Missense mutations in the 695-amino acid form of the amyloid precursor protein (APP695) cosegregate with disease phenotype in families with dominantly inherited Alzheimer's disease. These mutations convert valine at position 642 to isoleucine, phenylalanine, or glycine. Expression of these mutant proteins, but not of normal APP695, was shown to induce nucleosomal DNA fragmentation in neuronal cells. Induction of DNA fragmentation required the cytoplasmic domain of the mutants and appeared to be mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins).

APP is a transmembrane precursor of beta‐amyloid. In dominantly inherited familial Alzheimer's disease (FAD), point mutations V6421, V642F and V642G have been discovered in APP695. Here we show that expression of these mutants (FAD‐APPs) causes a clone of COS cells to undergo apoptosis associated with DNA fragmentation. Apoptosis by the three FAD‐APPs was the highest among all possible V642 mutants; normal APP695 had no effect on apoptosis, suggesting that apoptosis by APP mutants in this system is phenotypically linked to the FAD trait. FAD‐APP‐induced apoptosis was sensitive to bcl‐2 and most probably mediated by heteromeric G proteins. This study presents a model system allowing analysis of the mechanism for FAD‐APP‐induced cytotoxicity.

The tumor suppressor gene p53 is a potent transcriptional regulator of genes which are involved in many cellular activities including cell cycle arrest, apoptosis, and angiogenesis. Recent studies have demonstrated that the activation of the transcriptional factor nuclear factor kB (NF-kB) plays an essential role in preventing apoptotic cell death. In this study, to better understand the mechanism reponsible for the p53-mediated apoptosis, the eect of wild-type p53 (wt-p53) gene transfer on nuclear expression of NF-kB was determined in human colon cancer cell lines. A Western blot analysis of nuclear extracts demonstrated that NF-kB protein levels in the nuclei were suppressed by the transient expression of the wt-p53 in a dose-dependent manner. Transduced wt-p53 expression increased the cytoplasmic expression of IkBa as well as its binding ability to NF-kB, thus markedly reducing the amount of NF-kB that translocated to the nucleus. The decrease in nuclear NF-kB protein correlated with the decreased NF-kB constitutive activity measured by electrophoretic mobility shift assay. Furthermore, parental cells transfected with NF-kB were better protected from cell death induced by the wt-p53 gene transfer. We also found that the wtp53 gene transfer was synergistic with aspirin (acetylsalicylic acid) in inhibiting NF-kB constitutive activity, resulting in enhanced apoptotic cell death. These results suggest that the inhibition of NF-kB activity is a plausible mechanism for apoptosis induced by the wt-p53 gene transfer in human colon cancer cells and that anti-NF-kB reagent aspirin could make these cells more susceptible to apoptosis. Oncogene (2000) 19, 726 ± 736.

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In this study, we examined the distribution of heparanase protein in 75 esophageal squamous cell carcinomas by immunohistochemistry and analyzed the relationship between heparanase expression and clinicopathological characteristics. In situ hybridization showed that the mRNA expression pattern of heparanase was similar to that of the protein, suggesting that increased expression of the heparanase protein at the invasive front was caused by an increase of heparanase mRNA in tumor cells. Heparanase expression correlated significantly with depth of tumor invasion, lymph node metastasis, tumor node metastasis (TNM) stage and lymphatic invasion. Overexpression of heparanase in esophageal cancers was also associated with poor survival. In addition to its localization in the cytoplasm and cell membrane, heparanase was also identified in the nuclei of normal epithelial and tumor cells by immunohistochemistry. Furthermore, nuclear heparanase was detected in nuclear extract of cancer cell lines by Western blot and immunohistochemistry. Examination of the role of nuclear heparanase in cell proliferation and differentiation by double immunostaining for proliferating cell nuclear antigen (PCNA) and cytokeratin 10 (CK10) showed significant relationship between nuclear heparanase expression and differentiation (heparanase vs CK10), but not for proliferative state of esophageal cancer cells (heparanase vs PCNA). Our results suggest that cytoplasmic heparanase appears to be a useful prognostic marker in patients with esophageal cancer and that nuclear heparanase protein may play a role in differentiation. Inhibition of heparanase activity may be effective in the control of esophageal tumor invasion and metastasis.

in APP 695 (Hardy, 1992). These mutations co-segregate Narasimhan Gautam 8 and with the AD phenotype (Karlinsky et al., 1992), demon- Ikuo Nishimoto 1,2,9strating that V642 mutations in APP are established causes of AD. et al., 1996). Furtherprotein APP. Expression of these mutants causes a more, it has been found that the FAD-associated V642 COS cell NK1 clone to undergo pertussis toxin-sensitive mutants of APP cause cytotoxicity in cultured cells without apoptosis in an FAD trait-linked manner by activating Aβ mediation (Yamatsuji et al., 1996a,b). Thus, the the G protein G o , which consists of Gα o and Gβγ significance, as well as the role, of Aβ deposition for AD subunits. We investigated which subunit was responsdevelopment remains unclear. ible for the induction of apoptosis by V642I APP in NK1In its structure, orientation and localization, APP is cells. In the same system, expression of mutationally similar to cell surface receptors (Kang et al., 1987; Dyrks activated Gα o or Gα i induced little apoptosis. Apoptosis et al., 1988;Weidemann et al., 1989;Schubert et al., by V642I APP was antagonized by the overexpression Ferreira et al., 1993). The cytoplasmic domain of of the carboxy-terminal amino acids 495-689 of the APP binds Fe65 protein, which has a phosphotyrosine-β-adrenergic receptor kinase-1, which blocks the binding domain related to an oncogenic signal transducer, specific functions of Gβγ. Co-transfection of Gβ2γ2Shc (Fiore et al., 1995). It also binds APP-BP1, a gene cDNAs, but not that of other Gβxγz (x ϭ 1-3; z ϭ 2, product similar to AXR1 in Arabidopsis; AXR1 is required 3), induced DNA fragmentation in a manner sensitive for normal response to the plant growth hormone auxin to bcl-2. These data implicate Gβγ as a cell death (Chow et al., 1996). These observations suggest that APP mediator for the FAD-associated mutant of APP.has not only the structure but also the function of a cell Keywords: amyloid precursor protein/apoptosis/ surface receptor. Our own earlier study (Nishimoto et al., βγ complex/familial Alzheimer's disease/G protein 1993) found that APP 695 has an intrinsic G o -stimulating domain at His657-Lys676 and forms a complex with G o through this cytoplasmic domain. It has been confirmed that the synthetic His657-Lys676 peptide activates G o

Amino acids, especially branched-chain amino acids such as l-leucine, have been shown to regulate activation of p70 S6 kinase and phosphorylation of 4E-BP1 through the mTOR signaling pathway. In our recent study, l-arginine was also shown to activate the mTOR signaling pathway in rat intestinal epithelial cells. l-Glutamine is an amino acid that is required for culturing of numerous cell types, including rat intestinal epithelial cells. In this study, we showed that l-glutamine inhibited the activation of p70 S6 kinase and phosphorylation of 4E-BP1 induced by arginine or leucine in rat intestinal epithelial cells. Although the molecular mechanism of l-glutamine-induced inhibition of the mTOR signaling pathway is still unknown, the presence of this novel signal pathway may indicate that individual amino acids play specific roles for cellular proliferation and growth.

Heparanase is a marker for poor prognosis of patients with colon cancer and could be a suitable target for antitumor therapy in colon cancer.