Noboru Kuzumaki

Human gelsolin prevents apoptosis by inhibiting apoptotic mitochondrial changes via closing VDAC

Gelsolin is a Ca2+-dependent actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility through modulation of the actin network. Gelsolin was also recently suggested to be involved in the regulation of apoptosis: human gelsolin (hGsn) has anti-apoptotic activity, whereas mouse gelsolin (mGsn) exerts either proapoptotic or anti-apoptotic activity depending on different cell types. Here, we studied the basis of anti-apoptotic activity of hGsn. We showed that both endogenous and overexpressed hGsn has anti-apoptotic activity, that depends on its C-terminal half. We also found that hGsn and its C-terminal half but not mGsn could prevent apoptotic mitochondrial changes such as Δψ loss and cytochrome c release in isolated mitochondria to a similar extent as Bcl-xL, indicating that hGsn targets the mitochondria to prevent apoptosis via its C-terminal half. In the same way as anti-apoptotic Bcl-xL, which we recently found to prevent apoptotic mitochondrial changes by binding and closing the voltage-dependent anion channel (VDAC), hGsn and its C-terminal half inhibited the activity of VDAC on liposomes through direct binding in a Ca2+-dependent manner. These results suggest that hGsn inhibits apoptosis by blocking mitochondrial VDAC activity.

Inhibition of apoptosis by the actin-regulatory protein gelsolin

Gelsolin is an actin‐regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin‐regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti‐Fas antibody, C2‐ceramide or dexamethasone, without changing the F–actin morphology or the levels of Fas or Bcl‐2 family proteins. Upon the induction of apoptosis, an increase in CPP32(‐like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro‐CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti‐Fas treatment in the gelsolintransfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(‐like) proteases strongly inhibited Fas‐mediated apoptosis, but only partially suppressed both C2‐ceramide‐ and dexamethasone‐induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(‐like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

Detection of tyrosine hydroxylase mRNA and minimal neuroblastoma cells by the reverse transcription-polymerase chain reaction

To facilitate the diagnosis of bone marrow metastasis in neuroblastoma, we have developed a method of amplifying and detecting the tyrosine hydroxylase (TH) mRNA sequence in bone marrow cells using a combination of reverse transcription and the polymerase chain reaction (RT/PCR). By this method, the sequence of TH was detected clearly in the neuroblastoma tissues of all 6 patients and not detected in the bone marrow cells of any of the 9 negative control children. In a reconstitution experiment, 1 neuroblastoma cell per 100,000 normal bone marrow cells could be detected, thus indicating the great sensitivity of this method. Based on these results, this technique may be of value in the diagnosis and treatment follow-up of bone marrow metastasis of neuroblastoma.

Prognostic significance of the expression of ras oncogene product in non-small cell lung cancer

The clinical significance of ras oncogene expression in non‐small cell lung cancer was evaluated in 116 surgically treated patients. Archival paraffin sections of the tumors were analyzed immunohistochemically using anti‐ras p21 monoclonal antibody (MoAb) rp‐35, and p21 staining was correlated with clinicopathologic parameters and survival. Positive reactions (+ and ++) were observed in 72.5% of the adenocarcinomas and 55.6% of the squamous cell carcinomas studied. The T1 tumors showed a ++ reaction less frequently than T2 and T3 tumors (P < 0.05). Stage I tumors also were less reactive with MoAb rp‐35 than tumors in more advanced stages (P < 0.05). Survival analysis showed that patients with p21‐negative tumors had significantly longer survival times (a 5‐year survival rate of 64.1%) than those with p21 + tumors (38.0%, P < 0.05) or those with p21 ++ tumors (11.5%, P < 0.005). The significant correlation between p21 staining and patient survival was independent of histologic type, stage of disease, tumor or node status, and the resectability of tumors. On Coxs multivariate analysis, p21 staining was a major and independent prognostic determinant of survival. These results suggest that enhanced ras p21 expression may be one of the important biologic and clinical markers indicating the malignant potential of non‐small cell lung cancer.

Gelsolin functions as a metastasis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect

Gelsolin, an actin‐binding protein, is implicated as a critical regulator in cell motility. In addition, we have reported that cellular levels of gelsolin are decreased in various tumor cells, and overexpression of gelsolin by gene transfer suppresses tumorigenicity. We sought to assess the effects of gelsolin overexpression on metastasis and to determine the importance of a carboxyl‐terminus that confers Ca2+ dependency on gelsolin for effects of its overexpression. Expression vectors with cDNA encoding either full‐length wild‐type or His321 mutant form, isolated from a flat revertant of Ras‐transformed cells and a carboxyl‐terminal truncate, C‐del of gelsolin, were transfected into a highly metastatic murine melanoma cell line, B16‐BL6. Expression of introduced cDNA in transfectants was confirmed using Western blotting, 2‐dimensional gel electrophoresis and reverse transcription‐polymerase chain reaction (RT‐PCR). We characterized phenotypes of transfectants, such as growth rate, colony formation in soft agar, cell motility and metastasis formation in vivo. Transfectants expressing the wild‐type, His321 mutant and C‐del gelsolin exhibited reduced growth ability in soft agar. Although expression of integrin β1 or α4 on the cell surface of transfectants was not changed, wild‐type and His321 mutant gelsolin, except for C‐del gelsolin, exhibited retardation of cell spreading, reduced chemotatic migration to fibronectin and suppressed lung colonization in spontaneous metastasis assay. Gelsolin may function as a metastasis suppressor as well as a tumor suppressor gene. The carboxyl‐terminus of gelsolin is important for retardation of cell spreading, reduced chemotasis and metastasis suppression.

siRNA gelsolin knockdown induces epithelial-mesenchymal transition with a cadherin switch in human mammary epithelial cells

Epithelial‐mesenchymal transition (EMT) describes a process occurring during development and oncogenesis by which epithelial cells obtain fibroblast‐like properties and show reduced cell adhesion and increased motility. In this report, we demonstrated typical EMT in human mammary epithelial MCF10A small interfering (si)RNA gelsolin‐knockdown cells. EMT was characterized by fibroblastic morphology, loss of contact inhibition and focus formation in monolayer growth, enhanced motility and invasiveness in vitro, increased actin filaments, overexpression of RAC, activation of both extracellular signal‐regulated kinase and AKT, inactivation of glycogen synthase kinase‐3, conversion of cadherin from the E‐ to N‐type and induction of the transcription factor Snail. These results suggested that gelsolin functions as a switch that controls E‐ and N‐cadherin conversion via Snail, and demonstrated that its knockdown leads to EMT in human mammary epithelial cells and possibly to the development of human mammary tumors.

An improved intravesical model using human bladder cancer cell lines to optimize gene and other therapies

Orthotopic implantation of human bladder cancer cells into immunodeficient mice is an important tool for studying the biology and effects of therapy. Nevertheless, the incidence of tumor implantation and growth by transurethral instillation of the human bladder cancer cells into murine bladders has been low or not reproducible. However, using a modified intravesical technique and the human bladder cancer cell lines, KU-7 and UM-UC-2, we have been able to obtain a high and reproducible incidence of superficial bladder tumors. Furthermore, intravesical administration of the LacZ adenovirus vector resulted in significant β-galactosidase expression in these bladder tumors as well as the normal urothelium, which was associated with the removal of the glycosoaminoglycan layer. Because this modified technique produces a high incidence of superficial human tumor growth and allows the efficacy of gene transfer to be evaluated, it should be a useful model for the study of intravesical gene therapy for human bladder cancer. Cancer Gene Therapy (2000) 7, 1575–1580.

Inhibition of Alzheimer's amyloid-β peptide-induced reduction of mitochondrial membrane potential and neurotoxicity by gelsolin

Amyloid-beta (A beta) peptides play a central role in the development of Alzheimers disease. They are known to induce mitochondrial dysfunction and caspase activation, resulting in apoptosis of neuronal cells. Here we show that human cytoplasmic gelsolin inhibits A beta peptide-induced cell death of neuronally differentiated rat pheochromocytoma (PC-12) cells. We also show that the segment 5 but not 6 of human cytoplasmic gelsolin is the important region responsible for inhibition of A beta-induced cytotoxicity. Mitochondrial dysfunction associated with cell death, membrane potential loss and the release of cytochrome c are all abrogated in the presence of human full-length or segment 5 cytoplasmic gelsolin. Furthermore, RNA interference to reduce expression of endogenous gelsolin in PC-12 cells shows that rat gelsolin act as an inhibitor of A beta cytotoxicity. These results demonstrate that cytoplasmic gelsolin plays a important role in inhibiting Abeta-induced cytotoxicity by inhibiting apoptotic mitochondrial changes. The segment 5 of human cytoplasmic gelsolin is sufficient for the function.

Differential Phospholipase D Activation by Bradykinin and Sphingosine 1-Phosphate in NIH 3T3 Fibroblasts Overexpressing Gelsolin

Gelsolin, an actin-binding protein, shows a strong ability to bind to phosphatidylinositol 4,5-bisphosphate (PIP2). Here we showed in in vitroexperiments that gelsolin inhibited recombinant phospholipase D1 (PLD1) and PLD2 activities but not the oleate-dependent PLD and that this inhibition was not reversed by increasing PIP2concentration. To investigate the role of gelsolin in agonist-mediated PLD activation, we used NIH 3T3 fibroblasts stably transfected with the cDNA for human cytosolic gelsolin. Gelsolin overexpression suppressed bradykinin-induced activation of phospholipase C (PLC) and PLD. On the other hand, sphingosine 1-phosphate (S1P)-induced PLD activation could not be modified by gelsolin overexpression, whereas PLC activation was suppressed. PLD activation by phorbol myristate acetate or Ca2+ ionophore A23187 was not affected by gelsolin overexpression. Stimulation of control cells with either bradykinin or S1P caused translocation of protein kinase C (PKC) to the membranes. Translocation of PKC-α and PKC-β1 but not PKC-ε was reduced in gelsolin-overexpressed cells, whereas phosphorylation of mitogen-activated protein kinase was not changed. S1P-induced PLC activation and mitogen-activated protein kinase phosphorylation were sensitive to pertussis toxin, but PLD response was insensitive to such treatment, suggesting that S1P induced PLD activation via certain G protein distinct from Gi for PLC and mitogen-activated protein kinase pathway. Our results suggest that gelsolin modulates bradykinin-mediated PLD activation via suppression of PLC and PKC activities but did not affect S1P-mediated PLD activation.

Gelsolin suppresses tumorigenicity through inhibiting PKC activation in a human lung cancer cell line, PC10

Gelsolin expression is frequently downregulated in lung cancer and several types of different human cancers. To examine the effects of gelsolin restoration on tumorigenicity, we here stably expressed various levels of gelsolin via gene transfer in lung cancer cells (squamous cell carcinoma line, PC10). We observed the alterations in tumorigenicity in vivo when implanted in nude mice, and the changes in growth properties in vitro. As compared to parental cells and control clones, gelsolin transfectants highly reduced tumorigenicity and repressed cell proliferation. Moreover, we investigated bradykinin-induced responses in gelsolin-overexpressing clones, because agonist-stimulated activation of the phospholipases C (PLC)/protein kinase C (PKC) signal transduction pathway is critical for cell growth and tumorigenicity. Bradykinin promotes phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by PLC and translocation of various PKC isoforms from the cytosolic fraction to the particulate fraction. Bradykinin treatment did not increase inositoltriphosphate (IP3) production and induce the membrane fractions of PKCα and PKCγ in gelsolin tranfectants, while it induced PIP2 hydrolysis and increased the fractions in parental and control clones. These results suggest that gelsolin suppressed the activation of PKCs involved in phospholipid signalling pathways, inhibiting cell proliferation and tumorigenicity.