Tadashi Ishiyama

Phenotypic characterization of endometrial stromal sarcoma of the uterus

Endometrial stromal sarcoma (ESS) of the uterus is a rare uterine malignancy that has not been characterized in detail. To characterize the phenotype of ESS of the uterus, we extracted RNA from ESS and the stroma of normal endometrium using a tissue microdissection system and compared the expression profiles in the two tissues. After suppression subtractive hybridization and differential screening, we detected the metastasis‐associated lung adenocarcinoma transcript 1 (MALAT‐1) gene as one of the major genes upregulated in ESS, and a full‐length placental cDNA clone (CS0DI066YJ10) as one of the major genes downregulated. The results were confirmed by in situ hybridization in four resected specimens of ESS and 36 biopsy specimens of normal endometrial tissue. All ESS (4/4) and all cases of endometrial stromal cells in the proliferative phase (13/13) were positive for MALAT‐1, but samples of normal stroma in the secretory phase and menopausal state included some that were negative or weakly positive for MALAT‐1 (5/13 and 3/10, respectively). In contrast, all ESS and 12 of 13 cases of stromal cells in the proliferative phase were negative for the full‐length placental cDNA clone but 10 of 13 cases of endometrial stromal cells in the secretory phase were positive for transcripts of the gene (P < 0.05). These results indicated that endometrial stromal cells have different phenotypic characteristics between proliferative and secretory phases and the tumor cells of ESS have the phenotypic character of endometrial stromal cells in the proliferative phase. (Cancer Sci 2006; 97: 106  – 112)

Expression of HNFs and C/EBPα is correlated with immunocytochemical differentiation of cell lines derived from human hepatocellular carcinomas, hepatoblastomas and immortalized hepatocytes

Objective assessment of the differentiation grade of hepatocellular carcinomas (HCCs) is important for evaluation of the pathological diagnosis, prognosis and therapeutic treatment. Differentiation of hepatocytes is reflected by their expression of hepatic functional proteins in the mouse embryo, and liver‐enriched transcription factors (LETFs) have been shown to regulate hepatic functional genes strictly. Previous reports demonstrated that the level of LETF expression is altered in HCC or preneoplastic nodules compared with noncancerous tissues. Therefore, LETF expression levels might be useful as a measure of HCC maturation. In this study, to clarify the correlation between the expression of LETFs and the differentiation grade of HCCs, we performed a quantitative analysis of the mRNA expressions of HNFs and C/EBPα using real‐time reverse‐transcription PCR and immunocytochemical analysis for hepatic functional proteins in twelve cell lines. Furthermore, we examined orthotopic transplantations of the HCC cell lines in C.B‐17/Icrj‐scid/scid mice and characterized the histologic and cytologic differentiation of the tumors that developed. Our results showed that comprehensive expressions of HNF‐3β, HNF‐4α, HNF‐1α, and C/EBPα were specific to HCCs with well‐differentiated function and morphology. Furthermore, among these four transcription factors, HNF‐4α and HNF‐1α expressions showed synchronism and had a close relation with HCC differentiation. These in vitro results were confirmed in tumors developed in SCID mice in vivo. These findings suggested that HNF‐4α and HNF‐1α are useful markers to assess the degree of HCC differentiation, which we suggest could be evaluated objectively by the quantitative analysis of HNFs and C/EBPα in HCCs.

Establishment of an immortalized cell line from a precancerous lesion of lung adenocarcinoma, and genes highly expressed in the early stages of lung adenocarcinoma development

Atypical adenomatous hyperplasia (AAH) is classified as a precancerous lesion of lung adenocarcinoma. We established an immortalized AAH cell line (PL16T) and a human non‐neoplastic bronchial epithelial cell line (PL16B) from the same patient by transfection with the gene for SV40 large T antigen. The expression profile of PL16T was compared with that of PL16B by the suppression subtractive hybridization method. From 704 selectively hybridized clones, we finally selected 25 fragments of mRNA that showed transcription levels more than three times higher in PL16T than in PL16B. Thirteen (52%) and eight (32%) of them encoded tumor‐associated calcium signal transducer 2 (TACSTD2) and S100 calcium binding protein A2 (S100A2), respectively. The high transcription of TACSTD2 and S100A2 in PL16T was confirmed by in situ hybridization. In normal lung tissue, both TACSTD2 and S100A2 were expressed at very low levels, but seven and five of 14 AAH were positive for TACSTD2 and S100A2, respectively. The frequency of TACSTD2 positivity was increased in 16 of 22 bronchioloalveolar carcinomas (BAC) and adenocarcinoma with mixed subtype with BAC component (mixed BAC). Positivity for S100A2 occurred in four of 22 BAC and mixed BAC. The abnormal transcription of TACSTD2 and S100A2 are thought to be unique molecular markers of the preinvasive stage of lung adenocarcinoma.(Cancer Sci 2005; 96: 668 – 675)

ESTABLISHMENT OF HEPATIC STEM-LIKE CELL LINES FROM NORMAL ADULT PORCINE LIVER IN A POLY-D-LYSINE–COATED DISH WITH NAIR-1 MEDIUM

SummaryThe existence, origin, and bipotency of the hepatic stem cell (HeSC) have been investigated. However, the isolation and culture of HeSCs from adult liver tissue is not yet well established, and the mechanism by which HeSCs differentiate into mature cells remains unclear. On the other hand, the development of HeSC-isolating and-culturing methods and the in vitro clonal analysis of their mechanism of differentiation are required to enable clinical applications of regenerative medicine in the liver. For the purpose of providing HeSCs for these studies, we attempted to establish an HeSC line from a normal adult porcine liver using a unique culture system, a poly-D-lysine-coated culture dish with NAIR-1 medium (the PDL-NAIR-1 culture system). Moreover, we examined the differentiating capacity of HeSCs in vitro. We demonstrated that it was possible in the culture system that immature epithelial cells capable of proliferating grew selectively into aggregates and that two hepatic stem-like cell lines, PHeSC-A1 and PHeSC-A2, were established. The results from our data suggest that these hepatic stem-like cell lines were capable of self-renewing and differentiating into hepatocytes or biliary epithelial cells and show that the PDL-NAIR-1 culture system offers the immense advantage of isolating and culturing HeSCs from a normal adult liver. Furthermore, because of the ability to use a clonal analysis in vitro, these cell lines are useful for the investigation of various mechanisms in which HeSCs seem to participate and their application in the study of regenerative medicine in the liver.

OCIA domain containing 2 is highly expressed in adenocarcinoma mixed subtype with bronchioloalveolar carcinoma component and is associated with better prognosis

Although lung adenocarcinoma is a major cause of cancer death worldwide, details of its molecular carcinogenesis and stepwise progression are still unclear. To characterize the sequential progression from bronchioloalveolar adenocarcinoma of the lung (BAC, in situ carcinoma) to adenocarcinoma mixed subtype with BAC component, polymerase chain reaction‐based cDNA suppression subtractive hybridization (SSH) was carried out using two representative cases of BAC (non‐invasive tumors) and adenocarcinoma mixed subtype with BAC (invasive tumors). Through differential screening, virtual reverse northern hybridization and quantitative real‐time reverse‐transcription–polymerase chain reaction (qRT‐PCR) we selected five genes (TncRNA, OCIAD2, ANXA2, TMED4 and LGALS4) that were expressed at significantly higher levels in invasive adenocarcinoma mixed subtype with BAC than in BAC. After in situ hybridization and qRT‐PCR analyses, we confirmed that only the OCIAD2 gene showed significantly higher expression in the tumor cells of invasive adenocarcinoma mixed subtype with BAC than in BAC (P = 0.026). We then carried out in situ hybridization of OCIAD2 in 56 adenocarcinoma mixed subtype with BAC component and assessed the correlation between OCIAD2 expression and clinicopathological features. In contrast to our expectation, the patients with OCIAD2 expression showed a better clinical outcome than those without OCIAD2 expression, and OCIAD2 expression showed an inverse correlation with lymphatic invasion, blood vessel invasion and lymph node metastasis. These results suggest that OCIAD2 begins to express at the progression from in situ to invasive carcinoma, and is associated with the favorable prognosis of adenocarcinoma mixed subtype with BAC component. (Cancer Sci 2007; 98: 50–57)

Differentially expressed genes in a porcine adult hepatic stem-like cell line and their expression in developing and regenerating liver.

To identify differentially expressed genes in adult hepatic stem cells, we performed suppression-subtractive hybridization (SSH) between adult porcine hepatic stem-like cells (HSLCs) and hepatocytes, and the expression of selected genes was assessed in porcine fetal livers and regenerating liver in an 80% hepatectomy model. SSH and subsequent differential screening selected 39 clones that were expressed differentially in HSLCs, including six known genes, 10 unknown genes, one unidentified gene and some chimeric fragments. Four of these genes showed significantly higher expression in HSLCs than in mature hepatocytes: anti-leukoproteinase, matrix Gla protein, amyloid-β precursor protein (APP) and dickkopf-3 (DKK-3). Among them, the mRNA expression of APP and DKK-3 was significantly higher in fifth GW fetal liver than in seventh and thirteenth GW fetal and adult livers, unlike the expression patterns of α-fetoprotein (AFP) or albumin. These mRNAs were detected in the parenchyma of fifth GW fetal liver, whereas in normal adult liver possible expression was limited to the periportal area. On the other hand, immunohistochemistry, Massons trichrome staining and silver impregnation demonstrated APP and DKK-3 proteins in fifth GW fetal liver in which intralobular bile ducts and hepatic plates had not completely developed. DKK-3 and AFP mRNAs were upregulated on the seventh day (7D) after 80% hepatectomy. In the liver tissue, DKK-3 and AFP proteins were detected in mesenchymal cells in the periportal area and parenchyma, respectively. These data for DKK-3 expression in adult livers suggest the possible presence of adult HSLCs in the periportal area. The pattern of histological staining suggested that 7D liver was in the process of regeneration, showing a character similar to the fifth GW fetal liver. It is speculated that DKK-3 is upregulated in immature and developing livers, and has possible involvement in hepatic differentiation and liver regeneration.

The ACIN1 gene is hypermethylated in early stage lung adenocarcinoma.

Introduction and Hypothesis: In recent years, many studies have performed genome-wide searching for differentially methylated genes in cancer. We hypothesized that characteristic aberrant hypermethylation of CpG islands of certain genes may exist in the early stages of lung adenocarcinoma and that such alterations may be useful in the detection and treatment of early lung adenocarcinoma. Methods: A pair of immortalized cell lines originating from atypical adenomatous hyperplasia (PL16T) and from the resected end of the bronchus of the same patient (PL16B) was searched for aberrantly and differentially hypermethylated DNA fragments by a combination of the methylated CpG island amplification and suppression subtractive hybridization methods. Results: From 229 clones, we selected 15 fragments that had a genomic region meeting the criteria for a CpG island. We identified a gene, apoptotic chromatin condensation inducer 1 (ACIN1), that was hypermethylated in PL16T. A higher frequency of hypermethylation at a locus at the 5′: end of the DNA fragment isolated from the ACIN1 gene was found in small-sized adenocarcinoma (2 cm or less) (30/37, 81%) compared with normal lung tissue (9/37, 24%, p < 0.05). Interestingly, hypermethylation of ACIN1 was detected relatively frequently in the normal counterpart of adenocarcinoma without bronchioloalveolar carcinoma (BAC) component (7/16, 44%), but was rare in the normal counterpart of adenocarcinoma with BAC component (2/21, 10%, P < 0.05). Conclusions: We found hypermethylation of the ACIN1 gene in early stage lung adenocarcinoma. The role of methylation status in the development and malignant transformation of lung adenocarcinoma requires clarification.

Phenotypic characteristics of mouse lung adenoma induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The expression profile of adenoma induced by 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) in A/J mice was compared with that of normal lung tissue by suppression subtractive hybridization (SSH). The mRNAs of surfactant‐associated protein A (SP‐A) and lysozyme showed characteristically higher transcription in the adenoma tissue than in normal lung. High expression of both SP‐A and lysozyme in tumor cells was confirmed by in situ hybridization (ISH). In normal lung, alveolar type II pneumocytes were positive for both SP‐A and lysozyme, indicating that tumor cells retained the phenotypic characteristics of the murine alveolar type II pneumocytes. Previous studies of human adenocarcinomas have shown that the two proteins are expressed reciprocally; SP‐A and lysozyme are differential markers of atypical adenomatous hyperplasia (AAH) and non‐goblet cell type adenocarcinoma, and of goblet cell type adenocarcinoma, respectively. Thus, the present results indicate that the phenotype of NNK‐induced A/J mouse adenoma differs from that of AAH, which is thought to be a preinvasive lesion of human adenocarcinoma.