Kazuyasu Nakaya

Tumor metastasis suppressor nm23H1 regulates Rac1 GTPase by interaction with Tiam1

The putative tumor metastasis suppressor nm23H1 was originally identified in murine melanomas by subtraction cloning. It displays nucleoside diphosphate kinase activity and regulates cellular events, including growth and development. Recently nm23H1 has been reported to also act as a GTPase-activating protein of the Ras-related GTPase Rad. We attempted to determine whether nm23H1 also regulates Rho-family GTPases. Although we were unable to detect a direct association between nm23H1 and Rho-family GTPases, nm23H1 was shown to be associated with a Rac1-specific nucleotide exchange factor, Tiam1, by interaction with its amino-terminal region in extracts from the cells expressing exogenous Tiam1 and from native tissue. Overexpression of nm23H1 inhibited the Tiam1-induced production of GTP-bound Rac1 and activation of c-Jun kinase. On the other hand, forced overexpression of the wild type, but not the kinase-inactivated mutant of nm23H1, converted the GDP-bound forms of Rac1, Cdc42, and RhoA to their GTP-bound forms in vitro by its nucleoside diphosphate kinase activity, but nm23H1 alone apparently did not produce the GTP-bound form of these GTPases in vivo. These results suggest that nm23H1 negatively regulates Tiam1 and inhibits Rac1 activation in vivo. Moreover, adhesion-stimulated membrane ruffles of Rat1 fibroblasts were reduced by overexpression of nm23H1. Based on these observations, we concluded that we had identified a function of nm23H1 as a regulator of Rac1 and that it may be related to the effect of nm23H1 as a tumor metastasis suppressor.

Activation of AP-1 is required for bufalin-induced apoptosis in human leukemia U937 cells

In a previous study, we demonstrated that bufalin caused apoptosis in human leukemia U937 cells by the anomalous activation of mitogen-activated protein kinase (MAPK) via a signaling pathway that included Ras, Raf-1 and MAPK kinase-1. We report here the effect of bufalin on c-Jun N-terminal protein kinase (JNK), a member of the MAPK family, and on the signaling pathway downstream of MAPKs in U937 cells. When U937 cells were treated with 10−8 M bufalin, the activity of JNK1 was markedly elevated 3 h after the start of treatment and remained so for 9 h. This activation of JNK and the induction of apoptosis by bufalin were suppressed by expression of antisense mRNA for MAPK kinase-1. c-Jun was translocated from the cytoplasm to the nucleus after treatment of U937 cells with bufalin. The transcriptional activity of AP-1 was transiently enhanced by the treatment with bufalin and this activation was suppressed by the expression of antisense mRNA for MAPK kinase-1. Both curcumin (1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione), an inhibitor of the biosynthesis of AP-1, and the expression of dominant negative c-Jun inhibited the activation of AP-1 and the induction of apoptosis by bufalin. Expression of a constitutively active mutant form of MAPK kinase-1 induced the activation of AP-1 and subsequent apoptosis in U937 cells. These results suggest that the activation of AP-1 via a MAPK cascade that includes JNK is required for the induction of apoptosis by bufalin in U937 cells.

Bufalin induces apoptosis and influences the expression of apoptosis-related genes in human leukemia cells

A low concentration of bufalin, a component of bufadienoides in the traditional Chinese medicine chansu, was shown previously to induce differentiation of a broad range of human leukemia cell lines. In the present study, we found that bufalin at concentrations of 10(-7) M and higher induced apoptosis in human leukemia cells, such as HL60, ML1, but not in mouse leukemia M1 cells. A mere 15 min pretreatment of HL60 cells with 10(-6) M bufalin, followed by incubation for 15 h without bufalin, caused fragmentation of DNA and a decrease in cell viability, indicating that the signal for induction of apoptosis is triggered rapidly upon treatment with bufalin. Bufalin-induced apoptosis in HL60 cells was inhibited by ZnCl2, an inhibitor of endonuclease, but not by cycloheximide, an inhibitor of protein synthesis. Northern blot analysis revealed that the levels of expression of the c-myc and bcl-2 genes in HL60 cells decreased with time after treatment with bufalin. These results suggest that bufalin induces apoptosis specifically in human leukemia cells by altering the expression of these genes involved in apoptosis.

Apoptosis induced by arsenic trioxide in leukemia U937 cells is dependent on activation of p38, inactivation of ERK and the Ca2+-dependent production of superoxide

The mechanism of the induction of apoptosis by arsenic trioxide (As2O3), which was demonstrated recently to be an effective inducer of apoptosis in patients with leukemia, was examined in detail in human leukemia U937 cells. Upon treatment of U937 cells with 50 μM of As2O3, complete inactivation of the kinases ERK1 and ERK2 was detected within 30 min. p38 was activated within 3 hr, and the maximum activity was detected at 6 hr, when DNA fragmentation remained undetectable. Experiments with transfected cells that expressed constitutively activated MEK1 and a specific inhibitor of p38 also suggested that inactivation of ERKs and activation of p38 might be associated with the induction of apoptosis by As2O3. In contrast to the inactivation of ERKs and the activation of p38, activation of JNK by As2O3 appeared to protect cells against the induction of apoptosis. Treatment of U937 cells with As2O3 also caused the Ca2+‐dependent production of superoxide and intracellular acidification and a decrease in the mitochondrial membrane potential at the early stages of induction of apoptosis by As2O3. These changes preceded the release of cytochrome c from mitochondria and the activation of caspase‐3. It should be possible to exploit the unusual characteristics of the mechanism of induction of apoptosis by As2O3 in U937 cells by making use of synergistic effects of this compound with other inducers of apoptosis.

Cell and tissue distribution and developmental change of neuron specific 14 kDa protein (phosphoneuroprotein 14).

In the present paper, the distribution of a neuron-specific phosphoneuroprotein 14 (PNP 14) in cell and tissue was investigated in detail by the immunoblot method using affinity-purified antibody against this protein. The immunoblot of the supernatant fractions of various tissue homogenates of rat clearly demonstrated that PNP 14 was enormously rich in the brain. The content in rat brain was as much as 0.1% of the homogenate. The immunocytochemical study showed that the protein was localized at nerve endings in the cerebellum. Existence of the protein was also confirmed in cultured neuronal cells from postnatal rat midbrain, but not in glial cells. Examination of subcellular localization of PNP 14 indicates that the protein was present in synaptic plasma membranes and synaptic supernatant fractions, but not in synaptic vesicles. During the development of rat brain, PNP 14 came into existence after birth and its amount linearly increased to a maximum at 21-28 days after birth. The content of the protein then remained at the same level for more than 10 months. We concluded that this protein is neuron specific and supposed that it may be involved in neuronal formation and function.

Tiam1 is involved in the regulation of bufalin-induced apoptosis in human leukemia cells.

Bufalin, a component of the Chinese medicine chansu, induces apoptosis in various lines of human tumor cells, such as leukemia HL60 and U937 cells, by altering the expression of apoptosis-related genes, for example, bcl-2 and c-myc. In this study, we characterized a gene that is involved in bufalin-induced apoptosis by the differential display (DD) technique. The partial nucleotide sequence of one of the differentially expressed clones obtained after treatment with bufalin was identical to that of the human gene for Tiam1. When U937 cells were treated with 10−7 M bufalin, expression of both Tiam1 mRNA and the protein was induced 1 h after the start of the treatment. The increase of Tiam1 mRNA was transient but the level of Tiam1 protein continued to increase at least for 6 h. In addition, the activities of Rac1 and p21-activated kinase (PAK) were also stimulated by bufalin treatment. To evaluate the role of Tiam1 in the apoptotic process, we examined the effects of the expression of sense and antisense RNA for Tiam1 in U937 cells. Apoptosis was strongly induced by bufalin in cells that expressed sense RNA for Tiam1 as compared to apoptosis in control cells treated with bufalin only. Cells expressing antisense RNA for Tiam1 were significantly more resistant than the control bufalin-treated cells to induction of DNA fragmentation in response to bufalin. Moreover, sense transformants had elevated activities of PAK and c-Jun NH2-terminal kinase (JNK). These results suggest that Tiam1 might play a critical role in bufalin-induced apoptosis through the activation of Rac1, PAK, and JNK pathway.

Localization of phosphoneuroprotein 14 (PNP 14) and its mRNA expression in rat brain determined by immunocytochemistry and in situ hybridization.

Distribution and ultrastructural localization of a novel phosphoneuroprotein with a molecular mass of 14 kDa (PNP 14), and expression of its mRNA were studied in the adult rat central nervous system (CNS) by immunocytochemistry and in situ hybridization histochemistry. PNP 14 immunoreactivity was abundant in the molecular layer, present moderately in the granular layer, and rare in the Purkinje cell layer of the cerebellar cortex. No PNP 14 immunoreactivity was detected in the cerebellar medulla. In the forebrain, immunoreactivity was found in the hippocampus, striatum, and throughout the cerebral cortex, especially in layer V. Electron microscopic immunocytochemical observation in the cerebellar cortex revealed many PNP 14-immunoreactive axon terminals making synaptic contact with dendritic processes in both granular and molecular layers. PNP 14 immunoreactivity was present mainly in the cytoplasmic matrix in the presynaptic axon terminals. PNP 14 mRNA was localized in the granular layer of the cerebellar cortex, in the hippocampus, and in the cerebral cortex, suggesting that PNP 14 is synthesized in neurons of the granular layer and then transported to the molecular layer by axonal transport. These morphological findings suggest that PNP 14 is likely to modulate the function of selected CNS synapses.

Bufalin reduces the level of topoisomerase II in human leukemia cells and affects the cytotoxicity of anticancer drugs

When human leukemia HL-60 cells were treated with 10(-7) M bufalin, the amounts of both topoisomerase (topo) II alpha and II beta and the activity of topo II decreased markedly and were almost undetectable 18 h after the start of treatment. The level of topo II mRNA started to decrease immediately after the start of treatment with bufalin, with a subsequent decrease in the amount of topo II alpha protein. These changes preceded the fragmentation of DNA, a typical feature of apoptosis. The results suggest that bufalin caused a marked decrease in the steady-state level of topo II alpha mRNA, which led to a decrease in the amount and activity of the enzyme and to the induction of apoptosis. A reduction in the level of topo II alpha by bufalin was also observed in other lines of human leukemia cells such as ML1 and U937. The results were exploited to potentiate the effects of cisplatin and retinoic acid (RA) on HL-60 cells: pretreatment of HL-60 cells with 10(-7) M bufalin for 6 h increased the inhibitory effects of cisplatin and RA on cell growth and enhanced the induction of cell death.

Activation of protein kinase C by the 14-3-3 proteins homologous with exol protein that stimulates calcium-dependent exocytosis

The 14‐3‐3 proteins are a family of acidic proteins found mainly in the brain and are suggested to have a role in monoamine synthesis based on their ability to activate tyrosine and tryptophan hydroxylases in the presence of type II Ca2+/calmodulin‐dependent protein kinase. Recently, however, it has been demonstrated that a member of the 14‐3‐3 family, termed Exol, stimulates Ca2+‐dependent exocytosis in permeabilized adrenal chromaffin cells, suggesting that this protein family may influence the protein kinase C‐mediated control of Ca2+‐dependent exocytosis. Here we show that the 14‐3‐3 proteins activate protein kinase C at about 2‐fold more than the known level of the activated protein kinase, i.e. the activity of protein kinase C in the presence of Ca2+ and phospholipids. This raises the possibility that the cellular activity of protein kinase C is regulated by diverse members of the 14‐3‐3 family and that the reported ability of Exol to reactivate Ca2+‐dependent exocytosis is based on its stimulatory effect on protein kinase C activity. The 14‐3‐3 family, therefore, appears to be a multifunctional regulator of cell signalling processes mediated by two types of Ca2+‐dependent protein kinase, protein kinase C and type II calmodulin‐dependent protein kinase.

Selective Inhibitory Effect of Bufalin on Growth of Human Tumor Cells in vitro: Association with the Induction of Apoptosis in Leukemia HL‐60 Cells

We found that bufalin, an active principle of the Chinese medicine chansu, has selective inhibitory effects on the growth of various human cancer cells. In order to examine whether the growth‐inhibitory effect of bufalin on human cancer cells is associated with apoptosis, human leukemia cells were treated with bufalin. HL‐60, ML1, and U937 leukemia cells treated with bufalin at 10−8M and above had condensed and fragmented nuclei. Flow cytometric analysis of these cells treated with bufalin showed fragmented DNA smaller than that of the G1 phase. DNA of HL‐60 cells treated with bufalin showed a ladder pattern characteristic of apoptosis, as analyzed by agarose gel electrophoretic analysis. DNA synthesis and topoisomerase II activity of HL‐60 cells were markedly inhibited as the concentration of bufalin was increased. The concentration needed for inducing apoptosis of HL‐60 cells was 10−8M, which is comparable to that of camptothecin, but lower than those of other antitumor drugs such as cisplatin, VP16 and all‐trans retinoic acid. Apoptosis was not observed when human mononuclear and polymorphonuclear cells were treated with 10−6M bufalin for 24 h. These results indicate the association of the growth‐inhibitory effect of bufalin with the induction of apoptosis, at least in HL‐60 cells, and suggest the usefulness of bufalin for differentiation‐apoptosis‐inducing therapy for cancer.