Hisashi Hisatomi

Expression profile of a γ-deletion variant of the human telomerase reverse transcriptase gene

The human telomerase reverse transcriptase (hTERT) is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of chromosomes. The hTERT transcript has been shown to have two deletion type alternative splicing sites. One deletion site induces the alpha-deletion variant, lacking 36 bp from exon 6, and the other induces the beta-deletion variant, lacking 182 bp from exons 7 and 8. Here, we identified a novel deletion variant of the hTERT transcript in hepatocellular carcinoma cell lines. The deleted transcript was characterized by an in-frame deletion of 189 bp, spanning nucleotides 2710 to 2898, corresponding to the complete loss of exon 11 (gamma-deletion). The region lacking in the gamma-deletion lies within RT motifs D and E, suggesting that it is missing conserved residues from the catalytic core of the protein. Both gamma- and alpha-deletion variants were occasionally detected, but the beta-deletion variant was frequently observed. Our results may provide important information for more detailed studies on the regulation of telomerase activity.

Expression of survivin during liver regeneration.

Survivin functions to suppress cell death and regulate cell division, and is observed uniquely in tumor cells and developmental cells. However, the expression and regulation of survivin in non-transformed cells are not well elucidated. Therefore, we investigated the expression of survivin in a murine liver regeneration model after partial hepatectomy and intraperitoneal carbon tetrachloride (CCl(4)) injection. We found that the expression of survivin transcript and protein were markedly elevated with the onset of DNA synthesis and remained elevated during G2 and M phases during liver regeneration. In a normal mouse liver cell line, over-expression of survivin resulted in a decrease in the G0/G1 phase and an increase in the S and G2/M phases, resulting in Rb phosphorylation. These findings suggest that survivin is dramatically expressed in a cell cycle-dependent manner during liver regeneration and provide a new insight into the regulation of cell proliferation and differentiation.

ARHI/NOEY2 Inactivation May Be Important in Breast Tumor Pathogenesis

Allelic loss frequently occurs on the short arm of chromosome 1 in human breast carcinoma, suggesting that the ARHI/NOEY2 gene, an imprinted putative tumor suppressor gene, is involved in the pathogenesis of the tumor entity. To clarify the clinical importance of ARHI/NOEY2 mRNA in breast cancer, we studied whether ARHI/NOEY2 inactivation might contribute to tumors arising in the breast. An ARHI/NOEY2 message was detected by real-time PCR analysis in all noncancerous breast tissues, but was not detected in 2 of 26 breast cancer tissue samples. In 10 of 26 breast cancer tissue samples ARHI/NOEY2 mRNA was substantially reduced. ARHI/NOEY2 expression was lost or markedly reduced in 12 of 26 (46.15%) breast cancer tissue samples. In summary, we conclude that decreased ARHI/NOEY2 mRNA expression may play an important role in the pathogenesis of breast cancer.

Novel alternatively spliced variant with a deletion of 52 BP in exon 6 of the progesterone receptor gene is observed frequently in breast cancer tissues.

The human progesterone receptor (PR) is a ligand‐activated nuclear transcription factor that mediates progesterone action in target tissues. We found a novel alternatively spliced variant (ASV) of the PR mRNA in breast cancer tissues. The deleted transcript was characterized by an out‐of‐frame deletion of 52 bp in exon 6 (PR delta6/2 ASV). The PR delta6/2 ASV mRNA results in a partial defect in the region of the ligand‐binding domain of the hormone receptor, where conserved residues are missing from the core of the protein. To clarify the clinical significance of the PR delta6/2 ASV, we investigated the expression of this ASV in noncancerous and cancerous tissues from patients with breast cancer using RT‐PCR. The novel PR delta6/2 mRNA was detected in 24 of 39 (61.5%) cancerous tissues and in 3 of 39 (7.7%) noncancerous tissues from patients with breast cancer. PR delta6/2 ASV mRNA was expressed more frequently in breast cancer tissues than in noncancerous tissues (p < 0.0001), which suggests a possible relationship between the expression of PR delta6/2 and breast cancer. Our observations may provide a novel strategy for the genetic diagnosis of breast cancer.

Indomethacin Suppresses the Growth of Colon 26, Meth-A and FM3A Tumors in Mice by Reducing the Prostaglandin E2 Content and Telomerase Activity in Tumor Tissues

The antitumor effect of indomethacin on Colon 26, Meth‐A and FM3A tumors was investigated in mice. The prostaglandin E2 content in tumor tissues was assayed to find out if indomethacin acts on tumors, and the telomerase activity in tumors and somatic tissues (testis, liver, spleen and colon) was also monitored during indomethacin treatment. Growth of Colon 26, Meth‐A and FM3A tumors was significantly (P < 0.001‐0.05) suppressed by indomethacin compared to the untreated controls. The prostaglandin E2 content in the three tumors was markedly (P < 0.001) reduced by indomethacin. Telomerase activity in Colon 26 and FM3A tumors was significantly (P < 0.001) lower than that of untreated tumors (80% and 45% decrease versus the controls, respectively), and the activity in Meth‐A tumor was slightly decreased (10% decrease versus the control) by indomethacin. Telomerase activity in the somatic tissues was not significantly affected by indomethacin. In summary, this study shows the effectiveness of indomethacin as an antitumor agent against three types of tumors, and suggests that indomethacin affects telomerase activity in tumors in vivo.

Lipogenic gene expression profile in patients with gastric cancer

Numerous types of cancer exhibit increased lipogenesis and expression of lipogenic enzymes and transcription factors, including sterol regulatory element-binding protein-1. Lipogenic gene expression is upregulated at the mRNA level, in concert with metabolic pathways associated with changes in expression and/or activity of lipogenic transcription factors. However, this expression pattern in human gastric carcinoma has not been elucidated. In this study, lipogenic gene expression in cancer tissues was investigated using quantitative PCR. In patients with gastric cancer, carnitine O-palmitoyltransferase type I mRNA and miR-33b were significantly downregulated, suggesting that miR-33b downregulation is mediated by conditions that also affect the expression and/or activity of transcription factors involved in lipogenic gene expression. Consequently, the association between miR-33b and gastric cancer may provide a novel strategy for the genetic diagnosis of gastric cancer. However, additional studies including a larger number of samples are required to confirm these results.

Detection of low allele burden of JAK2 exon 12 mutations using TA-cloning in patients with erythrocytosis.

OBJECTIVE Polycythemia vera (PV) is a clonal myeloproliferative neoplasia associated with the activation of the Janus-activating kinase 2 (JAK2) mutation. The aim of this study is to identify clonal expansion of exon 12 mutations. METHODS We performed DNA sequencing of the JAK2 exon 12 after TA-cloning in JAK2-V617F-negative and JAK2-V617F-positive PV patients. RESULTS AND CONCLUSIONS We found clonal mutations (i.e. H538-K539delinsL and D544G) in 3 of 7 JAK2-V617F-negative PV patients, however, unlike JAK2-V617F, allele burden of JAK2 exon 12 mutation was low. Since allele-specific PCR is able to amplify only the limited region which contains known mutations with gain-of-function, we need to clarify the biological implications of unknown single nucleotide substitution of the JAK2 exon 12 with low clonal burden in erythrocytosis patients.

Mitochondrial DNA reduced by hypoxic conditions in three-dimensional (3D) spheroid cell cultures

Three-dimensional (3D) cell culture reflects many of the important properties of solid tumors, such as the inadequate diffusion of oxygen that results in hypoxia. To understand the mitochondrial states in cancer, we performed comparisons of the levels of mitochondrial DNA (mtDNA), fusion- and fission-related mitochondrial messenger RNA (mRNA), and mitochondrial protein expression between monolayer (2D)- and 3D-cultured cancer cells. The mtDNA levels were observed to be significantly lower in the 3D cells compared with the monolayer cells. In contrast, the differences in expression of the mitochondrial fusion- and fission-related mRNAs and mitochondrial proteins between 2D- and 3D-cultured cancer cells were not significant, as shown by real-time PCR and immunoblot analysis. Therefore, although mtDNA levels decrease as a whole during 3D culture, this does not appear to affect the fusion and fission of individual mitochondria. Indeed, the factors regulating mitochondrial dynamics during 3D cell culture remain unclear. This study provides the basis for future, more detailed studies on the regulation of mtDNA.

Survivin in liver

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Alternative splicing isoform in succinate dehydrogenase complex, subunit C causes downregulation of succinate-coenzyme Q oxidoreductase activity in mitochondria

Mitochondrial succinate dehydrogenase (SDH) is localized to the inner mitochondrial membrane and is responsible for the redox of succinic acid. SDH is a tetrameric iron-sulfur flavoprotein of the tricarboxylic acid cycle and respiratory chain. The SDH complex, subunit C (SDHC) transcript has deletion-type alternative splicing sites. Generally, alternative splicing produces variant proteins and expression patterns, as products of different genes. In certain cases, specific alternative splicing variants (ASVs) have been associated with human disease. Due to a frameshift mutation causing loss of the heme binding region, the SDHC Δ5 isoform (lacking exon 5) exhibits no SDHC activity. To investigate whether the SDHC splicing variants can function as dominant-negative inhibitors, SDHC ASVs were overexpressed in HCT-15 human colorectal cancer cells. Using real-time reverse transcription-polymerase chain reaction, a dominant-negative effect of the Δ5 isoform on SDHC mRNA was shown. In addition, Δ5 overexpression increased the levels of reactive oxygen species. Furthermore, in the Δ5 isoform-overexpressing cells, SDH activity was reduced. SDHC activation is a significant event during the electron transport chain, and the function of the SDHC Δ5 variant may be significant for the differentiation of tumor cells.