RESUMEN
We have studied by means of ultrastructural immunocytochemistry the localization of RNase A in nuclei of HeLa cells in control conditions and following cell ageing in culture. We have found that roundish, electron dense foci, which contain a significant amount of RNase A, can be detected within nucleoli of aged cells. These bodies also contain RNA and lack ribosomal S3 proteins, and may represent either simple storage sites or areas where RNA degradation takes place.
Asunto(s)
Nucléolo Celular/enzimología , Senescencia Celular/fisiología , Ribonucleasa Pancreática/metabolismo , Células HeLa , Humanos , ARN/metabolismo , Estabilidad del ARN/fisiologíaRESUMEN
It is well known that at the beginning of mitosis the nucleolus disassembles but then reassembles at the end of mitosis. However, the mechanisms of these processes are still unclear. In the present work, we show for the first time that selective inhibition of cyclin B-dependent kinase 1 (CDK1) by roscovitine induces premature assembly of the nucleolus in mammalian cells in metaphase. Treatment of metaphase cells with roscovitine induces formation of structures in their cytoplasm that contain major proteins of the mature nucleolus participating in rRNA processing, such as B23/nucleophosmin, C23/nucleolin, fibrillarin, Nop52, as well as partially processed (immature) 46-45S pre-rRNA. This effect is reproducible in cells of various types; this indicates that general mechanisms regulate early stages of the nucleolus reassembly with CDK1 participation in mammalian cells. Based on our and literature data, we suggest that inactivation of the CDK1-cyclin B complex at the end of mitosis results in dephosphorylation of B23/nucleophosmin and C23/nucleolin; this facilitates their interaction with pre-rRNA and leads to formation of insoluble supramolecular complexes--nucleolus-derived foci.
Asunto(s)
Antineoplásicos/antagonistas & inhibidores , Proteína Quinasa CDC2/antagonistas & inhibidores , Nucléolo Celular/química , Mitosis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Animales , Línea Celular , Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/ultraestructura , Ciclina B/antagonistas & inhibidores , Electroforesis en Gel de Poliacrilamida , Humanos , Ratones , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/metabolismo , Roscovitina , NucleolinaRESUMEN
Changes in the immunocytochemical status of the nucleoli during long-term (6-8 months) in vitro culturing of HeLa (carcinoma of the cervix uteri) cells were described using new A3 monoclonal antibodies selectively reacting with human cell nucleoli. The appearance of cells with abnormal location of A3 antigen was paralleled by a significant increase of culture sensitivity to some external factors (protein synthesis inhibition and oxidative stress). The data indicate that location of one of the nucleolar antigens is an indicator of the qualitative status of HeLa cells in the culture.