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Biomedical subjects

Akira Horii

Publications and source records attributed to Akira Horii.

9 recordsLinked to original sources
PubMed2003

APAF-1-ALT, a novel alternative splicing form of APAF-1, potentially causes impeded ability of undergoing DNA damage-induced apoptosis in the LNCaP human prostate cancer cell line.

We have found a novel alternative splicing product of the apoptotic protease activating factor 1 (APAF-1), termed APAF-1-ALT, in the LNCaP human prostate cancer cell line. APAF-1-ALT harbors the caspase recruitment domain and an incomplete CED-4 like/ATPase domain, but lacks the WD-40 repeat units. The LNCaP cell expressed the full-length APAF-1 weakly and APAF-1-ALT rather abundantly, especially after mycoplasma infection. LNCaP underwent a retarded DNA damage-induced apoptosis, which was independent of caspase 9 activity. APAF-1-ALT functioned less effectively in inducing apoptosis than did APAF-1-XL, the full-length APAF-1, in transient transfection. Moreover, APAF-1-ALT interfered with APAF-1-XL's ability to induce apoptosis in transient double transfection experiment. These results indicate that APAF-1-ALT is a molecule that is deficient and impeded for mediating apoptosis and that it may contribute to the resistance to DNA damage-induced treatment observed in LNCaP.

Actins↗
PubMed2003

A yeast two-hybrid assay provides a simple way to evaluate the vast majority of hMLH1 germ-line mutations.

Germ-line mutations in the hMLH1 gene are the most frequent cause of hereditary nonpolyposis colorectal cancer and are characterized by missense mutations at high frequency. We found a yeast two-hybrid assay to be an extremely useful and simple tool for evaluating the biological significance of such hMLH1 germ-line missense mutations; 78% (18 of 23) of the missense ones can be recognized as causative for nonpolyposis colorectal cancer. In addition, two of five variants not recognized as causative were thought to be rare polymorphisms. However, we could not detect any differences between wild-type hMLH1 and any of the nine already known polymorphisms causing amino acid alterations. Additional analysis demonstrated that the two-hybrid assay not only detected the dysfunctions at the COOH terminus of the hMLH1 protein necessary for the interaction with associated proteins but also detected a conformational change at the NH(2) terminus carrying ATPase activity. Thus, this method provides a simple and reliable system for accurate diagnosis of hMLH1 alterations.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Potential tumor suppressive pathway involving DUSP6/MKP-3 in pancreatic cancer.

We previously found frequent loss of heterozygosity at 12q21 and 12q22-q23.1 in primary pancreatic cancers, and the DUSP6/MKP-3 gene residing in this region at 12q22 lost its expression in the great majority of pancreatic cancer cell lines. The DUSP6/MKP-3 protein is a dual-specificity phosphatase that dephosphorylates the active form of ERK, making a feedback loop to control ERK activity. Gain-of-function mutations of KRAS2 occur in the great majority of pancreatic cancer cells, and loss of expression of DUSP6/MKP-3 may synergistically promote constitutive activation of ERK and uncontrolled cell growth. To study loss of the feedback pathway and its impact on pancreatic cancer cell growth, we first investigated the expression of DUSP6/MKP-3 in primary pancreatic cancer tissues immunohistochemically; we found up-regulation in mildly as well as severely dysplastic/in situ carcinoma cells and down-regulation in invasive carcinoma, especially in the poorly differentiated type. Adenovirus-mediated reintroduction of DUSP6/MKP-3 into cultured pancreatic cancer cells induced strong expression of recombinant DUSP6/MKP-3 and reduction of phosphorylated ERK in a dose-dependent manner based on the multiplicity of infection and resulted in suppression of cell growth. Moreover, analyses by flow cytometry and immunocytochemistry revealed that the exogenous expression of DUSP6/MKP-3 induced apoptosis. These results show that DUSP6 exerts apparent tumor-suppressive effects in vitro and suggest that DUSP6 is a strong candidate tumor suppressor gene at 12q22 locus.

Adenoviridae↗
PubMed2002

Suppression of the tumorigenic phenotype by chromosome 18 transfer into pancreatic cancer cell lines.

A number of lines of evidence have suggested that the long arm of chromosome 18 apart from SMAD4 may carry a tumor-suppressor gene(s) that plays a role in the early stage of pancreatic ductal carcinogenesis. Thus, adenovirus-mediated introduction of SMAD4 does not suppress in vitro growth in cells with completely inactivated SMAD4, and frequent loss of 18q at the SMAD4 locus is observed in pancreatic cancers but no abnormalities of the normal SMAD4 homolog have been detected. In this study, we introduced a normal copy of chromosome 18 into some pancreatic ductal carcinoma cells with and without a complete inactivation of SMAD4. Both anchorage-dependent and -independent proliferation as well as invasiveness were significantly suppressed in the hybrid clones compared with that of their parental cells. Moreover, significant suppression of tumorigenesis was observed after inoculation in nude mice, irrespective of the SMAD4 status. Our present study provides the first functional evidence of the existence of an additional tumor-suppressor gene(s), other than SMAD4 and DCC, that is responsible for the pathogenesis in the early stage of pancreatic ductal carcinogenesis.

Animals↗
PubMed2002

A microarray-based method for detecting methylated loci.

CpG island DNA methylation plays an important role in regulating gene expression in development and carcinogenesis. We developed a new microarray-based method called methylation amplification DNA chip (MAD) for detecting differences in methylation. In this method, only methylated CpG islands from the two samples that we wanted to compare were amplified and used for hybridization. The resource material for the microarray was derived from the methylated DNA library of the sample in which we wanted to detect hypermethylation. Choosing the methylated DNA library as the resource material of the microarray increased the percentage of DNA fragments derived from hypermethylated loci on the microarray.

Animals↗
PubMed2002

A novel target gene, SKP2, within the 5p13 amplicon that is frequently detected in small cell lung cancers.

We investigated DNA copy-number aberrations in 22 cell lines derived from small cell lung cancers (SCLCs) using comparative genomic hybridization. A minimal common region at 5p13, within the 5p11-p13 amplicon that was most frequently involved, harbored the CDH6, PC4, and SKP2 genes. These three genes showed amplification and consequent overexpression in the SCLC cell lines. SKP2 positively regulates progression of cell cycle by targeting several regulators, such as the cell-cycle inhibitor p27(KIP1), for ubiquitin-mediated degradation. SKP2 was amplified in 7 (44%) of 16 primary SCLC tumors, and consequently overexpressed in 10 (83%) of the 12 of those tumors we examined. Expression levels of SKP2 protein were cell cycle-dependent in SCLC cells as well as in normal cells, and were correlated with the DNA copy-number of the gene. There was an inverse correlation between the expression of SKP2 and p27(KIP1) proteins. Down-regulation of SKP2 using an anti-sense oligonucleotide remarkably suppressed the growth of SCLC cells. Our results indicate that SKP2 is likely to be a target of the 5p13 amplification and to play an important role in the growth of SCLC cells.

Carcinoma, Small Cell↗
PubMed2002

Gene therapy for pancreatic cancer targeting the genomic alterations of tumor suppressor genes using replication-selective oncolytic adenovirus.

In order to develop an effective therapeutic intervention for patients with pancreatic cancer, we examined the genetic alternations of pancreatic cancer. Based on these results, we are developing a new gene therapy targeting the genetic character of pancreatic cancer using mutant adenoviruses selectively replication-competent in tumor cells. Loss of heterozygosity (LOH) of 30% or more were observed on chromosome arms 17p (47%), 9p (45%), 18q (43%), 12q (34%), and 6q (30%). LOH of 12q, 17p, and 18q showed the significant association with poor prognosis. These data strongly suggest that mutation of the putative suppressor genes, TP53 and SMAD4 play significant roles in the disease progression. Based on this rationale, we are developing a new gene therapy targeting tumors without normal TP53 function. E1B-55kDa-deleted adenovirus (AxE1AdB) can selectively replicate in TP53-deficient human tumor cells but not cells with functional TP53. We evaluated the therapeutic effect of this AxE1AdB on pancreatic cancer without normal TP53 function. The growth of human pancreatic tumor in SCID mice model was markedly inhibited by the consecutive injection of AxE1AdB. Furthermore, AxE1AdB is not only the strong weapon but also useful carrier of genes possessing anti-tumor activities as a virus vector specific to tumors without normal TP53 function. It was reported that uracil phosphoribosyl transferase (UPRT) overcomes 5FU resistance. UPRT catalyzes the synthesis of 5-fluorouridine monophosphate (FUMP) from Uracil and phosphoribosylpyrophosphate (PRPP). The antitumor effect of 5FU is enhanced by augmenting 5-fluorodeoxyuridine monophosphate (FdUMP) converted from FUMP, which inhibits Thymidylate Synthetase (TS). The therapeutic advantage of restricted replication competent adenovirus that expresses UPRT (AxE1AdB-UPRT) was evaluatedin an intra-peritoneal disseminated tumor model. To study the anti-tumor effect of AxE1AdB-UPRT/5FU, mice with disseminated AsPC-1 tumors were administered the adenovirus, followed by the 5FU treatment. It was shown that the treatment with AxE1AdB-UPRT/5FU caused a dramatic reduction of the disseminated tumor burden without toxicity in normal tissues. These results revealed thatthe AxE1AdB-UPRT/5FU system is a promising tool for intraperitoneal disseminated pancreatic cancer.

Adenoviridae↗
PubMedDate not supplied

Loss of heterozygosity on chromosome 16p and 18q in anaplastic thyroid carcinoma.

The aim of this study was to clarify the genetic alterations in anaplastic transformation of the thyroid cancer. A total of 17 thyroid cancers including 7 anaplastic and 10 papillary cancers were analyzed for loss of heterozygosity (LOH) on chromosome 16p and 18q. All the samples from anaplastic cancer showed LOH at one or more loci out of ten markers on 16p, and only two showed one LOH at two loci out of five markers on 18q. No LOH was found on either 16p or 18q in papillary cancers. D16S423, D16S418 and D16s406 on 16p13.3 were the most frequently deleted loci in anaplastic cancers, and the region around these may harbor the putative tumor suppressor gene related to anaplastic transformation of thyroid cancer.

Carcinoma↗