Hox genes regulate the onset of Tbx5 expression in the forelimb.

Tbx4 and Tbx5 are two closely related T-box genes that encode transcription factors expressed in the prospective hindlimb and forelimb territories, respectively, of all jawed vertebrates. Despite their striking limb type-restricted expression pattern, we have shown that these genes do not participate in the acquisition of limb type-specific morphologies. Instead, Tbx4 and Tbx5 play similar roles in the initiation of hindlimb and forelimb outgrowth, respectively. We hypothesized that different combinations of Hox proteins expressed in different rostral and caudal domains of the lateral plate mesoderm, where limb induction occurs, might be involved in regulating the limb type-restricted expression of Tbx4 and Tbx5 and in the later determination of limb type-specific morphologies. Here, we identify the minimal regulatory element sufficient for the earliest forelimb-restricted expression of the mouse Tbx5 gene and show that this sequence is Hox responsive. Our results support a mechanism in which Hox genes act upstream of Tbx5 to control the axial position of forelimb formation.

Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells.

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family. Demethylation of Alu elements occurs in aging and cancer processes and has been associated with gene reactivation and genomic instability. By targeting the unmethylated SmaI site within the Alu sequence as a surrogate marker, we have quantified and identified unmethylated Alu elements on the genomic scale. Normal colon epithelial cells contain in average 25 486 +/- 10 157 unmethylated Alu's per haploid genome, while in tumor cells this figure is 41 995 +/- 17 187 (P = 0.004). There is an inverse relationship in Alu families with respect to their age and methylation status: the youngest elements exhibit the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional implications of atypical unmethylation states in Alu elements will surely contribute to decipher genomic organization and gene regulation in complex organisms.

Genomic and transcriptomic prognostic factors in R0 Dukes B and C colorectal cancer patients.

The advent of various 'omic' technologies has increased expectations in the field of biomarkers. In an attempt to clarify how different strategies may contribute to improving prognostic classification and to identify new predictors of patient outcome we analyzed genomic and transcriptomic profiles in a series of R0 Dukes B and C colorectal carcinomas. We have compared the predictive capability of each approach against conventional clinicopathological and molecular parameters. At a genomic level, gains at 11q including amplification at 11q13 were an indicator of poorer outcome. In transcriptomic analyses we identified 68 genes whose expression levels correlated with survival (p<0.01) and included overexpression of WASF1, NFE2L2, and MMP9, and underexpression of ITGAL, TSC2, and SDF2. Gene expression levels paralleled chromosomal changes only in 56% of the genes, suggesting that, as a general trend, the direct effect of chromosomal copy number changes on gene expression levels is minimal. Classification of tumors by genomic and transcriptomic signatures resulted in non-overlapping subgroups and was not of prognostic value. We conclude that genomic and transcriptomic profiling of colorectal carcinomas may contribute as novel prognostic markers, but it does not improve outcome prediction when global profiles or signatures are considered.

Genomic determinants of prognosis in colorectal cancer.

Colorectal cancer progression is characterized by the sequential acquisition of multiple genetic aberrations. Insights into the biology of cancer cell and the development of novel methodologies have open a new frontier in the search of independent molecular factors to better predict outcome. Besides the generation of a large list of candidate markers, their applicability in routine clinical settings has been hindered by the heterogeneity of the disease. The analysis of cumulated genetic damage offers a more comprehensive measure of the cancer cell's genomic disruption and appears as a gauge of malignant potential. The prognostic application of different determinants of genomic damage is reviewed.

Rapid gynogenetic mapping of Xenopus tropicalis mutations to chromosomes.

Pilot forward genetic screens in Xenopus tropicalis have isolated over 60 recessive mutations. Here we present a simple method for mapping mutations to chromosomes using gynogenesis and centromeric markers. When coupled with available genomic resources, gross mapping facilitates evaluation of candidate genes as well as higher resolution linkage studies. Using gynogenesis, we have mapped the genetic locations of the 10 X. tropicalis centromeres, and performed fluorescence in situ hybridization to validate these locations cytologically. We demonstrate the use of this very small set of centromeric markers to map mutations efficiently to specific chromosomes. Developmental Dynamics 238:1398-1406, 2009. (c) 2009 Wiley-Liss, Inc.

Genetic instability and divergence of clonal populations in colon cancer cells in vitro.

The accumulation of multiple chromosomal abnormalities is a characteristic of the majority of colorectal cancers and has been attributed to an underlying chromosomal instability. Genetic instability is considered to have a key role in the generation of genetic and phenotypic heterogeneity in cancer cells. To shed light on the dynamics of chromosomal instability in colon cancer cells, we have analyzed genetic divergence in clonal and subclonal derivates of chromosomally unstable (SW480) and stable (HCT116, LoVo) cell lines. Conventional G-banding karyotyping and arbitrarily primed PCR (AP-PCR) fingerprinting were used to calculate genetic distances among clones and parental cells, and to trace tree-type phylogenies among individual cells and clonal cell populations. SW480 cells showed enhanced karyotypic heterogeneity in clones as compared with parental cells. Moreover, genetic clonal divergence was also increased after two consecutive episodes of single-cell cloning, demonstrating that the homogeneity induced by the bottleneck of cloning is disrupted by genetic instability during clonal expansion and, as a consequence, heterogeneity is restored. These results demonstrate genetic drift in clonal populations originated from isolated cells. The generated cell heterogeneity coupled with selection provides the grounds for the reported feasibility of pre-neoplastic and neoplastic cells to generate new phenotypic variants with increased evolutionary potential.

Determination of genomic damage in neuroblastic tumors by arbitrarily primed PCR: MYCN amplification as a marker for genomic instability in neuroblastomas.

The aim of this study is to establish an estimation of the global genomic alteration in neuroblastic tumors (ganglioneuromas, ganglioneuroblastomas and neuroblastomas) and correlate them with different clinical parameters (age, sex, diagnosis, Shimada index, proliferation index, tumor location, and 1p and v-myc avian myelocitomatosis viral-related (MYCN) status) in order to find new molecular and/or prognostic markers for neuroblastoma. To assess the genomic damage in neuroblastic tumors, we used an arbitrarily primed PCR approach, a technique based on the reproducibility of band profiles obtained by a PCR with a low annealing temperature in its first cycles. Genomic damage was assessed by comparing band profiles of tumors and normal paired samples. Gains and losses in the intensity of the bands were computerized and referred to the total number of bands analyzed. We found a higher genomic damage fraction (GDF) in the female's group (U-Mann-Whitney, P = 0.025), but we could not find any association between GDF and tumor location, proliferation index, diagnosis or age of the patient. There was no relationship between 1p status and GDF, but tumors with MYCN amplification had a slightly higher GDF. MYCN amplification might in some way contribute to genomic instability of neuroblastomas.

Chromosomal instability correlates with genome-wide DNA demethylation in human primary colorectal cancers.

DNA hypomethylation is a common trait of colorectal cancer. Studies in tumor cell lines and animal models indicate that genome-wide demethylation may cause genetic instability and hence facilitate or accelerate tumor progression. Recent studies have shown that DNA hypomethylation precedes genomic damage in human gastrointestinal cancer, but the nature of this damage has not been clearly established. Here, we show a thorough analysis of DNA methylation and genetic alterations in two series of colorectal carcinomas. The extent of DNA demethylation but not of hypermethylation (both analyzed by amplification of intermethylated sites in near 200 independent sequences arbitrarily selected) correlated with the cumulated genomic damage assessed by two different techniques (arbitrarily primed PCR and comparative genomic hybridization). DNA hypomethylation-related instability was mainly of chromosomal nature and could be explained by a genome-wide effect rather than by the concurrence of the most prevalent genetic and epigenetic alterations. Moreover, the association of p53 mutations with genomic instability was secondary to DNA hypomethylation and the correlation between DNA hypomethylation and genomic instability was observed in tumors with and without mutation in the p53 gene. Our data support a direct link between genome-wide demethylation and chromosomal instability in human colorectal carcinogenesis and are consistent with the studies in model systems demonstrating a role of DNA demethylation in inducing chromosomal instability.

Validation of RNA arbitrarily primed PCR probes hybridized to glass cDNA microarrays: application to the analysis of limited samples.

BACKGROUND: The applicability of microarray-based transcriptome massive analysis is often limited by the need for large amounts of high-quality RNA. RNA arbitrarily primed PCR (RAP-PCR) is an unbiased fingerprinting PCR technique that reduces both the amount of initial material needed and the complexity of the transcriptome. The aim of this study was to evaluate the feasibility of using hybridization of RAP-PCR products as transcriptome representations to analyze differential gene expression in a microarray platform. METHODS: RAP-PCR products obtained from samples with limited availability of biological material, such as experimental metastases, were hybridized to conventional cDNA microarrays. We performed replicates of self-self hybridizations of RAP-PCR products and mathematical modeling to assess reproducibility and sources of variation. RESULTS: Gene/slide interaction (47.3%) and the PCR reaction (33.8%) accounted for the majority of the variability. From these observations, we designed a protocol using two pools of three independent RAP-PCR reactions coming from two independent reverse transcription reactions hybridized in duplicate and evaluated them in the analyses of paired xenograft-metastases samples. Using this approach, we found that HER2 and MMP7 may be down-regulated during distal dissemination of colorectal tumors. CONCLUSION: RAP-PCR glass array hybridization can be used for transcriptome analysis of small samples.