In cultured hamster melanoma lines, supplementation of L-tyrosine from 10 to 600 μM stimulated not only melanin synthesis but also tyrosinase activity in a dose- and time-dependent manner with kinetics defined by the original melanogenic potential of the cell line (Slominski et al., 1988). In melanotic and hypomelanotic lines, tyrosinase activity reached its peak at optimal media tyrosine concentration (200 or 400 μM, respectively) and decreased at pharmacologic concentrations (400 or 600 μM, respectively), whereas in amelanotic cells, there was a continued increase; this, however, slowed (reaching a plateau) when levels of melanization became high (Slominski et al., 1988). The most instructive results were obtained in lines of Bomirski hamster amelanotic melanoma cultured in Ham’s F10 media relatively low in tyrosine (10 μM). Increased L-tyrosine supplements produced concomitant induction and further increases melanin formation and stimulation of both the tyrosine hydroxylase and DOPA oxidase activities of tyrosinase in a process dependent on new protein synthesis (Slominski et al., 1988). This was later confirmed by showing increased production of tyrosinase protein without a significant mRNA expression, demonstrating translational regulation of the enzyme by its substrate in this model (Slominski 1989; Slominski and Costantino, 1991a). The effect was specific, as under the same conditions the enantiomers (D-isoform), related (L-phenylalanine and L-tryptophan) or unrelated (L-valine) amino acids or N-acetyl-tyrosine had little or no effect (Slominski et al., 1988), and this process was independent from L-tyrosine transformation to catecholamines, as both nor- and epinephrine as well as agonists of α and β adrenergic receptors had none or relatively lower effects on tyrosinase activity in comparison with their substrate (Howe et al., 1991). Importantly, induction of melanogenesis was preceded and accompanied by induction of melanosome synthesis and translocation of tyrosinase enzyme from trans-Golgi network (TGN) to premelanosomes with further enzymatic activation (Slominski et al., 1988, 1989b), and L-tyrosine-stimulated melanocyte-stimulating hormone (MSH) receptor expression and MSH receptor activity (Slominski and Pawelek, 1987; Slominski et al., 1989a). L-tyrosine also induced translocation from TGN to melanosomes of other enzymes such as acid phosphatase, indicating a more general effect on the intracellular transport without, however, changing the enzymatic activity of selected lysosomal enzymes (Slominski et al., 1988). This indicated a pleiotropic effect of L-tyrosine on intracellular transport and processing that was, however, specific for the stimulation of melanogenic protein activity. The fundamental role of L-tyrosine in induction of melanosome formation was confirmed by experiments with phenylthiourea (PTU), a non-toxic inhibitor of melanogenesis, which while inhibiting stimulation of tyrosinase by L-tyrosine, did not prevent L-tyrosine stimulation of melanosome synthesis (Slominski et al., 1989b). They presented as the premelanosome stage II with matrix as a multilamellar outer shell and paracrystalline core, or a vesicular matrix (Slominski et al., 1989b). In this model, MSH and agents that raise intracellular cyclic AMP, induced dendrite formation, inhibited cell growth, and caused substantial increases in tyrosinase activity without inducing melanin synthesis; tyrosinase accumulated solely in the TGN and the mature melanosomes were absent (Slominski et al., 1989c). Therefore, it was concluded that L-tyrosine played a crucial role in the induction of the melanotic phenotype through induction and enhancement of both melanosome synthesis/assembly and translocation of tyrosinase from the TGN to melanosomes, leading to in vivo activation of melanogenesis (Slominski and Paus, 1994; Slominski et al., 1988, 1989b). In parallel experiments performed with mouse Cloudman S91 melanoma cells L-tyrosine, while increasing melanin pigmentation, had no effect on or even decreased tyrosinase activity (Slominski et al., 1988), indicating that in this model the activities of the melanogenic apparatus are largely independent of extracellular tyrosine. This was most likely due to an endogenous production of L-tyrosine from L-phenylalanine (Slominski et al., 2004), as detection PH activity has been reported in these cells (Breakefield et al., 1978).
The confirmation for L-tyrosine function as a positive regulator of melanogenesis was also provided in B16 melanoma (Price et al., 1988). In this cell line, L-tyrosine and L-phenylanine not only stimulated melanogenesis but also increased dendrite formation and enhanced the metastatic capability of these cells (Prezioso et al., 1993), apparently through downregulation of protein kinase C (PKC)ζ (Sanz-Navares et al., 2001). Most recently, Zmijewski et al. (unpublished) have also shown that L-tyrosine induced melanin pigmentation in the amelanotic subline of B16 and that this induction was accompanied by the stimulation of tyrosinase mRNA, indicating a transcriptional mode of regulation that was absent in hamster melanoma. Similarly, other investigators demonstrated stimulation of tyrosinase by L-tyrosine in several human melanoma lines (Halaban et al., 2001, 2002a; Ramirez-Bosca et al., 1992; Slominski et al., 1999; Winder and Harris, 1992) in post-translational (Halaban et al., 2001), translational and/or transcriptional modes of action (Slominski et al., 1999).
The novel role for L-DOPA as a positive regulator of melanogenesis was shown for the first time in Cloudman S91 melanoma (McLane et al., 1987; Pawelek and Murray, 1986; Pawelek et al., 1988) and Bomirski hamster melanoma cells (Pawelek et al., 1988; Slominski et al., 1989b).Thus, the addition of phosphate at position C3 and/or C4 of L-DOPA, which produced relatively stable phospho-DOPA (P-DOPA), resulted in dose-dependent stimulatory effects on tyrosinase activity and melanin pigmentation through amplification of the MSH receptor system (McLane et al., 1987; Pawelek and Osber, 1992; Pawelek et al., 1988). L-DOPA by itself did not affect melanogenesis, being rapidly consumed by the melanogenic pathway in Cloudman melanoma cells; however, at micromolar or lower concentrations, it stimulated cell proliferation (McLane et al., 1987; Pawelek et al., 1988). In hamster amelanotic cells, L-DOPA induced rapid and dose-dependent increases of tyrosinase activity, being significantly more potent than its D- form, with a peak activity at 25 or 50 μM, which induced only moderate pigmentation and formation of predominantly immature melanosomes (Slominski et al., 1988). Tyrosinase accumulated predominantly in the TGN (Slominski et al., 1988) with increased concentration of the protein, as documented by Western blot, that was accompanied by an initial increase in tyrosinase mRNA, followed by a decrease below control levels (Slominski and Costantino, 1991b).