Claudins form a large family of tight junction proteins that have essential roles in the control of paracellular ion flux and the maintenance of cell polarity. Many studies have shown that several claudin family members are abnormally expressed in various cancers. In particular, CLDN4 (encoding claudin-4) is overexpressed in ovarian cancer. However, although CLDN4 overexpression is well established, the mechanisms responsible for this abnormal regulation remain unknown. In the present study, we delineate a small region of the CLDN4 promoter critical for its expression. This region contains two Sp1 sites, both of which are required for promoter activity. However, because of the ubiquitous expression of Sp1, these sites, although necessary, are not sufficient to explain the patterns of gene expression of CLDN4 in various ovarian tissues. We show that the CLDN4 promoter is further controlled by epigenetic modifications of the Sp1-containing critical promoter region. Cells that overexpress CLDN4 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN4 promoter region, and the reverse is observed in cells that do not express CLDN4. Moreover, the CLDN4-negative cells can be induced to express CLDN4 through treatment with demethylating and/or acetylating agents. Because CLDN4 is elevated in a large fraction of ovarian cancer, the mechanism leading to deregulation may represent a general pathway in ovarian tumorigenesis and may lead to novel strategies for therapy and an overall better understanding of the biology of this disease.Claudins are major components of tight junctions, which are essential in the control of paracellular ion flux and the maintenance of cell polarity. The presence of phosphorylation sites and PDZ-binding motifs in claudins suggest a role in signal transduction for these proteins (1-3). So far, 23 members of the claudin family have been identified, and the expression of the various claudins has been shown to vary among or even within tissues, probably depending on the exact physiological requirements (1). The gene for claudin-4, CLDN4, is normally expressed in the epithelial cells of colon mucosa, mammary gland, renal tubules, and thyroid gland.Recently, several claudin genes have been shown to be abnormally expressed in human malignancies (4). In particular, CLDN4 has now been shown to be significantly elevated in several malignancies such as breast, pancreas, prostate, and ovary (4). In ovarian cancer, we and several other groups have shown that the genes for CLDN3 and CLDN4, as well as their corresponding proteins (claudin-3 and -4) are frequently up-regulated (5-10). Because many CA125-negative ovarian cancers overexpress these claudins, they may represent diagnostically useful biomarkers for this disease (11). Furthermore claudin-3 and claudin-4 are receptors for the cytotoxic Clostridium perfringens enterotoxin (12), and intra-abdominal administration of Clostridium perfringens enterotoxin might represent an interesting new avenue for ovarian cancer therapy (13). Alt...

Fetal DNA in maternal plasma and serum has been shown to be a useful material for fetal gender determination and for screening tests for abnormal pregnancies except during early gestational ages. Maternal serum samples were obtained from 81 pregnant women during the 5th-10th weeks of gestation. Fetal gender was determined by conventional polymerase chain reaction (PCR) to detect a Y-chromosomal sequence (DYS14) in maternal serum during early gestation and confirmed by examination of the newborns after delivery. Real-time quantitative analyses of the SRY and beta-globin genes were also performed in order to determine fetal gender and to quantify fetal DNA concentration in maternal serum during early gestation. When using conventional PCR, the total sensitivity of identifying a male fetus was 95%, but its sensitivity after the 7th week was 100%, whereas in real-time quantitative PCR, the total sensitivity after the 5th week was 100%. Quantitative analyses of the SRY gene revealed that the mean concentration of fetal DNA in maternal serum was 30.55 copies/ml, that fetal DNA concentration showed a tendency to increase with the progression of pregnancy, and that it had a wide normal range. Thus, we could confidently determine fetal gender by using maternal serum samples taken as early as the 7th week.

Background: Endometriosis is a clinical condition that affects up to 10% of the women of reproductive age. Endometriosis is characterized by the presence of endometrial tissues outside the uterine cavity and can lead to chronic pelvic pain, infertility and, in some cases, to ovarian cancer.

AcKnowleDgeMenTSWe thank Dr. Ashani Weeraratna and the members of our laboratory for useful comments on the manuscript. This research was supported by the Intramural Research Program of the NIH, National Institute on Aging. Research PaperRegulation of the CLDN3 Gene in Ovarian Cancer Cells AbSTrAcTThe claudin (CLDN) genes encode a family of proteins involved in the formation and function of tight junctions. CLDN gene expression is frequently altered in several human cancers, and in particular, CLDN3 and CLDN4 are commonly overexpressed in ovarian cancer. However, the mechanisms leading to the deregulation of these genes in cancer remain unclear. In the present study, we have examined the CLDN3 promoter and have identified a minimal region containing an Sp1 site crucial for its activity. In addition, we find that the CLDN3 promoter is regulated through epigenetic processes. Cells that express high levels of CLDN3 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN3 promoter region, and the reverse is observed in cells that do not express this gene. CLDN3-negative cells can be induced to express CLDN3 through treatment with DNA methyltransferase or histone deacetylase inhibitors. Interestingly, in vitro binding experiments, as well as chip assays show that Sp1 binds the unmethylated promoter much more efficiently, providing a mechanism for CLDN3 silencing in non-expressing cells. Finally, siRNA-mediated knockdown of Sp1 led to a significant decrease of CLDN3 expression at both the mRNA and protein levels, demonstrating a crucial role for this transcription factor in the regulation of CLDN3. Our data provide a basis for CLDN3 expression in ovarian cancer cells, as well as a mechanism for the silencing of this promoter in tumors lacking expression of claudin-3.

Frequencies of disomy and diploidy in spermatozoa for chromosomes X, Y and 18 were compared among severe oligozoospermic men (<5x10(6) spermatozoa/ml), oligozoospermic men (5-20x10(6) spermatozoa/ml), and normospermic men using three-colour fluorescence in-situ hybridization (FISH). Semen samples were collected from 10 severe oligozoospermic men aged 26-49 years, 10 oligozoospermic men aged 27-48 years and seven normospermic men aged 25-31 years. Karyotypes in lymphocytes obtained from peripheral blood were all 46,XY. In severe oligozoospermic men, analysis of 200 interphases per individual using FISH showed XY constitutions for sex chromosomes in all cells. A minimum of 10 000 sperm nuclei per individual for each chromosome was evaluated in severe oligozoospermic men and oligozoospermic men, and a minimum of 6000 sperm nuclei per individual in normospermic men. In total, 245 707 sperm nuclei were evaluated. The hybridization efficiency was 99.8%. The severe oligozoospermic men showed significantly higher frequencies of XY disomy (0.41%) and diploidy (0.49%) compared with oligozoospermic men (0.16%, P < 0.01; 0.22%, P < 0.05) and normospermic men (0.18%, P < 0.05; 0.21%, P < 0.05) (Mann-Whitney U-test). The data suggest that when severe oligozoospermic men undergo intracytoplasmic sperm injection, there can be an increase in the rate of conceptuses with 47,XXY chromosomes.