NIOSHTIC-2 Publications Search
RhoGAP domain is required for nuclear translocation of DLC-1 tumor supressor and the subsequent induction of apoptosis in human lung adenocarcinoma cell line NCI-H358.
Yuan-BZ; Jefferson-AM; Millecchia-L; Reynolds-SH
Regulation of Cell Death in Oncogenesis, Waikoloa, Hawaii, January 26-30, 2005. Philadelphia, PA: American Association for Cancer Research, 2005 Jan; :A15
The DLC-l gene, determined recently to be a tumor suppressor gene in human non-small cell lung carcinomas (NSCLC), contains a RhoGAP domain which exhibits in vitro ability to inactivate RhoA and Cdc42 by converting them from the GTP-bound form into the GDP-bound form. Restoration of DLC-l expression in NCI-H358, a NSCLC cell line with no intrinsic expression of DLC-l, induced the inhibition of in vitro cell growth and suppression of in vivo tumorigenicity. A unique change in cell morphology, manifested mainly as a significant cytoplasmic extension, was observed in our recent studies in DLC-l-transfected but not in vector alone transfected NCI-H358 cells. To further investigate the morphological changes, we transfected NCI-H358 cells with green fluorescent protein (GFP)-tagged DLC-l cDNA, and observed consequences of gene transfection with a laser scanning confocal microscope. Cells cultured on coverslips were placed in a stage-mounted microincubator, enabling observation of the same cells for up to 48 hours. Images were captured at 20 minute intervals. Our observations showed that after the initial appearance of gene products in the cytoplasm, about half of GFP positive cells started to form long cytoplasmic extensions, followed by the appearance of swellings, or blebs, along the extended cytoplasm. The cells at this time point showed dramatic reduction of formation of stress fibers and filopodia, two actin cytoskeleton structures evoked by active RhoA d Cdc42, respectively. Following the formation of cytoplasmic extensions and cell blebs, the gene products started to migrate into the nucleus. This process was accompanied by gradual cell shrinkage and decomposition. The decomposed cells exhibited nuclear condensation and/or fragmentation, a typical characteristic of apoptosis. The apoptosis in DLC-l transfected cells was also evidenced by the significant increase of caspase-3 activity compared to vector alone transfected cells. To determine if the RhoGAP domain of DLC-l is responsible for DLC-l mediated cell growth inhibition, we constructed both domain deleted and point mutated (at codon 677, 714, 718) DLC-l expression vectors and transfected each of them into NCI-H358 cells. It was found that these RhoGAP mutant DLC-ls significantly reduced the ability of wild type DLC-l to inhibit in vitro colony formation and cell proliferation. Furthennore, we found that these mutant DLC-l s tagged with GFP did not induce formation of cytoplasmic extensions and blebs and did not inhibit stress fiber and filopodia formation. In addition, we noted, no nuclear migration of gene products of these mutant DLC-l s and significantly reduced the number of cellular decomposition during the entire observation period. Moreover, RhoGAP domain point mutations significantly reduced the wild type DLC-l induced caspase-3 activity. Collectively, these data suggest that the inhibitory effect of DLC-l on tumor cell growth is contributed by DLC-l-induced cell apoptosis following specific cell morphological changes and nuclear migration of DLC-l, which requires the integrity of RhoGAP domain in the DLC-l gene.
Tumors; Tumorigens; Tumorigenesis; Cell-growth; Cell-cultures; Cell-damage; Oncogenesis; Oncogenicity
Abstract; Conference/Symposia Proceedings
Regulation of Cell Death in Oncogenesis, Waikoloa, Hawaii, January 26-30, 2005