Recent clinical successes of small-molecule epidermal growth factor receptor (EGFR) inhibitors in treating advanced nonsmall cell lung cancer (NSCLC) have raised hopes that the identification of other deregulated growth factor pathways in NSCLC will lead to new therapeutic options for NSCLC. Met, the receptor for hepatocyte growth factor, has been implicated in growth, invasion, and metastasis of many tumors including NSCLC. To assess the functional role for Met in NSCLC, we evaluated a panel of nine lung cancer cell lines for Met gene amplification, Met expression, Met pathway activation, and the sensitivity of the cell lines to short hairpin RNA (shRNA)-mediated Met knockdown. Two cell lines, EBC-1 and H1993, showed significant Met gene amplification and overexpressed Met receptors which were constitutively phosphorylated. The other seven lines did not exhibit Met amplification and expressed much lower levels of Met, which was phosphorylated only on addition of hepatocyte growth factor. We also found a strong up-regulation of tyrosine phosphorylation in B-catenin and p120/D-catenin in the Met-amplified EBC-1 and H1993 cell lines. ShRNA-mediated Met knockdown induced significant growth inhibition, G 1 -S arrest, and apoptosis in EBC-1 and H1993 cells, whereas it had little or no effect on the cell lines that do not have Met amplification. These results strongly suggest that Met amplification identifies a subset of NSCLC likely to respond to new molecular therapies targeting Met.
The receptor tyrosine kinase c-Met is an attractive target for therapeutic blockade in cancer.
c-Met is a transmembrane tyrosine kinase that mediates activation of several signaling pathways implicated in aggressive cancer phenotypes. In recent years, research into this area has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt aberrant c-Met signaling. Screening efforts identified a unique class of 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one kinase inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81 (MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report details the development of 81 and provides a description of its unique biochemical properties.
Cellular glutathione peroxidase is a key intracellular antioxidant enzyme that contains a selenocysteine residue at its active site. Selenium, a selenocysteine incorporation sequence in the 3-untranslated region of the glutathione peroxidase mRNA, and other translational cofactors are necessary for "read-through" of a UGA stop codon that specifies selenocysteine incorporation. Aminoglycoside antibiotics facilitate read-through of premature stop codons in prokayotes and eukaryotes. We studied the effects of G418, an aminoglycoside, on cellular glutathione peroxidase expression and function in mammalian cells. Insertion of a selenocysteine incorporation element along with a UGA codon into a reporter construct allows for read-through only in the presence of selenium. G418 increased read-through in selenium-replete cells as well as in the absence of selenium. G418 treatment increased immunodetectable endogenous or recombinant glutathione peroxidase but reduced the specific activity of the enzyme. Tandem mass spectrometry experiments indicated that G418 caused a substitution of L-arginine for selenocysteine. These data show that G418 can affect the biosynthesis of this key antioxidant enzyme by promoting substitution at the UGA codon.Cellular glutathione peroxidase-1 (GPx-1) 2 is one of several mammalian glutathione peroxidases that reduce hydrogen and lipid peroxides using glutathione (GSH) as a reductant (1). GPx-1 is ubiquitously expressed and is localized mostly to the cytosol. Each monomer of the tetrameric enzyme contains a selenocysteine (SEC) residue at the active site of the protein (2, 3). This selenium-containing amino acid is incorporated during translation through the recognition of a UGA codon as a site for SEC incorporation rather than as a site for termination of translation.Unique mechanisms are involved in the biosynthesis of SEC as well as in the insertion of this selenium-containing amino acid into the nascent polypeptide chain (reviewed in Refs. 4 and 5). A special tRNA SEC that can recognizes the UGA codon is first charged with a serine that is enzymatically converted to a SEC (6). The specificity of SEC incorporation at a UGA codon in selenoprotein transcripts, rather than at UGA stop codons, is ensured by the presence of additional cis-sequences within the transcripts (7-9). Most widely studied is the SEC insertion sequence, or SECIS element, which forms a stem-loop structure in the 3Ј-untranslated region (3Ј-UTR) of the selenoprotein transcripts. Prior studies have shown that insertion of a UGA within the protein coding region of heterologous transcripts can be properly decoded as a selenocysteine codon provided that the SECIS element and surrounding sequences are present in the 3Ј-UTR of the transcript (10, 11). Recognition of the SEC codon also requires the presence of unique translational cofactors (12), such as the efSEC, and specific RNA-binding proteins, such as SBP2 (12, 13), which bind to the SECIS element and promote SEC insertion.Overall, SEC incorporation appears to be an inefficie...
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