RESUMEN
Expression of the human multidrug resistance 1 gene (MDR1), which encodes the P-glycoprotein transmembrane efflux pump, has been associated with treatment failure of some leukemias, primarily acute myeloid leukemia (AML). To elucidate the epigenetic events associated with overexpression of MDR1 in AML, we analyzed the methylation status of a 2000 bp region within the MDR1 locus using a bisulphite genomic sequencing technique. A CpG-rich domain, approximately 1 kb in size, encompasses the promoter region, exon I, and intron I. This domain was found to be relatively unmethylated in five out of six primary and cultured human hematopoietic cells, as well as five out of six AML patient samples, independent of the MDR1 phenotype. The data suggest that the methylation status of the CpG-rich domain does not act as a 'switch' to regulate expression of the MDR1 gene. In addition, we found an upstream Alu repeat sequence to be unmethylated in three out of five cultured hematopoietic cell lines, both MDR1 expressing and non-expressing. However, analysis of primary CD8-positive T cells and AML patient samples revealed dense methylation of this region which is consistent with methylation of Alu repeat sequences observed in somatic tissues.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Metilación de ADN , Leucemia Mieloide/genética , Enfermedad Aguda , Adulto , Elementos Alu/genética , Linfocitos T CD8-positivos/fisiología , Islas de CpG/genética , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Citometría de Flujo , Humanos , Leucemia Mieloide/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasAsunto(s)
Antígenos CD , Antígenos de Diferenciación/biosíntesis , N-Glicosil Hidrolasas/biosíntesis , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Antígenos de Diferenciación/genética , Clonación Molecular , Fermentación , Vectores Genéticos , Glicerol/metabolismo , Humanos , Glicoproteínas de Membrana , N-Glicosil Hidrolasas/genética , Pichia/genética , Pichia/crecimiento & desarrollo , Proteínas Recombinantes/biosíntesis , SolubilidadRESUMEN
Cyclic adenosine diphosphate (ADP)-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+), mobilizes calcium from intracellular stores in many cells. The synthesis of cADPR from NAD+ and its subsequent hydrolysis to ADPR is catalyzed by an ADP-ribosyl cyclase and a cADPR hydrolase, respectively. The ADP-ribosyl cyclase cloned from the ovotestis of the marine invertebrate Aplysia californica has amino acid sequence homology to the human lymphocyte surface antigen CD38. CD38 has been shown to catalyze both the formation and the hydrolysis of cADPR. In this study, we produced soluble, enzymatically active CD38 using recombinant expression techniques in bacteria and yeast. We engineered a gene coding for a soluble form of CD38 by excision of the region of the gene coding for the N-terminal amino acids representing the putative membrane spanning sequence and short putative intracellular sequence. For expression in bacteria (Escherichia coli), this construct was cloned into the pFlag-1 plasmid which allows induced, periplasmic expression and relatively simple purification of the soluble CD38. For expression in yeast (Pichia pastoris) the CD38 sequence was further modified to eliminate four putative N-linked glycosylation sites and the resulting construct was expressed as a secreted protein. Both systems produce soluble enzymes of approximately 30 kDa and both recombinant enzymes display similar cyclase and hydrolase activities.