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J R McCulloch

Publications and source records attributed to J R McCulloch.

15 recordsLinked to original sources
PubMed1988

Effect of retinoids on the proliferation, morphology and expression of glial fibrillary acidic protein of an anaplastic astrocytoma cell line.

We studied the effect of retinoids on the growth and differentiation of a cell line (U 343 MG-A) derived from a human malignant astrocytoma. Cultures treated with all-trans or 13-cis retinoic acid showed a dose-dependent inhibition of proliferation and a marked reduction in the mean cell number at the plateau phase of growth (3.5 x 10(6) vs. 1 x 10(7) cells/25 cm2) compared with untreated cultures. At confluence, cells treated with all-trans or 13-cis retinoic acid were contact-inhibited, whereas control cultures showed crowding, piling, and overgrowth. All-trans retinol or retinyl acetate did not inhibit growth. Astrocytoma cultures treated with all-trans retinoic acid (10(-6) M) for 5 days were modestly growth-inhibited but by day 16 had the same numbers of cells as controls; cultures that received all-trans retinoic acid for 9 days were markedly growth-inhibited for 7 days after the drug was removed. All-trans and 13-cis retinoic acid (10(-6) M) prevented the EDTA-induced cell detachment seen in control cultures. Strongly adherent all-trans retinoic-acid-treated astrocytoma cells grew at a slower rate than did readily detached all-trans retinoic-acid-treated or control cells. Cell spreading, an increased cytoplasmic:nuclear ratio, and greater numbers of broadly bipolar cells, some bearing thin cytoplasmic processes, were seen in cultures treated with 10(-6) M all-trans or 13-cis retinoic acid. Small tightly packed cuboidal cells and large broadly bipolar cells were seen in astrocytoma cultures from which all-trans retinoic acid was removed on days 5 and 9. Indirect immunofluorescence revealed more intense staining with antiserum to glial fibrillary acidic protein in cultures treated with 10(-6) M all-trans retinoic acid than in control cultures; electron-microscope examination of similarly treated cultures revealed more abundant 8-10 nm intermediate filaments than in control cultures. An enzyme-linked immunosorbent assay showed that all-trans or 13-cis retinoic acid caused a dose-dependent increase in the quantity of glial fibrillary acidic protein in the astrocytoma cells.

Astrocytoma↗
PubMed1988

The effects of human recombinant tumor necrosis factor on glioma-derived cell lines: cellular proliferation, cytotoxicity, morphological and radioreceptor studies.

To determine whether tumor necrosis factor is of potential value for the treatment of human malignant gliomas, we studied the effects of human recombinant tumor necrosis factor (rTNF-alpha) on the morphology, incorporation of tritiated thymidine, and proliferation of 5 established cell lines derived from human malignant gliomas and 3 normal human brain cell cultures. A radioreceptor analysis for rTNF-alpha was performed on all cell lines and cultures. Two of the 5 human glioma cell lines (SF-188 and U 343 MG-A) demonstrated a marked decrease (60% or less of untreated controls) in the uptake of tritiated thymidine when treated with rTNF-alpha at a concentration of 40 U/ml; rTNF-alpha at 100 U/ml had antiproliferative and cytotoxic effects on both cell lines. The growth and proliferation of cell lines SF-126 and U 251 MG were not affected by rTNF-alpha even at high concentrations (5,000 U/ml). The growth and proliferation of SF-539 were affected to an intermediate degree. A colony-forming efficiency assay corroborated the results of the proliferation studies: SF-126 was relatively resistant (surviving fraction of 0.9 at 500 U/ml) and SF-188 was relatively sensitive (surviving fraction of 0.08 at 500 U/ml) to the cytotoxic effects of rTNF-alpha. Time-sequence electron microscopy showed that rTNF-alpha at a concentration of 500 U/ml caused ultrastructural changes in SF-188, including increased intracytoplasmic vesiculation, swelling and degeneration of mitochondria, loss of cell:cell junctional complexes, and fragmentation of the plasma membrane. Studies with 125I-rTNF-alpha showed a variable degree of binding in all cell lines and cultures. SF-188, a highly sensitive cell line, demonstrated the strongest binding of 125I-rTNF-alpha (3,400 receptors/cell with high affinity; kd = 0.27 nM), while SF-126, a highly resistant cell line, had the weakest binding (809 receptors/cell; kd = 0.25 nM). We conclude that there is a spectrum of antiproliferative and cytotoxic activity among glioma-derived tumor cell lines exposed to rTNF-alpha. An increased number of rTNF-alpha receptors appears to be a necessary but insufficient condition to explain the antiproliferative effects observed in some glioma-derived cell lines.

Cell Division↗
PubMed1987

Establishment and characterization of five cell lines derived from human malignant gliomas.

We established and characterized five cell lines derived from human malignant gliomas (four glioblastomas multiforme and one highly anaplastic astrocytoma). All cell lines exhibited tumor cell morphology and growth kinetics, and anchorage-independent growth in soft agar. Cytogenetic analysis revealed significant aneuploidy in all five cases as well as clonal chromosomal alterations unique to each cell line. No cell line was tumorigenic in athymic mice. Two of the cell lines were sensitive to carmustine (BCNU) in monolayer and soft-agar cultures. Electron microscopy showed marked variability between cell lines in the number and structure of intracytoplasmic organelles; SF-126 formed collagen fibers in vitro. Immunohistochemical analysis of the surgical specimens showed variable expression of glial fibrillary acidic protein (GFAP) in malignant astrocytes; positive immunostaining for glycoproteins of the extracellular matrix was found predominantly in perivascular regions. In early-passage cultures, only cell line SF-295 expressed GFAP; at establishment, none of the cell lines expressed GFAF or glutamine synthetase. Fibronectin and laminin were expressed by all cell lines in early-passage culture, but expression of these glycoproteins at establishment was variable. Only SF-126 was positively identified by immunostains for procollagen III; this was also the only cell line in which DEAE-cellulose chromatography and SDS-PAGE demonstrated interstitial collagen synthesis. These well-characterized glioma-derived cell lines may now serve as useful tools with which to study the cell biology of gliomas. The synthesis of interstitial collagen by a glioma-derived cell line may suggest a derivation from vascular mesenchymal elements, either reactive or transformed, in the original heterogeneous malignant glioma, rather than from a glial precursor cell.

Adult↗
PubMed1987

Characterization of fetal human brain cultures. Development of a potential model for selectively purifying human glial cells in culture.

Seven fetal human brain and three fetal human leptomeningeal cultures were characterized according to cell morphology, ultrastructural features, antigen expression, and collagen biosynthesis capabilities. Primary cultures derived from mechanically and enzymatically dissociated samples of fetal human brain consisted of a heterogeneous cell population in which astrocytes, oligodendrocytes, neurons, mesenchymal (leptomeningeal) cells, and macrophages were identified by light and electron microscopy. With progressive subcultivation, a homogeneous, leptomeningeal cell-derived population predominated. Fetal human brain and leptomeningeal specimens embedded in paraffin were analyzed immunohistochemically for the distribution of glial fibrillary acidic protein (GFAP), vimentin, factor-VIII-related antigen, fibronectin, laminin, type IV collagen, and procollagen III. Only GFAP and vimentin identified astrocytes and radial glia in the developing human brain; fibronectin, laminin, and the collagen types were immunolocalized largely to the leptomeninges and to the cerebral vasculature. The percentage of cells positively identified by antiserum to GFAP was greatest in primary cultures of fetal human brain; by the fourth passage, none of the fetal brain cultures were GFAP positive. The progressive decrease in the percentage of GFAP-positive cells was accompanied by an increase in the percentage of cells identified by collagen immunomarkers. Furthermore, in double immunolabeling experiments, antibodies to GFAP recognized a population of cells that was not identified by antibodies to laminin, fibronectin, type IV collagen, or procollagen III. SDS-PAGE and DEAE-cellulose chromatography of [3H]-proline-labeled early-passage fetal human brain cultures revealed collagen profiles identical to those obtained from direct cultures of the leptomeninges. The characteristics of later-passage fetal human brain cultures were identical in all respects to those of the fetal human leptomeningeal cultures. The proliferation of leptomeningeal cells could be inhibited by exposing the cells to cis-hydroxyproline (200 micrograms/ml). Primary fetal human brain cultures similarly treated with the proline analogue were found to be highly enriched for glial cells; these cultures were more than 90% GFAP positive. We conclude that primary fetal human brain cultures consist of a heterogeneous population of cells, most of which under the present culture conditions can be identified as glial cells. Subcultivation of human fetal brain cultures results in the overgrowth of mesenchymal cells, which are presumably derived from the leptomeninges.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain↗
PubMed1987

Growth of a medulloblastoma on normal leptomeningeal cells in culture: interaction of tumor cells and normal cells.

Well-characterized medulloblastoma cells growing in suspension were placed on top of a confluent monolayer of leptomeningeal cells. In contrast to cells placed on plastic alone, which did not grow or attach, the medulloblastoma cells attached readily to the leptomeningeal cells and grew to form enlarging spheroids. The growth of these spheroids was supported with minimal essential medium containing 10% fetal calf serum or with human cerebrospinal fluid. Medulloblastoma cells grown on plastic remained viable for 7 to 10 days, whereas those grown on a monolayer of leptomeningeal cells remained viable for 40 days. Electron microscopy demonstrated increased interdigitation of the plasma membrane at the sites of contact between leptomeningeal and medulloblastoma cells, the deposition of fine, basement membrane-like material between the two cell types, and an increased number of cytoskeletal filaments in the leptomeningeal cells. We conclude that medulloblastoma cells can be grown on a leptomeningeal monolayer. This in vitro system may be useful in studying the mechanisms by which medulloblastoma cells attach to leptomeningeal elements and grow after dissemination in cerebrospinal fluid.

Arachnoid↗
PubMed1986

An ultrastructural and immunocytochemical analysis of leptomeningeal and meningioma cultures.

Using immunocytochemical methods, we localized several glycoproteins of the extracellular matrix to leptomeningeal cells and meningiomas in vitro. Three cell lines derived from normal human leptomeninges and seven from meningiomas were studied by indirect immunofluorescence to evaluate the cellular production of fibronectin, laminin, collagen type IV, and procollagen type III. All leptomeningeal cell lines stained intensely and uniformly for all matrix proteins; all meningioma cell cultures stained uniformly, but the intensity of staining varied considerably. After removal of the cells in culture adherent to glass with 25 mM ammonium hydroxide, indirect immunofluorescence demonstrated an exuberant residual extracellular residue enriched with fibronectin, laminin, collagen type IV, and procollagen type III. Electron microscopic examination of all leptomeningeal and meningioma cultures revealed desmosomes and dense tonofilament formation; in addition, granular, filamentous basement membrane-like material was abundant in the extracellular spaces of all cultures. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the cell layer of two leptomeningeal and four meningioma cultures showed production of interstitial collagen types I and III; diethylaminoethyl (DEAE)-cellulose chromatography of the medium demonstrated preferential production of procollagen type I. Our findings show conclusively that normal arachnoid cells in vitro synthesize several of the collagen subtypes and may be responsible for the "fibrous response" of the leptomeninges to trauma, infection, or infiltration by tumor. The similarities between leptomeningeal cells and meningiomas demonstrated by electron microscopy and by indirect immunofluorescence support the notion that meningiomas are derived from arachnoid cells. The localization of various mesenchymal glycoproteins within the intra- and extracellular spaces and the ubiquity of specialized intercellular junctions suggest that leptomeningeal cells in culture have the potential to behave like both stromal and epithelial cells.

Animals↗
PubMed1986

Establishment and characterization of a cell line from a human gliosarcoma.

A human gliosarcoma culture was characterized from the time of inception to the time of establishment of the cell line (SF-539 BT). Immunohistochemical analysis of the original tumor showed 2 distinct regions of cells. The gliomatous regions were identified by immunostains for glial fibrillary acidic protein and the sarcomatous regions by immunostains for laminin, collagen type IV, procollagen type III, and fibronectin. In early-passage culture, both types of cells maintained their characteristic immunohistochemical profiles; however, after the fourth subcultivation in monolayer culture, no cells expressing glial fibrillary acidic protein could be identified. All cells had become morphologically uniform and expressed laminin, collagen type IV, procollagen type III, and fibronectin only. The immunostaining profile of clones grown in soft agar was similar to that of cells in monolayer culture. At establishment, SF-539 BT has a saturation density of 1.3 X 10(6) cells/25 sq cm, a doubling time of 32 h, and a plating efficiency of 22% in monolayer culture. The tumor cell line is resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea, has an abnormal karyotype, grows anchorage independently, and forms a tumor that most closely resembles a spindle cell sarcoma in athymic mice. Its ultrastructure in monolayer culture consists of large cells with an expanded rough endoplasmic reticulum and abundant multivesicular bodies; in athymic mice, extracellular collagen fiber formation is prominent. DEAE-cellulose chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cultures labeled with [3H] proline demonstrated interstitial collagen formation. We conclude that the cell line at establishment is a collagen-producing spindle cell sarcoma that resembles the sarcomatous regions of the original mixed tumor. Further cell separation and characterization studies are needed to determine the pathogenesis of mixed tumors such as gliosarcoma.

Animals↗
PubMed1986

Characterization of normal human brain cultures. Evidence for the outgrowth of leptomeningeal cells.

To establish the histogenetic identity of the predominant cell type in monolayer cultures of normal human adult brain, eight brain specimens were placed into culture and characterized according to cell kinetics, karyotype, antigenic expression, and ultrastructural features. The protein profiles of both the cell layer and the medium were analyzed in selected cultures using sodium dodecyl sulfate polyacrylamide gel electrophoresis and diethylaminoethyl cellulose chromatography. All cultures displayed a limited life span in vitro; marked contact inhibition at confluence; a normal karyotype; an intracytoplasmic and extracellular glycoprotein profile consisting of fibronectin, procollagen type III, laminin, and collagen type IV; specialized intercellular junctions; and interstitial collagen chain synthesis. All of these features were identified in our previous study of human leptomeningeal cultures. The results of immunocytochemical staining for glial fibrillary acidic protein were negative in all cultures of normal human brain, except in early passages in two cultures, which lost the glial cell marker during subsequent passages; immunostains for vimentin were positive in all cells in all cultures. These results support the hypothesis that, in this study, cultures derived from normal human brain are not of glial origin. Our findings also suggest that glial cells are less well-suited to monolayer growth under our culture conditions than are other cell types in enzyme-dissociated brain tissue placed in culture, especially leptomeningeal cells. The identification of leptomeningeal cells as the predominant cell type in normal human brain cultures may prove useful in attempts to foster the growth of human glial cells by culturing brain samples under conditions that prohibit the growth of leptomeningeal cells. Under such conditions, astrocytes, oligodendroglia, and ependymal cells could be isolated with greater ease and cultured separately. These purified cultures of different glial cell types would then provide a more relevant in vitro model for studying human neurological diseases.

Animals↗