Epigenetics of lung cancer

Epigenetics is the study of heritable changes in gene expression that occur without changes in DNA sequence. It has a role in determining when and where a gene is expressed during development. Perhaps the most well known epigenetic mechanism is DNA methylation whereby cytosines at position 5 in CpG dinucleotides are methylated. Histone modification is another form of epigenetic control, which is quite complex and diverse. Histones and DNA make up the nucleosome which is the structural unit of chromatin which are involved in packaging DNA. Apart from the crucial role epigenetics plays in embryonic development, transcription, chromatin structure, X chromosome inactivation and genomic imprinting, its role in an increasing number of human diseases is more and more recognized. These diseases include cancer, and lung cancer in particular has been increasingly studied for the potential biological role of epigenetic changes with the promise of better and novel diagnostic and therapeutic tools.

Heterologous RNA encapsidated in pariacoto virus-like particles forms a dodecahedral cage similar to genomic RNA in wild-type virions

The genome of some icosahedral RNA viruses plays an essential role in capsid assembly and structure. In T=3 particles of the nodavirus Pariacoto virus (PaV), a remarkable 35% of the single-stranded RNA genome is icosahedrally ordered. This ordered RNA can be visualized at high resolution by X-ray crystallography as a dodecahedral cage consisting of 30 24-nucleotide A-form RNA duplex segments that each underlie a twofold icosahedral axis of the virus particle and interact extensively with the basic N-terminal region of 60 subunits of the capsid protein. To examine whether the PaV genome is a specific determinant of the RNA structure, we produced virus-like particles (VLPs) by expressing the wild-type capsid protein open reading frame from a recombinant baculovirus. VLPs produced by this system encapsidated similar total amounts of RNA as authentic virus particles, but only about 6% of this RNA was PaV specific, the rest being of cellular or baculovirus origin. Examination of the VLPs by electron cryomicroscopy and image reconstruction at 15.4-Angstrom resolution showed that the encapsidated RNA formed a dodecahedral cage similar to that of wild-type particles. These results demonstrate that the specific nucleotide sequence of the PaV genome is not required to form the dodecahedral cage of ordered RNA.

Probing the S100 protein family through genomic and functional analysis

The EF-hand superfamily of calcium binding proteins includes the S100, calcium binding protein, and troponin subfamilies. This study represents a genome, structure, and expression analysis of the S100 protein family, in mouse, human, and rat. We confirm the high level of conservation between mammalian sequences but show that four members, including S100A12, are present only in the human genome. We describe three new members of the S100 family in the three species and their locations within the S100 genomic clusters and propose a revised nomenclature and phylogenetic relationship between members of the EF-hand superfamily. Two of the three new genes were induced in bone-marrow-derived macrophages activated with bacterial lipopolysaccharide, suggesting a role in inflammation. Normal human and murine tissue distribution profiles indicate that some members of the family are expressed in a specific manner, whereas others are more ubiquitous. Structure-function analysis of the chemotactic properties of murine S100A8 and human S100A12, particularly within the active hinge domain, suggests that the human protein is the functional homolog of the murine protein. Strong similarities between the promoter regions of human S100A12 and murine S100A8 support this possibility. This study provides insights into the possible processes of evolution of the EF-hand protein superfamily. Evolution of the S100 proteins appears to have occurred in a modular fashion, also seen in other protein families such as the C2H2-type zinc-finger family. (C) 2004 Elsevier Inc. All rights reserved.

The molecular genetics of breast cancer: The contribution of comparative genomic hybridization

Comparative genomic hybridization (CGH) has been the technique of choice over the last 10 years for mapping DNA copy number changes in human tumors. Here we review the literature to demonstrate how CGH has contributed to the comprehension of molecular aspects of breast tumorigenesis. At least two distinct molecular pathways of breast cancer have been characterized that show a strong correlation with histological grade. It seems that grade I invasive ductal carcinomas (IDCs) arise from well-differentiated ductal carcinoma in situ (DCIS), whereas grade III IDCs come from poorly differentiated DCIS. In addition, dedifferentiation from a low- to a high-grade breast cancer has proven an unlikely phenomenon. CGH has been instrumental in dissecting distinct molecular pathways toward breast malignancy and in establishing a direct relationship between genotype and clinical pathological features. (C) 2005 Elsevier GrnbH. All rights reserved.

Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NATI (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NATI is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5'non-coding region of NATI. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NATI expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforrns were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NATI transcripts may contribute to the variation in NATI activity in vivo.

Functional characterization and genomic organization of the human Na+-sulfate cotransporter hNaS2 gene (SLC13A4)

Sulfate plays an essential role in human growth and development. Here, we characterized the functional properties of the human Na+-sulfate cotransporter (hNaS2), determined its tissue distribution, and identified its gene (SLC13A4) structure. Expression of hNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by thiosulfate, phosphate, molybdate. selenate and tungstate, but not by oxalate, citrate, succinate, phenol red or DIDS. Transport kinetics of hNaS2 determined a K, for sulfate of 0.38 mM, suggestive of a high affinity sulfate transporter. Na+ kinetics determined a Hill coefficient of 1.6 +/- 0.6, suggesting a Na: SO42- stoichiometry of 2:1. hNaS2 mRNA was highly expressed in placenta and testis, with intermediate levels in brain and lower levels found in the heart, thymus, and liver. The SLC13A4 gene contains 16 exons, spanning over 47 kb in length. Its 5'-flanking region contains CAAT- and GC-box motifs, and a number of putative transcription factor binding sites, including GATA-1, AP-1, and AP-2 consensus sequences. This is the first study to characterize hNaS2 transport kinetics, define its tissue distribution, and resolve its gene (SLC13A4) structure and 5' flanking region. (C) 2004 Elsevier Inc. All rights reserved.

Chloroplast and total genomic diversity in the endemic Costa Rican tree Lonchocarpus costaricensis (J.D. Smith) Pittier (Papilionaceae).

In Mesoamerica, tropical dry forest is a highly threatened habitat, and species endemic to this environment are under extreme pressure. The tree species, Lonchocarpus costaricensis is endemic to the dry northwest of Costa Rica and southwest Nicaragua. It is a locally important species but, as land has been cleared for agriculture, populations have experienced considerable reduction and fragmentation. To assess current levels and distribution of genetic diversity in the species, a combination of chloroplast-specific (cpDNA) and whole genome DNA markers (amplified fragment length polymorphism, AFLP) were used to fingerprint 121 individual trees in 6 populations. Two cpDNA haplotypes were identified, distributed among populations such that populations at the extremes of the distribution showed lowest diversity. A large number (487) of AFLP markers were obtained and indicated that diversity levels were highest in the two coastal populations (Cobano, Matapalo, H = 0.23, 0.28 respectively). Population differentiation was low overall, F-ST = 0.12, although Matapalo was strongly differentiated from all other populations (F-ST = 0.16-0.22), apart from Cobano (F., = 0.11). Spatial genetic structure was present in both datasets at different scales: cpDNA was structured at a range-wide distribution scale, whilst AFLP data revealed genetic neighbourhoods on a population scale. In general, the habitat degradation of recent times appears not to have yet impacted diversity levels in mature populations. However, although no data on seed or saplings were collected, it seems likely that reproductive mechanisms in the species will have been affected by land clearance. It is recommended that efforts should be made to conserve the extant genetic resource base and further research undertaken to investigate diversity levels in the progeny generation.

GONOME: measuring correlations between GO terms and genomic positions

Background: Current methods to find significantly under- and over-represented gene ontology (GO) terms in a set of genes consider the genes as equally probable balls in a bag, as may be appropriate for transcripts in micro-array data. However, due to the varying length of genes and intergenic regions, that approach is inappropriate for deciding if any GO terms are correlated with a set of genomic positions. Results: We present an algorithm - GONOME - that can determine which GO terms are significantly associated with a set of genomic positions given a genome annotated with (at least) the starts and ends of genes. We show that certain GO terms may appear to be significantly associated with a set of randomly chosen positions in the human genome if gene lengths are not considered, and that these same terms have been reported as significantly over-represented in a number of recent papers. This apparent over-representation disappears when gene lengths are considered, as GONOME does. For example, we show that, when gene length is taken into account, the term development is not significantly enriched in genes associated with human CpG islands, in contradiction to a previous report. We further demonstrate the efficacy of GONOME by showing that occurrences of the proteosome-associated control element (PACE) upstream activating sequence in the S. cerevisiae genome associate significantly to appropriate GO terms. An extension of this approach yields a whole-genome motif discovery algorithm that allows identification of many other promoter sequences linked to different types of genes, including a large group of previously unknown motifs significantly associated with the terms 'translation' and 'translational elongation'. Conclusion: GONOME is an algorithm that correctly extracts over-represented GO terms from a set of genomic positions. By explicitly considering gene size, GONOME avoids a systematic bias toward GO terms linked to large genes. Inappropriate use of existing algorithms that do not take gene size into account has led to erroneous or suspect conclusions. Reciprocally GONOME may be used to identify new features in genomes that are significantly associated with particular categories of genes.

Microarray-based comparative genomic hybridisation of breast cancer patients receiving neoadjuvant chemotherapy

We analysed the molecular genetic profiles of breast cancer samples before and after neoadjuvant chemotherapy with combination doxorubicin and cyclophosphamide (AC). DNA was obtained from microdissected frozen breast core biopsies from 44 patients before chemotherapy. Additional samples were obtained before the second course of chemotherapy (D21) and after the completion of the treatment (surgical specimens) in 17 and 21 patients, respectively. Microarray-based comparative genome hybridisation was performed using a platform containing approx5800 bacterial artificial chromosome clones (genome-wide resolution: 0.9 Mb). Analysis of the 44 pretreatment biopsies revealed that losses of 4p, 4q, 5q, 12q13.11–12q13.12, 17p11.2 and 17q11.2; and gains of 1p, 2p, 7q, 9p, 11q, 19p and 19q were significantly associated with oestrogen receptor negativity. 16q21–q22.1 losses were associated with lobular and 8q24 gains with ductal types. Losses of 5q33.3–q4 and 18p11.31 and gains of 6p25.1–p25.2 and Xp11.4 were associated with HER2 amplification. No correlations between DNA copy number changes and clinical response to AC were found. Microarray-based comparative genome hybridisation analysis of matched pretreatment and D21 biopsies failed to identify statistically significant differences, whereas a comparison between matched pretreatment and surgical samples revealed a statistically significant acquired copy number gain on 11p15.2–11p15.5. The modest chemotherapy-driven genomic changes, despite profound loss of cell numbers, suggest that there is little therapeutic selection of resistant non-modal cell lineages.

Lineages of acidophilic archaea revealed by community genomic analysis

Novel, low-abundance microbial species can be easily overlooked in standard polymerase chain reaction (PCR)-based surveys. We used community genomic data obtained without PCR or cultivation to reconstruct DNA fragments bearing unusual 16S ribosomal RNA ( rRNA) and protein-coding genes from organisms belonging to novel archaeal lineages. The organisms are minor components of all biofilms growing in pH 0.5 to 1.5 solutions within the Richmond Mine, California. Probes specific for 16S rRNA showed that the fraction less than 0.45 micrometers in diameter is dominated by these organisms. Transmission electron microscope images revealed that the cells are pleomorphic with unusual folded membrane protrusions and have apparent volumes of < 0.006 cubic micrometer.

Genomic and proteomic analysis of synaptic protein expression in the brains of human alcoholics

Alcoholism results in changes in the human brain which reinforce the cycle of craving and dependency, and these changes are manifest in the pattern of expression of mRNA and proteins in key cells and brain areas. Long-term alcohol abuse also results in damage to selected regions of the cortex. We have used cDNA microarrays to show that less than 1% of mRNA transcripts differ signifi cantly between cases and controls in the susceptible area and that the expression profi le of a subset of these transcripts is suffi cient to distinguish alcohol abusers from controls. In addition, we have utilized a 2D gel proteomics based approach to determine the identity of proteins in the superior frontal cortex (SFC) of the human brain that show differential expression in controls and long term alcohol abusers. Overall, 182 proteins differed by the criterion of > 2-fold between case and control samples. Of these, 139 showed signifi cantly lower expression in alcoholics, 35 showed signifi cantly higher expression, and 8 were new or had disappeared. To date 63 proteins have been identifi ed. The expression of one family of proteins, the synucleins, has been further characterized using Real Time PCR and Western Blotting. The expression of alpha-synuclein mRNA was signifi cantly lower in the SFC of alcoholics compared with the same area in controls (P = 0.01) whereas no such difference in expression was found in the motor cortex. The expression of beta- and gamma- synuclein were not signifi cantly different between alcoholics and controls. In contrast, the pattern of alphasynuclein protein expression differs from that of the corresponding RNA transcript. Because of the key role of synaptic proteins in the pathogenesis of alcoholism, we are developing 2-D DIGE based techniques to quantify expression changes in synaptosomes prepared from the SFC of controls and alcoholics.