Sherri Lynn Hubbard

Retinoic acid and the cyclin dependent kinase inhibitors synergistically alter proliferation and morphology of U343 astrocytoma cells

We have characterized the expression and activity of the cell cycle regulatory machinery and the organization of the cytoskeleton of the p16Ink4a-deficient astrocytoma cell line, U343 MG-a (U343), following retinoic acid (RA) treatment. RA causes cell cycle arrest at low cell density and significant morphological changes in U343 cells, reflected by reorganization of the intermediate filament, GFAP, and actin. RA-induced cell cycle arrest is also associated with induction of p27Kip1 expression, inhibition of cdk2-associated kinase activity and alteration of the phosphorylation state of the pRB-family proteins. We next determined the effect of inducing expression of the cyclin dependent kinase inhibitors (CKIs), p16Ink4a, p21Cip1/Waf1 or p27Kip1 on the proliferation and morphology of these malignant astrocytoma cells in the absence and presence of RA. Induction of p16, p21 or p27, using the tetracycline repressor system, potently inhibits proliferation of U343 cells. However, rather than resembling RA-treated cells, CKI-induced U343 cells become flat with abundant cytoplasm and perinuclear vacuolization. CKI-induced morphological alterations are accompanied by a significant reorganization of glial filaments within the cytoplasm. Interestingly, when U343 cells are growth arrested by p16, p21 or p27 induction and treated simultaneously with RA, a dramatic morphological change occurs, cells acquiring multiple long, tapering processes reminiscent of primary astrocytes. This rearrangement is accompanied by reorganization of GFAP, vimentin and actin. Vimentin specifically relocalizes to the tips of the long processes which form. The arrangement of intermediate filaments in these cells is, in fact, indistinguishable from their arrangement in primary human astrocytes. These data demonstrate that when a strong proliferative block, produced by CKI expression, occurs in conjunction with the morphogenic signals generated by RA, these p16-deficient malignant astrocytoma cells are induced to phenotypically resemble normal astrocytes.

Verotoxins inhibit the growth of and induce apoptosis in human astrocytoma cells

Verotoxin 1 (VT1) is an E. coli toxin comprising an A subunit with N-glycanase activity, and five smaller B subunits capable of binding to the functional receptor globotriaosylceramide (Galα1-4-Galβ1-4-Glcceramide-Gb3). VT is implicated in hemorrhagic colitis and the more serious hemolytic uremic syndrome. VT1 is active against various tumor cell lines in vitro and in vivo. To extend the anti-cancer spectrum of activity of VT to human brain tumors, in the present analysis we studied the effects of VT on the growth of 6 permanent human astrocytoma cell lines. All astrocytoma cell lines analyzed express Gb3 and were sensitive to VT-1 at a dose of 50 ng/ml, but sensitivity was not proportional to the relative Gb3 concentration. VT induced apoptosis in these cells was shown by electron microscopy. Morphological evidence (nuclear shrinkage and chromatin condensation) of apoptosis could be clearly distinguished 1.5 hrs after toxin addition. Ultrastructural preservation of organelles was observed in conjunction with blebbing of the plasma membrane, condensation of chromatin within the nucleus and nuclear shrinkage. Apoptosis was also induced by the recombinant toxin B subunit alone, suggesting that the ligation of Gb3 is the primary induction mechanism. These studies indicate that verotoxin/Gb3 targetting may provide a novel basis for the inhibition of astrocytoma tumour cell growth.

Astrocytoma Adhesion to Extracellular Matrix: Functional Significance of Integrin and Focal Adhesion Kinase Expression

Evidence is accumulating implicating a role for integrins in the pathogenesis of cancer, a disease in which alterations in cellular growth, differentiation, and adhesive characteristics are defining features. In the present report we studied a panel of 8 human astrocytoma cell lines for their expression of integrin subunits by RT-PCR, and of integrin heterodimers by immunoprecipitation analyses. The functionality of integrin heterodimers was assessed using cell attachment assays to plastic or single matrix substrates. Downstream effects of integrin activation were studied by western blot analyses of FAK expression in human astrocytoma cell lines growing on plastic and on a fibronectin matrix, and in 13 primary human brain tumor specimens of varying histopathological grade. Furthermore, we studied tyrosine phosphorylation of FAK in astrocytoma cells growing on plastic versus fibronectin. Finally, we analyzed the effects of intermediate filament gene transfer on FAK phosphorylation in SF-126 astrocytoma cells. Our data show that astrocytoma cell lines express various integrin subunits by RT-PCR, and heterodimers by immunoprecipitation analyses. The beta1 and alphav integrin subunits were expressed by all astrocytoma cell lines. The alpha3 subunit was expressed by all cell lines except SF-188. By immunoprecipitation, the fibronectin receptor (alpha5beta1 integrin heterodimer) and the vitronectin receptor (alphavbeta3) were identified in several cell lines. Astrocytoma cell attachment studies to human matrix proteins suggested that these integrin heterodimers were functional. Using confocal laser microscopy, we showed that FAK was colocalized to actin stress fibers at sites of focal adhesion complexes. By western blot, FAK was variably but quite ubiquitously expressed in human astrocytoma cell lines, and in several primary human astrocytoma specimens. When U373 and U87 MG astrocytoma cells bind to a fibronectin matrix, FAK is phosphorylated. GFAP-transfected SF-126 human astrocytoma cells were shown to overexpress the phosphorylated form of FAK only when these cells were placed on a fibronectin matrix. This result is of interest because it suggests that manipulations of the astrocytoma cytoskeleton per se can bring about potential signaling changes that channel through integrins and focal adhesion sites leading to activation of key kinases such as FAK.

Transfection of an invasive human astrocytoma cell line with a TIMP-1 cDNA: modulation of astrocytoma invasive potential.

Malignant astrocytomas are highly invasive tumors which infiltrate diffusely into regions of normal brain. The degradation of the extracellular matrix (ECM) by matrix metalloproteinases is thought to be one of the most important steps in the process of tumor invasion. However, the activity of most matrix metalloproteinases (MMPs) can be modulated by simultaneously secreted inhibitors (tissue inhibitors of metalloproteinases, TIMPs). We have previously shown that an imbalance between the levels of MMPs and TIMPs may be essential in the determination of the invasiveness of certain human malignant astrocytoma cell lines. To determine if the up-regulation of TIMP genes and gene products could modulate the invasiveness of human malignant astrocytoma cells, in the present study we have transfected a highly invasive astrocytoma cell line, SF-188, with an expression vector carrying a full-length TIMP-1 cDNA. The parental SF-188 astrocytoma cell line overexpresses the 72-kDa and 92-kDa type IV collagenases with little expression of TIMPs-1 and −2. Following transfection with TIMP-1, SF-188 astrocytoma clones expressed the 0.9 kb TIMP-1 message by northern analysis, and produced a 21 kDa metalloproteinase inhibitor by reverse zymography. The stable TIMP-1 SF-188 transformants demonstrated morphological changes and diminished growth rates in soft agar when compared to controls. The invasion of successfully TIMP-1 transfected astrocytoma cells across matrigel-coated filters was significantly decreased over controls. These results suggest that up- regulation of TIMP-1 expression in SF-188 astrocytoma cells has decreased their in vitro invasive potential

The E2F-family proteins induce distinct cell cycle regulatory factors in p16-arrested, U343 astrocytoma cells.

We previously demonstrated that P16Ink4a (p16) expression in p16-deficient U343 astrocytoma cells causes a G1 cell cycle arrest, profound changes in cytoskeletal proteins and alterations in expression and activity of the pRB and E2F family proteins. We examine here the effects of expressing wild type or mutant versions of the downstream targets of p16 in U343 astrocytomas. We first attempted to block proliferation of U343 cells using the dominant mutant of pRB, Δp34. Expression of this mutant in the human osteosarcoma, SAOS-2, potently blocked proliferation but did not affect the cell cycle of U343 cells. We next showed that expression of E2F-1, E2F-2, E2F-3 and E2F-4 are each able to overcome this p16-dependent cell cycle arrest but exhibit distinct biological activities. Adenoviral-mediated expression of E2F-1, E2F-2, E2F-3, or E2F-4 overcame the p16-dependent cell cycle block and induced alterations in cell morphology. E2F-5, only in conjunction with DP1, promoted cell cycle progression. For both E2F-1 and E2F-2, but not E2F-3 or E2F-5/DP1, cell cycle re-entry was associated with almost quantitative cell death. Only small numbers of dying cells were observed in E2F-4-expressing cultures. Expression of the different E2Fs altered the expression of distinct sets of cell cycle regulatory proteins. E2F-1 induced endogenous E2F-4 expression and also caused an increase in pRB, p107 and cyclin E levels. Expression of E2F-4 caused a weak increase in E2F-1 levels but also strongly induced pRB, p107, p130 and cyclin E. However, E2F-1 and E2F-4 clearly regulate expression of distinct genes, demonstrated when E2F-4 caused a threefold increase in the levels of cdk2 whereas E2F-1 failed to increase in this cyclin dependent kinase. Similarly, expression of E2F-1 or E2F-2 were shown to have distinct effects on the expression of cdk2, cyclin E and pRB despite both of these closely related E2F-family members potently inducing cell death. Thus, E2F-1, E2F-2, E2F-3 and E2F-4 are able to overcome the p16-dependent proliferative block in U343 astrocytoma cells. While overcoming this cell cycle block, each of the E2Fs uniquely affect the expression of a number of cell cycle regulatory proteins and have distinct abilities to promote cell death.

Astrocytoma cell interaction with elastin substrates: Implications for astrocytoma invasive potential

Elastin has been identified within the meninges and the microvasculature of the normal human brain. However, the role that elastin plays in either facilitating astrocytoma cell attachment to these structures or modulating astrocytoma invasion has not been previously characterized. We have recently shown that astrocytoma cell lines and specimens produce tropoelastin, and express the 67 kDa elastin binding protein (EBP*). In the present report, we have established that astrocytoma cells attach to elastin as a substrate in vitro. The U87 MG astrocytoma cell line demonstrated the greatest degree of adhesion. In addition, all astrocytoma cell lines examined were capable of penetrating and migrating through an intact elastin membrane, and of degrading tritiated‐elastin, a process that could be prevented by the pre‐incubation of astrocytoma cells with EDTA, but not with α1‐antitrypsin. Astrocytoma cells were also capable of penetrating 1 mm sections of human brain tissue maintained as organotypic cultures. Interestingly, the invasive potential of cultured astrocytoma cells plated on organotypic cultures of human brain was significantly increased after exposure to elastin degradation products (κ‐elastin), which interact with astrocytoma cell surface EBP. Our data show that astrocytoma cells express a functional 67 kDa EBP, enabling them to potentially recognize and attach to elastin as a substrate. These data also suggest that this elastin receptor may be involved in processes which regulate regional astrocytoma invasion. GLIA 25:179–189, 1999.

Cyclin and cyclin-dependent kinase expression in human astrocytoma cell lines.

The expression of cyclins (A, B1. D1, D3, E), cyclin-dependent kinases (CDK23, CDK4), and the cyclin-dependent kinase inhibitors (CDKIs) p16INK4A and p21CIPI was studied in 9 malignant human astrocytoma cell lines using northern blot analysis, immunocytochemistry, and immunoblotting to see if their altered expression contributed to astrocytoma proliferation. Steady state cell cycle mRNA expression was analyzed in unsynchronized tumor cells, and cell cycle phase-specific gene expression was analyzed in 3 synchronized cell lines. Analysis of steady state expression revealed increased levels of several different cyclin transcripts and CDKs in a number of astrocytoma cell lines compared with normal human brain tissue or cultured fibroblasts. We confirm previous reports identifying loss of p16INK4A in astrocytomas, as a p16INK4A transcript was identified in only 2 cell lines and protein in 1 cell line. However, we now show that p21CIPI expression was also diminished relative to normal fibroblasts in all astrocytoma cell lines studied regardless of p53 mutation status. Analysis of synchronized astrocytoma cells revealed altered timing of mRNA expression of several cyclins. Immunocytochemistry revealed a generalized increase in immunoreactivity of astrocytoma cells for most cyclins and CDKs compared with human fibroblasts. Immunoblotting also revealed increased expression of cyclin proteins in a number of astrocytoma cell lines. These data suggest that increased expression of cyclins and CDKs, and decreased expression of CDKIs by human astrocytoma cell lines may contribute to their increased proliferative state. In addition, our data show that alterations in cell cycle genes in astrocytomas are not confined to the cyclin D1-CDK4-pRb axis.

Expression of Nedd5, A Mammalian Septin, in Human Brain Tumors

The septins are a family of cytoskeletal GTPases that play an essential role in cytokinesis in yeast and mammalian cells. Nedd5 is a mammalian septin known to associate with. actin-based structures such as the contractile ring and stress fibers. In the present study, we examined the expression of Nedd5 in a series of human brain tumor cell lines and surgical specimens by northern and western analyses. The temporal expression of Nedd5 in U373 astrocytoma cells at various timepoints throughout the cell cycle was determined by an analysis of lovastatin- and nocodazole-treated, synchronized cell populations. The intracellular localization of Nedd5 was determined by immunocytochemistry of steady state cultures and nocodazole-treated cultures enriched in M phase cells. The effects of inhibiting Nedd5 expression in human brain tumors was determined by stably transfecting U373 astrocytoma cells with an antisense-Nedd5 cDNA expression vector and by analyzing clones for Nedd5 expression by immunocytochemistry, morphological changes, cell growth and nuclear content. All human brain tumor cell lines and surgical specimens expressed Nedd5 transcript and protein. Synchronized U373 astrocytoma cells showed a relative increase in Nedd5 transcript levels from late G1 to G2M phases; and an increase in Nedd5 protein levels from S to G2M phases. Maximum expression of both transcript and protein levels was observed at the G2M phase. By immunocytochemistry, Nedd5 was concentrated at the cleavage furrow of mitotic cells. Double staining with Nedd5 and F-actin showed co-localization of Nedd5 with actin filaments except during cytokinesis. Antisense-Nedd5 expression led to an accumulation of nuclear content. These data suggest that Nedd5 is involved in the process of cytokinesis in human brain tumours. Nedd5 expression may be cell cycle-dependent with increased levels found at G2M phase. Blocking Nedd5 expression in astrocytoma cells by antisense interferes with the process of cytokinesis during cell division.

Characterization of a Pineal Region Malignant Rhabdoid Tumor

The malignant rhabdoid tumor (MRT) of the central nervous system is a highly aggressive neoplasm of early infancy which is characterized by brain invasion and widespread dissemination along cerebrospinal fluid pathways. As the process of tumor invasion is mediated in part by the elaboration of proteases and protease inhibitors by tumor cells, we sought to determine the expression of the type-IV collagenases and their inhibitors (tissue inhibitors of metalloproteases, TIMPs) in an MRT from the pineal region of a 9-month-old male. In addition, as only a few reports exist concerning the cytogenetic abnormalities in MRTs, the cytogenetic features of this MRT were examined. When placed into tissue culture, the MRT grew vigorously in early passages. The cytogenetic analysis of the cells in passage one revealed a near diploid karyotype with some metaphases demonstrating monosomy 22. Northern analysis of type-IV collagenase transcripts revealed that the MRT expressed the highest levels of the 72- and 92-kD type-IV collagenase transcripts of any pediatric brain tumor examined. However, the MRT did not express any significant amounts of the TIMP-1 or TIMP-2 transcripts. By in situ hybridization analysis, the MRT demonstrated marked intratumoral expression of the type-IV collagenase but not TIMP transcripts. The results from this study suggest that this particular MRT may be a highly invasive brain tumor, at least in part on the basis of overexpression of the type-IV collagenases relative to the TIMPs.

CHARACTERIZATION OF GLIAL FILAMENT-CYTOSKELETAL INTERACTIONS IN HUMAN ASTROCYTOMAS : AN IMMUNO-ULTRASTRUCTURAL ANALYSIS

The role that glial filaments play in cells and tumors of glial origin is not well understood. We therefore undertook the present study to determine the relationships between glial and vimentin intermediate filaments (IFs), actin microfilaments, and CD44, a cell surface glycoprotein important in cell migration and invasion, in human astrocytoma cells. Three astrocytoma cell lines, U343 MG-A (U343), U251 MG (U251), and antisense GFAP-transfected U251 (asU251) were studied using immunofluorescence confocal and immunoelectron microscopy. Furthermore, we studied the phenotypic behaviour of these astrocytoma cell lines by analyzing their migration through Matrigel in vitro. U343 astrocytoma cells had the highest expression levels of glial fibrillary acidic protein (GFAP), whereas asU251 had virtually no expression of GFAP. Parental U251 cells had intermediate expression levels of GFAP. The elimination of GFAP expression in as U251 cells was accompanied by a marked increase in vimentin, actin microfilaments and CD44 levels. Gold labeling density counts of cytoskeletal and cell surface elements demonstrated that the differences between GFAP, actin, CD44 and vimentin levels in the different astrocytoma cell lines were statistically significant (p < 0.05). Results from the in vitro invasion assay revealed that U343 cells demonstrated the least invasive potential, whereas asU251 astrocytoma cells demonstrated the most. Our results show that elimination of GFAP expression by antisense leads to marked alterations in cell morphology and phenotypic behaviour. These data imply that GFAP may be linked spatially and functionally to cytoskeletal elements which may be altered when this IF is deleted in astrocytomas.