Takahisa Shimizu

Low-dose staurosporine suppresses proliferation and induces neurites in human prostatic cancer TSU-Pr1 cells

Despite the beneficial effects of androgen ablation therapy in patients with prostate carcinoma, advancing prostate cancer usually becomes hormone‐refractory. We attempted to establish a new prostate cancer therapy by controlling the malignancy of tumor cells through the induction of differentiation in vitro.

GM‐CSF induces expression of gp91phox and stimulates retinoic acid‐induced p47phox expression in human myeloblastic leukemia cells

Abstract:  All‐trans retinoic acid (ATRA) combined with granulocyte macrophage colony‐stimulating factor (GM‐CSF) synergistically increases superoxide‐generating activity in human myeloblastic leukemia ML‐1 cells. ATRA is known to increase the expression of some NADPH components; however, little is known about the effect of GM‐CSF on the expression of these components. We examined the expression of NADPH oxidase components in ML‐1 cells treated with ATRA, GM‐CSF, or a combination of ATRA and GM‐CSF. Expression of p47phox and gp91phox proteins increased markedly after treatment with both reagents. p47phox expression was increased by ATRA alone, and the expression was increased synergistically by the combination of ATRA with GM‐CSF. gp91phox was increased by ATRA or GM‐CSF alone. The expression of p47phox and gp91phox mRNA underwent similar changes to those seen in protein level. These results indicate that GM‐CSF induces expression of gp91phox and enhances ATRA‐induced p47phox expression. We speculate that the remarkable induction of gp91phox and p47phox protein is associated with an increase in superoxide‐generating activity due to the synergistic effect of ATRA plus GM‐CSF.

Upregulation of p21(WAF1/CIP1) leads to morphologic changes and esterase activity in TPA-mediated differentiation of human prostate cancer cell line TSU-Pr1.

We reported previously that human prostate cancer cell line TSU-Pr1 can differentiate into microglia-like cells by 12-O-tetra-decanoylphorbol-13-acetate (TPA) treatment. In this study, we identified a signal transduction pathway involved in TPA-induced TSU-Pr1 cell differentiation and investigated the mechanism of growth arrest that accompanies this differentiation. TPA-induced differentiation and growth arrest of TSU-Pr1 cells were inhibited by treatment with Protein kinase C (PKC) inhibitor GF109203X and mitogen-activated protein (MAP) kinase inhibitor PD98059. Treatment of TSU-Pr1 cells with TPA for 15 min or longer resulted in translocation of PKCα, PKCγ, and PKCε from cytosolic to membrane fraction. Our results suggest that TPA-induced TSU-Pr1 cell differentiation is associated with activation of MAP kinase and PKCα, PKCγ, and PKCε. The mechanism of growth arrest in TSU-Pr1 cells that underwent TPA-induced differentiation were examined for factors in the signaling pathway downstream of MAP kinase that control the cell cycle. Upregulation of p21WAF1/CIP1 cyclin-dependent kinase inhibitor protein was observed in a manner dependent on PKC or MAP kinase. Moreover, adenovirus-mediated overexpression of recombinant p21WAF1/CIP1 in TSU-Pr1 cells result in growth arrest, morphological change to microglia-like cells, and increased α-naphthyl acetate esterase activity, all of which are associated with cellular differentiation. Thus, our results indicate that p21WAF1/CIP1 mediates TPA-induced growth arrest and differentiation of TSU-Pr1 cells.

Involvement of diverse protein kinase C isoforms in the differentiation of ML-1 human myeloblastic leukemia cells induced by the vitamin D3 analogue KH1060 and the phorbol ester TPA

We reported previously that diverse combination of the vitamin D(3) analogue KH1060 together with 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically induces the differentiation of ML-1 cells into mature macrophages. To investigate the mechanism involved in their interaction, we examined the role of protein kinase C (PKC) in the differentiation of ML-1 cells to mature macrophages. We found that the specific PKC inhibitor GF109203 suppressed the morphological change and the alpha-naphthyl acetate esterase activity induced in ML-1 cells by treatment with KH1060 plus TPA. This treatment increased the translocation of PKC alpha, PKC epsilon, and PKC theta from cytosol to membranes. ML-1 cells treated with KH1060 alone increased translocation of PKC theta, whereas cells treated with TPA alone increased translocation of PKC alpha and PKC epsilon. These data showed that in human myeloblastic leukemia cells, diverse isoforms of PKC, including PKC alpha, epsilon, and theta, participate in the regulation of cell differentiation.

Complex regulation of CDKs and G1 arrest during the granulocytic differentiation of human myeloblastic leukemia ML-1 cells

We previously reported that all-trans retinoic acid (ATRA) and granulocyte-macrophage colony-stimulating factor (GM–CSF) synergistically induced granulocytic differentiation in human myeloblastic leukemia ML-1 cells. The combination of these agents also suppressed DNA-synthesis. In the present study, we investigated the suppression of cyclin dependent kinase (CDK) activities resulting in G1 arrest in differentiated ML-1 cells. We show that treatment of ML-1 cells with ATRA plus GM-CSF results in G1 arrest and suppression of CDK activities. Protein levels of the G1 CDKs were essentially unchanged during this time. However, we observed an increase in CDK2-bound p27 and CDK4-bound p18, and a decrease in CDK6-bound cyclin D3. These results suggest that complex regulation of CDKs play a key role in G1 arrest of ML-1 after treatment with ATRA and GM–CSF. We also showed that an increase in CDK2-bound p27 and CDK4-bound p18 are caused by treatment with ATRA and a decrease in CDK6-bound cyclin D3 is induced synergistically by treatment with both reagents. Furthermore, we propose that the changes in binding of p18 and cyclin D3 to CDKs are due to changes at the protein expression level and that the increase in p27 binding to CDK2 is due to a novel mechanism.

Hypophosphorylation of pRB and repression of cyclin D3 and cdc25A during the granulocytic differentiation of human myeloblastic leukemia ML-1 cells.

Recently we succeeded in inducing synergistic differentiation toward granulocytes in human myeloblastic leukemia ML-1 cells by treatment of ATRA in combination with GM-CSF. To research the mechanism of this differentiation process, we examined expression of cell cycle-related genes that are concerned with cell growth and differentiation. We detected change to the hypophosphorylated form of pRB and down-regulation of cyclin D3 and cdc25A during induced differentiation. Furthermore, these marked alterations were hardly detected in ML-1 cells treated with ATRA or GM-CSF alone. These results suggest that hypophosphorylation of pRB and repression of cyclin D3 and cdc25A are induced synergistically by treatment with ATRA plus GM-CSF in ML-1 cells.