SUPPRESSION OF PIGMENTATION IN MOUSE MELANOMA CELLS BY 5-BROMODEOXYURIDINE : Effects on Tyrosinase Activity and Melanosome Formation

Low concentrations (1–3 µg/ml) of 5-bromodeoxyuridine (BrdU) reversibly suppress pigmentation in a highly pigmented clone (B₅59) of cultured B16 mouse melanoma cells. We have found that unpigmented cells (clone C₃471), derived by long-term culture of B₅59 cells in 1 µg of BrdU/ml, were completely amelanotic with no biochemically or cytochemically detectable tyrosinase activity or ultrastructural evidence of premelanosomes. The process by which pigmentation is suppressed was studied in B₅59 cells during a 7-day period of growth with BrdU (3 µg/ml). Assays of tyrosinase activity showed that activity was reduced after 1 day and decreased progressively, approaching zero by 7 days. A quantitatively minor part of this reduction was directly attributable to the appearance of a dialyzable inhibitor of tyrosinase activity. Acrylamide gel electrophoresis revealed two bands of activity corresponding in Rx values to the T₁ and T₂ forms of soluble tyrosinase. Both were progressively reduced during growth with BrdU but one form (T₁) was consistently affected earlier than the other (T₂). Ultrastructural-cytochemical studies also showed an early effect on the localization of tyrosinase reaction product. At day 3, reaction product was no longer present in Golgi saccules and Golgi-associated smooth surfaced tubules, but was still seen within premelanosomes, compound melanosomes, and occasional Golgi-associated vesicles. By 7 days tyrosinase reaction product was usually not demonstrable. The number of premelanosomes was progressively decreased during growth with BrdU. Premelanosomes became concentrated in the juxtanuclear region and at day 3 many were contained within abnormally large and numerous compound melanosomes. Premelanosomes and compound melanosomes were rarely seen at 7 days, by which time the cultures were nearly amelanotic. The coordinated suppression of melanogenesis by BrdU may provide a useful model in which to study the normal regulation of this process.     

In vitro effects of hormonal stimuli upon tyrosinase and peroxidase activities in murine melanomas.

ACTH and corticosterone altered tyrosinase activity in the B-16, S-91 and Harding-Passey melanomas but did not evoke $\text{any}$ changes in peroxidase activity. Tyrosinase activity, but not peroxidase activity, was correlated with the degree of melanin pigmentation present in the melanomas. Thus, tyrosinase is the enzyme of physiological importance in the regulation of melanin pigmentation.     

Purification and Properties of Poly(A) Polymerase from Vigna unguiculata

Poly(A) polymerase was purified from germinating Vigna unguiculataseeds by successive column chromatography on phosphocellulose,Toyopearl HW-55S, heparin-Sepharose and TSKgel phenyl-5PW, whichyielded two activity fractions. The first fraction was purifiedas a single polypeptide with a mol wt of 63,000 as estimatedby SDS-PAGE. The enzyme activity was highly specific for ATPand required Mn²⁺ ion; an ATP-Mn complex may be the actual substrate.The polymerization reaction required a primer, with varioustypes of RNAs, poly(A) as well as dinucleoside phosphates having3'—OH, serving as efficient primers. The two forms ofthe enzyme had very similar properties with respect to divalentcation requirement and dependency on ion strength, but theyshowed some difference in primer preference. (Received March 4, 1988; Accepted May 2, 1988)     

Color reversion of the albino medaka fish associated with spontaneous somatic excision of the Tol‐1 transposable element from the tyrosinase gene

The medaka fish albino mutant, i¹ is one of the Tomita collection of medaka pigmentation mutants which exhibits a complete albino phenotype, because of inactivation of the tyrosinase gene due to insertion of a transposable element, Tol‐1. Recently, mosaic black‐pigmented i¹ medaka fish have arisen in one of our laboratory breeding populations. Their pigmented cells have been observed in all of the tissues, including the eye and skin, in which melanin is detectable in the wild type. In this study, we analyzed the tyrosinase gene of revertants and showed Tol‐1 to have been precisely excised from the gene, suggesting a causal relationship. Mosaic patterns of pigmentation indicate spontaneous somatic excision of the element from the tyrosinase gene. To our knowledge, this is the first transposable element with somatic excision activity demonstrated phenotypically in vertebrates. The pattern of pigmentation in mosaic revertants indicates frequencies of melanin pigments to be consistent with the numbers of melanophores per unit area of body sites, such as the eyes, head and dorsal trunk.     

Localization of 4-Chloroindole-3-acetic Acid in Seeds of Pisum sativum and Its Absence from All Other Organs

4-Chloroindole-3-acetic acid (4-Cl-IAA) was shown by GC-MS analysisto be present in immature and mature seeds of Pisum sativum,but not in any other organs of this plant. Its content was maximalat one week after anthesis and decreased as the seeds matured.Only indole-3-acetic acid (IAA) was detected in the other organsof P. sativum, its content being particularly high in the flowersand young pods during anthesis and the early pod set. (Received January 18, 1988; Accepted April 6, 1988)     

Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues

Abnormal melanogenesis results in excessive production of melanin, leading to pigmentation disorders. As a key and rate-limiting enzyme for melanogenesis, tyrosinase has been considered an important target for developing therapeutic agents of pigment disorders. Despite having an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which plays an important role in the potent inhibition of tyrosinase activity, cinnamic acids have not attracted attention as potential tyrosinase inhibitors, due to their low tyrosinase inhibitory activity and relatively high hydrophilicity. Given that cinnamic acids’ structure intrinsically features this (E)-scaffold and following our experience that minute changes in the chemical structure can powerfully affect tyrosinase activity, twenty less hydrophilic cinnamamide derivatives were designed as potential tyrosinase inhibitors and synthesised using a Horner-Wadsworth-Emmons reaction. Four of these cinnmamides (4, 9, 14, and 19) exhibited much stronger mushroom tyrosinase inhibition (over 90% inhibition) at 25 µM compared to kojic acid (20.57% inhibition); crucially, all four have a 2,4-dihydroxy group on the β-phenyl ring of the scaffold. A docking simulation using tyrosinase indicated that the four cinnamamides exceeded the binding affinity of kojic acid, and bound more strongly to the active site of tyrosinase. Based on the strength of their tyrosinase inhibition, these four cinnamamides were further evaluated in B16F10 melanoma cells. All four cinnamamides, without cytotoxicity, exhibited higher tyrosinase inhibitory activity (67.33 – 79.67% inhibition) at 25 μM than kojic acid (38.11% inhibition), with the following increasing inhibitory order: morpholino (9) = cyclopentylamino (14) < cyclohexylamino (19) < N-methylpiperazino (4) cinnamamides. Analysis of tyrosinase activity and melanin content in B16F10 cells showed that the four cinnamamides dose-dependently inhibited both cellular tyrosinase activity and melanin content and that their inhibitory activity at 25 μM was much better than that of kojic acid. The results of melanin content analysis well matched those of the cellular tyrosinase activity analysis, indicating that tyrosinase inhibition by the four cinnamamides is a major factor in the reduction of melanin production. These results imply that these four cinnamamides with a 2,4-dihydroxyphenyl group can act as excellent anti-melanogenic agents in the treatment of pigmentation disorders.     

Enzymes of the xylotrophic basidiomycete Lentinus edodes F-249 in the course of morphogenesis

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.     

INVESTIGATIONS OF THE ROLE OF MUCUS AND FAECES IN INTERSPECIFIC INTERACTIONS OF TWO LAND SNAILS

A previous report (Smallridge & Kirby, 1988) showed interspecificcompetition between the two land snails Theba pisana and Cernuellavirgata and suggested the mechanism of competition may be thatheterospecific mucus and excrement inhibits activity. We describea series of experiments in which we could find no consistentevidence for any short-term inhibition of activity in the presenceof heterospecific mucus. Furthermore snails did not consistentlyavoid mucus-treated surfaces or mucus-coated food. Cernuellastopped at and fed from snail faeces (both conspecific and heterospecific)more than did Theba. This suggests an alternate mechanism forinterspecific competition. (Received 9 April 1992; accepted 9 June 1992)     

Specific identification of an authentic clone for mammalian tyrosinase

Tyrosinase, the critical enzyme to melanin pigmentation in mammals, occurs as a series of isozymic forms, which have been previously regarded as different stages in processing of a single precursor form. Recently, three different cDNA clones have been identified which may encode tyrosinase, they share extensive sequence homology but are distinct; two of them have been mapped to genetic loci which regulate different aspects of melanogenesis. Since direct confirmation of the authentic tyrosinase sequence has proven impossible by conventional protein sequencing strategies, we have approached the identification of the tyrosinase gene by synthesizing peptides encoded by the putative genes and preparing antibodies to those peptides. By use of pulse-chase labeling and immunoprecipitation analyses, and by enzymatic determinations, pMT4 (which maps to the brown b locus in mice) is shown to encode a molecule with tyrosinase catalytic activity which is biochemically identical with authentic tyrosinase. However, our results raise the possibility that other gene products may contribute to melanogenesis by one or more melanogenic activities.     

Regulation of mammalian melanogenesis by tyrosinase inhibition

Melanocyte stimulating hormone (MSH) specifically induces differentiation of mammalian melanocytes. To further define the biochemical events elicited by this stimulus, we have cloned murine melanoma cells which are either highly responsive or nonresponsive to MSH, and have examined their ultrastructural appearance, their melanogenic activities, and also their expression of tyrosinase. We have found that the basal levels of melanogenic activity in pigmented and nonpigmented cells correlate with expression of surface MSH receptors rather than with production of tyrosinase. Nonpigmented cells produce a potent, highly stable inhibitor of melanogenesis; this inhibitor acts directly on tyrosinase to dramatically and abruptly suppress melanin production. This posttranslational control of tyrosinase activity may represent a critical regulatory point in mammalian pigmentation.     

Inhibitory effect of rose hip (Rosa canina L.) on melanogenesis in mouse melanoma cells and on pigmentation in brown guinea pigs

The compounds present in rose hips exerting an inhibitory action against melanogenesis in B16 mouse melanoma cells were investigated by dividing an aqueous extract of rose hips (RE) into four fractions. The 50% ethanol eluate from a DIAION HP-20 column significantly reduced the production of melanin and was mainly composed of procyanidin glycosides. We also found that this 50% ethanol eluate reduced the intracellular tyrosinase activity and also had a direct inhibitory effect on tyrosinase obtained as a protein mixture from the melanoma cell lysate. We also investigated the effect of orally administering RE on skin pigmentation in brown guinea pigs, and found that the pigmentation was inhibited together with the tyrosinase activity in the skin. These data collectively suggest that proanthocyanidins from RE inhibited melanogenesis in mouse melanoma cells and guinea pig skin, and could be useful as a skin-whitening agent when taken orally.     

Highly Repetitive Tandem Arrays of a Short 32 bp Unit in the Pharbitis nil Genome: the RsaI Family

A highly repetitive DNA sequence of Pharbitis nil, designatedthe RsaI family, was cloned, sequenced and analyzed with respectto its genomic organization. The RsaI family is arranged intandem arrays and composed of a 32 bp repeat unit, which isthe shortest unit thus far reported for plant repetitive sequences.The RsaI family represents 3% of the total genomic DNA and thecopy number of the 32 bp unit is estimated to be about 1 ? 10⁶per haploid genome. We suggest the existence of a higher orderrepeat unit, which may be composed of hundreds of the 32 bpunits and be repeated many times in the genome. (Received May 12, 1988; Accepted July 28, 1988)     

Stem cell factor rescues tyrosinase expression and pigmentation in discreet anatomic locations in albino mice

The K14‐SCF transgenic murine model of variant pigmentation is based on epidermal expression of stem cell factor (SCF) on the C57BL/6J background. In this system, constitutive expression of SCF by epidermal keratinocytes results in retention of melanocytes in the interfollicular basal layer and pigmentation of the epidermis itself. Here, we extend this animal model by developing a compound mutant transgenic amelanotic animal defective at both the melanocortin 1 receptor (Mc1r) and tyrosinase (Tyr) loci. In the presence of K14‐Scf, tyrosinase‐mutant animals (previously thought incapable of synthesizing melanin) exhibited progressive robust epidermal pigmentation with age in the ears and tails. Furthermore, K14‐SCF Tyr^c2j/c2j animals demonstrated tyrosinase expression and enzymatic activity, suggesting that the c2j Tyr defect can be rescued in part by SCF in the ears and tail. Lastly, UV sensitivity of K14‐Scf congenic animals depended mainly on the amount of eumelanin present in the skin. These findings suggest that c‐kit signaling can overcome the c2j Tyr mutation in the ears and tails of aging animals and that UV resistance depends on accumulation of epidermal eumelanin.     

A newly synthesized,potent tyrosinase inhibitor: 5-(6-Hydroxy-2-naphthyl)-1,2,3-benzenetriol

In searching for new agents with a depigmenting effect, we synthesized a derivative of resveratrol, 5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (5HNB) with a potent tyrosinase inhibitory activity. 5HNB inhibited mushroom tyrosinase with an IC₅₀ value of 2.95 μM, which is more potent than the well-known anti-tyrosinase activity of kojic acid (IC₅₀ = 38.24). The results of the enzymatic inhibition kinetics by Lineweaver–Burk analysis indicated 5HNB inhibits tyrosinase non-competitively when l-tyrosine was used as the substrate. Based on the strong inhibitory action of 5HNB, it is expected that 5HNB can suppress melanin production in which tyrosinase plays the essential role. Our expectation was confirmed by the experimentations with B16 melanoma cells in which 5HNB inhibited melanin production. We propose that 5HNB might have skin-whitening effects as well as therapeutic potential for treating skin pigmentation disorders.     

A Transgenic Mouse Model with Inducible Tyrosinase Gene Expression Using the Tetracycline (Tet‐on) System Allows Regulated Rescue of Abnormal Chiasmatic Projections Found in Albinism

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET‐ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.     

FOOD-INDUCED BODY PIGMENTATION QUESTIONS THE TAXONOMIC VALUE OF COLOUR IN THE SELF-FERTILIZING SLUG CARINARION SPP

Body pigmentation is a popular taxonomic marker in slugs to discriminateclosely related species. However, the genetic background ofbody pigmentation is known only for a few species, while inmany others body pigmentation is influenced by age, food and/orclimate. In this study, we investigated the effects of differentfood items on body pigmentation expression in two selfing pulmonategastropods, Arion (Carinarion) silvaticus and Arion (Carinarion) fasciatus.Both species mainly differ in the distribution of yellow-orangegranules on the body, which in A. fasciatus are concentratedin lateral bands, and in A. silvaticus are evenly scattered.Animals were raised individually under the same conditions, whilethey laid eggs as a consequence of selfing. This F₁ generation wasafterwards divided into two groups, which were fed with different fooditems. A diet of carrot, lettuce or paper had no effect on the distributionof the yellow-orange granules in A. silvaticus, but provokeda loss of the yellow-orange lateral bands in A. fasciatusso that externally these F₁ specimens became similar to A. silvaticus.In both species, a diet of nettle resulted in a strong yellow-orangepigmentation, which often formed yellow-orange lateral bands.These results indicate that food can probably influence the'species-specific' body pigmentation in Carinarion, and thusquestion the reliability of colour traits to distinguish A.silvaticus and A. fasciatus. (Received 5 May 2000; accepted 29 September 2000)     

Human Melanoma Cell Lines Show Little Relationship Between Expression of Pigmentation Genes and Pigmentary Behaviour In Vitro

Several laboratories are pursuing the question of whether the expression of pigment genes can be used as a useful marker for tumour progression. However, many melanoma tumours are amelanotic in vivo. The purpose of this study was to examine the relationship between the expression of tyrosinase-related genes [tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2)] and pigmentation of melanoma cells. Fourteen cutaneous melanoma cell lines were examined for visible pigment, melanin content, and dopa oxidase activity and findings were related to the previously determined expression of the three tyrosinase-related genes in these cells in culture. Four of the cell lines were also stimulated with α-MSH, isobutylmethylxanthine, and forskolin to examine the relationship between induced pigmentation and upregulation of pigmentation genes. There was no simple correlation between pigmentation gene expression and dopa oxidase activity or total melanin content of the 14 melanoma cell lines in culture. In the majority of cells, there was no appreciable pigment, whereas, in contrast, half of the cells showed significant dopa oxidase activity. Upregulation of dopa oxidase activity was achieved by α-MSH in two out of four cell lines examined in detail and with IBMX in three out of four of these cell lines. IBMX increased tyrosinase gene expression in all four cell lines; α-MSH was without effect; and TRP-1 and TRP-2 expression were largely unaffected by IBMX or α-MSH. Modest changes in morphology were noted in response to IBMX. Overall, however, human melanoma cell lines were, with two exceptions, amelanotic in culture despite the fact that 10 out of the 14 lines expressed tyrosinaserelated genes. We conclude that measurable pigmentation is not a necessary consequence of the expression of pigmentation genes. An implication of this work is that amelanotic tumours in vivo may nevertheless be positive for tyrosinase-related genes.