Yuji Yaginuma

Hypoxia-Independent Overexpression of Hypoxia-Inducible Factor 1α as an Early Change in Mouse Hepatocarcinogenesis

Hypoxia-inducible factor 1 (HIF-1) is involved in tumor progression/metastasis and activated in various cancers. Here we show that HIF-1alpha, which plays a major role in HIF-1 activation, is overexpressed in preneoplastic hepatocytic lesions from a very early stage during hepatocarcinogenesis in mice and man. Transcriptional targets of HIF-1, such as vascular endothelial growth factor, glut-1, c-met, and insulin-like growth factor II (IGF-II), were also overexpressed in mouse lesions. Oxygen tension within the lesions was not different from that of the normal hepatic tissues, indicating that HIF-1alpha expression was independent of hypoxia. On the other hand, Akt, the pathway of which can up-regulate HIF-1alpha expression, was activated in the mouse lesions, whereas HIF-1alpha was markedly down-regulated in the mouse hepatocellular carcinoma (HCC) cell lines after treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1alpha expression is dependent on PI3K/Akt signaling. Conversely, HIF-1alpha knockdown by short interfering RNA in the HCC cell line resulted in decreased expression of activated Akt together with the HIF-1 target genes, indicating that Akt activation is reversely dependent on HIF-1 activation. Treating the HCC cells with IGF-II or epidermal growth factor (EGF) up-regulated both phospho-Akt and HIF-1alpha, whereas inhibition of IGF-II or EGF signaling down-regulated them both, suggesting that IGF-II and EGF can, at least in part, mediate the activation of Akt and HIF-1alpha. However, Akt was not activated by IGF-II or EGF in the HIF-1alpha knockdown cells, indicating that expression of the HIF-1 target genes is necessary for the Akt activation. These findings suggest that the reciprocal activation of PI3K/Akt signaling and HIF-1alpha may be important in the progression of hepatocarcinogenesis.

Overexpression of N-Acetylglucosaminyltransferase III Enhances the Epidermal Growth Factor-induced Phosphorylation of ERK in HeLaS3 Cells by Up-regulation of the Internalization Rate of the Receptors

N-Acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension ofN-glycans by introducing a bisectingN-acetylglucosamine residue. In this study we investigated the effect of GnT-III on epidermal growth factor (EGF) signaling in HeLaS3 cells. Although the binding of EGF to the epidermal growth factor receptor (EGFR) was decreased in GnT-III transfectants to a level of about 60% of control cells, the EGF-induced activation of extracellular signal-regulated kinase (ERK) in GnT-III transfectants was enhanced to ∼1.4-fold that of the control cells. A binding analysis revealed that only low affinity binding of EGF was decreased in the GnT-III transfectants, whereas high affinity binding, which is considered to be responsible for the downstream signaling, was not altered. EGF-induced autophosphorylation and dimerization of the EGFR in the GnT-III transfectants were the same levels as found in the controls. The internalization rate of EGFR was, however, enhanced in the GnT-III transfectants as judged by the uptake of125I-EGF and Oregon Green-labeled EGF. When the EGFR internalization was delayed by dansylcadaverine, the up-regulation of ERK phosphorylation in GnT-III transfectants was completely suppressed to the same level as control cells. These results suggest that GnT-III overexpression in HeLaS3 cells resulted in an enhancement of EGF-induced ERK phosphorylation at least in part by the up-regulation of the endocytosis of EGFR.

α1,6fucosyltransferase is highly and specifically expressed in human ovarian serous adenocarcinomas

An elevated level of α1,6fucosylation in N‐glycans represents one of the cancer‐related alterations of oligosaccharides and is associated with the metastatic potential of hepatoma cells. However, expression of α1,6fucosyltransferase (α1,6FucT), which is involved in this aberrant glycosylation, has not been intensively explored in other malignant tumors. We report on a study of the expression of α1,6FucT in various types of epithelial ovarian carcinoma tissue, as well as normal ovary, benign and borderline ovarian tumors. The activity assay showed that α1,6FucT is highly and specifically elevated in serous adenocarcinomas but not in normal and other ovarian tumor tissues. This elevation was due to enhancement of mRNA expression, as evidenced by Northern blot analysis. Furthermore, we have shown immunohistochemically that α1,6FucT expression is localized predominantly in cancer cells. Lectin blot analysis using Lens culinaris agglutinin, which preferentially recognizes α1,6fucose residue, suggested that several glycoproteins were likely targets for modification by α1,6fucosylation in serous adenocarcinoma tissues. These findings suggest that the elevated expression of α1,6FucT and the resulting modification of N‐glycans are distinctive features of this type of ovarian cancer and may be related to the progression of this malignancy. Int. J. Cancer 88:914–919, 2000.

Activated hepatic stellate cells overexpress p75NTR after partial hepatectomy and undergo apoptosis on nerve growth factor stimulation.

Abstract: Background: Expression of neurotrophins (NTs) and their receptors is increased during hepatic regeneration, but their role is not well understood.

Neoplastic hepatocyte growth associated with cyclin D1 redistribution from the cytoplasm to the nucleus in mouse hepatocarcinogenesis

Cyclin D1 overexpression is a frequent change in hepatocellular carcinomas (HCCs). Our present study demonstrated that cyclin D1 overexpression with abundant cyclin E, cdk4, cdk2, and p27Kip1 (p27) occurred in neoplastic hepatocytes from the early stage of mouse hepatocarcinogenesis. While cyclin D1 expression was mainly found in the cytoplasm of the tumor cells, it shifted to the nucleus in association with cell proliferation after the animals were subjected to a partial hepatectomy (PH), and then returned once more to the cytoplasm when the cells became quiescent. Inhibition of PI3 kinase (PI3K) by Ly294002 in mouse HCC cells in vitro suppressed the nuclear shift of cyclin D1 as well as cell proliferation, while PI3K activation by PTEN suppression failed to induce nuclear shift of cyclin D1, suggesting that PI3K activation is essential but not sufficient for the cyclin D1 nuclear shift. While MEK‐ERK1/2 inhibition by PD98059 and mTOR inhibition by rapamycin affected the cyclin D1 nuclear shift and cell proliferation to a lesser extent, both these inhibitors reduced cyclin D1 levels. Finally, although p27, cdk4 and calmodulin (CaM) were detected in the cyclin D1 immunoprecipitates from both quiescent and proliferating HCC cells, Hsc70 and SSeCKS were detected only in the immunoprecipitate from quiescent cells, and p21Waf1/Cip1 (p21) was detected only in that from proliferating cells, suggesting that the cyclin D1 complex is different in quiescent and proliferating cells. These observations indicate that the nuclear/cytoplasmic localization of cyclin D1 plays an important role in the proliferation/quiescence of neoplastic hepatocytes.

Bilateral Oophorectomy in Asymptomatic Women over 50 Years Old Selected by Ovarian Cancer Screening

The purpose of this study was to evaluate bilateral oophorectomy in women over 50 years old found to have an adnexal mass using transvaginal ultrasonography (TVS) as a mass screening. With TVS a total of 23,451 women without symptoms were examined for ovarian cancer at annual screening for uterine cervical cancer. Two hundred fifty-eight women over 50 years old persistently had abnormal TVS results and 95 women gave informed consent for surgical tumor removal. In the 95 women operated, 7 malignant ovarian cancers were found. Especially adnexal masses which were thought to be benign were treated by laparoscopic surgery.

Abnormal structure and expression of PTEN/MMAC1 gene in human uterine cancers

The PTEN/MMAC1 gene, located on human chromosome 10q23, has recently been implicated as a candidate tumor suppressor gene in human cancers. In the present study, 12 uterine cancer cell lines and 87 uterine cancers of various grades and histological type were analyzed for PTEN/MMAC1 gene. Three of 44 endometrial carcinoma (7%) showed no PTEN/MMAC1 mRNA expression by RT–PCR analysis. Sequencing analysis of entire coding region of PTEN/MMAC1 gene revealed mutations in three of six endometrial cancer cell lines (50%) and 17 of 44 endometrial cancer tissues (39%). In contrast, for cervical cancers, only one of six cancer cell lines (2%) showed mutation, and one of 43 cancer tissues (2%) had an abnormality. Overall, 36% of the abnormal spots were located in exon 5, 24% were in exon 8, 16% were in exon 3, and 8% were in exon 6, and single cases of abnormality were found in exons 1, 4, and 7. Our results revealed that, in total, 60% of abnormalities were clustered in exons 5 and 8. Exon 5 is a functional domain of the PEN/MMAC1 gene, and therefore, abnormalities in this region may be important for loss of PTEN/MMAC1 gene function. Finally, we found a high frequency of PTEN/MMAC1 gene abnormalities in endometrial carcinomas but a low frequency in cervical carcinomas. These findings suggest that disruption of PTEN/MMAC1 by mutation or absence of expression may contribute to the pathogenesis or neoplastic evolution in a large proportion of endometrial carcinomas but in a small proportion of cervical carcinomas. Mol. Carcinog. 27:110–116, 2000.

Expression of NGF in hepatocellular carcinoma cells with its receptors in non-tumor cell components

Nerve growth factor (NGF) is suggested to have a role in tumor progression in addition to its role in differentiation and survival of neuronal cells. We investigated expression of NGF and its receptors, TrkA and p75NTR, in hepatocellular carcinomas (HCCs). Although hepatocytes and hepatic stellate cells (HSCs) showed respectively weak and intense NGF immunostaining in the background livers of patients suffering from liver cirrhosis (LC) or chronic hepatitis (CH), intense staining was demonstrated in HCC cells of 33 of 54 (61.1%) tumors. RT‐PCR detected NGF mRNA in 7 freshly‐isolated HCC samples, and in 2 of 4 cases, in which both background livers and tumors could be analyzed, NGF mRNA was more abundant in the tumors than the background livers. TrkA was detected in the smooth muscle cells of hepatic arteries, but it was negative in tumor cells as well as non‐neoplastic hepatocytes. p75NTR and α‐smooth muscle actin (αSMA) was expressed in HSCs in the background liver and fibroblast‐like cells in stromal septa, whereas HSCs within the HCC tissues were mostly negative for p75NTR but positive for αSMA. This suggests that HSCs in HCC have a different property from those in background livers. Furthermore, the stromal septa contained abundant nerve fibers, which may be related to the increased NGF expression in HCC cells. NGF and its receptors are then thought to have a role in cellular interactions involving HCC cells, HSCs, arterial cells and nerve cells in HCC tissues.

Analysis of the p53 gene in human choriocarcinoma cell lines

In the present study, we analysed human choriocarcinoma cell lines for abnormalities in the tumour-suppressor gene p53 by Southern blotting, Northern blotting, non-radioisotopic single-stranded conformational polymorphism (SSCP) and complementary DNA sequencing. In all cell lines (Bewo, GCH-1, GCH-2, SCH, JAR, JEG-3, NUC-1 and HCCM-5), no p53 gene abnormality was detected by using Southern blotting. p53 mRNA of the expected size was detected in all cell lines tested by Northern blotting. SSCP analysis revealed abnormalities of p53 cDNA in the SCH cell line. Sequencing analysis of the entire coding region of the p53 gene revealed that both alleles were expressed in the JEG-3 cell line, and one of the alleles contained a point mutation (G to T) in codon 167 (Gln to His). In the NUC-1 cell line both alleles were point mutated. One allele had a point mutation (A to T) that resulted in a codon 17 change (Glu to Asp), and another had a point mutation (A to T) that caused a codon 24 change (Lys to Asn). In the SCH cell line, AGG was inserted between codon 249 and 250; this insertion resulted in an abnormal structure of the p53 protein. In three out of eight human choriocarcinoma cell lines, a p53 gene abnormality was detected. Therefore our data demonstrate that p53 gene abnormalities are associated with choriocarcinoma cell lines.

Telomerase activity and human papillomavirus (HPV) infection in human uterine cervical cancers and cervical smears

Telomerase activity and human papillomavirus (HPV) infection were investigated in uterine cervical samples using molecular biology techniques. Thirteen cervical carcinomas and corresponding normal tissue from the same patient, and 102 cervical swabs were examined. Telomerase activity was detected in 12 of 13 cervical cancer tissues (92%). Of the 12 cases that showed telomerase activity, all were HPV positive, and the one case that did not show telomerase activity was HPV negative. A telomeric repeat amplification protocol assay detected telomerase activity in one out of seven normal cervical tissues (14%), and this one case was HPV positive. In cervical smear samples, telomerase activity was detected in two out of 36 normal smears (6%; both HPV positive), in 10 of 32 (31%) CIN1 (cervical intra-epithelial neoplasia) cases (three HPV positive), in four of five (80%) CIN2 cases (two HPV positive), in 15 of 21 (71%) CIN3 cases, (seven HPV positive) and in seven of eight (88%) squamous cell carcinoma cases (six HPV positive). These results suggest that telomerase activity may play some role in cervical carcinogenesis, and telomerase activity is associated with HPV infection in uterine cervical lesions.