Inhibition of DNA and Protein Synthesis in Melanocytes by a Melanoma Extract

Abstract

The alcohol precipitate (70–90%) prepared from a water extract of Harding-Passey mouse melanomas contained a melanoma melanocyte cell-cycle inhibitor. The incorporation of 14C-thymidine into the DNA of melanocytes was used as a measure of DNA synthesis by melanocytes and it appears to be valuable as a parameter of melanocyte proliferation. A single injection of melanoma extract into the peritoneal cavity of mice bearing Harding-Passey mouse melanoma caused 35–65 percent reduction of 14C-thymidine incorporation into the DNA of melanoma melanocytes. An in vitro assay method using 8–16 mouse melanoma melanocytes in tissue culture was established. Addition of melanocyte extract to the tissue culture medium containing adrenalin and hydrocortisone caused a reduction of mitotic index, of incorporation of 14C-thymidine into DNA, and of 3H-leucine incorporation into melanocyte protein. The inhibition of DNA and protein biosynthesis due to the melanocyte extract was well correlated. For inhibition of the melanocyte extract, which resembles epidermal chalone in activity, the presence of adrenalin and hydrocortisone was required. A procedure is described consisting of ethanol fractionation, gel filtration, and column chromatography which results in considerable purification of the melanoma melanocyte specific mitotic inhibitor. The biologically active material of the melanoma extract appears to consist of two different fractions; fraction II, which is heat labile and tissue nonspecific and consists mainly of protein with a molecular weight of 30,000–35,000, and fraction IV2 which is heat labile and tissue specific and consists of protein and RNA. The biologic activity of fraction IV2 seems to depend on both the protein and RNA moieties. These two active substances when isolated are still heterogeneous and further purification is required.