Keizo Kaba

Effects of protein kinase C modulators on multidrug resistance in human glioma cells.

To identify the role of protein kinase C (PKC) in multidrug resistance, the effects of phorbol-12-myristate-13-acetate (PMA), a PKC activator, or calphostin C, a PKC inhibitor, on intracellular vincristine accumulation and expression of P-glycoprotein phosphorylation were studied in one multidrug-resistant and three multidrug-sensitive human glioma cell lines. Basal PKC activities and immunoreactivities of PKC-alpha and -zeta were higher in multidrug-resistant cells than in multidrug-sensitive cells. There was no significant difference in the immunoreactivity of PKC-delta between multidrug-resistant and -sensitive cells, and immunoreactive PKC-beta, -gamma, and -epsilon were not detected in either multidrug-resistant or -sensitive cells. The treatment of multidrug-resistant cells with 100 nM PMA for 2 hours resulted in the activation not of PKC-zeta but of PKC-alpha, with concomitant decrease in vincristine accumulation and increase in P-glycoprotein phosphorylation. The exposure of multidrug-resistant cells to 100 nM PMA for 24 hours induced down-regulation not of PKC-zeta but of PKC-alpha, with concurrent decrease in vincristine accumulation, and reduced but still increased P-glycoprotein phosphorylation. The treatment of multidrug-resistant cells with 100 nM calphostin C for 2 hours decreased immunoreactive PKC-zeta and not immunoreactive PKC-alpha, inducing increase in vincristine accumulation, with concomitant decrease in P-glycoprotein phosphorylation. There was no evidence of significant change in vincristine accumulation in multidrug-sensitive cells treated with PMA or calphostin C. This may suggest that at least two isozymes of PKC, PKC-alpha and -zeta, are involved in P-glycoprotein phosphorylation and that vincristine efflux function in multidrug-resistant human glioma cells is closely associated with P-glycoprotein phosphorylation and is decreased by PKC inhibitor.

Lectin histochemistry of human gliomas

SummaryFormalin-fixed and paraffin-embedded specimens of 24 human gliomas were examined histochemically with five lectins: concanavalin A (Con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin 1 (RCA-1), peanut agglutinin (PNA), and Ulex europaeus agglutinin 1 (UEA-1). Although the staining intensity with lectins was variable, tumor cells in five astrocytomas, three oligodendrogliomas, six ependymomas, and one gliosarcoma, were generally positive for Con A, WGA, and RCA-1, and negative for PNA and UEA-1, whereas those in nine glioblastomas were usually positive for Con A and WGA and negative for RCA-1 and PNA as well as UEA-1. The malignancy in neoplastic astrocytes was correlated with the decrease in binding with lectins, especially RCA-1. Blood vessels, particularly the endothelial layers, in all gliomas were stained intensely with all lectins used. Macrophages showed two staining features with lectins; stippled and granular. The former macroplìages were positive for Con A, WGA, RCA-1, and PNA, and negative for UEA-1, whereas the latter macrophages were positive for all lectins used. Thus, the staining characteristics with lectins of macrophages were different from those of any glioma cells and very useful for identification of macrophages in gliomas.

Immunohistochemical study of fibronectin in hemangioblastomas and hemangiopericytomas

SummaryEight hemangioblastomas and two hemangiopericytomas were studied using indirect immunoperoxidase stains for fibronectin (FN) and glial fibrillary acidic protein (GFAP) in formalin-fixed, paraffinembedded surgical specimens. Stromal cells in hemangioblastomas were GFAP-negative and showed variable FN expression, while GFAP-positive cells were FN-negative, thus suggesting that the stromal cells are not derived from astrocytes. Hemangiopericytoma cells were poorly to intermediately FN-positive. The origin of stromal cells is discussed in the light of their fine structure and the immunohistochemical stains with other cell markers.

Plasma membrane structures of medulloblastoma and cerebellar sarcoma.

SummaryThree medulloblastomas and 1 cerebellar sarcoma were studied on their plasma membrane structures. The average number of membrane particles per μm2 plasma membrane was 710 on face A and 70 on face B of medulloblastoma and 1280 on face A and 160 on face B of cerebellar sarcoma. The membrane particles were often aggregated in medulloblastoma and diffusely scattered in cerebellar sarcoma. Small gap junctions were occasionally found in cerebellar sarcoma and not evident in medulloblastoma. Round membrane protrusions, about 0.5–0.6 μ in diameter and provided with several small depressions on their foot, were often observed in region of narrow perinuclear cytoplasm of cerebellar sarcoma and different in structure from cytoplasmic processes. The present series is too limited in number to allow a definite conclusion, but indicates that the plasma membrane structures are different in medulloblastoma and cerebellar sarcoma.

Interspecies comparison of c-myc gene in human and rat glioma cell lines

SummaryInterspecies difference in expression of the c-myc gene between two human and three rat glioma cell lines was studied with use of a human c-myc probe. The c-myc deoxyribonucleic acid (DNA) fragments detected at higher stringency in Southern blotting, showed a difference in size and gene copy number between human and rat glioma cells. The c-myc transcript was detected at both higher and lower stringencies in Northern blotting in human glioma cells, whereas it was demonstrated only at lower stringency in rat glioma cells, and the c-myc transcript was seen in cytoplasms of both glioma cells by in situ hybridization. The c-myc protein, if examined with anti-human c-myc protein monoclonal antibody, was observed as two separated components in Western blotting and localized immunocytochemically in nuclei in human glioma cells, whereas it was detected as three separate forms in Western blotting and shown in both nuclei and cytoplasm in rat glioma cells. The above discrepancy in manifestation of c-myc DNA fragments, transcript and protein could be due to the difference in nucleotide sequence of c-myc gene between human and rat glioma cells.