Biomedical subjects

R Hamanaka

Publications and source records attributed to R Hamanaka.

At least 19 recordsLinked to original sources

PubMed2001

Polo-like kinase (PLK) expression in endometrial carcinoma.

Polo-like kinase (PLK) is a cell cycle-regulated, cyclin-independent serine/threonine protein kinase. Recent reports have shown a critical role for PLK during tumorigenesis. To explore whether PLK plays a general role as a tumor marker of endometrial carcinomas, we examined the expression of PLK mRNA and protein in endometrial carcinomas and normal endometrium, and analyzed the relationship between PLK protein expression and malignant potential. We found that PLK mRNA was expressed in all specimens from endometrial carcinoma patients using RT-PCR methods, although some specimens from normal endometria were negative. Immunohistochemically, most of the PLK was found in the cytoplasm (around the nucleus), and partly in the nucleus of endometrial carcinoma glands and also secreted tissues from endometrial carcinoma glands. PLK was expressed at the basement membrane of carcinoma glands and partly expressed in the head portion of papillary carcinoma tissues. There was a significant correlation between percentages of PLK-positive cells and histological grade of endometrial carcinoma (P<0.0001). However, the expression of proliferating cell nuclear antigen and Ki-67 was independent of PLK expression. Moreover, we noted that PLK is strongly expressed in invading carcinoma cells. PLK expression could reflect the degree of malignancy and proliferation in endometrial carcinoma. Thus, in addition to being of diagnostic value, modulation of PLK activity in the tumors by chemotherapeutic agents or gene therapy may prove to be of therapeutic value.

Adult↗

PubMed2001

Expression of polo-like kinase in ovarian cancer is associated with histological grade and clinical stage.

Polo-like kinase (PLK), a cell cycle-regulated, cyclin-independent serine/threonine protein kinase, has been shown in recent reports to play a critical role during tumorigenesis. To investigate whether PLK plays a general role as a tumor marker of ovarian cancers, we examined the expression of PLK protein in ovarian cancers, and analyzed the relationship between PLK protein expression and histological grade. Immunohistochemically, the majority of PLK was found in the cytoplasm (around the nucleus), and a portion was found in the nucleus of ovarian cancer glands and also in the fluid secreted from these glands. PLK was expressed at the basement membrane of cancer glands and partly expressed in the head portion of papillary cancer tissues. A significant correlation was found between percentages of PLK-positive cells and histological grade of ovarian cancer (P<0.001). However, the expression of proliferating cell nuclear antigen, Ki-67, and cyclin B1 was independent of PLK expression. Taken together, these findings suggest that PLK expression may reflect the degree of malignancy rather than the degree of proliferation in ovarian cancer. Thus, in addition to being of diagnostic value, PLK activity in ovarian tumors may be modulated by chemotherapeutic agents or gene therapy to therapeutic effect.

Adenocarcinoma↗

PubMed2000

Polo-like kinase expression in normal human endometrium during the menstrual cycle.

The enzyme, polo-like kinase (PLK), is a mammalian serine/threonine kinase involved in cell cycle regulation. A great deal of evidence regarding the role of PLK in the cell cycle has been obtained through studies of cultured cells, though little is known about its function or even expression in vivo. The endometrium undergoes rapid proliferation and differentiation under ovarian steroid hormone control during the 28-day cycle. Thus, normal endometrium provides an excellent model in which to study the hormone dependency of PLK expression. In the present study, we examined the features of PLK expression in 20 samples of normal human endometrium during the menstrual cycle. The expression of Ki-67 and proliferating cell nuclear antigen (PCNA) were also examined as markers of proliferation. Immunohistochemical studies showed that PLK staining was detected in the basement membrane of many endometrial glands, stromal cells, and some endothelial cells. The number of PLK-positive endometrial gland cells was significantly higher in the late proliferative phase (19.16% +/- 4.98%) and the early secretory phase (19.28% +/- 4.99%) than in the early proliferative phase (2.60% +/- 2.33%) or the late secretory phase (5.76% +/- 2.16%) (P<0.0001). PLK expression seemed to be correlated with the expression of Ki-67 and PCNA in many endometrial glands and stromal cells particularly in the late proliferative phase, reflecting a role of PLK in cellular proliferation. Nevertheless, in the early secretory phase, at which point the expression of Ki-67 and PCNA decreased in endometrial glands, PLK was strongly expressed. This finding suggests that PLK may have some post-mitotic functions in certain specialized cell types. Although the highest expression of PLK was observed in the late proliferative and the early secretory phases, the expression drastically decreased in the late secretory phase. These findings, taken together, indicate that the expression of PLK in normal endometrium fluctuates over the course of the menstrual cycle, suggesting in turn that PLK is associated with hormone-dependent cellular proliferation and that hormone functions may be involved in its regulation.

Adult↗

PubMed1999

Expression of polo-like kinase (PLK) in the mouse placenta and ovary.

The polo-like kinase (PLK) is a mammalian serine/threonine kinase involved in cell cycle regulation. Much evidence for the role of PLK in the cell cycle has come from studies of cultured cells; however, little is known about its function or even expression in vivo. The present study examined the features of PLK expression in the mouse placenta and ovary. Immunohistochemical studies showed that PLK is highly expressed in the basement membrane of the endometrial gland, in some endothelial cells, in endometrium after embryo implantation, in trophoblastic tissue invading the decidua, in the ovarian stroma and in some lutein bodies. In contrast, PLK was not detectable by immunohistochemistry in endometrial stroma before decidualization, in decidua, in trophoblastic tissue not invading the decidua or in ovarian follicles. PLK expression seemed to be correlated with the expression of proliferation cellular nuclear antigen (PCNA) in many placental and ovarian cells, reflecting a role in cellular proliferation. Nevertheless, in ovarian stroma and lutein bodies where PCNA was not expressed, PLK was strongly expressed. This finding indicates that PLK may have some post mitotic functions in certain specialized cell types.

Amino Acid Sequence↗

PubMed1999

Immunohistochemical localization of polo like kinase in early human placenta.

Polo like kinase (PLK) is the kinase that was first cloned by us from the cDNA library of human placenta. It belongs to the serine/threonine kinase family and plays a very important role in cell proliferation. In this study, the localization of PLK in early human placenta in vivo was investigated. Immunostaining revealed PLK protein in syncytiotrophoblastic cells and extravillous trophoblastic cells; however proliferating cell nuclear antigen was not. It is known that cytotrophoblastic cells proliferate highly in early human placental villi; however, PLK was not detected in those cells. These results suggest that PLK plays a different role in syncytiotrophoblastic cells than it does in other proliferating cells.

Cell Cycle Proteins↗

PubMed1997

Malignant transformation of mammalian cells initiated by constitutive expression of the polo-like kinase.

Polo-like kinase (Plk) is the mammalian homologue of the Drosophila polo and Saccharomyces cerevisiae CDC5 genes, which are thought to be involved in regulating chromosomal segregation. Previously, we showed that transient ectopic expression of Plk could induce DNA synthesis in quiescent NIH 3T3 cells, suggesting that Plk might also have a function during G1 or S phase. Here we report that microinjection of Plk mRNA is sufficient to drive quiescent cells into mitosis and that constitutive expression of Plk in NIH 3T3 cells causes oncogenic focus formation. These transformed cells grow in soft agar and form tumors in nude mice. Because Plk expression has been shown to be high in various human tumors, we suggest that Plk may contribute to the promotion and/or progression of human cancers.

3T3 Cells↗

PubMed1995

Polo-like kinase is a cell cycle-regulated kinase activated during mitosis.

Previously, we demonstrated that expression of polo-like kinase (PLK) is required for cellular DNA synthesis and that overexpression of PLK is sufficient to induce DNA synthesis. We now report that the endogenous levels of PLK, its phosphorylation status, and protein kinase activity are tightly regulated during cell cycle progression. PLK protein is low in G1, accumulates during S and G2M, and is rapidly reduced after mitosis. During mitosis, PLK is phosphorylated on serine, and its serine threonine kinase function is activated at a time close to that of p34cdc2. The phosphorylated form of PLK migrates with reduced mobility on SDS-polyacrylamide gel electrophoresis, and dephosphorylation by purified protein phosphatase 2A converts it to the more rapidly migrating form and reduces the total amount of PLK kinase activity. Purified p34cdc2-cyclin B complex can phosphorylate PLK protein in vitro but causes little increase in PLK kinase activity.

Amino Acid Sequence↗

PubMed1994

Cloning and characterization of human and murine homologues of the Drosophila polo serine-threonine kinase.

We have cloned both human and murine complementary DNAs that are homologous to the Drosophila serine/threonine polo kinase and the recently cloned murine polo related kinase (PLK). Both the human and murine clones are about 2.1 kilobases with open reading frames of 1.8 kilobases, encoding proteins of 603 amino acids with a predicted size of 66 kilodaltons and an apparent size of 67 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. During embryonic development of the mouse, the mRNA was expressed in all tissues examined, whereas in adult tissues, expression was limited to thymus and ovaries. All cell lines examined also expressed mRNAs of similar size. Microinjection of in vitro transcribed sense mRNA into serum-starved murine NIH3T3 cells induced tritiated thymidine incorporation, whereas microinjection of antisense RNA into growing NIH3T3 cells blocked tritiated thymidine incorporation. When PC12 rat cells were induced to differentiate with nerve growth factor, gene expression of PLK was greatly reduced. Together, these results suggest that PLK expression is restricted to, and is perhaps required by, proliferating cells.

3T3 Cells↗

PubMed1993

Irsogladine is a potent inhibitor of angiogenesis.

We describe a novel inhibitor of angiogenesis, Irsogladine, an anti-ulcer drug. Irsogladine inhibited plasminogen activator synthesis of, and tube formation by, human microvascular endothelial cells in type 1 collagen gel treated with an angiogenic growth factor, EGF. Furthermore, Irsogladine administered orally significantly inhibited in vivo angiogenesis in mice. Irsogladine may be useful in the treatment of diseases associated with angiogenesis.

Animals↗

PubMed1993

Indispensable role of tissue-type plasminogen activator in growth factor-dependent tube formation of human microvascular endothelial cells in vitro.

Epidermal growth factor (EGF) stimulates the migration and proliferation of, and tissue-type plasminogen activator (tPA) synthesis in, human omental microvascular endothelial (HOME) cells in culture, as well as inducing the formation by these cells. In the present study, we examined the effects of various growth factors, i.e., transforming growth factor-alpha (TGF-alpha), insulin-like growth factor 1 (IGF-1), and hepatocyte growth factor (HGF) on HOME cells, and compared their effects with that of EGF. IGF-1 stimulated the proliferation and migration of these cells at a level comparable to EGF. EGF and TGF-alpha induced expression of tPA in HOME cells, while IGF-1 and HGF did not. EGF and TGF-alpha induced tube formation by HOME cells in type I collagen gel, while IGF-1 and HGF did not. The stimulatory effect of EGF on tube formation in the gel was blocked by anti-tPA antibody and by a serine protease inhibitor, aprotinin. When exogenous tPA and IGF-1 or HGF were added simultaneously to the culture, a marked induction of tube formation in the gel was observed. Exogenously added tPA alone, however, had no such inducible effect on tube formation. These results indicated an indispensable role of tPA in growth factor-dependent tube formation by HOME cells. Two subsets of growth factors appeared to modulate angiogenesis: One with fully active angiogenic activity which could induce PA (this included EGF and TGF-alpha), and the other, which could not induce PA and was not angiogenic, but could promote angiogenesis in the presence of PA. This subset included IGF-1 and HGF.

Cell Division↗

PubMed1993

Morphological change and destabilization of beta-actin mRNA by tumor necrosis factor in human microvascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) produced morphological changes from a cobblestone-like shape into a spindle shape in human omental microvascular endothelial (HOME) cells and also a drastic rearrangement of actin filaments. Expression of beta-actin gene was diminished in HOME cells treated with TNF-alpha for 24 h. Northern blot analysis of the beta-actin gene demonstrated that the cellular level of beta-actin mRNA was decreased at 6-12 h after exposure to TNF-alpha. However, there appeared to be no changes in cellular mRNA levels of beta-tubulin, fibronectin, laminin B1, laminin B2, and laminin binding protein genes after treatment with TNF-alpha. Nuclear run-on assays showed increased transcription of the low-density lipoprotein receptor gene, but not of the beta-actin gene. These data suggested that the TNF-alpha-induced inhibition of beta-actin gene expression was not due to altered transcription activity. The degradation rates of beta-actin, plasminogen activator inhibitor-1, and epidermal growth factor receptor mRNAs were examined in the presence of actinomycin D. beta-Actin mRNA was found to be specifically destabilized in TNF-alpha-treated HOME cells, while other mRNA species were not. Coadministration of cycloheximide blocked the TNF-alpha-induced degradation of beta-actin mRNA. The TNF-alpha-induced destabilization of beta-actin mRNA and rearrangement of actin filaments are discussed in relation to the morphological changes in human microvascular endothelial cells.

Actin Cytoskeleton↗

PubMed1992

A model system for tumor angiogenesis: involvement of transforming growth factor-alpha in tube formation of human microvascular endothelial cells induced by esophageal cancer cells.

Tumor growth is dependent on angiogenesis, which is thought to be mediated through growth factors, such as transforming growth factor-alpha (TGF-alpha) and -beta (TGF-beta), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), produced by tumor cells. We have developed a model system for tumor angiogenesis in vitro: tube formation of human omentum microvascular endothelial (HOME) cells in type I collagen gels when these cells are co-cultured with tumor cells. Exogenously added TGF-alpha induced tube formation of HOME cells in collagen gel. In contrast, TGF-beta inhibited the TGF-alpha-induced tube formation of endothelial cells. We investigated whether tube formation could be induced in HOME cells in collagen gel when the HOME cells were co-cultured with three esophageal cancer cell lines, TE1, TE2, and TE5. TE1 and TE2 cells expressed both TGF-alpha and TGF-beta mRNA, but the level of TGF-alpha mRNA in TE2 was found to be much lower than in TE1 cells. TE5 did not express either TGF-alpha or TGF-beta. The tube formation of HOME cell was induced when they were co-cultured with TE1 cells, while both TE2 and TE5 cell lines induced tube formation at much lower rates than TE1. TE1-induced tube formation of HOME cells was specifically blocked by co-administration of anti-TGF-alpha-antibody, but not by anti-bFGF-antibody. The present study suggests that, in our model system, esophageal tumor angiogenesis is partly controlled by TGF-alpha, possibly through a paracrine pathway.

Blotting, Northern↗

PubMed1992

Induction of low density lipoprotein receptor and a transcription factor SP-1 by tumor necrosis factor in human microvascular endothelial cells.

We have previously reported that tumor necrosis factor-alpha (TNF-alpha) enhances expression of interleukin-6, collagenase, plasminogen activator inhibitor-1, and basic fibroblast growth factor genes in human omental microvascular endothelial (HOME) cells in culture. In this study, we found that treatment of HOME cells with TNF-alpha or interleukin-1 (IL-1) caused enhanced expression of low density lipoprotein (LDL) receptor. A few-fold increase in both LDL binding activity and the receptor mRNA levels was observed when HOME cells were treated with either TNF-alpha or IL-1. Northern blot analysis showed that cellular expression of LDL receptor gene was significantly increased 12-24 h after exposure to TNF-alpha. No significant changes in the life-span of LDL receptor mRNA were observed in untreated and TNF-alpha-treated cells. Scatchard analysis showed an increased receptor number for LDL in TNF-alpha-treated cells. Parallel to increased LDL binding activity, internalization and degradation of LDL were also increased in HOME cells treated with TNF-alpha or IL-1. TNF-alpha-induced enhancement of LDL receptor gene expression was not observed when cycloheximide was present. Cellular mRNA level of SP-1 gene was increased about 3-4-fold at 12 h after treatment with TNF-alpha. Nuclear run-on assays showed increased transcription of LDL receptor gene as well as SP-1 gene by TNF-alpha. Gel retardation assay with the SP-1 consensus fragment showed that SP-1 binding activity was increased about 4-5-fold 12-24 h after treatment with TNF-alpha. NF-kB binding activity was also dramatically increased, but there is no NF-kB motif on the promoter for LDL receptor gene. The induction of LDL receptor by TNF might be mediated through a transcription factor, SP-1.

Base Sequence↗

PubMed1992

Effect of 15-deoxyspergualin, a microbial angiogenesis inhibitor, on the biological activities of bovine vascular endothelial cells.

We found recently that 15-deoxyspergualin, an analog of spergualin, which is an antibiotic and includes a spermidine moiety in its structure, exhibits anti-angiogenic activity. We have now carried out in vitro experiments with bovine vascular endothelial cells to determine which events occurring during angiogenesis are affected by this microbial angiogenesis inhibitor. 15-Deoxyspergualin did not inhibit the production of urokinase-type plasminogen activator (u-PA) or type IV collagenase by vascular endothelial cells. The direct inhibition of u-PA activity by 15-deoxyspergualin was not observed either. The angiostatic antibiotic neither affected the migration of vascular endothelial cells nor inhibited the endothelial cell proliferation in a two-dimensional culture system. We also examined the effect of 15-deoxyspergualin on the proliferation of endothelial cells in a three-dimensional culture system involving collagen gel, in which cell growth resembles more closely the endothelial cell proliferation during in vivo angiogenesis than that in a two-dimensional culture system without collagen gel. The antibiotic inhibited cell proliferation in a dose-dependent manner, indicating that the three-dimensional culture system is useful for finding a new angiogenesis inhibitor with a different mode of action from those of angiogenesis inhibitors found by using a two-dimensional assay system; however, no cause-effect relationship has yet been established. Taken together, these results suggest the possible involvement of the inhibition of vascular endothelial cell growth by 15-deoxyspergualin in its angiogenesis-inhibitory effect. 15-Deoxyspergualin appears to be a promising candidate as an angiogenesis inhibitor for controlling aberrant angiogenic responses occurring in different states, including tumor development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Rapid turnover of low density lipoprotein receptor in human monocytic THP-1 cells.

We examined whether human monocyte-derived macrophages had low density lipoprotein (LDL) receptors with a short life span. The human monocytic leukemia cell line, THP-1, was highly differentiated when treated with phorbol ester. LDL receptors degraded rapidly with half-lives of 3-4 h in THP-1 cells before phorbol ester treatment. During the transition into monocytic cells, expression of the LDL receptor gene was not affected. However, relative degradation rates of LDL receptors normalized by those of cellular total proteins were about twice as fast in phorbol ester-treated THP-1 cells compared to untreated cells.

Cell Differentiation↗

PubMed1991

The response to epidermal growth factor of human maxillary tumor cells in terms of tumor growth, invasion and expression of proteinase inhibitors.

Three cancer cell lines, IMC-2, IMC-3 and IMC-4, were established from a single tumor of a patient with maxillary cancer. We examined responses to epidermal growth factor (EGF) of these 3 cell lines with regard to cell growth and tumor invasion. The growth rate of IMC-2 in nude mice was markedly faster than that of the IMC-3 and IMC-4 cell lines. Assay for invasion through fibrin gels showed significantly enhanced invasive capacity of IMC-2 cells in response to EGF, but no change for IMC-3 and IMC-4 cells. We examined response to EGF of IMC-2 cells with regard to expression of a growth-related oncogene (c-fos), proteinases and their inhibitors. Expression of c-fos was transiently increased in IMC-2 cells at rates comparable to those seen in the 2 other lines in the presence of EGF. There was no apparent effect of EGF on the expression of urokinase-type plasminogen activator and 72-kDa type-IV collagenase in IMC-2 cells. In contrast, EGF specifically enhanced the expression of plasminogen activator inhibitor-I (PAI-I) and tissue inhibitor of metalloproteinases-I (TIMP-I) in IMC-2 cells. Our data suggest that proteinase inhibitors or other related factors may play an important role in tumor growth and invasion in response to EGF.

Animals↗

PubMed1991

Endogenous basic fibroblast growth factor-dependent induction of collagenase and interleukin-6 in tumor necrosis factor-treated human microvascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to enhance the synthesis of interleukin-6 (IL-6) and collagenase in human omental microvascular endothelial (HOME) cell (Mawatari, M., Kohno, K., Mizoguchi, H., Matsuda, T., Asoh, K., Van Damme, J. V., Welgus, H. G., and Kuwano, M. (1989) J. Immunol. 143, 1619-1627). In the present study, we have examined whether the TNF-alpha-induced synthesis of IL-6 or collagenase in HOME cells is mediated by an inducible growth factor. Among several growth factors examined, we found that the expression of basic fibroblast growth factor (bFGF) mRNA was the one most prominently enhanced when HOME cells were treated with TNF-alpha. The increase of bFGF mRNA by TNF-alpha in HOME cells was observed in both a dose- and time-dependent manner when assayed by Northern blot analysis. The induction of bFGF mRNA was observed by 3 h after incubation with TNF-alpha, and the maximal increase of 5-fold was obtained after 12 h of incubation with 100 units/ml TNF-alpha. Western blot analysis confirmed the enhanced synthesis of bFGF by TNF-alpha. Metabolic labeling and immunoprecipitation assays of bFGF showed that exposure to TNF-alpha enhanced secretion of bFGF into culture medium and also that TNF-alpha stimulated the production of bFGF molecules with molecular masses of 18, 21, and 22.5 kDa in HOME cells. TNF-alpha induced the expression of collagenase mRNA and IL-6 mRNA in HOME cells as well, and the coadministration of neutralizing anti-bFGF antibody almost completely blocked the effects of TNF-alpha. The treatment of HOME cells with exogenous bFGF significantly stimulated the expression of collagenase mRNA and IL-6 mRNA in HOME cells. Therefore, the biological effects of TNF-alpha on HOME cells may be mediated, at least in part, by TNF-alpha-induced bFGF.

Blotting, Northern↗

PubMed1991

Hepatocyte growth factor modulates migration and proliferation of human microvascular endothelial cells in culture.

Epidermal growth factor (EGF) induces tubular formation of cultured human omental microvascular endothelial (HOME) cells and EGF also stimulates cell migration as well as expression of tissue type plasminogen activator (t-PA). Here we studied the effects of hepatocyte growth factor (HGF) on cell proliferation, cell migration and expression of t-PA and other related genes. Migration of confluent HOME cells into the denuded space was stimulated by HGF after being wounded with razor blade, but at a reduced rate in comparison with EGF. HOME cells could be proliferated in response to exogenous 100 ng/ml of HGF at rates comparable to that of 20 ng/ml EGF. The chemotactic activity of HOME cells was significantly stimulated by HGF in a dose-dependent manner when assayed by Boyden chamber. HGF did not efficiently enhance expression of both the t-PA gene and a tissue inhibitor of metalloproteinase gene whereas it stimulated expression of plasminogen activator inhibitor-1. Our present study provides a new evidence that some of the biological effects of HGF on HOME cells in culture are similar to those of EGF.

Bilirubin↗