Vesicoenteric Fistula Arising from an Adenocarcinoma of Ectopic Pancreatic Tissue in a Meckel Diverticulum.

We report a case of a vesicoenteric fistula arising from an adenocarcinoma of ectopic pancreatic tissue in a Meckel diverticulum in a 58-year-old man. The patient suffered from refractory micturition pain and increased urinary frequency. Computerized tomography with a contrast agent showed a ring-shaped enhanced mass near the dome of the urinary bladder. Magnetic resonance imaging showed a cystic mass close to the urinary bladder with partly irregular wall and fistula formation to the urinary bladder. Surgical findings showed a Meckel diverticulum in the ileum, which formed a fistula with the urinary bladder, and Meckel diverticulectomy and partial cystectomy were performed. Histological findings revealed a vesicoenteric fistula arising from a papillary adenocarcinoma of ectopic pancreatic tissue in a Meckel diverticulum. The patient has survived without recurrence for more than 4 years since surgery.

Distinguishing testicular torsion from torsion of the appendix testis by clinical features and signs in patients with acute scrotum.

PURPOSE: Many physicians encounter confusion and difficulty in distinguishing testicular torsion (TT) from torsion of the appendix testis (TAT) in patients with acute scrotum because of the overlapping signs and symptoms. The objective of our study was to evaluate the clinical features and signs that can help distinguish TT from TAT. PATIENTS AND METHODS: We performed a retrospective study of patients with surgically confirmed TT and TAT at our institute from January 1990 to December 2013. Clinical findings, physical examination findings, climatic conditions, laboratory data, and color Doppler ultrasound (CDUS) findings were compared between the TT and TAT groups. RESULTS: Seventy patients were included in this study (49 with TT and 21 with TAT). Patients with TT were significantly older than those with TAT (p < 0.001). The ambient temperature at onset was significantly lower in patients with TT than in patients with TAT (p = 0.038). Testicular swelling, high-riding testes, onset during sleep, high leukocyte counts, and high creatine phosphokinase levels were significantly more common in patients with TT than with TAT (p = 0.021, 0.032, 0.006, 0.003, and 0.043, respectively). Multivariate analysis showed that age and onset during sleep were significant independent factors for detection of TT. Eight patients (16.3%) underwent preoperative CDUS evaluation, and an absent or decreased blood signal in the involved testes was significantly correlated with the presence of TT (p = 0.018). CONCLUSION: In clinical features, age and onset during sleep might be helpful to distinguish TT from TAT. When supported by findings, urgent surgical exploration is warranted in patients with suspected TT based on symptoms and CDUS features.

Serum level and immunohistochemical expression of vascular endothelial growth factor for the prediction of postoperative recurrence in renal cell carcinoma.

BACKGROUND: Vascular endothelial growth factor (VEGF) plays a major role in angiogenesis. One of the functions of VEGF is to regulate neovascularization in clear cell renal cell carcinoma (CCRCC). The objective of our study was to examine whether before nephrectomy serum levels of VEGF or expression of VEGF using immunohistochemistry (IHC) could predict postoperative recurrence in nonmetastatic CCRCC. RESULTS: Twelve patients (14.5%) had recurrence during a mean follow-up of 52.6 ± 31.2 months. The serum VEGF level was significantly higher in patients with recurrence than in those without recurrence (P = 0.038). High serum VEGF levels were above 416 pg/mL; this value was chosen based on a receiver operating characteristic analysis. The recurrence-free survival rate in patients with a high serum VEGF level was significantly lower than in those with a low serum VEGF level (P = 0.003). In total, tumors from 26 patients (31.3%) showed overexpression of VEGF using IHC. The recurrence-free survival rate in the IHC-positive group was significantly lower than that in the IHC-negative group (P = 0.044). Multivariate analysis indicated that preoperative serum VEGF levels (P = 0.013) and female gender (P = 0.004) were independent predictors of postoperative recurrence in nonmetastatic CCRCC. CONCLUSIONS: Preoperative serum VEGF levels is a useful predictor compared with IHC analysis of VEGF of postoperative recurrence in nonmetastatic CCRCC.

[Predicting postoperative recurrence of renal cell carcinoma using serum vascular endothelial growth factor].

BACKGROUND: Vascular endothelial growth factor (VEGF) is known as one of the key molecules in molecular targeting therapy for patients with renal cell carcinoma (RCC). Several studies have shown that VEGF might be useful for predicting prognosis in RCC. We examined whether pretreatment serum VEGF can be used as a predictor of recurrence-free survival in non-metastatic RCC. MATERIALS AND METHODS: We studied 85 patients with non-metastatic clear cell RCC who underwent nephrectomy between 2001 and 2010. Serum samples were collected for VEGF before operation. We evaluated the recurrence-free survival by univariate and multivariate analysis. RESULTS: 9 patients (10.6%) showed recurrence. Serum level of VEGF in patients with recurrence showed significantly higher than those in patients without recurrence (p = 0.0310). A cutoff level of 416 pg/mL for the separation of low and high serum VEGF levels was established based on the receiver operating characteristic (ROC) curve. The recurrence-free survival rate was significantly lower in patients with a high serum VEGF level (p = 0.0039). Multivariate analysis showed that pretreatment serum VEGF value was a significant predictor of postoperative recurrence in non-metastatic clear cell RCC (p = 0.0062). CONCLUSIONS: Pretreatment level of serum VEGF might be useful for prediction of postoperative recurrence in non-metastatic clear cell RCC.

[Case of congenital bladder diverticulum--cause of urinary retention in adulthood].

A 24-year-old man was referred to our department for urinary retention and urinary tract infection. He was pointed out a bladder diverticulum in childhood. Computerized tomography (CT) scanning and magnetic resonance imaging (MRI) showed a bladder diverticulum, 10 by 8 cm in size, which was located posteriorly in the bladder. He underwent resection of the bladder diverticulum and left ureterovesiconeostomy. The histopathologic finding showed a bladder wall with thin muscular layer. The operation made urination possible with successful resolution of voiding symptoms. Herein we report this rare case of congenital bladder diverticulum which was treated in adulthood, and present a review the literature.

Localization of nectin-free afadin at the leading edge and its involvement in directional cell movement induced by platelet-derived growth factor.

Afadin is an actin-filament-binding protein that binds to nectin, an immunoglobulin-like cell-cell adhesion molecule, and plays an important role in the formation of adherens junctions. Here, we show that afadin, which did not bind to nectin and was localized at the leading edge of moving cells, has another role: enhancement of the directional, but not random, cell movement. When NIH3T3 cells were stimulated with platelet-derived growth factor (PDGF), afadin colocalized with PDGF receptor, alphavbeta3 integrin and nectin-like molecule-5 at the leading edge and facilitated the formation of leading-edge structures and directional cell movement in the direction of PDGF stimulation. However, these phenotypes were markedly perturbed by knockdown of afadin, and were dependent on the binding of afadin to active Rap1. Binding of Rap1 to afadin was necessary for the recruitment of afadin and the tyrosine phosphatase SHP-2 to the leading edge. SHP-2 was previously reported to tightly regulate the activation of PDGF receptor and its downstream signaling pathway for the formation of the leading edge. These results indicate that afadin has a novel role in PDGF-induced directional cell movement, presumably in cooperation with active Rap1 and SHP-2.

MCAF1/AM is involved in Sp1-mediated maintenance of cancer-associated telomerase activity.

Telomerase maintains telomere length and is implicated in senescence and immortalization of mammalian cells. Two essential components for this enzyme are telomerase reverse transcriptase (TERT) and the telomerase RNA component (encoded by the TERC gene). These telomerase subunit genes are known to be mainly expressed by specificity protein 1 (Sp1). MBD1-containing chromatin-associated factor 1 (MCAF1), also known as ATFa-associated modulator (AM) and activating transcription factor 7-interacting protein (ATF7IP), mediates gene regulation, although the precise function of MCAF1 remains to be elucidated. Here, we report that MCAF1 is involved in Sp1-dependent maintenance of telomerase activity in cancer cells. Two evolutionarily conserved domains of MCAF1 directly interact with Sp1 and the general transcriptional apparatus. Selective depletion of MCAF1 or Sp1 down-regulates TERT and TERC genes in cultured cells, which results in decreased telomerase activity. The transcriptionally active form of RNA polymerase II and the general transcription factor ERCC3 decreased in the TERT promoter under the loss of MCAF1 or Sp1. Consistently, MCAF1 is found to be frequently overexpressed in naturally occurring cancers that originate in different tissues. Our data suggest that transcriptional function of MCAF1 facilitates telomerase expression by Sp1, which may be a common mechanism in proliferative cancer cells.

[Comparison of clinical results between TUR-P and holmium laser enucleation of the prostate (HoLEP) based on the initial experience].

OBJECTIVE: We compared the surgical results between holmium laser enucleation of the prostate (HoLEP) and transurethral resection of the prostate (TUR-P) for the treatment of men with benign prostatic hyperplasia (BPH). METHODS: A total of 87 patients with symptomatic BPH were analysed. HoLEP was performed on 46 men (mean age 68.2 +/- 7.5 years old) from December 2005 to February 2007, and TUR-P was performed on 41 men (mean age 69.2 +/- 7.3 years old) from April 2004 to March 2006. RESULTS: Both groups were comparable in terms of age, pre-operative IPSS, QOL index, urodynamic study results and prostate volume. During operation, decrease in hemoglobin was less in the HoLEP group than in the TUR-P group (1.15 +/- 1.2 vs 1.91 +/- 1.3 g/dl p < 0.05). The operation time was significantly longer in the HoLEP group than in the TUR-P group (161.9 +/- 65.0 vs. 118.3 +/- 36.9 minutes p < 0.001). Mean resected weight was 29.3 +/- 13.3 g (10-55) in the TUR-P group and 34.8 +/- 33.4 g (5-148) in the HoLEP group (p = 0.337). The catheterization period (52.1 +/- 29.6 vs. 115.2 +/- 27.5 hour p < 0.001) and hospital stay (6.6 +/- 2.3 vs. 9.4 +/- 2.2 days p < 0.001) were significantly shorter in the HoLEP group than in the TUR-P group. At follow up, Qmax, average flow rate and post void residual urine (PVR) in two groups improved significantly, and these parameters were not significantly different between the groups after 3 months. CONCLUSIONS: Both TUR-P and HoLEP were effective in relieving BOO. The estimated blood loss, a catheterization time and hospitalization were less or shorter in the HoLEP group. HoLEP may be a good alternative to the conventional transurethral electrocautery resection of the prostate for symptomatic BPH.

Regulation of platelet-derived growth factor receptor activation by afadin through SHP-2: implications for cellular morphology.

Upon binding of platelet-derived growth factor (PDGF), PDGF receptor is autophosphorylated at tyrosine residues in its cytoplasmic region, which induces the activation of diverse intracellular signaling pathways such those involving Ras-ERK, c-Src, and Rap1-Rac. Signaling through activated Ras-ERK promotes cell cycle and cell proliferation. The sequential activation of Rap1 and Rac affects cellular morphology and induces the formation of leading-edge structures, including lamellipodia, peripheral ruffles, and focal complexes, resulting in the enhancement of cell movement. In addition to the promotion of cell proliferation, the Ras-ERK signaling is involved in the regulation of cellular morphology. Here, we showed a novel role of afadin in the regulation of PDGF-induced intracellular signaling and cellular morphology in NIH3T3 cells. Afadin was originally identified as an actin filament-binding protein, which binds to a cell-cell adhesion molecule nectin and is involved in the formation of cell-cell junctions. When afadin was tyrosine-phosphorylated by c-Src activated in response to PDGF, afadin physically interacted with and increased the phosphatase activity of Src homology 2 domain-containing phosphatase-2 (SHP-2), a protein-tyrosine phosphatase that dephosphorylates PDGF receptor, leading to the prevention of hyperactivation of PDGF receptor and the Ras-ERK signaling. In contrast, knockdown of afadin or SHP-2 induced the hyperactivation of PDGF receptor and Ras-ERK signaling and consequently suppressed the formation of leading-edge structures. Thus, afadin plays a critical role in the proper regulation of the PDGF-induced activation of PDGF receptor and signaling by Ras-ERK. This effect, which is mediated by SHP-2, impacts cellular morphology.

The roles of nectins in cell adhesions: cooperation with other cell adhesion molecules and growth factor receptors.

Nectins are Ca(2+)-independent Ig-like cell adhesion molecules (CAMs) which homophilically and heterophilically interact in trans with nectins and form cell-cell adhesion. This cell-cell adhesion is involved in the formation of many types of cell-cell junctions such as adherens junctions, tight junctions, and synaptic junctions, cooperatively with other CAMs such as cadherins and claudins. Nectins transduce signals cooperatively with integrin alpha(v)beta(3), and regulate formation of cell-cell junctions. In addition, nectin interacts in cis with PDGF receptor and regulates its signaling for anti-apoptosis. Furthermore, nectin interacts in trans with nectin-like molecule-5 (Necl-5) and regulate cell movement and proliferation. We describe cooperative roles of nectins with other CAMs and growth factor receptors.

Cooperative roles of Par-3 and afadin in the formation of adherens and tight junctions.

Par-3 is a cell-polarity protein that regulates the formation of tight junctions (TJs) in epithelial cells, where claudin is a major cell-cell adhesion molecule (CAM). TJs are formed at the apical side of adherens junctions (AJs), where E-cadherin and nectin are major CAMs. We have revealed that nectin first forms cell-cell adhesions, and then recruits cadherin to nectin-based cell-cell adhesion sites to form AJs and subsequently recruits claudin to the apical side of AJs to form TJs. The cytoplasmic tail of nectin binds afadin and Par-3. Afadin regulates the formation of AJs and TJs cooperatively with nectin. Here, we studied the role of Par-3 in the formation of these junctions by using Par-3-knockdown MDCK cells. Par-3 was necessary for the formation of AJs and TJs but was not necessary for nectin-based cell-cell adhesion. Par-3 promoted the association of afadin with nectin, whereas afadin was not necessary for the association of Par-3 with nectin. However, the association of afadin with nectin alone was not sufficient for the formation of AJs or TJs, and Par-3 and afadin cooperatively regulated it. We describe here these novel roles of Par-3 in the formation of junctional complexes.

Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells.

The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Here, we studied the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associated with p120(ctn), beta-catenin, and alpha-catenin, at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly was mediated by the IQGAP1-dependent actin cytoskeleton, which was organized by Cdc42 and Rac small G proteins that were activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 bound to afadin, and this Rap1-afadin complex then interacted with p120(ctn) associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120(ctn) for the formation of AJs in Madin-Darby canine kidney cells.

Common signaling pathway is used by the trans-interaction of Necl-5/Tage4/PVR/CD155 and nectin, and of nectin and nectin during the formation of cell-cell adhesion.

Nectin is a Ca2+-independent Ig-like cell-cell adhesion molecule that forms homo- and hetero-trans-dimers (trans-interaction). Nectin first forms cell-cell adhesions and then recruits cadherin to the nectin-based cell-cell adhesion sites to form AJ cooperatively with cadherin. In addition, the trans-interaction of nectin and nectin induces the activation of Cdc42 and Rac small G proteins, which enhances the formation of AJ. The activation of Cdc42 and Rac by the trans-interaction of nectin and nectin is mediated by c-Src, another small G protein, Rap1, a Cdc42-GEF, FRG, and a Rac-GEF, Vav2. Necl-5/Tage4/PVR/CD155 is another Ca2+-independent Ig-like molecule, which does not homophilically trans-interact, but heterophilically trans-interacts with nectin-3, one member of the nectin family. We show here that the trans-interaction of Necl-5 and nectin-3 bidirectionally induces the activation of Cdc42 and Rac. Similarly to the activation of Cdc42 and Rac by the trans-interaction of nectin and nectin, the trans-interaction of Necl-5 and nectin-3 first recruits and activates c-Src at the Necl-5/nectin-3-based cell-cell contact sites. c-Src then phosphorylates FRG and Vav2, and the tyrosine-phosphorylated FRG and Vav2 are recruited to the Necl-5/nectin-3-based cell-cell contact sites. The trans-interaction of Necl-5 and nectin-3 also activates Rap1 through C3G, a Rap-GEF, and this activation of Rap1 is required for the activation of Cdc42 and Rac. These results indicate that the trans-interactions of Necl-5 and nectin-3 and of nectin and nectin induce the activation of Cdc42 and Rac through the common signaling molecules c-Src, Rap1, FRG, and Vav2.

Transcriptional repression and heterochromatin formation by MBD1 and MCAF/AM family proteins.

DNA methylation cooperates with methylation at lysine 9 of histone H3 (H3-K9), a modified histone molecule that is targeted by heterochromatin protein 1, to form a transcriptionally silent chromatin. Methyl CpG-binding protein MBD1 recognizes methylated CpG dinucleotide and recruits H3-K9 methyltransferases such as SETDB1 to genomic regions. Here we show that MBD1-containing chromatin-associated factor (MCAF) 1, also known as the human homologue of murine ATFa-associated modulator (AM), is required for transcriptional repression and heterochromatin formation by MBD1, together with the involvement of SETDB1. Moreover, the amino acid sequence of MCAF1 shows similarity to a number of sequences of the MCAF/AM-related proteins, resulting in the identification of a new member of the protein family, termed MCAF2. Immunoprecipitation and in vitro binding analyses reveal that both MCAF proteins interact with MBD1, SETDB1, and Sp1 via two evolutionarily conserved distinct domains. Furthermore, MCAF1 enhances transcriptional repression by MBD1 together with SETDB1, and exogenous expression of MCAF2 partly compensates for the repressive activity in MCAF1 knockdown HeLa cells. The expression of MBD1 mutant, which lacks interaction with MCAF proteins, perturbs heterochromatin protein 1-enriched heterochromatin formation at the MBD1-containing chromosomal loci. These data suggest that MBD1.MCAF1.SETDB1 complex facilitates the formation of heterochromatic domains, emphasizing the role of MCAF/AM family proteins in epigenetic control.

Hormonal regulation of metastasis-associated protein 3 transcription in breast cancer cells.

Metastasis-associated protein 3 (MTA3) is a cell type-specific subunit of the Mi-2/NuRD transcriptional corepressor complex. In breast cancer cells, MTA3 and the Mi-2/NuRD complex mediate repression of Snail, a transcription factor that promotes epithelial to mesenchymal transitions. Thus, MTA3 functions to maintain a differentiated, epithelial status in breast cancer. Interestingly, in mammary epithelial cells, MTA3 biosynthesis requires both functional estrogen receptor (ER) and estradiol. Here we have investigated the molecular basis for estrogen and ER-dependent expression of MTA3 in breast cancer cells. Molecular dissection of the MTA3 promoter using transient transfection assays identified a composite element required for high-level transcription consisting of an SP1 site in close proximity to a consensus estrogen response element half-site. Depletion of either SP1 or ER-alpha by RNA interference led to loss of MTA3 transcript in multiple breast cancer cell lines, indicating a requirement for both transcription factors in expression of endogenous MTA3. The MTA3 gene thus joins a growing list of loci regulated by both SP1 and ER.

MTA3 and the Mi-2/NuRD complex regulate cell fate during B lymphocyte differentiation.

The transcriptional repressor BCL-6 regulates B lymphocyte cell fate during the germinal center reaction by preventing terminal differentiation of B lymphocytes into plasma cells until appropriate signals are received. Here, we report a cofactor, MTA3, a cell type-specific subunit of the corepressor complex Mi-2/NuRD, for BCL-6-dependent cell fate determination. MTA3 is expressed in the same pattern in germinal centers as BCL-6. BCL-6 physically interacts with Mi-2/NuRD and this interaction is sensitive to BCL-6 acetylation status. Depletion of MTA3 by RNAi impairs BCL-6-dependent repression and alters the cell-specific transcriptional pattern characteristic of the B lymphocyte. Remarkably, exogenous expression of BCL-6 in a plasma cell line leads, in an MTA3-dependent manner, to repression of plasma cell-specific transcripts, reactivation of the B cell transcriptional program, expression of B lymphocyte cell surface markers, and reprogramming of cell fate.

Use of bifunctional cross-linking reagents in mapping genomic distribution of chromatin remodeling complexes.

Chromatin remodeling complexes consist of multiple subunits, some of which are in intimate contact with DNA while others are not. The ability to effectively cross-link proteins to DNA with formaldehyde is impacted by the average distance between the two species. Productive cross-linking of proteins not in direct contact with DNA is greatly facilitated by the inclusion of an initial cross-linking reaction using bifunctional imidoester cross-linking reagents.

Mi-2/NuRD: multiple complexes for many purposes.

The vertebrate Mi-2/NuRD complex is a multi-subunit protein complex containing both histone deacetylase and nucleosome-dependent ATPase subunits. Current models predict that this complex functions primarily in transcriptional repression. Surprisingly, every subunit of this complex presents heterogeneity at the protein and gene level. This raises the intriguing possibility of functional specialization resulting from incorporation of unique gene products into the complex. The MTA (metastasis-associated) proteins represent one class of alternative subunits of the human Mi-2/NuRD complex. The members of this family in human cells are differentially expressed depending on cell type and on physiologic parameters. We summarize evidence supporting the view that the alternative subunits of the complex that have arisen during vertebrate evolution endow unique functional properties.

Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link transcriptional repression and DNA repair in chromatin.

The methyl-CpG dinucleotide containing a symmetrical 5-methylcytosine (mC) is involved in gene regulation and genome stability. We report here that methylation-mediated transcriptional repressor methylated DNA-binding domain 1 (MBD1) interacts with methylpurine-DNA glycosylase (MPG), which excises damaged bases from substrate DNA. MPG itself actively represses transcription and has a synergistic effect on gene silencing together with MBD1. Chromatin immunoprecipitation analysis reveals the molecular movement of MBD1 and MPG in vivo: (i) The MBD1-MPG complex normally exists on the methylated gene promoter; (ii) treatment of cells with alkylating agent methylmethanesulfonate (MMS) induces the dissociation of MBD1 from the methylated promoter, and MPG is located on both methylated and unmethylated promoters; and (iii) after completion of the repair, the MBD1-MPG complex is restored on the methylated promoter. Mobility-shift and structural analyses show that the MBD of MBD1 binds a methyl-CpG pair (mCpG x mCpG) but not the methyl-CpG pair containing a single 7-methylguanine (N) (mCpG x mCpN) that is known as one of the major lesions caused by MMS. We further demonstrate that knockdown of MBD1 by specific small interfering RNAs significantly increases cell sensitivity to MMS. These data suggest that MBD1 cooperates with MPG for transcriptional repression and DNA repair. We hypothesize that MBD1 functions as a reservoir for MPG and senses the base damage in chromatin