Circadian Oscillations Persist in Cervical and Esophageal Cancer Cells Displaying Decreased Expression of Tumor-Suppressing Circadian Clock Genes.

There is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study, the circadian clock was investigated in cervical and esophageal cancers, to determine whether it is disrupted in these cancer types. Oncomine datamining revealed downregulation of multiple members of the circadian clock gene family in cancer patient tissue compared with matched normal epithelium. Real-time RT-PCR analysis confirmed significant downregulation of CLOCK, PER1, PER2, PER3, CRY1, CRY2, REV-ERBα, and RORα in esophageal tumor tissue. In cell line models, expression of several circadian clock genes was significantly decreased in transformed and cancer cells compared with noncancer controls, and protein levels were dysregulated. These effects were mediated, at least in part, by methylation, where CLOCK, CRY1, and RORα gene promoter regions were found to be methylated in cancer cells. Overexpression of CLOCK and PER2 in cancer cell lines inhibited cell proliferation and activation of RORα and REV-ERBα using agonists resulted in cancer cell death, while having a lesser effect on normal epithelial cells. Despite dysregulated circadian clock gene expression, cervical and esophageal cancer cells maintain functional circadian oscillations after Dexamethasone synchronization, as revealed using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are intact. IMPLICATIONS: This study is a first to describe dysregulated, yet oscillating, circadian clock gene expression in cervical and esophageal cancer cells, and knowledge of circadian clock functioning in these cancer types has the potential to inform chronotherapy approaches, where the timing of administration of chemotherapy is optimized on the basis of the circadian clock.

The Yin and Yang of cancer genes.

Cancer is caused by malfunctioning of genes that normally regulate cardinal processes including various nuclear functions, cell division and survival, cell surface to nucleus signaling cascades, etc. Cancer associated genes are often classified as oncogenes (OCGs) or tumor suppressor genes (TSGs) depending on whether they promote or suppress tumorigenesis, respectively. Such strict classification of cancer genes may however be an over-simplification. Several studies have highlighted a dual role for cancer genes, often impacting the same facet of tumorigenesis. Knowledge of a possible dichotomy of a cancer gene (particularly an OCG) is imperative when evaluating its possible utility as a therapeutic target. Though previous studies have extensively evaluated specific examples of cancer genes exhibiting a dual nature, efforts to unravel the molecular basis for such contrasting functions have been fewer. The current review is an attempt to delineate molecular events underlying the functional dichotomy of cancer genes at the DNA (mutations, gene fusions, etc.), RNA (alternative splicing, regulation through non-coding RNAs, etc.) and protein (isoforms, mis-localisation, post-translational modifications, proteolytic cleavage, etc.) levels.

MicroRNA-520c-3p functions as a novel tumor suppressor in lung adenocarcinoma.

Lung cancer is a malignancy with one of the highest incidence rates, and it is the leading cause of cancer-related death. To gain further insights into the underlying mechanisms of tumor growth and metastasis, we investigated the role and expression of microRNAs in lung adenocarcinoma (LUAD). We discovered a significantly lower expression level of microRNA-520c-3p (miR-520c-3p) in LUAD tissues than in nontumor tissues. miR-520c-3p is known to regulate multiple biological functions and cellular behaviors. In this study, we show that AKT1 and AKT2 are key direct targets of miR-520c-3p, which are required for its biological roles in LUAD. Mechanistically, downregulation of miR-520c-3p in LUAD is due to DNA methylation of the miR-520c-3p promoter region. Conversely, the activity of the transcription factor Yin Yang 1 (YY1) results in the upregulation of miR-520c-3p. Taken together, our results reveal methylation/YY1/miR-520c-3p/AKT1/AKT2 as a molecular axis with a potent biological function and highlight miR-520c-3p as a novel potent tumor suppressor in LUAD.

Sleep quality and methylation status of selected tumor suppressor genes among nurses and midwives.

Chronic sleep restriction may affect metabolism, hormone secretion patterns and inflammatory responses. Limited reports suggest also epigenetic effects, such as changes in DNA methylation profiles. The study aims to assess the potential association between poor sleep quality or sleep duration and the levels of 5-methylcytosine in the promoter regions of selected tumor suppressor genes. A cross-sectional study was conducted on 710 nurses and midwives aged 40-60 years. Data from interviews regarding sleep habits and potential confounders were used. The methylation status of tumor suppressor genes was determined via qMSP reactions using DNA samples derived from leucocytes. No significant findings were observed in the total study population or in the two subgroups of women stratified by the current system of work. A borderline significance association was observed between a shorter duration of sleep and an increased methylation level in CDKN2A among day working nurses and midwives. Further studies are warranted to explore this under-investigated topic.

Odd-skipped related 1 is a novel tumour suppressor gene and a potential prognostic biomarker in gastric cancer.

We report that the odd-skipped related 1 (OSR1) gene encoding a zinc-finger transcription factor was preferentially methylated in gastric cancer by genome-wide methylation screening. OSR1 expression was frequently silenced or down-regulated in gastric cancer cell lines. OSR1 expression was also significantly down-regulated at both mRNA and protein levels in primary gastric cancer tissues compared with adjacent normal tissues. The silencing or down-regulation of OSR1 was closely associated with promoter hypermethylation. Overexpression of OSR1 significantly inhibited cell growth, arrested the cell cycle, and induced apoptosis in the gastric cancer cell lines AGS, MKN28, and MGC803. Conversely, knockdown of OSR1 by OSR1-short hairpin RNA significantly enhanced cell growth, promoted the cell cycle, and inhibited apoptosis in the normal gastric epithelial cell line GES1. The dual-luciferase reporter assay revealed that OSR1 activated p53 transcription and repressed the T-cell factor (TCF)/lymphoid enhancer factor (LEF). Complementary DNA expression array and western blotting showed that OSR1 increased the expression of nuclear p53, p21, Fas, and death receptor-5, and suppressed the expression of cyclin D1 and cyclin-dependent kinase 4 in the p53 signalling pathway. In addition, OSR1 suppressed the expression of cytoplasmic ß-catenin, TCF-1, and LEF1 in the Wnt/ß-catenin signalling pathway. OSR1 methylation was detected in 51.8% of primary gastric cancer patients (85 of 164) by bisulphite genomic sequencing. Multivariate Cox regression analysis showed that OSR1 methylation was an independent predictor of poor survival. Kaplan-Meier survival curves revealed that OSR1 methylation was associated with shortened survival in TNM stage I-III patients. In conclusion, OSR1 acts as a functional tumour suppressor through the transcriptional activation of p53 and repression of TCF/LEF in gastric cancer. Detection of OSR1 methylation may serve as a potential biomarker of the early stage of gastric cancer.

Altered global methylation and hydroxymethylation status in vulvar lichen sclerosus: further support for epigenetic mechanisms.

BACKGROUND: Epigenetics refers to functionally relevant changes in the genome other than those of DNA sequence that can lead to changes in gene expression or cellular phenotype. There is evidence that epigenetics is relevant in the pathogenesis of autoimmune diseases such as vulvar lichen sclerosus (VLS), as well as in cancer, including cutaneous squamous cell carcinoma, which is frequently associated with VLS. OBJECTIVES: To study the global methylation and hydroxymethylation status in healthy controls and VLS lesions before and after long-term ultraviolet (UV)A1 treatment. METHODS: We studied 12 controls and 10 patients with VLS who were treated with medium-dose UVA1 four times weekly for 3 months. Immunohistochemistry and mutation analyses (polymerase chain reaction) were performed for 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), isocitrate dehydrogenases (IDHs) and the ten-eleven translocation (TET)2 enzyme. RESULTS: After 3 months of treatment, 5mC was significantly increased in VLS compared with baseline and controls. However, compared with controls 5hmC levels were significantly reduced in baseline VLS, but normalized after UVA1 treatment. Compared with controls, IDH1 expression was significantly higher in both treated and baseline VLS. By contrast, IDH2 levels were significantly reduced in baseline VLS compared with controls and UVA1-treated VLS. However, gene sequencing of the IDH1, IDH2 and TET2 genes did not reveal evidence of mutations. CONCLUSIONS: VLS is associated with altered expression of IDH enzymes and aberrant hydroxymethylation, indicating an epigenetic background for the pathogenesis of VLS. UVA1 phototherapy may cause normalization of 5hmC patterns, but also global DNA hypermethylation in VLS lesions, raising concerns with respect to an increased risk of photocarcinogenesis.

Curcumin and anthocyanin inhibit pepsin-mediated cell damage and carcinogenic changes in airway epithelial cells.

OBJECTIVES: Laryngopharyngeal reflux (LPR) is associated with inflammatory and neoplastic airway diseases. Gastric pepsin internalized by airway epithelial cells during reflux contributes to oxidative stress, inflammation, and carcinogenesis. Several plant extracts and compounds inhibit digestive enzymes and inflammatory or neoplastic changes to the esophagus in models of gastroesophageal reflux. This study examined the potential of chemoprotective phytochemicals to inhibit peptic activity and mitigate pepsin-mediated damage of airway epithelial cells. METHODS: Cultured human laryngeal and hypopharyngeal epithelial cells were pretreated with curcumin (10 micromol/L), ecabet sodium (125 microg/mL), and anthocyanin-enriched black-raspberry extract (100 microg/mL) 30 minutes before treatment with pepsin (0.1 mg/mL; 1 hour; pH 7). Controls were treated with media pH 7 or pepsin pH 7 without phytochemicals. Cell damage and proliferative changes were assessed by electron microscopy, cell count, thymidine analog incorporation, and real-time polymerase chain reaction array. Pepsin inhibition was determined by in vitro kinetic assay. RESULTS: Micromolar concentrations of curcumin, ecabet sodium, and black-raspberry extract inhibited peptic activity and pepsin-induced mitochondrial damage and hyperproliferation. Curcumin abrogated pepsin-mediated depression of tumor suppressor gene expression and altered the subcellular localization of pepsin following endocytosis. CONCLUSIONS: Several phytochemicals inhibit the pepsin-mediated cell damage underlying inflammatory or neoplastic manifestations of LPR. Dietary supplementation or adjunctive therapy with phytochemicals may represent novel preventive or therapeutic strategies for LPR-attributed disease.

Single-molecule and population probing of chromatin structure using DNA methyltransferases.

Probing chromatin structure with DNA methyltransferases offers advantages over more commonly used nuclease-based and chromatin immunoprecipitation methods for detection of nucleosomes and non-histone protein-DNA interactions. Here, we describe two related methods in which the readout of MTase accessibility is obtained by assaying 5-methylcytosine in DNA through the PCR-based technique of bisulfite genomic sequencing. The methyltransferase accessibility protocol (MAP) determines the relative frequency at which the enzyme accesses each of its target sites over an entire population of PCR amplified product. While MAP yields much quantitative information about relative accessibility of a region of chromatin, a complementary single-molecule view of methyltransferase accessibility, termed MAP for individual templates (MAP-IT), is provided by analysis of cloned PCR products. Absolute rather than relative methylation frequencies in a region are obtained by summing the methylation status at each site over a cohort of clones. Moreover, as the integrity of individual molecules is maintained in MAP-IT, unique information about the distribution of multiple footprints along continuous regions is gleaned. In principle, the population MAP and single-molecule MAP-IT strategies can be used to analyze chromatin structure in a variety of model systems. Here, we describe the application of MAP in living Saccharomyces cerevisiae cells and MAP-IT in the analysis of a mammalian tumor suppressor gene in nuclei. This application of MAP-IT provides the first means to simultaneously determine CpG methylation of mammalian genes and their overlying chromatin structure in the same single DNA molecule.

Soya phytonutrients act on a panel of genes implicated with BRCA1 and BRCA2 oncosuppressors in human breast cell lines.

Breast cancer is the most common cancer in women and a significant cause of death. Mutations of the oncosuppressor genes BRCA1 and BRCA2 are associated with a hereditary risk of breast cancer, and dysregulation of their expression has been observed in sporadic cases. Soya isoflavones have been shown to inhibit breast cancer in studies in vitro, but associations between the consumption of isoflavone-containing foods and breast cancer risk have varied in epidemiological studies. Soya is a unique source of the phytoestrogens daidzein (4',7-dihydroxyisoflavone) and genistein (4',5,7-trihydroxyisoflavone), two molecules that are able to inhibit the proliferation of human breast cancer cells in vitro. The aim of the present study was to determine the effects of genistein (5 microg/ml) and daidzein (20 microg/ml) on transcription in three human breast cell lines (one dystrophic, MCF10a, and two malignant, MCF-7 and MDA-MB-231) after 72 h treatment. The different genes involved in the BRCA1 and BRCA2 pathways (GADD45A, BARD1, JUN, BAX, RB1, ERalpha, ERbeta, BAP1, TNFalpha, p53, p21Waf1/Cip1, p300, RAD51, pS2, Ki-67) were quantified by real-time quantitative RT-PCR, using the TaqMan method and an ABI Prism 7700 Sequence Detector (Applied Biosystems). We observed that, in response to treatment, many of these genes were overexpressed in the breast cancer cell lines (MCF-7 and MDA-MB-231) but not in the dystrophic cell line (MCF10a).

Colocalization of kisspeptin and gonadotropin-releasing hormone in the ovine brain.

Kisspeptin is a peptide that has been implicated in the regulation of GnRH cells in the brain. Immunohistochemical studies were undertaken to examine the distribution of kisspeptin-immunoreactive (IR) cells in the ovine diencephalon and determine the effect of ovariectomy in the ewe. We report that kisspeptin colocalizes to a high proportion of GnRH-IR cells in the preoptic area, which is a novel finding. A high level of colocalization of kisspeptin and GnRH was also seen in varicose neuronal fibers within the external, neurosecretory zone of the median eminence. Apart from the kisspeptin/GnRH cells, a population of single-labeling kisspeptin-IR cells was also observed in the preoptic area. Within the hypothalamus, kisspeptin-IR cells were found predominantly in the arcuate nucleus, and there was an increase in the number of immunohistochemically identified cell within this nucleus after ovariectomy. Kisspeptin-IR cells were also found in the periventricular nucleus of the hypothalamus, but the number observed was similar in gonad-intact and ovariectomized ewes. The colocalization of GnRH and kisspeptin within cells of the preoptic area and GnRH neurosecretory terminals of the median eminence suggests that the two peptides might be cosecreted into the hypophyseal portal blood to act on the pituitary gland. Effects of ovariectomy on the non-GnRH, Kisspeptin-IR cells of the hypothalamus suggest that kisspeptin production is negatively regulated by ovarian steroids.

Tumor-suppressive maspin regulates cell response to oxidative stress by direct interaction with glutathione S-transferase.

Maspin, a novel serine protease inhibitor, suppresses tumor progression in several cancer models, including an in vivo model for prostate cancer bone metastasis. However, the molecular mechanism of maspin remains illusive, primarily because its molecular targets are unknown. To this end, we used a full-length maspin cDNA bait to screen against both a primary prostate tumor cDNA prey library and a HeLa cDNA prey library by the yeast two-hybrid method. We found that heat shock protein 90, glutathione S-transferase (GST), and heat shock protein 70 interacted with maspin with the highest frequencies. We confirmed the maspin/GST interaction using purified proteins, human epithelial cell lines, and human prostate tissues. A maspin variant that has a point mutation of Arg(340) to Ala (Mas(R340A)) showed a significantly decreased affinity for GST. Although purified maspin had no effect on the activity of purified GST in vitro, intracellular interaction between endogenous maspin and GST correlated with an elevated total GST activity in both MDA-MB-435- and DU145-derived stably transfected cells. Consistently, tumor cells treated with purified wild type maspin, but not Mas(R340A), enhanced cellular GST activity. Maspin expression in cancer cell lines also correlated with decreased basal levels of reactive oxygen species (ROS). Furthermore, H(2)O(2) treatment not only induced GST expression but also increased intracellular maspin/GST interaction, which was inversely correlated with the level of ROS generation. Conversely, maspin knockdown by small interfering RNA increased the basal, as well as H(2)O(2)-induced, ROS generation. Furthermore, the maspin effect on ROS generation was completely abolished by a GST inhibitor, indicating an essential role of GST in maspin-mediated cellular response to oxidative stress. Consistently, oxidative stress-induced vascular endothelial growth factor A expression was significantly inhibited in maspin-expressing cells. Together, our data suggest a new mechanism by which maspin, through its direct interaction with GST, may inhibit oxidative stress-induced ROS generation and vascular endothelial growth factor A induction, thus preventing further adverse effects on tumor genetics and stromal reactivity.

Maspin overexpression modulates tumor cell apoptosis through the regulation of Bcl-2 family proteins.

BACKGROUND: Maspin is a member of serpin family with tumor suppressing activity. Recent studies of maspin in animal models strongly support maspin's role as an inhibitor against the growth of primary tumor sand the process of metastasis. However, the molecular mechanism underlying this inhibition has not been fully elucidated. In this report, we analyze the effect of maspin on tumor cell apoptosis under several stress conditions. METHODS: Stable clones overexpressing maspin are established in the mouse mammary tumor TM40D cells. They are treated with staurosporine, TNF-alpha, and serum starvation. The rates of cell apoptosis are analyzed by TUNEL assay. Inhibitors against caspase 8 and 9 are used in the apoptosis assay. Western blot analysis and ribonuclease protection assay (RPA) are performed to examine the expression of Bcl2 family genes. RESULTS: We report that maspin expressing tumor cells have increased rate of apoptosis when they are treated with staurosporine and serum starvation. The effect is not through extracellular maspin. Maspin-mediated apoptosis is partially blocked by caspase 8 and 9 inhibitors, and is accompanied by changes in the Bcl-2 family proteins. Maspin-expressing tumor cells have a reduced level of anti-apoptotic protein Bcl-2, and an increased level of pro-apoptotic protein Bax. The regulation is not controlled at the transcriptional level but is through selective control of Bcl-2 and Bax protein stability. CONCLUSION: Maspin overexpression modulates tumor cell apoptosis through the regulation of Bcl2 family proteins. Such change results in an increased release of cytochrome c from mitochondria, thus the increased apoptosis in maspin-expressing cells. This evidence strongly suggests that the induction of apoptosis in maspin-overexpressing cells represents a major mechanism by which maspin inhibits breast tumor progression.

Hypermethylation of the RASSF1A gene in gliomas.

BACKGROUND: Promoter methylation is an important pathway in transcriptional silencing of tumor suppressor genes (TSGs) in brain tumors. The identified 3p21.3 tumor suppressor gene RAS association domain family protein 1A (RASSF1A) is highly methylated in primary lung, breast and other tumors. We investigated the promoter methylation and gene expression of RASSF1A in gliomas. METHODS: The methylation status of the promoter region of RASSF1A, p16INK4A and death-associated protein kinase (DAPK) genes was analyzed by methylation-specific PCR (MSP) in 41 surgically resected gliomas. RASSF1A expression was also detected by reverse-transcription polymerase chain reaction (RT-PCR) in 28 glioma tissues. RESULTS: The frequencies of RASSF1A, p16INK4A and DAPK promoter methylation were 13/41 (31.7%), 3/41 (7.3%) and 6/41 (14.6%) respectively. However, the methylations of those genes were not correlated with the clinical characteristics of patients (tumor grade, tumor types and sex). Among 28 glioma tissues, 6 showed the loss of the gene (21.4%). Promoter hypermethylation of RASSF1A is associated with loss of gene expression in glioma tissues. (p=0.022). CONCLUSIONS: Our results suggested that the RASSF1A gene might play an important role in glioma carcinogenesis. It also gives us an insight for future glioma medical therapy with a demethylating agent.

Mechanisms for pituitary tumorigenesis: the plastic pituitary.

The anterior pituitary gland integrates the repertoire of hormonal signals controlling thyroid, adrenal, reproductive, and growth functions. The gland responds to complex central and peripheral signals by trophic hormone secretion and by undergoing reversible plastic changes in cell growth leading to hyperplasia, involution, or benign adenomas arising from functional pituitary cells. Discussed herein are the mechanisms underlying hereditary pituitary hypoplasia, reversible pituitary hyperplasia, excess hormone production, and tumor initiation and promotion associated with normal and abnormal pituitary differentiation in health and disease.

Bracken fern-induced malignant tumors in rats: absence of mutations in p53, H-ras and K-ras and no microsatellite instability.

Bracken fern (genus Pteridium) has been shown to induce tumors in domestic and experimental animals. Epidemiological studies have also shown an association between human exposure to bracken toxins and increased risk for the development of upper gastrointestinal tract tumors. Our aim in this study was to investigate possible genomic alterations in bracken fern-induced tumors of experimental animals searching for molecular markers that might be used for human epidemiological studies. Using human colorectal carcinogenesis as a molecular model, we examined eight malignant bracken fern-induced tumors of rats for mutations in the genes associated with the "classic pathway" of colorectal cancer, i.e. p53 and ras, and also in the "mutator pathway" by evaluating microsatellite instability. Exons 5-9 of the p53 gene and exons 1 and 2 of the K-ras and H-ras genes were examined by DNA sequencing and no mutations were found in any of the eight tumors. Amplification of five previously validated microsatellite loci (one with mono-, three with di- and one with tetra-nucleotide repeat motifs) in the malignant tumors and in the surrounding normal tissue did not reveal any instability. The involvement of epigenetic alterations or of mutations in other tumor suppressor genes or oncogenes should be further investigated in the search for human epidemiological markers.

The molecular biology of laryngeal cancer.

Novel techniques have led to the discovery of many genes and gene products important in the development of HNSC and laryngeal cancer. Tumor suppressive genes and oncogenes have been identified, and many of their roles have been elucidated in a genetic progression model. As these molecular pathways become better understood, the information obtained will increasingly be used to guide patient therapy. Specifically, advances will probably be made in (1) molecular characterization of steps leading to laryngeal cancer; (2) molecular screening, staging, and surveillance; (3) molecularly based therapy, including gene transfer and small molecule therapy directed at specific molecular pathways involved in neoplasia; and (4) characterization of patients who are at high risk for laryngeal cancer. In the final analysis, however, smoking cessation for those at risk for head and neck cancer would have greater effect than all these efforts combined.

Injury-induced apoptosis of neurons in adult brain is mediated by p53-dependent and p53-independent pathways and requires Bax.

The mechanisms of injury-induced apoptosis of neurons within the CNS are not understood. We used a model of cortical injury in rat and mouse to induce retrograde neuronal apoptosis in thalamus. In this animal model, unilateral ablation of the occipital cortex causes unequivocal apoptosis of corticopetal projection neurons in the dorsal lateral geniculate nucleus (LGN) by 7 days postlesion. We tested the hypothesis that p53 and Bax regulate this retrograde neuronal apoptosis. We found, by using immunocytochemistry, that p53 accumulates in nuclei of neurons destined to undergo apoptosis. By immunoblotting, p53 levels increase ( approximately 150% of control) in nuclear-enriched fractions of the ipsilateral LGN by 5 days after occipital cortex ablation. p53 is functionally activated in nuclear fractions of the ipsilateral LGN at 5 days postlesion, as shown by DNA binding assay (approximately fourfold increase) and by immunodetection of phosphorylated p53. The levels of procaspase-3 increase at 4 days postlesion, and caspase-3 is activated prominently at 5 days postlesion. To identify whether neuronal apoptosis in the adult brain is dependent on p53 and Bax, cortical ablations were done on p53 and bax null mice. Neuronal apoptosis in the dorsal LGN is significantly attenuated (approximately 34%) in p53(-/-) mice. In lesioned p53(+/+) mice, Bax immunostaining is enhanced in the ipsilateral dorsal LGN and Bax immunoreactivity accumulates at perinuclear locations in dorsal LGN neurons. The enhancement and redistribution of Bax immunostaining is attenuated in lesioned p53(-/-) mice. Neuronal apoptosis in the dorsal LGN is blocked completely in bax(-/-) mice. We conclude that neuronal apoptosis in the adult thalamus after cortical injury requires Bax and is modulated by p53.