BACKGROUND The authors investigated whether the presence of matrix metalloproteinase‐2 (MMP‐2) and its inducer, CD147, in cancerous esophageal lesions and surrounding tissue might help to predict patient prognosis. METHODS Tissue samples from 101 patients with esophageal squamous cell carcinoma were stained with anti‐CD147 and anti–MMP‐2 antibodies for immunohistochemical analysis. RESULTS CD147 was expressed in cancerous and dysplastic lesions, but not in normal tissue. In contrast, MMP‐2 was detected mainly in normal interstitial tissue adjacent to cancerous lesions, but it was detected also in cancerous lesions in some patients. Pathologic findings demonstrated that the intensity of MMP‐2 staining in normal tissue was associated positively with the depth of tumor infiltration and the stage of disease, whereas MMP‐2 staining in cancerous tissue was associated positively with vascular and lymphatic vessel invasion as well as with immature differentiation of cancer cells. Using a proportional hazard model, including information on CD147 staining patterns within cancerous lesions along with clinical cancer staging, improved the accuracy of predicting patient prognosis. CONCLUSIONS These results suggested that measurement of CD147 and MMP‐2 expression with simple immunohistochemical staining may enhance further the understanding of the pathophysiology of invading tumor cells and, when used in combination with cancer staging, may increase the ability of investigators to predict prognosis in patients with esophageal squamous cell carcinoma. Cancer 2004. © 2004 American Cancer Society.

Ubiquitin, which can conjugate with cellular proteins, is classified into two forms: free ubiquitin and multiubiquitin chains. The latter is active as a signal for degradation of the targeted proteins. We found both forms in human serum and, using two immunoassays, quantitated them in sera from healthy subjects and patients with some diseases. Because of putative leakage of erythrocyte ubiquitin, hemolytic serum and serum obtained after long incubation (>1–2 h) of blood at room temperature were excluded. Serum concentrations of multiubiquitin chains and free ubiquitin were substantially higher in rheumatoid arthritis and hemodialysis patients, respectively, than healthy subjects. Additionally, in acute viral hepatitis, serum multiubiquitin chain concentrations were increased in the acute phase, decreased in the recovery phase, and correlated with alanine and aspartate aminotransferase activities (r = 0.676 and 0.610, P <0.0001 and <0.001, respectively). Therefore, serum ubiquitin may have prognostic value.

Abstract. Ovarian clear cell carcinoma (OCCC) has several significant characteristics based on molecular features that are distinct from those of ovarian high-grade serous carcinoma. Cellular glycogen accumulation is the most conspicuous feature of OCCC and in the present study its metabolic mechanism was investigated. The amount of glycogen in cells cultured under hypoxia increased significantly and approximately doubled after 48 h (P<0.01) compared to that under normoxic conditions. Periodic acid-Schiff positive staining also demonstrated intracellular glycogen storage. Western blot analysis revealed that HIF1α, which was overexpressed and stabilized under hypoxic conditions, led to an increase in the levels of cellular glycogen synthase 1, muscle type (GYS1), and conversely to a decrease in inactive phosphorylated GYS1 at serine (Ser) 641. Additional increases were observed in both protein phosphatase 1, which dephosphorylates and thereby induces GYS1 enzyme activity, and glycogen synthase kinase 3 beta (GSK3β) phosphorylated at Ser9, which is inactive on phosphorylation of GYS1 and subsequently induces its enzyme activity. By contrast, the level of PYGM-b decreased. These results indicated that the glycogen accumulation under a hypoxic environment resulted in the promotion of glycogen synthesis, but did not lead to inhibition of glycogen degradation and/or consumption. Under hypoxic conditions, HAC2 cells showed activation of the PI3K/AKT pathway caused by a mutation in exon 20 of PIK3CA, encoding the catalytic subunit p110α of PI3K. The resulting activation of AKT (phosphoSer473) also plays a role as a central enhancer in glycogen synthesis through suppression of GSK3β via phosphorylation at Ser9. Hypoxia decreased the cytocidal activity of cisplatin and doxorubicin to various degrees. In conclusion, the hypoxic conditions together with HIF1 expression and stabilization increased the intracellular glycogen contents and resistance to the anticancer drugs.

We report a case of PAX6 gene mutation with early-onset diabetes mellitus and aniridia. Low insulin secretory capacity in her parents suggested that her insulin secretory defect is as a result of not only PAX6 mutation but other genetic factors inherited from her parents.

A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus "'I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as a standard named the multi-ubiquitin-chain reference preparation 1 (MUCRPI). The concentration of MUCRPI was calculated from the recovered radioactivity of "'I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRPI. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays < 6%) and reproducibility (interassay < 9%). In addition, there was no substantial cross-reaction with mono-, di-and tri-ubiquitin, or mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains ( n > approximately 6 ) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi-ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea-soluble chain level was higher in the reticulocytes. Heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover.Keywords: ELISA ; heat shock ; multi-ubiquitin chain; reticulocyte; ubiquitin.Ubiquitin is found in either a free form or a form bound covalently to intracellular proteins in vivo (Hershko, 1988;Finley and Chau, 1991). In the latter form, the ubiquitin C-terminus is ligated to &-amino groups of appropriate Lys residues in target proteins by a family of ubiquitin-conjugating enzymes: a ubiquitin-activating enzyme (El), a ubiquitin-carrier protein (E2), and a ubiquitin-protein ligase (E3) (Hershko et al., 1983;Hershko and Ciechanover, 1992). In this process, several ubiquitin moieties are usually ligated sequentially to the initial acceptor protein, forming multi-ubiquitin chains in which the ubiquitin Cterminus is conjugated to the &-amino group of Lys48 of another ubiquitin moiety (Chau et al., 1989). The multi-ubiquitin chain acts as a signal to induce degradation of target proteins by 2 6 s proteasome (Chau et al., 1989;Hershko and Ciechanover, 1992). Therefore, the chain structure is essential for ubiquitin-dependent proteolysis.The ubiquitin-dependent proteolysis has been implicated in a variety of cellular processes, including stress response (Finley et al., 1987;Carlson et a]., 1987), cell-cycle control (Glotzer et al., 1991;Nishizawa et al., 1993) and apoptosis (Delic et al., 1993). Intracellular levels of ubiquitin and ubiquitin-protein conjugates are changed in response to heat shock (Parag et al., Abbreviations. MUCRPI , multi-ubiquitin chain reference preparation 1 ; E l , ubiquitin-activating enzy...