Kimihiro Kiyokane

Downregulation of TGFβ isoforms and their receptors contributes to keratinocyte hyperproliferation in psoriasis vulgaris

BACKGROUND Psoriasis vulgaris is a chronic inflammatory disorder characterized by epidermal hyperproliferation. Transforming growth factor beta (TGFbetas) have a major antiproliferative action in epidermis. OBJECTIVE We evaluated the distribution and levels of expression of TGFbeta isoforms and their receptors in psoriatic versus normal skin with the goal of discovering potential alterations in TGFbeta signal transduction associated with psoriasis. METHODS Expression of TGFbeta isoforms and their receptors was analyzed in normal and psoriatic skin using immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. Furthermore, DNA synthesis was measured in normal keratinocytes transfected with a dominant-negative TGFbeta receptor II (TbetaRII) vector that eliminated most of the cytoplasmic TbetaRII domain. RESULTS Marked elevations in DNA synthesis, as assessed by BrdU incorporation and proliferating cell nuclear antigen (PCNA) immunoreactivity, were confirmed in psoriatic epithelial cells. Using immunohistochemistry and RT-PCR analysis, expression of TGFbeta2 and 3 was diminished in the psoriatic epidermis as compared with those observed in normal skin. With respect to TGFbeta receptors, expression of TbetaRI and II was markedly decreased in the psoriatic epidermis. In addition, levels of Smad2 mRNA were also decreased in psoriatic skin. Transfection of normal keratinocytes with the dominant-negative TbetaRII vector significantly elevated DNA synthesis as compared with keratincoytes transfected with control vector (under condition of TGFbeta addition), suggesting that the dominant-negative TbetaRII mutant inhibits the antiproliferative effects of TGFbeta. CONCLUSION The present investigation strongly suggest that the TGFbeta signaling pathway is downregulated in psoriatic skin and this situation leads to abnormal cell proliferation due to a functional decrease in growth regulation.

The presence of h2-calponin in human keratinocyte

Calponin (h1 isoform) was characterized as a smooth muscle specific, actin-, tropomyosin-, calmodulin-binding protein and described as a factor which inhibits contraction. H2-calponin, encoded by a different gene from h1-calponin, was identified from the smooth muscles of mouse and pig. However, non-muscle calponin analogues have recently been reported in rat and pig brains. Here we show the presence of calponin expressed in human skin tissue and in cultured human keratinocytes using polyclonal antibodies to bovine aortic smooth muscle calponin. Western blot analysis demonstrated that calponin with a molecular weight around 36,000 existed in extracts of keratinocytes. Immunofluorescence microscopy displayed the localization of calponin in the cytoplasm of the basal cells in situ, and along the cell-to-cell borders in cultured human keratinocytes maintained in standard calcium medium. Furthermore, according to RT-PCR analysis using human h1- and h2-calponin-specific primers, calponin expressed by cultured human keratinocytes was identified as the h2 isoform. We demonstrated the presence of h2-calponin in human keratinocytes, and it might play a role in the structural organization of actin cytoskeleton at the cytoplasmic region of cell-to-cell junctions of keratinocytes.

A Case of Syringocystadenocarcinoma Papilliferum In situ Occurring Partially in Syringocystadenoma Papilliferum

We report a case of syringocystadenocarcinoma papilliferum in situ associated with syringocystadenoma papilliferum. The patient was a 64‐year‐old man with a red tumor that arose on top of his head two years before he consulted our department. The histological findings revealed a papillomatous growth on the epidermis forming several invaginations. Numerous papillary projections, lined by a two‐layered epithelium with a benign appearance and decapitations on the luminal surface of the cells, extended into the lumens of the invaginations. Some projections showed a disorderly arrangement of multilayered cells with atypical nuclei. No differences between findings in the syringocystadenocarcinoma papilliferum in situ and those in the syringocystadenoma papilliferum were observed histochemically (PAS) or immunohistochemically (cytokeratin, CEA, CA 19–9, S‐100, gross cystic disease fluid protein, lysozyme and Leu M1).

p53 gene mutations in squamous cell carcinoma occurring in scars: comparison with p53 protein immunoreactivity.

We determined the relationship between p53 expression and p53 gene mutations in squamous cell carcinoma occurring in scars and unrelated to UV light irradiation. We analyzed biopsy specimens obtained from three patients with squamous cell carcinoma. A monoclonal antibody against p53 (DO-7) was used for the immunohistochemical analysis. p53 gene mutations were detected by the polymerase chain reaction and single-strand conformation polymorphism analysis and direct DNA sequencing. p53 overexpression was observed in atypical squamous cells of one case. Those of two other cases, however, showed negative immunoreactivity to p53. Exon 6 of the p53 gene in all three cases and exon 7 in one case showed electrophoretic mobility shifts in polymerase chain reaction and single-strand conformation polymorphism analysis. DNA sequencing analysis showed a missense mutation and a silent mutation in exon 6 of the case with p53 overexpression, a three-base deletion in exon 6 of one case with no p53 overexpression, and a three-base deletion in exon 6 and a missense mutation in exon 7 of another such case. Although immunohistochemical overexpression of p53 has been thought to result from p53 gene mutations, our results suggest that negative immunoreactivity to p53 also can result from p53 gene mutations, for example, short gene deletions.

Metastatic epithelioid sarcoma with an N-ras oncogene mutation.

At age 25 a Japanese woman noticed an elastic-hard nodule 2 cm in diameter on the anterior side of her right leg. The nodule had developed an ulcer in its center. Simple resection was performed several times. However, the lesion recurred repeatedly. The patient underwent amputation of the right leg at the age of 34, because the diagnosis of epithelioid sarcoma was established histologically. No recurrence was observed for 9 years. Recently, the patient noticed multiple painful, ulcerative nodules about 1 cm in diameter on her scalp, trunk, and extremities. She refused extensive resection for a religious reason and died of massive hematemesis. Autopsy revealed metastatic epithelioid sarcoma in the skin, lungs, kidneys, pancreas, transverse colon, thyroid, and sternum. Chromosomal analysis of the tumor revealed various aberrations and an N-ras oncogene mutation.

Expression of lung resistance protein in epithelioid sarcoma in vitro and in vivo

Abstract The incidence of epithelioid sarcoma among patients with malignant soft tissue tumors is small, but the rates of recurrence and metastasis of this type of sarcoma are high. To date, effective chemotherapy for advanced epithelioid sarcoma has not been established and, furthermore, epithelioid sarcoma is known to exhibit multidrug resistance (MDR). The chemosensitivities to anticancer agents of two cell lines established from epithelioid sarcoma were examined in this study. The results showed that the ES-OMC-MN and SFT-8606 cell lines were resistant to vincristine (IC 50 1190 n M and 872 n M , respectively) and Adriamycin (IC 50 921 n M and 650 n M , respectively), but sensitive to actinomycin D (IC 50 < 10 n M ). P-glycoprotein (p-Gp) and MDR-associated protein (MRP) were not expressed in these cell lines, but a high expression level of lung resistance protein (LRP) was observed. The original tumor tissues from which the two cell lines were established were also found to be LRP-positive but not to express p-Gp or MRP. Their chemosensitivities to Adriamycin were not significantly altered in the presence of 2.5 μg/ml anti-LRP antibody (LRP-56), but the IC 50 of vincristine was much less (IC 50 128 n M and 27 n M , respectively) than that for an untreated cell line. It is thus suggested that the vincristine resistance in the two cell lines is LRP-mediated. Since cyclosporin A, known to be a modifier of p-Gp, also induced reversal of vincristine resistance in the ES-OMC-MN and SFT-8606 cell lines (IC 50 6.2 n M and 17 n M , respectively), it is suggested that cyclosporin A acts as a modifier of MDR mediated by LRP.

Establishment and characterization of an epithelioid sarcoma cell line with an autocrine response to interleukin-6

A novel epithelioid sarcoma (ES) cell line, designated as ES-OMC-MN, was established from a 44-year-old woman with recurrence and metastasis of ES. The cells were spindle-shaped or polygonal and were positive for cytokeratin and vimentin on immunohistochemical staining. Electron microscopy revealed desmosome-like structures between the cells. These characteristics were also noted in the original tumor. Southern blot analysis of HindIII digests showed an additional 8.0 kb band and an 8.8 kb band in DNA from the cultured cells and the original tumor as well as the peripheral blood cells of the patient, while only an 8.8 kb band was observed in control human placental DNA. There were no point mutations of N-ras codons 12, 13, and 61, suggesting that the abnormality of N-ras was not due to mutation but to polymorphism. Interleukin-6 (IL-6) was secreted into the culture medium at high levels. Recombinant IL-6 augmented the proliferation of these cells, while cell growth was inhibited by incubation with an anti-IL-6 antibody. The cells also expressed surface IL-6 receptors, indicating that IL-6 acted on this cell line in an autocrine manner. IL-6 and IL-6 receptors were also found in the original tumor. These results demonstrate that ES-OMC-MN cells retained all the morphological and biochemical characteristics of the original tumor and suggest that an autocrine effect of IL-6 may be involved in the development of ES.

Epidermal growth factor binding sites in the mouse exocrine and endocrine pancreas shown by in vivo quantitative microautoradiography and confocal laser scanning microscopy

Microautoradiography at 3, 6 and 15 min after intravenous injection of 125I-EGF was used to investigate the distribution of epidermal growth factor (EGF) binding sites in the pancreas of normal male mice. The autoradiographs were observed by confocal laser microscopy, which allows the quantification of silver grains. The results demonstrated that both endocrine and exocrine pancreatic cells exhibited substantial specific binding of 125I-EGF. The highest level of EGF binding was found in the duct cells of the exocrine pancreas followed by the acinar cells. The cells of the islets of Langerhans also showed substantial specific binding of 125I-EGF though the binding level was lower than that of the exocrine pancreas. In the control experiments, mice were injected with 125I-EGF and various amounts of unlabeled EGF.

Case of cutaneous malignant melanoma surviving 16 years with late recurrence

Late recurrence, defined as that occurring 10 or more years after diagnosis, is an unusual event in cutaneous malignant melanoma (MM). A 59‐year‐old woman presented with a black nodule measuring 10 mm × 9 mm on the sole of her right foot. She was diagnosed with MM and the tumor was totally excised with 5 cm of the normal surrounding skin. Eleven years after the operation, five in‐transit metastases were found in her right limb. They were all excised and β‐interferon (IFN‐β) was injected into the skin around the postoperative scars. However, numerous new in‐transit skin metastases have been emerging every year in her right limb. Fifty‐four in‐transit skin metastases have so far been found. Recently, there have been few in‐transit metastases. All in‐transit metastatic lesions were excised and local IFN‐β injections were conducted continuously. There is no evidence of metastases to the internal organs or lymph nodes. This report describes this case with a brief review of the published work concerning the rare late recurrences of MM.

Early blistering, poikiloderma, hypohidrosis, alopecia and exocrine pancreatic hypofunction: A peculiar variant of Rothmund-Thomson syndrome?

A 20-year-old male developed early blistering, poikiloderma, hypohidrosis, alopecia and exocrine pancreatic hypofunction caused by atrophy and fatty replacement of the pancreas. At 5 months of age, he initially presented at the hospital with numerous blisters on his extremities, inguinal and genital area. A biopsy specimen from a vesicular lesion showed a subepidermal bulla. Electron microscopic examinations of a vesicular lesion revealed vacuolar changes of the basal cells without hemidesmosomes. Subsequently, the blisters gradually resolved and healed without scars. At the age of 11, he was admitted for the treatment of cellulitis on his foot and at that time, laboratory examinations detected a decreased level of pancreatic enzymes due to exocrine pancreatic hypofunction. Abdominal ultrasonography and computed tomography (CT) showed the pancreas to be atrophic with fatty replacement. A genetic analysis revealed no mutation in his RECQL4 gene, which is responsible for the pathogenesis of Rothmund-Thomson syndrome (RTS). Although marked blister formation and exocrine pancreatic hypofunction are unusual complications of RTS, this case showed many typical clinical features of RTS. Therefore, this case was considered to be a peculiar variant of RTS.