The most severe form of brain glioma, glioblastoma (GBM), is highly malignant and usually resistant to chemotherapy. Therefore, discovery of new targets for gene therapy is important. Using subtraction cloning, we identified the human N-Myc downstream-regulated gene 2 (hNDRG2), located at chromosome 14q11.2, as a gene that is significantly suppressed in GBM tissues. Semiquantitative RT-PCR showed that the hNDRG2 gene transcript is expressed in normal brain tissue and low-grade gliomas but is present at low levels in 15 of 27 (56%) human GBM tissues and all of the 6 human glioblastoma cell lines examined. Furthermore, transfection of human glioblastoma U373 and U138 cells with a cDNA encoding hNDRG2 markedly reduced the cell proliferation. Our findings provide the first evidence to suggest that hNDRG2 may play a role in glioblastoma carcinogenesis.
Antisense therapy is limited in application in clinical therapy because of two existing problems: rapid degradation of antisense nucleic acids and poor diffusion across the cell membrane. Here we report the use of single-walled carbon nanotubes as delivery system for transporting antisense myc into HL-60 cells. Antisense-myc-conjugated single-walled carbon nanotubes were synthesized, characterized by atomic force microscopy (AFM), fluorescent microscopy, and Raman spectroscopy. Incubated with HL-60 cells at 37 degrees C, the single-walled carbon nanotubes (SWNTs) inside HL-60 cells were measured quantitatively by HPLC. Expression of c-myc gene and protein were analyzed by reverse transcriptase PCR and Western blot, respectively. Results showed that as-myc-conjugated SWNTs dropped character peaks at quadruple wavenumber (cm(-1)) compared with SWNTs, and could enter into HL-60 cells within 15 min after incubation, whose uptake amounts by HL-60 cells increased as the incubation time increased. After 48 hours, the amount of SWNTs in HL-60 cells began to decrease. Compared with as-myc and SWNTs, as-myc-conjuagted SWNTs exhibited the strongest inhibition on the proliferation of HL-60 cells, induced cell apoptosis, and down-regulated lowest expression of c-myc gene and C-MYC protein. The SWNTs can directly deliver as-myc into HL-60 cells, enhance the inhibition of as-myc on HL-60 cells, and is likely a better delivery system for antisense therapy. Antisense modified SWNTs can be used for intracellular gene regulation with potential applications in tumor therapy and drug delivery.
α1-Antitrypsin (AT) is a major elastase inhibitor within the lung. Oxidation of critical methionine residues in AT generates oxidized AT (Ox-AT), which has a greatly diminished ability to inhibit neutrophil elastase. This process may contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD) by creating a functional deficiency of AT permitting lung destruction. We show here that Ox-AT promotes release of human monocyte chemoattractant protein-1 (MCP-1) and IL-8 from human lung type epithelial cells (A549) and normal human bronchial epithelial (NHBE) cells. Native, cleaved, polymeric AT and secretory leukoproteinase inhibitor (SLPI) and oxidized conformations of cleaved, polymeric AT and SLPI did not have any significant effect on MCP-1 and IL-8 secretion. These findings were supported by the fact that instillation of Ox-AT into murine lungs resulted in an increase in JE (mouse MCP-1) and increased macrophage numbers in the bronchoalveolar lavage fluid. The effect of Ox-AT was dependent on NF-κB and activator protein-1 (AP-1)/JNK. These findings have important implications. They demonstrate that the oxidation of methionines in AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the antielastase lung protection, but also converts AT into a proinflammatory stimulus. Ox-AT generated in the airway interacts directly with epithelial cells to release chemokines IL-8 and MCP-1, which in turn attracts macrophages and neutrophils into the airways. The release of oxidants by these inflammatory cells could oxidize AT, perpetuating the cycle and potentially contributing to the pathogenesis of COPD. Furthermore, these data demonstrate that molecules such as oxidants, antiproteinases, and chemokines, rather than act independently, are likely to interact to cause emphysema.
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