Amelanotic Variant Research Articles

Chromatin repeat length correlates with phenotypic expression in hepatoma cells, their dedifferentiated variants, and somatic hybrids.

Chromatin repeat length is known to differ among the tissues of organisms. We have examined the repeat length of somatic cells in culture and have found an unexpected correlation between this parameter and the state of differentiation of the cells. Three sequentially derived subclones of a rat hepatoma, similar in phenotype, present a constant repeat length of 189 (+/- 1) base pairs. Three independent variant clones do not express hepatic functions and each has a different repeat length: 184 base pairs, 188 base pairs, and 192 base pairs. Somatic hybrids between the two types of cells in which liver functions are extinguished have a repeat length close or identical to that of the nonexpressing parent. In the case of one hybrid clone that reexpresses the liver phenotype and has undergone some chromosome loss, the 189-base-pair repeat length characteristic of the well-differentiated cells is reestablished. Finally, an amelanotic variant clone derived from a pigmented hamster melanoma clone also shows a signficantly altered repeat length. No correlations have been observed between repeat length and either generation time or chromosome number. Maintenance of the expression of differentiation therefore appears to be associated with a constant repeat length, and loss of the former with a modification in repeat length, suggesting that chromatin packaging is intimately involved in the regulation of gene expression.

Isolation and properties of amelanotic variants of a hamster melanoma

Stable amelanotic variants, isolated from a line of pigmented Syrian hamster melanoma cells, show a constant association of (1) dopa oxidase deficiency, (2) flattened epithelial or fibroblast morphology, (3) anchorage dependence, and (4) a long latent period in tumor formation, each of these properties distinguishing them from the dopa-oxidase-positive, anchorage-independent, highly malignant pigmented cells of origin. One of the variants is shown to have the properties of a pleiotropic conditional “mutant” since its cells show the simultaneous but transitory restoration of pigmentation and melanoma-like morphology when cloned at alkaline pH. Cells of this variant have been hybridized with pigmented cells, derived both from the line of origin and from a mouse melanoma. Hybrids from the first cross show all of the properties characteristic of the pigmented parental cells, but produce segregants reexpressing the ensemble of properties of the amelanotic variant, while those of the second cross show extinction (followed by reexpression) of pigmentation. It is therefore concluded that cells of the amelanotic variant produce a factor capable of extinguishing dopa oxidase activity, as revealed directly by the properties of the hamster-mouse hybrids and indirectly by the amelanotic segregants of the hamster-hamster hybrids.

Genetic control of melanization: isolation and analysis of amelanotic variants from cultured melanotic melanoma cells.

MELANOMA CELLS EXPRESS A PHENOTYPE THAT IS EASY TO RECOGNIZE: the synthesis of melanin. We used this marker to isolate clones of amelanotic variants from large populations of wild-type melanotic clones. Cloudman mouse melanoma (S91, clone M-3, CCL 53.1) was chosen as the parental line because the cells are highly pigmented, grow well as clones in soft agar, and fuse readily with Sendai virus. Subclones (10(7)) of this line were screened without prior mutagenesis, and nine amelanotic variants were isolated. The mutagen ethylmethanesulfonate increased the frequency of variants by three to four orders of magnitude. Wild-type cells had both basal and melanocyte stimulating hormone-inducible tyrosinase activities. The four amelanotic variants that we have examined to date all behaved similarly: they lost basal tyrosinase (EC 1.14.18.1; monophenol monooxygenase) activity but retained melanocyte stimulating hormone-inducible activity; they contained Stage-II melanosomes but no melanized melanosomes; they exhibited growth characteristics similar to those of wild-type cells in culture but produced fewer tumors in mice.