[A Case of Mucinous Adenocarcinoma Extending to Bladder Neck and Prostate].

A 56-year-old man visited a clinic with the chief complaint of frequent micturition and residual sensation of urine. He was referred to our hospital for close examination. Cystoscopy showed a tumor protruding toward the bladder neck from the prostate with stones and debris on the surface. Magnetic resonance imaging showed an encapsulated tumor of iso-intensity in the prostate in T2-weighed images. Prostate specific antigen was 0.88 mg/dl. Transurethral resection of prostate was performed under the diagnosis of benign prostate hyperplasia. During the operation, a solid tumor with mucus deposit was observed. Intraoperative rapid pathological diagnosis was mucinous adenocarcinoma. A radical cystectomy was performed. Pathologically, mucinous adenocarcinoma was distributed in the bladder neck, the prostate and surrounding tissue, but the prostatic urethra was intact. The surgery was assessed to be curative. Neither neoadjuvant nor adjuvant chemotherapy was performed, since the effectiveness of chemotherapy for mucinous adenocarcinoma arising from urothelial epithelium has not been established.

Rhodium(I)-Catalyzed Ring-Closing Reaction of Allene-Alkene-Alkynes: One-Step Construction of Tricyclo[6.4.0.02,6 ] and Bicyclo[6.3.0] Skeletons from Linear Carbon Chains.

Treatment of dodecatrienyne derivatives with [RhCl(CO)2 ]2 in refluxing toluene effected the cycloisomerization to produce tricyclo[6.4.0.02,6 ]dodecadienes. The one-carbon shortened undecatrienyne derivatives, however, afforded bicyclo[6.3.0]undecatriene derivatives instead of tricyclic compounds, the latter of which are well known as a basic skeleton of naturally occurring octanoids. On the basis of two experiments with deuterated substrates, a plausible reaction mechanism for the construction of these products was proposed.

Stereospecific and stereoselective rhodium(I)-catalyzed intramolecular [2+2+2] cycloaddition of allene-ene-ynes: construction of bicyclo[4.1.0]heptenes.

Treatment of the allene-ene-yne substrates with [{RhCl(CO)2}2] effected the intramolecular [2+2+2]-type ring-closing reaction to produce various of tri- and tetracyclic derivatives containing a cyclopropane ring. The reaction is highly stereoselective as well as stereospecific with good to excellent yields.

Analysis of DOK-6 function in downstream signaling of RET in human neuroblastoma cells.

Point mutations and structural alterations of the RET tyrosine kinase gene cause multiple endocrine neoplasia type 2 (MEN 2) and papillary thyroid carcinoma, respectively. RET activation by glial cell line-derived neurotrophic factor (GDNF) is essential for the development of the enteric nervous system and the kidney. The signal through RET tyrosine kinase requires several adaptor proteins including the DOK (downstream of kinase) family of proteins. Of the seven members of the DOK protein family, DOK-1, -4, -5, and -6 have been reported to play roles in the GDNF-RET signaling pathway. Although DOK-6 has been shown to bind to RET and promote GDNF-induced neurite outgrowth in mouse Neuro2A cells, DOK-6 function in human cells remains unclear. In the present study, we investigated the role of DOK-6 in GDNF-RET signaling in human cells including neuroblastoma cells. DOK-6 was constitutively localized to the plasma membrane via its pleckstrin homology (PH) domain, and was phosphorylated following RET activation via a MEN2A mutation or GDNF stimulation. However, DOK-6 could not significantly affect downstream signaling and neurite outgrowth in human neuroblastoma cells. The binding affinity of the DOK-6 phosphotyrosine-binding (PTB) domain to RET was much lower than that of the DOK-1, DOK-4, and SHC PTB domains to RET. These findings indicate that DOK-6 is involved in RET signaling with less influence when compared with DOK-1, DOK-4, and SHC.

Pleomorphic carcinoma with osteoclastic giant cells of the breast: immunohistochemical differentiation between coexisting neoplastic and reactive giant cells.

Herein is described a unique case of breast carcinoma with two different types of giant cells noted in both cytological and histological specimens. A 51-year-old Japanese woman noticed a hard mass in the upper outer quadrant of her left breast. Aspiration cytology exhibited numerous anaplastic giant cells; the cytological diagnosis was high-grade ductal carcinoma, although a few osteoclastic giant cells were also observed. A left simple mastectomy and sentinel lymph node biopsy were performed. Histologically, approximately 90% of the tumor was composed of giant cells; conventional invasive ductal carcinoma and ductal carcinoma in situ were found focally at the periphery of the tumor. The main part of the tumor contained both anaplastic, neoplastic giant cells and non-neoplastic, osteoclastic giant cells that were distinguishable from nuclear atypism. The presence of the two types of giant cells was also confirmed on immunohistochemistry using a histiocytic marker (CD68) and two epithelial markers (AE1/AE3 and CAM5.2). Based on the latest World Health Organization classification, the diagnosis was pleomorphic carcinoma with osteoclastic giant cells. To the authors' knowledge there has been no previous report on this subject except for a single case mentioned in Rosen's Breast Pathology.

A targeting mutation of tyrosine 1062 in Ret causes a marked decrease of enteric neurons and renal hypoplasia.

The Ret receptor tyrosine kinase plays a crucial role in the development of the enteric nervous system and the kidney. Tyrosine 1062 in Ret represents a binding site for the phosphotyrosine-binding domains of several adaptor and effector proteins that are important for the activation of intracellular signaling pathways, such as the RAS/ERK, phosphatidylinositol 3-kinase/AKT, and Jun-associated N-terminal kinase pathways. To investigate the importance of tyrosine 1062 for organogenesis in vivo, knock-in mice in which tyrosine 1062 in Ret was replaced with phenylalanine were generated. Although homozygous knock-in mice were born normally, they died by day 27 after birth and showed growth retardation. The development of the enteric nervous system was severely impaired in homozygous mutant mice, about 40% of which lacked enteric neurons in the whole intestinal tract, as observed in Ret-deficient mice. The rest of the mutant mice developed enteric neurons in the intestine to various extents, although the size and number of ganglion cells were significantly reduced. Unlike Ret-deficient mice, a small kidney developed in all knock-in mice, accompanying a slight histological change. The reduction of kidney size was due to a decrease of ureteric bud branching during embryogenesis. Thus, these findings demonstrated that the signal via tyrosine 1062 plays an important role in histogenesis of the enteric nervous system and nephrogenesis.

Dok-4 regulates GDNF-dependent neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein kinase.

During development of the central and peripheral nervous systems, neurite extension mediated via glial-cell-line-derived neurotrophic factor (GDNF) and its receptor RET is critical for neuronal differentiation. In the present study, we investigated the role of the RET substrate Dok-4 in neurite outgrowth induced by the GDNF/RET signaling pathway. In TGW neuroblastoma cells, which endogenously express both RET and Dok-4, depletion of Dok-4 through treatment with small interfering RNA resulted in a marked decrease in GDNF-stimulated neurite outgrowth. By contrast, exogenous expression of wild-type Dok-4 induced sustained p44/42 mitogen-activated protein kinase (ERK1/2) activation and enhanced neurite outgrowth. Expression of Dok-4 mutants in which the tyrosine residues at codons 187, 220 and 270, conserved between Dok-4, -5, and -6, were each replaced with a phenylalanine inhibited sustained ERK1/2 activation and neurite outgrowth. We also found that Dok-4 induced a significant activation of the small G protein Rap1 and that expression of a dominant active Rap1 mutant restored neurite outgrowth in Dok-4-depleted cells. By contrast, expression of a dominant negative Rap1 mutant impaired GDNF-stimulated neurite outgrowth from TGW cells. Finally, we found that neurite formation in cultured rat hippocampal neurons was enhanced by the expression of Dok-4. Together, our results suggest that Dok-4, through activation of the Rap1-ERK1/2 pathway, regulates GDNF-mediated neurite outgrowth during neuronal development.

Establishment and characterization of mouse mammary carcinoma cell lines expressing RET with a multiple endocrine neoplasia 2A mutation.

We recently generated transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation (RET-MEN2A). Mammary tumors with frequent lung metastasis were developed in 22% of female transgenic mice in a stochastic fashion. In the current study, we established two cell lines (named MKK-f and MKK-s) from mammary tumors developed in RET-MEN2A transgenic mice. MKK-f and MKK-s were derived from well-differentiated ductal carcinoma and sarcomatous spindle cell carcinoma, respectively. MKK-f cells show epithelial-like morphology with a doubling time of 19 h, and MKK-s cells show spindle-shaped morphology with a doubling time of 15 h. When inoculated in immunodeficient mice, both cell lines were tumorigenic, metastasized to the lung and displayed histological features similar to those of the primary tumors. They maintained a high level of RET expression and activation of signaling molecules downstream of RET. Consistent with the histological phenotype, expression of E-cadherin was almost undetectable in MKK-s cells, whereas its expression was very high in MKK-f cells. When the difference of gene expression between the two cell lines was analyzed using cDNA microarrays including approximately 900 genes/ESTs, a total of 21 up- or down-regulated (> 2.0-fold) genes were identified. Differentially regulated genes included thymosin beta-10, fibroblast growth factor receptor 4, aldo-keto reductase and caspase 6 genes, which are known to be associated with tumor development and progression. These results may reflect the profiles of the transcriptional changes associated with dedifferentiation or progression of mammary carcinomas developed in genetically engineered mice.

Role of Dok1 in cell signaling mediated by RET tyrosine kinase.

Using a yeast two-hybrid screen, we identified Dok1 as a docking protein for RET tyrosine kinase. Dok1 bound more strongly to RET with a multiple endocrine neoplasia (MEN) 2B mutation than RET with a MEN2A mutation and was highly phosphorylated in the cells expressing the former mutant protein. Analysis by site-directed mutagenesis revealed that tyrosine 361 in mouse Dok1 represents a binding site for the Nck adaptor protein and tyrosines 295, 314, 361, 376, 397, and 408 for the Ras-GTPase-activating protein. We replaced tyrosine 361 or these six tyrosines with phenylalanine (designated Y361F or 6F) in Dok1 and introduced the mutant Dok1 genes into the cells expressing the wild-type RET or RET-MEN2B protein. Overexpression of Dok1 or Dok1-Y361F, but not Dok1-6F, suppressed the Ras/Erk activation induced by glial cell line-derived neurotrophic factor or RET-MEN2B, implying that this inhibitory effect requires the Ras-GTPase-activating protein binding to Dok1. In contrast, overexpression of Dok1, but not Dok1-Y361F or Dok1-6F, enhanced the c-Jun amino-terminal kinase (JNK) and c-Jun activation. This suggested that the association of Nck to tyrosine 361 in Dok1 is necessary for the JNK and c-Jun activation by glial cell line-derived neurotrophic factor or RET-MEN2B. Because a high level of the JNK phosphorylation was observed in the cells expressing RET-MEN2B, its strong activation via Nck binding to Dok1 may be responsible for aggressive properties of medullary thyroid carcinoma developed in MEN 2B.