Differential expression patterns of Wnt and beta-catenin/TCF target genes in the uterus of immature female rats exposed to 17alpha-ethynyl estradiol.

To characterize the effects of an estrogen receptor (ER) agonist on the gene expressions in the uterus, immature female rats were administered once orally with 17alpha-ethynyl estradiol (EE, 3 mug/kg), a potent ER agonist. We focused on four categories of sex steroid hormone receptor genes: well-known estrogen target genes, Wnt genes, and beta-catenin/T-cell factor (TCF) target genes. ERalpha, ERbeta, progesterone receptor, and androgen receptor mRNAs were all downregulated at 24 and/or 48 h after EE administration. Complement C3 and insulin-like growth factor 1 mRNAs were markedly induced after EE administration. Although the time courses of Wnt4, Wnt5a, and Wnt7a mRNA status varied until 12 h after EE administration, all of them were simultaneously downregulated at 24 and 48 h. The remarkable downregulation of Wnt7a mRNA in response to EE was considered to be important to understand the various uterine phenomena affected by ER agonists. In the beta-catenin/TCF target genes, the downregulation of anti-Mullerian hormone type 2 receptor and bone morphogenetic protein 4 mRNA after EE administration appeared to be closely related to the downregulation of Wnt7a. The upregulation of cyclin D1 and follistatin mRNA at the early phase after EE administration was considered to have been affected by the upregulation of Wnt4. These results indicate that an ER agonist influences not only the mRNA expression of sex steroid hormone receptor genes and well-known estrogen target genes but also Wnt genes (Wnt4, Wnt5a, Wnt7a) and beta-catenin/TCF target genes in the uterus of immature rats, indicating that their molecules are the potential players affected by estrogenic stimuli.

Spaciotemporal alteration of 8-hydroxy-2'-deoxyguanosine levels in cardiomyocytes after myocardial infarction in rats.

Temporary or persistent heart failure is one of the major complications after myocardial infarction (MI). In order to elucidate the pathogenesis of MI, we studied the spaciotemporal alteration of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in cardiomyocytes in a rat model of ligation of the left anterior descending branch of the coronary artery. The lethality in this model was 18%. Hearts were dissected at 0, 3, 6, 12, 24, 48 h, and 1, 2, 4, 6 weeks after the operation. The cardiac level of 8-OHdG was evaluated biochemically as well as by immunohistochemistry with monoclonal antibody N45.1. Three to 6h after ligation, the 8-OHdG levels were increased in the cardiomyocytes of MI (six-fold) and peri-MI (four-fold) areas. After 24 h, the myocardium in the MI area was necrotized, and thereafter the 8-OHdG level decreased. 8-OHdG levels in the myocardium of peri-MI areas returned once to a normal level, but were significantly increased at 2-4 weeks along with the appearance of apoptotic cardiomyocytes in this area. The heart after MI has been generally considered as clinically stable after four weeks. However, cardiomyocytes near the infarcted area were oxidatively stressed even after four weeks when the affected lesion was extensive. The present data support the use of supplementary antioxidant therapies to save functional myocardium after MI. (213 words)

Novel surrogate end-point biomarker to evaluate agents for use in the chemoprevention of reactive oxygen species-associated cancer.

Cancer chemoprevention is the use of chemical agents to inhibit, delay or reverse carcinogenesis. We established a novel method to evaluate agents for use in the chemoprevention of reactive oxygen species (ROS)-associated cancer. Induction of renal cell carcinoma in rats by ferric nitrilotriacetate (Fe-NTA) is an established model of ROS-associated cancer. We recently identified the p16INK4A tumor suppressor gene as one of the major target genes in this model, and showed by the use of in situ hybridization that allelic loss of p16IK4A occurs in the increased fraction of renal tubular cells within a few weeks. In the present study, we tested whether diets including green tea powder or a processed grain food are effective chemopreventive agents in this animal model. Consumption of these modified diets led to a significant decrease in the fraction of aneuploid cells after 1 week of repeated Fe-NTA administration. A decrease in renal lipid peroxidation after a single administration of Fe-NTA was also observed. Therefore, intake of green tea or processed grain foods stabilizes p16INK4A in the genome, at least in this model, and might be helpful for the prevention of ROS-associated cancer. This novel method is versatile, and may work as a surrogate end-point biomarker for screening the usefulness of agents for cancer chemoprevention.

Detection of glyceraldehyde 3-phosphate dehydrogenase messenger RNA using a peptide nucleic acid probe in paraffin-embedded archival specimens.

Although the human genome project has been completed, the functions of many genes remain undetermined. In situ hybridization (ISH) is a key method for identifying cells in which a given messenger RNA is transcribed. Paraffin-embedded specimens remain precious materials for research, but preservation of high-quality RNA in these specimens is not expected unless ample caution was taken during fixation. Peptide nucleic acid (PNA) is a recently developed hybrid molecule with genetic information that has high stability and high affinity to the complementary DNA or RNA. We applied a PNA probe to mRNA ISH of liver specimens obtained by autopsy and embedded in paraffin 28-48 years ago. An 18-mer PNA probe for glyceraldehyde 3-phosphate dehydrogenase was used. Staining was then analyzed in association with morphology by hematoxylin and eosin staining, and with the time between death of the patient and tissue fixation. Notably, specimens fixed with formalin and embedded in paraffin 48 years ago yielded excellent results if the time before fixation was short enough (<8 h). There was a significant inverse correlation between the intensity of ISH staining and the time before fixation. Oligonucleotide PNA probe, albeit at high cost, would increase the value of paraffin-embedded specimens in storage for use in human medical research.

Specific allelic loss of p16 (INK4A) tumor suppressor gene after weeks of iron-mediated oxidative damage during rat renal carcinogenesis.

Oxidative tissue damage has been shown to be associated with carcinogenesis. In human cancers p16(INK4A) is one of the most frequently mutated tumor suppressor genes. The present study used the ferric nitrilotriacetate (Fe-NTA)-induced rat renal carcinogenesis model to determine whether oxidative damage can cause specific allelic loss of p16 (INK4A). By the use of fluorescent in situ hybridization in combination with imprint cytology at single-cell resolution, we found that the number of renal tubular cells with aneuploidy (1 or 3 signals) at the p16(INK4A) locus was significantly and specifically increased (1 week, 37.2 +/- 2.3%; 3 weeks, 37.8 +/- 1.3% vs control, 22.5 +/- 1.9%; mean +/- SE, N = 8; P < 0.001 and P < 0.0001, respectively) after repeated intraperitoneal administration of 5 to10 mg of iron/kg in the form of Fe-NTA for 3 weeks. No increase in aneuploidy was observed at the loci of either the p53 or vhl tumor suppressor gene. Furthermore, the increase in the cells with 3 signals was followed by a continuous increase in those with 1 signal. Therefore, the p16 (INK4A) locus is specifically vulnerable to oxidative damage, leading to its allelic loss within weeks, presumably due to a deficiency in the replication of both the alleles.