The INK4A/ARF locus: role in cell cycle control and apoptosis and implications for glioma growth.

The unique INK4A/ARF locus at chromosome 9p21 encodes two distinct proteins that intimately link the pRB and p53 tumour suppressor pathways. p16INK4A has been identified as an inhibitor of the cell cycle, capable of inducing arrest in G1 phase. p14/p19ARF on the other hand can induce both G1 and G2 arrest due to its stabilizing effects on the p53 transcription factor. In addition to their roles in growth arrest, both proteins are involved in cellular senescence and apoptosis. The frequent mutation or deletion of INK4A/ARF in human tumours as well as the occurence of tumours in the murine knockout models have identified both p16 and ARF as bona fide tumour suppressors.

Tracking the invasiveness of human astrocytoma cells by using green fluorescent protein in an organotypical brain slice model.

OBJECT: Although it is known that malignant astrocytomas infiltrate diffusely into regions of normal brain, it is frequently difficult to identify unequivocally the solitary, invading astrocytoma cell in histopathological preparations or experimental astrocytoma models. The authors describe an experimental system that facilitates the tracking of astrocytoma cells by using nonneoplastic cerebral tissue as the substrate for invasion. METHODS: Cerebral tissue was cut into 1-mm-thick slices and cultured in the upper chamber of a Transwell culture dish on top of a polyester membrane (0.4-mm pore size) that was bathed in medium supplied by the lower chamber. Two astrocytoma cell lines, U-87 MG (U87) and U343 MG-A (U343), were selected because of their differing basal cell motilities in monolayer cultures. The astrocytoma cells were stably transfected with vectors that expressed green fluorescent protein (GFP), either alone or as a fusion protein with the receptor for hyaluronic acid-mediated motility (RHAMM) in either sense or antisense orientations. Stably transfected clones that had high levels of GFP expression were selected using the direct visualization provided by fluorescence microscopy and fluorescence-activated cell-sorter analysis. The GFP-expressing astrocytoma cell clones were implanted into the center of the brain slice and the degree of astrocytoma invasion into brain tissue was measured at different time points by using the optical sectioning provided by the confocal laser microscope. The authors observed that GFP-expressing astrocytoma cells could be readily tracked and followed in this model system. Individual astrocytoma cells that exhibited green fluorescence could be readily identified following their migration through the brain slices. The GFP-labeled U87 astrocytoma cells migrated farther into the brain slice than the U343 astrocytoma cells. The RHAMM-transfected GFP-labeled astrocytoma cells also infiltrated farther than the GFP-labeled astrocytoma cells themselves. The expression of antisense RHAMM virtually abrogated the invasion of the brain slices by both astrocytoma cell lines. CONCLUSIONS: The authors believe that this organotypical culture system may be of considerable utility in studying the process of astrocytoma invasion, not only because it provides a better representation of the extracellular matrix molecules normally encountered by invading astrocytoma cells, but also because the GFP tag enables tracking of highly migratory and invasive astrocytoma cells under direct vision.

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.

Molecular biology and neurosurgery in the third millennium.

The application of techniques in molecular biology to human neurosurgical conditions has led to an increased understanding of disease processes that affect the brain and to novel forms of therapy that favorably modify the natural history of many of these conditions. Molecular strategies are currently being either used or sought for brain tumors, stroke, neurodegenerative diseases, vascular malformations, spinal degenerative diseases, and congenital malformations of the central nervous system. Considering that the structure of deoxyribonucleic acid was ascertained by Watson and Crick as recently as 1953, the progress that has been made to implement molecular medicine in clinical practice has been meteoric. More than 2000 patients have been treated in approved gene therapy trials throughout the world. Many of these patients have been treated for neurological diseases for which conventional medical therapies have been of limited utility. As part of this continuing series on advances in neurosurgery in the third millennium, we first reflect on the history of the nascent field of molecular biology. We then describe the powerful techniques that have evolved from knowledge in this field and have been used in many publications in Neurosurgery, particularly within the past decade. These methods include commonly used techniques such as advanced cytogenetics, differential display, microarray technology, molecular cell imaging, yeast two-hybrid assays, gene therapy, and stem cell utilization. We conclude with a description of the rapidly growing field of bioinformatics. Because the Human Genome Project will be completed within 5 years, providing a virtual blueprint of the human race, the next frontier (and perhaps our greatest challenge) will involve the development of the field of "proteomics," in which protein structure and function are determined from the deoxyribonucleic acid blueprint. It is our conviction that neurosurgeons will continue to be at the forefront of the treatment of patients with neurological diseases using molecular strategies, by performing essential research leading to increased understanding of diseases, by conducting carefully controlled studies to test the effects of treatments on disease processes, and by directly administering (by neurosurgical, endovascular, endoscopic, or stereotactic means) the treatments to patients.

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.

Co-expression of nestin and vimentin intermediate filaments in invasive human astrocytoma cells.

Intermediate filaments (IFs) are highly diverse intracytoplasmic proteins within the cytoskeleton which exhibit cell type specificity of expression. A growing body of evidence suggests that IFs may be involved as collaborators in complex cellular processes controlling astrocytoma cell morphology, adhesion and proliferation. As the co-expression of different IF subtypes has been linked to enhanced motility and invasion in a number of different cancer subtypes, we undertook the present study to examine the expression of vimentin and nestin in a panel of human astrocytoma cell lines whose tumorigenicity, invasiveness and cytoskeletal protein profiles are well known. Astrocytoma cells were examined for IF protein expression by immunofluorescence confocal and immunoelectron microscopy. The motility of all cell lines was determined by computerized time-lapse videomicroscopy. Invasive potential of astrocytoma cells was determined using Matrigel as a barrier to astrocytoma cell invasion in vitro. Vimentin was expressed by all astrocytoma cell lines. On the other hand, nestin was variably expressed among the different cell lines. The most motile and invasive astrocytoma cell line in our study was antisense GFAP-transfected U251 (asU251) astrocytoma cells which showed marked up-regulation of nestin expression compared to the U251 parental cell line and controls. The U87 astrocytoma cell line also demonstrated high nestin expression levels and was associated with an increased basal motility rate and a high degree of invasiveness through Matrigel. U343 astrocytoma cells did not express nestin, but had high levels of GFAP. It had the lowest motility rate and invasiveness of all the astrocytoma cell lines examined. Taken together, these data suggest that for the astrocytoma cell lines examined in this study, nestin and vimentin co-expression may serve as a marker for an astrocytoma cell type with enhanced motility and invasive potential. Further studies are required to determine the mechanism by which dual-IF protein expression alters other cytoskeletal or cell surface receptor protein components important in the process of astrocytoma invasion.

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.

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.

Investigation of the genes for RET and its ligand complex, GDNF/GFR alpha-I, in small cell lung carcinoma.

RET is a receptor tyrosine kinase expressed in neuroendocrine cells and in tumors of these cell types. RET activation may be mediated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor alpha-1 (GFR alpha-1). Activating RET mutations are found in the inherited cancer syndrome multiple endocrine neoplasia type 2 and in a subset of the related sporadic tumors, medullary thyroid carcinoma and pheochromocytoma, both being derived from neuroendocrine tissues. In one small study, mutations were identified in another tumor with neuroendocrine features, small cell lung carcinoma (SCLC). To determine whether RET mutations contribute to the pathogenesis of SCLC, we examined a panel of 54 SCLC cell lines. No mutations were identified in RET exons 10, 11, and 13-16, regions previously implicated in SCLC or other neuroendocrine tumors. We further examined the expression pattern of RET and the genes encoding the components of its ligand complex GDNF and GFR alpha-1, in 21 SCLC lines by using RT-PCR. Although we found no consistent pattern of expression for these three genes, RET was expressed in 57% of SCLC lines. Thus, although RET mutations appear unlikely to be an important step in the tumorigenesis of SCLC, the frequent expression of this gene suggests that RET may have a mitogenic role in a subset of SCLC cell lines.

Expression of RET 3' splicing variants during human kidney development.

The mature mammalian kidney arises through a series of reciprocal inductive interactions between two different cell groups, the ureteric bud epithelium and the metanephric mesenchyme. The RET receptor tyrosine kinase is required for induction and development of the metanephric kidney. Differential splicing at the 3' end of RET results in transcripts encoding three isoforms that differ with respect to their C-terminal 9 (RET9), 51 (RET51) or 43 (RET43) amino acids. In vitro assays have identified differences in the abilities of the RET9 and RET51 isoforms to induce differentiation suggesting functional differences between these proteins. We examined the relative expression levels of the three RET 3' splicing variants in developing human kidney using semi-quantitative RT-PCR. We observed consistent expression of the RET9 and RET43 variants in kidney samples spanning 7.5 through 24 weeks gestation. At early gestational ages (7.5-8.5 weeks), RET51 expression was very low (+/-5%) compared to RET9; however, a rapid seven fold increase in expression was detected by 9 weeks. Our data suggest that RET51 may contribute to differentiation-related events occurring after 8.5 weeks gestation rather than to induction of the human kidney.

The expression of RET and its multiple splice forms in developing human kidney.

A series of inductive events between two different cell groups, the ureteric bud epithelium and metanephric mesenchyme, gives rise to the functional mammalian kidney. These reciprocal inductive interactions involve a number of molecules, one of which is the RET receptor tyrosine kinase. The phenotype of mice lacking functional RET includes kidney agenesis or severe dysgenesis, indicating a requirement for RET in kidney organogenesis. To investigate RET expression in human kidney development, we used a semi-quantitative RT-PCR-based strategy to examine a panel of kidney RNA samples ranging from 8-24 weeks gestational age. We found RET expression was highest earlier in development (14 weeks) with expression decreasing through to 24 weeks gestation. While three alternative RET transcripts generated by exon skipping at the 5' end of the gene were all detected throughout kidney development, expression of one transcript, RET2/6, where exon 2 was spliced to exon 6, varied relative to full length RET during this period. Levels of RET2/6 were highest at the earliest age of fetal kidney examined (8 weeks) and decreased relative to all other RET transcripts to low adult levels. The period of high expression coincides with a period of rapid bud bifurcation. Thus, it is possible that RET2/6 has a role in the early growth and differentiation of the human kidney.

De novo mutation of GDNF, ligand for the RET/GDNFR-alpha receptor complex, in Hirschsprung disease.

Hirschsprung disease (HSCR) is a common congenital abnormality characterized by absence of the enteric ganglia in the hind gut. In 10-40% of HSCR cases, mutations of the RET receptor tyrosine kinase have been found. The recent identification of a multimeric RET ligand/receptor complex suggested that mutations of genes encoding other components of this complex might also occur in HSCR. To investigate this role, we examined the gene for glial cell line-derived neurotrophic factor (GDNF), the circulating ligand of the RET receptor complex, for mutations in a panel of sporadic and familial HSCR. We identified GDNF sequence variants in 2/36 HSCR patients. The first of these was a conservative change which did not affect the GDNF protein sequence. The second variant was a de novo missense mutation in a family with no history of HSCR and without mutation of the RET gene. Thus, our data are consistent with a causative role for GDNF mutations in some HSCR cases.