Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus.

Immunohistochemistry is widely used in biomedical research to localize specific epitopes of molecules in cells and tissues. The validity of interpretations based on immunohistochemistry requires appropriate positive and negative controls that are often not reported in publications. This omission may lead to incorrect interpretations and irreproducible results in the literature and contribute to wasted time, effort, and resources as well as erosion of confidence in scientific investigation by the general public, legislative bodies and funding agencies. The present article summarizes essential controls required for validation of immunohistochemical findings and represents a standard of practice for the use of immunohistochemistry in research and diagnostic investigations. Adherence to the guidelines described in the present article can be cited by authors as support for the validity of interpretations of the immunohistochemistry reported in their publications.

The epidermal growth factor receptor (EGFR) 1 transduces signals for proliferation and differentiation in neuronal and epithelial tissues. EGFR signal transduction is initiated by ligand binding, receptor dimerization, and inter-and intrareceptor activation of kinase activity, with concomitant phosphorylation of tyrosine residues (1). Assembly of SH2 containing signal transduction molecules ensues at the receptor with activation of downstream signaling pathways, several of which appear to converge at the MAPK/extracellular signal-related kinase kinase (MEK) with subsequent activation of the p42/p44 MAP kinases, a family of dual specificity serine/threonine kinases (2). MAP kinases phosphorylate a number of cytoplasmic substrates including cytoplasmic phospholipase A 2 (cPLA 2 ) as well as the nuclear ternary complex factor Elk-1 and Myc (3, 4, 29). Overexpression of intact EGFR in the presence of ligand or truncation of receptor by removal of the extracellular domain induces transformation in fibroblast systems (5, 6). The overexpression of intact EGFR has been documented in a variety of neuronal and epithelial neoplasms and carries a poor prognosis for patients with these malignancies, suggesting that overexpression of receptor may confer a proliferative advantage in the presence of limiting amounts of growth factor (7). The retroviral oncogene v-erbB encodes a form of the EGFR lacking the majority of the extracellular domain and containing mutations in the transmembrane region and carboxyl terminus. Mutational analysis has demonstrated that deletion of the extracellular domain causes downstream activation of MAP kinase and contributes significantly to transformation by v-erbB (6,8). In human tumors, various deletions of the extracellular domain of the EGFR have been identified in astrocytic neoplasms (9 -12). The most common of these alterations, termed the EGFRvIII, contains a deletion of nucleotides 275-1075 of the EGFR cDNA (9). The mutant gene product is a 145-kDa receptor that undergoes spontaneous homodimerization in transfected NIH3T3 cells and demonstrates increased receptor autophosphorylation.2 Expression of this mutant receptor in NIH3T3 cells is transforming, and a large proportion of cellular GRB2 associates with the EGFRvIII.2 This suggests a link with the Ras-MAP kinase pathway, which we have explored to delineate novel aspects of EGFRvIII signaling.In this study, we show that in NIH3T3 cells the EGFRvIII stimulates strong downstream activation of MEK, but the degree of p42 and p44 MAP kinase activation is more modest, recapitulating chronic EGF exposure in cells containing fulllength human EGFR. Down-regulation of protein kinase C by phorbol esters does not abrogate constitutive activation by

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxelmediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of ␤-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed ␤-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII-and HER2-expressing cells demonstrated equivalent total ␤-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2.95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both ␤-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.

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