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Uptake of melanosomes and increased melanin formation by cultured melanoma cells after treatment with isolated melanosomes.

Melanosomes isolated from a subcutaneous Harding-Passey mouse melanoma and purified by density gradient centrifugation were labelled in vitro with 14C-tyrosine or 3H-dihydroxyphenylalanine in the melanin portion. Incubation of monolayer cultures of Harding-Passey melanoma cells during exponential growth phase (wherin cells contained relatively few melanosomes) with isolated and labelled melanosomes during a time-period of up to 3 days resulted in rapid cellular uptake of label (reaching a saturation level after about half a day). Following a lag period of several hours, the melanin content rose near-linearly in the course of 3 days. Comparison of curves of uptake of radioactivity and melanin concentration indicates that the latter rise is due primarily to newly formed melanin. Ultrastructural studies revealed a strikingly increased number of melanosomes in melanosome-treated cells. Some of these appeared to be the result of phagocytotic uptake. In fact, invagigations of the plasma membrane containing exogenous melanosomes were observed. Since most intracellular melanosomes were localized directly in the cytoplasmic matrix, dissolution of the phagosome membrane appeared to have taken place. Aggregates of melanosomes either surrounded by a membrane or free in the cytoplasm were also observed. These bodies might represent phagolysosomes or/and centres for the formation of new melanosomes. The combined biochemical and ultrastructural findings suggest stimulated melanogenesis induced by phagocytized melanosomes.

Animals

Transfer mechanism of melanosomes in epidermal cell culture.

The mode of melanosome transfer from melanocytes to keratinocytes in epidermal cell cultures has been examined with time-lapse cinematography and electron microscopy. A tip of a melanocyte dendrite containing melanosomes became enfolded by a recipient keratinocyte. It was then pinched off to form a cluster of melanosomes which initially seemed to be surrounded by two layers of membranes. The phagocytized dendrite was gradually decomposed and became an aggregate of melanosomes surrounded by a single membrane of the keratinocyte. The individual melanosomes were dispersed from the aggregate into the keratinocyte cytoplasm, depending on the size of melanosomes. The larger ones were single and smaller ones were complex. The mechanism of melanosome transfer in vitro is a type of cytophagocytosis. The entire process consists of two steps: the first is a cytophagic process and the second a melanosome dispersion process. The process is influenced by various exogenous factors.

Cells, Cultured

Enhanced melanization of Harding-Passey mouse melanoma cells following treatment with exogenous melanosomes in monolayer culture.

Purified melanosomes isolated from subcutaneously growting Harding-Passey melanomas of NMRI-mice were labeled either in vitro with [14C]tyrosine or [14C]DOPA in the melanin portion, or in vivo in the melanin and protein portion following i. p. injection of [14C]tyrosine. Treatment of monolayer cultures of Harding-Passey melanoma cells (HPM-73 line) with such labeled melanosomes resulted in rapid uptake of label during the first 4 h which leveled off thereafter. A portion of the "incorporated" label could be removed by a 15 min chase with unlabeled melanosomes. Uptake of labeled melanosomes by HPM-73 cells was followed by increased cellular melanization which was not only due to melanin derived from incorporated melanosomes but primarily to newly formed melanin. Tyrosinase activity was elevated in melanosome-treated cells. Tyrosinase activity of control cells was significantly reduced following a 24 h exposure to actinomycin D or cycloheximide. On the other side, the same inhibitor treatment of melanosome-pretreated cells resulted in less inhibition of tyrosinase activity. The present findings suggest "melanophagic" properties of cultured melanoma cells resulting in enhanced melanogenesis after phagocytotic uptake of functionally active exogenous melanosomes.

Animals

Comparison of eumelanogenesis and pheomelanogenesis in retinal and follicular melanocytes; role of vesiculo-globular bodies in melanosome differentiation.

Vesiculo-globular bodies, 40 nm in diameter, are present in melanosomes. The mode of their involvement in melanosomal differentiation was studied by ultrastructural comparison of eu- and pheomelanogenesis occurring in retinal and follicular melanocytes. We found that the number and distribution of these bodies differ significantly with types of melanogenesis and tissues. They were not affected by physical stimuli nor by embryonic origin of melanocytes. The earliest form of melanosomes is identical in eu- and pheomelanogenesis. The vesiculo-globular bodies are involved in organization of melanosomal constituents. In eumelanogenesis, they are more numerous in feather than in retina and hair. They are least numerous in white hair and pink eyes where melanization is blocked. During melanosomal development, they become associated with melanosomal inner lamellae and their outer surface becomes melanized, but their core is hardly melanized, thus leaving small vesicular structures. In pheomelanogenesis, their number is almost equal in feather and hair. Lamellae are not formed, but these bodies fuse with each other to form an amorphous matrix on complete differentiation of melanosomes.

Animals

Ultrastructural analysis of gene interaction and melanosome differentiation in the retinal pigment cells of the albino goldfish.

The genotype of albino goldfish is represented by pp,cc, and the non-albino fish is PP,CC. The P gene begins to control the melanosome formation of retinal pigment cells at St-21; and the C, which is epistatic to P and p, governs melanosome formation in the dermal melanophores and in retinal pigment cells after St-22. Then in the pp,CC fish, between St-21 and St-22, melanosomes of the pigmented retina are albino in type, but after St-22 they become non-albino in type. From ultrastructural observations on the melanosomes of these late-melanizing fish, it is apparent that the imcompletely melanized albino pigment granules can differentiate into normal melanosomes under the control of the C gene. The processes of melanosome maturation in the late-melanizing goldfish are evident from these observations.

Albinism

Variation in melanosome numbers in cultured B-16 melanoma cells.

Melanosomes from B-16 mouse melanoma cells in culture were isolated by treatment of pigmented cells with 2% SDS, sonication, and heating at 100 degrees C. The total number of melanosomes in cultures of B-16 mouse melanoma cells increased exponentially during the rapid phase of sigmoid growth. The numbers of melanosomes per cell decreased during rapid phase of growth, and repigmentation was observed only when the cultures attained the stationary growth phase. BUdr at a minimum concentration of 0.5 mug/ml decreased both cell growth and numbers of melanosomes per cell, and completely inhibited repigmentation following a period of active growth. Cells cultured in 0.1 mug/ml BUdr grew at the same rate as untreated cells but contained fewer melanosomes/cell and lower total numbers of melanosomes during the late stages of the growth cycle.

Bromodeoxyuridine

The hair melanosome: another tissue reservoir of zinc.

Melanosomes were isolated from human and dog hairs and their Zn content was determined. The mean Zn concentrations, in mug Zn/g dry weight, were 687 (men's melanosomes), 641 (women's melanosomes) and 691 (dog melanosomes). These values rank melanosomes from pigmented keratinous structures among the structural elements with the highest Zn content. An interpretation of the possible causes of this accumulation was submitted. By means of the conversion factor between hair length and weight (determined at 0.004), it was estimated that daily Zn lossess via hair vary in the region of 19 mug. In detailed studies of Zn metabolism, excretion via pigmented keratinous structures ought not to be neglected.

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

Solubilizing effect of Trition X-100 on melanosome tyrosinase in hamster pigmented melanoma.

A study was made on the effect of Triton X-100 in different concentrations on the tyrosinase activity of two differently pigmented melanosome fractions of pigmented hamster melanoma, obtained by gradient centrifugation. Release of considerable tyrosinase activity was established spectrophotometrically in the supernatant of the treated melanosomes. The release of enzyme activity after treatment of the melanosome is probably due to a change in the permeability of the melanosome structures under the effect of the non-ionic detergent Trition X-100. A direct activating effect of Trition X-100 on the particulate enzyme should be excluded, since inhibition of DOPA-oxidase activity was established in the soluble tyrosinase fraction.

Animals

Human malignant melanoma. Melanosomal polymorphism and the ultrastructural dopa reaction.

An ultrastructural dopa reaction, with glutaraldehyde--formaldehyde prefixation, was carried out on ten specimens of malignant melanoma showing a wide variation in melanosomal morphology. All tumours, but only a minority of tumour cells, contained reaction product. In all tumours the reaction product was distributed similarly in the Golgi apparatus and Golgi associated endoplasmic reticulum (GERL). However, differences were noted in its deposition in melanosomes. Whereas vacuolar and lamellar profiles sometimes contained reaction product, it was not seen in normal, granular and abortive melanosomes. Tumour cells without melanosomes were also seen to contain reaction product.

Dihydroxyphenylalanine

Actin microfilaments in melanophores of Fundulus heteroclitus. Their possible involvement in melanosome migration.

In melanophores of Fundulus heteroclitus, hormone-stimulated melanosome aggregation is accompanied by cytoplasmic flow from the cellular processes to the perikaryon, and reversal of these events takes place upon hormone-induced melanosome dispersion. These cells contain parallel arrays of microtubules, the majority of which are located in the perikaryon and in cortical regions of the processes. Studies with heavy meromyosin binding demonstrated two types of actin filaments: 11 a decorated meshwork of filaments similar to those usually found in close association with plasma membranes, and 2) filaments decorated in a manner similar to that of stress fibers. There is an apparent increase in the amount of filaments during melanosome aggregation. These results are discussed in relation to intracellular movement.

Actins

Iontophoretic release of cyclic AMP and dispersion of melanosomes within a single melanophore.

Selective dispersion of melanosomes was often observed after iontophoretic injection of cyclic adenosine monophosphate (AMP) from a glass microelectrode positioned in a target melanophore in frog skin (as viewed from above through a microscope), with other melanophores in the field serving as controls. Because the skin has orderly arrays of several types of closely spaced cells, it is probable that at times the microelectrode also impales cells other than melanophores. When cyclic AMP injection inside a cell resulted in dispersion of melanosomes from a perinuclear position into dendritic processes, the onset of dispersion was relatively rapid, in many cases less than 4 min (mean time of onset, 5.3 +/- 2.9 [SD] min). A much slower dispersion (mean time of onset, 19.0 +/- 5.0 min) of melanosomes was observed when the microelectrode was positioned adjacent to a melanophore, and much larger quantities of cyclic AMP were released. In addition, no changes were observed for injections of 5'-AMP or cyclic guanosine monophosphate (GMP) through electrodes positioned inside or adjacent to melanophores. Potential measurements showed that after impaling a clell, a constant transmembrane potential could often be recorded over many minutes, indicating that the membrane tends to seal around the microelectrode. The results indicate that cyclic AMP acts more rapidly on the inside of a cell than when applied outside a cell and allowed to diffuse through the plasma membrane. This study introduces a model system whereby the properties of the plasma membrane and melanocyte-stimulating hormone (MSH) receptors can be studies within a single target cell.

Animals

Melanosomes of pigmented basal cell epithelioma.

Electron microscopic examination of a pigmented basal cell epithelioma of scrotal skin revealed many tumor cells and melanocytes which were laden with large packages of melanosomes. In tumor cells these packages were delimited by either single- or double-limiting membranes or were not membrane bound. The matrix of these large complexes was less electron dense and homogeneous than normal, containing electrontranslucent droplets besides melanosomes. No remnants of intracellular organelles such as nucleus, mitochondria or ribosomes could be found. The basal cell epithelioma cells in this case appear to have differentiated toward fetal basal cells and have developed the ability to phagocytize large packages of melanosomes.

Basal Cell Carcinoma

Nature of tyrosinase inactivation in melanosomes.

The decrease in tyrosinase activity following incubation of melanosomes isolated from mouse melanoma with dopa appeared to be related to the degree of in vitro melanization of melanosomes caused by incubation with dopa. The inactivation similarly occurred in the dopa-tyrosinase reaction system containing ascorbic aicd, although in the presence of ascorbic acid the dopa-quinone is immediately converted back to dopa and therefore melanin formation does not take place. The mode of inactivation of tyrosinase in melanosomes appeared to be similar to the reaction inactivation which is known to occur in the reaction of plant plant tyrosinase. Superoxide anion (O-2) and singlet oxygen (1O2) were estimated but undetectable during the dopa-tyrosinase reaction. Thus the inactivation of tyrosinase is not caused by the reaction products or the oxygen radicals.

Animals

Atypical melanosomal proteins in human malignant melanoma.

Isolated melanosomes from human malignant melanoma lesions and normal tissues were solubilized with Triton X-100, and proteins were fractionated by polyacrylamide gel electrophoresis. Determination of the relative mobilities of protein bands at different acrylamide gel concentrations allowed the calculation of the molecular size and charge of melanosomal proteins. Three classes of proteins were demonstrable as follows: (a) those found only in melanoma; (b) those found only in normal melanosomes; and (c) proteins common to malignant and control tissues. The similarity of human melanoma protein banding patterns to those previously described in B-16 murine melanoma reaffirms the importance of the latter as a biochemical model for human studies; in addition, the presence of unique proteins in malignant tissue paralleled reports of aberrant polypeptides in organells of several other tumor systems.

Aged

Electron microscopy of human melanosomes in unstained, fresh air-dried hair bulbs and their examination by electron probe microanalysis.

Electron microscopy of unstained, fresh air-dried spreads of plucked human hair bulbs revealed relatively well preserved melanocytes and dense storage granules of melanin. Energy dispersive X-ray microanalysis of melanosomes and premelanosomes from black facial hair bulbs disclosed high peaks for potassium and sulfur, intermediate peaks for magnesium and calcium, and low but distinct ones for phosphorus and chlorine. No peaks for magnesium were found in the central portion of mature melanosomes, but at their edges and in premelanosomes conspicuous peaks for this element were obtained.

Electron Probe Microanalysis

Inability of murine melanoma melanosomal "tyrosinase" (L-dopa oxidase) to oxidize tyrosine to melanin in polyacrylamide gel systems.

Melanosomal "tyrosinase" (L-dopa) was isolated from trypsin digest of B-16 mouse melanoma melanosomes, using polyacrylamide gel disc electrophoresis. The enzyme was represented by a single band, having characteristics similar to the T1 dopa-positive band observed when using supernatants of crude melanoma homogenates as the source. When gels with this band were incubated in solutions containing tyrosine and dopa in varying ratios , there was no enhancement of melanin formation by tyrosine when compared with incubations in corresponding concentrations of dopa alone. These data further support previous studies in our laboratory demonstrating an inability of so-called mamalian "tyrosinase" to convert tyrosine to melanin; since this enzyme readily converts L-dopa to melanin, it seems more reasonable to term this enzyme an L-dopa oxidase.

Animals