Tomokazu Fukuda

Effects of bisphenol A exposure on the proliferation and senescence of normal human mammary epithelial cells

The carcinogenic activity of bisphenol A (BPA) is responsible for stimulating growth in estrogen-dependent breast cancer tissues, cell lines and rodent studies. However, it is not fully understood how this compound promotes mammary carcinogenesis. In our study, we examined the effect of BPA on cellular proliferation and senescence in human mammary epithelial cells (HMEC). Exposure to BPA for 1 week at the early stage at passage 8 increased the proliferation and sphere size of HMEC at the later stage up to passage 16, suggesting that BPA has the capability to modulate cell growth in breast epithelial cells. Interestingly, the number of human heterochromatin protein-1γ positive cells, which is a marker of senescence, was also increased among BPA-treated cells. Consistent with these findings, the protein levels of both p16 and cyclin E, which are known to induce cellular senescence and promote proliferation, respectively, were increased in BPA-exposed HMEC. Furthermore, DNA methylation levels of genes related to development of most or all tumor types, such as BRCA1, CCNA1, CDKN2A (p16), THBS1, TNFRSF10C and TNFRSF10D, were increased in BPA-exposed HMEC. Our findings in the HMEC model suggested that the genetic and epigenetic alterations by BPA might damage HMEC function and result in complex activities related to cell proliferation and senescence, playing a role in mammary carcinogenesis.

BMP type I receptor inhibition reduces heterotopic ossification

Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2). Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I receptor kinases, LDN-193189 (ref. 6), inhibits activation of the BMP signaling effectors SMAD1, SMAD5 and SMAD8 in tissues expressing caALK2 induced by adenovirus specifying Cre (Ad.Cre). This treatment resulted in a reduction in ectopic ossification and functional impairment. In contrast to localized induction of caALK2 by Ad.Cre (which entails inflammation), global postnatal expression of caALK2 (induced without the use of Ad.Cre and thus without inflammation) does not lead to ectopic ossification. However, if in this context an inflammatory stimulus was provided with a control adenovirus, ectopic bone formation was induced. Like LDN-193189, corticosteroid inhibits ossification in Ad.Cre-injected mutant mice, suggesting caALK2 expression and an inflammatory milieu are both required for the development of ectopic ossification in this model. These results support the role of dysregulated ALK2 kinase activity in the pathogenesis of FOP and suggest that small molecule inhibition of BMP type I receptor activity may be useful in treating FOP and heterotopic ossification syndromes associated with excessive BMP signaling.

Distribution of Artificial Radionuclides in Abandoned Cattle in the Evacuation Zone of the Fukushima Daiichi Nuclear Power Plant

The Fukushima Daiichi Nuclear Power Plant (FNPP) accident released large amounts of radioactive substances into the environment. In order to provide basic information for biokinetics of radionuclides and for dose assessment of internal exposure brought by the FNPP accident, we determined the activity concentration of radionuclides in the organs of 79 cattle within a 20-km radius around the FNPP. In all the specimens examined, deposition of Cesium-134 (134Cs, half-life: 2.065 y) and 137Cs (30.07 y) was observed. Furthermore, organ-specific deposition of radionuclides with relatively short half-lives was detected, such as silver-110m (110mAg, 249.8 d) in the liver and tellurium-129m (129mTe, 33.6 d) in the kidney. Regression analysis showed a linear correlation between the radiocesium activity concentration in whole peripheral blood (PB) and that in each organ. The resulting slopes were organ dependent with the maximum value of 21.3 being obtained for skeletal muscles (R2 = 0.83, standard error (SE) = 0.76). Thus, the activity concentration of 134 Cs and 137Cs in an organ can be estimated from that in PB. The level of radioactive cesium in the organs of fetus and infants were 1.19-fold (R2 = 0.62, SE = 0.12), and 1.51-fold (R2 = 0.70, SE = 0.09) higher than that of the corresponding maternal organ, respectively. Furthermore, radiocesium activity concentration in organs was found to be dependent on the feeding conditions and the geographic location of the cattle. This study is the first to reveal the detailed systemic distribution of radionuclides in cattle attributed to the FNPP accident.

Chemopreventive efficacy of piroxicam administered alone or in combination with lycopene and β-carotene on the development of rat urinary bladder carcinoma after N-butyl-N-(4-hydroxybutyl)nitrosamine treatment

The effects of the non‐steroidal anti‐inflammatory drug (NSAID) piroxicam and the carotenoids lycopene and β‐carotcnc, alone or in combination, on the development of rat superficial urinary bladder carcinomas induced by N‐butyl‐N‐(4‐hydroxybutyI)nitrosamine (BBN) were studied. Male Fischer 344 rats, 6 weeks old, were given 0.05% BBN in the drinking water for 8 weeks followed by administration of piroxicam (0.0075% in the diet), lycopene (0.0025% in the drinking water) and/or β‐carotene (0.0025% in the drinking water) for 12 weeks, then killed for histological analysis of urinary bladder lesions. Cell proliferation potential was analyzed by immunohistochemical staining of the proliferative cell nuclear antigen (PCNA). Piroxicam alone, piroxicam+lycopene, and piroxicam +lycopene+β‐carotene all significantly decreased the incidences and numbers of transitional cell carcinomas (TCCs), but the combination of piroxicam with carotenoids did not result in a clear improvement in the preventive potential of piroxicam. Piroxicam +β‐carotene also caused a significant reduction and lycopene alone a slight but not significant reduction in the number of TCCs. In contrast, β‐carotene alone and lycopene +β‐carotene were without inhibitory influence on any of the lesion categories examined, and the latter significantly increased the proportion of high‐grade TCCs. Nevertheless, all of the chemopreventive agents, either alone or in combination, significantly decreased the TCC PCNA index, the effect extending to the surrounding epithelium in the piroxicam 4‐lycopene and piroxicam+lycopene+β‐carotene groups. These results indicate that the NSAID piroxicam may he a more effective chemopreventive agent than lycopene and β‐carotene for superficial urinary bladder carcinogenesis.

Augmentation of smad‐dependent BMP signaling in neural crest cells causes craniosynostosis in mice

Craniosynostosis describes conditions in which one or more sutures of the infant skull are prematurely fused, resulting in facial deformity and delayed brain development. Approximately 20% of human craniosynostoses are thought to result from gene mutations altering growth factor signaling; however, the molecular mechanisms by which these mutations cause craniosynostosis are incompletely characterized, and the causative genes for diverse types of syndromic craniosynostosis have yet to be identified. Here, we show that enhanced bone morphogenetic protein (BMP) signaling through the BMP type IA receptor (BMPR1A) in cranial neural crest cells, but not in osteoblasts, causes premature suture fusion in mice. In support of a requirement for precisely regulated BMP signaling, this defect was rescued on a Bmpr1a haploinsufficient background, with corresponding normalization of Smad phosphorylation. Moreover, in vivo treatment with LDN‐193189, a selective chemical inhibitor of BMP type I receptor kinases, resulted in partial rescue of craniosynostosis. Enhanced signaling of the fibroblast growth factor (FGF) pathway, which has been implicated in craniosynostosis, was observed in both mutant and rescued mice, suggesting that augmentation of FGF signaling is not the sole cause of premature fusion found in this model. The finding that relatively modest augmentation of Smad‐dependent BMP signaling leads to premature cranial suture fusion suggests an important contribution of dysregulated BMP signaling to syndromic craniosynostoses and potential strategies for early intervention.

Frequent mutations of Ki‐ras but no mutations of Ha‐ras and p53 in lung lesions induced by N‐nitrosobis(2‐hydroxypropyl)amine in rats

Point mutations of the Ki‐ras and p53 genes in rat lung lesions induced by N‐nitrosobis(2‐hydroxypropyl)amine (BHP) were investigated by polymerase chain reaction‐single‐strand conformation polymorphism analysis followed by direct sequencing using paraffin‐embedded tissues. Male Wistar rats 6 wk old were given 2000 ppm BHP in drinking water for 15 wk. Another group was given drinking water without BHP. The rats were killed 20–27 wk after the beginning of the experiment. Lung adenomatous and squamous lesions, including carcinomas, were induced. The frequencies of Ki‐ras mutations were 40% (six of 15) in alveolar hyperplasias, 36% (five of 14) in adenomas, 72% (18 of 25) in adenocarcinomas, 20% (three of 15) in squamous metaplasias, 50% (three of six) in squamous cell carcinomas, and 50% (five of 10) in adenosquamous carcinomas. The mutations were all G → A transitions at the second position of codon 12; no other mutations were detected. However, Ha‐ras mutations in exons 1 and 2 and p53 mutations in exons 5, 6, and 7 were not detected in adenocarcinomas and squamous cell carcinomas. These results indicate that Ki‐ras mutation is an early genetic event in some adenomatous and squamous lung carcinogenesis and that Ki‐ras mutations can cause benign lesions to convert to malignant lesions. The results also show that Ha‐ras and p53 mutations are not involved in rat lung carcinogenesis induced by BHP.

Conditional Transgenic System for Mouse Aurora A Kinase: Degradation by the Ubiquitin Proteasome Pathway Controls the Level of the Transgenic Protein

ABSTRACT Aurora A is a mitotic kinase that localizes to centrosomes. Expression of this protein is normally limited to the mitotic stage (G2-M) of the cell cycle, whereas human cancer cells frequently exhibit overexpression of Aurora A protein regardless of the cell cycle stage. In the present study, Aurora A transgenic mouse lines were generated with a new conditional expression system (cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter-Z-enhanced green fluorescent protein) in order to analyze the function of this protein. Although transcripts for Aurora A were elevated in multiple organs of the transgenic mice, the corresponding protein was not detected in extracts analyzed by immunoblotting. The treatment of transgenic-derived embryonic fibroblasts (MEF) with proteasome inhibitors markedly increased the protein level of transgenic Aurora A, indicating that the transgenic Aurora A protein is readily degraded in normal mouse tissues. Under the exponential growth conditions of MEF cells, transgenic Aurora A was detected within the mitotic stage of the cell cycle and localized to centrosomes. In contrast, the marker of the transgenic promoter (enhanced green fluorescent protein) was continuously expressed throughout the cell cycle, indicating the constitutive transcription of transgenic mRNA. These results indicate that transgenic Aurora A is protected from degradation within G2-M but is immediately degraded after translation in the G1-S stage of the cell cycle. The findings obtained with this transgenic model and derived cells support that the transition from protection to degradation by the ubiquitin proteasome system at the end of mitosis is an important step in controlling the level of Aurora A protein during the cell cycle.

Effects of radioactive caesium on bull testes after the Fukushima nuclear plant accident

We aimed to investigate the effect of chronic radiation exposure associated with the Fukushima Daiichi Nuclear Plant accident on the testis from 2 bulls. Estimated dose of internal exposure in one bull was 0.7–1.2 mGy (134Cs) and 0.4–0.6 mGy (137Cs) and external exposure was 2.0 mGy (134Cs) and 0.8 mGy (137Cs) (196 days). Internal dose in the other was 3.2–6.1 mGy (134Cs) and 1.8–3.4 mGy (137Cs) and external dose was 1.3 mGy (134Cs) and 0.6 mGy (137Cs) (315 days). Sperm morphology and spermatogenesis were within normal ranges. 134, 137Cs radioactivity was detected but Cs was not detectable in the testis by electron probe microanalysis. Thus, adverse radiation-induced effects were not observed in bull testes following chronic exposure to the above levels of radiation for up to 10 months. Since we could analyse a limited number of testes, further investigation on the effects of ionizing radiation on spermatogenesis should be extended to more animals.

Multifocal micronodular pneumocyte hyperplasia and lymphangioleiomyomatosis in tuberous sclerosis with a TSC2 gene.

A 45-year-old woman with a long-standing diagnosis of tuberous sclerosis (TSC) is presented. She has multifocal micronodular pneumocyte hyperplasia (MMPH) and lymphangioleiomyomatosis (LAM) of the lung, together with the detection of TSC2 gene mutation. During surgery for spontaneous pneumothorax, an open-lung biopsy was performed. Micronodules were well defined, measuring approximately 4 mm in diameter. These MMPHs were histologically composed of papillary proliferation of Type II pneumocytes, with positive immunoreactivity of keratin and surfactant apoprotein. The cystlike spaces, with dilatation and destruction of air spaces, were diffusely formed, and the walls were composed of the spindle cells. Such LAM showed positive immunoreactivity for HMB-45 (a monoclonal antibody specific for human melanoma) and tuberin (the gene product of TSC2). On germline mutation analysis using leukocytes of the present patient, a TSC2 gene mutation was confirmed as a deletion of G (or g) on Exon 9 by polymerase chain reaction–single-strand conformational polymorphism. However, no mutation was detected in her son. With microdissection analysis using paraffin-embedding lung tissues, LOH of the TSC2 gene preliminarily was detected in a LAM lesion but not in MMPH. It is suggested that MMPH, in addition to LAM, could be another pulmonary lesion in TSC patients and that the detection of TSC2 and/or TSC1 gene could essentially be useful for the pathogenesis of MMPH and LAM in TSC patients.

A Novel Gene “Niban” Upregulated in Renal Carcinogenesis: Cloning by the cDNA-amplified Fragment Length Polymorphism Approach

A modified AFLP (amplified fragment length polymorphism) method was employed to isolate genes differentially expressed in renal carcinogenesis of Tsc2 gene mutant (Eker) rats. One gene, selected for further investigation, was named “Niban” (“second” in Japanese), because it is the second new gene to be found after Erc (expressed in renal carcinoma) in our laboratory. Importantly, “Niban” is well expressed even in small primary rat Eker renal tumors, more than in progressed cell lines, and is also expressed in human renal carcinoma cells, but not in normal human or rat kidneys. Chromosome assignment was to RNO 13 in the rat, and HSA 1. This “Niban” gene is a candidate as a marker for renal tumor, especially early‐stage renal carcinogenesis.