ABSTRACT
Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a critical role in the inappropriate survival of various types of malignant cells. Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy in the Western world. Although overexpression and regulation of NF-kappaB has been described in CLL, its function remains unclear. Exposure of CLL cells to BAY117082 or Kamebakaurin, potent pharmacological inhibitors of the NF-kappaB pathway, accelerated apoptosis in approximately 70% of cases. Sensitivity to NF-kappaB pathway inhibitors was not related to the prognostic markers VH status, CD38 or Zap70 expression, or to the levels of nuclear NF-kappaB. Normal peripheral B cells were resistant to the apoptosis-inducing effects of these compounds. Cell death induced by the inhibitors was associated with activation of caspase-9 and -3, and loss of mitochondrial membrane polarization, but did not involve changes in the expression of Bcl-2 or Mcl-1. Inhibitors caused an increase in c-jun NH2-terminal kinase activity in CLL, but this did not appear to be important for apoptosis. Microarray analysis identified some potential novel NF-kappaB target genes, including interleukin-16- and the Bcl-2- related survival protein Bcl-w. These results demonstrate that a substantial proportion of CLL are dependent on NF-kappaB for enhanced survival and suggest that inhibition of NF-kappaB may have therapeutic potential.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , NF-kappa B/antagonists & inhibitors , ADP-ribosyl Cyclase 1/analysis , Aged , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Biomarkers, Tumor/analysis , Caspase 3/analysis , Caspase 3/metabolism , Caspase 9/analysis , Caspase 9/metabolism , Cell Nucleus/chemistry , Cell Survival/drug effects , Cell Survival/genetics , Diterpenes/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , MAP Kinase Kinase 4/metabolism , Male , Middle Aged , Myeloid Cell Leukemia Sequence 1 Protein , NF-kappa B/analysis , Neoplasm Proteins/metabolism , Nitriles/pharmacology , Prognosis , Proto-Oncogene Proteins c-bcl-2/metabolism , Sulfones/pharmacology , Tumor Cells, Cultured , ZAP-70 Protein-Tyrosine Kinase/analysisABSTRACT
X-linked adrenoleukodystrophy (ALD) is a degenerative neurological disease characterized by the accumulation of very long chain fatty acids in various tissues and demyelination of the central nervous system. The human gene responsible for the disease encodes a membrane-bound ATP-binding transporter protein that is located in peroxisomes. We isolated the mouse adrenoleukodystrophy gene, determined its structure, and mapped it both cytogenetically and genetically. The mouse gene is very similar in structure to the human gene, consisting of 10 exons arranged over a 22-kb genomic region. We localized it in band B of the mouse X chromosome by fluorescence in situ hybridization analysis and, using a new microsatellite repeat polymorphism, determined the map location as 47 cM from the X centromere. We found evidence for other sequences in the mouse genome related to the 3' end of Aldgh. This study paves the way for the construction of gene-targeting plasmids that may be used to develop an animal model of ALD.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Adrenoleukodystrophy/genetics , Chromosome Mapping , Genes/genetics , Membrane Proteins/genetics , X Chromosome , ATP Binding Cassette Transporter, Subfamily D, Member 1 , Animals , Base Sequence , Cloning, Molecular , DNA/analysis , Exons/genetics , Humans , Introns/genetics , Mice , Mice, Inbred C57BL , Microsatellite Repeats/genetics , Molecular Sequence Data , Polymorphism, Genetic , Restriction Mapping , Sequence Homology, Nucleic AcidABSTRACT
We studied the pathway of 5S ribosomal RNA (rRNA) during oogenesis in Xenopus from its storage in the cytoplasm to incorporation into ribosomes in the nucleus. Ribonucleoprotein particle (RNP) assembly assays reveal striking differences in the behavior of oocyte-type and somatic-type 5S rRNA after microinjection into stage II, III, or IV oocytes or into the cytoplasm of stage V-VI oocytes. Microinjected oocyte-type 5S rRNA predominantly interacts with the 5S rRNA gene-specific transcription factor IIIA (TFIIIA) to form storage 7S RNPs. In contrast, microinjected somatic-type 5S rRNA predominantly interacts with ribosomal protein L5 to form 5S RNPs, which are precursors to ribosome assembly. In addition, a greater amount of somatic-type 5S rRNA accumulates in the nucleus and is assembled into 60S ribosomal subunits. Thus, a slight difference in nucleotide sequence results in differential binding of 5S rRNA to TFIIIA and L5, specializing oocyte-type for storage in the oocyte cytoplasm and somatic-type for rapid mobilization and ribosome assembly. When oocyte-type and somatic-type 5S rRNA molecules were microinjected into the nucleus of stage V-VI oocytes in excess of other ribosomal components, the nucleocytoplasmic distribution of both types of RNA was similar, but the distinctive protein associations were maintained. In contrast, the behavior of oocyte-type and somatic-type 5S rRNA gradually synthesized in situ from microinjected cloned genes was similar, suggesting that nascent RNA is rapidly and directly recruited into ribosomes, thus bypassing an excursion into the cytoplasm prior to ribosome assembly.
