RNA干扰沉默缺氧诱导因子1α逆转肺癌细胞耐药性

目的：观察RNA干扰沉默缺氧诱导因子1α（HIF-1α）对肺癌细胞耐药性的影响。方法：构建靶向HIF-1α小干扰RNA基因,并转染到人肺腺癌耐顺铂细胞株A549／DDP细胞中。逆转录聚合酶链反应RT—PCR）检测细胞的HIF-1α、多药耐药基因-（MDR-1）以多药耐药相关蛋白基因（MRP）mRNA变化,免疫细胞化学法观察干扰后HIF-1α、P-糖蛋白以及MRP蛋白的变化。MTT法检测不同浓度的顺铂作用下细胞死亡率。结果：HIF-1αsiRNA组中H1F-1α、MDR—1、MRPmRNA水平显著降低（P〈0．05）。且蛋白水平也显著下降（P〈0．05）。HIF-1αsiRNA组细胞死亡率较未转染组均明显增高（P〈0．05）,转染siRNA阴性组不影响肿瘤细胞的耐药性。结论：HIF-1αsiRNA可显著降低A549／DDP细胞中H1F-1α、MDR-1、MRP表达,从而起到逆转肺腺癌A549／DDP细胞的耐药作用。     

Transient Expression of MDR-1/P-Glycoprotein in a Model of Partial Cortical Devascularization

1. MDR-1 gene product confer to expressing cells the multidrug resistance phenotype to a broad range of drugs and xenobiotics. 2. It is known that different stress signals are able to induce MDR-1 expression through different promoters. 3. In a rat model of ischemia by partial cortical devascularization we studied the expression profile and the cellular localization of MDR-1 after 1, 3, 7, 14 and 28 days post lesion (DPL). 4. Using two different antibody clones we found that MDR-1 is expressed in cortical and striatal neurons ipsilateral to the devascularizing lesion, starting at 1DPL, showing a maximum at 7DPL to be thereafter reduced until undetectable levels by 28DPL. 5. MDR-1 expression may be defining a neuronal subset with a particular pharmacological profile.     

PO(2)-dependent differential regulation of multidrug resistance 1 gene expression by the c-Jun NH2-terminal kinase pathway

Hypoxia-induced multidrug resistance 1 (MDR1) gene expression is known to be mediated by c-Jun NH(2)-terminal kinase (JNK) activation. However, the molecular mechanisms underlying this action of JNK remain elusive. On the contrary, there has been increasing evidence for a negative correlation of JNK activity with MDR1 expression under normoxic conditions. Here, we present evidence that the JNK pathway represses MDR1 expression in normoxia and activates MDR1 expression in hypoxia. Our data show that JNK pathway-induced MDR1 repression in normoxia is mediated by increased c-Jun binding to activator protein 1 site, located in the MDR1 promoter, and requires the activity of histone deacetylase 5. In contrast, JNK pathway-induced MDR1 activation in hypoxia is independent of the activator protein 1 site. Rather, this action is dependent on increased hypoxia-inducible factor 1 (HIF1) binding to the hypoxia response element in the MDR1 promoter, which is promoted by the interaction of HIF1alpha with c-Jun in the nucleus and requires the activity of the p300/CBP (CREB-binding protein) coactivator.     

Cytogenetic and FISH findings are complementary in childhood ALL

Primary genetic abnormalities of leukemia cells have important prognostic significance in childhood acute leukemia. In the last two years 30 newly diagnosed or recurrent childhood ALL bone marrow samples were analyzed for chromosomal abnormalities with conventional G-banding and interphase-fluorescence in situ hybridization (I-FISH) using probes to detect BCR/ABL fusions, cryptic TEL/AML1 and MLL rearrangements and p16(9p21) tumor suppressor gene deletions. G-banded karyotype analysis found clonal chromosomal aberrations in 50% of cases. With the use of complementary I-FISH techniques, ALL-specific structural and numerical changes could be identified in 70% of the patients. Nine cases (30%) had subtle chromosomal aberrations with prognostic importance that had not been detected in G-banded analysis. Conventional G-banding yielded additional information (rare and complex structural aberrations) in 19% of patients. The most common aberration (30%) was AML1 copy number increase present in G-banded hyperdiploid karyotype as a chromosome 21 tetrasomy in the majority of cases; one case displayed 5-6 copies and in another case amplification of AML1 gene on der(21) was combined with TEL/AML1 fusion of the homologue AML1 gene and deletion of the remaining TEL allele. High hiperdiploidy was detected in 6 cases, in one patient with normal G-banding karyotype. TEL/AML1 fusion signals were identified in four patients. Deletion of p16 locus was found in eight cases (23%), of which only two had cytogenetically visible rearrangements. G-banding in combination with I-FISH has produced major improvements in the sensitivity and accuracy of cytogenetic analysis of ALL patients and this method helps to achieve a more precise identification of different risk categories in order to choose the optimal treatment.     

RNA helicase A in the MEF1 transcription factor complex up-regulates the MDR1 gene in multidrug-resistant cancer cells

RNA helicase A (RHA) is a member of the DEAD/H family of RNA helicases and unwinds duplex RNA and DNA. Recent studies have shown that RHA regulates the activity of gene promoters. However, little information is available about the in vivo relevance of RHA in the regulation of natural genes. We previously characterized a nuclear protein (MEF1) that binds to the proximal promoter of the multidrug resistance gene (MDR1) and up-regulates the promoter activity. In the present study, we isolated and identified RHA as a component of the MEF1 complex by using DNA-affinity chromatography and mass spectrometry. The antibody against RHA specifically disrupted the complex formation in electrophoretic mobility shift assay, confirming the identity of RHA. Western blotting showed that RHA in drug-resistant cells had a higher molecular weight than that in drug-sensitive cells. Similar results were obtained when FLAG-tagged RHA was overexpressed in these cells. This size difference probably reflects posttranslational modification(s) of RHA in drug-resistant cells. Chromatin immunoprecipitation revealed that RHA occupies the MDR1 promoter in vivo. Overexpression of RHA enhanced expression of the MDR1 promoter/reporter construct and endogenous P-glycoprotein (P-gp), the MDR1 gene product, and increased drug resistance of drug-resistant cells but not the drug-sensitive counterpart. Introduction of short interfering RNA targeting the RHA gene sequence selectively knocked-down RHA expression and concomitantly reduced P-gp level. Thus, our study demonstrates, for the first time, the involvement of RHA in up-regulation of the MDR1 gene. Interactions of RHA with other protein factors in the MEF1 complex bound to the promoter element may contribute to P-gp overexpression and multidrug resistance phenotype in drug-resistant cancer cells.