Serum ceruloplasmin in melanoma patients.

We have studied the serum ceruloplasmin levels as a possible diagnostic factor or marker for detection, diagnosis and follow-up of patients with melanoma. Ceruloplasmin concentration was determined in 64 melanoma patients (MP) and in 37 healthy persons (HP) by nephelometry. We found a mean value of 29.85 +/- 5.47 mg/dl in MP and 26.10 +/- 5.22 mg/dl in HP. A significant increase in the levels of serum ceruloplasmin was observed in MP in comparison to those in HP (P = 0.0011). In order to check whether this test could discriminate between MP and HP, a complete statistical Receiver Operating Characteristic (ROC) curve analysis was performed. The cut-off value was 25.10 mg/dl. The area under the curve was 0.689. According to these results, the test could discriminate adequately between the two groups.

Melanization stimulating factors in the integument of the Mugil cephalus and Dicertranchus labrax.

The pigment pattern expression resides in the chromatoblasts of the embryonic skin. The differentiation of these chromatoblasts is influenced by specific local factors such a melanization inhibiting factor (MIF) and a melanization-stimulating factor (MSF). We reveal the presence of these factors by means of a series of experiments on the skin of the marine species of fish Dicertranchus labrax and Mugil cephalus, each with different pigment pattern, the former having a light skin and the latter a darker one. Media conditioned by exposure to dorsal and/or ventral skin, stimulates the melanization of Xenopus laevis neural crest cells throughout a 3 day assay period. Similarly conditioned culture media tested on B16-F10 murine malignant melanocytes, revealed a considerable influence in enzymatic activities: dopachrome tautomerase (DCT), tyrosine hydroxylase and dopa oxidase. The use of media in a dose response basis suggests that the conditioned media may contain both melanophore stimulating and inhibiting factors. The results obtained may actually reflect the resultant activity of the two factors present.

Whole blood glutathione peroxidase activity in melanoma patients.

We have studied whole blood glutathione peroxidase activity as a possible diagnostic factor or marker for detection and diagnosis of patients with melanoma. This activity was determined in 40 melanoma patients (MP) and in 40 healthy persons (HP) using an enzymatic method. We found a mean value of 17.90+/-6.82 units/ml in MP and 27.07+/-14.35 units/ml in HP. A very significant decrease in whole blood glutathione peroxidase activity was observed in MP in comparison to the enzymatic activity in HP (P = 0.0005). In order to check whether this test could discriminate between MP and HP, a complete statistical Receiver Operating Characteristic (ROC) curve analysis was performed. The cut-off value was 14.26 units/ml. The area under the curve was 0.737. According to these results, the test could discriminate adequately between both groups. However, the high specificity and low sensitivity values associated with that cut-off value would make this test a very valuable tool for confirming the detection, rather than for primary screening.

Serum sialic acid in malignant melanoma patients: an ROC curve analysis.

We have studied the total serum sialic acid as a possible diagnostic factor for the prognosis of patients with melanoma. The sialic acid concentration was determined in the sera of fifty melanoma patients (MP) and forty healthy persons (HP), by using an enzymatic method. We found a mean value of 67.52 +/- 17.32 mg/dl in MP and 57.11 +/- 12.98 mg/dl in HP (p < 0.005). In order to check whether this test could discriminate adequately between MP and HP, a complete statistical Receiver Operating Characteristic (ROC) curve analysis was performed. The cut-off value was 62.39 mg/dl. The area under the curve was 0.661. According to these results, the test could discriminate between both groups. However, due to the rise of sialic acid-rich acute-phase proteins in inflammatory processes, determination of total serum sialic acid would be more useful for diagnosis of melanoma stage and prognosis rather than for early detection and screening.

Serum tyrosine hydroxylase activity is increased in melanoma patients. An ROC curve analysis.

We have studied the serum tyrosine hydroxylase activity of tyrosinase, the key enzyme in the melanogenesis via, as a possible diagnostic factor or marker for detection, prognosis and follow-up of patients with melanoma. This activity was determined in 30 melanoma patients (MP) and in 30 healthy persons (HP) by using a radiometric method. We found mean values of 10.8+/-3.0 and 7.65+/-2.32 mU/l for serum tyrosine hydroxylase activity in MP and HP, respectively. A very significant increase in serum tyrosine hydroxylase activity was observed in MP in comparison to the enzymatic activity in HP (P < 0.00001). Although these data seem very conclusive, we wanted to know whether this test could discriminate adequately between MP and HP. In order to reach this aim, a complete statistical study was performed by using receiver operating characteristic (ROC) curve analysis. The cut-off value obtained was 8.47 mU/l. According to our results and after analytical treatment of the data, we can confirm that evaluation of tyrosine hydroxylase activity in serum could be a quick and reliable diagnostic method for detection, prognosis and follow-up in melanoma patients.

Serum zinc levels are increased in melanoma patients.

We studied the levels of serum copper and zinc as possible diagnostic factors or markers for the early detection of patients with melanoma. Levels were determined in 35 melanoma patients at various clinical stages and in 39 healthy persons. Measurements were performed by atomic absorption spectroscopy using 5100-PC-Perkin-Elmer equipment. We found that serum copper levels were very similar in the melanoma patients and the healthy individuals, the medium values being 118.32 +/- 25.32 micrograms/dl and 117.94 +/- 28.01 micrograms/dl, respectively. Therefore, no significant differences were observed with regard to copper levels. On the other hand, we obtained a medium value of 82.32 +/- 25.38 micrograms/dl for serum zinc levels in the melanoma patients and 56.72 +/- 11.79 micrograms/dl in the healthy persons, which represents a very significant increase in the serum levels of zinc in melanoma patients (P < 0.0001). A receiver operating characteristic (ROC) curve statistical analysis was also performed; the cut-off value obtained was 60.9 micrograms/dl. According to our results, zinc is increased in 86.5% of melanoma patients. Although further investigations are needed to assess its value in prognosis and follow-up, evaluation of serum zinc level could be a good tool to check for the presence of melanoma.

The amphibian melanization inhibiting factor (MIF) blocks the alpha-MSH effect on mouse malignant melanocytes.

We have found that a melanization inhibitory factor (MIF) extracted from the ventral skin of Rana forreri has a slight inhibitory effect on the activity levels of tyrosinase and dopachrome tautomerase in B16/F10 and Cloudman S-91 murine melanoma cell lines. Furthermore, this factor appears to block the effects of alpha-MSH on these enzymatic activities. However, MIF treatment does not affect the melanogenic action of theophylline on the same cells, suggesting that MIF acts proximal to MSH-mediated cAMP formation, possibly by interaction with the MSH receptor. In this way, we show that this amphibian factor has biological activity on mammalian melanocytes. This suggests the existence of mammalian counterparts of amphibian MIF in the mouse integument that might regulate epidermal melanocytes. These peptides might be related to the agouti protein, as they share similar mechanisms of action. The interaction of different peptides with the MSH receptor would be a complex but general mechanism responsible for many mammalian coat color variants.

Molecular mechanism for catalysis by a new zinc-enzyme, dopachrome tautomerase.

Dopachrome tautomerase (DCT; EC 5.3.3.12) catalyses the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid in the mammalian eumelanogenic biosynthetic pathway. This enzyme, also named TRP2, belongs to a family of three metalloenzymes termed the tyrosinase-related proteins (TRPs). It is well known that tyrosinase has copper in its active site. However, the nature of the metal ion in the active site of DCT is under discussion. Whereas theoretical predictions based on similarity between the protein sequences of the TRPs suggest the presence of copper, the different inhibition pattern of DCT with some metal chelators compared with that of tyrosinase suggests that the nature of the metal ion could differ. Direct estimations of the metal content in purified DCT preparations show the presence of around 1.5 Zn atoms/molecule and the absence of copper. Apoenzyme preparation by treatment of DCT with cyanide or o-phenanthroline followed by reconstitution experiments of tautomerase activity in the presence of different ions confirmed that the metal cofactor for the DCT active site is zinc. Our results are consistent with Zn2+ chelation by the highly conserved histidine residues homologous to the histidines at the classical copper-binding sites in tyrosinase. This finding accounts for the reaction catalysed by DCT, i.e. a tautomerization, versus the copper-mediated oxidations catalysed by tyrosinase. Based on the predicted tetrahedrical coordination of the zinc ions in the enzyme active site, a molecular mechanism for the catalysis of L-dopachrome tautomerization is proposed. From the present data, the existence of additional ligands for metal ions other than zinc in the DCT molecule, such as the proposed cysteine iron-binding sites, cannot be completely ruled out. However, if such sites exist, they could be subsidiary binding sites, whose function would be likely to stabilize the protein.

Effect of penicillin-streptomycin and other antibiotics on melanogenic parameters in cultured B16/F10 melanoma cells.

Penicillin and streptomycin, the most widely used antibiotics in mammalian cell cultures, caused a moderate stimulation in dopa oxidase and tyrosine hydroxylase activities, but a slight inactivation in the dopachrome tautomerase activity of B16/F10 melanoma cells at the routine concentration (100 units/ml penicillin and 100 micrograms/ml streptomycin) used for preventing bacterial growth in cultured animal cells. At these concentrations, tyrosinase activities and melanin content augmented with time during the first 24-48 hr. The opposite effect acted on cell viability. After withdrawal of the antibiotics from the culture medium, the recovery of melanogenic parameters to normal values was fully reached after few hours (around 10), and it was already noticeable as soon as 4 hr after removal. Other antibiotics used in cell culture, like kanamycin, gentamicin, and the antimicotic nystatin, exerted similar low effects at the recommended concentrations, always lower than two-fold and thus lower than those reported for amphotericin B. Taking into account these relatively low effects, and the high risk of contamination of mammalian cells culture without antibiotics, penicillin and streptomycin may still be routinely used in experiments leading to explore the melanogenic activity of malignant melanocytes in culture, unless very precise studies and strict conditions were needed.

Dopachrome tautomerase is a zinc-containing enzyme.

Dopachrome tautomerase (DCT) catalyzes the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid through the melanogenic biosynthetic pathway. This enzyme, also named TRP2, belongs to the family of the tyrosinase related proteins. The three members of the family contain two highly conserved metal-binding sites with three histidines on each. Tyrosinase has copper at its active site. It was assumed that although DCT might have copper in those metal binding sites, its active site could be related to other two putative iron-binding sites located in different positions. Based on apoDCT preparation with cyanide and reconstitution experiments, we propose that DCT have zinc instead of copper at the two metal-binding sites and that those sites actually correspond to the active site. The involvement of zinc, which cannot undergo redox reactions, accounts for the reaction that DCT catalyzes, a tautomerization versus the copper-mediated oxidations catalyzed by tyrosinase.

Levels of dopachrome tautomerase in human melanocytes cultured in vitro.

Several reports have been published about the level of activity and possible functions of dopachrome tautomerase (DCT) in mouse melanoma cells. Data about the levels of this activity in human melanocytes in culture are still scarce, and, as far as we know, a comparison between mouse and human melanocytes, or between normal and malignant melanocytes, has never been published. We have measured the tyrosinase and DCT activities, as well as the melanin content, in mouse Cloudman melanoma cells, two lines of human melanoma, and three lines of normal human melanocytes obtained from fetal skin. Although more cell lines should be tested to draw a general conclusion, our results suggest that normal melanocytes contained much higher tyrosinase activity and melanin content but lower DCT activity than malignant melanocytes. The two lines of human melanoma cells tested had lower levels of DCT activity than Cloudman melanoma cells. Finally, the low level of DCT activity found in normal human melanocytes cultured in vitro cannot be explained by any of the necessary stimulatory factors added to the cell culture media.

Melanization stimulating activity in the skin of the gilthead porgy, Sparus auratus.

The presence of a melanization-stimulating factor (MSF) was discovered in dorsal and/or ventral skin of Sparus auratus. Skin from this marine species was used to condition Steinberg's balanced salt solution (BSS), which was subsequently tested with the neural tube assay. BBS conditioned by dorsal and/or ventral skin of S. auratus at 25% and 50% concentrations had a profound stimulatory effect on the percentage of melanization of neural crest cells throughout the 3-day assay period. In some cases 90% melanization occurred within the first 24 hr. Such stimulated cells showed a doubling of the number of dendrites per cell. To assess the effects of MSF on other indices of melanization, dorsal and/or ventral skin was used to condition MEM used in the culture of B16-F10 murine melanoma cells. During the first 24 hr, B16-F10 murine melanoma cells responded to conditioned media by demonstrating a considerable increase in activities of tyrosine hydroxylase, dopa oxidase, and dopachrome tautomerase, but no effect was observed on melanin content. In contrast, melanin content increased after 48 hr of incubation, whereas the enzymatic activities were inhibited during this period. It seems that MSF activity, expressed in several ways, may be present generally among marine species.

Effect of amphotericin B on dopachrome tautomerase activity and other melanogenic parameters in cultured B16/F10 melanoma cells.

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173-175) for tyrosine hydroxylase.

Alpha-MSH and other melanogenic activators mediate opposite effects on tyrosinase and dopachrome tautomerase in B16/F10 mouse melanoma cells.

alpha-MSH was found to decrease the recently characterized dopachrome tautomerase activity in cultures of B16/F10 mouse melanoma cells. Other stimulating agents of melanogenesis, like dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, theophylline, retinol, and retinoic acid, caused the same effect. The grade of inhibition depended on the nature of the agent and the time of exposure. In all cases, both melanin production and tyrosinase activity were activated by these treatments, although the grade of tyrosine hydroxylase and dopa oxidase stimulation was different. Moreover, no correlation among the intensities of dopachrome tautomerase inhibition and tyrosinase activation by the tested agents could be obtained. The significance of these results in the regulation of mammalian melanogenesis is discussed.

Melanocyte stimulating hormone activation of tyrosinase in B16 mouse melanoma cells. Evidence for a differential induction of two distinct isoenzymes.

Tyrosinase induction in murine malignant melanocytes by alpha MSH is well known, but its molecular basis has not been characterized. Treatment of B16 melanoma cells with theophylline or alpha MSH mediates a larger induction of tyrosine hydroxylase than of dopa oxidase activity in total cell extracts, and in the melanosomal and microsomal fractions. No evidence for the modulation of a tyrosinase effector was found. SDS-PAGE and specific activity stain demonstrated two forms of tyrosinase, with different degrees of induction by theophylline. These results agree with the recent proposal that two tyrosinases, encoded by different genes, are present in murine melanocytes.

The action of glycosylases on dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase.

It is shown that dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase (DCT) is a glycoprotein containing N-linked oligosaccharides. The enzymic activity can be stimulated by partial deglycosylation with a number of glycosylases such as neuraminidase, beta-mannosidase and beta-galactosidase. However, the stability of the enzyme after the hydrolytic treatment becomes lower. Thus total deglycosylation with peptide N-glycosidase F directly provokes an inactivation of DCT. The native enzyme also shows a strong affinity for concanavalin A-Sepharose. This affinity decreases after treatment with neuraminidase and/or beta-mannosidase. The DCT associated with coated vesicles seems to be mostly glycosylated, since the action of glycosylases on the enzyme obtained from these vesicles produced a similar stimulation to that with the melanosomal enzyme. Treatment of cultured melanocytes with tunicamycin elicited a decrease in the amount of active DCT inside the cells. All data suggest that the structure of the carbohydrate moiety of DCT should be very similar to, if not identical with, the structure proposed for tyrosinase by Ohkura, Yamashita, Mishima & Kobata (1984) Arch. Biochem. Biophys. 235, 63-77.

Inorganic phosphate binding and electrostatic effects in the active center of aspartate aminotransferase apoenzyme.

The ionization state of the phosphate group bound at the aspartate aminotransferase apoenzyme's active site has been investigated utilizing Fourier-transform infrared spectroscopy following the band corresponding to the symmetric stretching of the dianionic phosphate. Unlike free phosphate, when inorganic phosphate is bound at the enzyme's active site, the integrated intensity value of the dianionic band does not change with pH within the studied range, and this value is similar to that for free dianionic phosphate at pH 8.3. From these results, we propose a dianionic state for the phosphate ion bound to cytosolic aspartate aminotransferase throughout the pH range of 5.7-8.3. The presence of other anions such as acetate and chloride or the substrate aspartate and its analogues produces a pH-dependent phosphate removal from the active site which is favored at low pH values. Elimination of the charged primary amine at the active-site Lys-258, through formation of a Schiff base with pyridoxal or chemical modification by carbamylation, also produces a pH-independent phosphate release. These results are interpreted as Lys-258 together with the active-site alpha-helix and other residues may be involved in stabilizing phosphate as a dianion in the apoenzyme phosphate pocket which anchors the phosphate ester of pyridoxal phosphate in the holoenzyme. It is proposed that the dianionic phosphate contributes to the apoenzyme's thermal stability through formation of strong hydrogen bond and salt bridges with the amino acid residues forming the phosphate binding pocket with assistance of Lys-258, and other active-site cationic components.

Inhibition by analogues of L-tyrosine transport by B16/F10 melanoma cells.

The effect of a number of L-tyrosine (L-Tyr) analogues on L-Tyr uptake by B16/F10 malignant melanocytes is reported. This amino acid can be taken up by two of the most ubiquitous transport systems found in animals cells, L and presumably ASC. L-Tyr analogues devoid of the amino group, like p-hydroxyphenyl pyruvic acid and related compounds, and L-Tyr analogues devoid of the carboxyl group, such as tyramine, do not affect L-Tyr uptake. The other aromatic amino acids, L-Phe and L-Trp, and the L-Tyr analogues DL-m-Tyr, L-diiodotyrosine and L-dopa, strongly inhibit the uptake of L-Tyr. This suggests that these chemicals are transported more efficiently than L-Tyr. The ASC system does not show stereospecificity, but the L system has greater affinity for L-Tyr than for D-Tyr. The ASC system also has greater affinity for tyrosine isomers with the hydroxyl group in the ortho and meta positions. The presence of a methyl group at the alpha-carbon of L-Tyr and L-dopa also increases the affinity of the ASC system for these agents. In contrast, alpha-methylation decreases the affinity of the L system in comparison to L-Tyr. Finally, L-Tyr esters do not inhibit, but stimulate the transport of L-Tyr, mainly by the ASC system.

Transport of L-tyrosine by B16/F10 malignant melanocytes: characterization of the process.

The main characteristics of L-tyrosine (L-Tyr) uptake by B16/F10 malignant melanocytes are reported. This amino acid can be taken up by two systems, both of them being saturable. The first one would be system L. This system can be studied in cells preloaded with amino acids that are a good substrate for system L, such as L-methionine or L-tryptophan. The kinetic parameters for L-Tyr uptake by this transport system are Vm = 6.5 pmol L-Tyr/10(3) cells.min and Km around 130 microM. The second system, probably the system ASC, shows lower capacity but higher affinity than the former. This system can be detected only in cells previously depleted of amino acids, showing approximate kinetic values of Vm 0.05 pmol L-Tyr/10(3) cells.min and Km around 5 microM. It is shown that the increase in cell density yields a decrease in the rate of L-Tyr uptake by system L, but this increase does not affect the high affinity system, alpha-MSH does not affect significantly the L-Tyr uptake by both systems. 2-Amino bicyclo-(2,2,1)-heptane-2-carboxylic acid produces a remarkable inhibition of the rate of L-Tyr uptake, but alpha-methylaminoisobutyric acid does not affect the rate of transport of this amino acid. The absence of sodium produces a slight but reliable decrease in the rate of L-Tyr uptake, supporting the involvement of two different transport systems. The ionophores monensin and nigericin enhance the transport by system L, but this effect is suppressed by the presence of ouabain. This finding indicates that the (Na+ -K+)-ATPase is essential for the stimulating action of ionophores.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport of L-tyrosine by B16/F10 melanoma cells: the effect of the intracellular content of other amino acids.

The uptake of L-Tyr by B16/F10 malignant melanocytes in culture has been studied. These melanoma cells can either be depleted of amino acids by 1 h preincubation in Hanks' isotonic medium or preloaded with a specific amino acid by 1 h preincubation in the same solution containing 2 mM of the amino acid to be preloaded. By means of these pretreatments, it is shown that the rate of L-Tyr uptake is greatly dependent on the content of other amino acids inside the cells. The L-Tyr uptake is higher in cells preloaded with amino acids transported by the L and ASC systems than in cells depleted of amino acids or preloaded with amino acids transported by the A system. It is concluded that L-Tyr is mainly taken up by an exchange mechanism with other amino acids mediated by the L1 system, although the ASC system can also participate in the process. In agreement with that, the homo-exchange performed by cells preloaded with unlabelled L-Tyr is more efficient than any other hetero-exchange, although L-Dopa, the product of tyrosine hydroxylation in melanin synthesis, is almost as efficient as L-Tyr. Apart from aromatic amino acids, melanoma cells preloaded with L-Met and L-His also yield a high initial rate of L-Tyr uptake. The results herein suggest that melanoma cells do not have transport systems specific for L-Tyr, even if this amino acid is needed to carry out the differential pathway of this type of cells, melanosynthesis.