Differentiation of new metastatic variants of B16 melanoma under different culture conditions.

The purpose of this study was to examine the differentiation of variant tumors of the B16 metastatic melanoma when tumors were grown serially under different culture conditions and transplanted into C57BL/6J black mice, lethal yellow Ay/a, albino c/c, and C+/c mutant mice. Morphological and biochemical markers of melanogenesis were examined in cells in culture and in the corresponding tumors. Cellular pigmentation was assessed in terms of the levels of DOPA and 5-S-CD and in terms of tyrosinase activity in the various cell lines and tumors. The observed change from high to low metastatic capacity, which was dependent on culture conditions, appeared to be unrelated to melanogenesis even though changes were observed in the biochemical melanotic phenotype. Overall, tumor cells from spontaneous pulmonary metastases appear to differentiate in ways that are unrelated to the instability of experimental metastatic capacity. The melanotic phenotype in albino c/c and C+/c mice was dependent on the phenotype of the parental tumors. A marked difference was observed between two pigmentation compartments, one of which was stable in the B16 control, while the other was unstable in YB16 and MB16 variant cells and in the tumors derived from them. It appears, therefore, that the metastatic capacity of B16 metastatic variants is changeable and is independent of the unstable melanogenic behavior. The production of metastases and the differentiation of tumors in the present experiments appeared to be related to the genetic background of the mice and the epigenetic metabolic environment of tumors and cells.

New variants of the B16 melanoma: tumorigenicity and metastatic properties under different culture conditions.

Variants of the B16 melanoma exhibiting markedly different tumorigenic and metastatic potential in Ay/a and a/a C57BL/6J syngeneic mice were investigated and compared, with respect to their relative growth and metastatic potential after culture under different conditions. Over 2 to 6 months of growth, in vitro cells demonstrated a rapid and significant decrease in their ability to form spontaneous lung colonies. Such a decrease depended on the conditions of culture. We suggest that the in vitro environment influences the phenotype of cells with respect to their capacity for spontaneous metastasis. This possibility must not be ignored, when conclusions obtained from studies of established cell lines are extended to cancers.

New variants of B16 mouse melanoma: differentiation and metastatic properties.

A system of tumor transplantation has been developed to select metastatic variants of B16 in mutants of the C57BL/6J black strain of mice. The effects of transplantation into nonagouti a/a and mutant recipients on the production of melanin and on the metastatic potential of tumors were investigated. Transplantation of the pigmented B16 melanoma from a nonagouti black a/a host to a yellow mutant Ay/a recipient resulted in an achromic and metastatic variant melanoma, designated YB16. The amelanotic phenotype occurred consistently after more than ten passages through yellow mice and simultaneously with an increase in the incidence of pulmonary metastases. When YB16 was transplanted back to the nonagouti black a/a host, a second variant, MB16, characterized by its variable pigmentation, was obtained. Pigmented and/or entirely achromic tumors were observed. MB16 was dramatically more metastatic than B16 and YB16 when injected s.c. or i.v. Metastases in the lungs were pigmented and/or achromic. The properties of tumor cells derived from artificially induced metastases were investigated after s.c. and i.v. injections. Whereas the metastatic cells expressed a potent ability to generate metastases when injected s.c., no differences in the incidence of metastases, as compared to the metastatic potential of cells of parental origin, were observed after i.v. injection. In the MB16 variant, there appeared to be an inverse relationship between differentiation (production of melanins) and malignancy. Our results demonstrate that differentiation and metastatic behaviour are dependent on specific mutations in the host environment which generate a pool of tumor cells from which highly metastatic variants can be selected.

[Antitumor activity of several new pentopyranosyl-chloroethylnitrosoureas]. / Activité antitumorale de quelques nouvelles chloroéthyl-nitrosourées pentopyranosiques.

Six N-(2-chloroethyl)-N-nitrosoureido derivatives of 4-deoxy-DL-pentopyranose have been synthesized and their antitumor activities were evaluated against melanoma MB16 in mice. The nitrosourea 38 was highly active in comparison with the clinically useful CCNU.

[Metastatic melanoma B16 of the C57Bl/6J mouse. Biological findings]. / Mélanome métastatique B16 de la souris C57Bl/6J. Données biologiques.

Biological study of a new metastatic variant of C57Bl/6J mice melanoma MB16 derived from a (poorly) invasive tumor (B16), are reported. We showed significative differences in tumor growth (female) and in pulmonary metastasis occurrence. Differentiation is weakly modified.

[Demonstration, in the black mouse, of a protein linked to the process of cancer formation in B16 melanoma]. / Mise en évidence chez la Souris "black" d'une protéine liée au processus de cancérisation par le mélanome B16.

One supplementary protein (PI = 3,5; molecular weight 70 000) was detected by electrofocusing in sera of black male and female Mice bearing the B16 melanoma. This protein was also present in the cytoplasmic fraction of tumoral cells, but not in the membrane fraction and cytoplasmic fractions of hepatic and spleen cells. The cancerous Mice presented an augmentation of the total sera proteins compared to the controls.

Role of thiol compounds in mammalian melanin pigmentation. II. Glutathione and related enzymatic activities.

Previously, we reported evidence suggesting that, in addition to tyrosinase, glutathione-reductase plays an important role in the regulation and control of the biosynthetic activity of melanocytes. Further investigations were performed on a mammal presenting a well-defined genotype for coat pigmentation, the mutant mouse [subline C57 BL (6J)], namely the nonagouti black (a/a) mutant and the yellow (Ay/a) mutant showing, respectively, pure uniform eumelanin and phaeomelanin pigmentation. Analysis of thiol compounds and glutathione-related enzyme levels in mouse skin gave similar results to those found in tortoise-shell guinea pig skin. The observed differences in the glutathione and glutathione-related enzyme content between black and yellow (or red) skin provide evidence that the increase of glutathione-reductase activity in the environment of the melanocytes may stimulate the pigment cells to produce phaeomelanin instead of eumelanin pigment.

Amelanotic changes in B 16 melanoma after transplantation to 'Yellow' Ay/a mice.

B 16 mouse melanoma maintained on nonagouti a/a mice (C 57 Bl 6j subline) was transplanted to 'Yellow' Ay/a mutants. B 16 melanoma has now been maintained for 1 year on the 'Yellow' strain. A microscopic and ultrastructural study of transplanted tumors is described. Several enzymatic activities including tyrosinases are investigated. A marked depigmentation of the B 16 melanoma is noted after its transplantation to the 'Yellow' strain, and melanogenic characteristics of the tumor are modified.

Role of thiol compounds in mammalian melanin pigmentation: Part I. Reduced and oxidized glutathione.

Evidence for the postulated role of glutathione reductase in melanin pigmentation has been obtained by determinations of the glutathione concentrations in Tortoiseshell guinea pig skin of different colors (black, yellow, red, and white). As expected, the lowest levels of reduced glutathione (GSH) were found associated with eumelanin type pigmentation, whereas the highest ones were found in the skin with phaeomelanin producing melanocytes. On the other hand, white skin of guinea pig having no active melanocytes showed GSH levels which were intermediate between those of the black and yellow areas. These results are consistent with the view that the activity of the enzyme glutathione reductase, though not primarily related to pigmentation, plays an important role in the regulation and control of the biosynthetic activity of melanocytes leading to various types of melanin pigments.

Cholinesterases isoenzymes: a comparative study in the skin and plasma.

Acetylcholinesterase (Ache) and pseudocholinesterase (BUche) activities were studied quantitatively in healthy skin by spectrophotometric methods and qualitatively by polyacrylamide gel electrophoresis. The results were compared to those obtained in plasma. The substrates used to reveal enzyme activities were acetylthiocholine (ATC) iodide and butyrylthiocholine (BTC) iodide, respectively. A linear relationship exists between the values of BUche and Ache activities in plasma and those in skin. Six isoenzymes of different electrophoretic mobility were observed in the skin. One of them, which is never found in plasma extracts, appears to be specific to the skin. On gradient gel electrophoresis, with both substrates (ATC and BTC), a single band of enzyme activity, corresponding to a molecular weight of 600,000 was observed. These results suggest that in the skin there is only one enzyme, most probably butyryl cholinesterase, which cleaves BTC somewhat faster than ATC. This methodology, when applied to the study of dermatoses in which abnormalities of cutaneous nerve terminals are suspected, should furnish precise functional pathophysiological details.

A rapid quantitative analysis of tyrosine and its oxidation products by tyrosinase.

A new technique based on liquid phase ion exchange chromatography on short column is proposed for quantitative determination of tyrosine and Dopa. alpha-Amino,beta-guanidinopropionic acid is used as an internal standard of coloration. The role of H2O2 and ascorbic acid on tyrosine and Dopa was checked. Ascorbic acid prevents the auto-oxidation of Dopa, H2O2 has no effect on tyrosine but oxidizes Dopa even in the presence of excess ascorbic acid. This method was tested in mushroom tyrosinase, with and without ascorbic acid. Assays performed with tyrosinase from rabbit ocular extracts clearly showed that they do oxidize tyrosine. Reliability of the method is comparable to radioassay.

Action of the "nude" gene on pigmentation of the coat in c57 black and c3 mice.

The authors have studied pigmentary changes of skin and hair due to the presence of the "nude" gene in pigmented species (C 57 Bl. 6J, C3H). Morphological (histology, electron microscopy) and biochemical (polyacrylamide gel electrophoresis of tyrosinase activities) studies showed that the pigmentary alterations of the "nude" mice were related to hair cycle and hair growth wave pattern. In fact, in the "nude" mice the hair follicles evolves cyclically similarly to mice of normal coat. In depigmented skin, the hair follicles were in the resting phase and no soluble tyrosinase could be demonstrated.

[Tyrosinases of the pigmented portions of the eye in 4 species of rodent]. / Les tyrosinases des parties pigmentées de l'aeil chez quatre espèces de Rongeurs

Ocular tyrosinases of choroid from adult Rabbit, Rat, Hamster and Guinea pig are separated by gel electrophoresis. A single tyrosinase is found in all mammalian species investigated, it is present in pigmented strains and lacking in albino ones. Molecular weights of those enzymes, as determined by polyacrilamide gradient gel electrophoresis, are about 67,000. Involvement of ocular mammalian tyrosinases in in vitro L tyrosine oxidation has been demonstrated.

The tyrosinases of normal and diseased human skin (naevi, malignant melanoma, vitiligo).

Extracts of healthy and diseased skin in vitiligo, naevi and malignant melanoma were electrophoresed on polyacrylamide gel. Melanin bands were visualized on the gel by Dopa reaction. The tyrosinases found in malignant melanoma were also present in naevocellular naevi. Additional dopa melanin bands were observed. Some bands which were not related to the presence of melanocytes varied with the level of the melanogenetic activity in the individual patients.