Based on gene expression patterns, we classified gastric cancers into 3 subtypes, and validated these in an independent set of tumors. The subgroups have differences in molecular and genetic features and response to therapy; this information might be used to select specific treatment approaches for patients with gastric cancer.

Engagement of the T cell antigen receptor (TCR)1 results in the sequential activation of the Src (p56 lck /p59 fyn ) and Syk (Syk/ZAP-70) families of protein-tyrosine kinases (PTKs) (1-3). Both families of PTKs are required for normal T cell development and function (4 -9). In resting T cells, the TCR chain is constitutively phosphorylated and associated with 11). Tyrosine phosphorylation of the receptor-associated ZAP-70 by p56 lck is a requisite modification resulting in the up-regulation of ZAP-70 catalytic activity (12, 13). Therefore, the recruitment of the CD4 and CD8 co-receptors into the TCR complex positions p56 lck to trans-phosphorylate ZAP-70. Phosphorylation and activation of ZAP-70, in turn, is required for both an increase in phosphoinositide metabolism and activation of the ras pathway.2 The integration of these downstream signals gives rise to transcriptional activation of cytokine genes and a resultant elevation in cytokine synthesis and secretion (15). While the up-regulation of ZAP-70 catalytic activity is required for TCR function, little is known about the cellular proteins which serve as substrates for this PTK.SLP-76 is a recently identified molecule which undergoes tyrosine phosphorylation upon TCR cross-linking (16 -19). This protein associates with both the SH3 domain of Grb2 and an SH2 domain of PLC␥. SLP-76 is structurally characterized by a C-terminal SH2 domain, a region enriched in proline residues which probably serves as the site for Grb2 binding, and an N-terminal motif which contains three tandemly repeated DYE(S/P)P sequences. We demonstrate here that SLP-76 is phosphorylated by ZAP-70 and that phosphorylation of these repeated tyrosine motifs is required for optimal generation of IL-2 in response to TCR ligation. In addition, overexpression of SLP-76 augments TCR-mediated transcriptional activation of the IL-2 gene, while mutation of the SH2 domain attenuates this response. Together, these studies identify SLP-76 as a physiologic substrate for ZAP-70 and suggest a mechanism by which TCR-induced activation of ZAP-70 regulates both the calcium and ras pathways. EXPERIMENTAL PROCEDURESCells and Antibodies-Jurkat and Sf9 cells (Pharmingen) were maintained as described previously (12). The mouse monoclonal antibody (mAb) H3 was generated against the SLP-76 SH2 domain. SLP-76 polyclonal antisera (22652) was generated against a peptide spanning amino acids 301-318 of human SLP-76. C305 is an anti-Jurkat Ti␣/␤-mAb (20); 4G10 (UBI) and PY20 (Santa Cruz) are anti-phosphotyrosine mAbs; 2F3.2 is an anti-ZAP-70 mAb (UBI), and 9E10 is an anti-myc mAb. Anti-GST mAb was purchased from Santa Cruz.Construction of Plasmids-A full-length SLP-76 cDNA was generated by reverse transcription PCR from murine T-cell mRNA. This cDNA was appended with a myc-epitope at the 3Ј end of the coding cDNA. Mutations were generated by PCR-directed mutagenesis and confirmed using standard dideoxy sequencing methods. These constructs were then subcloned into the pApuro vector (21) and the baculoviral vector pVL13...

The identification of small molecule inhibitors of antiapoptotic Bcl-2 family members has opened up new therapeutic opportunities, while the vast diversity of chemical structures and biological activities of natural products are yet to be systematically exploited. Here we report the identification of chelerythrine as an inhibitor of BclXL-Bak Bcl-2 homology 3 (BH3) domain binding through a high throughput screening of 107,423 extracts derived from natural products. Chelerythrine inhibited the BclXL-Bak BH3 peptide binding with IC 50 of 1.5 M and displaced Bax, a BH3-containing protein, from BclXL. Mammalian cells treated with chelerythrine underwent apoptosis with characteristic features that suggest involvement of the mitochondrial pathway. While staurosporine, H7, etoposide, and chelerythrine released cytochrome c from mitochondria in intact cells, only chelerythrine released cytochrome c from isolated mitochondria. Furthermore BclXL-overexpressing cells that were completely resistant to apoptotic stimuli used in this study remained sensitive to chelerythrine. Although chelerythrine is widely known as a protein kinase C inhibitor, the mechanism by which it mediates apoptosis remain controversial. Our data suggest that chelerythrine triggers apoptosis through a mechanism that involves direct targeting of Bcl-2 family proteins.

Polypeptide chain initiation factor eIF4GI undergoes caspasemediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m 7 GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-MouthDisease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed. Cell Death and Differentiation (2000) 7, 628 ± 636.