[show abstract][hide abstract] ABSTRACT: The brain-specific angiogenesis inhibitor 1 gene has been isolated in an attempt to find fragments with p53 "functional" binding sites. As reported herein and by others, brain-specific angiogenesis inhibitor 1 expression is present in some normal tissues, but is reduced or lost in tumour tissues. Such data and its particular structure prompted the hypothesis that brain-specific angiogenesis inhibitor 1 may act as a mediator in the local angiogenesis balance. We herein demonstrate that brain-specific angiogenesis inhibitor 1 over-expression suppresses tumour angiogenesis, delaying significantly the human tumour growth in immunodeficient mice. The inhibitory effect of brain-specific angiogenesis inhibitor 1 was documented using our intravital microscopy system, strongly implicating brain-specific angiogenesis inhibitor 1 as a mediator in the control of tumour angiogenesis. In contrast, in vitro tumour cell proliferation was not inhibited by brain-specific angiogenesis inhibitor 1 transfection, whereas some level of cytotoxicity was assessed for endothelial cells. Immunohistochemical analysis of tumour samples confirmed a reduction in the microvessel density index in brain-specific angiogenesis inhibitor 1-overexpressing tumours. At messenger level, moderate changes could be detected, involving the down-regulation of vascular endothelial growth factor and collagenase-1 expression. Furthermore, brain-specific angiogenesis inhibitor 1 expression that was lost in a selection of human cancer cell lines could be restored by wild-type p53 adenoviral transfection. Brain-specific angiogenesis inhibitor 1 should be considered for gene therapy and development of efficient drugs based on endogenous antiangiogenic molecules.
British Journal of Cancer 03/2002; 86(3):490-6. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to control in vitro and in vivo expression of the growth hormone (GH) gene using a glucocorticoid-sensitive promoter, the mouse mammary tumor virus long terminal repeat (MMTV LTR). We inserted the cDNA encoding the 20-kDa form of human GH (20K-GH) downstream of the MMTV LTR of plasmid pMSG, and used lipofection to transfer it to 3Y1 cells together with plasmid pMX, which contains a puromycin-resistant element. The secretion of GH from the selected transformants was dose-dependently augmented by the application of hydrocortisone, corticosterone, or dexamethasone, among which dexamethasone was the most potent. Analysis of the time course showed that 20K-GH secretion began to increase within 2 hours after the addition of glucocorticoid and reached a maximal level of about threefold over the unstimulated control at 3 hours; secretion then gradually declined and returned to near basal levels at 19 hours. Repeated glucocorticoid application led to repeated increases in GH secretion. When GH-producing cells were microcapsulated and transplanted into the abdominal cavities of rats, 20K-GH was detected in the plasma under control conditions and increased about 3.3-fold after administration of dexamethasone. We suggest that GH expression driven by the MMTV LTR promoter may be under the control of an endogenous glucocorticoid in vivo.
[show abstract][hide abstract] ABSTRACT: In order to determine the parameters that govern the activity of a ribozyme in vivo, we made a systematic analysis of chimeric tRNAVal ribozymes by measuring their cleavage activities in vitro as well as the steady-state levels of transcripts, the half-lives of transcribed tRNAVal ribozymes, and their activities in both HeLa and H9 cells. These analyses were conducted by the use of transient expression systems in HeLa cells and stable transformants that express ribozymes. Localization of transcripts appeared to be determined by the higher-order structure of each transcribed tRNAVal ribozyme. Since colocalization of the ribozyme with its target RNA is important for strong activity of the ribozyme in vivo, the best system for tRNA-based expression seems to be one in which the structure of the transcript is different from that of the natural tRNA precursor so that processing of the tRNAVal ribozyme can be avoided. At the same time, the structure of the transcript must be similar enough to allow recognition, probably by an export receptor, so that the transcript can be exported to the cytoplasm to ensure colocalization with its target. In the case of several tRNAVal ribozymes that we constructed, inspection of computer-predicted secondary structures enabled us to control the export of transcripts. We found that only a ribozyme that was transcribed at a high level and that had a sufficiently long half-life, within cells, had significant activity when used to withstand a challenge by human immunodeficiency virus type 1.
Journal of Virology 04/1999; 73(3):1868-77. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The AML1 and PEBP2beta/CBFbeta genes encode the DNA-binding and non-binding subunits, respectively, of the heterodimeric transcription factor, PEBP2/CBF. Targeting each gene results in an almost identical phenotype, namely the complete lack of definitive hematopoiesis in the fetal liver on embryonic day 11.5 (E11.5). We examined and compared the expression levels of various hematopoiesis-related genes in wild type embryos and in embryos mutated for AML1 or PEBP2beta/CBFbeta. The RNAs were prepared from the yolk sacs of E9.5 embryos, from the aorta-gonad- mesonephros regions of E11.5 embryos and from the livers of E11.5 embryos and RT-PCR was performed to detect various gene transcripts. Transcripts were detected for most of the hematopoiesis-related genes that encode transcription factors, cytokines and cytokine receptors, even in tissues from homozygously targeted embryos. On the other hand, PU.1 transcripts were never detected in any tissue of AML1(-/-) or PEBP2beta/CBFbeta(-/-) embryos. In addition, transcripts for the Vav, flk-2/flt-3, M-CSF receptor, G-CSF receptor and c-Myb genes were not detected in certain tissues of the (-/-) embryos. The results suggest that the expression of a particular set of hematopoiesis-related genes is closely correlated with the PEBP2/CBF function.
[show abstract][hide abstract] ABSTRACT: We have constructed an allosterically controllable novel enzyme (designated maxizyme) that can be transcribed in vivo under the control of a human tRNA(Val) promoter. The maxizyme has sensor arms that can recognize target sequences, and in the presence of such a target sequence only, it can form a cavity that can capture catalytically indispensable Mg2+ ions. As a target for a demonstration of the potential utility of the maxizyme, we chose BCR-ABL mRNA, the translated products of which cause chronic myelogenous leukemia. Only the maxizyme (but not conventional ribozymes) had extremely high specificity and high-level activity, not only in vitro but also in cultured cells including BV173 cells derived from a patient with a Philadelphia chromosome. The maxizyme induced apoptosis only in leukemic cells with this chromosome.
[show abstract][hide abstract] ABSTRACT: The Pebpb2 gene encodes a non-DNA binding subunit of the heterodimeric transcription factor, polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF), and is rearranged in inversion of chromosome 16 associated with human acute myeloid leukemia. To investigate its physiological function, Pebpb2 was mutated by a targeting strategy to generate a null mutant. The homozygous mutation in mice proved lethal in embryos around embryonic day 12.5, apparently due to massive hemorrhaging in the central nervous system. In addition, definitive hematopoiesis in the liver was severely impaired. The observed phenotype was indistinguishable from that reported for homozygous disruption of AML1, which encodes a DNA binding subunit of PEBP2/CBF. Thus, the results indicate that the two subunits function together as a heterodimeric PEBP2/CBF in vivo and that PEBP2/CBF plays an essential role in the development of definitive hematopoiesis.
Proceedings of the National Academy of Sciences 06/1997; 94(11):5697-702. · 9.74 Impact Factor