RESUMO
Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings. Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity. Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t(14;18). Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis. Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells. It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.
Assuntos
Linfócitos B/metabolismo , Membrana Celular/metabolismo , Leucemia de Células B/patologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apoptose , Linfócitos B/patologia , Membrana Celular/ultraestrutura , Células Cultivadas , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfoma de Células B/metabolismo , Microscopia Confocal , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Interleukin-10 (IL10), an anti-inflammatory cytokine, has been implicated in a variety of immune- and inflammatory-related diseases. We investigated the following SNPs: -1082, -819, -592 in the promoter region of IL10 in a normal (control) population and selected diseases: breast cancer (BrCa), systemic lupus erythematosus (SLE), and B-cell chronic lymphocytic leukemia (B-CLL) by denaturing high-performance liquid chromatography (DHPLC) and found distinct genotype and haplotype patterns. DHPLC was performed using the Transgenomic WAVE instrument, a mutational discovery tool that allows for high throughout analysis of SNPs. The principle of DHPLC is based on separation of homo- and heteroduplex formation of individual polymerase chain reaction products at specific melting temperatures and set gradients. The melting temperature selected for each SNP was based on size and sequence of the polymerase chain reaction product (for -1082, 57 degrees C; for -819, 58 degrees C; and for -592, 59.2 degrees C). Before fragment mutational analysis, all samples were denatured at 95 degrees C and slowly reannealed to allow for reassociation of different strands. Heteroduplex samples were easily distinguished from homoduplex samples. In order to identify wild type from homozygous mutant, two homoduplex polymerase chain reaction samples had to be mixed together, denatured at 95 degrees C and reannealed. The homozygous mutant, when combined with wild type, displayed a double peak on chromatogram. Once distinct chromatograms were established for each of the SNPs and the nucleotide changes confirmed by sequencing, genotype and haplotype frequencies were tabulated for the groups studied.