Twenty-four base pairs of the human antioxidant response element (hARE) are required for high basal transcription of the NAD(P)H:quinone oxidoreductase 1 (NQO 1 ) gene and its induction in response to xenobiotics and antioxidants. hARE is a unique cis-element that contains one perfect and one imperfect AP1 element arranged as inverse repeats separated by 3 bp, followed by a ''GC'' box. We report here that Jun, Fos, Fra, and
The transcription factor Pax6 plays a key role during development of various organs, including the brain where it affects cell fate, cell proliferation and patterning. To understand how Pax6 coordinates these diverse effects at the molecular level, we examined the role of distinct DNA-binding domains of Pax6, the homeodomain (HD), the paired domain (PD) and its splice variant (5a), using loss- and gain-of-function approaches. Here we show that the PD is necessary for the regulation of neurogenesis, cell proliferation and patterning effects of Pax6, since these aspects are severely affected in the developing forebrain of the Pax6Aey18 mice with a deletion in the PD but intact homeo- and transactivation domains. In contrast, a mutation of the HD lacking DNA-binding (Pax64Neu) resulted in only subtle defects of forebrain development. We further demonstrate distinct roles of the two splice variants of the PD. Retrovirally mediated overexpression of Pax6 containing exon 5a inhibited cell proliferation without affecting cell fate, while Pax6 containing the canonical form of the PD lacking exon 5a affected simultaneously cell fate and proliferation. These results therefore demonstrate a key role of the PD in brain development and implicate splicing as a pivotal factor regulating the potent neurogenic role of Pax6.
NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that catalyzes two-electron reductive metabolism and detoxification of quinones and their derivatives leading to protection of cells against redox cycling and oxidative stress. To examine the in vivo role of NQO1, a NQO1-null mouse was produced using targeted gene disruption. Mice lacking NQO1 gene expression showed no detectable phenotype and were indistinguishable from wild-type mice. However, NQO1-null mice exhibited increased toxicity when administered menadione compared with wild-type mice. These results establish a role for NQO1 in protection against quinone toxicity. The NQO1-null mice are a model for NQO1 deficiency in humans and can be used to determine the role of this enzyme in sensitivity to toxicity and carcinogenesis.
Medulloblastoma is the most malignant pediatric brain tumor. It is believed to originate from the undifferentiated external granule layer cells in the cerebellum, but the mechanism of tumorigenesis remains unknown. Here we studied three types of human medulloblastoma cells that express markers corresponding to different levels of neuronal differentiation. They expressed the neuronal repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF; refs. 7-10) at very high levels compared with either neuronal progenitor NTera2 (NT2) cells or fully differentiated human neuron teratocarcinoma (hNT cells). To counter the effect of REST/NRSF, we used a recombinant transcription factor, REST-VP16, constructed by replacing repressor domains of REST/NRSF with the activation domain of viral protein (VP16). Transient expression of REST-VP16 in medulloblastoma cells was able to compete with the endogenous REST/NRSF for DNA binding and stimulate neuronal promoters. High-efficiency expression of REST-VP16 mediated by adenovirus vectors (Ad.REST-VP16) in medulloblastoma cells was able to counter REST/NRSF-mediated repression of neuronal promoters, stimulate expression of endogenous neuronal genes and trigger apoptosis through the activation of caspase cascades. Furthermore, intratumoral injection of Ad.REST-VP16 in established medulloblastoma tumors in nude mice inhibited their growth. Therefore, REST/NRSF may serve as a new target for therapeutic interventions for medulloblastoma through agents such as REST-VP16.
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