A Novel Monoclonal Antibody Against Neuroepithelial and Ependymal Cells and Characteristics of Its Positive Cells in Neurospheres.

There are still few useful cell membrane surface antigens suitable for identification and isolation of neural stem cells (NSCs). We generated a novel monoclonal antibody (mAb), designated as mAb against immature neural cell antigens (INCA mAb), which reacted with the areas around a lateral ventricle of a fetal cerebrum. INCA mAb specifically reacted with neuroepithelial cells in fetal cerebrums and ependymal cells in adult cerebrums. The recognition molecules were O-linked 40 and 42 kDa glycoproteins on the cell membrane surface (gp40 INCA and gp42 INCA). Based on expression pattern analysis of the recognition molecules in developing cerebrums, it was concluded that gp42 INCA was a stage-specific antigen expressed on undifferentiated neuroepithelial cells, while gp40 INCA was a cell lineage-specific antigen expressed at the stages of differentiation from neuroepithelial cells to ependymal cells. A flow cytometric analysis showed that fetal and young adult neurospheres were divided into INCA mAb(-) CD133 polyclonal antibody (pAb)(-), INCA mAb(+) CD133 pAb(-), and INCA mAb(+) CD133 pAb(+) cell populations based on the reactivity against INCA mAb and CD133 pAb. The proportion of cells having the neurosphere formation capability in the INCA mAb(+) CD133 pAb(+) cell population was significantly larger than that of undivided neurospheres. Neurospheres formed by clonal expansion of INCA mAb(+) CD133 pAb(+) cells gave rise to neurons and glial cells. INCA mAb will be a useful immunological probe in the study of NSCs.

Luteolin attenuates doxorubicin-induced cytotoxicity to MCF-7 human breast cancer cells.

Luteolin, a flavone found in some vegetables, has been reported to exhibit antioxidant, antiinflammatory, and anticancer activities. In the present study, we found that luteolin has biphasic effects on the viability of the human breast cancer cell line MCF-7. That is, cell viability increased at relatively low luteolin concentrations and decreased at relatively high concentrations. Focusing on the proliferative effect at low concentrations, we showed that luteolin has a cytoprotective effect on MCF-7 cells when administered with doxorubicin. Moreover, luteolin attenuated doxorubicin-induced cytotoxicity even in the presence of the estrogen receptor (ER) antagonist ICI 182,780 and the ER-negative MDA-MB-453 human breast cancer cell line. Reactive oxygen species (ROS) were generated after doxorubicin treatment of MCF-7 cells. In contrast, luteolin attenuated doxorubicin-induced ROS generation. Levels of the antiapoptotic protein Bcl-2 in luteolin-treated MCF-7 cells were significantly higher than those in doxorubicin-treated MCF-7 cells. Our results suggest that a low concentration of luteolin attenuates doxorubicin-induced cytotoxicity to MCF-7 cells through a combination of antioxidant activity and an increase in levels of Bcl-2 protein.

Soluble matrix from osteoblastic cells induces mineralization by dental pulp cells.

Dental pulp cells have a capacity to differentiate into mineralization-inducing cells. To clarify the molecular mechanism, we established an in vitro mineralization-inducing system by rat clonal dental pulp cell line, RPC-C2A, and tried to purify a mineralization-inducing factor in conditioned medium (CM) from pre-osteoblastic MC3T3-E1 cells. The active factor was impermeable to an ultrafiltration membrane, and sedimented by ultracentrifugation. The sedimented factor was found as a needle-like structure about 1.3 microm in average length as observed by transmission electron microscopy. The factor contained type I collagen, suggesting not a matrix vesicle, but a soluble matrix. The mineralization-inducing activity was also detected in CM from primary culture of rat calvaria (RC) cells. These results suggested that the soluble matrices from osteoblastic cells serve, at least in part, as differentiation-inducing agents.

Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization.

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis.

[Effect of bcl-2 over-expression on the membrane structure of NG108-15 nerve cells: effectiveness of membrane-structure analysis using FACS for the first screening of apoptosis-inducing drugs].

Bcl-2 is a Bcl-2 family protein that is known to be anti-apoptotic and is predominantly localized to the mitochondria. We previously showed that an analgesic, buprenorphine hydrochloride (Bph), induces apoptosis in the rodent-derived nerve cell line, NG108-15, through the mitochondrial apoptotic route. A Bcl-2-overexpressing strain of NG108-15 cells, Bcl-2 (P2), was established, and the effect of Bcl-2 expression on Bph-induced apoptosis was compared between the mock vector-transfected NG108-15 cells and the Bcl-2 (P2) cells. The Bcl-2 (P2) cells died after treatment with Bph, and we observed all the biological and morphological markers of apoptosis that we tested for. In flow cytometric analysis, a difference in the cell membrane phospholipid flip-flop pattern-a feature of apoptosis- was observed between the NG108-15 cells and the Bcl-2 (P2) cells. Here, we show by flow cytometric analysis that Bcl-2 over-expression may affect the membrane structure of Bcl-2 (P2) cells. An increased fluorescein isothiocyanate (FITC) signal of annexin V-FITC, which typically represents phospholipid flip-flop of the cellular membrane in early apoptosis, was barely detected in the Bcl-2 (P2) cells. Since our previous study reported the localization of over-expressed Bcl-2 protein to the cell membrane of Bcl-2 (P2) cells, together these observations suggest that the Bcl-2 protein may affect the integrity of the structure of the NG108-15 cell membrane.

Over-expressed Bcl-2 cannot suppress apoptosis via the mitochondria in buprenorphine hydrochloride-treated NG108-15 cells.

We previously reported that the morphine alkaloid derivative buprenorphine hydrochloride (Bph) induces rapid apoptosis in NG108-15 nerve cells accompanied by the activation of caspase-3. Here, we found this kind of apoptosis was also accompanied by rapid loss of the mitochondrial membrane potential, followed by the efflux of cytochrome c from the mitochondria to the cytosol and the activation of caspases-9 and -3. Together, these results strongly suggested the Bph death signal was routed through the mitochondrial pathway in NG108-15 cells. In these cells, serum-starvation induces a different apoptosis, which we exploited to investigate Bcl-2's role as an apoptosis inhibitor. We made an NG108-15 transfectant, Bcl-2(P2), that stably expressed human Bcl-2, and used it to test Bcl-2's effect on the serum-starvation-induced apoptosis in NG108-15 cells. Cell viability, DNA-ladder formation, and efflux of cytochrome c from the mitochondria were all detected, showing that the human Bcl-2 functioned normally in the Bcl-2(P2) cells. Although the apoptotic events tested were identical in the parental cells and transformants, Bcl-2 expression completely failed to inhibit Bph-induced apoptosis in the Bcl-2(P2) cells.

Evaluation of cell death caused by CDF (cyclophosphamide, doxorubicin, 5-fluorouracil) multi-drug administration in the human breast cancer cell line MCF-7.

We evaluated the features of cell death induced by CDF (cyclophosphamide [CPA], doxorubicin [DOX], 5-fluorouracil [5-FU]) multi-drug administration in vitro using the human breast cancer cell line MCF-7. Used individually, DOX and 5-FU induced 60% cell death in MCF-7 cells, at 5 microg/ml and 25 microg/ml, respectively, by the 4th day following drug treatment. CPA was the least cytotoxic of the 3 drugs, causing only 20% cell death, even at the high concentration of 500 microg/ml. Treating cells with a mixture of all three anticancer drugs resulted in 60% cell death, on the second and third day following drug treatment. The nature of the cytotoxicity of CPA, DOX, and 5-FU was investigated, because these drugs are sometimes used to induce apoptosis. Biochemical analysis showed faint DNA fragmentation in the case of DOX or all three drugs, but not for treatment with CPA or 5-FU. In contrast, the morphological apoptotic feature of a condensed nucleus was observed only for CPA and 5-FU. Flow cytometric data agreed with the morphological results in that the FACS cytogram for DOX and for all three drugs was different from that for CPA or 5-FU given alone. These observations suggested that the cell death induced by these anticancer drugs in the human breast cancer cell line MCF-7 is a mixture of apoptotic and non-apoptotic, but it becomes completely non-apoptotic in the case of multi-drug administration.