Harding-Passey Mouse Melanoma Research Articles

We came over on the boat together.

We came over on the boat together.

Action of endogenous proteases on the distribution of tyrosinase isozymes in Harding–Passey mouse melanoma

A high percentage of the total tyrosinase found in Harding-Passey mouse melanoma occurs as a soluble form. This paper shows that melanosomal tyrosinase can be solubilized by several endogenous proteases to yield active tyrosinase. This enzyme, once proteolytically solubilized, can be further degraded, leading to enzyme inactivation. The nature and specificity of the main proteases involved in the solubilization process change depending on the size and necrosis stage of the tumour. Cathepsin B could be the main protease responsible for the solubilization in small tumours (less than 0.5 g). Large tumours are rich in necrotic cells, and cathepsin D and serine-proteases are the main hydrolytic enzymes involved in the proteolytic action on melanosomes. These results support the view that the high activity of tyrosinase found in the soluble fraction of malignant melanoma is mainly an artefact resulting from degradation of melanosomes by a variety of endogenous proteases, rather than the result of the actual occurrence of high levels of an independent cytosolic isozyme.

The role of sulfhydryl compounds in mammalian melanogenesis: the effect of cysteine and glutathione upon tyrosinase and the intermediates of the pathway

The effect of cysteine and glutathione on mammalian melanogenesis has been studied. It has been shown that their action is mediated by two different mechanisms. (a) The reaction of the thiol groups with dopaquinone after the tyrosinase-catalyzed oxidation of tyrosine and dopa. This mechanism leads to the formation of sulfhydryl-dopa conjugates and finally sulfur-containing pigments, phaeomelanins instead of eumelanins. This fact might produce an inhibition of melanogenesis due to the slower rate of chemical reactions involved in the polymerization of such thiol-conjugates when compared to that of indoles. (b) The direct interaction between the sulfhydryl compounds and the tyrosinase active site. This interaction may regulate the activity of the enzyme. It is shown that Harding-Passey mouse melanoma tyrosinase is more sensitive to sulfhydryl compounds than mushroom tyrosinase. Cysteine always produces an inhibition of the tyrosinase hydroxylase and dopa oxidase activities of melanoma tyrosinase, this inhibition becoming greater as the cysteine concentration increases. On the other hand, glutathione produces an activation of the tyrosine hydroxylase activity below 3 mM and an inhibition at higher concentrations. The limit between the enzymatic activation and inhibition appears at glutathione concentrations similar to the physiological levels of this compound found in melanocytes. Although the switch from eumelanogenesis to phaeomelanogenesis occurs at much lower concentrations of glutathione, taking into account these data it is discussed that this sulfhydryl compound may regulate not only the type but also the amount of melanin formed inside melanocytes.

The existence of apotyrosinase in the cytosol of Harding-Passey mouse melanoma melanocytes and characteristics of enzyme reconstitution by Cu(II)

This paper reports the effect of Cu(II) supplementation on the tyrosinase isozymes from Harding-Passey mouse melanoma. The dopa-oxidase activity of the microsomal and soluble isozymes is increased by incubation with Cu(II), whereas the activity of the unique ‘in vivo’ melanin-forming isozyme, bound to melanosomes, is not. Other divalent cations are ineffective in increasing the dopa-oxidase activity of tyrosinases. These results indicate the existence of a mixture of tyrosinase and apotyrosinase in the cytosol of melanocytes before reaching the melanosome. The paucity of Cu(II) in the cytosol could be one of the mechanisms of regulation contributing to avoid the formation of melanin outside the melanosome. Some kinetic characteristics of the enzymatic reconstitution of soluble and microsomal isozymes by Cu(II) are also studied, and the results suggest that the glycosylation of apotyrosinase during its maturation yields a conformational change favouring the binding of Cu(II) at the enzyme active site, by lowering the activation energy of the reconstitution reaction.

Aggregation equilibria of tyrosinase of Harding-Passey mouse melanoma.

The purification of two isoenzymes of tyrosinase has been carried out in Harding-Passey mouse melanoma. One is found in the cytosol and the other one bound to melanosomes. Both migrate as single bands on sodium dodecyl sulphate/polyacrylamide gels, having an apparent Mr of 58 000. Solubilized particulate tyrosinase showed an aggregation equilibrium involving a monomer, tetramer, octamer and a high-Mr micellar form with Brij 35, the solubilizing agent. H.p.l.c. studies indicated a interconversion between those species, the monomer contribution increasing with the sample dilution. The tetramer and the octamer probably represent the predominant forms in vivo. Soluble tyrosinase showed a simpler aggregation equilibrium, involving two forms, monomer and tetramer, with the same interconversion pattern. Fluorescence studies suggested that tryptophan residues were exposed to the aqueous environment when tyrosinase was dissociated by dilution. Tyrosinase shows a tendency to aggregate, at low protein concentration, and a resistance to dissociation by urea or SDS so remarkable that gel-permeation chromatography in 4M-urea does not affect the equilibrium, and the band obtained on SDS/polyacrylamide-gel electrophoresis is a dimer.

Regulation of the cytosolic and melanosome-bound tyrosinase activities in harding-passey mouse melanoma

1. 1. Mouse melanoma tyrosinases exist in the cytoplasm of melanocytes and also in a particulate form, bound to melanosomes. 2. 2. The cytosolic isoenzyme activity is not expressed in the melanocytes in vivo. One of the mechanisms for the activity regulation is the existence of a soluble inhibitor. This inhibition is non-competitive with regard to l-dopa. 3. 3. Particulate tyrosinase can be solubilized from the melanosome by several agents, Brij 35 and Triton X-100 being the most effective ones. Melanin accumulation in the organelle produces a competitive inhibition of the activity.

Scheduling of combination chemotherapy for a murine melanoma, with the subrenal capsular assay.

The effect of varying schedules for administration of two drugs, dimethyl triazeno imidazole carboxamide (DTIC) and methylcyclohexyl chloroethyl nitrosourea (MeCCNU), was studied using the Harding-Passey Mouse melanoma in the subrenal capsular assay. The results of the assay indicate that the responses of the tumor varied depending on the schedule of administration. The best response, with a mean decrease in dimensions of 3.0 ocular micrometer units (+/- 1.12 SD), was noted with administration of DTIC on day 1 after tumor implantation, followed by MeCCNU on day 2. These results are similar to previously reported data in CD2F1 mice bearing intraperitoneal Harding-Passey melanoma and suggest that the subrenal capsular assay may be of use in determining the best combinations and schedules of administration for treatment of tumors.

Transfer of tyrosinase to melanosomes in Harding-Passey mouse melanoma

The transfer of tyrosinase from microsomes into melanosomes, without passing through the cytosol in the Harding-Passey mouse melanoma cell, was confirmed by experiments carried out using a combination of radioisotope tracer techniques and immunoprecipitation. 3H-Labeled amino acid incorporation into tyrosinase present in the microsome, melanosome, and soluble fractions confirmed the precursor-product relationship of the enzyme in the microsome fraction and in the melanosome fraction. However, two forms of the enzyme, T s1- and T s2-tyrosinase, separated from the soluble fraction by polyacrylamide gel electrophoresis, were shown to play no role in the transfer since little or no incorporation of radioactivity into tyrosinase in this fraction was found. It is suggested that most tyrosinase observed in the soluble fraction does not leak from the melanosomes or the microsomes during homogenization, but comes from necrotic tumor cells. It appears that melanosomal and microsomal tyrosinase might be released from the membrane of necrotic cells modified by various degradation enzymes, considering the data on the recovery of tyrosinase from the soluble fraction, where one-third of total enzyme activity in the postnuclear fraction could not be increased, even when the postnuclear fraction of the tumor was further homogenized radically.

Characterization of Structural Properties for Morphologic Differentiation of Melanosomes. III. Free and Protein-bound Dopa and 5-S-Cysteinyldopa in B16 and Harding-Passey Melanomas

This study analyzed the free and protein-bound forms of dopa, 5-S-cysteinyldopa (5-S-CD), and 5-S-glutathionedopa (5-S-GD) in B16 and Harding-Passey (HP) mouse melanomas to investigate the role of these catechols for melanogenesis and melanosome morphogenesis, inasmuch as these tumors produce melanosomes different in color and ultrastructure, i.e., eumelanosome type in B16 and pheomelanosome type in HP. Between B16 and HP mouse melanomas, however, we found (a) no significant difference in the level of free dopa and 5-S-CD in melanosomes and tumors, although the levels of these catechols reflected well the type of melanogenesis in control hair of normal mice, (b) a significant difference in free 5-S-GD level, which might, in part, reflect the observed difference in melanogenesis, and (c) no apparent difference in the level of bound dopa and 5-S-CD in either melanosomes or tumors. Thus, the striking difference in the color of melanosomes between B16 and HP melanomas seems to be related primarily to the content--not the type--of melanin pigments.

Establishment of pigmented melanocyte culture strain from Harding-Passey melanoma.

A cultured cell strain of melanotic melanoma was established from Harding-Passey mouse melanoma. The established cells proliferated as a monolayer culture and actively produced melanin pigment. Under an electron microscope, these cells showed the typical characteristic features of the melanocyte. When a cell suspension was inoculated into Swiss mice, they developed typical melanocytic tumors which closely resembled the original melanomas.

The properties of antibody against smooth endoplasmic reticulum of melanocytes.

The smooth endoplasmic reticulum (smooth-surface membrane, SSM) is quite similar in properties to the cytoplasmic which is known to be antigenic. SSM of the melanoma melanocytes can be isolated from the mouse melanoma as a reasonably pure preparation and it contains tyrosinase. Rabbits were immunized by the SSM isolated from Harding-Passey mouse melanomas. Formation of the antibody was detected by the double immuno-diffusion method. Anti-SSM-serum thus formed was found to react with cytoplasmic membrane in the immunofluorescent test, and also showed the specific cytotoxicity to the mouse melanoma cells. On the other hand, the tyrosinase activity of SSM was not inhibited by this antiserum.

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.

Pigmented Iris Malignant Melanomas in New Zealand White Rabbits

Harding-Passey mouse melanoma, maintained in tissue culture for 3 months, was injected into the anterior chamber of anesthetized New Zealand white rabbits. The rabbits were immunosuppressed with depot

Isolation and characterization of a Golgi-rich fraction from the Harding-Passey mouse melanoma.

Golgi-rich fraction was isolated from Harding-Passey mouse melanoma by centrifugation through the discontinuous sucrose density gradient and its properties were compared with those of the same fraction isolated from rat liver. The specific activity of UDP-galactose: N-acetylglucosamine galactosyltransferase was 35 times higher in the melanoma Golgi fraction than in the melanoma homogenate and was a half that in the rat liver Golgi fraction. The specific activities of marker enzymes for other subcellular components such as 5'-nucleotidase, acid phosphatase and glucose-6-phosphatase in the melanoma Golgi fraction were all one-third those in the melanoma homogenate. Electron micrographs of the negatively-stained Golgi fractions of melanoma and liver revealed the presence of a system of tubules, vesicles and plate-like center regions which are known as components of Golgi apparatus. Tyrosinase activity was found to be present in this fraction of mouse melanoma, but its specific activity was lower than that in the rough or smooth surface membrane fraction or in the melanosome fraction.

The identification of a cell culture inhibitor in a tumour extract

An extract of Harding-Passey mouse melanoma was found to inhibit the proliferation of the same cell line grown in culture when measured by both thymidine incorporation and by the increase in cell number. The active ingredient was purified by ultrafiltration, column chromatography and preparative electrophoresis. The purified material was identified by mass spectrometry and by gas chromatography as spermidine. The partially purified material reversed the methylglyoxal Bis-guanyl hydrazone (Methyl-GAG) inhibition of cultured cells to the same extent as spermidine. Very low concentrations of Methyl-GAG were found to suppress the spermidine inhibition of thymidine incorporation into cells. The inhibitor extracted from melanoma tumours behaved in the same way as spermidine at all Methyl-GAG concentrations. Thus, it is unlikely that the active ingredient in the biological extract is anything other than spermidine.

Melanosome: An unusually resistant subcellular particle

Melanosomes isolated from human, horse and Harding-Passey mouse melanomas, from human hair and from bovine eye pigment tissues were hydrolyzed with 6N HCl 24hours at 110 °C. An electron microscopic comparison of hydrolyzed and untreated melanosomes showed that the hydrolyzed melanosomes retained their size, shape, and, to a great extent, their ultrastructural features. The results 1/suggest that melanin constitutes the rigid framework of the particles providing them with unusual resistibility; 2/raise the question of the nature of their lysosomal degradation/reported by some authors/ if extreme acid hydrolysis has remained without any effect. Melanosomes are special subcellular particles of pigment cells ( Seiji et al., 1961) originating by apposition of polymerizing melanin precursors on primary protein polymer — either on premelanosome matrix protein exhibiting tyrosinase activity or on matrix protein under tyrosinase catalysis. The synthetized melanoprotein complex is a tough substance, which represents the major melanosome component. Unlike the biosynthesis, the melanosome degradation has remained obscure. Electron microscopy revealed that in vivo melanosomes are disintegrated in lysosomes ( Mishima, 1966; Kawamura et al., 1967; Sato et al.,1969; Olson et al., 1970; Wolff and Hönigsmann, 1972; Wolff, 1974). The in vitro model experiments confirmed that the protein moiety of melanosomes could be hydrolyzed by lysosomal enzymes ( Ohtaki, 1970; Ohtaki and Seiji, 1971; Saito and Seiji, 1973), however, the melanin moiety appeared to be very resistant to lysosomal digestion. In this study the isolated melanosomes were, therefore, exposed to extreme hydrolytic treatment — acid hydrolysis and its influence was studied by means of electron microscopy.

Action of trypsin and detergents on tyrosinase of normal and malignant melanocytes.

The T1 variety of tyrosinase is present in both particulate and soluble or readily solubilized forms in the pigmented hypodermis (hair bulbs) of C57BL mice and Harding-Passey mouse melanoma. Trypsin treatment of 35,000g supernatants containing the microsomal (small granule) fraction of gentle homogenates of hair bulbs and melanoma results in significantly increased T1 activity within polyacrylamide gels. Similar treatment of 100,000g supernatants results in a slight increase in T1 activity. Addition of Triton-X or DOC to 35,000g supernatants of hair bulb and melanoma homogenates followed by centrifugation at 100,000g results in a marked enhancement of T1 when the latter supernatants are treated with trypsin. In the absence of trypsin treatment, T1 activity is comparable to nondetergent-treated controls. A slow-moving dopa-reactive band (Ts) is found in electropherograms of the nontrypsinized 100,000g supernants of detergent-treated 35,000g supernatants. It is absent in those treated with trypsin. The slow-moving enzyme appears to give rise to T1 molecules when eluted from acrylamide gels and even to a greater extent when elution is combined with trypsin treatment prior to reelectrophoresis. In mammals, tyrosinase apparently is not derived by a proteolytic activation of protyrosinase.

Morphologic and microspectrophotometric studies on spontaneous melanomas in Xiphophorus helleri.

Melanomas developed in both sexes of a strain of Tuxedo variety of the swordtail (Xiphophorus helleri) at a relative frequency of 10-15%. They did not metastasize. However, the tumor margin had infiltratitive growth and subsequently ulcerated. This feature, together with the histologic and cytologic features and apparent heteroploidy of the tumors, as revealed by their DNA content, indicated that the tumors were indeed neoplastic. Electron microscopic findings on the melanosomes in these melanomas at various stages of development were comparable with those on the Harding-Passey mouse melanoma, which contains granular premelanosomes.

Melanogenesis from tryptophan. Biogenetic experiments with Harding-Passey mouse melanoma.

Il triptofano14C-uniformemente marcato nell'anello benzenico e il triptofano marcato14C nel metilene sono incorporati in modo significativo nella melanina del melanoma di Harding-Passey del topo.5-ossitriptofano e 5-ossitriptamina non mostrano invece una significativa incorporazione nella stessa melanina. L'incorporazione ottenuta con il triptofano marcato sia nel nucleo che nel metilene e all'incirca uguale a quella ottenuta per somministrazione di tirosina-2-C14, per cui anche il triptofano deve essere considerato un precursore delle melanine.

Ultrastructural studies on tumours and cell cultures of the Harding--Passey mouse melanoma.

SUMMARY Ultrastructural studies have been carried out on tumours and monolayers of cells of the transplantable Harding-Passey mouse melanoma, grown in tissue culture. Tumours were found to consist of melanin-producing, melanin-producing-and-containing and melanin-containing cells. Melanosomal complexes found in melanin-containing cells appeared to arise by fusion of solitary melanosomes either with each other or with lysosomes. Melanophages, previously regarded as macrophages which have migrated into the tumour, are probably altered and older melanoma cells. Melanosomal complexes contained in these cells have formed by autophagocytosis. Tissue culture studies support this view. Melanoma cells grown in vitro were invariably found to contain melanosomal complexes as well as solitary melanosomes, and older cells in culture contained numerous melanosomal complexes. Virus-like particles resembling immature C-type virus particles were found in all the tumours examined and were especially abundant in cultured cells. These particles were found in the cisternae of rough endoplasmic rcticulum. The significance of these virus particles is still a matter for speculation.