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Overexpression of the epidermal growth factor receptors (EGFRs) and human epidermal growth factor receptor 2 occurs frequently in human cancers and is associated with aggressive tumor behavior and poor patient prognosis. We have investigated the effects in vitro and in vivo of a new class of inhibitor molecules on the growth of several human cancer cell lines. BIBX1382 [N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine] and BIBU1361 [(3-chloro-4-fluoro-phenyl)-[6-(4-diethylaminomethyl-piperidin-1yl)-pyrimido[5,4-d]pyrimidin-4-yl]-amine] are two new selective EGFR kinase inhibitors that do not block the activity of other tyrosine kinases. BIBU1361 blocked epidermal growth factor-induced phosphorylation of EGFR and also prevented downstream responses such as mitogen-activated protein kinase kinase (MAPK/extracellular signal-regulated kinase kinase) and MAPK activation in cells. In accordance with these observations thymidine incorporation into EGFR-expressing KB cells was selectively and potently inhibited by BIBX1382 and BIBU1361 with half-maximally effective doses in the nanomolar range. Oral administration of these compounds inhibited the growth of established human xenografts in athymic mice, including vulval and head and neck squamous cell carcinomas. Tumor growth inhibition by BIBX1382 coincided with reduced pEGFR and Ki-67 levels in vivo, which is in accordance with the expected effect of EGFR inhibitors. Collectively, these results show that the structural class of pyrimidopyrimidines, exemplified here by BIBX1382 and BIBU1361, represents an interesting scaffold for the design of EGFR inhibitors.

We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

The identification, purification, and biochemical characterization of specific markers for neuroglial cells in the central nervous system is an essential step toward a better understanding of the function of glial cells. This manuscript reports the identification and purification of a neuroglia-associated protein (NAP-185) with an apparent molecular mass of 185 kDa. While its expression is not restricted to the brain, it was first identified in a specific subpopulation of glial cells when chick brain stem sections were analyzed with an affinity-purified rabbit antiserum raised against the catalytic domain of the T-cell protein tyrosine phosphatase. This 185-kDa antigen was purified to apparent homogeneity and confirmed to be responsible for the neuroglial staining observed. In spite of its immunological relation to T-cell protein tyrosine phosphatase, purified NAP-185 failed to display tyrosine phosphatase activity. The primary sequence of five NAP-185-derived peptides shows that this protein has not yet been characterized and that it is possibly related to AP180, a clathrin-associated protein.

The two mouse melanoma cell lines B16-F1 and B16-G4F retain their melanogenic capacity when cultured in vitro. Melanotropic peptides such as alpha-melanocyte-stimulating hormone (alpha-MSH) induce formation and release of melanin pigment in B16-F1 cells. In contrast, B16-G4F cells do not respond to alpha-MSH. Using receptor-binding analysis and photoaffinity crosslinking we demonstrate that the lack of response of B16-G4F cells to alpha-MSH is due to the absence of functional MSH receptors from the cell surface. Northern blot analysis of receptor mRNA revealed that MSH receptor mRNA is not expressed in B16-G4F cells. These cells represent a new tool for the study of signal pathways related to the control of melanogenesis in melanoma cells.