The epidermal growth factor receptor (EGFR) gene is amplified or mutated in 30%-50% of human gliobastoma multiforme (GBM). These mutations are associated usually with deletions of the INK4a-ARF locus, which encodes two gene products (p16 INK4a and p19 ARF ) 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 expressing tv-a, a gene encoding the retrovirus receptor. TVA, under 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. These lesions occur more frequently with gene transfer to mice expressing tv-a from the progenitor-specific nestin promoter than to mice expressing tv-a from the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, suggesting that tumors arise more efficiently from immature cells in the glial lineage. Furthermore, EGFR-induced gliomagenesis appears to require additional mutations in genes encoding proteins involved in cell-cycle arrest pathways. We have produced these combinations by simultaneously infecting tv-a transgenic mice with vectors carrying cdk4 and EGFR or by infecting tv-a transgenic mice bearing a disrupted INK4a-ARF locus with the EGFR-carrying vector alone. Moreover, EGFR-induced gliomagenesis does not occur in conjunction with p53 deficiency, unless the mice are also infected with a vector carrying cdk4. The gliomagenic combinations of genetic lesions required in mice are similar to those found in human gliomas.[Key Words: EGFR; gliomagenesis; cell cycle arrest] Received July 29, 1998; accepted in revised form October 14, 1998. Gliomas are the most common forms of primary brain tumors and are classified into four clinical grades (Kleihues et al. 1993). The most aggressive tumors, grade 4, also known as glioblastoma multiforme (GBM), appear to arise either de novo as GBMs or present initially as lower grade tumors that progress to higher grades over time . Certain mutations and other properties are highly correlated with one or the other of these two pathogenic pathways. Gliomas that are first detected as grade 4 tumors usually lack an intact INK4a-ARF tumor suppressor locus (Schmidt et al. 1994;He et al. 1995;Ichimura et al. 1996); and they are thus unable to make the two INK4a-ARF gene products, p16 INK4a and p19 ARF , which arrest the cell cycle by different pathways in G 1 and in both G 1 and G 2 , respectively ( Fig. 1; Serrano et al. 1993;Quelle et al. 1995). In addition, these tumors occur frequently in older patients, contain mutations or amplification of the EGFR gene, tend to be more aggressive, and are generally diploid He et al. 1994;Schlegel et al. 1994;Ono et al. 1996;Hayashi et al. 1997). In contrast, gliomas that progress from lower to higher grade lesions are usually wild type for INK4a-ARF , but have an ampl...

Wnt-1 was ®rst identi®ed as a protooncogene activated by viral insertion in mouse mammary tumors. Transgenic expression of this gene using a mouse mammary tumor virus LTR enhancer causes extensive ductal hyperplasia early in life and mammary adenocarcinomas in approximately 50% of the female transgenic (TG) mice by 6 months of age. Metastasis to the lung and proximal lymph nodes is rare at the time tumors are detected but frequent after the removal of the primary neoplasm. The potent mitogenic e ect mediated by Wnt-1 expression does not require estrogen stimulation; tumors form after an increased latency in estrogen receptor a-null mice. Several genetic lesions, including inactivation of p53 and over-expression of Fgf-3, collaborate with Wnt-1 in leading to mammary tumors, but loss of Sky and inactivation of one allele of Rb do not a ect the rate of tumor formation in Wnt-1 TG mice.

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 virusderived gene vectors. TVA-based retroviral gene transfer oers 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 dierent ASLV vectors. We discuss the theoretical and practical aspects of using recombinant avian retroviruses with TVA transgenic mice to generate cancer models.

Nearly all human gliomas exhibit alterations in one of three genetic loci governing G 1 arrest: INK4a-ARF, CDK4, or RB. To discern the roles of CDK4 amplification and INK4a-ARF loss in gliomagenesis, we compared the behavior of astrocytes lacking a functional INK4a-ARF locus with astrocytes overexpressing CDK4. Either a deficiency of p16 INK4a and p19 ARF or an increase in Cdk4 allows cultured astrocytes to grow without senescence. Astrocytes overexpressing CDK4 grow more slowly than INK4a-ARF-deficient astrocytes and convert to a tetraploid state at high efficiency; in contrast, INK4a-ARF-deficient cells remain pseudodiploid, consistent with properties observed in human gliomas with corresponding lesions in these genes. Received July 20, 1998; revised version accepted October 14, 1998. Over half of high-grade human gliomas lack a functional INK4a-ARF locus (Jen et al. 1994;Schmidt et al. 1994) and hence can produce neither p16 INK4a nor p19 ARF , the two proteins encoded by this locus (Quelle et al. 1995). Most of the remaining gliomas either lack the RB gene or demonstrate a 10-to 100-fold amplification of the CDK4 locus (He et al. 1994(He et al. , 1995Ichimura et al. 1996). We are developing animal models for gliomagenesis in hopes of understanding these patterns and discerning the contributions made to tumor formation by each abnormality . We have taken advantage of two genetic alterations in mice: disruption of the INK4a-ARF locus by targeted mutation (Serrano et al. 1996) and astrocyte-specific expression of a transgene encoding TVA, the receptor for subgroup A avian leukosis viruses (ALV) (Holland and Varmus 1998). Production of TVA molecules by these cells makes them susceptible to infection by RCAS vectors carrying coding domains for CDK4 and other genes .We have used this gene transfer system to investigate the effects of INK4a-ARF loss and CDK4 overexpression in astrocyte cell culture and to determine whether similarities exist between the cultured cells and human gliomas with similar abnormalities. Results and Discussion Loss of INK4a-ARF and overexpression of CDK4 both immortalize astrocyte culturesTo compare the growth properties of INK4a-ARF-deficient and CDK4-overexpressing astrocytes, we subjected appropriately selected cultures to repeated passage at standard density and counted the number of cells at each passage. Astrocytes overexpressing CDK4 were prepared by infecting primary brain cultures from Gtv-a mice [carrying a tv-a transgene under the control of the astrocytespecific glial fibrillary acidic protein (GFAP) promoter] with an RCAS vector carrying the human CDK4 cDNA (RCAS-cdk4). Cultures of INK4a-ARF-deficient astrocytes were prepared by infecting primary brain cell cultures from Gtv-a transgenic; INK4a-ARF −/− mice with an RCAS vector bearing the puro-R gene (RCAS-puro) ) and selecting for resistance to puromycin. Parallel cultures were prepared by infecting brain cells from Gtv-a transgenic mice with RCAS vectors carrying the alkaline phosphatase (AP) or the basic fibroblast grow...