Periostin is a secreted protein that is highly expressed in early osteoblastic cells in vitro and in periosteum and periodontal ligament tissues in vivo. It is known that periostin supports cellular adhesion and spreading in vitro. Although, the mechanisms of transcriptional regulation of periostin are poorly understood, gene-profiling data have revealed that overexpression of Twist, a basic helix-loop-helix (bHLH) transcription factor, resulted in increased periostin expression as validated by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Twist is an important transcription factor for cell type determination and differentiation and has been shown to play an important regulatory role in early osteogenesis. In situ hybridization of mouse calvarial bones indicated that periostin and Twist mRNA are co-localized at the osteogenic fronts of calvarial bones. To characterize the 5' flanking region of the periostin gene, primer extension was carried out to identify the transcription start site, and DNA sequence analysis confirmed the presence of a 'Twist-box' response element. The results of electrophoretic mobility shift assay (EMSA) using nuclear extracts of MC3T3-E1 cells revealed that Twist bound to the Twist-box sequence on the periostin promoter. In vivo footprinting experiments using ligation-mediated PCR (LM-PCR) indicated that the Twist-box sequence was protected in undifferentiated MC3T3-E1 preosteoblasts but not in differentiated MC3T3-E1 osteoblasts. To determine whether Twist actually regulates the periostin expression, 293T cells were transiently co-transfected with the periostin promoter construct and the human Twist expression vector. Reporter analysis indicated that the periostin promoter activities were enhanced by overexpression of Twist. These data suggest that Twist can bind to the periostin promoter in undifferentiated preosteoblasts and up-regulate periostin expression, consistent with the up-regulation of periostin expression by Twist as observed in the gene-profiling data.

Purpose: CLIC4, a member of a family of intracellular chloride channels, is regulated by p53, c-Myc, and tumor necrosis factor-a. Regulation by factors involved in cancer pathogenesis, together with the previously shown proapoptotic activity of CLIC4, suggests that the protein may have a tumor suppressor function. To address this possibility, we characterized the expression profile, subcellular localization, and gene integrity of CLIC4 in human cancers and determined the functional consequences of CLIC4 expression in tumor epithelium and stromal cells. Experimental Design: CLIC4 expression profiles were analyzed by genomics, proteomics, bioinformatics, and tissue microarrays. CLIC4 expression, as a consequence of crosstalk between stroma and epithelium, was tested in vitro by coculture of breast epithelial tumor cells and normal fibroblasts, and the functional consequences of CLIC4 expression was tested in vivo in xenografts of human breast tumor cell lines reconstituted with CLIC4 or mixed with fibroblasts that overexpress CLIC4 transgenically. Results: In cDNA arrays of matched human normal and tumor tissues, CLIC4 expression was reduced in renal, ovarian, and breast cancers. However, CLIC4 protein levels were variable in tumor lysate arrays. Transcript sequences of CLIC4 from the human expressed sequence tag database and manual sequencing of cDNA from 60 human cancer cell lines (NCI60) failed to reveal deletion or mutations in the CLIC4 gene. On matched tissue arrays, CLIC4 was predominantly nuclear in normal human epithelial tissues but not cancers.With advancing malignant progression, CLIC4 staining became undetectable in tumor cells, but expression increased in stromal cells coincident with up-regulation of a-smooth muscle actin, suggesting that CLIC4 is up-regulated in myofibroblasts. Coculture of cancer cells and fibroblasts induced the expression of both CLIC4 and a-smooth muscle actin in fibroblasts adjacent to tumor nests. Introduction of CLIC4 or nuclear targeted CLIC4 via adenovirus into human breast cancer xenografts inhibited tumor growth, whereas overexpression of CLIC4 in stromal cells of xenografts enhanced tumor growth. Conclusion: Loss of CLIC4 in tumor cells and gain in tumor stroma is common to many human cancers and marks malignant progression. Up-regulation of CLIC4 in tumor stroma is coincident with myofibroblast conversion, generally a poor prognostic indicator. Reactivation and restoration of CLIC4 in tumor cells or the converse in tumor stromal cells could provide a novel approach to inhibit tumor growth.

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To date, proteomic analyses on gastrointestinal cancer tissue samples have been performed using surgical specimens only, which are obtained after a diagnosis is made. To determine if a proteomic signature obtained from endoscopic biopsy samples could be found to assist with diagnosis, frozen endoscopic biopsy samples collected from 63 gastric cancer patients and 43 healthy volunteers were analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. A statistical classification model was developed to distinguish tumor from normal tissues using half the samples and validated with the other half. A protein profile was discovered consisting of 73 signals that could classify 32 cancer and 22 normal samples in the validation set with high predictive values (positive and negative predictive values for cancer, 96.8% and 91.3%; sensitivity, 93.8%; specificity, 95.5%). Signals overexpressed in tumors were identified as α-defensin-1, α-defensin-2, calgranulin A, and calgranulin B. A protein profile was also found to distinguish pathologic stage Ia (pT1N0M0) samples (n = 10) from more advanced stage (Ib or higher) tumors (n = 48). Thus, protein profiles obtained from endoscopic biopsy samples may be useful in assisting with the diagnosis of gastric cancer and, possibly, in identifying early stage disease.

Silent (painless) thyroiditis has been recognized as a clinical entity for over a decade and is characterized by spontaneously resolving thyrotoxicosis. Its etiology is uncertain; however, a few reports have indicated the occurrence of TSH binding-inhibiting immunoglobulins (TBII) and thyroid-stimulating antibodies (TSAb) in some of the patients. The present study was undertaken to evaluate thyroid function and the occurrence of TBII and TSAb and thyroid autoantibodies (antithyroglobulin and antimicrosomal) in 53 patients with silent thyroiditis during the course of their disease. The patients were divided into 2 major groups: I) those who developed transient hypothyroidism and II) those who did not. All patients initially had significantly increased concentrations of serum T4, free T4, and free T3, suppressed TSH levels, and decreased thyroid radioiodine uptake. TBII and TSAb were initially positive in 8 (15.1%) and 10 patients (18.9%), respectively. Forty patients were available for follow-up. TBII was positive in 6 of 24 (25.0%), and TSAb was positive in 8 of 24 (33.3%) of the patients who developed transient hypothyroidism during the course of their disease. Among the patients who did not become hypothyroid at any time, TBII was positive in only 2 of 16 (12.5%), and none of the patients became TSAb positive. The findings indicate that increased TSAb and TBII activity may be detected in patients with silent thyroiditis and, when present, are associated with transient hypothyroidism during the course of the disease.