Expression of the tumor suppressor gene maspin and its significance in intraductal papillary mucinous neoplasms of the pancreas

BACKGROUND: Maspin is a member of the serpin family of protease inhibitors and is thought to inhibit carcinoma invasion, metastasis, and angiogenesis and induce apoptosis. We examined maspin expression immunohistochemically and assessed its significance in intraductal papillary mucinous neoplasm (IPMN) of the pancreas. METHODS: We examined 39 surgically resected specimens of IPMN that included 17 adenomas (IPMAs), 5 borderline tumors (IPMBs), 4 non-invasive carcinomas (non-invasive IPMCs), and 13 invasive carcinomas (invasive IPMCs). Immunostaining was performed according to the EnVision ChemMate method. The degree of maspin expression was scored and assessed according to the percentage and staining intensity of positive cells. RESULTS: Maspin expression was minimal in normal pancreatic duct epithelium, whereas in IPMNs, maspin was expressed in neoplasms of all stages. Maspin expression increased with increasing grade from IPMAs, IPMBs, to non-invasive IPMCs but decreased significantly in invasive IPMCs. No specific association between maspin expression and mucin type was found. Analysis of maspin expression with respect to clinicopathologic factors in cases of invasive IPMC revealed a greater extent of invasion in cases of low maspin expression and significantly fewer apoptotic cells in the tumor.CONCLUSIONS: Maspin was expressed at high levels in IPMNs at various stages from adenoma to invasive carcinoma, and our results suggest that maspin may be involved in the occurrence and progression of IPMN. In addition, our data suggest that the apoptosis-inducing action of maspin suppresses invasion and progression of IPMN.

Combination of simvastatin and hydroxyapatite fiber induces bone augmentation

This study evaluated the capability of hydroxyapatite fiber (HAF) as a carrier and the bone formation by blending simvastatin. The mixture of HAF and simvastatin (0.15, 0.45, 0.75 mg) was placed in 1 ml of tris-buffer and the release of simvastatin from HAF was calculated per 24 hours for 10 days. Bilateral 5 mm-diameter and 3 mm-hight Teflon chambers were fixed on calvaria of adult Japanese white rabbits and filled with 40 mg HAF which containing simvastatin (0, 0.15, 0.45, 0.75 mg). The animals were sacrificed at 4 and 8 weeks and calculated radiologically by Micro-CT. After dyeing by toluidine blue the samples were analyzed histologically. In all of the study groups approximately 25% of simvastatin was released until 10 days. The new bone volume ratio measured by Micro-CT of 4 and 8 weeks group was (22.4%, 21.3%, 41.6%, 26.3%) and (20.2%, 11.7%, 42.1%, 31.2%) in different doses respectively. The 0.45 mg group showed significantly higher new bone volume ratio than 0 mg group and 0.15 mg group. The histological measurement and observations also supported these results. In conclusion, the HAF could be used as a carrier for simvastatin. Combinations of HAF and simvastatin have the potentiality to stimulate new bone formation and approximately 0.45 mg simvastatin in 40 mg HAF is the optimal dose in rabbit chamber model.

Identification and Characterization of Human Genomic Binding Sites for Retinoic Acid Receptor/Retinoid X Receptor Heterodimers

All-trans retinoic acid (ATRA) triggers a wide range of effects on vertebrate development by regulating cell proliferation, differentiation, and apoptosis. ATRA activates retinoic acid receptors (RARs) which heterodimerize with retinoid X receptors (RXRs). RAR/RXR heterodimers function as ATRA-dependent transcriptional regulators by binding to retinoic acid response elements (RAREs). To identify RAR/RXR heterodimer-binding sites in the human genome, we performed a modified yeast one-hybrid assays and identified 193 RAR/RXR heterodimer-binding fragments in the human genome. The putative target genes included genes involved in development process and cell differentiation. Gel mobility shift assays indicated that 160 putative RAREs could directly interact with the RAR/RXR heterodimer. Moreover, 19 functional regulatory single nucleotide polymorphisms (rSNPs) on the RAR/RXR-binding sequences were identified by analyzing the difference in the DNA-binding affinities. These results provide insights into the molecular mechanisms underlying the physiological and pathological actions of RAR/RXR heterodimers.

Identification of CAR/RXR<i>α</i>Hetero-dimer Binding Sites in the Human Genome by a Modified Yeast One-Hybrid Assay

The constitutive androstane receptor (CAR) is a transcription factor that belongs to the nuclear receptor superfamily. CAR binds as a heterodimer with the retinoid X receptor α (RXRα) to CAR response elements (CAREs) and regulates the expression of various drug metabolizing enzymes and transporters. To identify CAR/RXRα binding sites in the human genome, we performed a modified yeast one-hybrid assay that enables rapid and efficient identification of genomic targets for DNA-binding proteins. DNA fragments were recovered from positive yeast colonies by PCR and sequenced. A motif enrichment analysis revealed that the most frequent motif was a direct repeat (DR) of RGKTCA-like core sequence spaced by 4 bp. Next, we predicted 149 putative CAR/RXRα binding sites from 414 unique clones, by searching for DRs, everted repeats (ERs) and inverted repeats (IRs) of the RGKTCA-like core motif. Based on gel mobility shift assays, the CAR/RXRα heterodimer could directly interact with the 108 predicted sequences, which included not only classical CAREs but also a wide variety of arrangements. Furthermore, we identified 17 regulatory polymorphisms on the CAR/RXRα-binding sites that may influence individual variation in the expression of CAR-regulated genes. These results provide insights into the molecular mechanisms underlying the physiological and pathological actions of CAR/RXRα het-erodimers.