Mitotic arrest caused by an X-ray microbeam in a single cell expressing EGFP-aurora kinase B.

Although the highest radiosensitivity of cells in the M phase among the other cell phases, such as the G(1), S and G(2) phases, has been known, the exact mechanism of radiosensitivity in mitotic cells remains unclear. Recently, mitotic arrest caused by DNA-damaging reagents has been shown, and the molecular mechanism in the arrest has been discussed in detail. In this study, abnormal cell-cycle progression in the M phase was investigated when a single mitotic cell in each mitotic stage was irradiated with a 5.35 keV X-ray microbeam focused on the cell nucleus. An X-ray microbeam irradiation system installed at BL-27 in Photon Factory, High Energy Accelerator Research Organization (HEARO, Tsukuba) was used. HeLa cells, genetically modified and expressing enhanced green fluorescent protein-tagged aurora kinase B, were used as irradiated samples in order to recognise the stage of each cell in the M phase. Thus, 10 Gy irradiation concentrated at the nucleus of a single cell elongated the cell-cycle progression in the M phase by delaying the metaphase/anaphase transition. The dose dependence of the elongation of the M phase was also examined. An irregular distribution of DNA in anaphase cells was observed after irradiation.

Anticarcinogenic effect and enhancement of metastatic potential of BALB/c 3T3 cells by ginsenoside Rh(2).

It has been reported that ginsenoside Rh(2), 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 neoplastic transformation assay, the initiating activity of 3-methylcholanthrene (3-MCA) was suppressed by Rh(2) (>or= 1 x 10(-5) M) 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 Rh(2) even if the transformation frequency was enhanced by a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). To examine the other effects of Rh(2) on carcinogenesis, we turned our attention to the metastatic phenotype. Using metastatic src-transformed A31-1-1 cells, we found that Rh(2) augmented the metastatic potential in an experimental metastasis assay. These data indicate that Rh(2) 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 Rh(2).

Interaction and feedback regulation between STK15/BTAK/Aurora-A kinase and protein phosphatase 1 through mitotic cell division cycle.

STK15 is an Aurora/Ipl-1 related serine/threonine kinase that is associated with centrosomes and induces aneuploidy when overexpressed in mammalian cells. It is well known that phosphorylation and dephosphorylation of kinases are important for regulation of their activity. But mechanisms by which STK15 activity is regulated have not been elucidated. We report that STK15 contains two functional binding sites for protein phosphatase type 1 (PP1), and the binding of these proteins is cell cycle-regulated peaking at mitosis. Activated STK15 at mitosis phosphorylates PP1 and inhibits PP1 activity in vitro. In vivo, PP1 activity co-immunoprecipitated with STK15 is also reduced. These data indicate that STK15 inhibits PP1 activity during mitosis. Also, PP1 is shown to dephosphorylate active STK15 and abolish its activity in vitro. Furthermore, we show that non-binding mutants of STK15 for PP1 are superphosphorylated, but their kinase activities are markedly reduced. Cells transfected with these non-binding mutants manifest aberrant chromosome alignment during mitosis. Our results suggest that a feedback regulation through phosphorylation/dephosphorylation events between STK15 kinase and PP1 phosphatase operates through the cell cycle. Deregulation of this balance may contribute to anomalous segregation of chromosomes during mitotic progression of cancer cells.

Functional suppression of integrin beta 4-mediated adhesion caused by in vivo sequential selection for cancer cell intravasation.

Intravasation is essential for hematogenous metastasis in cancer cells, but its cellular determinants have not been well elucidated because of a lack of suitable experimental cell systems. Int-3LL was originally developed by in vivo sequential selection for intravasation from Lewis lung carcinoma (3LL) cells. Here, we found that these variant cells showed a highly penetrating ability in vitro as well as an augmented intravasating potential in vivo. In three-dimensional collagen-gel, Int-3LL cells formed diffusive colonies with less plating efficiency than their parental cells. Despite these properties, Int-3LL cells showed an ability of invasive migration in vitro similar to parental cells. On the other hand, a reduced adhesiveness and less spreading on extracellular matrices were revealed in Int-3LL cells. Analyses using anti-integrin antibodies indicated that the dysadhesion phenotype in Int-3LL cells was associated with integrin beta 4 dysfunction, which is known to produce epithelial detachment. Also, the types and the levels of integrins were not indistinguishable between Int-3LL and parental 3LL cells. Thus, the impaired function of integrin beta 4-mediated adhesion is considered to be an important factor in intravasation during metastasis.

Downregulation of an AIM-1 kinase couples with megakaryocytic polyploidization of human hematopoietic cells.

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3-dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.

Myosin II regulatory light chain as a novel substrate for AIM-1, an aurora/Ipl1p-related kinase from rat.

Previous studies demonstrated that the phosphorylated myosin II regulatory light chain (MRLC) is localized at the cleavage furrow of dividing cells, suggesting that phosphorylation of MRLC plays an important role in cytokinesis. However, it remains unclear which kinase(s) phosphorylate MRLC during cytokinesis. AIM-1, an Aurora/Ipl1p-related kinase from rat, is known as a serine/threonine kinase that is required for cytokinesis. Here we examined the possibility that AIM-1 is a candidate for a kinase that phosphorylates MRLC during cytokinesis. As a result, we showed that AIM-1 monophosphorylated MRLC at Ser19 using two-dimensional phosphopeptide mapping analysis and several MRLC mutants. Furthermore, AIM-1 was colocalized with monophosphorylated MRLC at the cleavage furrow of dividing cells. We propose here that AIM-1 may participate in monophosphorylation of MRLC during cytokinesis.

Involvement of cyclin-dependent kinases in doxorubicin-induced apoptosis in human tumor cells.

Apoptotic cell death caused by doxorubicin, a chemotherapeutic agent, is suppressed by expression of p21 (waf1/cip1/sdi1), a cyclin-dependent kinase (cdk) inhibitor. To examine cdk activity required for doxorubicin-induced apoptosis, we transfected p21-deficient human tumor DLD1(p21-/-) cells with plasmids carrying wild-type p21 and mutated p21 unable to bind to cdks or proliferating cell nuclear antigen. The apoptosis induced at the G(2)/M phase after doxorubicin treatment was suppressed by transient expression of the p21 with cdk-binding activity but not by the p21 lacking the activity. We also transfected cells with plasmids carrying wild-type, dominant negative and constitutively active mutants of cdk2 or cdk4. The apoptosis was suppressed by transient expression of dominant negative mutants of cdk2 or cdk4. These findings indicate that cdk is involved in the doxorubicin-induced apoptosis pathway.

Molecular cloning of mouse thioredoxin reductases.

We screened clones for thioredoxin reductase genes with a degenerate PCR-based strategy and have isolated two novel cDNA clones from a mouse thymocyte cDNA library. These encode two distinct thioredoxin reductases (TrxR1 and TrxR2) with 499 and 527 amino acid (aa) residues and calculated molecular masses of 54.5 kDa and 56.8 kDa respectively. These proteins share 90% and 50% aa sequence identity with those of previously cloned human TrxR, containing the redox-active cysteines, FAD binding domain, and the selenocysteine (SeCys) insertion sequence, which is composed of a putative stem-loop sequence located in the 3'-untranslated region (UTR). TrxR2 showing less homology to human TrxR has a mitochondrial translocation signal and a mitochondrial prepeptide protease cleavage site in the N-terminal domain. Transient expression experiments of each gene as fusion proteins with Xpress-tagged protein in NIH 3T3 cells indicated that TrxR1 was localized in the nucleus and cytoplasm and TrxR2 in the mitochondria. Furthermore, we mapped the TrxR1 gene to chromosome 10 (placed 1.71 cR from D10Mit42, lod>3.0) and the TrxR2 gene to chromosome 16 (placed 22.56 cR from D16Mit34, lod>3.0). Thus, the mouse has at least two distinct nuclear genes for TrxR that have different translocation sites in the cell.

Decrease of metastatic ability after selection for intravasating ability in Lewis lung carcinoma (3LL) cell line.

The release of cancer cells from the primary site and penetration into blood vessels are obligatory preliminary steps for metastasis. To investigate the mechanism of such steps we isolated variant cells (designated as Int-3LL) possessing enhanced intravasating ability from Lewis lung carcinoma (3LL) cells by in vivo selection. In spite of the enhanced intravasating ability of Int-3LL, the spontaneous and experimental metastatic abilities of Int-3LL decreased significantly compared to parent cells. Such a cell line has never been reported so far. The matched pair of cell lines described in this report provides a useful system for investigating the primary steps of metastasis.

Down-regulation of nuclear factor kappaB is required for p53-dependent apoptosis in X-ray-irradiated mouse lymphoma cells and thymocytes.

Transcription factors p53 and nuclear factor kappaB (NF-kappaB) have been implicated in apoptosis induced by DNA-damaging agents, but the relationship between these two factors at the molecular level is largely unknown. We have isolated apoptosis-resistant mutant sublines from a radiosensitive mouse lymphoma 3SB cell line that undergoes p53-depen-dent apoptosis after X-ray irradiation, and we have analyzed the NF-kappaB activity. Two of these apoptosis-resistant sublines expressed mutant p53 protein and exhibited a defect in the induction of cyclin-dependent kinase inhibitor p21 after X-ray irradiation. A decrease in the DNA binding activity of NF-kappaB was observed in the parental 3SB cells after exposure to X-rays, whereas the same activity was unaffected by radiation in the two mutant sublines. A similar down-regulation of NF-kappaB activity by X-rays was observed in thymocytes derived from p53 wild-type and heterozygous mice, but not in thymocytes from p53 homozygous knock-out mice. These results suggest that NF-kappaB inactivation is p53 dependent and is required for X-ray-induced apoptosis in thymic lymphoma cells and normal thymocytes.

Multinuclearity and increased ploidy caused by overexpression of the aurora- and Ipl1-like midbody-associated protein mitotic kinase in human cancer cells.

Aurora- and Ipl1-like midbody-associated protein (AIM-1) is a serine/ threonine kinase that is structurally related to Drosophila aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. A kinase-negative form of AIM-1 inhibits the formation of cleavage furrow without affecting nuclear division, indicating that the gene controls entry into cytokinesis during M phase in mammalian cells. A human gene that encodes the protein AIM-1 was overexpressed in colorectal and other tumor cell lines. The regulation of AIM-1 expression was cell cycle dependent in normal and tumor cells, and the maximum accumulation was observed at G2-M. Exogenous overexpression of wild-type AIM-1 produced multinuclearity in human cells, suggesting that the excess amount of AIM-1 had a dominant-negative effect on the overexpressing cells. In long-term culture of AIM-1-overexpressing cells, multiple nuclei in a cell were occasionally fused, and then an increased ploidy and aneuploidy were induced. Thus, the overexpression of AIM-1 in colorectal tumor cell lines is thought to have a causal relationship with multinuclearity and increased ploidy. Cytokinesis error caused by AIM-1 overexpression is a major factor in the predisposition of tumor cells to the perturbation of chromosomal integrity that is commonly observed in human neoplasia. Thus, defects of pathways essential for mitotic regulation are important during human cancer development.

AIM-1: a mammalian midbody-associated protein required for cytokinesis.

Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation. Errors in this process result in aneuploidy which can lead to cell death or oncogenesis. In this paper we describe a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G2/M phase. The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis. Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division. Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death. These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells.

Isolation and characterization of apoptosis-resistant mutants from a radiosensitive mouse lymphoma cell line.

To analyze specific genes related to radiation-induced apoptosis, 12 apoptosis-resistant clones were isolated from cells of the radiosensitive mouse thymic lymphoma 3SB line after treatment with ethyl methanesulfonate. Five of 12 clonal cell lines were recloned and were examined for their susceptibility to X-ray-induced apoptosis. A cell survival assay showed that all five secondary cell lines were two to three times more resistant to X rays than 3SB cells. When 3SB cells were exposed to 5 Gy of X rays, the fraction of cells stained with erythrosin B increased quickly within 8 h of incubation after irradiation. However, no apoptosis occurred in these secondary mutant cells. In particular, the percentage of cells undergoing apoptosis in one clone, 1B1C4, was low even after incubation for 48 h. In contrast to X rays, after exposure to 20 J/m2 UV radiation, the proportion of apoptotic cells in these mutant cells increased and reached about 60 to 100% at 24 h, indicating a difference in the ability of X rays and UV radiation to induce apoptosis. A similar radioresistance was observed using agarose gel electrophoresis of DNA from cells of all X-irradiated secondary lines. Western blot analysis and a sequence-specific DNA-binding assay demonstrated that 1B1C4 cells had a functional defect in p53 protein, but the other four cell lines displayed wild-type p53 after X irradiation. Our results suggest the existence of separate radiation-specific p53-dependent and independent apoptosis in thymic lymphoma cells. Thus these apoptosis-resistant cell lines provide a useful tool to identify the genes involved in the signaling pathways leading to X-ray-specific apoptosis.

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).

Molecular cloning and characterization of the promoter of the human N-methylpurine-DNA glycosylase (MPG) gene.

The promoter region of the human N-methylpurine-DNA glycosylase (MPG) gene was cloned and characterized. The cloned segment contains two first exons that were earlier identified and named exons 1a and 1b. These were found to be separated by approximately 800 bp. The minimal promoter region was identified upstream to the distal exon 1a, by transient transfection, and no promoter activity was found in the region in between exons 1a and 1b, suggesting that transcription starts at a single site which is then processed to generate mRNAs of the isoforms. The promoter sequence is G and C rich and contains neither TATA box, nor apparent CAAT sequences, although a partially matched CAAT sequence was identified just downstream to the minimal promoter.

A BALB/c 3T3-transformed cell line suitable for transfection assay of metastasis-inducing genes.

A clonal cell line, 1-1ras1000, transformed by the activated c-Ha-ras oncogene, does not form metastases after i.v. injection into mice (experimental metastasis assay). Here, we show that this cell line is useful as a recipient to detect metastasis-inducing genes, using a transfection assay. Cells (1-1ras1000) were susceptible to metastasis induction by transfection with either v-src or genomic DNA from a v-src-and v-fos-transferred highly metastatic rat cell line (SR202). The susceptibility of 1-1ras1000 cells for lung metastasis induction was suitable for a genomic transfection assay to detect a metastasis-inducing gene in the transfected cells which had incorporated genomic DNA from donor metastatic tumor cells. When DNAs extracted from 7 human tumors were tested for metastasis induction, 2 DNAs from nonmalignant tumors (non-tumorigenic tumors in athymic nude mice) (2/2) were negative and 4 DNAs from malignant tumors (4/5) were positive in 1-1ras1000 cells for primary transfection. in one of the resulting metastases, the ability to metastasize was also transferred in the second and third cycles of genomic DNA transfection at high frequencies. All of the resulting metastases carried the human repetitive Alu sequence. Neither re-arrangements of the endogenous c-Haras nor changes of protein amounts were detected. Recipient 1-1ras1000 cells had a negligible rate of spontaneously metastatic conversion during in vitro cultivation and transfection processes. The resulting metastasized cells were easily isolated from the lung after culturing in selection medium containing G418 (geneticin). Isolated cells stably retained the ability to form metastatic lung nodules when re-injected into mice. Thus, 1-1ras1000 cells appear to be a useful system for the isolation of metastasis-inducing genes from human metastatic tumors.

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.

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 modules 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.

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations.

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.