MelanomaCells Resistant toInhibition ofGrowthbyMelanocyte Stimulating Hormone

Melanocyte stimulating hormone(MSH) enhancesmelanizationbut inhibitsproliferation of CloudmanS91melanoma cellsinculture. We haveiso- lated variants ofthesecells thatcangrowinthepresence ofMSH. Theconclusions we havereachedfromanalyses ofthesecells arethefollowing: (1)Basaltyrosinase activ- ity(monophenolmonooxygenase; monophenol,dihy- droxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1), i.e., theactivity thatispresentintheabsenceofadded MSH,isrelated throughacommon biochemical pathway toMSH-mediatedcontrol ofgrowth.(2)MSH-inducible tyrosinase activity doesnotappeartoberelated toMSH control ofgrowth.(3)The morphological changesthat occurfollowing theaddition ofMNSHorcAMP arerelated tocontrols ofgrowthandnottothoseofmelanization. Melanocyte stimulating hormone(MSH)enhances melaniza- tion butinhibits theproliferation ofcells fromtheCloudman S91mousemelanoma inculture (1). Botheffects result from anincrease intheintracellular levels ofcyclic AMP which occurs within minutes after theaddition ofMSH tothecul- tures (1-3). Theconcentration ofcyclic AMP inside acell iscrucial to thecontrol ofproliferation ofthatcell(4). Forexample, rapidly dividing cells generally havelowerlevels ofcyclic AMP thanslowly dividing orstationary cells (5-9); some tumorviruses bring aboutarapid dropincyclic AMP in normal cells before thecells undergo. amalignant transfor- mation (10, 11); andawidevariety oftransformed cells ap- peartorevert tonormal whencyclic AMP oroneofitsan- alogues isaddedtothemediumofthecells inculture (1, 12-24), orinjected intoanimals harboring tumors ofthose cells (25, 26).However, little isknownofthemechanisms inthecontrol processes. Daniel etal.(27) andSibley etal.(28) selected formutantmouselymphoma cells thatcould growin concentrations ofcyclic AMP thatwerelethal tothewild- typecells. Byanalyzing these mutants theyhopetofindout moreabout therole ofcyclic AMP inthecontrol ofgrowth. Similarly, wehaveisolated andstudied clones ofmelanoma cells thatareresistant totheinhibitory effects ofMSH on growth. Inthis report wedescribe experiments whichclarify therelationships ofcyclic AMP tothecontrol ofboththe growth andmelanization ofthese cells. METHODS Isolation ofMSH-Resistant Cells. Cells werecultured ina mannerdescribed previously (29)except thattheculture mediumcontained MSH (0.2 MM). Melanotic andamelanotic clones thatgrewinthepresence ofMSH wereisolated and subeloned further. Mostofthedatapresented wereobtained fromtheanalyses oftwoclones. Oneclone wasmelanotic inthepresence ofMSH andwasdesignated asPS1-mel-1. Theother wasamelanotic inthepresence ofMSH andwas called PS1-amel-1. Theseclones will bereferred toasmel-i andamel-1. Theparental strain fromwhichthese lines were derived isdesignated PS1-wt(wild type) andisapigmented subelone oftheCloudman S91cell line. Analytical Procedures. Tyrosinase activity (monophenol monooxygenase; monophenol, dihydroxyphenylalanine: oxy-