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Melanoma cells resistant to inhibition of growth by melanocyte stimulating hormone.

Melanocyte stimulating hormone (MSH) enhances melanization but inhibits proliferation of Cloudman S91 melanoma cells in culture. We have isolated variants of these cells that can grow in the presence of MSH. The conclusions we have reached from analyses of these cells are the following: (1) Basal tyrosinase activity (monophenol monooxygenase; monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1), i.e., the activity that is present in the absence of added MSH, is related through a common biochemical pathway to MSH-mediated control of growth. (2) MSH-inducible tyrosinase activity does not appear to be related to MSH control of growth. (3) The morphological changes that occur following the addition of MSH or cAMP are related to controls of growth and not to those of melanization.

Animals

Reductant-dependent electron distribution among redox sites of laccase.

Rhus laccase (monophenol monooxygenase, monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) an O2/H2O oxidoreductase containing four copper ions bound to three redox sites (type 1, type 2, and type 3 Cu pair), was titrated anaerobically with several reductants having various chemical and thermodynamic properties. The distribution of electron equivalents among the redox sites was found to be reductant dependent. When the data for titration by various reductants of the type 3 site were plotted against those of the type 1 site according to the Nernst formalism, the slope n varied from 2.0 to 1.0. The redox potential of the reductant's first oxidation step is qualitatively correlated with the value of n and is suggested as the factor that modulates the electron distribution. Such a behavior implies a nonequilibrium situation. A very good simulation of the data was provided by an analysis assuming a formally variable cooperativity between the two type 3 copper ions. This apparent variability is suggested to result from a process whereby sufficiently strong reductants induce a transition of the type 3 site from a cooperative two-electron acceptor to a pair of independent one-electron acceptors. This uncoupled state of the type 3 site is considered metastable. Other possible models were also investigated. Summarizing the available data, we conclude that the two-electron accepting behavior of the 330-nm chromophore is the exception rather than the rule.

Anaerobiosis

Ultrastructure and cytochemical localization of laccase in two strains of Leptosphaerulina briosiana (Pollaci) Graham and Luttrell.

Substrate specificity tests were used to identify the presence of laccase in two strains of Leptosphaerulina briosiana (Poll.) Graham and Luttrell, an ascomycete which causes leaf spot in alfalfa. Cytochemical localization of monophenol monooxygenase (laccase) as well as the ultrastructures of the two strains were investigated. Laccase was observed in the outer layers of the cell walls of both strains. The ultrastructures of vegetative hyphae of both strains were typical of those found in most ascomycetes.

Ascomycota

Light-induced enzyme system leading to pigmentation.

Alpha-Chymotrypsin was light sensitized by acylating with cis-cinnamoyl ester, a substrate interconvertible to the trans form by ultraviolet (UV) light. The degree of acylation by this method was complete leaving no residual activity of the enzyme. Upon UV irradiation the inhibited enzyme regained about 70% of its original activity, thereby adding light-sensitiveness to the proteolytic enzyme. In seeking a photographic application of the light-sensitized enzyme, a pigmenting enzyme was incorporated with it. The coupled enzyme system was shown to exhibit a light signal in the form of dark pigment slurry.

Catechol Oxidase

Metabolism of diethylstilbestrol: identification of a catechol derived from dienestrol.

The enzymatic oxidation of E-3,4-bis-(p-hydroxyphenyl)-hex-3-ene (diethylstilbestrol) by either mushroom tyrosinase or rat liver microsomes in the presence of NADPH and air yields a catechol. Upon further oxidation of both compounds with periodate and condensation of the resulting o-quinones with o-phenylenediamine, phenazines are produced. The phenazines derived from the products of both the plant and animal enzyme systems are identical to the product obtained by oxidation of diethylstilbestrol with potassium nitrosodisulfonate and condensation of the o-quinone produced with o-phenylenediamine. High and low resolution mass spectra of the phenazine are consistent with its derivation from a catechol having two fewer hydrogens than diethylstilbestrol.

Animals

Genetic control of retinal ganglion cell projections.

We have assessed the effects of 15 pigmentation mutations on the development of retinal ganglion cell projections in mice in two ways: (1) by analyzing the pattern of innervation of the ipsilateral lateral geniculate nucleus as mapped in autoradiograms of brains of animals killed 12 days after intravitreal injection of 3H-proline into one eye and (2) by determining the ratio of axonally transported radioactive protein in the contralateral and ipsilateral optic tracts after similar intravitreal injections. Analysis of the ratio of transported protein in the two optic tracts provides a new and useful assay of the degree of decussation in experimental animals. The effects of the mutations on eye pigmentation, whole eye melanin content and relative tyrosinase activity also were examined. The degree of ipsilateral innervation generally correlates with the degree of pigmentation of the retinal pigment epithelium and with tyrosinase activity. However, discrepancies have been found in ch and ce mutants. In these animals the pigment epithelium is well pigmented, and the area of ipsilateral innervation in the lateral geniculate nucleus is extensive, despite a high ratio of label in contralateral to ipsilateral optic tracts and low tyrosinase activity. Furthermore, mice heterozygous for the c2J allele have pigmentation and optic projections that are normal even though tyrosinase is reduced to 40% of normal. The few anomalous results suggest that alternative or additional factors may control optic axon projections.

Alleles

Tyrosinase activity in the sera of patients with malignant melanoma: method and specificity.

Serum tyrosinase activity has been measured by adapting the [3]tyrosine assay for tyrosinase and significant elevations of serum tyrosinase activity were found in patients with malignant melanoma. In contrast to findings in a study which utilized [14C]tyrosine, augmented levels of tyrosinase activity were not observed in sera from patients with other malignancies, including subjects with carcinoma of the breast. The results of the examinations for soluble tyrosinase activity in human malignant melanoma tissue-cultured lines were all positive, whereas human cell lines from carcinoma of the breast, carcinoma of the colon and sarcoma uniformly showed no activity. The method employed for detecting tyrosinase activity holds promise as a specific diagnostic test and may be valuable for monitoring the response to clinical treatment of patients with malignant melanoma.

Breast Neoplasms

Effects of sugars on melanogenesis in cultured melanoma cells.

A permanent cell line C2M of mouse melanoma B16 was highly melanized in a modified Eagle's MEM supplemented with 10% calf serum, when the medium contained 1 mM galactose and 10 mM pyruvate instead of 5.5 mM glucose. The activity of the key anzyme for melanogenesis, tyrosinase (EC 1.14.18.1), of living cells cultured in the galactose-pyruvate medium was consistently 27 times higher than that of cells in normal MEM. This high level of tyrosinase activity was maintained in the stationary phase, in contrast to the activity of cells in normal medium, which decreased sharply in the stationary phase. It seems likely that tyrosinase activity is suppressed by the presence of glucose rather than stimulated by galactose. This modified medium should be useful obtaining a high level of tyrosinase activity in living cells in culture and in cell-free extracts.

Animals

Effects of pH and type of sugar in the medium on tyrosinase activity in cultured melanoma cells.

The tyrosinase (EC 1.14.18.1) activity of cultured mouse melanoma cells B16 in the stationary phase of growth, depends greatly on the pH of the medium and the kind of sugar present. The enzyme activity of a homogenate of cells grown at pH 7.2 in Eagles's MEM supplemented with 10% new born calf serum and con taining galactose in place of glucose, was about ten times that of a homogenate of cells cultured at pH 6.3 in the same medium. The tyrosinase activity changed reversibly on changing the pH of the culture medium. When cultured at a constant pH of 7.2, cells grown with 1 mM galactose had about five times higher tyrosinase activity than cells grown with 1 mM glucose. Only a small amount of lactate accumulated in cultures with glucose and it had little effect on the enzyme activity. These two findings explain the very low tyrosinase activity of cells cultured in medium with 5 mM glucose: the low activity is due to the presence of glucose and to the low pH resulting from conversion of glucose to lactic acid.

Catechol Oxidase

The role of adenosine 3',5'-cyclic monophosphate in the density-dependent regulation of growth and tyrosinase activity of B-16 melanoma cells.

Incubation of cultured B-16 melanoma cells with 1-methyl-3-isobutyl xanthine (MIX) produced a sustained rise in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) which preceded an increase in the specific activity of tyrosinase (EC 1.10.3.1). Cultures of two clones of melanoma cells, one having a mean population doubling time twice that of the other, showed density-dependent inhibition of growth. The tyrosinase activity of each line increased progressively during logarithmic growth, reaching maximal values shortly after the cultures achieved confluence. Intracellular cAMP levels fell during logarithmic growth, being minimal in confluent cultures. The stimulatory effects of MIX and confluence on tyrosinase activity were additive. Cells plated at high density had a lower tyrosinase activity than cells allowed to achieve a similar density by successive division from sparsely planted cultures although the intracellular cAMP levels of such cultures were not different. We support the observations of other investigators that agents which increase intracellular cAMP concentrations can both inhibit cell division and stimulate tyrosinase activity. There are, however, mechanisms for increasing tyrosinase activity and inhibiting cell division which are expressed as B-16 melanoma cells approach confluence and which are not mediated by an increase in intracellular cAMP concentrations.

2',3'-Cyclic-Nucleotide Phosphodiesterases

Phenylalanine hydroxylase in melanoma cells.

A pigmented subclone of Cloudman S91 melanoma cells, PS1-wild type, can grow in medium lacking tyrosine. This ability is conferred by phenylalanine hydroxylase activity, and not by tryptophan hydroxylase, tyrosine hydroxylase or tyrosinase activities, although the latter activity is also present in these cells. Conversion of phenylalanine to tyrosine was measured in living cells by chromatographic identification of the metabolites of [14C]phenylalanine and in cell extracts using a sensitive assay for phenylalanine hydroxylase. Phenylalanine hydroxylase activity in melanoma cell extracts was identified by its inhibition with p-chlorophenylalanine and not with 6-fluorotryptophan, 3-iodotyrosine, phenylthiourea, tyrosine or tryptophan; and by adsorption with antiserum prepared against purified rat liver phenylalanine hydroxylase, and migration of immunoprecipitable activity with authentic phenylalanine hydroxylase subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Animals

Tyrosinase maturation and pigment expression in B16 melanoma: relation to theophylline treatment and intracellular cyclic AMP.

We have studied the effects of theophylline treatment on pigmentation characteristics and growth of two B16 melanoma cell lines, HFH-18 and P/140. Cell counts of control and theophylline-treated cultures confirmed that the drug inhibits cell growth. Light and electron microscope cytochemistry with the L-dopa reaction indicated that the two cell lines differ in their ability to transfer Golgi-associated tyrosinase to developing premelanosomes. The results of these experiments, considered with results of electrophoretic analyses and activity measurements by the Pomerantz method, also provide evidence that increased tyrosinase synthesis occurs in response to theophylline treatment. In addition, results indicate that theophylline induces changes in the rate of synthetic or degradative posttranslational modification of tyrosinase. Measurements of intracellular cyclic AMP levels by radioimmunoassay in control cultures and in theophylline- and alpha-MSH-treated cultures were made. Although the hormone induced spectacular increases in cyclic AMP levels, theophylline produced no detectable change. These results indicate that theophylline differs from alpha-MSH because theophylline-induced changes in pigmentation may not require the participation of intracellular cyclic AMP.

Animals

Segregation during cleavage of a factor determining endodermal alkaline phosphatase development in ascidian embryos.

Localized alkaline phosphatase activity (EC 3.1.3.1) develops progressively in endodermal tissues of the presumptive digestive system in Ciona intestinalis embryos. It was first detected histochemically at late gastrulation, and a puromycin sensitivity period coincident with this time suggests that new alkaline phosphatase is synthesized. Embryos in which cell division was blocked with cytochalasin B at early cleavage stages up to the 64-cell stage, eventually differentiated strong alkaline phosphatase activity in certain cells at each cleavage-arrested stage. The maximum cell numbers and their positions were identical to those of the previously known endodermal cell lineage. Actinomycin D did not prevent development of endodermal alkaline phosphatase when administered from fertilization onwards, nor did other inhibitors of RNA synthesis (chromomycin A3, cordycepin, and daunomycin). There is probably a preformed maternal mRNA for endodermal alkaline phosphatase present in the unfertilizec Ciona egg. Either this RNA itself, or some related translation factor, is localized in the egg cytoplasm and segregated during early cleavages into the endodermal cell lineage of the embryo.

Acetylcholinesterase

Isolation and characterization of a variant of B16-mouse melanoma resistant to MSH growth inhibition.

A variant of B-16 F1 mouse melanoma was selected for its ability to survive and replicate in the presence of melanocyte-stimulating hormone (MSH). Although the variant (MR-4) was completely resistant to growth inhibition of MSH, cyclic AMP was still able to block cell replication. Tyrosinase activity in MR-4 cells was considerably lower than in B-16 F1 cells. MSH induced a two fold to three-fold increase in tyrosinase activity in both cell types, but the absolute activity in MR-4 remained significantly less than in the parental cells. MR-4 cells were also found to have a markedly depressed cyclic AMP-dependent protein kinase activity relative to B-16 F1 cells. The protein kinase from both cell types was stimulated by cyclic AMP, but the level of MR-4 kinase activity at maximal cyclic AMP concentrations remained considerably lower than B-16 F1 kinase activity under the same conditions. In both cell types adenylate cyclase activity was markedly stimulated by MSH. When equal numbers of viable F1 and MR-4 cells were injected subcutaneously into C57/B1 mice, the MR-4 cells formed tumors earlier and killed the host sooner than the parental F1 cells. We conclude that the biochemical alteration which allows MR-4 cells to replicate in the presence of MSH is a low level of tyrosinase activity, which in turn may be the result of low cyclic AMP-dependent protein kinase activity.

Animals

Hydroxyanisole-induced regression of the Harding-Passey melanoma in mice.

The drug p-hydroxyanisole (OHA) was found to inhibit the incorporation of 3H-thymidine in the Harding-Passey melanoma cells in culture. Because the cultured cells had lost some of their pigment-forming capacity, the enzyme tyrosinase was added to the culture. This greatly increased the sensitivity of the cells to OHA, strongly suggesting that cells producing the enzyme would be preferentially killed by the drug. An in-vivo study of the effect of OHA injected into tumour-bearing mice showed a beneficial effect, including increased survival time, reduction in tumour size and in many cases complete loss of tumour and no recurrence. An experiment with animals immunologically suppressed by radiation suggests that the effect is not an immunological one.

Animals

Trysinase positive oculocutaneous albinism in the goldfish, Carassius auratus l., and ultrastructural and biochemical study of the eye.

Ultrastructural studies, and cytochemical and biochemical determinations of tyrosinase activity were conducted on the pigment epithelium of albino and xanthic goldfish eyes. In eyes of xanthic goldfish, two types of melanosomes are present, spherical and elongated. Melanized melanosomes are absent in the eyes of the albino goldfish, but elongated lamellar premelanosomes are observed. Internal vesicles are present in both melanosome types in the pigment epithelium of the xanthic goldfish but are absent in premelanosomes of the albino. There are also differences in the distribution of lipid droplets, smooth endoplasmic reticulum and Golgi complexes with the latter two being more abundant in the albino. Tyrosinase was not identified cytochemically; however, the enzyme was demonstrated biochemically in the pigment epithelia of both albino and xanthic goldfish. The enzyme is associated with the particulate and soluble fractions fo both types of eyes. Particulate albino tyrosinase may be solubilized by triton X-100 treatment. Tyrosinase inhibitors are present in the particulate fractions of both albino and xanthic goldfish eyes. Thus, in the goldfish, ocular albinism appears to be a multiple defect at the molecular and ultrastructural levels.

Albinism

Melanogenesis in genetically determined pigment cell tumors of platyfish and platyfish-swordtail hybrids: correlation between tyrosine activity and degree of malignancy.

In the genetically determined pigment cell tumors of platyfish and platyfish-swordtail hybrids, the degree of malignancy of pigment cells which have been neoplastically transformed by a tumor gene (Tu) depends on the type and number of certain regulating genes (R). In the present study, the tyrosinase activities in tumors of different degrees of malignancy (black spots, premelanomas, melanomas) have been determined. The results demonstrate a close correlation between the level of tyrosinase activity and the degree of malignancy. Spot patterns consisting of completely differentiated (benign) Tu-transformed cells show no tyrosinase activity. Premelanomas containing a few incompletely differentiated (malignant) Tu-transformed cells in addition to many differentiated ones show moderate tyrosinase activities. Melanomas which contain increasing numbers of incompletely differentiated cells with increasing growth rates show high to extremely high tyrosinase activities. Thus, the tyrosinase levels present in these tumors can be used as an indicator for the degree of differentiation and, thereby, for the degree of malignancy of the neoplastically transformed pigment cells.

Animals

Chymotrypsin activation of melanosome tyrosinase in hamster melanotic melanoma.

The tyrosinase activity in two sucrose gradient isolated melanosome fractions from a melanotic hamster melanoma was found to increase after alpha-chymotrypsin treatment. The enhancement in tyrosinase activity had its maximum at a concentration of 1 mg/ml alpha-chymotrypsin after 120 min incubation at 37 degrees C. No direct activating effect of alpha-chymotrypsin was found either on the soluble tyrosinase fraction from freshly prepared untreaed whole-tumor homogenate or on purified mushroom tyrosinase. The activating effect of alpha-chymotrypsin upon the melanosome tyrosinase is believed to be due to the endopeptidic hydrolysis of the--CO--NH--bound existing between tyrosinase and tyrosine and phenylalanine residues in the melanin molecule. Although alternative interpretations are not excluded, the observed enhancement in tyrosinase activity after alpha-chymotrypsin treatment of melanosomes might indicate the existence of an "enzyme liberating" mechanism in the melanosomes.

Animals