Identification and validation of colorectal neoplasia-specific methylation markers for accurate classification of disease

Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis.     

VEGF receptor 2/‐3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/‐3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/‐3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.     

Epidermal growth factor receptor-dependent control of keratinocyte survival and Bcl-xL expression through a MEK-dependent pathway

Previous work has shown that the epidermal growth factor receptor (EGFR) tyrosine kinase moiety provides protection to normal human keratinocytes against apoptosis. This protection is, at least in part, due to EGFR-dependent expression of the antiapoptotic Bcl-2 family member, Bcl-x(L). Here we focused on intracellular signaling pathways relevant to keratinocyte survival and/or Bcl-x(L) expression. By using pharmacological inhibitors and dominant negative expression constructs, we observed that phosphatidylinositol 3-kinase/AKT and phospholipase C gamma/protein kinase C alpha activation were required for keratinocyte survival independently of EGFR activation or Bcl-x(L) expression. By contrast, MEK activity required EGFR activation and, as shown by use of the MEK inhibitor PD98059 and a dominant negative MEK construct, was necessary for Bcl-x(L) expression and survival. Consistent with an earlier study, blocking SRC kinase activities similarly led to down-regulation of Bcl-x(L) protein expression and impaired keratinocyte survival. In conclusion, our results demonstrate that EGFR-dependent MEK activity contributes to both Bcl-x(L) expression and survival of normal keratinocytes. Other signaling pathways (i.e. phosphatidylinositol 3-kinase/AKT and phospholipase C gamma/protein kinase C alpha) are obligatory to keratinocyte survival but not to Bcl-x(L) expression, and control of these pathways by EGFR activation is not rate-limiting to normal keratinocyte survival.     

The evolution of cellular computing: nature's solution to a computational problem

How do cells and nature 'compute'? They read and 'rewrite' DNA all the time, by processes that modify sequences at the DNA or RNA level. In 1994, Adleman's elegant solution to a seven-city directed Hamiltonian path problem using DNA launched the new field of DNA computing, which in a few years has grown to international scope. However, unknown to this field, two ciliated protozoans of the genus Oxytricha had solved a potentially harder problem using DNA several million years earlier. The solution to this problem, which occurs during the process of gene unscrambling, represents one of nature's ingenious solutions to the problem of the creation of genes. RNA editing, which can also be viewed as a computational process, offers a second algorithm for the construction of functional genes from encrypted pieces of the genome.     

Genome-wide linkage analysis replicates susceptibility locus for fasting plasma triglycerides: NHLBI Family Heart Study

Recent reports implicate chromosomal regions linked to inter-individual variation in plasma triglycerides. We conducted genome-wide scans to replicate these linkages and/or identify other loci influencing plasma triglycerides in the NHLBI Family Heart Study (FHS). Data were obtained for 501 three-generational families. Genotyping was done by the Utah Molecular Genetics Laboratory and NHLBI Mammalian Genotyping Service; markers from both were placed on one genetic map. Analysis was done using multipoint variance components linkage. Fasting plasma triglycerides were log-transformed and age-, sex-, and field center-adjusted; suggestive linkage evidence was found on chromosome 8 (LOD=2.80 at 89 cM, marker D8S1141). Further adjustment for waist girth, BMI, diabetes, hypertension, and lipid-lowering drugs suggested linkage regions on chromosomes 6 (LOD=2.29 at 79 cM, marker D6S295) and 15 (LOD=1.85 at 43 cM, marker D15S659). Since HDL is correlated with triglycerides and because it was linked to this region on chromosome 15 in FHS, we created a composite triglyceride–HDL phenotype. The combined phenotype LOD score was 3.0 at the same marker on chromosome 15. Chromosome 15 likely harbors a susceptibility locus with an influence on triglycerides and HDL. Regions on chromosomes 6 and 8 may also contain loci contributing to inter-individual variation in plasma triglycerides.     

A locus for circadian period of locomotor activity on mouse proximal chromosome 3

Lengthened circadian period of locomotor activity is a characteristic of a congenic strain of mice carrying a nonsense mutation in exon 5 of the carbonic anhydrase II gene, car2. The null mutation in car2 is located on a DBA/2J inbred strain insert on proximal chromosome 3, on an otherwise C57BL/6J genomic background. Since reducing the size of the congenic region would narrow the possible candidate genes for period, two recombinant congenic strains (R1 and R2) were developed from the original congenic strain. These new congenic strains were assessed for period, genetic composition, and the presence of immunoreactive carbonic anhydrase II. R1 mice were homozygous DBA/2J for the distal portion of the original DBA/2J insert, while R2 mice were homozygous DBA/2J for the proximal portion. R1 mice had a significantly lengthened period compared to R2 mice and wild-type C57BL/6J mice, indicating that the gene(s) affecting period is likely found within the reduced DBA/2J insert (~1 cM) in the R1 mice. The R1 mice also possessed the null mutation in car2. This study confirmed the presence of a gene(s) affecting period on proximal chromosome 3 and significantly reduced the size of the congenic region and the number of candidate genes. Future studies will focus on identifying the gene influencing period.