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T Tamaoki

Publications and source records attributed to T Tamaoki.

At least 55 records · Page 3Linked to original sources
PubMed1998

A useful method to evaluate bone resorption inhibitors, using osteoclast-like multinucleated cells.

Since excessive bone resorption in postmenopausal osteoporosis is a pathological manifestation of the disease, it is important to control bone resorption activity of osteoclasts as a means of controlling the disease. To screen bone resorption inhibitors, a simple and quantitative method to assay bone resorption is needed. However, it has been difficult to prepare a lot of osteoclasts required for the screening of many compounds in vitro. We used the method for the preparation of mouse osteoclast-like multinucleated cells in vitro and developed a useful method in which osteoclast-like multinucleated cells were placed on dentin slices and the calcium released from dentin into the culture medium was measured. Under the optimal conditions, the increase of the calcium concentration caused by bone resorption activity of the osteoclast-like cells was significant and inhibited by calcitonin in a dose-dependent manner. We also examined the inhibitory effect of calcitonin on the pit area caused by bone resorption and found that the decrease of the calcium released from dentin was correlated with that of the pit area. Furthermore, several known bone resorption inhibitors such as bisphosphonate, bafilomycin A1, and herbimycin A showed inhibitory effects on the calcium release from dentin slices. Thus, this simple method provides us a useful screening system to find bone resorption inhibitors with novel mechanisms.

Animals↗
PubMed1998

Expression of HNF-1 alpha and HNF-1 beta in various histological differentiations of hepatocellular carcinoma.

Hepatic nuclear factor 1 (HNF-1) regulates genes in a hepatocyte-specific manner. It has been previously reported that the ratio of HNF-1 alpha and HNF-1 beta mRNA is related to histological differentiation hepatocellular carcinoma (HCC). In this study, the expression levels of the HNF-1 alpha and HNF-1 beta proteins were analysed relatively and quantitatively in various histologically differentiated HCC and surrounding non-cancerous tissues, and HNF-1 alpha binding activity for the AT element of the B domain of the human alpha-fetoprotein enhancer was examined. Western blot analysis demonstrated that HNF-1 alpha protein was expressed at a higher level in well-differentiated HCC tissues than in the surrounding non-HCC tissues; on the other hand, the HNF-1 alpha protein was expressed at lower levels in moderately and poorly differentiated HCCs than in the surrounding non-HCC tissues. The levels of HNF-1 beta expression in well-differentiated and poorly differentiated HCCs were similar to and higher than those found in the respective surrounding non-cancerous portions. In binding assays, HNF-1 binding activity was high in well-differentiated HCC and lower in moderately and poorly differentiated HCCs. Most well-differentiated HCC cases showed immunohistochemical expression of HNF-1 alpha. These findings show that poor histological differentiation of HCC correlates with decreases in the level and activity of HNF-1 alpha proteins.

Blotting, Western↗
PubMed1998

Metabolism and ribonucleotide reductase inhibition of (E)-2'-deoxy-2'-(fluoromethylene)cytidine, MDL 101,731, in human cervical carcinoma HeLa S3 cells.

UNLABELLED: (E)-2'-Deoxy-2'-(fluoromethylene)cytidine, MDL 101,731, has shown potent antitumor activity against various human xenograft models. PURPOSE: The purpose of this study was to elucidate the mechanism of the antitumor activity of MDL 101,731 against human carcinoma cells through investigating metabolism and the target enzyme of MDL 101,731. METHODS: In respect of the intracellular metabolism of MDL 101,731, the effect on enzymes in the pyrimidine salvage pathway and the intracellular metabolites of MDL 101,731 were investigated. In respect of the target enzyme, the effect on intracellular deoxyribonucleoside triphosphate (dNTP) pools and the inhibition of the enzyme activity were investigated. RESULTS: MDL 101,731 which shows antiproliferative activity against human cervical carcinoma HeLa S3 cells at nanomolar concentrations (IC50, 30-50 nM), was hardly metabolized to (E)-2'-deoxy-2'-(fluoromethylene)uridine (FMdU) which had no antiproliferative activity below 100 microM because of resistance to human cytidine deaminase. MDL 101,731 showed low activity against murine lymphocytic leukemia P388R cells (Ara-C-resistant cells) which contained lower deoxycytidine kinase activity than parental P388 cells. In addition, the antiproliferative activity of MDL 101,731 against HeLa S3 cells was reversed by deoxycytidine. Studies of the intracellular metabolism of 3H-MDL 101,731 demonstrated that it was rapidly metabolized to the diphosphate and the triphosphate forms without the other metabolites in HeLa S3 cells. A 3-h treatment with 0.1-10 microM MDL 101,731 decreased intracellular dNTP pools. The recovery of dNTP pools decreased by treatment with 2 microM MDL 101,731 was much slower than the recovery following treatment with 10 mM hydroxyurea, a reversible ribonucleotide reductase inhibitor. At a dose of 250 mg/kg, MDL 101,731 continuously inhibited ribonucleotide reductase activity up to 72 h in a HeLa S3 xenograft model. CONCLUSIONS: This study suggests that the prolonged ribonucleotide reductase inhibition by rapidly activated metabolites of MDL 101,731 in part contributes to the potent antitumor activity of this drug against various xenografts.

Antineoplastic Agents↗
PubMed1998

Hair follicle elongation in organ culture of skin from newborn and adult mice.

By means of skin organ culture, some biological characteristics of hair follicle elongation were examined. When skin sections from 6-day-old C3H mice were cultured, spontaneous elongation of hair follicles was maintained. Without insulin, hair follicle elongation was poorly maintained irrespective of the presence of serum at 20%. Insulin could be replaced by IGF-I at 100 ng/ml. During in vitro elongation of hair follicles, bromodeoxyuridine was incorporated into germinal epithelial cells around dermal papillae. Skin sections from 4-week-old C3H mice did not show hair follicle elongation in complete medium. However, when 0.5 mM minoxidil was added to the medium, concentration dependent thickening and elongation of hair follicles was observed. In contrast, in vitro elongation of newborn pelage hair follicles was not enhanced by minoxidil. These results suggest that this system with the use of skin sections from 4-week-old C3H mice would be a potential in vitro model of human androgenic alopecia in which the anagen phase is suppressed but the suppression is partially released by minoxidil.

Aging↗
PubMed1998

Combined effect of navelbine with medroxyprogesterone acetate against human breast carcinoma MCF-7 cells in vitro.

Navelbine (NVB, vinorelbine ditartrate, KW-2307), a new vinca alkaloid analogue, has been shown to be clinically effective against advanced breast cancer. In this report, the combined effect of NVB with medroxyprogesterone acetate (MPA), a synthetic progesterone derivative, was examined in vitro against human breast carcinoma MCF-7 cells. The combined effect was demonstrated to be synergistic using the isobologram and median-effect plot analyses. To elucidate the mechanism of action, we further examined effects of both drugs on cell cycle distribution of the cells in combination and/or alone. NVB at 2 nM induced apparent G1-phase accumulation as well as the induction of cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1) protein and the dephosphorylated form of retinoblastoma protein (pRb). In contrast, MPA at 0.1 microM also induced G1-phase accumulation as well as the reduced expression of cyclin D1 protein. In addition, the combination of both drugs induced augmented G1-phase accumulation, which occurred along with p21(WAF1/CIP1) protein induction, cyclin D1 protein reduction and pRb dephosphorylation. These results demonstrate that the synergistic combined effect of NVB with MPA was mediated through enhancement of G1-phase accumulation that resulted from the different action point(s) of each drug. Furthermore, the synergistic combined effect of NVB with MPA was also observed in other human breast carcinoma cell lines, such as T-47D and ZR-75-1. These results suggest that combination therapy of NVB with MPA in breast cancer might be effective in clinical studies.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

The relationship between the antitumor activity and the ribonucleotide reductase inhibitory activity of (E)-2'-deoxy-2'-(fluoromethylene) cytidine, MDL 101,731.

(E)-2'-deoxy-2'-(fluoromethylene) cytidine (MDL101,731) is a new deoxycytidine analog which shows potent antitumor activity against several human tumor models. We previously showed that MDL101,731 inhibited human ribonucleotide reductase (RNR) in HeLa S3 human cervical carcinoma cells. Recently, it has been reported that another deoxycytidine analog, 2'-deoxy-2'-methylidenecytidine (DMDC) which also inhibits RNR from Escherichia coli, does not inhibit RNR in intact L1210 murine leukemia cells. MDL101,731 was designed as an inhibitor of RNR, so it is important to know the contribution of the RNR inhibitory activity of the drug on its antitumor efficacy in vivo. Therefore, we examined the relationship between the antitumor activity and RNR inhibitory activity of MDL101,731 using LX-1 human lung carcinoma which was highly sensitive to this drug. MDL101,731 showed strong inhibition of RNR activity in LX-1 lung carcinoma by both i.v. and p.o. administration. Administration of 15 mg/kg i.v. and 30 mg/kg p.o. of MDL101,731, doses which showed almost the same degree of antitumor activity against LX-1 lung carcinoma on a daily 5 day schedule, caused a similar degree and similar kinetics of inhibition of RNR in LX-1 lung carcinoma at least for 12 h after administration. On the other hand, DMDC as well as 1-beta-D-arabinofuranosyl-cytosine (ara-C), which is a well-known deoxycytidine analog and inhibits DNA polymerase alpha, did not inhibit RNR in LX-1 lung carcinoma at doses demonstrating antitumor activity. These results indicate that MDL101,731 exhibited antitumor activity through inhibition of RNR activity in tumor cells in vivo and the mechanism of antitumor action of MDL 101,731 might be different from those of DMDC and ara-C, at least in part.

Animals↗
PubMed1998

In vitro and in vivo effects of KT6352, a derivative of indolocarbazole compounds, on murine megakaryocytopoiesis.

We investigated the in vitro and in vivo effects of KT6352, a derivative of indolocarbazole compound, on murine megakaryocytopoiesis. When serum-free megakaryocyte (Meg) colony assay was performed with 100 U/mL of recombinant mouse interleukin-3 (rmIL-3), the addition of 1x10(-11)M to 1x10(-9)M of KT6352 increased the number of Meg colonies. An additional increase of Meg colonies by KT6352 was observed in the serum-free culture containing rmIL-3 plus recombinant mouse interleukin-6 or rmIL-3 plus recombinant mouse stem cell factor. KT6352 did not stimulate Meg colony formation without rmIL-3. When KT6352 was administered intraperitoneally to normal BALB/c male mice at a dose of 10 mg/kg daily for 5 consecutive days, a 2.1-fold increase in the platelet count was observed on day 14, and the prolonged thrombocytopoiesis was detectable from 9 to 27 days after KT6352 administration. A marked increase in the white blood cell count was also observed from 5 to 14 days after KT6352 treatment. Before the gradual increase of platelet counts, 8 days after KT6352 administration, a marked increase in the number of colony-forming units of megakaryocytes (CFU-Megs) in bone marrow and spleen was observed, and a substantial increase in the number of splenic CFU-Megs was observed 14 and 23 days after KT6352 administration. Bone marrow Meg ploidy analysis by two-color flow cytometry showed a shift in the modal ploidy class from 16 to 32 and an increase in the frequency of 64 cells in KT6352-treated mice. These results suggest a possible therapeutic benefit of KT6352 in the management of thrombocytopenia.

Animals↗
PubMed1997

In vivo gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene.

The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but overexpressed in human hepatocellular carcinoma (HCC). Here, we demonstrate that replication defective recombinant adenoviral vectors, containing the human AFP promoter/enhancer, can be used to express the Escherichia coli cytosine deaminase (CD) gene (AdAFPCD) and the beta-galactosidase gene (AdAF-PlacZ) in AFP-producing HCC cell lines. Expression of the CD gene by adenovirus from the AFP promoter/enhancer (AdAFPCD) induced cells sensitive to 5-fluorocytosine (5FC) in the AFP-producing cells but not in the AFP-nonproducing cells. Transduction by an adenoviral vector harboring an ubiquitous strong promoter and CD gene showed enzymatic activity and 5FC killing in all cell lines. When AdAFPlacZ was injected into the s.c. established hepatoma in vivo, expression of the beta-galactosidase gene was confined to AFP-producing HCC xenografts. Moreover, HCC xenografts regressed by transduction with AdAFPCD and subsequently with 5FC treatment in vivo. These findings suggest that utilization of the AFP promoter/enhancer in an adenoviral vector can confer selective expression of a heterologous suicide gene in hepatocellular carcinoma cells in vitro and in vivo.

Adenoviridae↗
PubMed1997

Characterization of the molecules involved in the hematopoietic microenvironment provided by mouse stromal cell line MC3T3-G2/PA6 using a unique reporter system that analyzes the direct cell-to-cell interaction.

As an approach to characterizing the molecules involved in the hematopoietic microenvironment provided by a murine clonal preadipose cell line MC3T3-G2/PA6 (PA6), we developed a unique system to detect the early phase of signal transduction caused by the direct cell-to-cell interaction using the reporter plasmid pfosluc2 with the c-fos enhancer/promoter linked with the Photinus pyralis luciferase gene. The plasmid pfosluc2 was genetically introduced into a mouse myeloid leukemia cell line NFS-60 which showed a growth dependency on contact with PA6 cells, and the mechanism by which stromal PA6 cells promote the proliferation of NFS-60 cells through the direct cell-to-cell interaction was analyzed. The direct cell-to-cell interaction with PA6 cells was found to cause a significant c-fos induction to NFS-60 cells within 1 h. Approximately 10(5) cDNA clones prepared from PA6 cells were screened for their activity to promote the c-fos expression in NFS-60 cells through the direct cell-to-cell interaction, and 13 positive clones were obtained. Of these positive clones, five clones encoded the stem cell factor, and the others encoded the hepatocyte growth factor (HGF). The c-fos induction caused by the contact with PA6 cells in NFS-60 was completely inhibited by addition of both antagonistic anti-c-kit and anti-HGF antibodies. These results represent direct evidence for the action of HGF on the proliferation of hematopoietic cells through direct cell-to-cell interaction with stromal cells. Thus, our developed reporter system can be useful in investigating the direct cell-to-cell interaction between stromal and hematopoietic cells.

Animals↗
PubMed1997

Lymphostin (LK6-A), a novel immunosuppressant from Streptomyces sp. KY11783: taxonomy of the producing organism, fermentation, isolation and biological activities.

In the course of screening for inhibitors of the lymphocyte kinase, Lck (p56lck), aiming at novel immunosuppressants, we isolated a novel alkaloid, lymphostin (LK6-A), from the culture broth of Streptomyces sp. KY11783. Lymphostin was produced in a fermentation medium supplemented with a highly porous polymer resin, which prevented the degradation of this compound in the culture broth. Lymphostin inhibited the kinase activity of Lck with an IC50 value of 0.05 microM, and exhibited potent inhibitory activity against the mixed lymphocyte reaction (MLR) with an IC50 value of 0.009 microM.

Chromatography, High Pressure Liquid↗
PubMed1997

Mouse bone marrow stromal cell line MC3T3-G2/PA6 with hematopoietic-supporting activity expresses high levels of stem cell antigen Sca-1.

The murine clonal preadipose cell line, MC3T3-G2/PA6 (PA6), has the ability to support in vitro proliferation of hematopoietic stem cells defined as colony-forming units in spleen (CFU-S). In order to ascertain the relationship between the hematopoietic-supporting activity of PA6 cells and their expression, we cultured a number of these cells for over 45 weeks and investigated the level at which they expressed several cell surface markers and membrane-bound growth factors. Besides expressing stem cell factor (SCF) and macrophage colony-stimulating factor (M-CSF), PA6 cells were found by flow cytometry analysis to express high levels of stem cell antigen-1 (Sca-1). The expression level of Sca-1 in PA6 cells correlated with the ability of the latter to support hematopoiesis, whereas no such correlation was observed in the case of SCF and M-CSF expression. A cDNA clone encoding the protein recognized by anti-Sca-1 antibody was isolated from PA6 cells by expression cloning, so that its nucleotide sequence encoded the protein identical to mouse alloantigen Ly-6A.2. Genetically engineered COS-7 cells, transformed by the expression vector carrying the Ly-6A.2 gene, suppressed proliferation of murine lineage marker-negative (Lin) bone marrow cells by themselves and synergistically augmented proliferation of these cells in the presence of SCF. These results suggest that Ly-6A.2 regulates the proliferation of hematopoietic progenitor cells, and is one of the molecules organizing the hematopoietic microenvironment provided by stromal cells.

Animals↗
PubMed1996

Cloning of the cDNA encoding the mouse ATBF1 transcription factor.

We have isolated a mouse ATBF1 cDNA which is 12-kb long and capable of encoding a 406-kDa protein containing four homeodomains and 23 zinc-finger motifs. Mouse ATBF1 is 94% homologous to the human ATBF1-A transcription factor. Northern blot and RNase protection analysis showed that levels of ATBF1 transcripts were low in adult mouse tissues, but high in developing brain, consistent with a role for ATBF1 in neuronal differentiation.

Amino Acid Sequence↗
PubMed1996

Gene therapy for alpha-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.

We have developed a recombinant replication-defective adenovirus containing human alpha-fetoprotein (AFP) promoter/enhancer to direct cell type-specific expression of the herpes simplex virus thymidine kinase (HSVtk) gene to AFP-producing hepatocellular carcinoma (HCC) cells. After an in vitro infection by a recombinant adenovirus carrying the lacZ gene under the control of human AFP promoter/enhancer (AdAFPlacZ), an expression of the lacZ gene was demonstrated efficiently in AFP-producing HuH-7 and HepG2 cell lines, but not in AFP-nonproducing HLE and HLF cell lines, although lacZ gene expression was demonstrated in all these cell lines when infected with adenovirus vector carrying lacZ gene driven by the beta-actin-based promoter. Expression of the HSVtk gene by adenovirus, from AFP promoter/enhancer (AdAFPtk) induced the cells sensitive to ganciclovir (GCV) in the AFP-producing cell line efficiently, but not in AFP-nonproducing HLF hepatoma cells. An in vitro bystander effect was observed when only 10% of the cells were infected with AdAFPtk. These findings suggest that the AFP promoter/enhancer sequence can provide the tumor-specific activity for the therapeutic gene expression, and that the AdAFPtk vector induces the selective growth inhibition by GCV in the adenovirus-infected human hepatoma cells in vitro. Recombinant adenovirus transfer of the HSVtk gene under the control of tumor-specific promoter followed by GCV may have promise as a targeted in situ treatment for solid neoplasms.

Adenoviridae↗
PubMed1996

A new alternative splice variant of the mouse Fas antigen with a deletion in the N-terminal portion of the extracellular domain.

The Fas antigen (Fas/APO-1/CD95) has been shown to induce apoptosis when bound to a monoclonal anti-Fas antibody or Fas ligands. Recently, a new soluble human Fas isoform which lacks the transmembrane domain due to alternative splicing has been isolated; however, no mouse Fas isoforms have been reported so far. Analysis of Fas transcripts by RT-PCR detected no Fas transcripts corresponding to the human soluble Fas isoform in mouse thymus, spleen and liver. However, we detected a new isoform with a 117-bp deletion in the second exon in various mouse tissues and cell lines. This isoform, termed truncated Fas (T-Fas), can be generated by alternative splicing and lacks the N-terminal portion of the extracellular domain just after the signal sequence. Since the deletion involves the first cysteine-rich motif believed to be necessary for binding to the Fas ligand, the T-Fas protein may lack the ability to induce apoptosis. The expression of T-Fas relative to that of the normal Fas varies considerably among mouse tissues and cell lines, suggesting preferential transcription of the T-Fas isoform in certain cell types.

Alternative Splicing↗
PubMed1996

Expression ratio of hepatocyte nuclear factor-1 to variant hepatocyte nuclear factor-1 in differentiation of hepatocellular carcinoma and hepatoblastoma.

BACKGROUND/AIMS: Liver-specific protein genes have multiple cis-/trans-acting elements, but those accountable for hepatocytic differentiation are unclear. An AT-rich core sequence (AT motif) is essential as a cis-acting element for the hepatic transcription. Homologous proteins hepatocyte nuclear factor-1 (HNF-1) and variant HNF-1 (vHNF-1) bind to this motif. The ratio of HNF-1 to vHNF-1 mRNA was examined in various liver tissues with respect to their differentiation. METHODS: The competitive reverse transcriptional polymerase chain reaction was employed to amplify HNF-1 and vHNF-1 mRNA simultaneously and to examine their expression ratio in total RNA extracted from frozen liver tissues of 37 patients with hepatocellular carcinoma, five patients with hepatoblastoma, and 15 non-neoplastic liver tissues. RESULTS: The ratio of HNF-1 to vHNF-1 mRNA was higher in well-differentiated cases than in poorly-differentiated and undifferentiated cases, except that one poorly-differentiated hepatoblastoma displayed a high ratio. Non-neoplastic liver tissues had low ratios similar to poorly-differentiated hepatocellular carcinoma, the reason for which remained unknown. However, chronic hepatitis and liver cirrhosis cases also demonstrated low ratios, and hence degenerative changes themselves displayed no obvious influence on such ratios. Thus, the gene expression of HNF-1 and vHNF-1 seemed to be differentially regulated in neoplastic and non-neoplastic hepatocytes. CONCLUSIONS: These results suggested that the ratio of HNF-1 to vHNF-1 mRNA correlated with histological differentiation of HCC and hepatoblastoma.

Adult↗
PubMed1996

Developmental changes in expression of the ATBF1 transcription factor gene.

The expression of the ATBF1 gene in developing brain was analyzed in mice from 13 days of gestation to 28 days after birth using RNase protection and in situ hybridization methods. The level of ATBF1 transcripts was the highest on embryonic day 13-15 and then progressively decreased to a hardly detectable level on postnatal day 28. Throughout the period examined, ATBF1 mRNA was expressed consistently in the basal telencephalon, diencephalon, and mesencephalon, with the highest levels in the inferior colliculus and thalamus. On the other hand, no significant expression was observed in cerebellum, neocortex, hippocampus, and olfactory bulb. These results illustrate significant regional differences in the ATBF1 expression and suggest a role of the ATBF1 gene in the formation of some specific cell populations in developing central nervous system.

Animals↗