Joan Rankin Shapiro

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.

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.

Genetics of nervous system tumors.

Genetic aberrations are being defined for the various glial tumors. Astrocytic tumors can evolve by two different pathways. The genetic aberrations now being defined for these two pathways are different and can be associated with the grade of malignancy. In oligodendrogliomas, the genetic lesions differ from the astrocytic tumors, and several markers have been linked to chemosensitivity response and survival. Genetic aberrations in ependymomas also differ from the astrocytic tumors or oligodendrogliomas. Although additional cases are needed to study the genetic aberrations, the abnormalities identified suggest that spinal cord tumors carry different markers than intracranial tumors and that the markers within the cranium may be different based on their location.

BCNU-sensitivity in parental cells and clones from four freshly resected near-diploid human gliomas: an astrocytoma, an anaplastic astrocytoma and two glioblastomas multiforme.

SummaryWe compared the BCNU sensitivity of 4 freshly resected tumors (astrocytoma WM, and malignant gliomas MK, MB, and AM) and their clones to their karyology. The majority of primary cells in all 4 tumors had near-diploid chromosome numbers (2n±) and all were resistant to concentrations of BCNU exceeding 10μg/ ml. Followingin vitro cultivation, the cells from tumors WM and MB retained their 2n ± modal chromosome number with little change in the complexity of the karyotype. In contrast, tumors MK and AM demonstrated a more unstable genome. The modal chromosome number of MK shifted from 45 to 86 and that of tumor AM from 45 to 90. Karyotyping demonstrated additional ploidy changes and new marker chromosomes in both tumors. The colony forming assay (CFA) performed on thein vitro cultivated cells demonstrated little change in the sensitivity to BCNU in tumors WM and MB, while tumors MK and AM exhibited greater than a one log cell kill at 10.0μg/ml and 15.0μg/ml BCNU, respectively. The modal chromosome number and BCNU sensitivity followed a similar pattern in the 30 clones that were isolated; 21 clones with near-diploid and pseudodiploid chromosome numbers were all resistant to BCNU doses at or greater than 10μ/ml. In contrast, 9 clones isolated from the 3 malignant gliomas with 3n ± and 4n ± modal chromosome numbers were sensitive to this concentration of BCNU. The karyotypes of the hyperdiploid clones were more complex; they contained 5 or more ploidy changes and/or had marker chromosomes. These studies confirm the association of diploidy and BCNU-resistance in freshly resected malignant gliomas.

Over-representation of specific regions of chromosome 22 in cells from human glioma correlate with resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea

BackgroundGlioblastoma multiforme is the most malignant form of brain tumor. Despite treatment including surgical resection, adjuvant chemotherapy, and radiation, these tumors typically recur. The recurrent tumor is often resistant to further therapy with the same agent, suggesting that the surviving cells that repopulate the tumor mass have an intrinsic genetic advantage. We previously demonstrated that cells selected for resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) are near-diploid, with over-representation of part or all of chromosomes 7 and 22. While cells from untreated gliomas often have over-representation of chromosome 7, chromosome 22 is typically under-represented.MethodsWe have analyzed cells from primary and recurrent tumors from the same patient before and after in vitro selection for resistance to clinically relevant doses of BCNU. Karyotypic analyses were done to demonstrate the genetic makeup of these cells, and fluorescent in situ hybridization analyses have defined the region(s) of chromosome 22 retained in these BCNU-resistant cells.ResultsKaryotypic analyses demonstrated that cells selected for BCNU resistance were near-diploid with over-representation of chromosomes 7 and 22. In cells where whole copies of chromosome 22 were not identified, numerous fragments of this chromosome were retained and inserted into several marker and derivative chromosomes. Fluorescent in situ hybridization analyses using whole chromosome paints confirmed this finding. Additional FISH analysis using bacterial artificial chromosome probes spanning the length of chromosome 22 have allowed us to map the over-represented region to 22q12.3–13.32.ConclusionCells selected for BCNU resistance either in vivo or in vitro retain sequences mapped to chromosome 22. The specific over-representation of sequences mapped to 22q12.3–13.32 suggest the presence of a DNA sequence important to BCNU survival and/or resistance located in this region of chromosome 22.

Molecular Biological Events in the Selection of Chemotherapy Resistant-Cells in Human Malignant Gliomas

Within the cellular heterogeneity of freshly resected human gliomas we have observed cells with a specific non-random karyotypic deviation to be intrinsically chemoresistant to the chemotherapeutic agent, BCNU (1,3 bis-(2 chloroethyl)-1-nitrosourea). The BCNU-resistant cells were near-diploid (2n± with chromosome complements of 35–57 chromosomes/metaphase) and resistant to concentrations of ≥7.5 μg/ml BCNU. In contrast, the hyperdiploid populations from the same tumor (3n± and 4n± with 58 or more chromosomes/metaphase) were sensitive. Cytogenetic analysis of the BCNU-resistant cells demonstrated that more than 90% of the karyotypes depicted cells with an over-representation of chromosomes 7 and 22, the chromosomes to which platelet-derived growth factor (PDGF) chains A and B were mapped. Further analyses determined that these cells secreted a factor(s) that stimulated 3H-thymidine incorporation in PDGF receptor-positive cells, and that this factor competed with 125I-PDGF in receptor binding experiments. Southern, northern and western blot analyses demonstrated amplification and over-expression of the genes encoding both the A and B chains, as well as aberrant forms of PDGF. Phosphorylation analyses demonstrated that these cells were autocrine. To determine if this same cell persisted in vivo following a patient’s treatment with BCNU, we analyzed recurrent tumors from 5 patients whose original tumors were known to have this cell as a minor subpopulation and who required a second surgical resection. The cytogenetic and bioassay analyses demonstrated that in all 5 patients this BCNU-resistant cell was the dominant cell type in the recurrent tumor. In situ hybridization studies of paraffin-embedded sections from primary and recurrent samples of one tumor further substantiated that there is an increase in the proportion of cells that were producing PDGF-A chain mRNA. These data suggest that intrinsically resistant cells can survive currently available chemotherapeutic treatments and repopulate the tumor mass.