This study describes genomic rearrangements near the 3' end of the epidermal growth factor receptor (EGFR) gene in eight glioblastomas displaying coamplification and expression of both normal and rearranged EGFR. In four of these cases, it was possible by PCR to amplify tumor EGFR cDNA, which allowed sequence determination of the 3' transcript alterations associated with the rearrangements. Such analysis revealed that the four cases have in common a deletion of 255 bases that encode a portion of the receptor's cytoplasmic domain. The remaining four cases revealed genomic rearrangements in the same region of the gene as those described above and revealed aberrant EGFR transcripts lacking the same 255 bases determined to be missing in the sequenced EGFR cDNAs as well as large regions of contiguous downstream sequences. Therefore, all of the eight cases described here express transcripts that do not encode large C-terminal, intracellular portions of the receptor. In three of the eight cases, the EGFR transcripts displaying a 3' alteration also displayed a 5' inframe deletion of sequences encoding a portion of the extracellular domain, and for one of the corresponding patients it was possible to determine that the two transcript alterations were acquired as separate events. We have now detected the 5' and/or 3' alterations in 21 of 32 cases of glioblastoma with EGFR amplification; no genetic alterations have been detected in glioblastomas without EGFR amplification. In combination with previously published reports, these data suggest the in vivo evolution of EGFR toward an increasingly oncogenic potential through gene amplification with subsequent and successive gene alterations.

The epidermal growth factor receptor gene has been found to be amplified and rearranged in human glioblastomas in vivo. Here we present the sequence across a splice junction of aberrant epidermal growth factor receptor transcripts derived from corresponding and uniquely rearranged genes that are coamplified and coexpressed with nonrearranged epidermal growth factor receptor genes in six primary human glioblastomas. Each of these six tumors contains aberrant transcripts derived from identical splicing of exon 1 to exon 8 as a consequence of a deletion-rearrangement of the amplified gene, the extent of which is variable among these tumors. In spite of this intertumoral variability, each intragenic rearrangement results in loss of the same 801 coding bases (exons 2-7) and creation ofa new codon at the novel splice site in their corresponding transcripts. These rearrangements do not, however, affect the mRNA sequence for the signal peptide, the first five codons, or the reading frame downstream of the rearrangement.The normal epidermal growth factor receptor (EGFR) gene product is a 170-kDa transmembrane glycoprotein found on many normal and malignant cells (1-7). The extracellular binding of one of its two known endogenous ligands, epidermal growth factor and transforming growth factor a, results in conformational changes of the extracellular domain (8), the activation of the receptor's intracellular tyrosine kinase activity (9,10), and the stimulation of DNA synthesis. A constitutively activated and cell-transforming variant of this receptor, with most of the extracellular domain deleted and further carboxyl-terminal deletions and mutations, is encoded by the v-erbB oncogene of avian erythroblastosis virus (11,12). In brain tumors, EGFR gene amplification is exclusively seen in the most malignant variants of adult gliomas, especially the glioblastomas (13). Studies of DNA, mRNA, and protein from primary human glioblastomas and xenografted glioblastomas indicate that EGFR gene rearrangements are frequently associated with EGFR amplification in such tumors (1, 14-21); most results suggest rearrangements causing the loss of coding sequences for the extracellular domain (17,18,20).We have studied the DNA and RNA from primary tumor tissue from six patients with brain tumors histopathologically classified as glioblastomas (22,23) where the tumors were determined to have amplification of the EGFR gene (13).Here we demonstrate that each of these tumors has highly expressed aberrant EGFR transcripts resulting from identical coerced splicing of uniquely rearranged and amplified EGFR genes.

Neuropeptide Y (NPY), a 36-amino-acid peptide widely expressed in the brain is involved in many physiological responses, including hypothalamic control of food intake and cardiovascular homeostasis. NPY mediates its effects through binding to the Y1, Y2 and Y5 G-protein-coupled receptors. Little is known of the role of the Y2 receptor in mediating the different NPY effects. We inactivated the Y2 receptor subtype in mice and found that these mice developed increased body weight, food intake and fat deposition. The null mutant mice showed an attenuated response to leptin administration but a normal response to NPY-induced food intake and intact regulation of re-feeding and body weight after starvation. An absence of the Y2 receptor subtype also affected the basal control of heart rate, but did not influence blood pressure. These findings indicate an inhibitory role for the Y2 receptor subtype in the central regulation of body weight and control of food intake.

Neuropeptide Y (NPY), a 36-aa peptide, is widely distributed in the brain and peripheral tissues. Whereas physiological roles of NPY as a hormone͞neurotransmitter have been well studied, little is known about its other peripheral functions. Here, we report that NPY acts as a potent angiogenic factor in vivo using the mouse corneal micropocket and the chick chorioallantoic membrane (CAM) assays. Unlike vascular endothelial growth factor (VEGF), microvessels induced by NPY had distinct vascular tree-like structures showing vasodilation. This angiogenic pattern was similar to that induced by fibroblast growth factor-2, and the angiogenic response was dose-dependent. (2). Although the neuropeptides were isolated and characterized several decades ago and the NPY receptor cDNAs have been cloned for 5-10 yr, surprisingly little is known about the molecular mechanisms that regulate NPY receptor activity and the biological significance of NPY in the periphery.It has been found that NPY regulates the vascular tone by inducing contractions of blood vessels (3). NPY also stimulates growth of vascular smooth muscle cell and hypertrophy of ventricular cardiomyocytes (4, 5). The trophic effect of NPY on blood vessels does not seem to be limited to vascular smooth muscle cells.