We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively. Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts. The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets. The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy. Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME). The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c. Neither line is tumorigenic in nude mice. Hetero-karyons between the two lines can be constructed and form wild-type, black pigment. Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations. These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.

Coat-colour mutations account for over 60 of the known genetic loci of the mouse, and include mutations acting at various stages of development from the neural crest stage onwards (Silvers, 1979). The mechanisms of action of many of these mutations are unknown, but progress in understanding them has already been accelerated by the development of techniques for the selective cell culture of mouse neural crest (Ito & Takeuchi, 1984), mouse melanoblasts (Mayer & Oddis, 1977; Mayer, 1982; Bennett et al. 1987) and postnatal mouse melanocytes (Sato et al. 1985; Tamura et al. 1987). Melanoblasts or melanocytes can now be selected readily in primary cultures by the use of media supplemented with TPA and cholera toxin, as originally used for human melanocytes (Eisinger & Marko, 1982).

A further technical development, which will greatly facilitate biochemical and molecular-genetic studies, is the isolation of immortal lines of mouse melanocytes. Two clonal lines have been derived from black (a/a) mice (Sato et al. 1985; Bennett et al. 1987). Other melanocyte lines have been established from the dermis of mice carrying mutations including several at the albino (c) locus, although these lines were not cloned and no evidence was presented that the lines contained no other dermal cells such as fibroblasts (Tamura et al. 1987; Halaban et al. 1988). Albino melanocyte lines were also reported in an abstract (Abe & Takeuchi, 1985). The C locus is thought to code for the pigment-synthesizing enzyme tyrosinase (Silvers, 1979; Kwon et al. 1987; Halaban et al. 1988). Here we describe the establishment of two new cloned melanocyte lines homozygous respectively for albino (c/c) and for brown (b/b), another locus at which the expression appears to be developmentally controlled and melanocyte-specific. A tyrosinase-related protein, possibly another enzyme or a melanosomal structural protein, maps at or near the B locus (Jackson, 1988).

We also describe somatic complementation between the b and c mutations in heterokaryons.

Materials

Tissue-culture media and plastics (Nunc) were obtained from Gibco Europe (Uxbridge, UK), foetal calf serum from Tissue Culture Services (Slough, UK) and cholera toxin from Schwarz-Mann (Orangeburg, NY, USA). Polystyrene latex beads, phytohaemagglutinin (PHA/P), TPA, mitomycin C, 2-ME and 4-norleucine, 7-D-phenylalanine-a--melanocyte stimulating hormone (N-MSH) were from Sigma Chemical Co. (Poole, UK). TPA, cholera toxin and N-MSH were dissolved and stored as previously described (Bennett et al. 1985). Polyethylene glycol (PEG) 1540 was from Koch-Light (Haverhill, Suffolk, UK).

Media

The basic culture medium was a supplemented Eagle’s minimal essential medium (SMEM) containing penicillin, streptomycin, sodium pyruvate and nonessential amino acids (Kreider et al. 1975); and prepared with only 25mm-sodium bicarbonate as previously described (Bennett et al. 1987) to give a pH of 6η9 with 10% CO2. Ham’s F10 medium was supplemented with 18 mm bicarbonate to give the same pH.

Animals

Albino embryos were obtained from a female mouse of the outbred LAC-MF1 strain (B/B,c/c), which was maintained at St George’s Hospital Medical School. Brown embryos were from a mating of a brown male and female from the partially inbred ‘Q’ strain, maintained at the MRC Mammalian Development Unit, London NW1, and the pregnant female was kindly donated by Dr Ian Jackson. Random-bred nude {nu/nu) mice were maintained at the Imperial Cancer Research Fund Animal Breeding Unit, South Mimms.

Keratinocyte feeder cells

The XB2 immortal mouse keratinocyte line (Rheinwald & Green, 1975) was kindly provided by Dr J. G. Rheinwald and was adapted in our laboratory to grow in the absence of ?T? feeder cells, in Dulbecco’s modified Eagle’s medium (DEM) and 10% fetal calf serum (FCS). To prepare keratinocyte feeder cells, nearly confluent cultures of XB2 cells on 85 mm plates were growth-inactivated by incubation for 2h in 5 ml DEM and 10% FCS containing 4 μg ml−1 mitomycin C, then washed in DEM and incubated for 10 min in fresh DEM+FCS without mitomycin. They were then subcultured and either replated at 3×104 cells ml1 or frozen in liquid nitrogen and replated when needed at 5×104ml−1 (allowing for reduced viability).

Primary cultures of melanoblasts

These were prepared essentially as described previously (Mayer & Oddis, 1977; Bennett et al. 1987). The albino embryos were 16η5 days old and the brown embryos were 18η5 days old. In brief, the epidermis was separated with trypsin, washed in medium (SMEM+10% FCS), minced in a drop of medium using two scalpels, and pipetted vigorously in about 2ml medium with a siliconized Pasteur pipette. The suspension was suitably diluted with the same medium, then supplemented with cholera toxin (10 nM) and N-MSH (100 pm) and plated into 50 mm culture plates containing XB2 feeder cells, at 4 ml medium per dish. The cultures were incubated at 37°C with 10 % CO2. TPA (200 nM) was added after 3–4 days. The medium was initially changed twice weekly, but this was adjusted according to the number of cells present.

The most usual medium during the early passages was SMEM with 10% FCS, TPA (200 nm), N-MSH (100 pm) and cholera toxin (10 nm, reduced to 1 nM after the first 1 – 2 passages). Preliminary work has indicated that MSH increases the yield of diploid mouse melanocytes (unpublished data). With immortal mouse melanocytes, MSH stimulates both proliferation and pigment synthesis (Tamura et al. 1987 and our unpublished data), but this is not known for diploid cells. In our current method for culture of diploid melanocytes, the concentration of FCS has been reduced to 5 % throughout. For subculture, cell suspensions were made as for the established lines (see below), but were replated on to fresh XB2 feeder cells.

Culture of established lines

The final growth medium used for both lines was SMEM with 5% FCS, TPA (200 nm), and 2-ME (100 μm). Both were subcultured approximately every 7 – 10 days, by the procedure described previously for melan-a cells (Bennett et al. 1987). Cells were replated at 2 – 3× 104ml−1,10 ml per 85 mm dish, in growth medium without feeder cells.

Frozen cell stocks in liquid nitrogen were prepared by standard procedures except that the freezing medium was SMEM with 5% FCS, 100 μM-2-ME and 7 · 5% dimethyl sulphoxide (DMSO), and care was taken to avoid a rise in pH through loss of CO2. We now use this method for all melanocyte cultures.

Cloning of melanocytes

A cell suspension was prepared as for subculture. Cells were plated, either by limiting dilution or by manual selection with a drawn-out Pasteur pipette and mouth tube, into 6-mm culture wells containing XB2 feeder cells. The cloning medium was 50 % SMEM and 50 % Ham’s F10 medium, pH6η9 (Materials and methods), with 5 % FCS, conditioned for 1 day by the feeder cells and then supplemented with TPA and cholera toxin. This method gave consistently high cloning efficiencies of up to 80 %.

Light microscope photography

Cultures were washed in Dulbecco’s phosphate-buffered saline lacking calcium and magnesium chlorides (PBSA), fixed in 4% formalin in PBSA and washed with water. They were photographed with water in the dishes, using an Olympus inverted microscope with planachromat objectives.

Electron microscopy

Cell suspensions were prepared as above from two confluent 85 mm plates of melan-c cells at passage 26. The cells were centrifuged, washed with PBSA and resuspended in fixative: 2% glutaraldehyde in 0·1 M-cacodylate buffer, pH7η2. They were fixed at refrigerator temperature for 1 h, rinsed twice in cacodylate buffer, centrifuged at 4000 revs min−1 and the cell pellet postfixed for lh in osmium tetroxide. The pellet was dehydrated and embedded in Araldite. Thin sections were cut, and examined and photographed with a Philips EM301 electron microscope.

Tumorigenicity tests

Melan-b and melan-c cells were harvested as above, washed in PBSA and resuspended at the required concentrations in PBSA. They were injected in 0 · 2ml volumes into female, thymus-deficient nude (nu/nu) mice aged 16 weeks. 10 mice received 2 × 106 cells subcutaneously in the flank region and 5 received 106 cells intravenously in the tail vein.

Preparation of heterokaryons

Polystyrene latex beads, diameter 1 μm, were suspended and stored at 4×109ml−1 in 70% ethanol, and were then pre-sumed to be sterile. Melan-c cells were labelled by growth for 10 days in the presence of beads (2×107ml−1 medium). Suspensions of melan-b and bead-labelled melan-c cells in complete medium were prepared as above and plated on 33-mm dishes at 10s ml−1, 2 ml per dish, both separately (controls) and in a 1:1 mixture. The cells were allowed to attach and spread, then fused in situ by the following series of washes (1ml per dish): (1) Three washes in serum-free SMEM; (2) PHA/P, 100 μgmG1 in serum-free SMEM, 15 min; (3) 10% DMSO in SMEM, 10s; (4) PEG 1540, 45% w/v in SMEM with 10% DMSO, final pH approximately 7 · 6, 1 min; (5) 10% DMSO in PBSA, 10 s; (6) SMEM+lη8m?-EGTA, a few seconds, and (7) complete growth medium + lη8mm-EGTA, 20 min in incubator. See Shay (1982) for reviews on the use of bead-labelling, PHA, DMSO and EGTA in PEG-mediated cell fusion. PHA and DMSO increase efficiency of fusion, while EGTA increases viability.

Derivation of melan-b

Primary cultures were plated in February, 1986. Many groups of unpigmented melanoblasts were seen within a few days; as usual these proliferated and matured to pigmented melanocytes over the first few weeks, after which no melanoblasts were detected. The epidermal keratinocytes failed to proliferate in the selective medium, and these and the feeder cells died out over about 2 weeks. Typical melanoblasts and melanocytes are shown for reference in Fig. 1; these are from black (a/a) mice and therefore resemble wild-type cells, as mutations at the A locus do not directly affect melanocytes (Silvers, 1979). Living b/b melanocytes, where locally crowded, were visibly brown by bright-field microscopy; elsewhere the pigment was very faint.

Fig. 1.

Diploid melanocytes and melanoblasts in a primary culture. These are black (a/a) ceils, as a general illustration. (A) Phase-contrast; (B) bright-held optics. The melanin pigment is seen clearly in B. The cells can be arranged along a morphological spectrum, as exemplified by the series 1—4, so that the cells with appearance 1 are presumed to be melanoblasts. These emerge and proliferate from expiants before melanocytes are seen. The melanoblasts are very small, bipolar and/or crescent-shaped with a ruffled membrane on the convex side. Very few other cells (primary or XB2 keratinocytes or fibroblasts) are present. Scale bar, 200μm.

Fig. 1.

Diploid melanocytes and melanoblasts in a primary culture. These are black (a/a) ceils, as a general illustration. (A) Phase-contrast; (B) bright-held optics. The melanin pigment is seen clearly in B. The cells can be arranged along a morphological spectrum, as exemplified by the series 1—4, so that the cells with appearance 1 are presumed to be melanoblasts. These emerge and proliferate from expiants before melanocytes are seen. The melanoblasts are very small, bipolar and/or crescent-shaped with a ruffled membrane on the convex side. Very few other cells (primary or XB2 keratinocytes or fibroblasts) are present. Scale bar, 200μm.

The cells were subcultured after 3 weeks, and in total 8 times over the first 7 months. Proliferating melanocytes could be observed throughout this period, as judged by the presence of colonies of small, healthy-looking, pigmented cells with some mitoses (see Bennett et al. 1987 for an illustration of pigmented melano-cytes in mitosis). However, growth was balanced by cellular senescence and death so that the number of cells showed no net increase during this time. Fibro-blast-like cells also grew but did not outgrow the melanocytes so long as cholera toxin was present.

From passage 8, melanocyte growth accelerated and XB2 feeder cells and N-MSH were now omitted. At passage 19 and 10 · 5 months of culture, single-cell clones were prepared, by limiting dilution (Materials and methods). Numerous proliferating clones were obtained. Two pigmented melanocyte clones were selected, from wells containing no unpigmented (fibro-blast-like) cells, and subcultured. They now grew well and growth was not noticeably impaired by simplification of the medium to SMEM, 5 % FCS, 200 nM-TPA and 100 μM-2-ME. 2-ME was now added because of results obtained with the albino cell line (see below). Frozen stocks from both clones were prepared and one was selected for characterization and named ‘melan-b’.

Since the cells had emerged from a phase of senescence they were presumed to be established and immortal (Pollack, 1981).

Derivation of melan-c

To identify and purify albino melanocytes, as they lack pigment, we could use only morphological criteria together with the knowledge that an almost-pure melanocyte population is normally selected under the conditions used. Cells which were small, elongated, epithelioid to dendritic and having nuclei which were dark by phase-contrast optics (Bennett et al. 1987) were provisionally taken to be melanocytes.

The primary culture (May, 1986) was passaged after 3 weeks and the resulting plates were kept for 7 weeks, after which large, healthy, melanocyte-like colonies were observed. One of these colonies, assumed to be a clone, was subcultured by local application of trypsin and EDTA solution into two 16 mm wells containing XB2 feeder cells. This was counted as passage 2. No fibroblast-like cells were observed in this culture sub-sequently, but net growth was very poor, largely because many cells died after subculture or addition of fresh medium, even though feeder cells were present. The total cell number increased only slightly during a further 7 subcultures and 5 months. At this point improved growth was observed on reducing the serum concentration from 10 % to 5 %, suggesting an inhibitory effect of FCS. Therefore some cultures were transferred to medium with the reducing agent 2-ME (100 μM), which might alter some serum components. 2-ME is used in the culture of other cell types such as hybridomas (Langone & Van Vunakis, 1986) and early mouse embryo cells and embryonal carcinoma cells, which it enables to grow in the absence of feeder cells (Smith & Hooper, 1987). A marked effect was noticed: cell death ceased immediately and within a few days there were substantially more cells than in control cultures. Quantitative studies at a later passage confirmed this effect (see below).

From passage 9, 5% FCS and 2-ME were used routinely and the cells grew progressively. Feeder cells could now be omitted. Frozen stocks were prepared from passage 12 onwards. The cells now behaved as an established line and were named ‘melan-c’. Subclones of the line have also been prepared (Materials and methods).

General characteristics of melan-b and melan-c cells

All information from this section onwards relates to the established lines grown in the absence of feeder cells.

Fig. 2 shows the appearance of the two lines by light microscopy, with melan-a melanocytes for comparison. All three populations have a similar appearance, except that melan-a cells are less aligned than the others and melan-b cells are relatively flat. Melan-c cells also became flatter at later passages. Melan-b cells have only light pigmentation, and melan-c cells are unpigmented although granular at very high magnifications (not shown). The granules are probably premelanosomes (see below). The pigmentation of melan-b cells is visibly brown, both by light microscopy of dense cultures and as seen macroscopically in pelleted cells (Fig. 3). This is different from a low density of black pigment, which gives a grey cell pellet.

Fig. 2.

Cloned, immortal melanocytes by phase-contrast optics. (A) melan-a, passage 17; (B) melan-b, passage 10; (C) melan-c, passage 13. At this magnification, melan-a cells show abundant pigment granules, melan-b cells have fine, inconspicuous granules and melan-c do not appear granular. Bar, 200 μm.

Fig. 2.

Cloned, immortal melanocytes by phase-contrast optics. (A) melan-a, passage 17; (B) melan-b, passage 10; (C) melan-c, passage 13. At this magnification, melan-a cells show abundant pigment granules, melan-b cells have fine, inconspicuous granules and melan-c do not appear granular. Bar, 200 μm.

Fig. 3.

Pelletted melanocytes. From left to right: melan-a, melan-b and melan-c (107cells each). The cells were fixed in 4% formalin in suspension before centrifugation.

Fig. 3.

Pelletted melanocytes. From left to right: melan-a, melan-b and melan-c (107cells each). The cells were fixed in 4% formalin in suspension before centrifugation.

Both lines grow slowly in the earlier passages, with population doubling times of approximately 2 · 5 – 3 days as estimated from cell yields at subculture. As with melan-a (Bennett et al. 1987), the growth rate increases gradually with passage number.

Ultrastructure of melan-c cells

Albino melanocytes in vivo produce premelanosomes, the unpigmented precursors of melanosomes (pigment organelles) (Parakkal, 1967; Hearing et al. 1973). These organelles are unique to pigment cells and are readily identifiable by electron microscopy. Ultrastructural examination of melan-c cells revealed abundant premelanosomes, especially in the peripheral cytoplasm as expected (Fig. 4). Most of them were classifiable as stage II melanosomes, which contain longitudinal filaments with cross-striations (Quevedo et al. 1987).

Fig. 4.

Electron micrographs of melan-c cells. (A) Part of a typical cell showing many premelanosomes in the peripheral cytoplasm. They are the pale organelles containing longitudinal filaments. (B) Premelanosome at higher magnification showing transverse striations. Bars, (A) 500 nm and (B) 100 nm.

Fig. 4.

Electron micrographs of melan-c cells. (A) Part of a typical cell showing many premelanosomes in the peripheral cytoplasm. They are the pale organelles containing longitudinal filaments. (B) Premelanosome at higher magnification showing transverse striations. Bars, (A) 500 nm and (B) 100 nm.

Growth responses to 2-ME and TPA

The effects of 2-ME on the growth of melan-c cells were retested at later passages. Fig. 5 shows a typical experiment, in which the effect of heat-inactivation of the FCS was also tested. Heated FCS inhibited proliferation, but 2-ME (100 μM) produced a marked increase in cell yield over 8 days. Addition of 2-ME at 100 μM to the FCS before use was less stimulatory (Fig. 5). From these observations, it is unlikely that the favourable action of 2-ME was due to inactivation of complement, or any other effect on the serum. When the concentration of 2-ME was varied, the best growth was obtained with concentrations between 30 and 300 μM. (further data not shown), so routine use at 100 μm was continued. A small, but statistically significant, improvement in the growth of melan-b cells (about 40 % more cells in 6 days) was obtained with the same supplementation.

Fig. 5.

Proliferation of melan-c cells in media with and without 2-ME or heat-inactivated serum. SMEM was supplemented with 5 % FCS which was either untreated or heated for 1 h at 56°C. 2-ME was added to media the day before use to allow equilibration. All media contained TPA (200 nm). A melan-c cell suspension was prepared as for subculture in medium with 1 % FCS, diluted into each test medium at 2×104 cells ml−1, plated at 2 ml per 33 mm dish and incubated. On the specified days, cells were resuspended and counted by haemocytometer. The media were renewed after 4 days. Each point show the mean and standard error of 6 counts, 2 from each of 3 dishes. Key: •, SMEM+untreated FCS; ▀, same+2-ME, 100 μ?; □, SMEM+FCS to which 100 μ?-2-ME had been added, giving a final concentration of 5μ?-2-ME, ▽, SMEM+ heated FCS.

Fig. 5.

Proliferation of melan-c cells in media with and without 2-ME or heat-inactivated serum. SMEM was supplemented with 5 % FCS which was either untreated or heated for 1 h at 56°C. 2-ME was added to media the day before use to allow equilibration. All media contained TPA (200 nm). A melan-c cell suspension was prepared as for subculture in medium with 1 % FCS, diluted into each test medium at 2×104 cells ml−1, plated at 2 ml per 33 mm dish and incubated. On the specified days, cells were resuspended and counted by haemocytometer. The media were renewed after 4 days. Each point show the mean and standard error of 6 counts, 2 from each of 3 dishes. Key: •, SMEM+untreated FCS; ▀, same+2-ME, 100 μ?; □, SMEM+FCS to which 100 μ?-2-ME had been added, giving a final concentration of 5μ?-2-ME, ▽, SMEM+ heated FCS.

Both cell lines grew poorly in the absence of TPA. In a typical experiment the numbers of melan-c cells obtained after 7 days’ growth in different media were as follows (mean and standard error in units of 104 cells ml−1; other details as for Fig. 5): complete growth medium (GM), 16 · 19 ±0-86; GM minus ME, 6 · 38 ± 0 · 37; GM minus TPA, 4 · 87 ± 0 · 20; GM minus ME and TPA, 2 · 24 ± 0 · 38. Both melan-b and melan-c cells became flattened and polygonal instead of elongated when TPA was not included, as previously described and illustrated for melan-a cells (Bennett et al. 1987). Diploid, non-immortal mouse and human melanocytes show a similar proliferative and morphological response to TPA, which all three cell lines have thus retained.

Karyology

Chromosome spreads (Rothfels & Siminovitch, 1958) were prepared from melan-b cells at passage 15 and melan-c cells at passage 17. The modal chromosome numbers were 39 for melan-b (30/50 cells) and 40 (diploid number) (38/50 cells) for melan-c. A subpopulation of 10/50 melan-b cells (20%) had 40 chromosomes.

Tumorigenicity tests

As neither line was from fully inbred mice, their tumorigenicity was tested in immunodeficient (nude) mice (Materials and methods). Neither line formed any tumour or transient nodule in any of 15 injected mice per cell line. The mice were killed after 6 months, when histological examination of the injected sites, in those animals that had received subcutaneous injections, revealed no evidence of tumour initiation.

Somatic complementation test

The b and c mutations are recessive and complement each other in heterozygotic animals. Therefore it is reasonable to expect complementation in hetero-karyons between these b and c melanocytes in vitro, given that melan-c cells are from MFI (B/B) mice. Expression of a gene believed to be the B gene is limited to melanocytes and melanoma (Jackson, 1988), and this specificity is also indicated by the fact that mutations at the b locus do not appear to affect anything but pigmentation in mice (Silvers, 1979). Thus complementation would also be an indication of normal, melanocytic differentiation in melan-c cells.

Melan-c cells were prelabelled with polystyrene beads and fused to melan-b cells with PEG 1540, PHA/P and DMSO (Materials and methods). 3 days later the cultures were examined. The results are shown in Fig. 6. Fused (and other) cells in the control cultures of melan-c or melan-b alone had no pigment or the inconspicuous brown pigment, respectively, but numerous cells in the mixed cultures contained conspicuous pigment similar to that of black melanocytes (see Bennett et al. 1987). All such dark cells contained beads, providing evidence that they were hybrid cells. To assess the proportion of heterokaryons that expressed black pigment, a count was made of the proportion of cells with prominent pigment out of 200 cells with beads, pigment (faint or prominent) and more than 1 nucleus. The ratio was 182/200 or 91 %. In fused melan-b cultures, the corresponding proportion of cells with two or more nuclei that had prominent pigment was 0/200. The 9% of apparent heterokaryons that contained only faint pigment may alternatively have been brown homokaryons which had adsorbed or phagocytosed beads from dead melan-c cells, of which many were present. Thus somatic complementation was observed in at least 90% of heterokaryons.

Fig. 6.

Fused melanocytes. Cultures of melan-b, melan-c or a 1:1 mixture of both were plated at 105 cells ml−1 and fused in situ as described (Materials and Methods). The cultures were then incubated for 3 days in growth medium with 1 n?-N-MSH. MSH was included in case it would increase the rate of pigment synthesis as it does in melanoma cells (reviews: Pawelek, 1979; Bennett, 1988) and in melan-a melanocytes (unpublished data). In each case, the same field is shown by phase-contrast (left) and bright-field optics. Care was taken that photographic procedures were identical in all cases. All cultures contain dead cells killed by the fusion treatment. (A,B) melan-b only. Only a faint granulation is visible in either mono- or multinucleate living cells. Dead, rounded-up cells and fragments look more granular; this was a consistent property of melan-b cells. (C,D) melan-c only. Polystyrene beads in living and dead cells appear prominent and dark by bright-field or refractile by phase-contrast (arrowheads); no pigment is present. (E –H): melan-b+melan-c, two fields. Many cells in these cultures contained heavy, dark pigment like that of black melanocytes. Such cells were typically giant and tortuous in shape, and contained beads (arrowheads); they were thus presumably hybrid cells. Scale bar, 200 μm. Inset in G and H: part of cell on left at ?× higher magnification, for clearer images of beads (bright by phase contrast) and melanin.

Fig. 6.

Fused melanocytes. Cultures of melan-b, melan-c or a 1:1 mixture of both were plated at 105 cells ml−1 and fused in situ as described (Materials and Methods). The cultures were then incubated for 3 days in growth medium with 1 n?-N-MSH. MSH was included in case it would increase the rate of pigment synthesis as it does in melanoma cells (reviews: Pawelek, 1979; Bennett, 1988) and in melan-a melanocytes (unpublished data). In each case, the same field is shown by phase-contrast (left) and bright-field optics. Care was taken that photographic procedures were identical in all cases. All cultures contain dead cells killed by the fusion treatment. (A,B) melan-b only. Only a faint granulation is visible in either mono- or multinucleate living cells. Dead, rounded-up cells and fragments look more granular; this was a consistent property of melan-b cells. (C,D) melan-c only. Polystyrene beads in living and dead cells appear prominent and dark by bright-field or refractile by phase-contrast (arrowheads); no pigment is present. (E –H): melan-b+melan-c, two fields. Many cells in these cultures contained heavy, dark pigment like that of black melanocytes. Such cells were typically giant and tortuous in shape, and contained beads (arrowheads); they were thus presumably hybrid cells. Scale bar, 200 μm. Inset in G and H: part of cell on left at ?× higher magnification, for clearer images of beads (bright by phase contrast) and melanin.

Proliferating mouse melanocyte cultures can readily be derived through differentiation in vitro of embryonic mouse melanoblasts. From such cultures, we have established two new melanocyte lines carrying homozygous recessive mutations which produce clearly observable phenotypes in the cells. The methods described yield immortal melanocyte lines at a high frequency (approximately one in two attempts). The isolation of further mutant lines is in progress. A method for cloning mouse melanocytes is also described. The cloning efficiency is high and further subclones of both lines have been readily obtained.

Several observations identify the unpigmented melan-c cells firmly as melanocytes. The most conclusive is the presence of many premelanosomes in the cytoplasm. In addition, the cells contain the B gene in an expressible state – not, for example, repressed by methylation – as shown by somatic complementation; they grow well in the presence of TPA and cholera toxin and very slowly in the absence of TPA; they originate from epidermis and they have the correct morphology.

The reducing agent 2-ME proved essential for the establishment of the melan-c line, which still grows poorly without it; however, its stimulation of the growth of melan-b cells is slight and preliminary tests on primary melanoblast/melanocyte cultures indicate an inhibition of growth (unpublished data). The reason is unknown, but 2-ME should evidently be used with caution in pigment cell culture.

The melan-b and melan-c lines will be useful as further lines of non-tumorigenic melanocytes, for experimental comparisons with melanoma cells. Moreover, they provide excellent vehicles for the study of mammalian gene expression. We have shown that each of the two mutations can be directly complemented by fusion with a melanocyte carrying the wild-type gene. We have now obtained efficient transfection and integration of exogenous DNA carrying drug-resistance markers, with both melan-b and melan-c cells. These lines thus permit gene transfer experiments aimed at the identification of the B and C genes, which appear to be developmentally controlled. Putative sequences for both these genes have recently been cloned (Shibahara et al. 1986; Kwon et al. 1987; Yamamoto et al. 1987; Jackson, 1988; reviewed by Bennett, 1988), including one sequence asserted to be either the tyrosinase gene (Shibahara et al. 1986), or a sequence at or near the B locus (Jackson, 1988). Once genomic B and C sequences are identified, it will be possible to study the expression of manipulated constructs in melan-b and melan-c cells, using the very prominent natural marker, black melanin.

We are most grateful to Dr Ian Hart of the Imperial Cancer Research Fund for carrying out the tumorigenicity tests, and to Drs Ian Jackson and Ruth Halaban for communication of unpublished work. This research was supported by the Cancer Research Campaign and a Wellcome Vacation Scholarship and Foulkes Foundation Fellowship to P.J.C.

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