RESUMEN
BACKGROUND: Comparisons of platelet RNAs could provide crucial information on platelet function, thrombopoiesis and the etiology of megakaryocyte (MK) or platelet disorders. OBJECTIVES: We developed a method for stringent purification of platelets from small blood samples from single donors. Purity of the platelet preparations was verified by an RT-PCR assay. We tested three methods to identify the differences in RNA between platelet sources. METHODS: Differential hybridization to cDNA macro-arrays and suppressive-subtractive hybridization PCR (SSH-PCR) were used to compare RNAs from normal platelets to those from a Bernard-Soulier syndrome (BSS) patient. Affymetrix GeneChip U133 plus 2.0 arrays were used to compare male and female platelet RNAs. RESULTS: Macroarrays identified approximately 7500 platelet transcripts, but failed to identify differentially expressed transcripts with confidence. SSH-PCR produced libraries almost exclusively of mitochondrial-derived transcripts, but included nuclear-encoded genes that could not be confirmed by immunoblotting of normal and BSS platelet lysates. The Affymetrix platform gave reproducible profiles from our small-scale purified platelet preparations, whereas a partially purified platelet preparation produced a drastically skewed transcript profile. The microarray analysis identified the heparanase precursor transcript as overexpressed in female platelets, and we observed variable yet consistently higher levels of heparanase protein in female platelets compared with male platelets in four independent donor pairs. CONCLUSIONS: This demonstrates for the first time that differential platelet transcript levels can identify changes in expression level of platelet proteins. Combined with our small-scale platelet preparation method, this establishes a system to compare platelets from the limited clinical sources to help elucidate molecular bases for platelet or megakaryocyte pathologies.
Asunto(s)
Plaquetas/metabolismo , ARN/sangre , ARN/genética , Separación Celular/métodos , Perfilación de la Expresión Génica , Humanos , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Glucocorticoid resistance was investigated in human leukemic CCRF-CEM cells. A mutation (L753F), which renders the human glucocorticoid receptor (hGR) gene functionally hemizygous, was identified in all CEM-derived cell lines analyzed. Allele-specific PCR identified the same mutation in lymph node biopsy material from patient CEM cells. Given the correlation between hGR concentration and glucocorticoid sensitivity, this suggests that loss of functional heterozygosity may result in resistance to glucocorticoid-based chemotherapy. The L753F mutation was probably not responsible for the ontogeny of the disease because it did not appear to be present in all leukemic cells. Thus, it is unlikely that hGR mutations would be detected in leukemic patients at presentation, but they may occur, and be selected for, during treatment. Deletions and point mutations in the hGR gene of cells selected for steroid resistance in vitro were investigated by PCR-single strand conformation polymorphism analysis. Loss of hGR mRNA expression resulted from 5'-deletion of the hGR gene and nonsense mutations in exon 6. These results provide the first evidence for somatic mutation in the hGR gene of a patient with acute lymphoblastic leukemia, offer a potential in vivo mechanism for acquisition of steroid resistance in leukemia, and suggest that screening for additional in vivo mutations will require analysis of genomic DNA.
Asunto(s)
Leucemia/genética , Mutación , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo Conformacional Retorcido-Simple , Receptores de Glucocorticoides/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Desoxirribonucleasas de Localización Especificada Tipo II , Humanos , Leucemia/patología , Ganglios Linfáticos/patología , Mutación Puntual , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T , Células Tumorales CultivadasRESUMEN
The role of the ligand in glucocorticoid receptor-mediated transactivation and transrepression of gene expression was investigated. Half-maximal transactivation of a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene in transfected cells expressing the human glucocorticoid receptor mutant GRL753F, from which the rate of ligand dissociation is four to five times higher than the rate of dissociation from normal receptors, required a 200- to 300-fold-higher concentration of dexamethasone than was required in cells expressing the normal receptor. Immunocytochemical analysis demonstrated that this difference was not the result of a failure of the mutant receptor to accumulate in the nucleus after steroid treatment. In contrast, in cells cotransfected with a reporter gene containing the AP-1-inducible collagenase gene promoter, the concentration of dexamethasone required for 50% transrepression was the same for mutant and normal receptors. Efficient receptor-mediated transrepression was also observed with the double mutant GRL753F/C421Y, in which the first cysteine residue of the proximal zinc finger has been replaced by tyrosine, indicating that neither retention of the ligand nor direct binding of the receptor to DNA is required. RU38486 behaved as a full agonist with respect to transrepression. In addition, receptor-dependent transrepression, but not transactivation, was observed in transfected cells after heat shock in the absence of the ligand. Taken together, these results suggest that unlike transactivation, transrepression of AP-1 activity by the nuclear glucocorticoid receptor is ligand independent.
Asunto(s)
Colagenasas/genética , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/biosíntesis , Chlorocebus aethiops , Proteínas de Unión al ADN/biosíntesis , Calor , Humanos , Riñón , Cinética , Ligandos , Virus del Tumor Mamario del Ratón , Mifepristona/farmacología , Mutagénesis Sitio-Dirigida , Mutación Puntual , Regiones Promotoras Genéticas/efectos de los fármacos , Receptores de Glucocorticoides/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
The molecular basis for the receptorless (r-) and activation-labile (act1) phenotypes of glucocorticoid-resistant mutants isolated from glucocorticoid-sensitive human leukemic CEM-C7 cells was determined. Clones isolated from a complementary DNA library prepared from r- ICR27TK.3 cells, in which one glucocorticoid receptor (GR) gene has been deleted, contained a single adenosine to thymidine transversion in the third position of codon 753, resulting in the substitution of phenylalanine for leucine. This mutant gene (GR753F) had only 13% of the trans-activating activity of the normal gene and produced a M(r) 92,000 receptor protein with the same r- phenotype seen in ICR27TK.3 cells. Analysis of complementary DNA clones isolated from a library prepared from parental glucocorticoid-sensitive 6TG1.1 cells showed that these cells express both a normal GR gene (GR+) and the GR753F gene. Thus, their genotype is GR+/GR753F. Analysis of clones isolated from a complementary DNA library prepared from glucocorticoid-resistant activation-labile 3R7. 6TG.4 cells revealed the presence of the GR753F gene and a second mutant gene (GR421Y) containing a guanosine to adenosine transition in the second position of codon 421, resulting in the replacement of the first cysteine of the proximal zinc finger of the DNA-binding domain by tyrosine. This mutant had no trans-activating activity but normal ligand-binding characteristics. Thus, the genotype of act1 3R7.6TG.4 cells is GR421Y/GR753F. Consequently, the sequence-specific DNA-binding activity of receptors in act1 cells is attributable to the GR753F gene, while the ligand-binding activity seen in intact cells is attributable to the GR421Y gene. These results provide a direct explanation for the r- and act1 phenotypes of glucocorticoid-resistant cells and demonstrate that glucocorticoid-sensitive cells derived from CEM-C7 cells contain a heterogeneous population of normal and mutant receptors.