Reduction of Melanogenic Activity and Responsiveness to α-Melanocyte-Stimulating Hormone during Serial Passage of Melanoma Cells

Abstract

French

Background:

Murine melanoma cells such as Cloudman S91 or B16 mouse melanoma cells have been used extensively to study mechanisms involved in pigmentation because these cells have tyrosinase, the key enzyme in pigmentation, and produce pigment. We have observed that serial passaged S91 cells tend to decrease their basal pigment content and to lose their responsiveness to α-melanocyte-stimulating hormone (α-MSH).

Objective:

Because this reduction of melanogenic capacity is a widely acknowledged but virtually unstudied characteristic of both human and murine melanoma cell lines in culture, we wished to document and quantify the phenomenon.

Methods:

Commercially attained S91 melanoma cells were serially passaged. Basal pigmentation as well as α-MSH responsiveness and expression of protein kinase C-beta (PKC-β) were assessed.

Results:

S91 cells progressively lost their basal pigmentation under standardized conventional conditions of culture, from an initial melanin content of 20 ± 4 pg/cell content to 12 ± 5 pg/cell within 70 population doublings (16 passages) and to 4.5 ± 6 pg/cell, a level at or below the detectable level of our melanin assay, by 110 population doublings (28 passages). When responsiveness to α-MSH was assessed, a 6-day treatment with 10⁻⁶M α-MSH initially induced the pigment content four-fold from 20 ± 4 to 82 ± 2 pg/cell. In contrast, after 110 population doublings, identical treatment with α-MSH induced pigment content less than two-fold from 4.5 ± 6 to 7.5 ± 2 pg/cell. PKC-β expression was readily detected by immunofluorescence in early passage pigmented cells, but not in late passage nonpigmented cells.

Conclusion:

These results confirm that while murine melanoma cells are a useful model system for pigmentation studies, it is important to monitor changes in the cells' basal abilities to pigment and to respond to exogenous pigment-inducing factors. They further suggest that factors in the culture environment or the internal milieu of melanoma cells exposed to continuous mitogenic stimulation inhibit melanogenesis. One candidate mechanism is down regulation of PKC-β.

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References

Pawelek

. Studies on the Cloudman melanoma cell line as a model for the action of MSH. Yale J Biol Med 1985; 58:571–578.

Pawelek

Wong

Sansone

Morowitz

. Molecular controls in mammalian pigmentation. Yale J Biol Med 1973; 46:430–443.

Pawelek

. Factors regulating growth and pigmentation of melanoma cells. J Invest Dermatol 1976; 66:201–209.

Lerner

Fitzpatrick

. Biochemistry of melanin formation. Physiol Rev 1950; 30:91–126.

Ito

. Biochemistry and physiology of melanin. In: Levine

, ed. Pigmentation and pigmentary disorders. Boca Raton, FL: CRC Press, 1993:33–59.

Jackson

. A cDNA encoding tyrosinase-related protein maps to the brown locus in mice. Proc Natl Acad Sci U S A 1988; 85: 4392–4396.

Halaban

Moellmann

. Murine and human b locus pigmentation genes encode a glycoprotein (pg75) with catalase activity. Proc Natl Acad Sci U S A 1990; 87:4809–4813.

Vijayasaradhi

Bouchard

Houghton

. The melanoma antigen pg75 is the human homologue of the mouse b (brown) locus gene product. J Exp Med 1990; 171:1375–1380.

Jackson

Chambers

Tsukamoro

. A second tyrosinase-related protein, TRP2, maps to and is mutated at the mouse slaty locus. EMBO J 1992; 11:527–535.

10.

NIGMS human genetic mutant cell repository. Catalog of cell lines, 1993.

11.

Park

Russakovsky

Ohno

Gilchrest

. The β isoform of protein kinase C stimulates human melanogenesis by activating tyrosinase in pigment cells. J Biol Chem 1993; 268:11742–11749.

12.

Gordon

Gilchrest

. Human melanogenesis is stimulated by diacylglycerol. J Invest Dermatol 1989; 93:700–702.

13.

Wade

Burkart

. The role of adenosine 3″,5″-cyclic monophosphate in the density-dependent regulation of growth and tyrosinase activity of B-16 mouse melanoma cells. J Cell Physiol 1978; 94:265–274.

14.

Fuller

Lunsford

Inman

. α-Melanocyte-stimulating hormone regulation of tyrosinase in Cloudman S-91 mouse melanoma cell culture. J Biol Chem 1987; 262:4024–4033.

15.

Korner

Pawelek

. Activation of melanoma tyrosinase by a cyclic AMP-dependent protein kinase in a cell-free system. Nature 1977; 267:444–447.

16.

Halaban

Pomerantz

Marshall

Lerner

. Tyrosinase activity and abundance in Cloudman melanoma cells. Arch Biochem Biophysiol 1984; 230:383–387.

17.

Wong

Pawelek

. Melanocyte-stimulating hormone promotes activation of pre-existing tyrosinase molecules in Cloudman S91 cells. Nature 1975; 255:644–646.

18.

Fuller

Viskochil

. The role of RNA and protein synthesis in mediating the action of MSH on mouse melanoma cells. Life Sci 1979; 24:2404–2416.

19.

Lotan

. Enhancement of melanotic expression in cultured mouse melanoma cells by retinoids. J Cell Physiol 1981; 106:179–189.

20.

Imokawa

Mishima

. Loss of melanogenic properties in tyrosinase induced by glycosylation inhibitors within malignant melanoma cells. Cancer Res 1982; 42:1994–2002.

21.

Varga

DiPasquale

Pawelek

. Regulation of melanocyte-stimulating hormone (MSH) action at the receptor level: Discontinuous binding of MSH to synchronized melanoma cells during the cell cycle. Proc Natl Acad Sci U S A 1974; 71:1590–1593.

22.

Lambert

Lerner

. Optimization of a melanotropin-receptor binding assay using reversed high performance liquid chromatography. J Chromatog 1983; 226:567–576.

23.

Park

Russakovsky

Fernandez

Gilchrest

. Protein kinase C stimulates melanogenesis through phosphorylation of tyrosinase. J Invest Dermatol 1993; 100:37.

24.

Park

Russakovsky

Gilchrest

. Depletion of protein kinase C blocks α-melanocyte stimulating hormone-induced melanogenesis. J Invest Dermatol 1992; 98:562.

25.

Park

Russakovsky

Fernandez

Gilchrest

. Pigmentation of S91 cells by α-melanocyte stimulating hormone requires protein kinase C-β mediated induction of tyrosinase. J Invest Dermatol 1994; 102:553.

26.

Niles

Loewy

. Induction of protein kinase C in mouse melanoma cells by retinoic acid. Cancer Res 1989; 49:4483–4487.

27.

Gruber

Ohno

Niles

. Increased expression of protein kinase C-α plays a key role in retinoic acid-induced melanoma differentiation. J Biol Chem 1992; 267:13356–13360.

28.

Fuller

Niekrasz

Hoganson

. Down-regulation of tyrosinase mRNA levels in melanoma cells by tumor promoters and by insulin. Mol Cell Endocrinol 1990; 72:81–87.

29.

Silagi

. Control of pigment production in mouse melanoma cells in vitro. J Cell Biol 1969; 43:263–274.

30.

Abdel-Malek

Kreutzfeld

Hadley

. Long-term and residual melanotropin-stimulated tyrosinase activity in S91 melanoma cells is density dependent. In Vitro Cell Dev Biol 1986;22:75–81.

31.

Reenstra

Yaar

Gilchrest

. Effect of donor age on epidermal growth factor processing in man. Exp Cell Res 1993; 209: 118–122.

32.

Gordon

Peacocke

Gilchrest

. Induction of c-fos but not c-myc in S-91 cells by melanization signals. J Dermatol Sci 1992; 3:35–41.

33.

Bradford

. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 1976; 72:248–254.

34.

Liscovitch

Cantly

. Lipid second messengers. Cell 1994; 77:329–334.

35.

Naeyaert

Eller

Park

Gilchrest

. Pigment content of cultured human melanocytes does not correlate with tyrosinase message level. Br J Dermatol 1991; 125:297–303.

36.

Yamanish

Graham

Buckmeier

Meyskens

Jr . The differential expression of protein kinase C genes in normal human neonatal melanocytes and metastatic melanomas. Carcinogenesis 1991; 42:105–109.

37.

Veronesi

Cascinelli

Santinami

. Cutaneous melanoma: Status of knowledge and future perspective. New York: Academic Press, 1987.

38.

Hoganson

Ledwitz-Rigby

Davidson

Fuller

. Regulation of tyrosinase mRNA levels in mouse melanoma cell clones by melanocyte-stimulating hormone and cyclic AMP. Somat Cell Mol Genet 1989; 15:255–263.

39.

Bitensky

Demopoulos

Russell

. Pigmentation: Its genesis and biological control. Riley

, ed. New York: Appleton-Century-Crofts, 1972:247–255.

40.

Deschatrette

Weiss

. Characterization of differentiated and dedifferentiated clones of a rat hepatoma. Biochimie 1974; 56:1603–1611.

41.

Kuo

Conley

Chen

. A transcriptional hierarchy involved in cell-type specification is selectively inhibited by extinguishing lock. Nature 1992; 335:457–461.

42.

Edelman

Blumenthal

Krebs

. Protein serine/threonine kinases. Annu Rev Biochem 1987; 56:567–613.

43.

Svoboda

KKH

. Intracellular localization of types I and II collagen mRNA and endoplasmic reticulum in embryonic corneal epithelia. J Cell Science 1991; 100:23–33.