Eric C. Holland

Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice.

Gliomas are the most common primary malignant brain tumours and are classified into four clinical grades, with the most aggressive tumours being grade 4 astrocytomas (also known as glioblastoma multiforme; GBM). Frequent genetic alterations in GBMs (refs 2–5) result in stimulation of common signal transduction pathways involving Ras, Akt and other proteins. It is not known which of these pathways, if any, are sufficient to induce GBM formation. Here we transfer, in a tissue-specific manner, genes encoding activated forms of Ras and Akt to astrocytes and neural progenitors in mice. We found that although neither activated Ras nor Akt alone is sufficient to induce GBM formation, the combination of activated Ras and Akt induces high-grade gliomas with the histological features of human GBMs. These tumours appear to arise after gene transfer to neural progenitors, but not after transfer to differentiated astrocytes. Increased activity of RAS is found in many human GBMs (ref. 11), and we show here that Akt activity is increased in most of these tumours, implying that combined activation of these two pathways accurately models the biology of this disease.

Development of a flexible and specific gene delivery system for production of murine tumor models.

To develop models of human cancer we have expressed the avian retroviral receptor, TVA, under a variety of mammalian promoters in transgenic mice, thus rendering mice susceptible to infection with avian leukosis virus-derived gene vectors. TVA-based retroviral gene transfer offers advantages over current murine models of human cancer. A single transgenic mouse line can be used to evaluate multiple genetic lesions, individually and in combination. Furthermore, mutant genes are introduced somatically into animals, as occurs in the majority of naturally occurring tumors. Because the avian viral vectors replicate only in avian cells, the viral receptor in infected transgenic mouse cells remains available for multiple rounds of infection with different ASLV vectors. We discuss the theoretical and practical aspects of using recombinant avian retroviruses with TVA transgenic mice to generate cancer models.

Astrocytes Give Rise to Oligodendrogliomas and Astrocytomas after Gene Transfer of Polyoma Virus Middle T Antigen in Vivo

The cells of origin for oligodendrogliomas and astrocytomas are not known but are presumed to be oligodendrocyte and astrocyte precursors, respectively. In this paper we report the generation of mixed gliomas from in vivo transformation of glial fibrillary acidic protein (GFAP)-positive cells (differentiated astrocytes) with polyoma virus middle T antigen (MTA). MTA is a powerful oncogene that activates a number of signal transduction pathways, including those proposed to be involved in gliomagenesis, and has been shown to induce tumors in many cell types. We have achieved transfer of MTA expression specifically to GFAP(+) cells in vivo using somatic cell gene transfer, and find resultant formation of anaplastic gliomas with mixed astrocytoma and oligodendroglioma morphological features. We conclude that GFAP- expressing astrocytes, with appropriate signaling abnormalities, can serve as the cell of origin for oligodendrogliomas, astrocytomas, or mixed gliomas.

A Mouse Model for Glioma: Biology, Pathology, and Therapeutic Opportunities

The epidermal growth factor receptor (EGFR) gene is amplified or mutated in 30-50% of human glioblastoma multiforme. These mutations are usually associated with deletions of the INK4α-ARF locus, which encodes 2 gene products (p16INK4a and p19ARF) involved in cell cycle arrest and apoptosis. We have investigated the role of EGFR mutation in gliomagenesis using avian retroviral vectors to transfer a mutant EGFR gene to glial precursors and astrocytes in transgenic mice. These mice express tv-a, a gene encoding the retrovirus receptor TVA, which is under the control of brain cell type-specific promoters. We demonstrate that expression of a constitutively active, mutant form of EGFR in cells in the glial lineage can induce lesions with many similarities to human gliomas, including increased cell density, vascular proliferation, and immunohistochemical staining for glial fibrillary acidic protein (GFAP) and nestin. We also demonstrate that primary astrocytes cultured from transgenic mice expressing tv-a from the GFAP promoter are efficiently infected in culture, and such genetically modified cell cultures can be tumorigenic in nude mice. The combinations of genetic lesions (eg, mutated EGFR, INK4a-/-) leading to tumor formation in these 2 mouse systems are similar to those found in human gliomas. These genetically defined animal models for gliomas will allow for the testing of therapies that are targeted specifically at the gene products involved in the pathogenesis of gliomas.

Signing on to expression profiles

Unique expression patterns of a large number of genes can now define cell specific signatures under a given context. Cell specific signatures will be invaluable to understanding differentiation, cell type specificity and the cellular biology of environmental responses. Expression signatures could greatly enhance drug development by increasing efficacy and decreasing toxicity of therapeutic compounds. We have used (Affymetrix) oligonucleotide microarrays to survey and compare the expression profiles of 7000 human genes from cells of various origins, including lymphocytes, umbilical vein endothelial cells (HUVEC), a breast adenocarcinoma cell line (MCF7) and a glioblastoma cell line (U373MG). Environmental context such as time in culture, state of differentiation, media composition, CO2 content and culture confluency may alter cell signatures. Chemical compound manipulation of cell lines provides one mechanism to dissect function at the gene expression level. For example, extended treatment of endothelial cells with vascular endothelial growth factor (VEGF) results in greater than two-fold changes in expression level of approximately 100 genes, while extended treatment of glioma cell lines with platelet derived growth factor (PDGF) results in greater that two-fold changes in 39 genes. Analysing expression data from multiple cell lines can also be used to identify similarities of expression profiles for all cell lines. We have observed that an average cell type expresses 2335 out of 7000 genes assayed (approxiamately 33%). Sixty-four percent of the 2335 genes are expressed in endothelial, U373MG, and MCF7 cells, with a total of 630 expressed at similar levels in all three cell types despite variation in growth media. Additional cell types are being analyzed to refine the universal and cell-specific signatures.