To clarify the roles of integrin and extracellular matrix (ECM) in the process of non-small cell lung cancer (NSCLC) brain metastasis, we established an in vivo model of brain metastasis of human NSCLC cell line EBC-1/original in athymic mice, and established highly brain metastatic subclone EBC-1/brain and highly bone metastatic subclone EBC-1/bone. Integrin expression of these subclones was evaluated by flow cytometry. In vitro cell attachment, migration and proliferation assays with ECMs were performed using these subclones. Expression of integrin α α α α3 subunit was higher in EBC-1/brain than in both EBC-1/original and EBC-1/ bone. In vitro cell attachment, migration, and proliferation assays revealed that EBC-1/brain had higher affinity and higher reactivity to laminin than EBC-1/original and EBC-1/bone. Blocking of integrin α α α α3β β β β1 significantly (P < ung cancer patients often suffer from brain metastasis. Indeed, clinical data in Japan show that more than half of metastatic brain tumors originates from lung cancer, 1) implying that most lung cancers have high potential for metastasis to brain.

In order to investigate whether c-Src is involved in carcinogenesis and progression of breast carcinoma, we examined the expression of activated c-Src in tissue sections from surgically resected human breast specimens. First, we confirmed the specificity of the antibody against activated c-Src (Clone 28) using six cell lines established from human breast carcinomas by western blotting. As expected, activated c-Src was detected as a 60 kDa band in all cell lines tested. Immunofluorescence analysis demonstrated that the activated c-Src was mainly observed in cytoplasms of these cells. Then, we designed an immunohistochemical study with 73 human breast carcinoma tissues. Glandular epithelial and myoepithelial cells in normal mammary glands adjacent to carcinoma nests and infiltrating stromal cells were negative for activated c-Src. In contrast, 37 of the 73 breast carcinoma tested (50.7%) were positive for activated c-Src, and this positive staining was inversely correlated with Ki-67 labeling index (p < 0.0001), TNM stage (p < 0.0001), tumor size (p < 0.0001), an d histological grade (p = 0.0002). These results strongly suggest that the activation of c-Src would be related to the progression of breast carcinomas with low aggressiveness.

Tumour metastasis is known clinically to have organ specificity. We hypothesised that integrins might be involved in determining the organ specificity of tumour metastasis. Here, we report the results of spontaneous metastasis tested in nude mice that were inoculated with Chinese hamster ovary (CHO) cells expressing integrin a5b1 at various levels. The growth of the primary tumour inversely correlated with the a5 expression level on CHO cells, which is consistent with a previous report (Schreiner et al, 1991). The rates of pulmonary, lymph node, and adrenal metastases that developed in nude mice were not related to changes of the a5 expression level on CHO cells. Kidney metastasis developed in 40% of nude mice inoculated with a5B2 cells (CHO cells overexpressing a5) and in 20% of mice with CHO-K1 cells (CHO cells expressing native a5), whereas inoculation with CHO-B2 cells (a5-defective mutants) and a5CHO cells with the highest expression of a5 did not lead to development of kidney metastasis. Furthermore, a5CHO, which shows the slowest growth of these cell types, did not lead to primary tumours in nude mice. These findings suggest that there is an appropriate level of a5 expression on tumour cells that leads to metastasis. Microscopic observations revealed that micrometastasis in the kidney was formed in glomeruli. An adhesion assay using frozen sections of the kidney demonstrated that a5B2 cells, but not CHO-B2 cells, effectively adhered to glomeruli. Kidney metastasis in vivo and the adhesion of a5B2 to glomeruli shown ex vivo were significantly suppressed by the administration of GRGDS peptide. Finally, we conclude that the interaction of a5b1 on tumour cells with fibronectin in kidney glomeruli is involved in kidney metastasis and that the tumour has appropriate levels of integrins crucial for metastasis.

Modulation of mast‐cell activation may provide novel ways to control allergic diseases. Here, we show that protein tyrosine phosphatase ε (PTPε; Ptpre) plays key regulatory roles during mast‐cell activation mediated by the high‐affinity IgE receptor (FcεRI). Bone marrow‐derived mast cells (BMMC) from Ptpre−/− mice exhibited enhanced FcεRI‐induced Ca2+ mobilization and mitogen‐activated protein kinase (MAPK) (JNK and p38) activation, and showed corresponding enhancement of evoked degranulation and cytokine production, but not leukotriene production. Examination of proteins linking tyrosine kinase activation and Ca2+ mobilization revealed that the absence of PTPε leads to increased phosphorylation of the linker for activation of T cells and SH2 domain‐containing leucocyte phosphoproteins of 76 kDa, but not Grb2‐associated binder‐2 (Gab2). Because Gab2 is considered to be situated downstream of Fyn kinase, we reasoned that Fyn may not be a target of PTPε. In the event, Syk but not Lyn was hyperphosphorylated in PTPε‐deficient BMMC. Thus, PTPε most likely exerts its effects at the level of Syk, inhibiting downstream events including phosphorylation of SLP‐76 and linker of activated T cells and mobilization of Ca2+. Consistent with the in vitro data, antigen‐ and IgE‐mediated passive systemic anaphylactic reactions were augmented in Ptpre−/− mice. Given that the number of mast cells is unchanged in these mice, this observation most likely reflects alterations of mast cell‐autonomous signalling events. These data suggest that PTPε negatively regulates FcεRI‐mediated signalling pathways and thus constitutes a novel target for ameliorating allergic conditions.