Yoshihiro Ami

Promoting effects and mechanisms of action of androgen in bladder carcinogenesis in male rats.

OBJECTIVE It has been reported that blocking of testosterone production inhibits bladder carcinogenesis in various animal models. We investigated how testosterone acts on rat bladder carcinogenesis using an antiandrogen, flutamide, and a 5 alpha-reductase inhibitor, finasteride. METHODS Experiment 1: we administered 0.05% BBN [N-butyl-N-(4-hydroxybutyl)nitrosamine] orally to 117 Wistar rats for 10 weeks, divided them into seven groups-control, surgical castration, finasteride (2 mg/kg), luteinizing hormone releasing hormone (LH-RH) agonist (1 mg/kg) flutamide (50 mg/kg), LH-RH agonist plus finasteride, and LH-RH agonist plus flutamide-, and then cystectomized them to investigate the incidence of bladder cancer on week 21; experiment 2: we administered 0.05% BBN to 154 Wistar rats for 7 weeks, divided them into seven groups-control, finasteride 2, 4, and 8 mg/kg, and flutamide 50, 100, and 200 mg/kg-, and then we cystectomized them to investigate the dose-dependent influence on bladder carcinogenesis of these drugs on week 20, and experiment 3: we investigated the presence of androgen receptors in rat and mouse normal bladder mucosa using a monoclonal antibody. RESULTS AND CONCLUSIONS Experiment 1: Surgical castration and LH-RH agonist treatment significantly reduced the occurrence of carcinomas. There was no significant additive effect of coadministered finasteride or flutamide with LH-RH agonist. Finasteride or flutamide monotherapy showed no statistically significant effects on the results of experiment 1 at the doses used. Experiment 2: Flutamide showed a dose-dependent effect on reducing the number of rats with bladder cancer, and at a dosis of 200 mg/kg twice a week, the difference was statistically significant when compared with the control group, whereas finasteride had no statistically significant suppressing effect at any dose. Experiment 3: Mouse and rat bladder urothelium expressed the androgen receptor. Our results indicate that testosterone itself might have a more potent action on bladder carcinogenesis rather than its converting form, 5 alpha-dihydrotestosterone.

Isolation of the poly(ADP-ribose) polymerase-encoding cDNA from Xenopus laevis: phylogenetic conservation of the functional domains

The complete nucleotide (nt) sequence of the Xenopus laevis poly(ADP-ribose) polymerase (PARP)-encoding cDNA was determined. The putative X. laevis PARP protein consists of 1008 amino acids (aa) with a molecular weight of 113 kDa. X. laevis PARP shares 74, 83, 73, 78 and 42% aa sequence homology with the human, bovine, mouse, chicken and Drosophila melanogaster PARPs, respectively. Comparison of the PARP aa sequences among these species showed conservation of two zinc-finger motifs in the DNA-binding domain, and an NAD-binding motif and a Rossmann fold in the catalytic domain. The first Leu of the putative leucine zipper of D. melanogaster PARP is substituted to Lys in X. laevis PARP. All the Glu residues in the leucine zipper are conserved in these six species.

Telomerase Is Upregulated in Irreversible Preneoplastic Lesions during Bladder Carcinogenesis in Rats

Multiple occurrence or recurrence after transurethral resection is an important characteristic of superficial bladder tumors. To study bladder carcinogenesis, we focused on detection of telomerase activation, which was investigated in several human cancers, including bladder tumors. We experimentally examined the telomerase activity during bladder carcinogenesis, especially in precancerous lesions, induced by N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) in rats. Male Wistar rats were given 0.05% BBN in water from the age of 8 weeks to 24 weeks. Subgroups were euthanized at 4, 8, 10, 12, 18, and 24 weeks after BBN administration. Using the stretch PCR method, telomerase activity was semiquantified in exfoliated bladder epithelial cells. In addition, telomere length in each subgroup was measured by southern hybridization for the terminal restriction fragment using a (TTAGGG)4 probe. Statistical analyses were performed using analysis of variance and Fishers PLSD test. Epithelial cells of normal bladder in the control groups and those of diffuse hyperplasia, which was a reversible change at 4 weeks, expressed no telomerase activity. In contrast, telomerase activity significantly increased in the stage after nodular hyperplasia, an irreversible change at 8 weeks, then elevated with carcinogenesis. However, telomere length was still preserved by the 12th week, and was shortened at 18 and 24 weeks. These results suggest that telomerase activation is probably induced independent of telomere shortening during bladder carcinogenesis in the rat, and might be a biological tumor marker of irreversible preneoplastic lesions, which evolve into bladder tumors in the rat.

Prediction of in vitro response to interferon‐α in renal cell carcinoma cell lines

We analyzed the correlation between interferon‐α (IFNα) response and gene expression profiles to predict IFNα sensitivity and identified key molecules regulating the IFNα response in renal cell carcinoma (RCC) cell lines. To classify eight RCC cell lines of the SKRC series into three subgroups according to IFNα sensitivity, that is, sensitive, resistant and intermediate group, responses to IFNα (300–3000 IU/mL) were quantified by WST‐1 assay. Microarray, followed by supervised hierarchical clustering analysis, was applied to selected genes according to IFNα sensitivity. In order to find alteration of expression profiles induced by IFNα, sequential microarray analyses were performed at 3, 6, and 12 h after IFNα treatment of RCC cell lines and mRNA expression level was confirmed using quantitative real time polymerase chain reaction. According to the sequential microarray analysis between IFNα‐sensitive and ‐resistant line, seven genes were selected as candidates for IFNα‐sensitivity‐related genes in RCC cell lines. Among these seven genes, we further developed a model to predict tumor inhibition with four genes, that is, adipose differentiation‐related protein, microphthalmia associated transcription factor, mitochondrial tumor suppressor 1, and troponin T1 using multiple linear regression analysis (coefficient = 0.948, P = 0.0291) and validated the model using other RCC cell lines including six primary cultured RCC cells. The expression levels of the combined selected genes may provide predictive information on the IFNα response in RCC. Furthermore, the IFNα response to RCC might be modulated by regulation of the expression level of these molecules. (Cancer Sci 2007; 98: 529–534)

Low expression of microphthalmia-associated transcription factor, a potential molecular target for interferon-alpha susceptibility, is associated with metastasis in renal cell carcinoma

We previously reported that microarray expression profiling identified several candidate genes in association with interferon‐α (IFN‐α) response in renal cell carcinoma (RCC) cell lines (Cancer Sci 2007; 98: 529). Among them, we focused on microphthalmia‐associated transcription factor (MITF), because its expression profile correlated well with IFN‐α‐response status. In addition, we investigated the clinical significance of the expression level of MITF using surgical specimens. RNA was extracted from 14 RCC cell lines and 65 RCC samples and was used in this study. Transfection of MITF cDNA into IFN‐α‐resistant RCC cell lines resulted in elevation of MITF expression and acquisition of IFN‐α‐sensitivity by quantitative PCR and WST‐8 assay, respectively. Statistical analysis revealed that low MITF mRNA expression in RCC samples was significantly correlated with the presence of metastasis and poor survival of the patient. However, the correlation between MITF expression and IFN‐α response was not obvious in the clinical cases. MITF gene transfection elevated IFN‐α‐sensitivity in RCC cell lines, suggesting that this gene is a target molecule for modulation of the IFN‐α response. Quantification of MITF mRNA expression might be clinically useful to predict metastasis and survival of patients with RCC. (Cancer Sci 2009; 100: 1714–1718)

Cloning of Poly(ADP-ribose) Polymerase cDNA from Lower Eukaryotes

Poly(ADP-ribose) polymerase (EC 2.4.4.30) catalyzes DNA-dependent ADP-ribosylation of nuclear proteins with NAD as a substrate (1-8). Poly(ADP-ribose) polymerase activity was reported from various eukaryotic cells but not from prokaryotic cells (1-8).