Amelanotic Cells Research Articles

Clonal variation in the sensitivity of B16 melanoma to m-AMSA.

A hypothesis that m-AMSA may have greater cytotoxicity in melanin-containing tumour tissues, because it may reversibly bind to melanin, leading to prolonged drug exposure, was examined. Clonal lines of B16 melanoma which differed widely in pigmentation level were selected by isolating artificial lung colonies and in vitro soft-agar colonies, and implanting them into mice. Excision cell-survival assays performed 24 h after drug administration showed that in vivo sensitivity to m-AMSA progressively increased as pigmentation level decreased, but that m-AMSA drug levels measured 24 h after treatment were much lower in amelanotic than in melanotic lines. In dose-survival studies the reduced sensitivity of melanotic cell lines was revealed as a large shoulder (Dq = 27 mg/kg) though the terminal slopes for melanotic and amelanotic cell lines were similar (D10 approximately 31 mg/kg). Time-course studies indicated that there was no significant loss of drug from a melanotic cell line for 72 h after drug administration, though in an amelanotic cell line drug levels fell 10-fold in 10 h. There was, however, no evidence for prolonged drug cytotoxicity in the melanotic cell line. Using a fractionated drug-treatment regime, the greater cytotoxicity of m-AMSA to amelanotic tumour tissue was confirmed in a non-invasive regrowth-delay assay.

Effects of dopamine agonists and antagonists on murine melanoma:Correlation with dopamine binding activity

The biosynthetic pathway melanin is present in many melanomas. Previous investigations have suggested that pharmacologic levels of intermediates in this pathway (L-dopa, dopamine, and their analogues) may inhibit macromolecular synthesis in some tissue culture melanoma cell lines and prolong survival in tumor-bearing mice. Recently, a potent antidopamine drug (pimozide) has been developed. This study was designed to investigate the effects of these drugs on murine melanomas and to correlate effects on macromolecular synthesis with competitive dopamine binding activity (receptors) and melanin synthesis. Three murine melanomas (F1, F10, B16) were studied. The amelanotic B16 cell line showed no inhibition by dopa, dopamine, or pimozide when assayed for 14C-leu or 3H-TdR incorporation. Using a competitive binding assay, only low levels of dopamine binding were present. The very melanotic F1 cell line showed no inhibition by dopa or dopamine, but pimozide inhibited 14C-leu and 3H-TdR incorporation in a dose-response fashion; 50% inhibition was noted at 10(-9) M concentration with no loss in cell viability as tested by trypan blue exclusion or cell counting. Competitive dopamine binding was present (19 pmoles per g of wet tissue) with a Kd of 0.2 nM, figures approximating those seen in normal dog caudate nucleus controls. The F10 line, with melanin production between the B16 and F1, was intermediate in terms of inhibition of 14C-leu and 3H-TdR incorporation and dopamine binding. Purified melanin did not bind and, thus, does not appear to explain these binding results. These data suggest that pimozide, a drug which is a potent dopamine antagonist, may inhibit 14C-leu and 3H-TdR incorporation in murine melanoma cells and that competitive dopamine binding (receptor) appears to correlate with this inhibition. This drug and the dopamine binding assay may be useful in the study of human melanoma.

Bromodeoxyuridine-induced molecular species conversion of sialic acids of gangliosides and the alteration of cellular phenotypic expression in B16 mouse melanoma cells.

Gangliosides of B16 mouse melanoma cells were characterized and the effects of 5-bromodeoxyuridine (BrdU) on gangliosides were examined in relation to the alteration of cellular phenotypic expression. In melanotic cells, gangliosides were shown to be composed of both hematosides containing N-acetylneuraminic acid (NeuAc-hematoside) and N-glycolylneuraminic acid (NeuGc-hematoside) as determined by thin-layer chromatography, gas chromatography-mass spectrometry and the use of anti-NeuGc-hematoside antiserum. Gangliosides other than hematosides were not detectable under the conditions of thin-layer chromatography employed. BrdU treatment of the melanotic cells induced about a two-fold increase in the ratio of NeuGc-hematosides to the total hematosides without any significant change in the cellular ganglioside content. The treatment concomitantly induced a change in cellular morphology, an increase in cell-to-substrate adhesiveness, and the suppression of tyrosinase activity. Amelanotic cells, which showed cellular phenotypes similar to those of BrdU-treated melanotic cells, were demonstrated to have over twice as much NeuGc-hematosides as compared with the melanotic cells. These results indicate a possible relationship in melanoma cells between the molecular species conversion of sialic acids of hematosides and the alteration of cellular phenotypic expression by BrdU treatment or spontaneous amelanization.

Effect of estradiol and degree of cell differentiation on human melanoma susceptibility to killing by monoclonal antibodies

An estradiol non-responsive melanotic (E −M +ShA) was derived from an estradiol-responsive melanotic (E +M +ShA), and an estradiol-responsive amelanotic (E +M −SR) was derived from estradiol non-responsive amelanotic (E −M −SR) cell lines by deprivation and supplementation of the culture medium with estradiol, respectively, E +M +ShA cells became E −M +ShA after 5 weeks, i.e. 2 subcultures, of culture in the absence of estrogen, and E −M −SR cells became E +M −SR after 7 weeks, i.e. 3 subcultures, of culture in the presence of 10 −7 M 17β-estradiol. Spleen lymphocytes from mice pre-immunized with E +M +ShA, E −M +ShA, E −M −SR and E +M −SR have been fused with murine non-producer myeloma cells to obtain mouse mouse hybridoma cultures that synthesize monoclonal antibodies against human malignant melanoma. The complement-dependent cytotoxic activities of these antibodies linked responsiveness to estradiol with melanotropin biosynthesis.

Collagenolytic Activities of Cultured Human Malignant Melanoma Cells1

Three human malignant melanomas were cultured in pure populations and one tumor was cloned into melanotic and amelanotic cell lines. In the homogenates of these cultured cells, specific collagenase activities were demonstrated by isotope release from 14C-labeled collagen, disc electrophoresis, and specific cleavage of collagen molecules as demonstrated in the segment long spacing form. No significant collagenase activity was observed in the culture media. Interestingly, early cultures had a high collagenase activity in the cells and as they were successively subcultured, the activity diminished. Cysteine completely inhibited the degradation of tropocollagen as determined by disc electrophoresis and EDTA partially inhibited the degradation. It is concluded that human malignant melanoma cells produce a specific collagenase in vitro which can be extracted in early culture directly from the homogenate.

Peroxidases in the neural retina and pigment epithelium.

AbstractPeroxidase activity, assayed with 2 mM‐H2O2 and suitable hydrogen donors (either p‐phenyl‐enediamine or diaminobenzidine), was demonstrated in homogenates of neural retina and pigment epithelium of both the dog and the cow. The enzyme is particle‐associated in the native state, but is readily extractable by brief sonication or freeze‐thawing. At optimum pH, which is between 4.0 and 4.5 for both sources, the specific activity is up to 40 times greater in pigment epithelial cells than in neural retina. Some catalase activity was detected in extracts from both bovine and canine neural retina, but catalase was essentially absent in pigment epithelium.Fractionation of bovine pigment epithelial cells showed that peroxidase activity is associated mainly with heavy organelles sedimenting at low centrifugal forces. Melanosomes, nuclei, melanolysosomes and plasma membranes were the principal organelles identified in these low speed sediments. It was not possible to separate them either by differential centrifugation or on discontinuous sucrose gradients. However, melanosomes were excluded as the only source of peroxidase activity by isolating separately the melanotic and amelanotic cell populations; equal peroxidase was found in both cell types. Since nuclei are not a likely source of this enzyme, it is suggested that most of the peroxidase activity in bovine pigment epithelial cells is localized in either the melanolysosomes, plasma membranes, or both.

Spontaneous maturation and differentiation of B16 melanoma cells in culture.

B16 melanoma tumors and cultures are composed of cells with different melanin contents and replicative activities. The hypothesis was tested in vitro that these various cells constituted a population in the process of differentiation and maturation. Early cultures were predominantly composed of small, amelanotic cells with high replicative activity. Older cultures contained mostly larger and heavily melanotic cells with little or no replicative activity. Replicative activity, as measured by the uptake of tritiated thymidine, was inversely proportional to cell size and melanin content. Colony-forming ability was impaired if the original cells were melanotic. Tumorigenicity was unaffected except in very old (9-day) cultures. Our results support the concept that malignant melanocytes undergo a sequence of developmental changes which eventuates in the production of mature cells characterized by enlargement, elevated melanin content, and reduced replicative and colony-forming capacity.

Differentiation of lens-like structures from newt iris epithelial cells in vitro.

Dissociated cells of pigmented iris epithelium from adult newts grew intensively in monolayer cultures after a lag of two to three weeks. During the lag period, depigmentation occurred in many cells. When cultures became confluent five to six weeks after seeding, many tiny lens-like structures (30-70 per plate) differentiated from dense foci of amelanotic epithelial cells. These lens-like structures appeared in all cultures originated from cells of ventral as well as dorsal iris. The identification of these structures as lens was established by both immunological and ultrastructural techniques.

The melanocyte system of cattle skin. I. Amelanotic dendritic cells of epidermis.

The amelanotic dendritic cells of cattle skin have been studied using the histochemical reactions for non-specific alkaline phosphatase and specific cholinesterase. These epidermal cells in cattle skin show no reaction for adenosine triphosphatase.

Pigment Transformation and Induction in Hamster Malignant Amelanotic Melanocytes

Hamster malignant amelanotic melanocytes were incubated in vitro with RNA and DNA from DMBA-induced hamster benign Blue Nevi. Of 6 experiments, temporary pigment induction occurred in 4, and permanent pigment transformation once. In the transformed culture, following initial pigmentation of more than 90% of cells, most of the pigment transformants subsequently died, but a stable population containing 1% heavily pigmented cells survived. The pigmented survivors exhibited markedly decreased in vitro growth capacity, proliferating only ⅕ as rapidly as the amelanotic cells. Amelanotic survivors showed features of increased contact inhibition including parallel orientation, decreased maximum plate density, and increased adherence to the culture plate. The latter feature alone also occurred temporarily in 4 cultures showing only pigment induction. It appears that benign Blue Nevi of hamsters contain genetic and epigenetic determinants of pigmentation lacking in malignant amelanotic melanocytes of this species. In addition, other genetic determinants seem to be present which are capable of modifying in vitro growth and surface properties of the malignant cells in the direction of more normal behavior. Under the conditions of these experiments, transfer both of a characteristic differentiated function, pigmentation, and of more normal growth and surface behavior characteristics from a benign melanoma to a malignant melanoma cell line has been observed. These findings suggest need for further experimental investigations to determine whether the neoplastic character of malignant melanoma is necessarily irreversible.

Cellular blue nevus. Melanogenic activity and malignant transformation.

Benign and malignant blue nevi have been studied cytopathologically, enzymatically and electron microscopically. Scarcely pigmented, Schwann-cell-like, spindle-shaped cells which characterize cellular types of blue nevus have been found to possess tyrosinase activity and can form premelanosome systems. These spindle-shaped cells can undergo melanization, at least in vitro, in the vesicles and cisternae of the smooth endoplasmic reticulum in the vicinity of the Golgi apparatus as well as in the intermediate vesicles and premelanosomes. It is concluded that the characteristic spindle-shaped cells of blue nevus represent a different melanogenic activity but retain the cytological and enzymic properties of melanin synthesizing blue nevus It is therefore proposed that in the future these spindle-shaped cells characterizing blue nevus be considered amelanotic blue nevus cells.

Control of pigment production in mouse melanoma cells in vitro. Evocation and maintenance.

A clonally derived amelanotic melanoma cell line repeatedly has been forced to produce pigment by the inhibitor of DNA synthesis, I-beta-D-arabinofuranosylcytosine (ara-C) at sublethal levels. One ara-C-derived melanotic line has been cloned, and has continued to produce pigment for 2 years on normal medium. The inhibitor is most effective when administered to synchronized cells in four pulses on successive days at 1.8 x 10(-5)M during the S phase of the cell cycle. Colcemid at a sublethal concentration, and growth on medium solidified with agar also evoked pigment production in this line, but a large number of other inhibitors of biosynthetic processes did not, under the conditions tested. The melanotic lines are active producers of tyrosinase (DOPA oxidase), whereas the amelanotic line produces an inhibitor of tyrosinase activity. Both enzyme and inhibitor are labile at 4 degrees C and -20 degrees C, and decay of the inhibitor in homogenates of amelanotic cells reveals a low level of residual DOPA oxidase activity. The mean population doubling time of a cloned melanotic line is 23 hr, and that of a cloned amelanotic line 16.5 hr. A similar decrease in rate of growth is found in other melanotic lines and is believed to be a significant factor in maintaining this differentiated function. Rapid growth may be related to the production of an inhibitor by the amelanotic cells.

MELANOMA IN AN ALBINO.

The diagnosis of a malignant melanoma in a pure albino patient was clearly established by electronmicroscopic demonstration of premelanosomes and melanosome structures in the amelanotic cancer cells. It appears that the melanocyte is necessary for the development of this cancer, but the metabolic pathway for melanin production is not. The latter is a guide to the chemotherapeutic control of melanomas. Removal of nevi or birthmarks in albinos may prevent the development of this cutaneous malignancy.