RESUMO
Murine melanoma cells provide an excellent system for studying the proposed role of nuclear nonhistone proteins (NHP's) as regulators of gene expression. Cloudman mouse melanoma cells (S91, NCTC 3960, CCL 53), grown in culture, are normally lightly pigmented, but in the presence of melanocyte stimulating hormone (MSH) show a large increase in melanin content. Cells were grown in medium with and withoug MSH and labeled with either 14C- or 3H-leucine, respectively. Following 48 hr of incubation, the cells were harvested, combined, and nuclei isolated. The NHPs were extracted from these nuclei in a series of steps which yielded 4 major fractions. Each fraction was further separated on DEAE cellulose columns into a total of 40 subfractions, each of which was electrophoresed on SDS gels. Each gel was sliced and counted and the 14C/3H ratio was determined for each slice. A number of differences in 14C/3H ratios were observed between the NHPs isolated from MSH-treated and control cells which reflect changes in the synthesis and/or transport of NHPs in MSH-treated cells.
Assuntos
Proteínas Cromossômicas não Histona/genética , Genes Reguladores , Hormônios Estimuladores de Melanócitos/farmacologia , Melanoma/genética , Neoplasias Cutâneas/genética , Animais , Células Cultivadas , Ativação Enzimática , Técnicas In Vitro , Melaninas/metabolismo , Melanoma/enzimologia , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/genética , Receptores de AMP Cíclico , Neoplasias Cutâneas/enzimologiaRESUMO
Nuclear nonhistone proteins (NHP's) have been implicated as regulatory agents involved in controlling genetic expression. Utilizing murine melanoma cells, we describe a method for isolating and fractionating NHP's which greatly increases the yield of these proteins as well as the level of resolution required for detecting small differences in particular NHP's. Mouse melanoma cells were grown in medium labeled with [(3)H]leucine. Following 48 hr of incubation, the cells were harvested and nuclei isolated. The NHP's were extracted from the nuclei in a series of steps which yielded four major fractions: NHP(1), NHP(2), NHP(3), NHP(4). This method solubilized 80-90% of the protein from the nuclear homogenate. The NHP fractions were then separated on DEAE-cellulose columns in a series of salt steps increasing in concentration from 0.05 to 0.50 M NaCl, followed by steps of 2 M NaCl and 4 and 7 M guanidine-hydrochloride. The 40 NHP fractions eluted from these columns were further separated on polyacrylamide-SDS gels and ranged in molecular weight from 9000 to 110,000 daltons. Differences were observed in the electrophoretic pattern of each of these 40 fractions. The high resolution of these fractionation procedures greatly enhances the possibility of observing small changes in proteins which may play a role in gene regulation.
