Michael E. Berens

DICHOTOMY OF ASTROCYTOMA MIGRATION AND PROLIFERATION

Astrocytomas often show high rates of local invasion that lead to local recurrence of the disease. Histologically, the most highly invasive astrocytoma cells are detected in isolation rather than as nests of tumor. Our study attempted to determine whether the migratory response to extracellular substrates influences the proliferative behavior of these highly invasive cells. The preferential and specific migratory response of human astrocytoma cells to extracellular matrix proteins was assessed by a microliter scale migration assay. Growth curve studies on protein ligands permissive (merosin) for cell migration indicated that the lag phase was protracted compared with cells seeded on non‐permissive proteins (vitronectin). Once a certain cell density was reached, logarithmic proliferation was indistinguishable on the different proteins. The proliferation index of populations of cells migrating on merosin and vitronectin was measured by both BrdU incorporation and MIB‐I immunocytochemistry labeling. Cells seeded on vitronectin showed higher proliferation throughout the population than cells seeded on merosin. On merosin, the more migratory cells at the periphery were less proliferative than non‐migratory cells in the central region of that population. The integrin‐associated signal transduction protein, p125FAK, was heavily localized in the membrane of non‐migrating cells and largely absent in migrating astrocytoma cells. We conclude that temporally, proliferation and migration are mutually exclusive behaviors. Cell density or non‐permissive substrates that inhibit cell motility favor a more proliferative phenotype. Conversely, active migration suppresses cell proliferation.

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 5700 and 7000 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), PLCγ, and CD44, were over expressed; other genes, like adducin 3γ 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.

Migration of Human Glioma Cells on Myelin

Histoanatomically invading astrocytoma cells appear to migrate along distinct structures within the brain. Astrocytoma invasion may occur along extracellular matrix (ECM) protein-containing structures, such as blood vessels, but most frequently occurs along tracts of myelinated fibers. This behavior most likely is a consequence of the use of constitutive extracellular ligands expressed along the pathways of preferred dissemination. Enzymatic modification of the extracellular space or deposition of ECM by the tumor cells may also create a more permissive environment. Established human glioma cell lines and two preparations of primary cells isolated from glioblastoma biopsies were studied with the use of cell adhesion and monolayer migration assays to investigate whether crude human central nervous system myelin extracts present specific cell adhesion ligands that promote glioma attachment and cell migration. Two cell lines showed high levels of adhesion and migration on central nervous system myelin similar to levels of migration on the ECM protein merosin, which has previously been shown to be a highly permissive substrate for cultured astrocytoma cells. Two other cell lines showed lower but specific migratory response; one cell line did not attach or specifically migrate on crude myelin extracts. For both glioblastoma primary cell preparations, myelin and merosin were the most permissive substrates for attachment and migration. Other ECM proteins (collagen type IV, fibronectin, and vitronectin) were moderate or nonpermissive substrates. Our findings indicated that astrocytoma cells may be able to use oligodendrocyte membrane-associated ligands as well as ECM proteins of the basement membranes for invasion of normal brain.

Substrates for astrocytoma invasion

A better understanding of the influences of specific extracellular substrates, including proteins, glycosaminoglycans, and parenchymal cells, on the invasive behavior of glioma cells would potentially lead to novel forms of treatment aimed at confining the tumor. A monolayer, microliter scale assay was used to investigate how different substrates influenced glioma migration. Basal or unspecific movement (range, 10-260 microns/d) was determined by observing a panel of seven established human glioma cell lines. Migration rates two to five times higher than this basal activity were referred to as preferential and specific glioma migration; these rates generally occurred on merosin and tenascin. Collagen, fibronectin, or vitronectin were less supportive of migration. The glioma cells migrated on hyaluronic acid, but they did not migrate to the extent generally found on the extracellular matrix proteins. Glioma-derived extracellular matrix also served to promote cell migration. This finding implicates a role for either glioma remodeling or synthesis of a permissive environment for local dissemination that may be independent of the constitutive matrix proteins normally found in the brain. Although the glioma cells were able to migrate over monolayers of other glioma cells, they were unable to migrate over astrocytes and fibroblasts. Our findings indicate that the invasive behavior of glioma cells in situ is most likely a consequence of the interplay between the cells manipulation of the environment and the constitutive ligands associated with specific regions or structures of the brain.

The role of cultured Schwann cell grafts in the repair of gaps within the peripheral nervous system of primates

With recent advances in cell culture techniques it is possible to isolate human SCs from adult peripheral nerves, expand and purify their number in cell culture, and construct a cellular prosthesis from the cultured cells. The current study was designed to ascertain whether these techniques could be used to repair nonhuman primate nerve injuries. In 12 adult female cynomologous monkeys, the musculocutaneous (msk) nerve was divided and prevented from regenerating and the brachioradialis nerve (brach) was exposed bilaterally (n = 24 nerves) and injured so that a 15-mm gap existed within the nerve. The brach nerves were either repaired with sural nerve autografts (n = 6), guidance channels which contained monkey SCs (120 x 10(6) cells/ml; n = 6), or guidance channels without SCs (n = 6). The remaining brach nerves (n = 6) had either no injury or an injury to the nerve without a repair. Autologous expanded primate SCs were increased in number at least 10-fold over a 2-week period at which time the SC purity exceeded 99.9%. Monkeys in each group, including the control group, regained some degree of elbow flexion after 3 months despite sectioning both the mask nerve and the brach nerve; therefore, we were unable to determine simply on clinical grounds which repair was the most effective in promoting functional recovery. Brach nerves repaired with sural nerve grafts were superior to both the channels which contained SCs and empty channels in regards to the number of myelinated axons proximal, within, and distal to the repair site (P < 0.05). Electrophysiologic results closely paralleled the histologic data with evidence of reinnervation of the brachioradialis muscle based on the compound muscle action potential in both sural nerve graft and monkey SC channel repair groups.

Altered Gene Expression in Human Astrocytoma Cells Selected for Migration: I. Thromboxane Synthase

Human glioma cells from a long-term cell line were selected for their ability to migrate on a glioma-derived extracellular matrix. When tested over 28 serial passages, the migration-selected strain showed a genetically stable, enhanced migration rate compared with the parental cells. Proliferation studies demonstrated that the growth rate of migration-selected cells was slightly arrested. Both the selected strain and the parental culture showed anchorage-independent growth in soft agarose and were tumorigenic in athymic mice. Using molecular genetic strategies display to isolate genes expressed differentially between the 2 populations, a 300-bp sequence homologous to thromboxane synthase was upregulated in the migration-selected cells relative to the parental cells. Expression levels of thromboxane synthase were highly elevated in the migration-selected cells when assessed by RNAse-protection assay and by flow cytometry. Two specific thromboxane synthase inhibitors, Dazmegrel and Furegrelate, reduced the migration rate of the migration-selected cells to a rate equal to or less than the rate exhibited by the parental cells, respectively. The inhibitors effect on the parental cells was inconsequential. These results suggest that aberrations in the regulation of thromboxane synthase expression or activity may influence the motility of human glioma cells.

Proliferation and motility responses of primary and recurrent gliomas related to changes in epidermal growth factor receptor expression

Astrocytic neoplasms show a high incidence of elevated or mutated epidermal growth factor receptor (EGFR) expression. Although proliferative effects from EGFR activation are well described, the role that changes in this receptor play in glioma growth and migration remain poorly addressed. This report characterizes changes in the levels of EGFR expression in three glial tumors at initial presentation (resection) and at the time of recurrence. By quantitative flow cytometry the mean level of EGFR expression increased, decreased, or remained the same in different recurrent astrocytomas relative to their primary tumor cells. Immunocytochemistry for EGFR on monolayer cells corroborated the level of expression in the recurrent tumors relative to their matched primary specimen. Immunoprecipitation indicated that 170 kd EGFR was expressed in each of the tumors, and showed normal down regulation following treatment with EGE Proliferation response to EGF was seen in only 1/6 instances, but was concentration-dependent when observed. Stimulated migration of the cells was frequently seen and was also concentration-dependent on EGF; the magnitude of response was related to the relative level of 170 kd EGFR expression in the cells. EGFR immunostaining of tissue sections from the tumors confirmed the levels of EGFR expressed in primary and recurrent astrocytomas as was seen in the cultured cells. These results indicate that the relative levels of EGFR in early passage cell cultures from glioma specimens concurs with the measured tissue levels of expression. Human glioma cells are more responsive to migration induction than proliferation induction by EGF.

Migration arrest in glioma cells is dependent on the αv integrin subunit

Local invasion is a hallmark of gliomas. Infiltrating tumor cells establish sites of persistent and recurrent lesions that ultimately prove fatal. Determinants of glioma cell migration include integrins and their ligands within the matrix. In contrast to the response to other matrix proteins, glioma cells migrating on tenascin do not follow a characteristic dose‐dependent pattern. For two of four glioma cell lines tested, tenascin acts as both a permissive and a nonpermissive motility substrate, i.e., low densities of tenascin are permissive substrates, whereas high densities are nonpermissive. Specific antisense oligonucleotides directed at the αv integrin subunit effectively suppress the anti‐migratory phenotype of glioma cells at high tenascin densities. The two cell lines that fail to demonstrate this unusual biphasic pattern do not endogenously express the αv subunit, whereas the cell lines for which high densities of tenascin are anti‐migratory are found to express αv. We conclude that loss of the αv integrin subunit may be associated with the invasive behavior of gliomas, along vascular channels that express tenascin. GLIA 24:236–243, 1998.

Interferon-β inhibits proliferation and progression through S phase of the cell cycle in five glioma cell lines

SummaryThe growth inhibitory effect of IFN-β was evaluated in 5 human glioma cell lines (AO2V4, GJC, GJR, NN and NNR) and in normal astrocyte cultures (SC and TM). All 5 glioma cell lines showed an anti-proliferative response to IFN-β whereas normal glial cells were non-responsive. IFN-β at 10, 100 and 500 U/ml lead to a 30%,70% and 80% relative decrease in cell number after 12 days, respectively in AO2V4 cells. GJC and GJR cell lines also responded significantly to the lowest concentration of IFN-β tested and at 500 U/ml the relative cell number decreased 55%. The NN and NNR cells were the least responsive to IFN-β with maximum growth inhibition of 30% at 500 U IFN-β/ml. Following treatment with IFN-β, AO2V4, GJC, GJR and normal astrocytes all expressed mRNA encoding the anti-viral protein, 2-5A synthetase demonstrating that IFN-β bound to receptors on all four cell lines and activated signal transduction pathways required for induction of an anti-viral protein. A determination of the relative number of viable cells showed that none of these cells exhibited a significant decrease in cell viability. Since the antiproliferative response to IFN-β was not primarily due to cell death, the effect of IFN-β on cell cycle progression was evaluated by flow cytometry. All treated glioma cell lines showed a relative increase in proportion of cells in S phase. AO2V4 cells had a 50%–80% increase in the percentage of cells in S phase, whereas GJC, GJR and NNR had percentage increases of 20%–40%. IFN-β treatment of normal astrocytes did not significantly alter their cell cycle profile. These data suggest that IFN-β exerts its antiproliferative effect on glioma cells by arresting the ordered progression through S phase or decreasing entry into G2/M phase of the cell cycle.

Tumorigenic, invasive, karyotypic, and immunocytochemical characteristics of clonal cell lines derived from a spontaneous canine anaplastic astrocytoma

SummaryTumor cells from a spontaneously arising canine astrocytoma were isolated and cloned. Three clonally derived cell lines (DL3580 clone 1, DL3580 clone 2, and DL3580 clone 3) were developed and found to express glial fibrillary acidic protein (GFAP) as well as epidermal growth factor receptor (EGFR/c-erbB1). The cell lines were tumorigenic as subcutaneous xenografts or as intracranial implants in athymic mice, or both. Both the monolayer astrocytoma cells and the xenograft tumor cells from clone 2 were aneuploid, with a modal number of 84 chromosomes per metaphase; clones 1 and 3 were also aneuploid with modal numbers of 82 and 75/79, respectively. The histology of both the initial spontaneously occurring tumor in the dog and the intracranial astrocytoma in athymic mice demonstrated features of diffuse infiltration into normal brain. These newly developed canine glioma cell lines are karyotypically stable for 1 yr in culture and carry the same marker chromosomes as the parental lines. These glioma cell lines may serve as models for investigating mechanisms of glioma invasion into brain. Additionally, clonal cell lines with divergent properties isolated from the same tumor may assist in studies of the molecular basis of astrocytoma progression and heterogeneity.