Inducible expression of p57KIP2 inhibits glioma cell motility and invasion.

To examine the role of p57KIP2 in human malignant glioma cells, we studied its expression in a panel of human malignant glioma specimens by western blot and immunohistochemical analysis. To determine the effects of p57KIP2 expression on the phenotype of glioma cells, we analyzed two inducible stably transfected p57KIP2 expressing glioma cell lines. Expression of p57KIP2 was induced in U373 and U87 malignant glioma cells with doxycycline using the tetracycline repressor system. A phagokinetic track assay on gold particles was used to investigate differences in cell migration between p57KIP2 expressing and non-expressing control cells. The effects of the extracellular matrix (ECM) on U373 motility was determined in p57+ and p57-cells on surfaces coated with 5 microg/cm2 of fibronectin, laminin, type I and type IV collagens. The invasion of p57+ and p57- glioma cells across BD Biocoat Matrigel invasion chambers was then determined. p57KIP2 was weakly expressed in 4/6 glioblastoma (GBM) specimens by western blot. By immunohistochemistry, p57KIP2 immunoreactivity was positive in 8/40 GBMs, and was primarily nuclear in location. The motility of U373 glioma cells was significantly reduced after p57KIP2 induction. The presence of ECM proteins did not further alter the motility of p57+ and p57- glioma cells. The results of the invasion chamber assay showed that p57+ cells exhibited a 35% reduction in their invasive capacity as compared to p57- cells. These data suggest that p57KIP2 is expressed in at least some malignant gliomas. Inducible expression of 57KIP2 in cell lines deficient in this cyclin-dependent kinase inhibitor reduces their otility and invasiveness.

Novel laser system and laser irradiation method reduced the risk of carbonization during laser interstitial thermotherapy: assessed by MR temperature measurement.

BACKGROUND AND OBJECTIVE: To establish laser interstitial thermotherapy (LITT) for intracranial tumors, the authors investigated a method to regulate localized temperature generated by interstitial laser irradiation using magnetic resonance (MR) temperature mapping. STUDY DESIGN/MATERIALS AND METHODS: A diode laser system and six different types of optical-fiber system were developed for LITT. The characteristics of temperature profiles produced by each laser-fiber system were investigated with MR temperature measurement (the water proton chemical technique), and differences in the temperature profile induced by two laser-irradiation methods (continuous and intermittent) were observed. RESULTS: All fiber systems with the exception of the diffuse-projection fiber system, created a spherical temperature profile. Carbonization sometimes occurred around the bare-end fiber tip upon high power laser irradiation. The diffuse-projection fiber system produced a cylindrical temperature distribution, and the temperature profile showed a more gradual temperature elevation than the bare-end fiber. No carbonization occurred at the tip of the diffuse-projection fiber system. In addition, the utilization of the intermittent irradiation method also increased temperature gradually. Fiber-system modification and intermittent irradiation reduced laser-beam intensity and the risk of carbonization. CONCLUSION: The use of a diffuse-projection fiber system which intermittently transmits a reduced intensity laser beam is an effective tool to regulate temperature during LITT using MR temperature measurement.

Gene expression of angiogenesis related factors in glioma.

Angiogenesis plays an important role in growth and proliferation of cancer. Various angiogenic and angiostatic factors regulate angiogenesis. In this study, we examined gene expression of the angiopoietin family including angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2) in 39 gliomas and 5 glioma-xenografts by RT-PCR. Ang1 and Ang2 genes were expressed in 54%, and 77% of gliomas, respectively. The expression of Ang1 was significantly correlated with the expression of Ang2. Both Ang1 and Ang2 were shown to be expressed in the glioma cells. Ang2 gene expression was correlated with VEGF gene expression. Angiopoietin molecules may synergistically cooperate in growth and vascularization in glioma.

Expression of p57(KIP2) potently blocks the growth of human astrocytomas and induces cell senescence.

Astrocytic tumors frequently exhibit defects in the expression or activity of proteins that control cell-cycle progression. Inhibition of kinase activity associated with cyclin/cyclin-dependent kinase co-complexes by cyclin-dependent kinase inhibitors is an important mechanism by which the effects of growth signals are down-regulated. We undertook the present study to determine the role of p57(KIP2) (p57) in human astrocytomas. We demonstrate here that whereas p57 is expressed in fetal brain tissue, specimens of astrocytomas of varying grade and permanent astrocytoma cell lines do not express p57, and do not contain mutations of the p57 gene by multiplex-heteroduplex analysis. However, the inducible expression of p57 in three well-characterized human astrocytoma cell lines (U343 MG-A, U87 MG, and U373 MG) using the tetracycline repressor system leads to a potent proliferative block in G(1) as determined by growth curve and flow cytometric analyses. After the induction of p57, retinoblastoma protein, p107, and E2F-1 levels diminish, and retinoblastoma protein is shifted to a hypophosphorylated form. Morphologically, p57-induced astrocytoma cells became large and flat with an expanded cytoplasm. The inducible expression of p57 leads to the accumulation of senescence-associated beta-galactosidase marker within all astrocytoma cell lines such that approximately 75% of cells were positive at 1 week after induction. Induction of p57 in U373 astrocytoma cells generated a small population of cells ( approximately 15%) that were nonviable, contained discrete nuclear fragments on Hoechst 33258 staining, and demonstrated ultrastructural features characteristic of apoptosis. Examination of bax and poly-(ADP ribose) polymerase levels showed no change in bax, but decreased expression of poly-(ADP ribose) polymerase after p57 induction in all astrocytoma cell lines. These data demonstrate that the proliferative block imposed by p57 on human astrocytoma cells results in changes in the expression of a number of cell cycle regulatory factors, cell morphology, and a strong stimulus to cell senescence.

Alterations of the p53 and pRB pathways in human astrocytoma.

Human astrocytomas are characterized by a number of molecular changes affecting two critical tumor suppressor pathways: the pRB and the p53 pathways. Genetic alterations functionally eliminate pRB and p53 themselves or upstream and/or downstream molecules such as products of the Ink4a/ARF locus, p16Ink4a and p14ARF. As a result, malignant cells are defective in critical cell cycle and apoptosis regulatory elements contributing to unrelenting tumour growth and invasion. Current research aims to discover effective means of reconstituting p53 and pRB pathway components in an effort to attenuate the aggressive phenotype of astrocytoma.

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.

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 alpha1-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 (kappa-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.

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.

Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression.

BACKGROUND: Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene is frequently associated with malignant gliomas. One type of EGFR mutation in primary gliomas results in overexpression of an aberrant EGFR messenger RNA (mRNA) that lacks sequences of exons II through VI of the human EGFR gene. We observed that the aberrantly spliced EGFR mRNA contains a ribozyme cleavable sequence (5'-AAG GUA AUU-3') created by the joining of EGFR exon I to exon VII. We hypothesized that an appropriately designed ribozyme RNA could mediate site-specific cleavage of the aberrant EGFR mRNA and reduce the growth of aberrant EGFR-producing tumor cells. METHODS: We synthesized aberrant EGFR mRNA substrates and a sequence-specific hammerhead ribozyme (abEGFR-rib) to examine the ribozyme's activity in vitro. We also constructed an abEGFR-rib plasmid and introduced it into ERM5-1 cells, which are murine NIH3T3 cells transfected to express an aberrant EGFR complementary DNA. We measured the growth potential of the cotransfected cells in culture and in nude mice. RESULTS: The synthesized abEGFR-rib efficiently and specifically cleaved aberrant EGFR mRNA substrates in vitro. Expression of the transfected abEGFR-rib suppressed expression of aberrant EGFR mRNA in ERM5-1 cells and reduced the growth of tumors formed by the cotransfected cells in nude mice. Finally, the incorporation of bromodeoxyuridine, a measure of mitotic activity, was also decreased in abEGFR-rib-producing ERM5-1 cells in vivo. CONCLUSION: Ribozymes targeted to aberrant EGFR mRNA can inhibit the growth of tumors formed by cells that express this mRNA.

Role of glial filaments in cells and tumors of glial origin: a review.

In the adult human brain, normal astrocytes constitute nearly 40% of the total central nervous system (CNS) cell population and may assume a star-shaped configuration resembling epithelial cells insofar as the astrocytes remain intimately associated, through their cytoplasmic extensions, with the basement membrane of the capillary endothelial cells and the basal lamina of the glial limitans externa. Although their exact function remains unknown, in the past, astrocytes were thought to subserve an important supportive role for neurons, providing a favorable ionic environment, modulating extracellular levels of neurotransmitters, and serving as spacers that organize neurons. In immunohistochemical preparations, normal, reactive, and neoplastic astrocytes may be positively identified and distinguished from other CNS cell types by the expression of the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). Glial fibrillary acidic protein is a 50-kD intracytoplasmic filamentous protein that constitutes a portion of, and is specific for, the cytoskeleton of the astrocyte. This protein has proved to be the most specific marker for cells of astrocytic origin under normal and pathological conditions. Interestingly, with increasing astrocytic malignancy, there is progressive loss of GFAP production. As the human gene for GFAP has now been cloned and sequenced, this review begins with a summary of the molecular biology of GFAP including the proven utility of the GFAP promoter in targeting genes of interest to the CNS in transgenic animals. Based on the data provided the authors argue cogently for an expanded role of GFAP in complex cellular events such as cytoskeletal reorganization, maintenance of myelination, cell adhesion, and signaling pathways. As such, GFAP may not represent a mere mechanical integrator of cellular space, as has been previously thought. Rather, GFAP may provide docking sites for important kinases that recognize key cellular substrates that enable GFAP to form a dynamic continuum with microfilaments, integrin receptors, and the extracellular matrix.

Localization of aberrant messenger RNA of epidermal growth factor receptor (EGFR) in malignant glioma.

Human gliomas occasionally show rearrangements with deletions (exons II to VII) in the epidermal growth factor receptor (EGFR) gene, resulting in the expression of aberrant EGFR mRNA. This abnormality of EGFR gene expression is closely related to the malignancy of glioma. However, this EGFR gene abnormality has not been demonstrated directly in the glioma cells themselves, as gliomas consist of heterogeneous tissue components. In this study, we used in situ hybridization (ISH) to detect aberrant EGFR mRNA in the tumor cells in 26 human gliomas and 19 human glioma xenografts. We used digoxigenin (DIG)-labeled antisense oligonucleotide probes for ISH, which demonstrated aberrant EGFR mRNA in 2 of the 26 gliomas and in 3 of the 19 human glioma xenografts. ISH with an aberrant EGFR-specific probe (oligo-PA) revealed that EGFR mRNA was absent from multinucleated giant cells and from proliferating endothelial cells, but this transcript was present in small glioma cells. Identical aberrant EGFR mRNA was confirmed in these glioma and human glioma xenografts by Southern blotting. Northern blotting, and by reverse transcription-polymerase chain reaction (RT-PCR). These findings suggest that small tumor cells specifically express the aberrant EGFR mRNA in certain high grade gliomas.

Murine P-glycoprotein on stromal vessels mediates multidrug resistance in intracerebral human glioma xenografts.

Human glioma usually shows intrinsic multidrug resistance because of the blood-brain barrier (BBB), in which membrane-associated P-glycoprotein (P-gp), encoded by the human multidrug resistance gene MDR1, plays a role. We studied drug sensitivity to vincristine (VCR), doxorubicin (DOX) and nimustine (ACNU) in both intracerebrally and subcutaneously xenotransplanted human glioma. We examined the levels of MDR1 and murine mdr3 gene expression in the xenografts by reverse transcriptase polymerase chain reaction and the localization of P-gp by immunohistochemistry. Six of seven subcutaneously transplanted xenografts (scX) were sensitive to the above three drugs. In contrast, all three intracerebrally transplanted human glioma xenografts (icX) were resistant to P-gp-mediated drugs VCR and DOX, but were sensitive to the non-P-gp-mediated drug ACNU. Neither icX nor scX showed any MDR1 expression. Intracerebrally transplanted human glioma xenografts showed an increased level of murine mdr3 gene expression, whereas scX showed only faint expression. The localization of P-gp was limited to the stromal vessels in icX by immunohistochemistry, whereas scX expressed no P-gp. Our findings suggest that the P-gp expressed on the stromal vessels in icX is a major contributing factor to multidrug resistance in human glioma in vivo.

Advantage of in vivo chemosensitivity assay to detect vincristine-resistance in a human epidermoid carcinoma xenograft.

We examined both in vitro and in vivo chemosensitivity of the human epidermoid carcinoma xenograft xeKB3-1-R which shows overexpression of the multidrug resistance gene (MDR1). XeKB3-1-R was sensitive to vincristine (VCR, 6%) and doxorubicin (DOX, 9%) in the adhesive tumor cell culture system in vitro. However, this xenograft showed decreased sensitivity to VCR (65%) and DOX (42%) in an in vivo chemosensitivity assay. The in vivo resistance of xeKB3-1-R to both VCR and DOX was reversed by coadministration of cyclosporin A (VCR 22%, DOX 11%). The xenograft xeKB3-1-R expressed significantly higher levels of MDR1 than xeKB3-1. The results confirmed that multidrug resistance in xeKB3-1-R was related to enhanced MDR1 expression in vivo. The observed discrepancies between in vitro and in vivo chemosensitivities suggest that the in vivo sensitivity assay more accurately reflects drug resistance as a result of low-level MDR1 overexpression in solid tumors.

Marked elevation of carcinoembryonic antigen and carbohydrate antigen 19-9 in the peripheral blood as an initial manifestation of meningeal carcinomatosis.

Marked elevation of tumor markers in the peripheral blood was initially a sole manifestation of meningeal carcinomatosis in a man with gastric carcinoma. In addition to extensive meningeal carcinomatosis, no metastatic lesions were found at autopsy other than microscopic infiltration into a tiny paraaortic lymph node. Elevation of these markers in the peripheral blood is best explained by meningeal carcinomatosis. When elevation of these markers is otherwise unexplainable, meningeal carcinomatosis should be considered as a diagnostic possibility even in the absence of neurological symptoms.

Ultrastructural localization of P-glycoprotein on capillary endothelial cells in human gliomas.

The P-glycoprotein (P-Gp) encoded by the human multidrug-resistance gene MDR1 has been suggested to play certain roles in the blood-brain barrier (BBB). However, the detailed mechanism of the activity of P-Gp in multidrug-resistance (MDR) remains unclear in human glioma. We examined the localization of P-Gp in human glioma by immunohistochemical (IHC) and immunoelectron microscopic (IEM) methods with anti P-Gp monoclonal antibodies (C219, MRK16). We also examined MDR1 expression in primary glioma and xenografts by reverse transcription-polymerase chain reaction (RT-PCR) with human MDR1-specific primers. The IHC study showed no P-Gp expression on tumour cells but it was present on capillary endothelial cells and IEM analysis showed definitive localization on their luminal surface. MDR1 gene expression was detected in eight primary glioma and three normal brain specimens by RT-PCR, but not in glioma xenografts. The lack of MDR1 expression in these cells appears to be a consequence of the replacement of the original human stroma, including blood vessels, by murine stroma in glioma xenografts. The unique distribution of P-Gp on the capillary blood vessels was confirmed in human glioma by the results of immunohistochemical and molecular biological studies.

Development of akinetic mutism and hyperphagia after left thalamic and right hypothalamic lesions.

A case of childhood post-traumatic akinetic mutism is presented. The patient showed a hyperphagic condition while recovering from akinetic mutism. He had lesions in the left interlaminal nucleus of the thalamus, right globus pallidus, and right dorsomedial nucleus of the hypothalamus. Laboratory data indicated slightly disturbed hypothalamic functions. In general, akinetic mutism can be seen with bilateral destructive lesions, while hyperphagia may occur after destruction of dorsomedial hypothalamic nucleus, but it is very rare. This is the first reported case of akinetic mutism caused by a unilateral lesion.

[A case report: persistent primitive hypoglossal artery aneurysm].

The authors describe a case of persistent primitive hypoglossal artery aneurysm in a 42-year-old woman who had complained of headache, mainly in the occiput, for 5 days prior to admission. Because of a sudden exacerbation of the headache associated with vomiting, she was hospitalized on July 31, 1988. On admission, a cranial computed tomography scan demonstrated a high density lesion in the basal cisterns which suggested subarachnoid hemorrhage (SAH). Right carotid angiography revealed a persistent primitive hypoglossal artery and an aneurysm arising from this artery at the junction of the posterior inferior cerebellar artery. Bilateral vertebral arteries were shown to be hypoplastic. This was followed by a right suboccipital craniectomy on the 6th hospital day at which time a neck clipping was made. Her postoperative course was uneventful. On discharge on August 22, she was ambulatory and had no neurological deficit except for a mild hoarseness which developed after surgery. Well over one hundred cases of persistent primitive hypoglossal artery aneurysm have been reported. However, as far as we could discern, there have been only 9 cases of persistent primitive hypoglossal artery aneurysm including this present case. Most of the cases had SAH due to the rupture of these aneurysms with favorable outcome after the surgery. In addition, some embryological considerations were made.