neurotransmitters, acetycholine and dopamine, have been shown to modulate gap junction function from several organisms (Iwatsuki and Pet- ersen, 1978; Findlay and Petersen, 1982; Ter- anishi et al.. 1983; Piccolino et al.. 1984; La- sater and Dowling, 1985; Neyton and Traut- mann, 1986). Since it would be hard to imagine, in evolutionary terms, that gap junctional communication plays no role in this highly specialized tissue. modulation of gap junction function in brain cells by diel- drin might be expected to play some role in its neurotoxicity. Finally, as a note of speculation, one could imagine that, in the brain. chemical neuro- transmission and gap junction transfer of ions and small molecular weight molecules comprise a highly coordinated and integrated intercellular communication network (Ben- nett er al.. 1985). Conceivably, gap junctional communication provides a means to regulate growth control and differentiation of premi- totic cells, as well as a means to provide “nu- trients” and regulatory signals to postmitotic neural cells. Endogenous and exogenous modulation of gap junctional communica- tion in either pre- or postmitotic brain cells could have both adaptive, as well as toxic, consequences. depending on the nature of the inhibition. SUTER ET AL. REFERENCES ANDREW, R. D.. MACVICAR, B. A.. DUDEK, F. F... AND HATI‘ON, G. I. (1981). Dye transfer through gap junc- tions between neuroendocrine cells of rat hypothala- mus. Science 211, 1 187-1 189. ASHWOOD-SMITH, M. .1. (1981). 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P., WARREN, S. T.. TSUSHI- MOTO, G., AND CHANG, C. C. (1982). Inhibition of cell-cell communication by tumor promoters. In Car- cinogenesis (E. Hecker. N. E. Fusenig, W. Kunz, F. Marks, and H. W. Thielman. Eds), Vol. 7. pp. 565- 585. Raven Press. New York. WADE. M., TROSKO, J. E.. AND SCHrNDLER. M. (1986). A fluorescence photobleaching assay of gap junction mediated cell-cell communication between human cells. Science 232, 429-552. Yorr'rr, L P.. CHANG. C. C.. AND TRosrto. J. E. (1979). Elimination of metabolic cooperation in Chinese hem- ster cells by a tumor promoter. Science 206, [089-1091. DISGISSICN The data obtained frail the reported studies verified the fact that thewildtypedtinesehamsterfibrcblasts (V79) havecarpetentgap junctional intercellular camamicaticn. ‘Ihis assessment was determined by both the metabolic cooperation and the scrape-loading/dye transfer assays. 'Ihese cells were stably tramformed following transfection with recanbinant plasmids: the control psvzneo, containing the nearly- cinrmisrtancegeneasaselectivemarker, andthepsm4 plasmid. The latterccrrtairsbcththeselectivenarkerandtheentirelnmanc—Ha— ras-1 oncogene derived fran the EJ/T4 bladder carcinana (Shih and Weinberg 1982). Southern blot analysis showed the presence of a faint 6.6Kbbardinthewildtypeccrrtmlcellsarrimraintensebarflsof similar and variable sizes in all Banal-digested INA fran clones trans- fected with pswo4 , indicating variable integration sites (Figure 4) . 'Il're stability of transformation was verified, and maintained, by selec- tion in G418 antibiotic. The expression of c—Ha-ras-l was tested by indirect immofluores— cence using macelcnal antibody, clone Y13-259 (mrth et: a1 1982). Intense flmmnemsdetectedinflnsecells transfectedwithc—Ha- ras-1, ascarparedtoabarelydetectabletrace inthewildtypecells and in cells transfected with the ccrrtrcl plasmid psvzneo (Figure 5). 'nrecellsexprassingthelerasproteinacquiredamretransfomed mrpholcgyascarparedtocartmlwildtypeatflthcsetransfectedwith psvzneo alme. 'Ihe alterations included spindle-shape mrpholcgy, 80 81 refractilecellcutlixewithanirregulargrcwthpatternandlackof cmtact irhibitim. The cells appeared overlapping and piling in clmpswithnecrcticca'rters. ‘Ihiswasinadditicntoamrked decreaseofcelladheramcetothesubstrateand, insanecells,the appearance of cytoplasmic vacuoles. In the netabolic cooperatim assays, the variety of the co-cnl- tnned combinations resulted in a remarkable recovery of 6'16r colmies vhereverthec—Ha-ras—lanogenewasexpressedineitherorboflmco— cultured cell types. Statistical analysis of three different experi- ments of netabolic cooperatim showed reproducible results (p < 0.0005 top< 0.00001). ‘Iheresults obtainedbyassayim forgapjunctiamal cammication utilizing the scrape-loading/dye transfer technicpe were in agreement with those of the netabolic cocperation assays. All cells transfected with c-Ha-ras-l showed cmplete inhibition of Iucifer yel- lowdyetransferascmparedtothewildtypeandpsvzneotransfected cartrcl cells. In the control series, the hanogeneity of gap juncticn- al camunication ability ammg the pooled wild type cells was verified in eighteen different subclcnes by scrape-loading/dye transfer and six ofthenwerereportedintvnofthemetaboliccoopemtimassaysper— formed. Hunthedatadvtainedintlnseriesofeaperimentsreportedhexe, astxulgcorrelatimappearstoeocistbetweentheexmessimofthec- Ha-ras-l product i.e., the p21 polypeptide, as detected by monoclcnal antibody against p21, and the inhibition of cell-cell carmmication via the maIbrane gap jumtions. This correlation resalbles those effects anertedbyeamgewnisd'micnltmnorpmters (Yattietal 1979, nix-ray and Fitzgerald 1979, m et a1 1987), by certain growth factors 82 (Madlmkar et a1 1988), metabolites (Malcolm et a1 1985), hornranes (Mark et a1 1972, Decker 1976, 1981, [hhl ard Berger 1978, Garfield et a1 1980), and by nembrane bound oncogenic products (Azarnia and Ioewenstein 1984b, Chang et a1 1985). This end point parallelism be- tween the p21 effects and those of factors that modulate gap jtmcticnal intercellular cammmicntion lerd support to the hypothesis under study, i.e. that the oncogenic precinct of the H-ras my play a role in the process of tlmlor pranotion by inhibition of junctional coupling. ‘mesymetrybetweentheendoganzsempressimofraspnardthe exogenous euposuretotmmrpranoters inmdulatingcellulargapjmuc— tiorshasitsgramdsinseverelraterkableprepertiesoftherasgene product. ‘Ihecncogenic, aswellasthemrnalcellularraspreto—onoo— genes, products share a cannon intracellular translocntion site, i.e. the inner side of theplasma membrane (Willumsen et a1 1984), andthsy only differ by a single point nutaticn at specific mcleotides (Barbacid1987). Boththeancogenearrithepmto-aaoogenehavestnxc- tural, as well as biochanical similarities to the G-prcteins which are cmponerrtsoftmmajornmbrare signal tramduction systens, theaden- ylate cyclase (Wakelam et a1 1986) and PIPZ (Scolnick et a1 1979, Willingham et a1 1980) . 'Ihe G-preteins, also lawn as guanine-nucleo- tide-birding proteins, are coupled to cell surface receptors and assist in the process of mediating a mitogenic signal to an effector molecule (3) via modulating adenylate cyclase. ‘Ihis modulation alters the intracellular levels of cAMP which, when elevated mhame gap junc- tion-mediated munication (Saez et a1 1986, Wiener and Loewenstein 1983, Johnson et a1 1985) . Activation of G—proteins subsequently lead to intracellular signal transduction (Sweet et a1 1984, McGrath et a1 83 1984, Iacal et al 1986, Gibbs et a1 1934, Colby et al 1986). Recently, ras expression was found to correlate with decreased adenylate cyclase activity and low cAMP levels in NIHBTB cells (Hiwasa and Sakiyama 1986) . Given that low (NIP is associated with decreased gap junctional cammication (Saez et al 1986, Wiener and Ioewenstein 1983, Jdmson et al 1985), those observations do not contradict the results obtained here, i.e. that ras expressim is acoatpanied by decreased cellular coupling. 'n'xerasproductmsfan'ritobeanintegralcmpaaentoftlnvery versatile mamrane signaling pathway, the inositol [mosphate system (Fleishman et a1 1986). 11113 pathway transmits signals induced by growth factors and other mitogenic agents intracellularly, whidi irdtrce an action that results in [NA synthesis and cell proliferatim (Feramisco et a1 1984, Stacey and 10mg 1984). In this systen (see Fig- ure 14), the manbrane phosphoinositides (PIP) are cleaved by phospho- lipase c, whidacmldbeactivatedbytheG—prcteins, intotwobypro— ducts, the diacylglyoerol (ms) and inositol triumphate (193) (Berridge 1983) . 'Ihe diacylglyoerol is an essential co-factor for PKC activation (Nishizuka 1984) ard the IP3 is known to mdailize the cal- cium ions fran intracellular, non mitochondrial stores (Berridge et a1 1984). 'Ihe H-ras expression was reported to stinulate an increased production of DAG, as well as inositol phosphates in ras transformed cells (Lacal et a1 1987, Wolfinan and Macara 1987, Chiantgi et a1 1986, Preiss et al 1986, Fleischnan et a1 1986, Wakelam et a1 1986). The ms is known to activate PKC (Berridge 1984, Kikkawa et al 1983, Sharkey et a1 1984) whichalsoactsasareoeptorto, andisactivatedby, the potert um pranoter, TPA (castagna et al 1982, Niedel et a1 1983, 84 ..cemd Adam scan cognates. souaau cauuosoucouu anemia ocnuoEoEmcouu a no noduoucoaoueou Owuoaozom .vaousmah 0 ounces :wououe «use coercmorcdc m.v Acuqmocwocemose “mum uncommon unasaaoo muozemoze scauocsu _ aqua douamocm "nee on. use rllll. taboos ++ rezone Acauccecm “mom \\a . Acuooaac~>oofic uoeuoe. AaooHoeo: maze cwououe o mmmewa c norcmore .xmo~oeo:. 000.. uom.o.ov acueoooz ' 8533 233 oxm 85 Nishizuka 1986) . Recently, the activated PKC was found to be involved durim the cellular respmse to H-ras expression (Iacal et a1 1987). Furthermore, thedcwnregulationofPKbephorbolesters (Pastietal 1986) eliminates the cellular mitogenic response to ras p21 (Iacal et a1 1987) . 'Ihe activaticn of PKC was inplicated in the blockage of gap jtmctiasarflwasreoentlyamntodirectlymosmorylatethejmic- tionalprcteins ofrathepatocytesinacell-freesystan ('I‘akedaetal 1987) . In addition to being strongly involved in tramnenbrane signal- ingsystets, boththecellulararriauoogenicrasledirectlyindwe umbranechanges. 'Ihesediangesarebothrapidarrilonglastingwith the cncogenic p21 (Bar-Sagi and Feramisco 1986) and they occur follow- ing its microinjection into qriescent, contact irhibited cells. 'Ihe umbrane alteratims include Wm rufflin; and pinocytosis (Bar- SagiandFeramisco1986). Werufflingdistortsthecellmrfir- ologyarditsmrmalbamdaryanisurfacesofcattactvmilepinocytosis affects intracellular haneostasis, particularly by increasing ca++ in- flux. Calcium ions are knom to activate the calcium sensitive PRC (May et a1 1985) , an effect associated with the blockage of jmcticnal coupling (Rose and Ioewenstein 1975, Rose et a1 1977). 'Ihe cell sur- facerufflirgandpinocybosiswerealso fourritobeinducedbyeocposing cells maintained in serum—free medium to either growth factors e.g., EBF (Haiglar et a1 1979) or poor (Davies and Ross 1978), or serum (Brink at al 1976) , DEF (Camolly et a1 1979) or insulin (Goshima et a1 1984). 'Ihe fact thattherasprotein directly induces similarchangss might indicate its ability to bypass the cellular need for external mitogenic stimli in order for the cell to proliferate. Anotherdiaracteristicoftherasgenepmductisthatit 86 initiates INA synthesis and cell proliferatim (Feramisco et al 1984) and confers bmorigenic prcper'ties on preneoplastic or inmortalized cell lines or cells expressing certain nuclear oncogene products e.g. myc, p53, Ela, or the large T antigen of SV40 virus (Land et al 1983a, 1983b, 1986, Parada et al 1984, Van Roy et a1 1986, Beer at al 1986, Yanoopoulous et al 1985, Ruley 1983, Segana and Yamguchi 1987, Connan et a1 1985). Interestingly, this latter property is shared by the phorbol ester, TPA, which irriuced tramformed foci in cells inmortal- izedbythemyccncogene (Oamanetal1985). Anadditionalanalogy vasreportedbetueenrasaniTPAvmentheybouiactedsyrergistically during in gitrg transformation, thus suggesting a cannon cellular ef- fect of both of them (Dotto et al 1985). Althoughtheras expression has been correlated with cellular transformation and proliferation, it wasfamdtoprmotedifferentiatimincertaincellsystais (Nodaet a1 1985, Bar-Sagi and Feramisco 1985, Hagag at al 1986), an effect similartothatinducedby‘I'PAinsalecells (Yamasaki1984b). 'Ihis differential effect could depend partially, or entirely, on the bio- dmicalormlecularbackgramofthehostcell. Inotherwords, the rasgeneregulaticn, incertainhostcells, maybealteredbysuppres- sorgenesor"anti-oncogenes" or itsproductmaymtundergocertain post-tramlaticnal modifications necessary for its activity. Iheaforelentia'edpmpertiesoftherasaicogeneproductbythan- selvesconstitutestrongbasistosuspectaportential rolethep21 might play during the stages of tumor pranotion, i.e. the induction of proliferation and the inhibition of gap junctional camunication. 'Ihis rolevmldbesimilarinsanerespectstothatplayedbyemogelw tmnorprunoterswiththedifferencebeingthatrasleis 87 constitutively expressed in the cell. If this particular cell were initiated, or to be initiated, then, most of the requirenents for cel- lular transformation and proliferation could be met and clonal expan- sim would follow. As mentioned earlier, the rapid proliferatim of initiated "inmrtalized" cells increases their dances for adiiticnal geneticdanageandttmscontributestottmorprogressim, andmetas- tasis, aswellasheterogeneity. 'merasaicogenewasfommdhighly expressedinseveralnetastatictmnrcellsandlessexpressedinmal- ignant tutors before invasion ard netastasis (Barfly et a1 1985, Bradley et a1 1986, Tahara et al 1986, Egan 1987, Collard et al 1987). This fact might indicate that the ras oncogene, in sane system, might play a more inportant role during tumor progression and metastasis rather than an earlier one in the initiation phase. Reportstothecontraryarguedthattherasmcogenemayinflterne the initiation stages of carcinogenesis (Balmin et a1 1984, Brown et a1 1986). In the reported experiments, the mouse skin keratinocytes were exposed i_n_v_i_g_p_ to 7,12-dimethylbenz[a]anthracene (EMBA), a chan- ical known to reproducibly activate the H-ras cnoogene, or were sub- jected_i£yiv_otoHarveym1riresarcalavimscmtaininganactivated ras. These cells acqrired tmnorigenic properties within few weeks fol- lmingexpoairetothepotentumorpranoter'rPA. However,thej.n gimmmyMassaysutilizedforthoseuperimsnts failedto demonstrate whether the hmnrigenic cells expressing the ras a'noogene were not inmortalized or initiated before hand. It thus appears that thepossible role oftherasoncogeneinmitogenesisani "unnorprano- tion" isirducedbymatbrare-depenientreactiorsthataretrarsduoed intracellularly, processed at various levels and lead to the observed 88 biological responses. ‘Ine major consequences are, therefore, the loss of contact inhibition, alteration or loss of differentiation, blockage of gap jmxztional communication, stimlaticn of INA synthesis ard cell proliferation. Despite the positive correlation between the expression of the ras oncogene and the iflfibitim of gap junction cammication that is rqaortedhere, adirecteause/effectargmnentcanmtbenadeatthis stage. Saneofthecellulardiangesseconiarytotransfectionwithc- Ha-ras-l could cimmstantially interfere with cellular coupling. Anongthose, thealteratimofmenbranepinocytosisnayhaveaninporb tant effect an cellular homeostasis by allowing a perturbation in the canartration of iais, e.g. Ca‘H', or other factors that may block gap junctions. Moreover, the transformed mrphology, i.e. membrane ruf- flirgandardiorageirdeperdence, canactas mechanical factorslimit- irg intercellular contact. In addition, auxillary cellular changes acompanyin; the transfection with, and the expression of, the ras cmoogene, e.g. increase of pH (Hagag et al 1987), alteration of the rucleotide pool or gene expression, could act, individually or in can- binaticn, to inhibit gap junction coupling as well. Furthermre, the mitogenic effect of ras p21 that results in cell division can, by it- self, lead to an inevitable \mcoupling in the early stages of mitosis. Furtherirwestigaticmsarerequiredtodeterminethemleofthe rasamcogeneinumorigenesisingereralandinthenndulaticnofgap jmctiors in mecific. (mcogenes linked to irducible pranoters, e.g. thatofthsmirinemamarytmmrvirus (INN) orthemetalthioninepro- mters, irriucible by dexamethazone or zinc respectively, might be use- fulintheelucidatimofamredirectcorrelatimbehveenthe 89 expression of the H—ras p21 protein and the inhibition of gap junctions. Cells transformed by ras, with constitutive or inducible expression, canbe injectedinrudemice, ormnccnpranisedanimals, to test for correlation betweai the p21 expressim, irhibitim of gap junction and their tumorigenic capability in yjyg. Also, antisense mmtorasoranti-p21antibodiescouldbeintroducedintocells transformed with ras and these cells evaluated for their ccnmmicaticn cmpetence. 'Ihe mechanisms of pherotypic transformation, membrane ruf- fling, pinocytosis or even blockage of gap junctiors by the ras onco— geneshavenotyetbeenelucidated. Itisthereforenecessarytostudy the various cellular factors suspected to play a role in mediating the ras effect. Prolonged down-regulation of PKC e.g. by rhorbol ester treatment, before and after transfection with ras oncogene, might dir- ectly verify the role of PKC in the process of tramformation by ras. In addition, microinjection of ras p21 in the presence of protein syn- thesis inhibitors, e.g. cyclohexamide, and testing for modulation of gap junction coniuctance might help explore whether the production of newly synthesized proteins is required. Also, the role of intracel- lular Ca“ in ras-induced effects can be studied using 1143-3, a can- pound known to inhibit mobilization of calcium fran intracellular stores (Chiou and Malagodi 1975) . Calcium is one of the factors needed to activate me (May et a1 1935). Anctherintriguirgaspectforreseardiwouldbetocharacterize the intermediate biochenical pathways that intervenebetweentheac- tivated moogenic p21 and the blockage of gap junctions. 'Ihis charac- terization might lead to the identification of cmpamds that may potentially reverse the inhibition of gap junctions and induce cell 90 differentiatim, e.g. CAMP (Saez et a1 1986, Veld et a1 1985, Johnson et a1 1935, Wiener and Ioeuenstein 1933) and retinoic acid (Bollag 1972 , Sdiiff ard lbore 1985) . Efforts need also be directed to eluci- date the physiologic functim of the normal ras proto-oncogene and its role in cell cycle, nenbrare function and cell proliferation. Progress inanyofthoseaspectsoutlinedheremightprcvevaluableardcould open up further possibilities for an in depth characterization of the role of gap junctions, tumor pranoters and oncogenes in carcinogenesis. MICE The results described in the reported stiflies exhibit significant correlation between the expression of the H-ras product p21 and the in- hibition of gap junctional caummication.'1hese data are in agreement with the proposed hypothesis, i.e. that ras may mimick the effects of certain tumor praroters by blocking the direct intercellular carmmicn- ticn during carcinogenesis. ‘Ihis hypothesis thus integrates gap junc- tion function, tamer prunotion and oncogene expression during the proc- ess of malignant transformaticm. 'Ihe results are not in contradiction with the known biochenical duaracteristics of the ras oncogene product. P21 has been reported to induce a constitutive activation of PKC through the elevation of intracellular DAG levels. 'Ihis PKC activation alsoocwrsdurirgcell—exposmetoghorbolesterunmrprawtersani in cells expressing the src oncogene product. PKC activation leads to the phosphorylation of gap junction proteins in sane cell system and positively correlates with the inhibition of gap junctional camunica- tion. 'meinteroellularcwplingnedxanismappearstohaveafimdamental role in maintaining cellular hareostasis, control of differentiation, proliferation and contact inhibition. ‘Ihe interference with this junc- tional permeability was found to be associated with cellular exposure to mitogenic agents and minor pranoters. In addition, inhibited junc- tional ccmnmicaticn correlates with certain malignant pl'xenotypes and netastasizing cells and with those expressing oncogenic products. The 91 92 data presented here have elucidated an inportant potential function of the ras oncogene, i.e. the interference with gap junction conductance presumably acting through the nenbrane signal transduction mechanism. 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