Masato Takimoto

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.

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.

Suppression of Erk activation and in vivo growth in esophageal cancer cells by the dominant negative Ras mutant, N116Y.

Our previous studies demonstrated that introduction of a dominant negative H‐ras mutant, N116Y, inhibits the growth of various types of cancer cells in vitro. In this study, we tested the efficacy of N116Y in blocking the growth of esophageal cancer cells using an adenoviral vector. Infection with N116Y adenovirus, (AdCMV‐N116Y), in which N116Y expression is driven by the cytomegalovirus promoter, significantly reduced the in vitro growth of all esophageal cancer cell lines studied. Esophageal cancer cells that contained wild‐type K‐ras and H‐ras (TE8, SGF3, SGF7) were more sensitive to AdCMV‐N116Y than HEC46 cells that expressed mutant K‐ras protein. Most importantly, direct injection of AdCMV‐N116Y into TE8‐ or SGF3‐induced tumors in nude mice suppressed their growth significantly. To examine the suppressive mechanism of N116Y, cell cycle profile and the activation of extracellular signal‐regulated kinase 2 (Erk2) were examined by flow cytometry and Western blot analysis, respectively. In TE8 cells, progression into S phase was clearly blocked after infection with AdCMV‐N116Y. Infection with AdCMV‐N116Y did not strongly suppress the activation of Erk2 after EGF stimulation in serum‐starved HEC46 cells, whereas it completely suppressed activation in TE8, SGF3 and SGF7 cells. Our observations suggest that N116Y reduces growth of human esophageal cancer cells and suppresses the activation of Erk2; they also indicate that N116Y is a potential candidate gene for human esophageal cancer gene therapy. Int. J. Cancer 78:366–371, 1998.© 1998 Wiley‐Liss, Inc.

Depression of T Cell-Mediated Immunity and Enhancement of Autoantibody Production by Natural Infection with Microorganisms in Spontaneously Hypertensive Rats (SHR)

We studied the effects of breeding conditions on the development of immunological abnormalities in spontaneously hypertensive rats (SHR) with congenital T cell depression. The depression of T cell functions, the production of natural thymocytotoxic autoantibody (NTA), and the development of polyarteritis nodosa were more evident in SHR reared under a conventional (CV) environment than in specific‐pathogen‐free (SPF) SHR bred in a semi‐barrier system. Enhancement of these immunologic abnormalities was also observed by the conventionalization of SPF‐SHR. A high frequency of antibodies to mouse hepatitis virus (MHV), Sendai virus, and Mycoplasma pulmonis was detected in CV rat sera, whereas no antibodies were detected in SPF‐SHR. The experimental infection of Sendai virus induced the enhancement of T cell depression and of NTA production in SPF‐SHR. We interpret these results to mean that the natural infection of microorganisms causes an acceleration of immunologic abnormalities in SHR reared in a CV environment.

Adenovirus-mediated gene therapy for bladder cancer in an orthotopic model using a dominant negative H-ras mutant

It has been suggested that abnormal Ras function is important in the carcinogenesis and progression of bladder cancer. Our aim was to investigate the efficacy of transurethral inoculation of an adenovirus expressing the dominant negative H‐ras mutant N116Y against orthotopically implanted human bladder‐cancer cells in nude mice. We used a replication‐defective adenovirus vector containing the β‐galactosidase gene (AdCMV‐LacZ) as a control and the N116Y gene (AdCMV‐N116Y) as the therapeutic vector under the transcriptional control of the cytomegalovirus promoter. We initially investigated the in vitro growth‐suppressive effects of AdCMV‐N116Y on 2 human bladder‐cancer cell lines, KU‐7 and UMUC‐2. Thereafter, we examined the inhibitory effects of AdCMV‐N116Y on the 2 orthotopically implanted cell lines in nude mice. Intravesically created, orthotopic human bladder cancers were established in female KSN athymic nude mice with 1× 107 cancer cells. Then, 2, 3 and 4 days following implantation, 1 × 109 pfu of AdCMV‐LacZ or AdCMV‐N116Y were administered transurethrally. In vitro growth assays revealed significant growth suppression (>95%) with apoptosis of target cells treated with AdCMV‐N116Y compared to AdCMV‐LacZ. Transurethral inoculation of AdCMV‐N116Y into the bladder brought about a significant reduction in size (73% to 90%) and number (47% to 78%) of orthotopically implanted human bladder tumors compared to AdCMV‐LacZ or PBS. Normal mucosa in nude mice had minor inflammation with the infiltration of mononuclear cells. Our results suggest that gene therapy via transurethral inoculation of AdCMV‐N116Y holds promise for the treatment of human bladder cancer.

Gelsolin gene silencing involving unusual hypersensitivities to dimethylsulfate and KMnO4 in vivo footprinting on its promoter region

We previously reported that gelsolin gene expression is reduced in various tumors. In an effort to gain further insights into the mechanism of gelsolin downregulation in tumors, we examined the in vivo properties of the gelsolin promoter in urinary bladder cancer cell lines. Neither mutation nor hypermethylation was responsible for gene silencing at the promoter. After exposure to trichostatin A (TSA), a histone deacetylase inhibitor, gelsolin promoter activity was markedly enhanced in the cancer cells, not in cells derived from normal tissue. Chromatin immunoprecipitation assays revealed that both histones H3 and H4 were hypoacetylated in the promoter region of the cancer cells, and the accumulation of acetylated histones was detected by TSA treatment. In vivo footprinting analysis revealed the presence of dimethylsulfate (DMS) hypersensitive site in the untranslated region around nucleotide −35 only in the cancer cells but not in cells derived from normal tissue, and analysis of KMnO4 reactive nucleotides showed that the stem loop structure could be formed in vivo of the cancer cells. This novel stem loop structure may play a part in regulating the transcription of the gelsolin gene in the cancer cells. These results suggest that nucleosome accessibility through histone deacetylation and structural changes (DMS hypersensitivity and stem loop structure) in the promoter region form the basis of the mechanism leading to the silencing of gelsolin gene in human bladder cancer.

Targeted Knockdown of the Kinetochore Protein D40/Knl-1 Inhibits Human Cancer in a p53 Status-Independent Manner

The D40 gene encodes a kinetochore protein that plays an essential role in kinetochore formation during mitosis. Short inhibitory RNA against D40, D40 siRNA, has been shown to deplete the D40 protein in the human cancer cell line HeLa, which harbors wild-type p53, and this activity was followed by the significant inhibition of cell growth and induction of apoptotic cell death. The p53-null cancer cell line, PC-3M-luc, is also sensitive to the significant growth inhibition and cell death induced by D40 siRNA. The growth of PC-3M-luc tumors transplanted into nude mice was inhibited by the systemic administration of D40 siRNA and the atelocollagen complex. Furthermore, D40 siRNA significantly inhibited growth and induced apoptotic cell death in a cell line with a gain-of-function (GOF) mutation in p53, MDA-MB231-luc, and also inhibited the growth of tumors transplanted into mice when administered as a D40 siRNA/atelocollagen complex. These results indicated that D40 siRNA induced apoptotic cell death in human cancer cell lines, and inhibited their growth in vitro and in vivo regardless of p53 status. Therefore, D40 siRNA is a potential candidate anti-cancer reagent.

Gelsolin Induces Promonocytic Leukemia Differentiation Accompanied by Upregulation of p21CIP1

Tumor suppressor genes have received much attention for their roles in the development of human malignancies. Gelsolin has been found to be down-regulated in several types of human cancers, including leukemias. It is, however, expressed in macrophages, which are the final differentiation derivatives for the monocytic myeloid lineage, implicating this protein in the differentiation process of such cells. In order to investigate the role of gelsolin in leukaemic cell differentiation, stable clones over-expressing ectopic gelsolin, and a control clone were established from U937 leukaemia cells. Unlike the control cells, both gelsolin-overexpressing clones displayed retarded growth, improved monocytic morphology, increased NADPH and NSE activities, and enhanced surface expression of the β-integrin receptor, CD11b, when compared with the parental U937 cells. Interestingly, RT- PCR and western blot analysis also revealed that gelsolin enhanced p21CIP1 mRNA and protein expression in the overexpressing clones. Moreover, transient transfection with siRNA silencing P21CIP1, but not the control siRNA, resulted in a reduction in monocytic differentiation, accompanied by an increase in proliferation. In conclusion, our work demonstrates that gelsolin, by itself, is capable of inducing monocytic differentiation in U937 leukaemia cells, most probably through p21CIP1 activation.

Suppressive effect of modified dominant negative RAS mutant on human cancer by gene transfer with non-viral vector

N116Y, H-RAS mutant, has dominant negative activity in the RAS function and a suppressive effect on the growth of various types of cancer cells. However, a replication error of N116Y is of potential concern for carcinogenesis in clinical application. To decease the concern, we constructed modified N116Y by deleting the carboxyl terminus, which is necessary for the oncogenic function of Ras. One of the C-terminal deletion mutants of N116Y, N116Y-Cdel2 showed a growth-suppressing effect on various human cancer cell lines in vitro: the cervical cancer cell line HeLa, the pancreatic cancer cell line PCI43, the colon cancer cell lines SW480 and LoVo, and the tongue cancer cell line SAS. In addition, the suppressive effect of N116Y-Cdel2 on LoVo cells was also observed in vivo using a non-viral gene transfer vector, HVJ envelope. Our experiments suggest that the modified N116Y is a potential candidate gene for human cancer gene therapy.

Testis cancer gene D40 expression and its relationship with clinicopathological features in infertile men.

We previously identified a novel human cancer/testis gene, D40, which is dominantly expressed in testicular germ cells, various cancer cell lines and primary human tumors. The expression of D40 mRNA and proteins in various testicular tissues was quantified using the conventional reverse transcription polymerase chain reaction (RT–PCR), real‐time quantitative RT–PCR and western blot analysis. The relationship between levels of D40 expression, serum follicle stimulating hormone (FSH) level and Johnsens score was examined. D40 mRNA expression was observed in the testes of infertile men, except those with Sertoli‐cell‐only syndrome or Klinefelters syndrome. The quantity of D40 mRNA and protein was correlated with Johnsens score and inversely correlated with serum FSH level. The present results show that the expression levels of D40 mRNA and proteins decrease according to the degree of spermatogenesis impairment in male infertile patients.