Antiproliferative Effects of DNA Methyltransferase 3B Depletion Are Not Associated with DNA Demethylation

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs

Participacija in vsebina nizozemskih strankarskih razpravljalskih skupin na Internetu

Članek obravnava dve politični razpravljalski skupini, povezani z nizozemskimastrankama D66 in GroenLinks, in njun prispevek k demokraciji. Razpravljanje naj bi bilo v demokraciji posvetovalno: temeljilo naj bi na racionalni argumentaciji, naj bi ne bilo podvrženo monopolu posameznikov, povezano naj bi bilo z javnimi zadevami. Članek proučuje posvetovalnost političnega razpravljanja na internetu, tako da meri prispevek posameznega udeleženca. Rezultati kažejo, da sta imeli obe listi le majhen delež zelo pogostih udeležencev. Prispevki razpravljavcev so bili usmerjeni k drugim razpravljavcem v skupini. Mnenja so bila običajno izražena brez argumentov, čepa so bili argumenti že izraženi, so bili zdravorazumski ali pa so izhajali iz časopisov ali teleteksta. Razpravljalna foruma nista upravičila pričakovanjglede pospeševanja demokracije. Lahko pa zapolnjujeta prostor med institucionaliziranim javnim razpravljanjem, ki poteka v strankarskih elitah, in neinstitucionaliziranim, neformalnim javnim razpravljanjem, ki izvira iz drugih javnih in zasebnih sfer.This study examines two political discussion lists affiliated with the Dutch political parties D66 and GroenLinks, and the possible contribution of these lists to democracy. Discussion in a democracy, it is argued, should be deliberative: based on rational argumentation, not monopolised by any particular individuals, and related to public affairs. The aim of this study is to assess the deliberativeness of political discussion lists on the Internet. To this end, the degree of contribution from participants to the lists is measured. The findings from this study suggest that, whereas the discussions on the lists as a whole were not monopolised by any individual, both lists had only a small number of very frequent participants. The contributions of members of this in-group were oriented towards one another. Opinions were mainly expressed without argumentation, and when argumentation was given it was predominantly based on common sense or on external sources such as newspapers or teletext. These two discussion lists do not live up to the expectations of furthering democracy. They may, however, eventually serve to fill the gap between the institutionalised public discussion that exists within the party elite and the uninstitutionalised, informal public discussion that transpires in other public and private domains

The hypermethylation phenotype is maintained in DNMT3B knockout cells.

<p>(A) Heatmap based on Infinium 27 k data illustrating the methylation levels of 67 CIMP marker genes in HCT-116 colon cancer cells and HCT-116 DNMT3B knockout cells (3BKO). (B) Boxplot illustrating methylation levels of 67 CIMP marker genes. Lines in boxes denote medians, boxes the interquartile range, and whiskers the 2.5th and 97.5th percentiles, respectively. (C) Heatmap based on Infinium 27 k data illustrating the methylation levels of 543 hypermethylated PRC2 target genes in HCT-116 colon cancer cells and HCT-116 DNMT3B knockout cells (3BKO). (D) Boxplot illustrating methylation levels of PRC2 target genes. AVB, Average Beta value.</p

The hypermethylation phenotype is reversed in HCT-116 DNMT1; DNMT3B double knockout cells.

<p>(A, B) Boxplots illustrating the methylation levels of CIMP (A) and PRC2 (B) marker genes in parental HCT-116 cells (HCT-116), HCT-116 cells lacking DNMT3B (3BKO), HCT-116 cells with a hypomorphic DNMT1 allele (1KO), and HCT-116 DNMT1; DNMT3B double knockout cells (DKO) on Infinium 27 k methylation arrays. Lines in boxes denote medians, boxes the interquartile range, and whiskers the 2.5th and 97.5th percentiles. ***P<0.001 relative to parental HCT116 cells, determined by a Wilcoxon rank sum test. (C) Model for the role of DNMT3B during cancer development and progression. In normal cells, CpG islands are generally unmethylated and the corresponding genes are actively transcribed. During cancer development, certain regions become methylated by de novo methyltransferases such as DNMT3A and DNMT3B. Once the hypermethylation phenotype is established, the methylation pattern is maintained by the maintenance methyltransferase DNMT1 and becomes independent of DNMT3 enzymes. Only a strong reduction of overall methyltransferase activity causes the demethylation of hypermethylated cancer genes and inhibits the re-establishment by de novo methyltransferases.</p

Efficient depletion of DNMT3B has no effect on DNA methylation.

<p>(A) DNMT3B mRNA levels were determined after siRNA knockdown with DNMT3B siRNA #4 by quantitative RT-PCR analysis. Expression values are means of triplicates and were calculated relative to Lamin B1 expression. Error bars represent standard errors. ***P<0.001 relative to control siRNA, determined by student's t-test prior to normalization. (B) Comparison of Infinium 27 k methylation profiles of HCT-116 cells transfected with DNMT3B siRNA #4 or control siRNA. (C) Efficient depletion of DNMT3B protein in stably shRNA-transduced HCT-116 cells. DNMT3B protein levels were determined by immunoblot analysis using ß-actin as a loading control. The double band presumably reflects the expression of two (or more) DNMT3B isoforms. (D) Comparison of Infinium 27 k methylation profiles between HCT-116 cells stably transduced with lentiviruses containing DNMT3B shRNA #1 and control cells transfected with a control shRNA. (E) Infinium 450 k methylation analysis comparing methylation profiles of HCT-116 cells transduced with DNMT3B-shRNA #1 and control cells transduced with a non-targeting control shRNA. (F) Heatmap based on the Infinium 450 k data, showing hypermethylated CIMP markers in HCT-116 cells stably transduced with shRNA #1 or #2 targeting DNMT3B or with a non-targeting control shRNA. AVB, Average Beta value.</p

DNMT3B expression in human cancer tissue and colon cancer cell lines.

<p>(A) Array Northern analysis of DNMT3B mRNA expression on Affymetrix HGU133Plus2.0 arrays in tumor (T) tissue samples compared to normal (N) tissues of the same organ. n indicates the number of analyzed samples for the specified tissue type. Data are shown as mean±SD; *P<0.05 relative to normal tissue (N), **P<0.01 relative to normal tissue (N), ***P<0.001 relative to normal tissue (N), determined by student's t-test. (B) Quantitative real-time PCR analysis of DNMT3B mRNA expression in colon cancer cell lines and normal cells (HMEC-T53, WI-38). Expression values are means of triplicates and were calculated relative to GAPDH expression. Error bars represent standard errors. (C) Immunoblot of DNMT3B protein expression in lysates from various colon cancer cell lines and normal cells (HMEC-T53, WI-38). ß-actin was used as a loading control.</p

TRANSFORMATION AND RESOURCES IN THE "NEW" GEO-ECONOMY

The essay addresses the big changes that took place in the geo-economy of manufacturing as a result of the amazing growth of the Asian economies, especially China’s, over the last three decades. The indicators used in order to assess the extent of the change are the consumption data of 11 industrial raw materials (the 6 main non-ferrous metals: copper, aluminium, lead, zinc, tin and nickel; plus crude steel, rubber, plastic materials, wood-based panels, paper and paperboard) in the G-20 countries. The figures were compared to the data of the evolution of the manufacturing output worldwide to measure the degree of shifting of its center of gravity from North America and Europe towards Asia. Both statistical methods lead to the conclusion that the distribution of the manufacturing output in the world has been changing remarkably in the last 10 to 15 years, moving eastward: while in the year 2000 the United States still held 24.8% of the world output in current dollars, in 2007, though still at the top, its share dropped to 18.2% and in 2010 China ranked first with a 21.7% share. After Japan and Germany, that retain the third and fourth positions respectively among manufacturing producers, India and South Korea rose sharply to the fifth and sixth positions. Italy ranked seventh. At the same time a progressive rise was observed in basic product consumption in industrial processing. The United States, the first world consumer in 9 basic products out of 11 in 1980, lost its record in the use of industrial materials in 2010, replaced by China. As regards Italy, in Europe our country was second after Germany in the use of industrial materials throughout the period from 1980 to 2010