Glioma cell motility is associated with reduced transcription of proapoptotic and proliferation genes: a cDNA microarray analysis.

Microarray analysis of complementary DNA (cDNA) allows large-scale, comparative, gene expression profiling of two different cell populations. This approach has the potential for elucidating the primary transcription events and genetic cascades responsible for increased glioma cell motility in vitro and invasion in vivo. These genetic determinants could become therapeutic targets. We compared cDNA populations of a glioma cell line (G112) exposed or not to a motility-inducing substrate of cell-derived extracellular matrix (ECM) proteins using two sets of cDNA microarrays of 5,700 and 7,000 gene sequences. The data were analyzed considering the level and consistency of differential expression (outliers) and whether genes involved in pathways of motility, apoptosis, and proliferation were differentially expressed when the motility behavior was engaged. Validation of differential expression of selected genes was performed on additional cell lines and human glioblastoma tissue using quantitative RT-PCR. Some genes involved in cell motility, like tenascin C, neuropilin 2, GAP43, PARG1 (an inhibitor of Rho), PLCy, and CD44, were over expressed; other genes, like adducin 3y and integrins, were down regulated in migrating cells. Many key cell cycle components, like cyclin A and B, and proliferation markers, like PCNA, were strongly down regulated on ECM. Interestingly, genes involved in apoptotic cascades, like Bcl-2 and effector caspases, were differentially expressed, suggesting the global down regulation of proapoptotic components in cells exposed to cell-derived ECM. Overall, our findings indicate a reduced proliferative and apoptotic activity of migrating cells. cDNA microarray analysis has the potential for uncovering genes linking the phenotypic aspects of motility, proliferation, and apoptosis.

Death-associated protein 3 (Dap-3) is overexpressed in invasive glioblastoma cells in vivo and in glioma cell lines with induced motility phenotype in vitro.

PURPOSE: To discover the genetic determinants of glioma invasion in vivo, we compared the mRNA expression profiles of glioblastoma cells residing at the tumor core versus those at the invasive rim of a human tumor resection. EXPERIMENTAL DESIGN: From a single glioblastoma specimen, 20,000 individual cells from each region (core and invasive rim) were collected by laser capture microdissection and analyzed by mRNA differential display. Differential expression of gene candidates was confirmed by laser capture microdissection and quantitative reverse transcription-PCR in additional glioblastoma multiforme specimens, and the role in migration was further evaluated in glioma cell lines in vitro. RESULTS: Reproducible overexpression the death-associated Protein 3 (Dap-3) mRNA (NM 004632, GenBank; also reported as human ionizing resistance conferring protein mRNA, HSU18321, GenBank) by invasive cells was identified. Although the full-length Dap-3 protein has been described as proapoptotic, the NH(2)-terminal fragment can act in a dominant negative way resulting in protection from programmed cell death. In glioma cell lines T98G and G112 with an induced motility phenotype, Dap-3 was up-regulated at the mRNA and protein level as assessed by quantitative reverse transcription-PCR, cDNA microarray, and Western blot analysis. These cells showed an increased resistance to undergo camptothecin-induced apoptosis, which was overcome by effective Dap-3-antisense treatment. Antisense treatment also decreased the migration ability of T98G cells. CONCLUSIONS: Dap-3 is up-regulated in invasive glioblastoma multiforme cells in vivo and in glioma cells with an induced motility phenotype in vitro. When migration is activated, Dap-3 is up-regulated and cells become resistant to apoptosis. These findings suggest that Dap-3 confers apoptosis-resistance when migration behavior is engaged.

Identification and validation of P311 as a glioblastoma invasion gene using laser capture microdissection.

The mRNA expression profiles from glioblastoma cells residing at the tumor core and invasive rim of a human tumor resection were compared. From a single tumor specimen, 20,000 single cells from each region were collected by laser capture microdissection. Differential expression of 50-60 cDNA bands was detected. One of the sequences overexpressed by the invasive cells showed 99% homology to the P311 gene, the protein product of which is reported to localize at focal adhesions. Relative overexpression of P311 by invading glioblastoma cells compared with tumor core was confirmed by quantitative reverse transcription-PCR of six glioblastoma specimens after laser capture microdissection collection of rim and core cells. In vitro studies using antisense oligodeoxynucleotides and integrin activation confirmed the role of P311 in supporting migration of malignant glioma cells. Immunochemistry studies confirmed the presence of the P311 protein in tumor cells, particularly at the invasive edge of human glioblastoma specimens.

Gap junction intercellular communication in gliomas is inversely related to cell motility.

Gliomas are lethal because of local invasion into brain parenchyma. Glioma cells were isolated from different regions (white matter, gray matter and tumor core) of a glioma-bearing dog brain. Individual clonal cell lines were established from each area, and characterized for growth, migration and gap junctions. The regional clonal cell lines differed in rates and preferred substrate for migration. Cell lines generated from invaded white matter showed stimulated migration on collagen and variable migration on merosin, whereas migration of cell lines derived from invaded gray matter showed the reciprocal responses: stimulation on merosin and inhibition on collagen. Gap junctional communication showed significant degrees of variation between the different clones. A direct inverse relationship between the number of cells demonstrating gap junctional communication and migration rate of cells away from multicellular spheroids was evident. Glioma cells which have a reduced capacity to connect to each other have an accelerated migration rate onto autologous, glioma-derived matrix. These results suggest that invasive glioma cells suppress autologous cell-to-cell cohesion, partly evident as reduced formation of gap junctions. In addition, glioma cells were stimulated to migrate in a dose-dependant manner in response to epidermal growth factor (EGF) coincident with the reduction of Cx43 levels and increased serine phosphorylation. We speculate that in order for glioma cells to invade locally into brain parenchyma they must first detach from neighboring cells ("let go...let's go" paradigm of invasion).