AbstractNormal melanosome biogenesis requires the association of structural proteins with tyrosinase. 3T3 Swiss fibroblasts transfected with mouse tyrosinase cDNA (line 13.4, clone c) are a unique system in which melanogenesis takes place in the absence of melano- somal structural proteins. Our study confirmed that transfected fibroblasts displayed tyrosinase activity and some of them produced pigment granules. In the ab- sence of melanosomal structural proteins the granules failed both to show a typical ultrastructure and to un- dergo the usual melanosome ontogenesis. The differen- tiating agent – dimethyl sulfoxide- increased phaeome- lanin production. Pigment was localized in membrane- bound vesicles which were identified as lysosomes by means of immunogold electron microscopy. Cell line 13.4 had higher levels of lysosomal enzymes (â-hex- osaminidase, α-mannosidase) than both parental 3T3 cells and clone pKG4 (fibroblasts transfected with the G418 resistance plasmid). Melanosomal proteins act as scavengers of toxic products of melanogenesis, and our results suggest that in their absence cells may employ an alternative mechanism to sequester injuri- ous products. Key words Melanogenesis · Transfection · Lysosome · Tyrosinase · Melanosome Int r oduct ion The best differentiated function of pigment cells is lanin production (melanogenesis). Since cytotoxic speci are produced during melanogenesis, it is strictly compa mentalized to melanosomes [28]. Melanosome morp genesis requires fusion of melanosomal structural prote with tyrosinase [15] and includes characteristic develop mental stages [30]. Mouse fibroblasts transfected with mouse tyrosin cDNA are a unique system in which melanogenesis take place in the absence of melanosomal structural protein Such a system offers a unique opportunity to obtain inf mation on the roles of melanosomal nontyrosinase teins both in melanosome ontogenesis and in coping wi the toxic intermediates of melanogenesis. Materials and methods Cell cultures Three related cell lines were studied [33, 35]. Line 13.4 was es lished by cotransfecting 3T3 Swiss mouse fibroblasts with the rosinase expression vector pHDmcTyr1 and a plasmid conferri G418 resistance [33]. Pigmented clone c, used in our study, wa derived from the original line 13.4 by two rounds of subcloning [35]. Clone pKG4 is the 3T3 Swiss mouse fibroblast line fected with the vector pKG4 only [35]. Line 3T3 represen original 3T3 Swiss fibroblasts. All the lines were grown routinely in Ham’s F10 mediu (Gibco, Paisley, UK) supplemented with 10% fetal calf serum, 2 mM L-glutamine and penicillin (100 IU/ml) and streptomycin (10 µ g/ml) in a humidified atmosphere containing 5% CO 2 at 37°C. Mycoplasma tests were performed with negative results usi Gen-Probe Mycoplasma T.C. Rapid Detection System (Euro-DP Glyn Rhonwy, Llanberis, UK) Enzyme assays The dopa oxidase activity of tyrosinase (EC 1.14.18.1) was dete mined using the MBTH assay of Winder and Harris [34] based Jan Borovanský · A. Mieke Mommaas · Nico P. M. Smit · Denise Eygendaal · Alison J. Winder · Bert Jan Vermeer · Stan Pavel Melanogenesis in transfected fibroblasts induces lysosomal acti Arch Dermatol Res (1997) 289: 145–150 © Springer-Verlag 1997 Received: 13 May 1996 ORIGINAL PAPER J. Borovanský · N. P. M. Smit · A. M. Mommaas · D. Eygendaal · B. J. Vermeer · S. Pavel Department of Dermatology, University Hospital, 2300 RC Leiden, The Netherlands J. Borovanský (Y) IInd Department of Medical Biochemistry, 1st Faculty of Medicine, Charles University, U nemocnice 5, CZ-128 53 Prague 2, Czech Republic Fax (+422)-24915449 A. J. Winder 1 Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK Present address: 1 Glaxo Research & Development Ltd., Greenford Road, Greenford, Middlesex, UB6 OHE, UK