RESUMEN
The CD44+ and CD44- subpopulations of the colorectal cancer cell line Caco2 were analyzed separately for their sensitivities to the antitumor drug camptothecin. CD44+ cells were less sensitive to camptothecin than CD44- cells. The relative resistance of CD44+ cells was correlated with (i) reduced activity of the nuclear enzyme topoisomerase I and (ii) insensitivity of this enzyme to camptothecin when analyzed in extracts. In contrast, topoisomerase I activity was higher in extracts from CD44- cells and the enzyme was camptothecin sensitive. Topoisomerase I from the two subpopulations were differentially phosphorylated in a manner that appeared to determine the drug sensitivity and activity of the enzyme. This finding was further supported by the fact that phosphorylation of topoisomerase I in CD44+ cell extract by protein kinase CK2 converted the enzyme to a camptothecin sensitive, more active form mimicking topoisomerase I in extracts from CD44- cells. Conversely, dephosphorylation of topoisomerase I in extracts from CD44- cells rendered the enzyme less active and camptothecin resistant. These findings add to our understanding of chemotherapy resistance in the Caco2 CD44+ cancer stem cell model.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Camptotecina/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Resistencia a Antineoplásicos , Células CACO-2 , Humanos , Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Fosforilación/efectos de los fármacosRESUMEN
Heat shock genes respond to moderate heat stress by a wave of transcription. The induction phase is accompanied by the massive eviction of histones, which later reassemble with DNA during the ensuing phase of transcription downregulation. In this article, we identify determinants of this reassembly throughout the heat shock protein 104 gene (HSP104) transcription unit. The results show that, although histone H3 lacking amino acids 4-30 of its N-terminal tail (H3Delta4-30) is normally deposited, reassembly of H3Delta4-40 is obliterated with an accompanying sustained transcription. On mutation of the histone chaperones Spt6p and Spt16p, but not Asf1p, reassociation of H3 with DNA is compromised. However, despite a lasting open chromatin structure, transcription ceases normally in the spt6 mutant. Thus, transcriptional downregulation can be uncoupled from histone redeposition and ongoing transcription is not required to prevent chromatin reassembly.
Asunto(s)
Cromatina/química , ADN/química , Regulación Fúngica de la Expresión Génica , Proteínas de Choque Térmico/genética , Histonas/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Transcripción Genética , Eliminación de Gen , Regulación de la Expresión Génica , Genotipo , Proteínas de Choque Térmico/química , Hibridación Fluorescente in Situ , Mutación , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Factores de TiempoRESUMEN
Members of the family of adenosine deaminases acting on RNA (ADARs) can catalyze the hydrolytic deamination of adenosine to inosine and thereby change the sequence of specific mRNAs with highly double-stranded structures. The ADARs all contain one or more repeats of the double-stranded RNA binding motif (DRBM). By both in vitro and in vivo assays, we show that the DRBMs of rat ADAR2 are necessary and sufficient for dimerization of the enzyme. Bioluminescence resonance energy transfer (BRET) demonstrates that ADAR2 also exists as dimers in living mammalian cells and that mutation of DRBM1 lowers the dimerization affinity while mutation of DRBM2 does not. Nonetheless, the editing efficiency of the GluR2 Q/R site depends on a functional DRBM2. The ADAR2 DRBMs thus serve differential roles in RNA dimerization and GluR2 Q/R editing, and we propose a model for RNA editing that incorporates the new findings.
Asunto(s)
Adenosina Desaminasa/metabolismo , ARN Bicatenario/metabolismo , Adenosina Desaminasa/química , Adenosina Desaminasa/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dimerización , Proteínas Fluorescentes Verdes/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Edición de ARN , ARN Bicatenario/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas de Unión al ARN , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
We describe a new human isoform, GFAP epsilon, of the intermediary filament protein GFAP (glial fibrillary acidic protein). GFAP epsilon mRNA is the result of alternative splicing and a new polyadenylation signal, and thus GFAP epsilon has a new C-terminal protein sequence. This provides GFAP epsilon with the capacity for specific binding of presenilin proteins in yeast and in vitro. Our observations suggest a direct link between the presenilins and the cytoskeleton where GFAP epsilon is incorporated. Mutations in GFAP and presenilins are associated with Alexander disease and Alzheimer's disease, respectively. Accordingly, GFAP epsilon should be taken into consideration when studying neurodegenerative diseases.
