The association between the T309G polymorphism of the MDM2 gene and sensitivity to anticancer drug is dependent on the p53 mutational status in cellular models.

BACKGROUND: We investigated, in the panel of 60 human tumour cell lines of the National Cancer Institute (NCI-60), whether the R72P polymorphism of TP53 and the T309G polymorphism of MDM2 were associated to the in vitro cytotoxicity of anticancer agents, extracted from the NCI database. For validation, the same study was performed independently on a second panel of tumour cell lines, JFCR-45. METHODS: Both SNPs were identified in cell DNA using PCR-RFLP techniques confirmed by direct sequencing and by pyrosequencing. For the analysis of the results, the mutational status of p53 was taken into account. RESULTS: In the NCI-60 panel, the TP53 rare-allele frequency was 32% and the MDM2 rare-allele frequency 39%. The MDM2 alleles were distributed according to Hardy-Weinberg equilibrium whereas this was only found, for the TP53 alleles, in p53 non-mutated cell lines. Comparable results were obtained in the JFCR-45 validation set. The TP53 SNP had low impact on anticancer drug cytotoxicity in either panel. In contrast, the MDM2 gene polymorphism had a major impact on anticancer drug cytotoxicity, essentially in p53 non-mutated cell lines. Presence of the rare allele was associated to significantly higher MDM2 protein expression and to increased sensitivity to DNA-interfering drugs. In the JFCR-45 panel, a similar effect of the MDM2 gene polymorphism was observed, but was less dependent on the p53 mutational status. CONCLUSIONS: We hypothesised that cell lines harbouring the MDM2 G allele presented a lower availability of p53 for DNA repair, translating into higher sensitivity to DNA-damaging agents.

Identification of genes that restrict astrocyte differentiation of midgestational neural precursor cells.

During development of the mammalian CNS, neurons and glial cells (astrocytes and oligodendrocytes) are generated from common neural precursor cells (NPCs). However, neurogenesis precedes gliogenesis, which normally commences at later stages of fetal telencephalic development. Astrocyte differentiation of mouse NPCs at embryonic day (E) 14.5 (relatively late gestation) is induced by activation of the transcription factor signal transducer and activator of transcription (STAT) 3, whereas at E11.5 (mid-gestation) NPCs do not differentiate into astrocytes even when stimulated by STAT3-activating cytokines such as leukemia inhibitory factor (LIF). This can be explained in part by the fact that astrocyte-specific gene promoters are highly methylated in NPCs at E11.5, but other mechanisms are also likely to play a role. We therefore sought to identify genes involved in the inhibition of astrocyte differentiation of NPCs at midgestation. We first examined gene expression profiles in E11.5 and E14.5 NPCs, using Affymetrix GeneChip analysis, applying the Percellome method to normalize gene expression level. We then conducted in situ hybridization analysis for selected genes found to be highly expressed in NPCs at midgestation. Among these genes, we found that N-myc and high mobility group AT-hook 2 (Hmga2) were highly expressed in the E11.5 but not the E14.5 ventricular zone of mouse brain, where NPCs reside. Transduction of N-myc and Hmga2 by retroviruses into E14.5 NPCs, which normally differentiate into astrocytes in response to LIF, resulted in suppression of astrocyte differentiation. However, sustained expression of N-myc and Hmga2 in E11.5 NPCs failed to maintain the hypermethylated status of an astrocyte-specific gene promoter. Taken together, our data suggest that astrocyte differentiation of NPCs is regulated not only by DNA methylation but also by genes whose expression is controlled spatio-temporally during brain development.

PTOV1: a novel testosterone-induced atherogenic gene in human aorta.

There are gender differences in the development of atherosclerosis, possibly owing to differences in sex steroid hormone action and/or metabolism. One of the atherogenic effects of testosterone is thought to be androgen receptor (AR)-mediated vascular smooth muscle cell (VSMC) proliferation. However, the detailed mechanism of this effect, particularly the identity of the genes associated with VSMC proliferation, remains largely unknown. Therefore, we first employed microarray analysis and, subsequently, quantitative RT-PCR to analyse RNA expression in AR-positive human VSMCs treated with testosterone in order to detect testosterone-induced genes associated with cell proliferation. We further examined whether the genes identified were involved in cell proliferation using small interfering RNA (siRNA) transfection. Expression of the gene products was then evaluated in human aorta with various degrees of atherosclerosis in order to evaluate the clinical relevance of the findings. Both microarray and quantitative RT-PCR analyses demonstrated marked induction of the human prostate overexpressed protein 1 (PTOV1) gene by testosterone in the cell lines: this gene was recently identified as a novel androgen-induced gene involved in prostate tumour cell proliferation. Inhibition of PTOV1 by transfection of its corresponding siRNA suppressed testosterone-induced cell proliferation. In human aorta, PTOV1 immunoreactivity in the nuclei of neointimal VSMCs was abundantly detected in male aorta with mild atherosclerotic changes compared with female aorta or male aorta with severe atherosclerotic changes. These findings indicate that the PTOV1 gene is androgen-responsive in VSMCs and that it may play an important role in androgen-related atherogenesis in the human aorta, particularly early atherosclerosis in the male aorta, through regulating proliferation of neointimal VSMCs.

The OECD program to validate the rat uterotrophic bioassay to screen compounds for in vivo estrogenic responses: phase 1.

The Organisation for Economic Co-operation and Development has completed the first phase of an international validation program for the rodent uterotrophic bioassay. This uterotrophic bioassay is intended to identify the in vivo activity of compounds that are suspected agonists or antagonists of estrogen. This information could, for example, be used to help prioritize positive compounds for further testing. Using draft protocols, we tested and compared two model systems, the immature female rat and the adult ovariectomized rat. Data from 19 participating laboratories using a high-potency reference agonist, ethinyl estradiol (EE), and an antagonist, ZM 189,154, indicate no substantive performance differences between models. All laboratories and all protocols successfully detected increases in uterine weights using EE in phase 1. These significant uterine weight increases were achieved under a variety of experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). For each protocol, there was generally good agreement among laboratories with regard to the actual EE doses both in producing the first significant increase in uterine weights and achieving the maximum uterine response. Furthermore, the Hill equation appears to model the dose response satisfactorily and indicates general agreement based on calculated effective dose (ED)(10) and ED(50) within and among laboratories. The feasibility of an antagonist assay was also successfully demonstrated. Therefore, both models appear robust, reproducible, and transferable across laboratories for high-potency estrogen agonists such as EE. For the next phase of the OECD validation program, both models will be tested against a battery of weak, partial estrogen agonists.

Transgene expression of thioredoxin (TRX/ADF) protects against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hematotoxicity.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) has a variety of toxic effects on a number of organs, including the hematopoietic system. The importance of TCDD-induced oxidative stress has been evaluated in several target organs. However, its role in hematotoxicity remains poorly understood, although bone marrow is known to produce reactive oxygen species. The aim of this study is to evaluate not only the contribution of oxidative stress to TCDD-induced hematotoxicity but also the protective function of TRX/ADF, a known anti-oxidative stress agent, on the hematotoxicity of TCDD in ADF wild-type (WT) and transgenic (Tg) mice. WT and Tg mice received a single intraperitoneal injection of 20 microg TCDD/kg. One day after the treatment, blood and bone marrow cellularity was measured and bone marrow levels of granulotyce/macrophage colony-forming units were determined in the in vitro colony assay. The expression of human TRX transgene by their bone marrow cells was analyzed by Western blot electrophoresis. Our results showed that overexpression of TRX/ADF protects against TCDD-induced hematotoxicity, indicating that induction of oxidative stress that results in disruption of redox regulation may be an important mechanism in TCDD-induced bone marrow toxicity. Moreover, we detected a significant decrease of AhR mRNA levels in bone marrow cells of Tg mice following TCDD treatment, suggesting a biological role of TRX/ADF in the AhR-mediated pathway through which TCDD induces oxidative stress.

Hemopoietic cell kinetics after intraperitoneal single injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely spread environmental pollutant. Homopoietic system is one of the targets of TCDD in laboratory animals including monkeys. The present study is the hemopoietic cell kinetics in mice, from the severe depression in cellularity of bone marrow and CFU-GM, to their recovery after the intraperitoneal injection of high dosage of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The bone-marrow cellularity and CFU-GM were severely decreased to 37.8% and 48% of the control, respectively until day 1 after exposure to TCDD. They were, however, soon recovered, even overshot the control value. Subsequently, they tended to show decrease and oscillation again to and under the control value. In conclusion, our cell kinetic study has proven the oscillation in bone-marrow cellularity and CFU-GM during the recovery period, of which the observation seems to be useful to extend our understanding in the hematotoxicity of TCDD.

Prevention of the down-regulation of gap junctional intercellular communication by green tea in the liver of mice fed pentachlorophenol.

Much evidence has been documented supporting the hypothesis that the down-regulation of gap junctional intercellular communication (GJIC) is a cellular event underlying the tumor promotion process and that treatment to prevent the down-regulation or to up-regulate GJIC is important in preventing tumor promotion. We explored the potential preventive effects of green tea against the promoting action of pentachlorophenol (PCP) in mouse hepatocarcinogenesis, examining whether drinking green tea prevents the down-regulation of GJIC inhibition in the liver caused by tumorigenic doses of PCP. We used a modified in vivo GJIC assay, the incision loading/dye transfer method. Male B6C3F1 mice were given a green tea infusion for 1 week and then PCP was fed at a dose of 300 or 600 p.p.m. in the diet for the following 2 weeks, along with green tea treatment. A dose-related inhibition of GJIC in the hepatocytes was evident in the mice treated with PCP alone that was associated with a reduction in connexin32 (Cx32) plaques in the plasma membrane and an increase in the cell proliferation index. Drinking green tea significantly protected mice against GJIC inhibition, the reduction in Cx32 and the elevation of the labeling index. These findings suggest that green tea might act as an anti-promoter against PCP-induced mouse hepatocarcinogenesis via its ability to prevent down-regulation of GJIC.

Development of invasive adenocarcinoma in a long-standing diverted ileal J-pouch for ulcerative colitis: report of a case.

We report a case of a 50-year-old male with ulcerative colitis who developed well-differentiated adenocarcinoma in the ileal J-pouch, which had been defunctioning for 18 years. The extension of the carcinoma in the pouch suggested that it had recently appeared in the pouch. Monitoring by endoscopic examination and biopsy or pouch excision seems to be an appropriate action if a pouch is out of the fecal stream.

Significant increase in prostaglandin E-main urinary metabolite by laxative administration: comparison with ulcerative colitis.

OBJECTIVE: To assess the production of prostaglandin E(2), an important chemical mediator in diarrhea induced by laxative administration, a prostaglandin E-main urinary metabolite (7alpha-hydroxy-5,11-diketotetranor-prosta-1,16-dioic acid, PGE-MUM) was measured in healthy volunteers and compared with the values of patients with ulcerative colitis. METHODS: PGE-MUM was determined by a simplified immunoassay of bicyclic PGE-MUM and analyzed for the influence of laxative administration and active/remission phases of ulcerative colitis. RESULTS: Administration of laxatives induced a significant increase in PGE-MUM in healthy volunteers. A significant elevation was also found in the active as compared with the remission phase of ulcerative colitis. The PGE-MUM levels were significantly correlated with our modified Talstad scores, clinical disease activity indices in ulcerative colitis. It was confirmed by time course studies of individual patients that changes in PGE-MUM correlated well with colitis activity. CONCLUSION: Laxative administration induces production of prostaglandin E(2) as one of the chemical mediators, although its production grade is relatively low as compared with ulcerative colitis in the active phase.

[In vivo test for endocrine disruptors].

The Endocrine Disruptor Issue has been brought up widely to the public by a book titled "Our stolen future"(Theo Colborn et al., 1996), in which the reproductive impairment of a variety of wild life was documented in relation to chemical exposures. The fear that the similar adverse effects could have been observed in humans is based on the understandings that estrogens and androgens are highly conserved signaling molecules among species. In addition, the estrogen receptors are known to have abilities to bind a variety of chemicals. Although such chemicals bind weakly to the receptor, it is also known that, at least in some assay systems, high concentration of these chemicals can elicit effects that are quite similar to those of the natural hormones. Thus, such "hermonally active agents, HAAs" can be ligands to the hormone receptors in an organism, and may cause adverse effects, namely the "receptor-mediated toxicity", which is a relatively new concept in the field of toxicology. This new aspects of toxicology is briefly discussed in relation to the reversible/irreversible effects in developing organisms and the shape of dose-response curves, both especially in the low-dose ranges, now often referred to as "low-dose effects".