ABSTRACT
The p53 protein accumulates rapidly through post-transcriptional mechanisms following cellular exposure to DNA damaging agents and is also activated as a transcription factor leading to growth arrest or apoptosis. Phosphorylation of p53 occurs after DNA damage thereby modulating its activity and impeding the interaction of p53 with its negative regulator oncogene Mdm2. The serines 15 and 37 present in the amino terminal region of p53 are phosphorylated by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. In order to verify if specific p53 mutations occur in the multi-drug resistance phenotype, we analysed the p53 gene in two T-lymphoblastoid cell lines, CCRF-CEM and its multi-drug-resistant clone CCRF-CEM VLB100, selected for resistance to vinblastine sulfate and cross-resistant to other cytotoxic drugs. Both cell lines showed two heterozygous mutations in the DNA binding domain at codons 175 and 248. The multi-drug resistant cell line, CCRF-CEM VLB100, showed an additional mutation that involves the serine 37 whose phosphorylation is important to modulate the protein activity in response to DNA damage. The effects of these mutations on p53 transactivation capacity were evaluated. The activity of p53 on pro-apoptotic genes expression in response to DNA damage induced by (-irradiation, was affected in the vinblastine (VLB) resistant cell line but not in CCRF-CEM sensitive cell line resulting in a much reduced apoptotic cell death of the multi-drug resistant cells.
Subject(s)
Apoptosis/genetics , Drug Resistance, Multiple/genetics , Gene Expression Regulation, Leukemic/genetics , Leukemia, T-Cell/genetics , Mutation, Missense , Tumor Suppressor Protein p53/genetics , Amino Acid Substitution , Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Base Sequence , Cell Survival/radiation effects , Conserved Sequence , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , DNA, Neoplasm/radiation effects , Dactinomycin/pharmacology , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/genetics , Exons , Genes, p53/genetics , Humans , Leukemia, T-Cell/metabolism , Leukemia, T-Cell/pathology , Polymorphism, Single-Stranded Conformational , Radiation Tolerance/genetics , Serine/genetics , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/radiation effects , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/physiology , Vinblastine/pharmacologyABSTRACT
Human sex chromosomes, which are morphologically and genetically different, share few regions of homology. Among them, only pseudoautosomal regions (PARs) pair and recombine during meiosis. To better address the complex biology of these regions, we sequenced the telomeric 400 kb of the long arm of the human X chromosome, including 330 kb of the human Xq/YqPAR and the telomere. Sequencing reveals subregions with distinctive regulatory and evolutionary features. The proximal 295 kb contains two genes inactivated on both the inactive X and Y chromosomes [ SYBL1 and a novel homologue ( HSPRY3 ) of Drosophila sprouty ]. The GC-rich distal 35 kb, added in stages and much later in evolution, contains the X/Y expressed gene IL9R and a novel gene, CXYorf1, only 5 kb from the Xq telomere. These properties make Xq/YqPAR a model for studies of region-specific gene inactivation, telomere evolution, and involvement in sex-limited conditions.
Subject(s)
Proteins/genetics , Telomere/genetics , X Chromosome/genetics , Y Chromosome/genetics , Base Composition , Blotting, Southern , Cell Line , Chromosome Mapping , Chromosomes, Artificial, Yeast , Dosage Compensation, Genetic , Humans , In Situ Hybridization, Fluorescence , Intracellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Proteins/metabolism , R-SNARE Proteins , Repetitive Sequences, Nucleic Acid , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Telomere/metabolism , X Chromosome/metabolism , Y Chromosome/metabolismABSTRACT
SYBL1 is a gene in the 320kb human pseudo-autosomal region at the terminus of Xq and Yq. In contrast to other pseudoautosomal genes, SYBL1 is inactivated on one X in every female cell, and is also inactive on the Y of male cells. Hypermethylation of the CpG island associated with the human gene is involved in this phenomenon. In an attempt to further examine its regulation, the genomic organization of the X-linked mouse Sybl1 homolog was analyzed and compared with the human gene. Human and mouse show the same exon number, exon-intron junctions and a highly conserved basal promoter. The structural and functional conservation of basal regulatory regions suggests that inactivation is imposed by similar auxiliary epistatic regulatory mechanism.
