RESUMEN
The presence of DNA topoisomerase IIalpha was investigated in interphase and metaphase mouse erythroleukemia (MEL) Friend-S cells, and in extracted with 25 mM lithium diiodosalicylate buffer (Lis) nuclei using indirect immunofluorescence. The results showed that DNA topoisomerase IIalpha is localised in the nuclei. In the metaphase cells, we found high concentrations of this enzyme in the mitotic chromosomes. Our results support the idea of the accumulation of DNA topoisomerase IIalpha at the end of the cell cycle. The extractions of nuclei with 25 mM Lis led to the complete depletion of DNA topoisomerase IIalpha from the residual nuclear matrix. Using a high dilution of the first antibody, we established that the high level of heterochromatin compactisation in the interphase nuclei is caused by the high concentration of DNA topoisomerase IIalpha.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , Isoenzimas/metabolismo , Leucemia Eritroblástica Aguda/enzimología , Animales , Antígenos de Neoplasias , Núcleo Celular/enzimología , Proteínas de Unión al ADN , Técnica del Anticuerpo Fluorescente Indirecta , Virus de la Leucemia Murina de Friend , Heterocromatina/enzimología , Interfase , Leucemia Eritroblástica Aguda/patología , Metafase , Ratones , Células Tumorales CultivadasRESUMEN
Endogenous proteolysis in chromatin of terminally differentiated, quiescent, and actively proliferating cells was studied by measuring the released acid-soluble radioactivity of [3H]tryptophan-prelabelled nuclear proteins, and by following the specific quantitative and qualitative changes in electrophoregrams of chromosomal proteins. The experiments suggest that the chromatin of differentiated mouse kidney and liver cells, as well as chromatin from Friend cells induced to commit terminal differentiation, exhibit increased proteolysis in comparison with that of chromatin isolated from actively proliferating cells. Enhanced proteolysis was found also for the slowly renewing and quiescent cells from adult mice. The control experiments designated to discriminate between the two possible alternatives explaining the difference--increased activity of the proteolytic enzymes associated with chromatin, or increased susceptibility of the chromosomal proteins to proteases--supported the latter alternative.
Asunto(s)
Diferenciación Celular , División Celular , Cromatina/metabolismo , Interfase , Animales , Células de la Médula Ósea , Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Riñón/citología , Leucemia Eritroblástica Aguda , Hígado/citología , Ratones , Ratones Endogámicos C57BL , Péptido Hidrolasas/metabolismo , Bazo/citologíaRESUMEN
In contrast to the widely accepted idea of the nearly absolute metabolic stability of histones, our experiments support the view that the histones of nonproliferating, terminally differentiated cells undergo continuous replacement. This conclusion is based on the incorporation of labeled amino acids into the histones of mouse kidney and liver cells after their intraperitoneal introduction. We have found that the intranuclear uptake of the histones made in the absence of replicative synthesis and their integration into chromatin proceed with striking delay. The metabolic rates of individual histones measured by calculating their half-lives suggest that each histone turns over at a specific rate. With regard to the basic chromatin structure, the nucleosome, such unequal turnover should mean that the histone core does not participate in this process as a single unit but rather as a protein mosaic in which each partner follows its own rate of removal. Additional experiments suggested that intact nucleosomes take part in the replacement, but the relative proportion of the nucleosomes involved should be limited. The nonnucleosomal H1A and H1 degree histones have been found to undergo faster replacement than the core histones. Moreover, in comparison to each other, these two histone subfractions are also replaced at a different rate. The results of autoradiography of isolated kidney and liver nuclei after continuous labeling with [3H]-thymidine suggest that the histone replacement is not associated with the repair of DNA.
Asunto(s)
Diferenciación Celular , Histonas/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Animales , Cromatina/metabolismo , Replicación del ADN , Histonas/aislamiento & purificación , Cinética , Lisina/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Nucleosomas/metabolismo , TritioRESUMEN
The metabolism of nonhistone chromosomal proteins was studied in two lines of cells showing a different degree of contact inhibition: human diploid fibroblasts, which are easily contact-inhibited, and Chinese hamster fibroblasts, which had been made to stop proliferating by fasting. By following the 3H414C ratio of [3H]tryptophan-labelled nonhistone chromosomal proteins and [14C]thymidine-labelled DNA in chase experiments three main groups of these proteins could be detected with respect to their metabolic behaviour: (a) a metabolically stable group which is acid-insoluble and represents the bulk of nonhistone chromosomal proteins in proliferating cells; this group is conserved when the cells enter a resting phase; (b) a metabolically labile group which is acid-soluble and is observed as a minor fraction in proliferating cells; (c) a metabolically labile group which is acid-insoluble and accumulates in resting cells; this fraction is much larger in contact-inhibited cells. Stimulation of cell proliferation by trypsinization decreases the amount of nonhistone chromosomal proteins in resting cells to the basic level observed in proliferating cells.