Modification of tryptophan and methionine residues is implicated in the oxidative inactivation of surfactant protein B

Exposing BLES (bovine lipid extract surfactant), a clinical surfactant, to reactive oxygen species (ROS) alters surfactant protein B (SP-B), as indicated by Coomassie Blue staining, silver staining, and Western analysis. Hypochlorous acid (HOCl) treatment leads to elevated maximum surface tension (gammamax) and a deterioration in minimum gamma (gammamin) during surface area cycling. Fenton reaction resulted in immediate increases in gammamin and gammamax. Intrinsic fluorescence measurements indicated Fenton, but not HOCl, induced conversion of Trp9 of SP-B to hydroxyTrp (OHTrp), N-formylkynurenine (NFKyn), and kynurenine (Kyn). Electrospray ionization mass spectrometry (ESI-MS) revealed molecular weight alterations consistent with oxidation of Met (HOCl, Fenton) and Trp (Fenton) residues. Oxidative alterations to Met29 and Met65 (HOCl, Fenton) and to Trp9 (OHTrp with HOCL and NFKyn plus Kyn with Fenton) were confirmed by matrix-assisted laser desorption mass spectrometry (MALDI-MS) studies on SP-B tryptic fragments. Some Met oxidation was observed with control SP-B. When taken together with captive bubble tensiometer measurements, these studies suggest that Met oxidation of SP-B by HOCl or Fenton interferes with phospholipid respreading during compression-expansion of surfactant films, while Fenton oxidation, which produces more extensive Met oxidation and disruption of the indole ring of Trp9, further abrogated the ability of such films to attain low surface tensions during compression. These studies provide insight into the manner by which ROS generated during acute lung injury and the acute respiratory distress syndrome act to inhibit not only endogenous surfactant but also therapeutic surfactants administered to counteract these conditions.     

Estimating genomic instability mediated by Alu retroelements in breast cancer

Alu-PCR is a relatively simple technique that can be used to investigate genomic instability in cancer. This technique allows identification of the loss, gain or amplification of gene sequences based on the analysis of segments between two Alu elements coupled with quantitative and qualitative analyses of the profiles obtained from tumor samples, surgical margins and blood. In this work, we used Alu-PCR to identify gene alterations in ten patients with invasive ductal breast cancer. Several deletions and insertions were identified, indicating genomic instability in the tumor and adjacent normal tissue. Although not associated with specific genes, the alterations, which involved chromosomal bands 1p36.23, 1q41, 11q14.3, 13q14.2, occurred in areas of well-known genomic instability in breast and other types of cancer. These results indicate the potential usefulness of Alu-PCR in identifying altered gene sequences in breast cancer. However, caution is required in its application since the Alu primer can produce non-specific amplification.     

Iron and carcinogenesis: from Fenton reaction to target genes

Abstract

Reactive oxygen species (ROS) have been shown to be associated with a wide variety of pathological phenomena such as carcinogenesis, inflammation, radiation and reperfusion injury. Iron, the most abundant transition metal ion in our body, may work as a catalyst for the generation of ROS in pathological conditions. In the past few years, there have been great advances in the understanding of iron metabolism. These include the discoveries of iron transporters and the gene responsible for hereditary hemochromatosis. Iron overload has been shown to be associated with carcinogenesis. We recently identified the major target genes (p16^INK4A and p15^INK4B tumor suppressor genes, which encode cyclin-dependent kinase inhibitors) in a ferric nitrilotriacetate-induced rat renal carcinogenesis model, in which the Fenton reaction is induced in the renal proximal tubules. Allelic loss of the p16 gene occurs early in carcinogenesis and specifically at the p16 loci as compared with other tumor suppressor genes. This led to the novel concept of 'genomic sites vulnerable to the Fenton reaction'. Here, recent new findings on iron metabolism are reviewed and the concept of the vulnerable sites explored. More effort to link iron metabolism with human carcinogenesis is anticipated.     

The enhancer of zeste homolog 2 (EZH2), a potential therapeutic target, is regulated by miR-101 in renal cancer cells

We investigated a prognostic significance and the mechanism of aberrant nuclear expression of EZH2, a histone methyltransferase, in human renal cell carcinoma (RCC). We found nuclear EZH2 in 48 of 100 RCCs and it was significantly correlated with worse survival in RCC patients. We detected a decreased expression of miR-101 in 15 of 54 RCCs. We found that re-expression of miR-101 resulted in EZH2 depletion and decreased renal cancer cell proliferation. Our results show nuclear EZH2 as a prognostic marker of worse survival in human RCC, and identify miR-101 as a negative regulator of EZH2 expression and renal cancer cell proliferation.     

Met Is the Most Frequently Amplified Gene in Endometriosis-Associated Ovarian Clear Cell Adenocarcinoma and Correlates with Worsened Prognosis

Clear cell adenocarcinoma of the ovary (OCC) is a chemo-resistant tumor with a relatively poor prognosis and is frequently associated with endometriosis. Although it is assumed that oxidative stress plays some role in the malignant transformation of this tumor, the characteristic molecular events leading to carcinogenesis remain unknown. In this study, an array-based comparative genomic hybridization (CGH) analysis revealed Met gene amplification in 4/13 OCC primary tumors and 2/8 OCC cell lines. Amplification of the AKT2 gene, which is a downstream component of the Met/PI3K signaling pathway, was also observed in 5/21 samples by array-based CGH analysis. In one patient, both the Met and AKT2 genes were amplified. These findings were confirmed using fluorescence in situ hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemistry. In total, 73 OCC cases were evaluated using real-time quantitative PCR; 37.0% demonstrated Met gene amplification (>4 copies), and 8.2% had AKT2 amplification. Furthermore, stage 1 and 2 patients with Met gene amplification had significantly worse survival than patients without Met gene amplification (p<0.05). Met knockdown by shRNA resulted in reduced viability of OCC cells with Met amplification due to increased apoptosis and cellular senescence, suggesting that the Met signaling pathway plays an important role in OCC carcinogenesis. Thus, we believe that targeted inhibition of the Met pathway may be a promising treatment for OCC.     

Iron and carcinogenesis: from Fenton reaction to target genes

Reactive oxygen species (ROS) have been shown to be associated with a wide variety of pathological phenomena such as carcinogenesis, inflammation, radiation and reperfusion injury. Iron, the most abundant transition metal ion in our body, may work as a catalyst for the generation of ROS in pathological conditions. In the past few years, there have been great advances in the understanding of iron metabolism. These include the discoveries of iron transporters and the gene responsible for hereditary hemochromatosis. Iron overload has been shown to be associated with carcinogenesis. We recently identified the major target genes (p16(INK4A) and p15(INK4B) tumor suppressor genes, which encode cyclin-dependent kinase inhibitors) in a ferric nitrilotriacetate-induced rat renal carcinogenesis model, in which the Fenton reaction is induced in the renal proximal tubules. Allelic loss of the p16 gene occurs early in carcinogenesis and specifically at the p16 loci as compared with other tumor suppressor genes. This led to the novel concept of 'genomic sites vulnerable to the Fenton reaction'. Here, recent new findings on iron metabolism are reviewed and the concept of the vulnerable sites explored. More effort to link iron metabolism with human carcinogenesis is anticipated.     

Integration of DNA copy number alterations and transcriptional expression analysis in human gastric cancer

Background

Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level.

Principal Findings

We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridization (aCGH). Statistical analysis was applied to correlate previously published gene expression data obtained from cDNA microarrays with corresponding DNA copy number variation data to identify candidate oncogenes and tumor suppressor genes. We found that gastric cancer samples showed recurrent DNA copy number variations, including gains at 5p, 8q, 20p, 20q, and losses at 4q, 9p, 18q, 21q. The most frequent regions of amplification were 20q12 (7/72), 20q12–20q13.1 (12/72), 20q13.1–20q13.2 (11/72) and 20q13.2–20q13.3 (6/72). The most frequent deleted region was 9p21 (8/72). Correlating gene expression array data with aCGH identified 321 candidate oncogenes, which were overexpressed and showed frequent DNA copy number gains; and 12 candidate tumor suppressor genes which were down-regulated and showed frequent DNA copy number losses in human gastric cancers. Three networks of significantly expressed genes in gastric cancer samples were identified by ingenuity pathway analysis.

Conclusions

This study provides insight into DNA copy number variations and their contribution to altered gene expression profiles during human gastric cancer development. It provides novel candidate driver oncogenes or tumor suppressor genes for human gastric cancer, useful pathway maps for the future understanding of the molecular pathogenesis of this malignancy, and the construction of new therapeutic targets.     

Detection of DNA abnormalities by arbitrarily primed PCR fingerprinting: amplification of the MDM2 gene in a mediastinum fibrosarcoma.

Arbitrarily primed PCR (AP-PCR) fingerprinting method is easy and useful for analysis of genetic alterations in anonymous chromosomal regions. We applied this technology to analysis of DNA from human primary cancers and found amplification of a DNA fragment in a mediastinum fibrosarcoma. PCR-based analysis of radiation hybrid panels following cloning and nucleotide sequence determination of the fragment revealed that it was derived from a region of chromosome 12q13-q15. In this region, the MDM2 and IFNG genes were noted as known genes that could be involved in human carcinogenesis. Southern blot analysis of genomic DNA of the tumor revealed the amplification of the MDM2 gene together with the fragment locus, but not the IFNG gene. Our results demonstrated that detection of DNA alterations by AP-PCR fingerprinting without any previous knowledge of the genes and subsequent analysis of radiation hybrid panels could lead to easy identification of candidates for genes involved in carcinogenesis.     

Progression elevated gene-3, PEG-3, induces genomic instability in rodent and human tumor cells

Genomic instability is a fundamental component of cancer progression. Subtraction hybridization identified a novel rodent gene, progression elevated gene-3 (PEG-3) whose expression directly correlates with cancer aggressiveness and progression. Moreover, ectopic expression of PEG-3 in rodent or human tumor cells produces an aggressive transformed phenotype. We demonstrate that PEG-3 expression in rodent tumor cells correlates directly with genomic instability as characterized by alterations in chromosome composition and structure. Additionally, elevated endogenous or ectopic expression of PEG-3 in rodent and human tumor cells, respectively, enhances gene amplification, as monitored by resistance to methothrexate (MTX) and amplification of the dihydrofolate reductase (dhfr) gene. Stable expression of PEG-3 in normal cloned rat embryo fibroblast (CREF) cells marginally elevates MTX resistance, but morphology remains unaltered and anchorage independence is not induced, suggesting that these phenotypes are separable in immortal cells and gene amplification may precede the acquisition of morphological and oncogenic transformation. The present studies document that stable, inducible, and transient expression of PEG-3 in cancer cells augments genomic instability. In these contexts, one mechanism by which PEG-3 influences cancer progression may be by preferentially facilitating the development of genomic changes in evolving cancer cells.     

Multistep renal carcinogenesis in the Eker (Tsc 2 gene mutant) rat model

Cancer is a heritable disorder of somatic cells. Environment and heredity both contribute to the origin of human cancer. The Eker (Tsc 2 gene mutant) rat model of hereditary renal carcinoma (RC) is an example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal. To the best of our knowledge, this was the first isolation of a Mendelian dominantly predisposing cancer gene in a naturally occurring animal model. Carcinogenesis looks like an opened Japanese fan, because initiated cells growing in several directions will develop into tumors having many gene abnormalities, and this is suggested by the edge of the fan. To search for such genetic alterations, we identified genes (Niban and Erc) that were expressed more abundantly in renal tumors than in the normal kidney.I will review this unique model for the study of multistep renal carcinogenesis and discuss cancer prevention and delay of carcinogenesis.     

Allele-specific disparity in breast cancer

Background

Molecular alterations critical to development of cancer include mutations, copy number alterations (amplifications and deletions) as well as genomic rearrangements resulting in gene fusions. Massively parallel next generation sequencing, which enables the discovery of such changes, uses considerable quantities of genomic DNA (> 5 ug), a serious limitation in ever smaller clinical samples. However, a commonly available microarray platforms such as array comparative genomic hybridization (array CGH) allows the characterization of gene copy number at a single gene resolution using much smaller amounts of genomic DNA. In this study we evaluate the sensitivity of ultra-dense array CGH platforms developed by Agilent, especially that of the 1 million probe array (1 M array), and their application when whole genome amplification is required because of limited sample quantities.

Methods

We performed array CGH on whole genome amplified and not amplified genomic DNA from MCF-7 breast cancer cells, using 244 K and 1 M Agilent arrays. The ADM-2 algorithm was used to identify micro-copy number alterations that measured less than 1 Mb in genomic length.

Results

DNA from MCF-7 breast cancer cells was analyzed for micro-copy number alterations, defined as measuring less than 1 Mb in genomic length. The 4-fold extra resolution of the 1 M array platform relative to the less dense 244 K array platform, led to the improved detection of copy number variations (CNVs) and micro-CNAs. The identification of intra-genic breakpoints in areas of DNA copy number gain signaled the possible presence of gene fusion events. However, the ultra-dense platforms, especially the densest 1 M array, detect artifacts inherent to whole genome amplification and should be used only with non-amplified DNA samples.

Conclusions

This is a first report using 1 M array CGH for the discovery of cancer genes and biomarkers. We show the remarkable capacity of this technology to discover CNVs, micro-copy number alterations and even gene fusions. However, these platforms require excellent genomic DNA quality and do not tolerate relatively small imperfections related to the whole genome amplification.     

Genome-wide profiling of gene amplification and deletion in cancer

Accumulations of genetic changes in somatic cells induce phenotypic transformations leading to cancer. Among these genetic changes, gene amplification and deletion are most frequently observed in several kinds of cancers. Amplification of oncogene and/or deletion of tumor suppressor gene, together with dysfunction of the gene by point mutation, are the main causes of cancer. Genome-wide analysis of amplification and deletion of genes in cancers is basic to resolving the mechanisms of carcinogenesis. Comparative genomic hybridization (CGH) developed in 1992 has been utilized to identify DNA copy number abnormalities in various kind of cancers and several reports have shown its usefulness in screening of the genes involved in carcinogenesis, and also in the identification of prognostic factors in cancer. We have shown that 1q23 gain is associated with neuroblastomas that are resistant to aggressive treatment, and have poor prognosis, and 1q and 13q gains are possibly related to drug resistance in ovarian cancers. Recently, the "rough draft" of the human genome was reported and we are ready to utilize the vast information on genomic sequences in cancer research. Moreover, microarray technology enables us to analyze more than ten thousand genes at a time and revealed genetic abnormalities in cancers at a genome-wide level. By combination of microarray and CGH, a powerful screening method for oncogenes and tumor suppressor genes in cancers, called array-CGH, has been developed by several groups. In this article, we overview these genome-wide analytical methods, CGH and array-CGH, and discuss their potential in molecular characterization of cancers.     

Cell cycle alterations and lung cancer

It is now widely accepted that human carcinogenesis is a multi-step process and phenotypic changes during cancer progression reflect the sequential accumulation of genetic alterations in cells. The recent progress of scientific research has notably increased knowledge about biological events involved in lung cancer pathogenesis and progression, thanks to the use of molecular biology and immunohistochemistry techniques. Lots of the genetic alteration found in small cells lung cancer (SCLC) and in not small cells lung cancer (NSCLC) concern the expression of cell cycle genes, actually recognized as onco-suppressor genes and the lack of equilibrium between oncogenes and oncosuppressor genes. The present review of literature widely describes the cell cycle control, the lung cancer molecular pathogenesis, the catalog of known genetic alterations and the recent advances in global expression profiles in lung tumors, on the basis of the various hystological types too. Such data suggest the potential use of this knowledges in clinical practice both as prognostic factors and innovative therapeutic possibilities and they impose the necessity of new studies about cell cycle control and lung carcinogenesis.     

Stage-specific roles of fibulin-5 during oxidative stress-induced renal carcinogenesis in rats

By using a rat model of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA), this study performed genome-wide analysis to identify target genes during carcinogenesis. It screened for genes with decreased expression in RCCs, with simultaneous loss of heterozygosity, eventually to focus on the fibulin-5 (fbln5) gene. Oxidative damage via Fe-NTA markedly increased Fbln5 in the proximal tubules. RCCs presented lower levels of Fbln5. However, a fraction of RCCs presenting pulmonary metastasis revealed significantly higher levels of Fbln5 than those without metastasis, accompanied by immunopositivity of RCC cells and myofibroblast proliferation. Experiments revealed that RCC cell lines showed lower expression of fbln5 than its non-transformed counterpart NRK52E, but that fbln5 transfection to RCC cell lines changed neither proliferation nor migration/invasion. The data suggest that Fbln5 plays a role not only in the tissue repair and remodelling after renal tubular oxidative damage but also in RCC metastasis, presumably as a cytokine.     

Comprehensive genetic analysis of cancer cells

Human cancer is viewed as a disorder of genes originating from the progeny of a single cell that has accumulated multiple genetic alterations. The genetic alterations include point mutation, chromosomal rearrangements and imbalances. Amplifications primarily involve oncogenes whose overexpression leads to growth deregulation, while deletions commonly target tumor suppressor genes that control cell cycle checkpoints and DNA repair mechanisms. With the advent of molecular cytogenetics procedures for global detection of genomic imbalances and for multicolor visualization of structural chromosome changes, as well as the completion of human genome mapping and the development of microarray technology for serial gene expression analysis of the entire genomes, a significant progress has been made in uncovering the molecular basis of cancer. The major challenge in cancer biology is to decipher the molecular anatomy of various cancers and to identify cancer-related genes that now comprise only a fraction of human genes. The complete genetic anatomy of specific cancers would allow a better understanding of the role of genetic alterations in carcinogenesis, provide diagnostic and prognostic markers and discriminate between cells at different stages of progression toward malignancy. This review highlights current technologies that are available to explore cancer cells and outlines their application to investigations in human hepatocellular carcinoma.     

Genetic identification of multiple loci that control breast cancer susceptibility in the rat.

We have used a rat model of induced mammary carcinomas in an effort to identify breast cancer susceptibility genes. Using genetic crosses between the carcinoma-resistant Copenhagen (COP) and carcinoma-sensitive Wistar-Furth rats, we have confirmed the identification of the Mcs1 locus that modulates tumor number. We have now also identified two additional loci, Mcs2 and Mcs3. These three loci map to chromosomes 2, 7, and 1, respectively, and interact additively to suppress mammary carcinoma development in the COP strain. They are responsible for a major portion of the tumor-resistant phenotype of the COP rat. No loss of heterozygosity was observed surrounding the three loci. A fourth COP locus, Mcs4, has also been identified on chromosome 8 and acts in contrast to increase the number of carcinomas. These results show that mammary carcinoma susceptibility in the COP rat is a polygenic trait. Interestingly, a polymorphism in the human genomic region homologous to the rat Mcs4 region is associated with an increased breast cancer risk in African-American women. The isolation of the Mcs genes may help elucidate novel mechanisms of carcinogenesis, provide information important for human breast cancer risk estimation, and also provide unique drug discovery targets for breast cancer prevention.     

Polymorphisms,genomic imprinting and cancer susceptibility

Polymorphisms have been identified in proto-oncogenes and tumor suppressor genes that predispose people to cancer. Recent evidence indicates that genomic imprinting, an epigenetic form of gene regulation that results in uniparental gene expression, can also function as a cancer predisposing event. Thus, cancer susceptibility is increased by both Mendelian inherited genetic and non-Mendelian inherited epigenetic events. Consequently, chemical and physical agents cannot only induce cancer through the formation of genetic mutations but also through epigenetic changes that result in the inappropriate expression of imprinted proto-oncogenes and tumor suppressor genes. The role of genomic imprinting in carcinogenesis and cancer susceptibility is examined in this review.     

Differential gene expression of the key signalling pathway in para‐carcinoma,carcinoma and relapse human pancreatic cancer

Pancreatic cancer (PC) has a high rate of mortality and a poorly understood mechanism of progression. Investigation of the molecular mechanism of PC and exploration of the specific markers for early diagnosis and specific targets of therapy are key points to prevent and treat PC effectively and to improve their prognosis. In our study, expression profiles experiment of para‐carcinoma, carcinoma and relapse human PC was performed using Agilent human whole genomic oligonucleotide microarrays with 45 000 probes. Differentially expressed genes related with PC were screened and analysed further by Gene Ontology term analysis and Kyoto encyclopaedia of genes and genomes pathway analysis. Our results showed that there were 3853 differentially expressed genes associated with pancreatic carcinogenesis and relapse. In addition, our study found that PC was related to the Jak–STAT signalling pathway, PPAR signalling pathway and Calcium signalling pathway, indicating their potential roles in pancreatic carcinogenesis and progress. Copyright © 2013 John Wiley & Sons, Ltd.     

Array CGH analysis reveals chromosomal aberrations in mouse lung adenocarcinomas induced by the human lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Exposure to genotoxic carcinogens in tobacco smoke is a major cause of lung cancer. However, the effect this has on DNA copy number and genomic stability during lung carcinogenesis is unclear. Here we used bacterial artificial chromosome array-based comparative genomic hybridization to examine the effect of NNK, a potent human lung carcinogen present in tobacco smoke, on the major genomic changes occurring during mouse lung adenocarcinogenesis. Observed were significantly more gross chromosomal changes in NNK-induced tumors compared with the spontaneous tumors. An average of 5.6 chromosomes were affected by large-scale changes in DNA copy number per NNK-induced tumor compared with only 2.0 in spontaneous lung tumors (p = 0.017). Further analysis showed that gains on chromosomes 6 and 8, and losses on chromosomes 11 and 14 were more common in NNK-induced tumors (p 相似文献