The Tumor Growth Inhibitory Effect of a Standardized Extract of Cultured Lentinula edodes Mycelia Using Patient Derived Xenograft Model.

Patient derived xenograft (PDX) is a powerful tool to confirm pharmacological efficacy in non-clinical studies for the development of various drugs including anti-cancer agents and therapeutic research. A standardized extract of cultured Lentinula edodes mycelia, a product name AHCC® is produced by Amino Up Co., Ltd. (Sapporo, Japan). In this study, we investigated the inhibitory effect of AHCC® on the growth of tumor PDX in Super SCID (severe combined immunodeficiency) mice. Effects of AHCC® and BCG administration on the growth of renal cancer PDX implanted in Super SCID mice were evaluated by PDX growth curve. Tendency for the effects on the growth of renal cancer PDX in Super SCID by administration of AHCC® and BCG before implanting the PDX were demonstrated. The effects of the oral administration of AHCC® on the growth of renal, invasive and non-invasive breast cancer PDX in Super SCID mice were studied. In Super SCID mice transplanted with renal cancer PDX, AHCC® significantly suppressed tumor proliferation from the day 48 to 83 after transplantation. In two types of breast cancer PDX, tendency of the growth inhibitory effects of AHCC® were shown by PDX growth curve. Significant inhibitory effect was found at only one time point for during proliferation in each PDX. Super SCID-PDX model has the potential to be a useful tool to investigate for the effect of functional foods.

Altered Actions of Memantine and NMDA-Induced Currents in a New Grid2-Deleted Mouse Line.

Memantine is a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, and is an approved drug for the treatment of moderate-to-severe Alzheimer's disease. We identified a mouse strain with a naturally occurring mutation and an ataxic phenotype that presents with severe leg cramps. To investigate the phenotypes of these mutant mice, we screened several phenotype-modulating drugs and found that memantine (10 mg/kg) disrupted the sense of balance in the mutants. Moreover, the mutant mice showed an attenuated optokinetic response (OKR) and impaired OKR learning, which was also observed in wild-type mice treated with memantine. Microsatellite analyses indicated that the Grid2 gene-deletion is responsible for these phenotypes. Patch-clamp analysis showed a relatively small change in NMDA-dependent current in cultured granule cells from Grid2 gene-deleted mice, suggesting that GRID2 is important for correct NMDA receptor function. In general, NMDA receptors are activated after the activation of non-NMDA receptors, such as AMPA receptors, and AMPA receptor dysregulation also occurs in Grid2 mutant mice. Indeed, the AMPA treatment enhanced memantine susceptibility in wild-type mice, which was indicated by balance sense and OKR impairments. The present study explores a new role for GRID2 and highlights the adverse effects of memantine in different genetic backgrounds.

p53-Dependent suppression of genome instability in germ cells.

Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in γ-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2(-/-) males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2(-/-) and wild-type fish. By contrast, irradiated p53(-/-) fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2(-/-) fish, but negligible levels in p53(-/-) fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells.

Effects of fission neutrons on human thyroid tissues maintained in SCID mice.

Morphology and function (secretion of thyroid hormone) of human thyroid tissues from Graves' disease patients are well maintained in C57BL/6J-scid mice. Serum level of thyroid hormone was reduced by fission neutrons from the nuclear reactor UTR-KINKI, and changes in thyroid hormone by fission neutrons were bigger than those by low LET radiations, X-rays and (137)Cs gamma-rays, suggesting high relative biological effectiveness (RBE; 6.5) of fission neutrons. Microarray analyses revealed that about 3% of genes showed more than 4-fold change in gene expression in the unexposed thyroid tissues against surgically resected thyroid tissues from the same patient, probably due to the difficult oxygen and nutrient supply shortly after transplantation. Dose-dependent changes in gene expression against unexposed concurrent controls were observed with increasing doses of fission neutrons (0.2-0.6Gy) and (137)Cs gamma-rays (1.0-3.0Gy) and showed high RBE (4.2). Furthermore, there were some specific genes which showed more than 4-fold change in gene expression in all the thyroid tissues exposed to higher doses of radiation, especially neutrons (0.4 and 0.6Gy), but none at lower doses (0.2Gy of neutrons and 1.0 and 2.0Gy of gamma-rays). These genes related to degeneration, regeneration, apoptosis, and transcription, respond specifically and very sensitively to neutron injury in human thyroid tissues. This is the first experimental report that fission neutrons can induce some morphological and functional disorders in human tissues, showing high RBE against gamma-ray exposure. These results are useful to evaluate the risks of fission neutrons and cosmic rays to humans.

Differential radiation sensitivity to morphological, functional and molecular changes of human thyroid tissues and bone marrow cells maintained in SCID mice.

Morphology and function of human organs and tissues are well maintained in the improved SCID (severe combined immunodeficient) mice for a long period (approximately 3 years). To study the radiation-induced damage on human thyroid gland, human thyroid tissues transplanted to SCID mice were consecutively exposed to X-rays or 137Cs gamma-rays at high and low dose rates for approximately 2 years. Consecutive irradiation resulted in the disappearance of follicles and significant decrease of thyroid hormone secretion. Mutations in p53 and c-kit genes were induced significantly in human thyroid tissues from old head and neck cancer patients (av. 56.8 years, 4 males) and a Graves' disease patient (20 years, male) over the dose of 24 Gy (44.7+/-5.9 Gy, mean+/-S.E) and 11 Gy (20.2+/-7.8 Gy), respectively, while mutations were not detected at lower doses nor in unexposed matched controls (p < 0.01). There were significant differences in mutation frequency in the transplanted human thyroid tissues (31 years, female) between high dose rate (1.19 Gy/min; 8 in 20 tissues) and low dose rate (0.00023 Gy/min; 0 in 14 tissues) exposures (p < 0.01). Mutations were not detected in RET, K-ras and beta-catenin genes. Expression analysis by GeneChip indicated that gene expression was also well maintained in the transplanted human thyroid tissues. However, lower doses (1 or 3 Gy) of 137Cs gamma-rays can induce changes in gene expression in the transplanted human thyroid tissues. Furthermore, fatally irradiated SCID mice could survive with human bone marrow cell transplantation. When about half of mouse bone marrows were replaced by human bone marrow cells, the human bone marrow cells showed high sensitivity to gamma-irradiation; 28.0% and 0.45% survival after 0.5 and 2.0 Gy exposures, respectively.

Germ-line mutations at a mouse ESTR (Pc-3) locus and human microsatellite loci.

We examined the use of the mouse Pc-3 ESTR (expanded simple tandem repeat) locus and 72 human microsatellite loci as potentially sensitive biomarkers for mutagenic exposures to germ cells in mice and humans respectively. In the mouse work, we treated male mice with TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; a chemical known to induce congenital anomalies in humans and mice) and, analysed the F(1) fetuses for Pc-3 mutations. Although the incidence of anomalies was higher in the TCDD group, there were no induced mutations. However, respiratory distress syndrome (RDS) was observed in 3 of 7 fetuses born to male mice which were treated with TCDD and which showed abnormal length of Pc-3 allele. In the human studies, the children of Chernobyl liquidators were examined for mutations at a total of 72 (31 autosomal, 1 X-linked and 40 Y-linked) microsatellite loci. This study was prompted by earlier findings of increases in microsatellite mutations in barn swallows and wheat in the highly contaminated areas after the Chernobyl accident. We examined 64 liquidator families (70 children) and 66 control families (70 children). However, no increases in mutation rates were found. The estimated mean dose to the liquidators was about 39 mSv and this might be one possible reason why no increases of mutations could be found.

Distribution of genotype/subtype and mutational spectra of the surface gene of hepatitis B virus circulating in Hanoi, Vietnam.

In order to ascertain the molecular epidemiological features and mutational spectra of hepatitis B virus (HBV) in Hanoi, Vietnam, direct sequencing of the 219-nucleotide fragment of the surface (S) gene of HBV from the sera of 40 patients mostly with chronic hepatitis were carried out. The samples were classified into genotypes by phylogenetic and genotype-specific analysis, and subtypes by the deduced amino acid sequences. The results showed that genotype B with ayw1 was predominant genotype/subtype (63%), followed by genotype C with adr (18%). The quasi-species nature of the HBV in the sera was observed in 24 of 40 samples examined. One sample (HN109) showed mixture of genotypes B and C. Among 26 amino acid substitutions, 16 were the variants and the remainders were mutations. In the "a" determinant region, three mutations with methionine to leucine (L) changes at the 133 amino acid residue were in the first loop and no mutations were in the second loop. A new mutation, threonine to methionine at 126 amino acid residue, was observed in one sample. In conclusion, the analysis of the S gene region of HBV showed that in Hanoi, genotype B with ayw1 was prevalent and the quasi-species nature of HBV was also common.

Microsatellite mutations show no increases in the children of the Chernobyl liquidators.

We performed a study on Belarusian "liquidators", exploring whether increase in the frequencies of germline mutations at microsatellite loci could be found in their progeny. The liquidators, mostly young males, were those involved (during 1986 and 1987) in clean-up operations in the radioactively contaminated area following the Chernobyl nuclear power plant accident in 1986. Many liquidators fathered children during the clean-up period and after the work had been terminated. The numbers of families studied were 64 (liquidators) and 66 (controls). A total of 72 loci (31 autosomal, one X-linked and 40 Y-linked) were used. DNA was isolated from peripheral blood lymphocytes and the microsatellite loci were amplified by the polymerase chain reaction with fluorescence-labelled primers. Mutations were detected as variations in the length of the loci. At the Y-linked loci, the mutation rates (expressed as number of mutations among the total number of loci for the individuals included) are 2.9 x 10(-3) (4/1392) and 2.1 x 10(-3) (3/1458) in the children of exposed and control parents, respectively. This difference is not statistically significant. At the autosomal loci, the corresponding estimates are 5.9 x 10(-3) (11/1862; exposed group) and 8.5 x 10(-3) (18/2108; control). Again, the difference is not significant. The possibility that the Belarusian population might have been unexpectedly exposed to some chemical contaminants in the environment appears unlikely in view of the finding that the spontaneous mutation rates at the same set of loci in several non-Belarusian populations were similar to those in Belarus. The estimated mean radiation dose to the liquidators was small, being about 39 mSv, and this might be one reason why no increases in mutation rates due to radiation could be found.