DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines

<p>Abstract</p> <p>Background</p> <p>DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines.</p> <p>Results</p> <p>The relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by methylation-specific PCR, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity and expression of DNMT1, DNMT3a, and DNMT3b proteins) were examined in 12 breast cancer cell lines. Unsupervised cluster analysis of the expression of 64 methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (<it>CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A</it>), whereas the low-frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein levels compared to normal breast epithelial cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Microarray data mining identified a strong cluster of primary breast tumors that express the hypermethylation signature defined by <it>CDH1</it>, <it>CEACAM6, CST6, ESR1, LCN2</it>, and <it>SCNN1A</it>. This subset of breast cancers represents 18/88 (20%) tumors in the dataset analyzed, and 100% of these tumors were classified as basal-like, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers.</p> <p>Conclusion</p> <p>These observations combine to strongly suggest that: (a) a subset of breast cancer cell lines express a hypermethylator phenotype, (b) the hypermethylation defect in these breast cancer cell lines is related to aberrant overexpression of DNMT activity, (c) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in tumor cells expressing this phenotype, and (d) the six-gene hypermethylator signature characterized in breast cancer cell lines defines a distinct cluster of primary basal-like breast cancers.</p

Treatment with 2-AAF blocks the small hepatocyte-like progenitor cell response in retrorsine-exposed rats

Liver regeneration after partial hepatectomy (PH) in retrorsine-exposed rats is accomplished through proliferation and differentiation of small hepatocyte-like progenitor cells (SHPCs). The cells of origin of SHPCs are not known. We investigated the possibility that SHPCs are directly derived from oval cells, a known liver progenitor cell, by combining the retrorsine/PH (RP) model with 2-acetamidofluorene (2-AAF), an anti-mitotic agent that elicits an oval cell reaction in response to liver deficit

Re-expression of tumorigenicity after attenuation of human synaptotagmin 13 in a suppressed microcell hybrid cell line

Human chromosome 11p11.2 contains a putative liver tumor suppressor locus that was identified using a microcell hybrid cell line-based model of tumor suppression. Transcription mapping of suppressed microcell hybrid cell lines suggests that human SYT13 represents a strong candidate for the 11p11.2 tumor suppressor gene. Other evidence suggests that the putative 11p11.2 tumor suppressor gene (SYT13 or some other) may modulate the tumorigenic potential of neoplastic liver cell lines through direct or indirect regulation of the rat Wt1 tumor suppressor gene. To characterize a functional role for SYT13 in liver tumor suppression, we employed RNAi to attenuate SYT13 expression in a suppressed microcell hybrid cell line (GN6TF-11neoCX4). SYT13-attenuated cells display aggressive phenotypic properties that are similar to or indistinguishable from the parental tumor cells (GN6TF), including altered cellular morphologies, disrupted contact inhibition, elevated saturation densities, restoration of anchorage-independent growth and increased tumorigenicity in vivo. Moreover, SYT13 attenuation and re-expression of tumorigenicity in GN6TF-11neoCX4-derived cell lines was accompanied by a significant decrease of Wt1 expression. In contrast, the phenotypic properties of scrambled-control cells were similar to the suppressed microcell hybrid cells and Wt1 expression was unaffected. These observations combine to establish that: i) human SYT13 functions as a liver tumor suppressor gene that complements a molecular defect in GN6TF rat liver tumor cells resulting in a normalized cellular phenotype in vitro and suppression of tumorigenicity in vivo; ii) RNAi-mediated attenuation of SYT13 expression restores the neoplastic phenotype of GN6TF-11neoCX4 microcell hybrid cells, consistent with the function of a liver tumor suppressor gene; and iii) loss of Wt1 expression is important for the re-establishment of tumorigenic potential by GN6TF-11neoCX4 microcell hybrid cells after attenuation of SYT13

Loss of post-transcriptional regulation of DNMT3b by microRNAs: A possible molecular mechanism for the hypermethylation defect observed in a subset of breast cancer cell lines

A hypermethylation defect associated with DNMT hyperactivity and DNMT3b overexpression characterizes a subset of breast cancers and breast cancer cell lines. We analyzed breast cancer cell lines for differential expression of regulatory miRs to determine if loss of miR-mediated post-transcriptional regulation of DNMT3b represents the molecular mechanism that governs the overexpression of DNMT3b that drives the hypermethylation defect in breast cancer. MicroRNAs (miRs) that regulate (miR-29a, miR-29b, miR-29c, miR-148a, miR-148b) or are predicted (miR-26a, miR-26b, miR-203, miR-222) to regulate DNMT3b were examined among 10 hypermethylator and 6 non-hypermethylator breast cancer cell lines. Hypermethylator cell lines express diminished levels of miR-29c, miR-148a, miR-148b, miR-26a, miR-26b, and miR-203 compared to non-hypermethylator cell lines. miR expression patterns correlate inversely with methylation-sensitive gene expression (r=−0.66, p=0.0056) and directly with the methylation status of these genes (r=0.72, p=0.002). To determine the mechanistic role of specific miRs in the dysregulation of DNMT3b among breast cancer cell lines, miR levels were modulated by transfection of pre-miR precursors for miR-148b, miR-26b, and miR-29c into hypermethylator cell lines (Hs578T, HCC1937, SUM185) and transfection of antagomirs directed against miR-148b, miR-26b, and miR-29c into non-hypermethylator cell lines (BT20, MDA-MB-415, MDA-MB-468). Antagomir-mediated knock-down of miR-148b, miR-29c, and miR-26b significantly increased DNMT3b mRNA in non-hypermethylator cell lines, and re-expression of miR-148b, miR-29c, and miR-26b following transfection of pre-miR precursors significantly reduced DNMT3b mRNA in hypermethylator cell lines. These findings strongly suggest that: i) post-transcriptional regulation of DNMT3b is combinatorial, ii) diminished expression of regulatory miRs contributes to DNMT3b overexpression, iii) re-expression of regulatory miRs reduces DNMT3b mRNA levels in hypermethylator breast cancer cell lines, and iv) down-regulation of regulatory miRs increases DNMT3b mRNA levels in non-hypermethylator breast cancer cell lines. In conlcusion, the molecular mechanism governing the DNMT3b-mediated hypermethylation defect in breast cancer cell lines involves the loss of post-transcriptional regulation of DNMT3b by regulatory miRs

Molecular and cellular heterogeneity in breast cancer: Challenges for personalized medicine

Breast cancer is noted for disparate clinical behaviors and patient outcomes, despite common histopathological features at diagnosis. Molecular pathogenesis studies suggest that breast cancer is a collection of diseases with variable molecular underpinnings that modulate therapeutic responses, disease-free intervals, and long-term survival. Traditional therapeutic strategies for individual patients are guided by the expression status of the estrogen and progesterone receptors (ER and PR) and human epidermal growth factor receptor 2 (HER2). Although such methods for clinical classification have utility in selection of targeted therapies, short-term patient responses and long-term survival remain difficult to predict. Molecular signatures of breast cancer based on complex gene expression patterns have utility in prediction of long-term patient outcomes, but are not yet used for guiding therapy. Examination of the correspondence between these methods for breast cancer classification reveals a lack of agreement affecting a significant percentage of cases. To realize true personalized breast cancer therapy, a more complete analysis and evaluation of the molecular characteristics of the disease in the individual patient is required, together with an understanding of the contributions of specific genetic and epigenetic alterations (and their combinations) to management of the patient. Here, we discuss the molecular and cellular heterogeneity of breast cancer, the impact of this heterogeneity on practical breast cancer classification, and the challenges for personalized breast cancer treatment

Anomalous Condensates and the Equivalence Theorem

A recently published report has called into question the validity of the equivalence theorem in dynamically broken gauge theories in which the fermions making up the symmetry breaking condensate lie in an anomalous representation of the broken gauge group. Such a situation can occur if the gauge anomaly is cancelled by another sector of the theory. Using the example of the one family Standard Model without scalar Higgs structure, we analyze a low energy effective theory which preserves the symmetries of the fundamental theory and demonstrate the validity of the equivalence theorem in this class of models.Comment: 14 Pages (Plain TeX + Harvmac), 2 uuencoded PostScript figures appended (figures also available by mail or fax), LBL-3490

Newtonian Hydrodynamics of the Coalescence of Black Holes with Neutron Stars I: Tidally locked binaries with a stiff equation of state

We present a detailed study of the hydrodynamical interactions in a Newtonian black hole-neutron star binary during the last stages of inspiral. We consider close binaries which are tidally locked, use a stiff equation of state (with an adiabatic index Gamma=3) throughout, and explore the effect of different initial mass ratios on the evolution of the system. We calculate the gravitational radiation signal in the quadrupole approximation. Our calculations are carried out using a Smooth Particle Hydrodynamics (SPH) code.Comment: Replaces previous version which had figures separate from the text of the paper. Now 47 pages long with 19 embedded figures (the figures are the same, they were renumbered) Uses aaspp4.st

Epstein-Barr virus-specific methylation of human genes in gastric cancer cells

<p>Abstract</p> <p>Background</p> <p>Epstein-Barr Virus (EBV) is found in 10% of all gastric adenocarcinomas but its role in tumor development and maintenance remains unclear. The objective of this study was to examine EBV-mediated dysregulation of cellular factors implicated in gastric carcinogenesis.</p> <p>Methods</p> <p>Gene expression patterns were examined in EBV-negative and EBV-positive AGS gastric epithelial cells using a low density microarray, reverse transcription PCR, histochemical stains, and methylation-specific DNA sequencing. Expression of PTGS2 (COX2) was measured in AGS cells and in primary gastric adenocarcinoma tissues.</p> <p>Results</p> <p>In array studies, nearly half of the 96 human genes tested, representing 15 different cancer-related signal transduction pathways, were dysregulated after EBV infection. Reverse transcription PCR confirmed significant impact on factors having diverse functions such as cell cycle regulation (<it>IGFBP3</it>, <it>CDKN2A, CCND1, HSP70, ID2, ID4)</it>, DNA repair <it>(BRCA1, TFF1</it>), cell adhesion (<it>ICAM1</it>), inflammation (<it>COX2</it>), and angiogenesis (<it>HIF1A</it>). Demethylation using 5-aza-2'-deoxycytidine reversed the EBV-mediated dysregulation for all 11 genes listed here. For some promoter sequences, CpG island methylation and demethylation occurred in an EBV-specific pattern as shown by bisulfite DNA sequencing. Immunohistochemistry was less sensitive than was western blot for detecting downregulation of COX2 upon EBV infection. Virus-related dysregulation of COX2 levels <it>in vitro </it>was not recapitulated <it>in vivo </it>among naturally infected gastric cancer tissues.</p> <p>Conclusions</p> <p>EBV alters human gene expression in ways that could contribute to the unique pathobiology of virus-associated cancer. Furthermore, the frequency and reversability of methylation-related transcriptional alterations suggest that demethylating agents have therapeutic potential for managing EBV-related carcinoma.</p

Discovering the Data of Safety: Embry-Riddle’s Aviation Safety and Security Archives

The path to the sky and beyond has not been simple or obstacle-free, but dedicated dreamers have worked to overcome obstacles, learn from mishaps, and develop new technologies to achieve their goals. As the leading university for aviation and aerospace education, Embry-Riddle Aeronautical University maintains a firm commitment to the practice and study of safety. As part of this mission, the university has established the Aviation Safety and Security Archives (ASASA) which is a national treasure of aviation safety history and information