Glucocorticoids alter neuronal differentiation of human neuroepithelial-like cells by inducing long-lasting changes in the reactive oxygen species balance.

Prenatal exposure to excess glucocorticoid has been shown to have adverse effects on the developing nervous system that may lead to alterations of fetal and adult neurogenesis, resulting in behavioral changes. In addition, an imbalance of the redox state, with an increased susceptibility to oxidative stress, has been observed in rodent neural stem cells exposed to the synthetic glucocorticoid analog dexamethasone (Dex). In the present study, we used the induced pluripotent stem cells (IPSC)-derived lt-NES AF22 cell line, representative of the neuroepithelial stage in central nervous system development, to investigate the heritable effects of Dex on reactive oxygen species (ROS) balance and its impact on neuronal differentiation. By analysing gene expression in daughter cells that were never directly exposed to Dex, we could observe a downregulation of four key antioxidant enzymes, namely Catalase, superoxide dismutase 1, superoxide dismutase 2 and glutathione peroxidase7, along with an increased intracellular ROS concentration. The imbalance in the intracellular REDOX state was associated to a significant downregulation of major neuronal markers and a concomitant increase of glial cells. Interestingly, upon treatment with the antioxidant N-acetyl-cysteine (NAC), the misexpression of both neuronal and glial markers analyzed was recovered. These novel findings point to the increased ROS concentration playing a direct role in the heritable alterations of the differentiation potential induced by Dex exposure. Moreover, the data support the hypothesis that early insults may have detrimental long-lasting consequences on neurogenesis. Based on the positive effects exerted by NAC, it is conceivable that therapeutic strategies including antioxidants may be effective in the treatment of neuropsychiatric disorders that have been associated to increased ROS and impaired neurogenesis.

Tumor cell-specific BRCA1 and RASSF1A hypermethylation in serum, plasma, and peritoneal fluid from ovarian cancer patients.

Because existing surgical and management methods can consistently cure only early-stage ovarian cancer, novel strategies for early detection are required. Silencing of tumor suppressor genes such as p16INK4a, VHL, and hMLH1 have established promoter hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer and as a promising target for molecular detection in bodily fluids. Using sensitive methylation-specific PCR, we screened matched tumor, preoperative serum or plasma, and peritoneal fluid (washes or ascites) DNA obtained from 50 patients with ovarian or primary peritoneal tumors for hypermethylation status of the normally unmethylated BRCA1 and RAS association domain family protein 1A tumor suppressor genes. Hypermethylation of one or both genes was found in 34 tumor DNA (68%). Additional examination of one or more of the adenomatous polyposis coli, p14ARF, p16INK4a, or death associated protein-kinase tumor suppressor genes revealed hypermethylation in each of the remaining 16 tumor DNA, which extended diagnostic coverage to 100%. Hypermethylation was observed in all histologic cell types, grades, and stages of ovarian tumor examined. An identical pattern of gene hypermethylation was found in the matched serum DNA from 41 of 50 patients (82% sensitivity), including 13 of 17 cases of stage I disease. Hypermethylation was detected in 28 of 30 peritoneal fluid DNA from stage IC-IV patients, including 3 cases with negative or atypical cytology. In contrast, no hypermethylation was observed in nonneoplastic tissue, peritoneal fluid, or serum from 40 control women (100% specificity). We conclude that promoter hypermethylation is a common and relatively early event in ovarian tumorigenesis that can be detected in the serum DNA from patients with ovary-confined (stage IA or B) tumors and in cytologically negative peritoneal fluid. Analysis of tumor-specific hypermethylation in serum DNA may enhance early detection of ovarian cancer.

Detection of breast cancer in nipple aspirate fluid by CpG island hypermethylation.

PURPOSE: New approaches to the early detection of breast cancer are urgently needed as there is more benefit to be realized from screening. Nipple aspiration is a noninvasive technique that yields fluid known to contain breast epithelial cells. Silencing of tumor suppressor genes such as p16(INk4a), BRCA1, and hMLH1 have established hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer and as a promising target for molecular detection. EXPERIMENTAL DESIGN: Using sensitive methylation-specific PCR, we searched for aberrant promoter hypermethylation in a panel of six normally unmethylated genes: glutathione S-transferase pi 1 (GSTP1); retinoic acid receptor-beta2 (RARbeta2); p16(INk4a); p14(ARF); RAS association domain family protein 1A (RASSF1A); and death-associated protein kinase (DAP-kinase) in 22 matched specimens of tumor, normal tissue, and nipple aspirate fluid collected from breast cancer patients. RESULTS: Hypermethylation of one or more genes was found in all 22 tumor DNAs (100% diagnostic coverage) and identical gene hypermethylation detected in 18 of 22 (82%) matched aspirate fluid DNAs. In contrast, hypermethylation was absent in benign and normal breast tissue and nipple aspirate DNA from healthy women. CONCLUSIONS: Promoter hypermethylation of important cancer genes is common in breast cancer and could be detected in matched aspirate DNAs from patients with ductal carcinoma in situ or stage I cancer. Promoter hypermethylation represents a promising marker, and larger studies may lead to its useful application in breast cancer diagnosis and management.

Promoter hypermethylation of tumor suppressor genes in urine from kidney cancer patients.

Kidney cancer confined by the renal capsule can be surgically cured in the majority of cases, whereas the prognosis for patients with advanced disease at presentation remains poor. Novel strategies for early detection are therefore needed. Molecular DNA-based tests have successfully used the genetic alterations that initiate and drive tumorigenesis as targets for the early detection of several types of cancer in bodily fluids, including urine. Using sensitive methylation-specific PCR, we screened matched tumor DNA and sediment DNA from preoperative urine specimens obtained in 50 patients with kidney tumors, representing all major histological types, for hypermethylation status of a panel of six normally unmethylated tumor suppressor genes VHL, p16/CDKN2a, p14ARF, APC, RASSF1A, and Timp-3. Hypermethylation of at least one gene was found in all 50 tumor DNAs (100% diagnostic coverage) and an identical pattern of gene hypermethylation found in the matched urine DNA from 44 of 50 patients (88% sensitivity), including 27/30 cases of stage I disease. In contrast, hypermethylation of the genes in the panel was not observed in normal kidney tissue or in urine from normal healthy individuals and patients with benign kidney disease (100% specificity). Hypermethylation of VHL was found only in clear cell, whereas hypermethylation of p14ARF, APC, or RASSF1A was more frequent in nonclear cell tumors, which suggested that the panel might facilitate differential diagnosis. We conclude that promoter hypermethylation is a common and early event in kidney tumorigenesis and can be detected in the urine DNA from patients with organ-confined renal cancers of all histological types. Methylation-specific PCR may enhance early detection of renal cancer using a noninvasive urine test.