RESUMEN

Fanconi anemia (FA) is a genetic disorder characterized by congenital abnormalities, bone marrow failure, and increased susceptibility to cancer. Of the fifteen FA proteins, Fanconi anemia group C (FANCC) is one of eight FA core complex components of the FA pathway. Unlike other FA core complex proteins, FANCC is mainly localized in the cytoplasm, where it is thought to function in apoptosis, redox regulation, cytokine signaling, and other processes. Previously, we showed that regulation of FANCC involved proteolytic processing during apoptosis. To elucidate the biological significance of this proteolytic modification, we searched for molecular interacting partners of proteolytic FANCC fragments. Among the candidates obtained, the transcriptional corepressor protein C-terminal binding protein-1 (CtBP1) interacted directly with FANCC and other FA core complex proteins. Although not required for stability of the FA core complex or ubiquitin ligase activity, CtBP1 is essential for proliferation, cell survival, and maintenance of chromosomal integrity. Expression profiling of CtBP1-depleted and FA-depleted cells revealed that several genes were commonly up- and down-regulated, including the Wnt antagonist Dickkopf-1 (DKK1). These findings suggest that FA and Wnt signaling via CtBP1 could share common effectors.

Asunto(s)

Oxidorreductasas de Alcohol/metabolismo , Apoptosis , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/genética , Animales , Biomarcadores/metabolismo , Western Blotting , Diferenciación Celular , Proliferación Celular , Inestabilidad Cromosómica , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Ensayo de Inmunoadsorción Enzimática , Proteínas del Grupo de Complementación de la Anemia de Fanconi/antagonistas & inhibidores , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Mapas de Interacción de Proteínas , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos Híbridos

RESUMEN

The main cause of morbidity and mortality in Fanconi anemia patients is the development of bone marrow (BM) failure; thus correction of hematopoietic stem cells (HSCs) through gene transfer approaches would benefit FA patients. However, gene therapy trials for FA patients using ex vivo transduction protocols have failed to provide long-term correction. In addition, ex vivo cultures have been found to be hazardous for FA cells. To circumvent negative effects of ex vivo culture in FA stem cells, we tested the corrective ability of direct injection of recombinant lentiviral particles encoding FancC-EGFP into femurs of FancC(-/-) mice. Using this approach, we show that FancC(-/-) HSCs were efficiently corrected. Intrafemoral gene transfer of the FancC gene prevented the mitomycin C-induced BM failure. Moreover, we show that intrafemoral gene delivery into aplastic marrow restored the bone marrow cellularity and corrected the remaining HSCs. These results provide evidence that targeting FA-deficient HSCs directly in their environment enables efficient and long-term correction of BM defects in FA.

RESUMEN

Treatments of the hematological manifestation in Fanconi anemia (FA) are first supported by attempts to stimulate hematopoiesis with androgens or hematopoietic growth factors. However, the long-term curative treatment of the hematological manifestation in FA patients is bone marrow (BM) or cord blood stem cell transplantation. The success rate for BM transplantation is fairly high with HLA-matched sibling donors but is, unfortunately, low with HLA-matched unrelated donors. An alternative curative treatment for those patients with no sibling donors might be gene transfer into hematopoietic stem cells. Because FA patients have reduced numbers of stem/progenitor cells, ex vivo expansion of hematopoietic stem cells would be a crucial step in gene transfer protocols. Using the FA mouse model, Fancc-/-, we tested the ability of CD34- hematopoietic stem cells to support ex vivo expansion. We determined that Fancc-/- CD34- stem cells have reduced reconstitution ability and markedly reduced self-renewal ability after culture, as shown by secondary transplants. These results indicate that FA stem cells may not be well suited for ex vivo expansion before gene transfer or transplantation protocols.

Asunto(s)

Anemia de Fanconi/patología , Hematopoyesis , Células Madre Hematopoyéticas/patología , Animales , Antígenos CD34 , Apoptosis , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Madre Hematopoyéticas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados

RESUMEN

The function of the Fanconi anemia group C protein (FANCC) is still unknown, though many studies point to a role in damage response signaling. Unlike other known FA proteins, FANCC is mainly localized to the cytoplasm and is thought to act as a messenger of cellular damage rather than an effector of repair. FANCC has been shown to interact with several cytoplasmic and nuclear proteins and to delay the onset of apoptosis through redox regulation of GSTP1. We investigated the fate and function of FANCC during apoptosis. Here we show that FANCC undergoes proteolytic modification by a caspase into a predominant 47-kDa ubiquitinated protein fragment. Lack of proteolytic modification at the putative cleavage site delays apoptosis but does not affect MMC complementation. These results suggest that FANCC function is regulated through proteolytic processing.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Proteínas Nucleares , Proteínas/química , Proteínas/metabolismo , Apoptosis/efectos de los fármacos , Sitios de Unión , Caspasas/metabolismo , Línea Celular , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación C de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Células HeLa , Humanos , Mitomicina/farmacología , Peso Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transfección

RESUMEN

Sudden infant death syndrome (SIDS) has been associated with the volatilization of arsenic, antimony or phosphorus compounds from infants' bedding material by micro-organisms, the so-called 'toxic gas hypothesis'. The volatilization of arsenic by aerobic micro-organisms isolated from new sheepskin bedding material, as well as on material used by a healthy infant and by an infant who perished of SIDS, was examined. Three fungi were isolated from a piece of sheepskin bedding material on which an infant perished of SIDS, which methylated arsenic to form trimethylarsenic(V) species, precursors to volatile trimethylarsine. These three fungi were identified as Scopulariopsis koningii, Fomitopsis pinicola and Penicillium gladioli by their 26S-ribosomal RNA polymerase chain reaction products. These fungi were not previously known to methylate arsenic. The volatilization of arsenic by these three fungi was then examined. Only P. gladioli volatilized arsenic and only under conditions such that the production of sufficient trimethylarsine to be acutely toxic to an infant is unlikely. S. brevicaulis grew on the sheepskin bedding material and evolved a trace amount of trimethylarsine. Known human pathogens such as Mycobacterium neoaurum and Acinetobacter junii were isolated from used bedding.

Asunto(s)

Arsénico/toxicidad , Arsenicales/metabolismo , Ropa de Cama y Ropa Blanca/microbiología , Lana/microbiología , Animales , Arsénico/metabolismo , Ascomicetos/metabolismo , Bacillus/metabolismo , Humanos , Lactante , Metilación , Penicillium/metabolismo , Polyporaceae/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ovinos , Muerte Súbita del Lactante/etiología , Volatilización

RESUMEN

BACKGROUND: Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow failure (BM) and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells. METHODS: Since genes involved in stem cell differentiation and/or maintenance are usually regulated at the transcription level, we used a semiquantitative RT-PCR method to evaluate FA gene transcript levels in purified hematopoietic stem cells. RESULTS: We show that most FA genes are highly expressed in primitive CD34-positive and negative cells compared to lower levels in more differentiated cells. However, in CD34- stem cells the Fancc gene was found to be expressed at low levels while Fancg was undetectable in this population. Furthermore, Fancg expression is significantly decreased in Fancc -/- stem cells as compared to wild-type cells while the cancer susceptibility genes Brca1 and Fancd1/Brac2 are upregulated in Fancc-/- hematopoietic cells. CONCLUSIONS: These results suggest that FA genes are regulated at the mRNA level, that increased Fancc expression in LTS-CD34+ cells correlates with a role at the CD34+ differentiation stage and that lack of Fancc affects the expression of other FA gene, more specifically Fancg and Fancd1/Brca2, through an unknown mechanism.