Takahide Ota

Overexpression of Aurora-A potentiates HRAS-mediated oncogenic transformation and is implicated in oral carcinogenesis.

Aurora kinases are known to play a key role in maintaining mitotic fidelity, and overexpression of aurora kinases has been noted in various tumors. Overexpression of aurora kinase activity is thought to promote cancer development through a loss of centrosome or chromosome number integrity. Here we observed augmentation of G12V-mutated HRAS-induced neoplastic transformation in BALB/c 3T3 A31-1-1 cells transfected with Aurora-A. Aurora-A-short hairpin RNA (shRNA) experiments showed that the expression level of Aurora-A determines susceptibility to transformation. Aurora-A gene amplification was noted in human patients with tongue or gingival squamous carcinoma (4/11). Amplification was observed even in pathologically normal epithelial tissue taken at sites distant from the tumors in two patients with tongue cancer. However, overexpression of Aurora-A mRNA was observed only within the tumors of all patients examined (11/11). Our data indicate that Aurora-A gene amplification and overexpression play a role in human carcinogenesis, largely due to the effect of Aurora-A on oncogenic cell growth, rather than a loss of maintenance of centrosomal or chromosomal integrity.

Inhibitory effect of RNAi on Japanese encephalitis virus replication in vitro and in vivo.

Flaviviruses include many insect‐mediated small viruses and still cause serious problems in the world. In humans, JEV can cause acute meningioencephalomyelitis, resulting in fatality rates of 5 to 40%. RNA‐interference (RNAi) as an antiviral mechanism was originally discovered in plants and then found in the specific suppression of gene expression of other organisms such as Caenorhabditis elegans, Drosophila and vertebrates. As JEV is an RNA virus, RNAi could be a reasonable approach for therapeutic purposes to use against Japanese encephalitis. In this study, we examined the effect of RNAi on JEV replication. Viral reproduction in Vero cells was decreased to 7.2% and 39.0% of control by the transfection of small interference RNAs, JCR and JN3R at 250 NM, respectively. Under the transfection of 5 μg/ml pJRi which produces stem‐loop RNAi, viral reproduction was decreased to about 10% of control. Western blot analysis indicated that RNAi inhibited the translation level. We used pJRi in the animal experiment. After the inoculation of viruses at 5× 103 PFU, pJRi at 1.0 and 5.0 μg/g was injected into mice i.p. JEV‐infected control mice (n=5) died within 15 days. pJRi (1.0 or 5.0 μg/g)‐medicated mice survived 40 or 80% at 15 days. The data clearly indicate that pJRi has highly potent inhibitory activity against JEV replication in vivo. The results in vivo and in vitro provide evidence that JEV replication was efficiently inhibited by RNAi and RNAis could be used as an antiviral drug against JEV infection.

LyGDI functions in cancer metastasis by anchoring Rho proteins to the cell membrane.

Rho family GTPases play an important role in a number of processes related to metastasis, and RhoGDP dissociation, inhibitors (RhoGDIs) regulate Rho family proteins. We cloned genomic DNA from colon carcinoma SW480 cells capable of transforming nonmetastatic ras‐transformed 1‐1ras1000 cells into metastatic cells. This DNA contained a truncated human ras homolog gene family GDP dissociation inhibitor beta (ARHGDIB) gene, resulting in a C‐terminal truncated form of LyGDI (ΔC‐LyGDI, 166–201 deletion), a member of the RhoGDIs. The stable expression of ΔC‐LyGDI induced pulmonary metastasis in 1‐1ras1000 cells, whereas expression of full‐length LyGDI did not induce metastasis. ΔC‐LyGDI was preferentially localized in the membrane, detected in a NP‐40‐insoluble fraction, and co‐purified with radixin, moesin, Rac1, Cdc42, and RhoA. In ΔC‐LyGDI transfectant, an activation state of Rac1 was elevated and ΔC‐LyGDI was associated with Rac1‐GTP. In keeping with the observed localization of Rac1 to the cell membrane and the elevated level of Rac1‐GTP, ΔC‐LyGDI transfectants were found to be more invasive than mock transfectant. These results suggest that LyGDI functions in the cell membrane to afford spatial regulation of Rho family GTPase signaling through ezrin radixin moesin (ERM) proteins during metastasis.

Anticarcinogenic Effect and Enhancement of Metastatic Potential of BALB/c 3T3 Cells by Ginsenoside Rh2

It has been reported that ginsenoside Rh2, a purified ginseng saponin with a dammarane skeleton, has anticarcinogenic effects on mammalian cells. To determine the significance of these effects on multistage carcinogenesis, we utilized the BALB/c 3T3 cell system. In an in vitro two–stage neoplas–tic transformation assay, the initiating activity of 3–methylcholanthrene (3–MCA) was suppressed by Rh2 (>lxlO–5M) in both BALB/c 3T3 A31–1–1 cells and the more carcinogen–susceptible variant A31–1–13 cells. The suppressive effects in this concentration range were thought to be caused by suppression of DNA replication via indirect Cdk2 inhibition. On the other hand, the promotion steps of both the target cells were not affected by Rh2 even if the transformation frequency was enhanced by a tumor promoter, 12–O–tetradecanoylphorbol–13–acetate (TPA). To examine the other effects of Rh2 on carcinogenesis, we turned our attention to the metastatic phenotype. Using metastatic src–transformed A31–1–1 cells, we found that Rh2 augmented the metastatic potential in an experimental metastasis assay. These data indicate that Rh2 has diverse effects on the expression of the transformed phenotype in BALB/c 3T3 cells, but support the idea that growth suppression is likely to be a major mechanism of the anticarcinogenic effects of Rh2.

Different metastatic potentials of ras- and src-transformed BALB/c 3T3 A31 variant cells.

The metastatic phenotype of tumor cells is thought to be induced by an aberrant signaling cascade or cascades that are different from those required for tumorigenicity. Oncogene‐transfected cells with different tumorigenicities and metastatic potentials have been used to identify such pathways and responsible molecules. However, oncogenes that can induce tumorigenicity in recipient cells also frequently induce the metastatic phenotype at the same time. The difficulty in obtaining cell lines that are tumorigenic but not metastatic has hampered such studies. In this report, we transfected the activated c‐Ha‐ras oncogene into BALB/c 3T3 A31 variant cells and found that the transfectants were tumorigenic but they did not form metastatic lung nodules in the experimental metastasis assay. The phenotype was very stable and was maintained during cultivation. On the other hand, the metastatic potentials of either the transfected cells or the original variant cells could be induced by transfection of the v‐src oncogene. The src transfectants formed extensive nodules in lung when injected into the tail veins of congeneric mice. The cell motility of the metastatic src transfectants on Matrigel‐coated dishes was greater than that of the ras transfectants. The src transfectants were also invasive in Matrigel when analyzed on a filter. These variant cells transformed by the ras and src oncogenes will be a useful system for identifying the signaling cascades responsible for the metastatic potential of tumors.

Isolation of a highly metastatic cell line to lymph node in human oral squamous cell carcinoma by orthotopic implantation in nude mice

To isolate a variant cell line with increased lymph node metastasising potential from human oral squamous cell carcinoma (SCC) we performed in vivo selection using orthotopic implantation in nude mice. Human oral SCC cells (HSC-3) were injected orthotopically into the tongue of nude mice. After 3 weeks their cervical lymph nodes were excised and the tumour cells metastasised to the lymph nodes were isolated and injected into the tongue of nude mice again, this procedure was performed three times. The resultant cells, designated as HSC-3-M3, metastasised to cervical lymph nodes in 90% of mice, while parental HSC-3 cells metastasised in only 30% of mice after injection into the tongue. HSC-3 and HSC-3-M3 cell lines which have the same origin but different lymphatic metastatic capacities could be a useful model system for studying mechanisms involved in lymph node metastasis of human oral SCC.

HUMAN AIM-1 : CDNA CLONING AND REDUCED EXPRESSION DURING ENDOMITOSIS IN MEGAKARYOCYTE-LINEAGE CELLS

The rat AIM-1 gene encoding an Aurora- and Ipl1-like midbody-associated protein serine/threonine kinase has a mitotic regulator function playing a key role in the onset of cytokinesis during mitosis. This report presents a cDNA sequence and megakaryocytic differentiation-dependent expression profile of the human AIM-1 gene. The nucleotide sequences of the human AIM-1 were identified from cDNAs of three cell lines, including cervical carcinoma HeLa cells, colorectal tumor SW480 cells, and normal human diploid skin fibroblast NHDF cells, and no mutation was found. The expression levels of AIM-1 transcript were markedly reduced during differentiation into megakaryocytic cell lineage in human leukemia cells induced by 12-o-tetradecanoyl-phorbol-13-acetate (TPA), suggesting that the downregulation of AIM-1 contributes to the differentiation by repeated duplication of DNA without cytokinesis (endomitosis).

Promotion or suppression of experimental metastasis of B16 melanoma cells after oral administration of lapachol.

Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] is a vitamin K antagonist with antitumor activity. The effect of lapachol on the experimental metastasis of murine B16BL6 melanoma cells was examined. A single oral administration of a high toxic dose of lapachol (80-100 mg/kg) 6 h before iv injection of tumor cells drastically promoted metastasis. This promotion of metastasis was also observed in T-cell-deficient mice and NK-suppressed mice. In vitro treatment of B16BL6 cells with lapachol promoted metastasis only slightly, indicating that lapachol promotes metastasis primarily by affecting host factors other than T cells and NK cells. A single oral administration of warfarin, the most commonly used vitamin K antagonist, 6 h before iv injection of tumor cells also drastically promoted the metastasis of B16BL6 cells. The promotion of metastasis by lapachol and warfarin was almost completely suppressed by preadministration of vitamin K3, indicating that the promotion of metastasis by lapachol was derived from vitamin K antagonism. Six hours after oral administration of lapachol or warfarin, the protein C level was reduced maximally, without elongation of prothrombin time. These observations suggest that a high toxic dose of lapachol promotes metastasis by inducing a hypercoagulable state as a result of vitamin K-dependent pathway inhibition. On the other hand, serial oral administration of low non-toxic doses of lapachol (5-20 mg/kg) weakly but significantly suppressed metastasis by an unknown mechanism, suggesting the possible use of lapachol as an anti-metastatic agent.

G1 phase-specific suppression of the Cdk2 activity by ginsenoside Rh2 in cultured murine cells.

Ginsenoside Rh2, a plant glycoside with a dammarane skeleton resembling a steroid skeleton as an aglycone, has anticancer potentials in vitro or in vivo. To elucidate the molecular mechanisms of the effects of Rh2, we have examined the Cyclin-dependent kinase-2 (Cdk2) activity in G1 arrested B16 melanoma cells and in S phase-arrested Meth-A sarcoma cells, that have been treated with Rh2. The kinase activity was suppressed in B16 cells but not in Meth-A cells. In addition, Rh2 was found to induce G1 arrest and concomitantly suppress the Cdk2 activity in carcinogen-susceptible BALB/c 3T3 A31-1-1 and A31-1-13 cell lines. Thus, Rh2 has a G1 phase-specific suppressive effect on the Cdk2 activity, supporting further evaluation of Rh2 and its related compounds in cancer chemoprevention studies.

tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.

Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.