Motoi Fukumoto

The distribution of vascular endothelial growth factor-producing cells in clinical radiation necrosis of the brain: pathological consideration of their potential roles.

The cell type and localization of vascular endothelial growth factor (VEGF)-producing cells in human radiation necrosis (RN) are investigated from a histopathological and immunohistochemical standpoint using clinical specimens. Eighteen surgical specimens of symptomatic RN in the brain were retrospectively reviewed. These cases included different original histological tumor types and were treated with different radiation modalities. Histological analyses were performed using hematoxylin and eosin (H&E) staining, and anti-VEGF and anti-hypoxia-inducible factor (HIF)-1α immunohistochemistry. H&E staining showed marked angiogenesis and reactive astrocytosis at the perinecrotic area. The most prominent vasculature in this area was identified as telangiectasis. Immunohistochemistry indicated that HIF-1α was expressed predominantly in the perinecrotic area and that a large majority of VEGF-expressing cells were reactive astrocytes intensively distributed in this area. VEGF produced by the reactive astrocytes localized mainly in the perinecrotic area might be a major cause of both angiogenesis and the subsequent perilesional edema typically found in RN of the brain. The benefits of anti-VEGF antibody (bevacizumab) treatment in RN may be that VEGF secretion from the perinecrotic tissue is inhibited and that surgery would remove this tissue; both of these benefits result in effective reduction of edema associated with RN.

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.

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.

Targeting of tumor endothelial cells combining 2 Gy/day of X-ray with Everolimus is the effective modality for overcoming clinically relevant radioresistant tumors.

Radiotherapy is widely used to treat cancer because it has the advantage of physically and functionally conserving the affected organ. To improve radiotherapy and investigate the molecular mechanisms of cellular radioresistance, we established a clinically relevant radioresistant (CRR) cell line, SAS‐R, from SAS cells. SAS‐R cells continue to proliferate when exposed to fractionated radiation (FR) of 2 Gy/day for more than 30 days in vitro. A xenograft tumor model of SAS‐R was also resistant to 2 Gy/day of X‐rays for 30 days. The density of blood vessels in SAS‐R tumors was higher than in SAS tumors. Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, sensitized microvascular endothelial cells to radiation, but failed to radiosensitize SAS and SAS‐R cells in vitro. Everolimus with FR markedly reduced SAS and SAS‐R tumor volumes. Additionally, the apoptosis of endothelial cells (ECs) increased in SAS‐R tumor tissues when both Everolimus and radiation were administered. Both CD34‐positive and tomato lectin‐positive blood vessel densities in SAS‐R tumor tissues decreased remarkably after the Everolimus and radiation treatment. Everolimus‐induced apoptosis of vascular ECs in response to radiation was also followed by thrombus formation that leads to tumor necrosis. We conclude that FR combined with Everolimus may be an effective modality to overcome radioresistant tumors via targeting tumor ECs.

Guanine nucleotide-binding protein 1 is one of the key molecules contributing to cancer cell radioresistance

Standard fractionated radiotherapy for the treatment of cancer consists of daily irradiation of 2‐Gy X‐rays, 5 days a week for 5–8 weeks. To understand the characteristics of radioresistant cancer cells and to develop more effective radiotherapy, we established a series of novel, clinically relevant radioresistant (CRR) cells that continue to proliferate with 2‐Gy X‐ray exposure every 24 h for more than 30 days in vitro. We studied three human and one murine cell line, and their CRR derivatives. Guanine nucleotide‐binding protein 1 (GBP1) gene expression was higher in all CRR cells than their corresponding parental cells. GBP1 knockdown by siRNA cancelled radioresistance of CRR cells in vitro and in xenotransplanted tumor tissues in nude mice. The clinical relevance of GBP1 was immunohistochemically assessed in 45 cases of head and neck cancer tissues. Patients with GBP1‐positive cancer tended to show poorer response to radiotherapy. We recently reported that low dose long‐term fractionated radiation concentrates cancer stem cells (CSCs). Immunofluorescence staining of GBP1 was stronger in CRR cells than in corresponding parental cells. The frequency of Oct4‐positive CSCs was higher in CRR cells than in parental cells, however, was not as common as GBP1‐positive cells. GBP1‐positive cells were radioresistant, but radioresistant cells were not necessarily CSCs. We concluded that GBP1 overexpression is necessary for the radioresistant phenotype in CRR cells, and that targeting GBP1‐positive cancer cells is a more efficient method in conquering cancer than targeting CSCs.

A comprehensive dose evaluation project concerning animals affected by the Fukushima Daiichi Nuclear Power Plant accident: its set-up and progress

It is not an exaggeration to say that, without nuclear accidents or the analysis of radiation therapy, there is no way in which we are able to quantify radiation effects on humans. Therefore, the livestock abandoned in the ex-evacuation zone and euthanized due to the Fukushima Daiichi Nuclear Power Plant (FNPP) accident are extremely valuable for analyzing the environmental pollution, its biodistribution, the metabolism of radionuclides, dose evaluation and the influence of internal exposure. We, therefore, sought to establish an archive system and to open it to researchers for increasing our understanding of radiation biology and improving protection against radiation. The sample bank of animals affected by the FNPP accident consists of frozen tissue samples, formalin-fixed paraffin-embedded specimens, dose of radionuclides deposited, etc., with individual sampling data.

Analysis of Plasma Protein Concentrations and Enzyme Activities in Cattle within the Ex-Evacuation Zone of the Fukushima Daiichi Nuclear Plant Accident

The effect of the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on humans and the environment is a global concern. We performed biochemical analyses of plasma from 49 Japanese Black cattle that were euthanized in the ex-evacuation zone set within a 20-km radius of FNPP. Among radionuclides attributable to the FNPP accident, germanium gamma-ray spectrometry detected photopeaks only from 134Cs and 137Cs (radiocesium) commonly in the organs and in soil examined. Radioactivity concentration of radiocesium was the highest in skeletal muscles. Assuming that the animal body was composed of only skeletal muscles, the median of internal dose rate from radiocesium was 12.5 μGy/day (ranging from 1.6 to 33.9 μGy/day). The median of external dose rate calculating from the place the cattle were caught was 18.8 μGy/day (6.0–133.4 μGy/day). The median of internal and external (total) dose rate of the individual cattle was 26.9 μGy/day (9.1–155.1 μGy/day). Plasma levels of malondialdehyde and superoxide dismutase activity were positively and glutathione peroxidase activity was negatively correlated with internal dose rate. Plasma alanine transaminase activity and percent activity of lactate dehydrogenase (LDH)-2, LDH-3 and LDH-4 were positively and LDH-1 was negatively correlated with both internal and total dose rate. These suggest that chronic exposure to low-dose rate of ionizing radiation induces slight stress resulting in modified plasma protein and enzyme levels.

TRPV4-dependent induction of a novel mammalian cold-inducible protein SRSF5 as well as CIRP and RBM3

Cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) are two evolutionarily conserved RNA-binding proteins that are structurally related to hnRNPs and upregulated in response to moderately low temperatures in mammalian cells. Although contributions of splicing efficiency, the gene promoters activated upon mild hypothermia and the transcription factor Sp1 to induction of CIRP have been reported, precise mechanisms by which hypothermia and other stresses induce the expression of mammalian cold-inducible proteins (CIPs) are poorly understood. By screening the serine/arginine-rich splicing factors (SRSFs), we report that the transcript and protein levels of SRSF5 were increased in mammalian cells cultured at 32 °C. Expression of SRSF5 as well as CIRP and RBM3 were also induced by DNA damage, hypoxia, cycloheximide and hypotonicity. Immunohistochemical studies demonstrated that SRSF5 was constitutively expressed in male germ cells and the level was decreased in human testicular germ cell tumors. SRSF5 facilitated production of p19 H-RAS, and increased sensitivity to doxorubicin in human U-2 OS cells. Induction of CIPs was dependent on transient receptor potential vanilloid 4 (TRPV4) channel protein, but seemed independent of its ion channel activity. These findings indicate a previously unappreciated role for the TRP protein in linking environmental stress to splicing.

Effect of aging on norepinephrine-related proliferative response in primary cultured periportal and perivenous hepatocytes

Norepinephrine (NE) amplifies the mitogenic effect of EGF in a rat liver through the adrenergic receptor coupled with G protein, Ghα. Ghα is also known as a transglutaminase 2 (TG2), whose cross-linking activity is implicated in hepatocyte growth. Recently, we found that NE-induced amplification of EGF-induced DNA synthesis in hepatocytes obtained from perivenous regions of liver is caused by inhibiting the downregulation of EGF receptor (EGFR) by TG2. In the present study, we investigated the effect of aging on NE-related proliferative response. Hepatocytes were obtained from the liver of 7- and 90-wk-old rats. To examine this in detail, periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated using the digitonin/collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, G protein activity, and TG2 activity were measured. NE slightly potentiated [125I]EGF binding to EGFR, and EGF-induced DNA synthesis in PVH but not in PPH. [3H]NE binding studies indicated that PVH have a greater number of receptors than PPH, and that the number of receptors in both subpopulations increased with aging. NE-induced changes in G protein activity and TG2 activity in 90-wk-old rats were slight compared with 7-wk-old rats. These results suggest that NE results in a slight recovery effect on the age-related decline in EGF-induced DNA synthesis because of incomplete switching of the function from TG2 to Ghα.

A case of hepatocellular carcinoma developed after allogeneic bone marrow transplantation.

To the Editor: Allogeneic bone marrow transplantation (BMT) and peripheral blood stem cell transplantation (PBSCT) have been widely adopted as a curative treatment modality for patients with various hematological malignancies and non-malignant diseases. Bone marrow (BM)-derived cells and peripheral blood stem cells have been noted to have the potential to transdifferentiate or dedifferentiate into various epidermal or endothelial cells. Tran et al. were the first to report a transplantation case where male BM-derived cells could migrate into the cheek of a female and differentiate into epithelial cells. In this study, we attempted to determine whether liver cancer that occurred in a recipient after allogeneic BMT was of recipient origin or donor origin. A 40-year-old man had suffered from acute promyelocytic leukemia and received a BMT from his human leucocyte antigen (HLA)-matched younger brother when he was 22 years old; he had maintained complete remission of leukemia. The patient’s blood type converted from O to AB which was his brother’s blood type. He was thought to be infected with HCV by blood transfusions received before the BMT. Ultrasound examination revealed a solitary tumor in the S7 segment of the liver 18 years after the BMT. The tumor was completely resected and was a moderately differentiated hepatocellular carcinoma (HCC) accompanied by chronic hepatitis (A1/F1) without liver cirrhosis. Lymphocyte infiltrates were not evident in the tumor tissue (Fig. 1a,b). Sequence-specific primer-based polymerase chain reaction (PCR) amplified short tandem repeat (STR) analysis was performed to determine the origin of cells from the liver cancer, peripheral blood leukocytes (PBL), hair follicles, fingernails and buccal swabs of the recipient. DNA extraction was carried out using a QIAamp DNA Blood Mini Kit (QIAGEN Japan, Tokyo, Japan). DNA from nails was extracted using ISOHAIR (Nippon Gene, Tokyo, Japan). STR were PCR amplified using an AmpFlSTR Identifiler Kit (Applied Biosystems, Foster City, CA, USA) and AmpFlSTR Yfiler PCR Amplification Kit (Applied Biosystems). PCR products were analyzed using ABI Prism 310 Genetic Analyzer (Applied Biosystems) and GeneMapper ID (ver.3.2) software (Applied Biosystems). After screening for suitable STR loci which could differentiate between the donor and the recipient we analyzed seven highly polymorphic autosomal unlinked STR loci and a Y chromosomal STR locus (Table 1). The STR profile of PBL, hair follicles and fingernails from the donor was completely matched. The representative result of STR analysis of DNA from the non-tumor part of the recipient liver is shown in Fig. 1c. As well as the peaks from the donor DNA, peaks not observed in the donor DNA were detected, which were apparently from the recipient DNA. These indicate that STR analysis can differentiate between DNA from the donor and that from the recipient. The result of STR analysis in this study is summarized in Table 1. PBL from the recipient were completely converted to donor type while pubic hairs and fingernails were recipient-derived. STR analysis showed that buccal mucosa was the chimera of the recipient and the donor. Neutrophilic leukocytes were observed in the smear specimen from buccal swabs of the recipient (Fig. 1d), indicating that the buccal swabs were not truly chimerical but contaminated with leukocytes. The nontumor part of the liver was chimerical whereas HCC was completely recipient-derived. Histologically leukocyte infiltrates were not obvious in the HCC part, but were observed in the non-tumor part of the liver. Both tumor and non-tumor tissues were considered to be recipient in origin and contamination of the donor’s blood cells resulted in the chimeric profile of the recipient’s liver tissues. From the ratio of each peak area, a contamination ratio of about 50% both in buccal smears and the non-tumor part of the liver was evaluated. The source of leukocyte contamination in buccal smears was later revealed to be dental caries. Three populations of stem cells which can respond to liver tissue renewal or damage, that is, hepatocytes, oval cells and BM-derived cells, were considered. However, the exact significance of BM-derived cells is not fully elucidated. Liver damage is required for the formation of hepatocytes from BM cells. The frequency of BM-derived hepatocytes is reportedly one out of 250 000 hepatocytes, furthermore, BM-derived hepatocytes are the result of cell fusion between the donor’s BM cells and the recipient’s hepatocytes. HCC developed in the recipient was associated with HCV infection. Although HCV NS5A protein promotes chromosome instability and aneuploidy the STR profile examined in HCC was completely identical to the PBL of the recipient. This indicates that STR analysis is reliable for personal identification or kindred study, even from DNA that was prepared from cancerous tissues. HCV infection leads to HCC through hepatitis and cirrhosis. Recently BM-derived stem cells have been considered to play a role in liver regeneration and fibrosis. Mouse experiments revealed that injections of BM-derived stem cells can stimulate liver regeneration during chronic liver injury by enhancing the degradation of liver fibrosis. It would Pathology International 2010; 60: 795–797 doi:10.1111/j.1440-1827.2010.02601.x