Combined effects of a third-generation bisphosphonate, zoledronic acid with other anticancer agents against murine osteosarcoma.

Bisphosphonates (BPs) are widely used to treat bone diseases and also appear to possess direct antitumour activity. We have previously reported that third-generation BPs such as zoledronic acid (ZOL) and minodronic acid (YM529) synergistically augment the effects of anticancer agents in various cancer cells. Recently, we have also reported the antitumour effects of YM529 on murine osteosarcoma cells. As YM529 has not been clinically available, we herein focused on the anti-osteosarcoma effects of ZOL which is clinically available. In addition to ZOL alone, we evaluated the concurrent or sequential combined effects of ZOL with other anticancer agents against murine osteosarcoma cell lines. ZOL showed almost same anti-osteosarcoma activity compared with YM529 and more sensitive growth inhibitory effects against osteosarcoma cells than normal cells. Moreover, ZOL acted synergistically in vitro when administered concurrently with paclitaxel (PAC) or gemcitabine (GEM), not only in wild-type osteosarcoma cells but also in P-glycoprotein (P-gp)-overexpressing osteosarcoma cells, which were much less sensitive against each anticancer agent. Furthermore, 24 h of ZOL pretreatment significantly augmented the sensitivity of doxorubicin (DOX), PAC or GEM against osteosarcoma cells. These findings suggest that combined administration of ZOL with other anticancer agents may improve the osteosarcoma treatment.

Induction of apoptosis in an estrogen-responsive mouse Leydig tumor cell by leukotriene.

For estrogen-responsive B-1F cells, established from estrogen-responsive mouse Leydig cell tumor, it has been reported that the 5-lipoxygenase (5-LOX) metabolic pathway appears to be associated with cell growth. The addition of 5-LOX inhibitor 2-(12-hydroxydodeca-5,10-diyl)-3,5,6-trimethyl-1,4-benzoquinone (AA861) to the medium resulted in a dose-dependent increase in cell yield as described previously. When the growth of the palpable tumors was measured, AA861 had stimulated in vivo tumor growth in adult male mouse inoculated B-1F cells. The effects of AA861 and 17beta-estradiol (E2) on the contents of various arachidonic acid metabolites in B-1F cells and their conditioned medium were examined. Although AA861 and E2 decreased the contents of leukotrienes (LTs), the two did not significantly change those of prostaglandins, thromboxan, prostacyclin, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE. In immunohistochemical study B-1F cells show positive staining for 5-LOX in the E2-depleted condition, while E2 decreased the expression of 5-LOX. The decrease of the intensities of 79-kDa 5-LOX protein and 403-bp RT-PCR product bands was observed. The growth of Morpholino-anti oligo delivered B-1F cells was higher than that of Standard control oligo delivered cells. The delivery of Morpholino-anti oligo into B-1F cells caused the decrease of contents of LTs and 5-HETE in the cells and medium, and the reduction of 5-LOX activity. When LTD4 was added in the culture medium, the increasing concentrations of LTD4 resulted in a significant inhibition of cell yields of E2-treated B-1F cells. Morphological changes such as nuclear condensation and fragmentation, and DNA ladder pattern were demonstrated in E2-stimulated B-1F cells treated with LTD4 as well as in control cells cultured in the basal medium. These results implicate that 5-LOX at least plays an important role in the growth of B-1F cells and LD4 induces the apoptosis of B-1F cells.

Functional characterization of Musca glutamate- and GABA-gated chloride channels expressed independently and coexpressed in Xenopus oocytes.

Ligand-gated chloride channels (LGICs) are important targets for insecticides and parasiticides. Genes encoding subunits of two LGICs, a glutamate-gated chloride channel (MdGluCl-alpha) and a gamma-aminobutyric acid (GABA)-gated chloride channel (MdRdl), were cloned from house-flies (Musca domestica L.). These genes were first expressed independently in Xenopus laevis oocytes by cRNA injection in order to investigate the pharmacology of these ligand-gated channels using two-electrode voltage-clamp electrophysiology. It was found that L-glutamate and GABA activated the MdGluCl-alpha homo-oligomers with an EC(50) value of 30 microM and the MdRdl homo-oligomers with an EC(50) value of 101 microM, respectively. Both channels were chloride ion-permeable, and the MdRdl channel was more sensitive to chloride channel blockers, such as gamma-hexachlorocyclohexane (gamma-HCH), fipronil and picrotoxinin, than the MdGluCl-alpha channel. MdGluCl-alpha required only 1-2 days of incubation after cRNA injection to be expressed in oocytes, whereas 4-7 days of incubation was necessary to achieve MdRdl expression. However, when the cRNA of MdGluCl-alpha was injected at a dose of 1% (w/w) 1 day after the injection of the cRNA of MdRdl, a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdl expression became shorter. These results suggest that MdGluCl-alpha assists in the expression of MdRdl when the two are coexpressed.

Juvenile form of Alexander disease with GFAP mutation and mitochondrial abnormality.

The authors report a 29-year-old woman with marked atrophy of the cerebellum, medulla oblongata, and spinal cord, dementia, diffuse white matter abnormality on MRI, ragged-red fibers, and R88C mutation in the human glial fibrillary acidic protein (GFAP). Mitochondria DNA (mtDNA) analysis showed a rare polymorphism at A8291G. This mtDNA polymorphism, which has been associated with limb-girdle type mitochondrial myopathy, may modify the clinical symptoms of this juvenile form of Alexander disease with GFAP mutation.

An autopsy case of bacterial septic shock 12 years following ABO-incompatible renal transplantation.

We report the case of an ABO-incompatible kidney transplant recipient who died suddenly following a good transplant course of 12 years. For 10 years after transplantation, the graft function had been stable (s-Cr: 1.0-1.5 mg/dL), although chronic hepatitis C had developed, with elevation of transaminase. In the 11th year, he was admitted into the hospital with low-grade fever and general fatigue. Jaundice and anaemia progressed, and he died 2 months after admission. The autopsy diagnosis was: (1) post-renal transplantation state, (2) phlegmonous enterocolitis with septic infarction, (3) cellulitis and necrotic myositis, and (4) sepsis. The transplanted kidney graft showed well-preserved glomeruli and tubules, corresponding to chronic allograft nephropathy (CAN) grade Iota (ci1, ct1, cv1), according to the Banff classification. The pathological changes observed in this long-surviving ABO-incompatible kidney graft were similar to those of an ABO-compatible graft, although its degree was milder.

Prenatal and neonatal exposure to bisphenol-A enhances the central dopamine D1 receptor-mediated action in mice: enhancement of the methamphetamine-induced abuse state.

Bisphenol-A (BPA), one of the most common environmental endocrine disrupters, has been extensively evaluated for toxicity in a variety of tests in rodents, including developmental and reproductive toxicity, and carcinogenicity. However, little is known about its action on the CNS. In this report, we show that prenatal and neonatal exposure to BPA in mice leads to the enhancement of the dopamine D1 receptor-dependent rewarding effect induced by a psychostimulant methamphetamine. Furthermore, this treatment with BPA markedly enhanced hyperlocomotion and its sensitization induced by methamphetamine, which reflects extensive abuse associated with sociological and psychiatric problems. We also demonstrated that chronic exposure to BPA produced an up-regulation of dopamine D1 receptor function to activate G-protein in the mouse limbic forebrain, which is thought to be a critical site for the expression of rewarding effects by abuse drugs. Additionally, chronic BPA exposure produced a significant increase in levels of the dopamine D1 receptor mRNA in the whole brain. In contrast, no change in protein levels of methamphetamine-targeted proteins, dopamine transporter or the type 2 vesicle monoamine transporter in the brain was observed by prenatal and neonatal exposure to BPA. The present data provide the first evidence that prenatal and neonatal exposure to BPA can potentiate the central dopamine D1 receptor-dependent neurotransmission, resulting in supersensitivity of methamphetamine-induced pharmacological actions related to psychological dependence on psychostimulants.

Investigation of parenchymal cell differentiation in organotypic slice culture of mouse fetal liver under administration of sodium butyrate.

The fetal mouse liver tissues in our organotypic slice culture were spread and flattened for at least 3 weeks; small, round cells were distributed in the center and polygonal cells were seen in the periphery. Ultrastructurally, polygonal cells showed abundant rough endoplasmic reticulum and mitochondria. They expressed albumin (ALB) and alpha-fetoprotein (AFP) for at least 3 weeks, and Cx32-immunoreactivity was also seen in a plaque on the cells. Many proliferating cell nuclear antigen (PCNA)-positive cells were observed at the periphery, and there were scattered CK-19-positive cells. The spreading of the fetal liver tissue in organotypic slice culture was reduced in medium containing sodium butyrate (SB). The expression of ALB was well maintained in polyglonal cells of the SB(+) group 3 weeks after culture and AFP-immunoreactivity was decreased in the SB(+) group. The concentration of ALB in the medium was significantly higher in the SB(+) than in the SB(-) group. CK-19-positive cells in the SB(+) group were increased in number more than those in the SB(-) group. PCNA-positive cells were less numerous in the SB(+) group, and Cx32-positive plaques were increased. SB can help immature hepatocytes to differentiate into the mature type and the cholangiocytic lineage, reducing their proliferation. These findings suggest that parenchymal cells in our organotypic slice culture of the fetal mouse liver can maintain structure and function as in vivo for the long term, and SB is shown to be a differentiation inducer of parenchymal cells in the slice culture.

In utero exposure to brief hyperthermia interferes with the production and migration of neocortical neurons and induces apoptotic neuronal death in the fetal mouse brain.

To investigate the pathogenetic mechanisms of brain maldevelopment induced by maternal hyperthermia, we exposed pregnant ICR mice to 43 degrees C for 12.5 min on day 13.5 or 14.5 of gestation and examined the proliferation and migration of neuronal precursor cells in the telencephalon of their fetuses. The brain weight was significantly decreased in heat-stressed fetuses when examined at 72 h after treatment. Histological examination revealed that the thickness of the neopallium, especially that of the intermediate (migratory) zone and the cortical plate, was decreased in the heated group. BrdU/anti-BrdU immunohistochemistry showed that cell proliferation in the matrix cell zone was suppressed for up to 8 h after hyperthermia and that the migration of BrdU-labeled neurons from the matrix cell zone to the primordial cortex was decelerated significantly. In addition, apoptotic cell death which is rarely observed in the brain of control animals increased in the brain of heat-stressed fetuses at 8-12 h after treatment. Thus, it seems that brief hyperthermia at critical stages of neuronal differentiation can interfere with the production and migration of neuronal precursor cells and result in abnormal brain development and neurobehavioural disturbances.

Musashi1, an evolutionarily conserved neural RNA-binding protein, is a versatile marker of human glioma cells in determining their cellular origin, malignancy, and proliferative activity.

Tumor cells often express phenotypic markers that are specific to the cells from which they originated. A neural RNA-binding protein, Musashil, is an evolutionarily well-conserved marker for neural stem cells/ progenitor cells. To examine the origin of gliomas, we examined the expression of the human Musashil homolog, MSI1, in human glioma tissues and in normal human adult and fetal brains. As we had seen previously in rodents, in the normal human brain, MSI1 was expressed in cells located in the ventricular and subventricular zones, in GFAP-negative glial cells, and in GFAP-positive astrocytes. In glioblastomas, MSI1 was expressed in GFAP-negative tumor cells forming foci that were clearly demarcated and surrounded by GFAP-positive cells. Tumor cells arranged in pseudopalisades were also strongly immunoreactive with MSI1 antibodies. The percentage of MSI1-labeled tumor cells increased in higher-grade astrocytomas and correlated with proliferative activity, as estimated by an MIB-1 staining index. Our results indicate that MSI1 is an excellent marker for neural progenitor cells including neural stem cells in normal human brains. Furthermore, the expression of MSI1 correlates well with the immature nature as well as the malignancy of tumor cells in human gliomas. Thus, we expect the analysis of MSI1 expression to contribute to the understanding of the cellular origin and biology of human gliomas.

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein-Barr virus (EBV)-based plasmid vectors.

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein-Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 microg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 microg of DNA. More than 70% of liver cells stained with X-gal when beta-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver.

Developmental expression of a unique carbohydrate antigen, Tn antigen, in mouse central nervous tissues.

Using an anti-Tn monoclonal antibody, the Tn antigen was detected immunohistochemically in prenatal and early postnatal central nervous tissues. On embryonic day 9 (E9), the antigen was distributed throughout the single neuroepithelial layer in the neocortex and then became more prominent in the preplate than in the ventricular zone along with formation of the preplate. Following division of the preplate and concomitant formation of the cortical plate, distinct labeling of the neocortex occurred in the marginal, subplate and intermediate zones, whereas in the cortical plate and ventricular zone were virtually not immunostained. It is notable that thalamocortical afferent fibers were also immunostained specifically on E14. After birth, the localization of the antigen became less noticeable and by 3 weeks after birth, the antigen had substantially disappeared. In the developing cerebellum, prominent labeling was also observed in the molecular layer and outskirts of the cerebellar nuclei on early postnatal days. To characterize the glycoprotein bearing the Tn antigen biochemically, immunoblot analysis was performed. The glycoprotein, most of which was extracted with a salt solution, migrated as a broad smeared band corresponding to a molecular weight of about 250 kDa on SDS-PAGE. Among the various tissues examined, this glycoprotein was only detected in the brain and its amount increased until an early postnatal stage with a peak on postnatal day 3 (P3), and then decreased gradually with age. This spatially and developmentally regulated expression of the Tn antigen suggests that this antigen plays a significant role in brain development.

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis.

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.

The cerebrospinal fluid level of glial fibrillary acidic protein is increased in cerebrospinal fluid from Alzheimer's disease patients and correlates with severity of dementia.

To gain insight of the underlying mechanisms of astroglial response to Alzheimer's disease (AD), the level of glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) from controls and AD subjects were immunochemically determined, and the correlation between that level and dementia severity of AD patients was evaluated. Means and SD of CSF levels of GFAP for the young control group (from 1 to 25 years, mean +/- SD 14.2 +/- 5.0, n = 13) adult control (from 26 to 55 years, 41.6 +/- 10.1, n = 9) and senescent control (older than 56 years, 65.4 +/- 8.0, n = 8) were 2.96 +/- 1.04, 2.80 +/- 1.46 and 3.99 +/- 1.55 ng/ml, respectively, and the CSF level of GFAP was not dependent on age (ANOVA, p = 0.17). While that of the AD patient group (n = 27, 70.8 +/- 8.0 years) was 8.96 +/- 7.80 ng/ml, significantly higher than that of both the all-control (3.19 +/- 1.39 ng/ml, t test, p < 0.001) and age-matched senescent (3.99 +/- 1.55 ng/ml, t test, p < 0.005) control groups. The receiver-operator characteristic (ROC) curve revealed that the GFAP concentration at 5 ng/ml in CSF could serve as a cutoff value. The CSF level of GFAP in the moderately to severely demented patients (MMSE /= 18, 6.85 +/- 5.76 ng/ml, n = 18; ANOVA, p < 0.05). These findings together with our previous report on an increase in the CSF level of apolipoprotein E suggest that degeneration and stimulation of astrocytes takes place concurrently in the AD brain.