Biomedical subjects
Publications and source records attributed to Chitra F Manohar.
Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.
Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,670 aligned transcripts, 19 transfer RNA genes, 341 pseudogenes and three RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukaemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. Whereas the segmental duplications of chromosome 16 are enriched in the relatively gene-poor pericentromere of the p arm, some are involved in recent gene duplication and conversion events that are likely to have had an impact on the evolution of primates and human disease susceptibility.
In the childhood cancer neuroblastoma (NB), the level of expression of the multidrug resistance-associated protein (MRP1) gene is strongly correlated with expression of the MYCN oncogene in primary NB tumors, suggesting that MRP1 may be a target for MYCN-mediated gene regulation. In this study, we show that MYCN induction in human NB cells results in increased MRP1 mRNA and protein levels, which in turn is accompanied by increased drug resistance and enhanced MRP1-mediated drug efflux. Furthermore, luciferase activity from MRP1 promoter/luciferase gene reporter constructs was significantly increased in NB cells with exogenous overexpression of MYCN, whereas activity was decreased in NB cells stably transfected with MYCN-antisense vectors. Decreased luciferase activity was observed with promoter constructs that lacked one or two E-box sequences or had E-box double point mutations, while a truncated MRP1 promoter lacking all three E-boxes exhibited only basal levels of activity. Specific electrophoretic mobility shifts of MRP1 E-box sequences were detected with nuclear extracts from NB cells with MYCN overexpression, and complex formation was inhibited with the addition of antibodies directed against MYCN or MYC. These findings indicate that by interacting with E-box elements within the promoter, MYCN can upregulate MRP1 expression and modulate drug resistance in NB.
MYCN amplification and consequent deregulated expression plays a crucial role in determining the clinical behavior of neuroblastoma. Enhanced expression of MYCN confers growth potential to neuroblastoma cells, and a direct link between MYCN expression and the development of neuroblastoma has been demonstrated in transgenic mice studies. Although the molecular pathways underlying the regulation of MYCN have not been fully elucidated, post-transcriptional mechanisms appear to be important. Previously, we reported that an embryonic lethal abnormal vision-like (ELAV) protein binds with high specificity to at least two AU-rich elements within the MYCN 3'-untranslated region. In this study, we characterized the ability of cis-acting elements within the MYCN 3'-untranslated region to destabilize mRNA in cells and examined the functional consequences of its interactions with the ELAV protein HuD. We show that at least 4 cis-acting elements within the MYCN 3'-untranslated region are able to signal the degradation of stable heterologous mRNA. Ectopic overexpression of HuD dramatically inhibits RNA decay mediated by the full-length MYCN 3'-untranslated region and cis-acting destabilizing elements that harbor HuD binding sites in vivo. HuD may contribute to the malignant phenotype of neuroblastoma cells by stabilizing MYCN mRNA, thereby enhancing steady-state levels of expression of this oncogene.