Establishment and characterization of a new squamous cell carcinoma cell line, TMUU-08, derived from human bladder cancer

Human squamous cell carcinoma (SCC) of the bladder is a rare malignancy that represents less than 5% of bladder tumors. In contrast to non-bilharzial SCC, bilharzial SCC is a distinct pathological disease that is rarely encountered in Japan. The majority of patients with non-bilharzial SCC present with a poorly differentiated, muscle-invasive tumor with no previous episode of urothelial carcinoma (UC). Even in the absence of distant metastases, the prognosis of patients with non-bilharzial SCC of the bladder remains dismal because patients die of localized recurrence. This is in contrast to UC in which distant metastasis accounts for the great majority of recurrence. The 5-year survival rate of the patients treated for non-bilharzial SCC of the bladder was only about 10%. To date, large numbers of reports have examined the establishment of a human bladder cancer cell line with UC. However, few reports exist regarding the establishment of the human bladder cancer cell line with SCC. In the present study, we established a new cell line (TMUU-08) from the metastatic lymph node of a patient with SCC of the bladder. The TMUU-08 cell line of human bladder SCC was characterized. These results indicate that TMUU-08 cells might be useful in basic studies not only in the treatment but also etiology of human bladder SCC.     

Inhibitory effect of luteolin on osteoclast differentiation and function

Osteoclasts are multinucleated cells that play a crucial role in bone resorption, and are formed by the fusion of mononuclear osteoclasts derived from osteoclast precursors of the macrophage lineage. Compounds that specifically target functional osteoclasts would be ideal candidates for anti-resorptive agents for clinical applications. In the present study, we investigated the effects of luteolin, a flavonoid, on the regulation of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, functions and signaling pathway. Addition of luteolin to a coculture system of mouse bone marrow cells and ST2 cells in the presence of 10⁻⁸ M 1α,25(OH)₂D₃ caused significant inhibition of osteoclastogenesis. Luteolin had no effects on the 1α,25(OH)₂D₃-induced expressions of RANKL, osteoprotegerin and macrophage colony-stimulating factor mRNAs. Next, we examined the direct effects of luteolin on osteoclast precursors using bone marrow macrophages and RAW264.7 cells. Luteolin completely inhibited RANKL-induced osteoclast formation. Moreover, luteolin inhibited the bone resorption by mature osteoclasts accompanied by the disruption of their actin rings, and these effects were reversely induced by the disruption of the actin rings in mature osteoclasts. Finally, we found that luteolin inhibited RANKL-induced osteoclastogenesis through the suppression of ATF2, downstream of p38 MAPK and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) expression, respectively. Taken together, the present results indicate that naturally occurring luteolin has inhibitory activities toward both osteoclast differentiation and functions through inhibition of RANKL-induced signaling pathway as well as actin ring disruption, respectively.     

Screening system for <Emphasis Type="SmallCaps">d</Emphasis>-Asp-containing proteins using <Emphasis Type="SmallCaps">d</Emphasis>-aspartyl endopeptidase and two-dimensional gel electrophoresis

d-Asp-containing proteins have been implicated in many aging-related diseases. To clarify the role of d-Asp-containing proteins in such diseases, we developed a screening system for these proteins using a d-aspartyl endopeptidase that specifically cleaves the proteins at the C-terminus. The digested proteins were detected by means of two-dimensional gel electrophoresis and identified using nano-liquid chromatography/tandem mass spectrometry. We were able to detect myelin basic protein, a known d-Asp-containing protein, in the brain tissues of mice; this indicates that our system is effective for screening d-Asp-containing proteins.     

USP14 inhibits ER-associated degradation via interaction with IRE1α

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) lumen induces ER stress. Eukaryotic cells possess the ER quality control systems, the unfolded protein response (UPR), to adapt to ER stress. IRE1α is one of the ER stress receptors and mediates the UPR. Here, we identified ubiquitin specific protease (USP) 14 as a binding partner of IRE1α. USP14 interacted with the cytoplasmic region of IRE1α, and the endogenous interaction between USP14 and IRE1α was inhibited by ER stress. Overexpression of USP14 inhibited the ER-associated degradation (ERAD) pathway, and USP14 depletion by small interfering RNA effectively activated ERAD. These findings suggest that USP14 is a novel player in the UPR by serving as a physiological inhibitor of ERAD under the non-stressed condition.