We report the identification and characterization of mouse matriptase-2 (m-matriptase-2), an 811-amino-acid protein composed of an N-terminal cytoplasmic domain, a membrane-spanning domain, two CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains, three LDLR (low-density-lipoprotein receptor class A) domains and a C-terminal serine-protease domain. All m-matriptase-2 protein domain boundaries corresponded with intron/exon junctions of the encoding gene, which spans approx. 29 kb and comprises 18 exons. Matriptase-2 is highly conserved in human, mouse and rat, with the rat matriptase-2 gene ( r-maltriptase-2 ) predicted to encode transmembrane and soluble isoforms. Western-blot analysis indicated that m-matriptase-2 migrates close to its theoretical molecular mass of 91 kDa, and immunofluorescence analysis was consistent with the proposed surface membrane localization of this protein. Reverse-transcription PCR and in-situ -hybridization analysis indicated that m-matriptase-2 expression overlaps with the distribution of mouse hepsin (m-hepsin, a cell-surface serine protease identified in hepatoma cells) in adult tissues and during embryonic development. In adult tissues both are expressed at highest levels in liver, kidney and uterus. During embryogenesis m-matriptase-2 expression peaked between days 12.5 and 15.5. m-hepsin expression was biphasic, with peaks at day 7.5 to 8.5 and again between days 12.5 and 15.5. In situ hybridization of embryonic tissues indicated abundant expression of both m-matriptase-2 and m-hepsin in the developing liver and at lower levels in developing pharyngo-tympanic tubes. While m-hepsin was detected in the residual embryonic yolk sac and with lower intensity in lung, heart, gastrointestinal tract, developing kidney tubules and epithelium of the oral cavity, m-matriptase-2 was absent in these tissues, but strongly expressed within the nasal cavity by olfactory epithelial cells. Mechanistic insight into the potential role of this new transmembrane serine protease is provided by its novel expression profile in embryonic and adult mouse.

p14 Arf ͉ apoptosis ͉ cancer ͉ extracellular matrix

Conventional cancer therapies are based on preferential killing of tumor cells by DNA damage. Previous work showed that, for certain cell types, loss of integrin-mediated adhesion decreased the apoptotic response to DNA damage because of decreased p53 levels after detachment from the extracellular matrix. Integrin ligation restored p53 and sensitivity to DNA damage. In this study, we show that c-Abl mediates a second pathway by which adhesion to extracellular matrix regulates cell killing by chemotherapeutic agents 5-arabinofuranosylcytosine, cisplatin, and camptothecin. Activation of c-Abl tyrosine kinase by DNA damage requires cell adhesion. Abl-dependent stabilization of p73, a p53-related proapoptotic transcription factor, is also adhesion-dependent. Sensitivity to the Abl inhibitor STI571 suggests differential utilization of the p53 and c-Abl͞p73 pathways by different tumor cell lines. These data suggest that killing of p53-negative tumor cells by chemotherapy would be enhanced by integrin ligation to activate the alternative c-Abl͞p73 pathway.C urrent cancer therapies are based primarily on radiation and chemotherapy that damage DNA to selectively kill fastgrowing tumor cells. This strategy is efficacious in the treatment of childhood leukemia and solid tumors at early stages, but suffers from the limitation that a small number of cancer cells commonly evade therapy and accumulate additional mutations, leading to recurrence of therapy-resistant tumors. The tumor suppressor gene p53 provides a key pathway that mediates cell death after DNA damage (1, 2). DNA damage increases the level and the transcriptional activity of p53, resulting in induction of cell cycle arrest genes such as p21 Waf1 and proapoptotic genes such as Noxa, Puma, and Bax. Inactivation of p53 is one of the most common genetic alterations in human cancer and is associated with progression to metastatic disease, resistance to therapy, and genomic instability.We recently described an effect of integrin-mediated adhesion on p53 levels and subsequent sensitivity of cells to DNA damage (3). For susceptible cell types, loss of adhesion to extracellular matrix (ECM) caused a decrease in p53 levels, leading to decreased apoptosis after exposure to ionizing radiation or chemotherapeutic drugs. The change in p53 was caused by a more rapid decline in levels of p19Arf, which inhibits MDM2 to prevent ubiquitin-and proteosome-dependent degradation of p53. This behavior was cell type specific. It was observed in a melanoma, a rhabdomyosarcoma and a fibrosarcoma line, as well as primary mouse embryonic fibroblasts (MEFs). However, several other tumor cell types either died directly after detachment or showed no decrease in sensitivity to DNA damage in suspension.A second pathway that mediates cellular responses to DNA damage involves the c-Abl tyrosine kinase. Activation of c-Abl by DNA damage contributes to cell cycle arrest and apoptosis via the p53 homolog p73 (4-6). Interestingly, c-Abl is also regulated by integrins (7,8). Detachment of cells from th...