RESUMEN
Mature erythrocytes in mammals have no nuclei, although they differentiate from nucleated precursor cells. The mechanism by which enucleation occurs is not well understood. Here we show that deoxyribonuclease II (DNase II) is indispensable for definitive erythropoiesis in mouse fetal liver. No live DNase II-null mice were born, owing to severe anemia. When mutant fetal liver cells were transferred into lethally irradiated wild-type mice, mature red blood cells were generated from the mutant cells, suggesting that DNase II functions in a non-cell-autonomous manner. Histochemical analyses indicated that the critical cellular sources of DNase II are macrophages present at the site of definitive erythropoiesis in the fetal liver. Thus, DNase II in macrophages appears to be responsible for destroying the nuclear DNA expelled from erythroid precursor cells.
Asunto(s)
Endodesoxirribonucleasas/metabolismo , Eritropoyesis , Hematopoyesis Extramedular , Hígado/embriología , Hígado/fisiología , Macrófagos/enzimología , Animales , Apoptosis , Diferenciación Celular , Trasplante de Células , ADN/análisis , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Eritroblastos/citología , Eritroblastos/metabolismo , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Feto/enzimología , Marcación de Gen , Globinas/genética , Globinas/metabolismo , Factores de Transcripción de Tipo Kruppel , Hígado/citología , Hígado/enzimología , Lisosomas/enzimología , Macrófagos/química , Macrófagos/ultraestructura , Ratones , Ratones Noqueados , Mutación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Apoptosis is often accompanied by degradation of chromosomal DNA. CAD, caspase-activated DNase, was identified in 1998 as a DNase that is responsible for this process. In the last several years, mice deficient in the CAD system have been generated. Studies with these mice indicated that apoptotic DNA degradation occurs in two different systems. In one, the DNA fragmentation is carried out by CAD in the dying cells and in the other, by lysosomal DNase II after the dying cells are phagocytosed. Several other endonucleases have also been suggested as candidate effectors for the apoptotic degradation of chromosomal DNA. In this review, we will discuss the mechanism and role of DNA degradation during apoptosis.
Asunto(s)
Apoptosis/genética , Cromosomas/genética , Fragmentación del ADN/genética , ADN/metabolismo , Desoxirribonucleasas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Desoxirribonucleasas/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Humanos , Proteínas/genética , Proteínas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Caspase-activated DNase (CAD) degrades chromosomal DNA during apoptosis, whereas ICAD (inhibitor of CAD) inhibits the CAD's DNase by binding to it. Here, we describe the assignment of murine CAD and ICAD genes to the distal part of murine chromosome 4. Molecular cloning and structural analysis indicated that CAD and ICAD genes are comprised of 7 and 6 exons, respectively. Two different ICAD mRNAs coding for two forms of ICAD proteins (ICAD-S and ICAD-L) were found to be produced by alternative splicing of intron 5. The CAD and ICAD mRNAs were detected ubiquitously in various murine tissues. Analyses of the promoter activity with a series of deletion mutants of their 5' flanking regions indicated that a 190-bp 5' flanking region of the CAD gene was sufficient to promote the transcription. Whereas, a 120-bp flanking region of ICAD gene was required to promote its transcription. These regions do not show similarity between CAD and ICAD genes, suggesting that expression of CAD and ICAD genes is regulated by different mechanisms.
Asunto(s)
Desoxirribonucleasas/genética , Regiones Promotoras Genéticas , Proteínas/genética , Animales , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario , Desoxirribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos , Femenino , Regulación de la Expresión Génica , Células HeLa , Humanos , Células Jurkat , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN Mensajero , Análisis de Secuencia de ADN , Distribución TisularAsunto(s)
Apoptosis/inmunología , Fragmentación del ADN/inmunología , Desoxirribonucleasas/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Linfocitos/enzimología , Proteínas/metabolismo , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis , Células Cultivadas , Fragmentación del ADN/genética , Desoxirribonucleasas/genética , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Ratones Noqueados , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/genética , Bazo/citología , Bazo/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
Many cells die during mammalian development and are engulfed by macrophages. In DNase II(-/-) embryos, the TUNEL-positive DNA of apoptotic cells is left undigested in macrophages, providing a system for studying programmed cell death during mouse development. Here, we showed that an Apaf-1-null mutation in the DNase II(-/-) embryos greatly reduced the number of macrophages carrying DNA at E11.5. However, at later stages of the embryogenesis, a significant number of macrophages carrying undigested DNA were present in Apaf-1(-/-) embryos, indicating that cells died and were engulfed in an Apaf-1-independent manner. In most tissues of the Apaf-1(-/-) embryos, no processed caspase-3 was detected, and the DNA of dead cells accumulated in the macrophages appeared intact. Many nonapoptotic dead cells were found in the tail of the Apaf-1(-/-) embryos, suggesting that the Apaf-1-independent programmed cell death occurred, and these dead cells were engulfed by macrophages. In contrast, active caspase-3 was detected in E14.5 thymus of Apaf-1(-/-) embryos. Treatment of fetal thymocytes with staurosporine, but not etoposide, induced processing of procaspases 3 and 9, indicating that the E14.5 thymocytes have the ability to undergo caspase-dependent apoptosis in an Apaf-1-independent manner. Thus, programmed cell death in mouse development, which normally proceeds in an efficient Apaf-1-depenent mechanism, appears to be backed up by Apaf-1-independent death systems.