Shingo Kamoshida

Mediator Subunits MED1 and MED24 Cooperatively Contribute to Pubertal Mammary Gland Development and Growth of Breast Carcinoma Cells

ABSTRACT The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ER-targeted genes encoding E2F1 and cyclin D1, which promote progression through the G1/S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells.

Clinicopathological implications of expressions of hypoxia-related molecules in esophageal superficial squamous cell carcinoma.

This study was conducted to clarify whether or not expressions of hypoxia-related molecules would have clinicopathological significance in squamous cell carcinoma (SCC) of the esophagus. Expressions of hypoxia inducible factor-1 alpha (HIF-1alpha), glucose transporter 1 (GLUT-1) and RAC-1 were immunohistochemically analyzed in 96 surgically resected SCCs at pT1b (sm1, 12 cases; sm2, 35 cases; sm3, 49 cases). They were divided into a lymph node metastasis (LNM)-positive group composed of 44 cases and an LNM-negative group composed of 52 cases. Immunohistochemical profiles were estimated based on the staining extent (score: 1+, 2+, 3+) and intensity (score: 1+, 2+, 3+). A significant expression pattern was found in the nucleus for HIF-1alpha, cell membrane for GLUT-1 and cytoplasm for RAC-1. The cases were categorized into a high score group (total score of 4 or more) and a low score group (total score of 3 or less) in each maker, respectively. A comparison made between the LNM-positive group and the LNM-negative group showed that the proportion of cases with a high score was larger in the LNM-positive group than in the LNM-negative group (HIF-1alpha, P = .02; GLUT-1, P = .008; RAC-1, P = .001). Among them, HIF-1alpha was found to be significantly related to the disease-free survival (P = .019) and overall survival (P = .034) as well as LNM (disease-free survival, P = .030; overall survival, P = .030). The multivariate analysis demonstrated that the HIF-1alpha expression would be an independent indicator for prognosis. In the superficial SCCs of the esophagus, GLUT-1 and RAC-1 may be involved in LNM, and HIF-1alpha overexpression is expected to predict an unfavorable clinical outcome.

Expression of organic anion-transporting polypeptide 1A2 and organic cation transporter 6 as a predictor of pathologic response to neoadjuvant chemotherapy in triple negative breast cancer

Organic anion-transporting polypeptide 1A2 (OATP1A2) and organic cation transporter 6 (OCT6) are involved in the uptake of taxanes and anthracyclines, respectively. The aim of this study was to evaluate expression levels of OATP1A2 and OCT6 as a predictor of response to neoadjuvant chemotherapy (NAC) in breast cancer. A total of 124 patients who received anthracycline/taxane-based NAC were included. Expression levels of OATP1A2 and OCT6 were immunohistochemically assessed in core needle biopsies obtained prior to NAC. A pathologic good response (pGR) and a pathologic complete response (pCR) were achieved in 24 and 10xa0% of patients, respectively. In univariate analysis of the entire cohort, negative hormone receptor (HR) status (pGR and pCR, Pxa0<xa00.001), high Ki-67 level (pGR, Pxa0=xa00.03; pCR, Pxa0=xa00.02), triple negative (TN) subtype (pGR, Pxa0=xa00.001; pCR, Pxa0<xa00.001), and high OCT6 (pGR, Pxa0=xa00.003) were associated with the response. In combined analysis, high OATP1A2/high OCT6 level was also a significant factor for pGR (Pxa0=xa00.001) and pCR (Pxa0=xa00.001). Two separate multivariate analyses showed that HR status, TN subtype and combined high OATP1A2/high OCT6 level were significant independent predictors. When TN and non-TN tumors were assessed separately in univariate analysis, high Ki-67 level (Pxa0=xa00.04) were associated with pGR and combined high OATP1A2/high OCT6 level was associated with both pGR (Pxa0=xa00.005) and pCR (Pxa0=xa00.03) in the TN group. Multivariate analysis identified the combined high OATP1A2/high OCT6 level as the sole independent predictor of pGR. In the non-TN group, negative HR status (Pxa0=xa00.03) and positive HER2 status (Pxa0=xa00.005) were associated with pGR, but HER2 status was the sole independent predictor of pGR. These results suggest that response-associated predictors may differ between the TN and non-TN tumors. Combined high OATP1A2/high OCT6 may be a potential predictor of response to anthracycline/taxane-based chemotherapy in breast cancer, especially in TN tumors.

Significance of cell proliferation markers (Minichromosome maintenance protein 7, topoisomerase IIα and Ki-67) in cavital fluid cytology: Can we differentiate reactive mesothelial cells from malignant cells?

The aim of this study was to evaluate whether immunocytochemical expressions of proliferation markers, such as minichromosome maintenance protein 7 (MCM 7), topoisomerase IIα (topo IIα), and Ki‐67, in reactive mesothelial cells and malignant cells obtained from cavital fluids could be useful for their differential diagnosis. Samples diagnosed as reactive mesothelial cells (14 cases) or malignant tumors (28 cases) in cavital fluids were examined. Immunocytochemical staining of MCM 7, topo IIα, and Ki‐67 was performed with the universal immunoperoxidase polymer method. In reactive mesothelial cells, MCM 7 was stained in a fine granular pattern and its distribution was uniform in the nuclei. Topo IIα and Ki‐67 were stained in a coarse granular pattern and the distributions were the same as MCM 7. In contrast, in malignant cells, MCM 7 was stained in an irregular and fine granular pattern, and topo IIα and Ki‐67 were stained in a uniform and coarse granular pattern. Labeling indices of MCM 7 (cut‐off value; 30%, sensitivity; 100%, and specificity; 100%), topo IIα (cut‐off value; 15%, sensitivity; 89.3%, and specificity; 92.9%) and Ki‐67 (cut‐off value; 30%, sensitivity; 64.3%, and specificity; 92.9%) of malignant cells were significantly higher than those of reactive mesothelial cells. MCM 7, topo IIα, and Ki‐67 are different types of cell proliferation markers. MCM 7 and topo IIα, in particular, could be reliable tools for differential diagnosis between reactive mesothelial cells and malignant cells. Diagn. Cytopathol. 2010.

Effects of Combined Administration of DPD-Inhibitory Oral Fluoropyrimidine, S-1, Plus Paclitaxel on Gene Expressions of Fluoropyrimidine Metabolism-Related Enzymes in Human Gastric Xenografts

BackgroundS-1 is the most effective oral fluoropyrimidine derivative widely used for patients with gastric carcinoma in Japan. Although S-1 plus taxane has been a promising candidate as an effective chemotherapeutic regimen, the mechanisms of its additive or synergistic anticancer effects and changes in gene expression after the administration of these agents have not yet been fully elucidated.MethodsExperimental chemotherapy was performed using human gastric carcinoma xenografts, MKN-45 and TMK-1, to examine anticancer effects and gene expressions of fluoropyrimidine metabolism-related enzymes including thymidine phosphorylase (TP), thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and uridine phosphorylase (UP). Nude mice were treated with S-1, paclitaxel, and their combination. After treatment, inxa0vivo antitumor effects of S-1, paclitaxel alone, and their combination and the effects on gene expressions of enzymes involved in 5-fluorouracil metabolism were examined using the RT-PCR method.ResultsThe combined use of S-1 and paclitaxel showed additive to synergistic antitumor effects on both gastric cancer xenografts. While consistent upregulation of dThPase and DPD gene expression was exhibited after administration of S-1, no further increase of dThPase gene expression after combined use of S-1 with paclitaxel was observed. There was no increase in TS gene expression after the administration of either S-1 alone or paclitaxel alone.ConclusionThese results provide some insight into the mechanism and/or rationale underlying the additive to synergistic effect of combined administration of S-1 and paclitaxel in gastric carcinoma.

Optimal Antigen Retrieval for Ethanol-Fixed Cytologic Smears

Antigen retrieval, a crucial technique for immunostaining, is often carried out on formalin‐fixed, paraffin‐embedded (FFPE) tissue sections. The role of antigen retrieval in immunostaining of ethanol‐fixed smears remains unclear. The authors evaluated the effects of 2 common antigen retrieval procedures, heat‐induced antigen retrieval and protease‐induced antigen retrieval, for immunostaining using a broad panel of antibodies.

Immunohistochemical analysis of organic anion transporter 2 and reduced folate carrier 1 in colorectal cancer: Significance as a predictor of response to oral uracil/ftorafur plus leucovorin chemotherapy

Colorectal cancer is one of the most common malignancies in developed countries and chemotherapy is the standard treatment option for advanced colorectal cancer. Identification of biomarkers for predicting response to uracil/ftorafur plus leucovorin (UFT/LV) chemotherapy is an important issue in colorectal cancer treatment. Organic anion transporter 2 (OAT2) and reduced folate carrier 1 (RFC1) are the major uptake transporters of 5-fluorouracil (5-FU) and LV, respectively. In the present study, the correlation between OAT2 and RFC1 expression and histological response to preoperative UFT-based (UFT or UFT/LV) chemotherapy was investigated. Pre-treatment biopsy specimens obtained from 45 patients were evaluated for OAT2 and RFC1 expression levels by using an immunohistochemical approach. A high expression of OAT2 and RFC1 was significantly correlated with good response to UFT-based chemotherapy (P<0.0001 and P= 0.002, respectively). In multivariate logistic regression analysis, a high OAT2 expression was an independent predictor of good response to UFT-based chemotherapy (P=0.02), unlike RFC1 expression. High expression levels of OAT2 were significantly correlated with a good response in the UFT-treated (P= 0.04) as well as the UFT/LV-treated (P<0.0005) groups; however, RFC1 expression levels were significantly correlated with a good response only in the UFT/LV-treated group (P=0.02). Therefore, immunohistochemical analysis of OAT2 and RFC1 may serve as a useful tool for predicting the efficacy of UFT/LV treatment regimens in colorectal cancer patients.

Endogenous Interleukin-18 Regulates Testicular Germ Cell Apoptosis during Endotoxemia

Orchitis (testicular swelling) often occurs during systemic inflammatory conditions, such as sepsis. Interleukin 18 (IL18) is a proinflammatory cytokine and is an apoptotic mediator during endotoxemia, but the role of IL18 in response to inflammation in the testes was unclear. WT and IL18 knockout (KO) mice were injected lipopolysaccharide (LPS) to induce endotoxemia and examined 12 and 48u200a h after LPS administration to model the acute and recovery phases of endotoxemia. Caspase activation was assessed using immunohistochemistry. Protein and mRNA expression were examined by western blot and quantitative real-time RT-PCR respectively. During the acute phase of endotoxemia, apoptosis (as indicated by caspase-3 cleavage) was increased in WT mice but not in IL18 KO mice. The death receptor-mediated and mitochondrial-mediated apoptotic pathways were both activated in the WT mice but not in the KO mice. During the recovery phase of endotoxemia, apoptosis was observed in the IL18 KO mice but not in the WT mice. Activation of the death-receptor mediated apoptotic pathway could be seen in the IL18 KO mice but not the WT mice. These results suggested that endogenous IL18 induces germ cell apoptosis via death receptor mediated- and mitochondrial-mediated pathways during the acute phase of endotoxemia and suppresses germ cell apoptosis via death-receptor mediated pathways during recovery from endotoxemia. Taken together, IL18 could be a new therapeutic target to prevent orchitis during endotoxemia.

Smac/DIABLO expression in human gastrointestinal carcinoma: Association with clinicopathological parameters and survivin expression

Lack of apoptosis is a key factor in carcinogenesis and tumor progression. Survivin is a member of the inhibitor of apoptosis protein (IAP) family. Second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) is an antagonist of IAPs. Recently, Smac/DIABLO was identified as a potent therapeutic target. However, the clinical significance of Smac/DIABLO in gastrointestinal carcinomas remains unclear. In the present study, Smac/DIABLO expression was analyzed by immunohistochemistry in 72 gastric adenocarcinomas and 78 colorectal adenocarcinomas. The expression of Smac/DIABLO was significantly higher in colorectal carcinoma than in gastric carcinoma. Additionally, a correlation was found between the expression of Smac/DIABLO and nuclear survivin in well- to moderately-differentiated colorectal adenocarcinomas (r=0.245; P<0.01). Based on these results, it was hypothesized that gastric and colorectal carcinomas differ in the level of Smac/DIABLO expression. Our previous studies revealed that the expression of cleaved caspase-9 was significantly lower in colorectal carcinoma than in gastric carcinoma (P<0.0001). Conversely, the expression levels of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and survivin were significantly higher in colon cancer than in gastric cancer (P<0.0001 and P<0.01, respectively). Taken together, these results indicate that not only LC3 and survivin expression, but also Smac/DIABLO expression, are significantly higher in colorectal carcinoma than in gastric carcinoma. We hypothesize that the analysis of Smac/DIABLO, survivin and LC3 expression in colorectal carcinoma is likely to aid cancer therapy due to the involvement of these markers in apoptosis and/or autophagy.

Immunohistochemical expression profiles of solute carrier transporters in alpha‐fetoprotein‐producing gastric cancer

Alpha‐fetoprotein (AFP)‐producing gastric cancer (GC) is an aggressive tumour with high rates of liver metastasis and poor prognosis, and for which a validated chemotherapy regimen has not been established. Drug uptake by solute carrier (SLC) transporters is proposed as one of the mechanisms involved in sensitivity to chemotherapy. In this study, we aimed to develop important insights into effective chemotherapeutic regimens for AFP‐producing GC.