Presencia de metales pesados en insectos comestibles

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New Topoisomerase I mutations are associated with resistance to camptothecin

<p>Abstract</p> <p>Background</p> <p>Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance.</p> <p>Methods</p> <p>We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of <it>TOP1 </it>mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing.</p> <p>Results</p> <p>In SN38 resistant HCT116 clones we identified three new <it>TOP1 </it>mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these <it>TOP1 </it>mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells.</p> <p>Conclusions</p> <p>These results indicate that the <it>TOP1 </it>mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.</p

Specific extracellular matrix remodeling signature of colon hepatic metastases.

To identify genes implicated in metastatic colonization of the liver in colorectal cancer, we collected pairs of primary tumors and hepatic metastases before chemotherapy in 13 patients. We compared mRNA expression in the pairs of patients to identify genes deregulated during metastatic evolution. We then validated the identified genes using data obtained by different groups. The 33-gene signature was able to classify 87% of hepatic metastases, 98% of primary tumors, 97% of normal colon mucosa, and 95% of normal liver tissues in six datasets obtained using five different microarray platforms. The identified genes are specific to colon cancer and hepatic metastases since other metastatic locations and hepatic metastases originating from breast cancer were not classified by the signature. Gene Ontology term analysis showed that 50% of the genes are implicated in extracellular matrix remodeling, and more precisely in cell adhesion, extracellular matrix organization and angiogenesis. Because of the high efficiency of the signature to classify colon hepatic metastases, the identified genes represent promising targets to develop new therapies that will specifically affect hepatic metastasis microenvironment

Clinical characteristics of patients included in the training set.

<p>Clinical characteristics of patients included in the training set.</p

Expression of identified genes in three studies.

<p>Datasets from three intendant studies were renormalized together using an empirical Bayes method. Among the 33 genes of the gene signature, 32 genes were present in our (red), Sheffer (green) and Kleivi (blue) datasets (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone-0074599-t004" target="_blank">Table 4</a>). The log2 ratio of HM to CT is plotted. Genes were ordered from the most downregulated to the most upregulated gene in HM versus CT in our study.</p

Functional annotation enrichment analysis of the 33-gene signature.

<p>Gene Ontology terms significantly over-represented in the 33-gene signature were identified and plotted using ClueGO. A) The size of the nodes are inversely proportional to the pvalue in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone-0074599-g005" target="_blank">Fig. 5B</a>. Line widths between GO terms are proportional to the kappa scores used to define the categories. B) Table giving the results of the ClueGO analysis. Nr: Number of genes in our 33-gene signature associated with the GO term.%: Percentage of the genes of the considered GO term presents in our signature. Pvalue: pvalue of the GO term, corrected for multiple testing. C) Relation between the GO annotations of the 11 “cell adhesion”-associated genes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone-0074599-g005" target="_blank">Fig. 5B</a> and GO:0007155 term. Black arrows: “is a”. Green arrows: “Positively regulates”.</p

External validation.

<p>nc: non correctly classified</p>1<p>Number of the genes of our 33 gene signature present on the used platform.</p>2<p>Number of samples correctly classified using our signature (restricted to the number of genes in column “Nb genes”).</p>3<p>Genes in common between our 33 gene signature and those published in each study (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone-0074599-t002" target="_blank">Table 2</a> in Ki, Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone-0074599-t002" target="_blank">Table 2</a> in Koh, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone-0074599-g002" target="_blank">Fig. 2</a> in Kleivi, Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone-0074599-t002" target="_blank">Table 2</a> in Lin).</p>4<p>Ten of the CN samples are from the 13 paired patients used to identify the 34-probe signature.</p>5<p>CT and CP are not clearly separated and are in a single class (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone-0074599-g003" target="_blank">Fig. 3</a>).</p>6<p>LN and LM clustered in independent groups but were not separated from CT (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone-0074599-g003" target="_blank">Fig. 3</a>).</p>7<p>27 CT with synchronous and 20 CT with metachronous metastases.</p>8<p>12 rectum and 3 colon tumors.</p>9<p>HM and LM are not separated and are in a single class.</p>10<p>8 right and 5 left colon tumors. 5 rectum tumors.</p>11<p>Only SPP1 is in the top 35 ranking genes in Lin's study (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone-0074599-g001" target="_blank">Fig. 1</a> in Lin).</p

Identification of the 34-probe gene signature.

<p>The CT/HM values for the 13 pairs of samples and the 34 probes identified using paired SAM analysis were plotted versus the CT/HN values obtained in one paired sample from Sheffer's study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone.0074599-Sheffer1" target="_blank">[7]</a>. The horizontal axis and the diagonal (HM = HN) separate the graph in four quadrants. (a) Genes over-expressed in HM versus CT, and downregulated in HM versus HN. (b) Genes over-expressed in HM versus CT, and in HM versus HN. (c) Genes downregulated in HM versus CT, and over-expressed in HM versus HN. (d) Genes downregulated in HM versus CT, and in HM versus HN. The red dashed line corresponds to a simulated contamination of a CT sample with 50% of a HN sample (see equation 1 in results section). The hatched region corresponds to HM samples that either are expressed at a comparable level (less than a 2-fold difference) in HM and CT or HM and HN, or whose variation between CT and HM can be explained by a contamination of HM by HN.</p

Hierarchical clustering of colon validation set.

<p>Data collected, hybridized and normalized by Sheffer et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074599#pone.0074599-Sheffer1" target="_blank">[7]</a> were clustered using our 34-probe signature.</p