Cause and Consequences of Genetic and Epigenetic Alterations in Human Cancer

Both genetic and epigenetic changes contribute to development of human cancer. Oncogenomics has primarily focused on understanding the genetic basis of neoplasia, with less emphasis being placed on the role of epigenetics in tumourigenesis. Genomic alterations in cancer vary between the different types and stages, tissues and individuals. Moreover, genomic change ranges from single nucleotide mutations to gross chromosomal aneuploidy; which may or may not be associated with underlying genomic instability. Collectively, genomic alterations result in widespread deregulation of gene expression profiles and the disruption of signalling networks that control proliferation and cellular functions. In addition to changes in DNA and chromosomes, it has become evident that oncogenomic processes can be profoundly influenced by epigenetic mechanisms. DNA methylation is one of the key epigenetic factors involved in regulation of gene expression and genomic stability, and is biologically necessary for the maintenance of many cellular functions. While there has been considerable progress in understanding the impact of genetic and epigenetic mechanisms in tumourigenesis, there has been little consideration of the importance of the interplay between these two processes. In this review we summarize current understanding of the role of genetic and epigenetic alterations in human cancer. In addition we consider the associated interactions of genetic and epigenetic processes in tumour onset and progression. Furthermore, we provide a model of tumourigenesis that addresses the combined impact of both epigenetic and genetic alterations in cancer cells.     

Diagnostics of Epigenetic Alterations in Hereditary and Oncological Disorders

The review considers the epigenetic defects and their diagnostics in several hereditary disorders and tumors. Aberrant methylation of the promoter or regulatory region of a gene results in its functional inactivation, which is phenotypically similar to structural deletion. Screening tests were developed for Prader–Willi, Angelman, Wiedemann–Beckwith, and Martin–Bell syndromes and mental retardation FRAXE. The tests are based on allele methylation analysis by methylation-specific or methylation-sensitive PCR. Carcinogenesis-associated genes (RB1, CDKN2A, ARF14, HIC1, CDH1, etc.) are often methylated in tumors. Tumors differ in methylation frequencies, allowing differential diagnostics. Aberrant methylation of tumor suppressor genes occurs in early carcinogenesis, and its detection may be employed in presymptomatic diagnostics of tumors.     

Precision Glycoproteomics Reveals Distinctive N-Glycosylation in Human Spermatozoa

Spermatozoon represents a very special cell type in human body, and glycosylation plays essential roles in its whole life including spermatogenesis, maturation, capacitation, sperm–egg recognition, and fertilization. In this study, by mapping the most comprehensive N-glycoproteome of human spermatozoa using our recently developed site-specific glycoproteomic approaches, we show that spermatozoa contain a number of distinctive glycoproteins, which are mainly involved in spermatogenesis, acrosome reaction and sperm:oocyte membrane binding, and fertilization. Heavy fucosylation is observed on 14 glycoproteins mostly located at extracellular and cell surface regions in spermatozoa but not in other tissues. Sialylation and Lewis epitopes are enriched in the biological process of immune response in spermatozoa, while bisected core structures and LacdiNAc structures are highly expressed in acrosome. These data deepen our knowledge about glycosylation in spermatozoa and lay the foundation for functional study of glycosylation and glycan structures in male infertility.     

Reproduction in mice: Spermatozoa as factors in the development and implantation of embryos

The effects on mouse embryo development in vivo of varying the numbers of spermatozoa used in artificial inseminations was studied. The two criteria used in the evaluation of the progress of embryo development were 1) ability to reach the two-cell stage and 2) success of development from the two-cell stage through implantation. A 44% reduction in the yield of two-cell embryos and a 67% reduction in the number of implants was observed when C3HeB/FeJ females were inseminated with one-twentieth the number of spermatozoa estimated to be present in a typical ejaculate. The reduction in the yield of two-cell embryos was substantially reversed by a second insemination of a large number of heat-inactivated spermatozoa 12 hr after the first insemination. The sperm-dependent reduction in development from the two-cell stage through implantation was prevented only by normal viable (unheated) spermatozoa. These results were rationalized by the hypothesis that in female C3HeB/FeJ mice spermatozoa serve physiological functions beyond the fertilization of ova and that spermatozoa may act to foster early embryo development through modulation of the environments embryos experience as they move through the reproductive tract.     

Genetic and Epigenetic Alterations in Primary–Progressive Paired Oligodendroglial Tumors

The aim of the present study was to identify genetic and epigenetic alterations involved in the progression of oligodendroglial tumors. We characterized 21 paired, World Health Organization (WHO) grade II and III oligodendroglial tumors from patients who received craniotomies for the partial or complete resection of primary and secondary oligodendroglial tumors. Tumor DNA was analyzed for alterations in selected genetic loci (1p36, 9p22, 10q23–24, 17p13, 19q13, 22q12), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2) and the CpG island methylation status of critical tumor-related genes (MGMT, P16, DAPK, PTEN, RASSF1A, Rb1). Alterations of these markers were common early in the tumorigenesis. In the primary tumors we identified 12 patients (57.1%) with 1p36 deletions, 17 (81.0%) with 19q13 deletions, 9 (42.9%) with 1p36/19q13 codeletions, 11 (52.3%) with 9p22 deletions, and 12 (57.1%) with IDH1 mutation. Epigenetic analysis detected promoter methylation of the MGMT, P16, DAPK, PTEN, RASSF1A, and Rb1 genes in 38.1%, 19.0%, 38.1%, 33.3%, 66.7%, and 14.3% of primary tumors, respectively. After progression, additional losses of 1p, 9p, 10q, 17p, 19q and 22q were observed in 3 (14.3%), 1 (4.8%), 3 (14.3%), 2 (9.5%), 1 (4.8%) and 3 (14.3%) cases, respectively. Additional methylations of the MGMT, P16, DAPK, PTEN, RASSF1A, and RB1 promoters was observed in 4 (19.0%), 2 (9.5%), 0 (0%), 6 (28.6%), 2(9.5%) and 3 (14.3%) cases, respectively. The status of IDH1 mutation remained unchanged in all tumors after progression. The primary tumors of three patients with subsequent progression to high-grade astrocytomas, all had 9p deletion, intact 1p, intact 10q and unmethylated MGMT. Whether this may represent a molecular signature of patients at-risk for the development of aggressive astrocytomas needs further investigation.     

Integrative Analysis Reveals Relationships of Genetic and Epigenetic Alterations in Osteosarcoma

Background

Osteosarcomas are the most common non-haematological primary malignant tumours of bone, and all conventional osteosarcomas are high-grade tumours showing complex genomic aberrations. We have integrated genome-wide genetic and epigenetic profiles from the EuroBoNeT panel of 19 human osteosarcoma cell lines based on microarray technologies.

Principal Findings

The cell lines showed complex patterns of DNA copy number changes, where genomic copy number gains were significantly associated with gene-rich regions and losses with gene-poor regions. By integrating the datasets, 350 genes were identified as having two types of aberrations (gain/over-expression, hypo-methylation/over-expression, loss/under-expression or hyper-methylation/under-expression) using a recurrence threshold of 6/19 (>30%) cell lines. The genes showed in general alterations in either DNA copy number or DNA methylation, both within individual samples and across the sample panel. These 350 genes are involved in embryonic skeletal system development and morphogenesis, as well as remodelling of extracellular matrix. The aberrations of three selected genes, CXCL5, DLX5 and RUNX2, were validated in five cell lines and five tumour samples using PCR techniques. Several genes were hyper-methylated and under-expressed compared to normal osteoblasts, and expression could be reactivated by demethylation using 5-Aza-2′-deoxycytidine treatment for four genes tested; AKAP12, CXCL5, EFEMP1 and IL11RA. Globally, there was as expected a significant positive association between gain and over-expression, loss and under-expression as well as hyper-methylation and under-expression, but gain was also associated with hyper-methylation and under-expression, suggesting that hyper-methylation may oppose the effects of increased copy number for detrimental genes.

Conclusions

Integrative analysis of genome-wide genetic and epigenetic alterations identified dependencies and relationships between DNA copy number, DNA methylation and mRNA expression in osteosarcomas, contributing to better understanding of osteosarcoma biology.     

Evaluation of Uridine Metabolism in Human and Animal Spermatozoa

The objective of this study was to elucidate the role of uridine for spermatozoa, since this pyrimidine nucleoside was found in millimolar concentration in human seminal plasma. Here, the degradative activity of uridine-phosphorylase [EC 2.4.2.3] and the salvage activity of uridine kinase [EC 2.7.1.48] were detected in human spermatozoa. HPLC analysis depicted the uptake of exogeneous ¹⁴C-labelled adenine, but not of uridine and of hypoxanthine, into nucleotide pools of boar spermatozoa. On addition of uridine, the computer-assisted semen analysis (CASA) of human cells revealed a reduction of the percentage of motile spermatozoa in contrast to an elevation of some velocity parameters. It is concluded that exogeneous uridine could function as suppressor for early capacitation and as a substrate for phosphorolysis, if ribose is needed, rather than to satisfy a demand for intracellular pyrimidine nucleotides.     

Epigenetic Alterations in Fanconi Anaemia: Role in Pathophysiology and Therapeutic Potential

Fanconi anaemia (FA) is an inherited disorder characterized by chromosomal instability. The phenotype is variable, which raises the possibility that it may be affected by other factors, such as epigenetic modifications. These play an important role in oncogenesis and may be pharmacologically manipulated. Our aim was to explore whether the epigenetic profiles in FA differ from non-FA individuals and whether these could be manipulated to alter the disease phenotype. We compared expression of epigenetic genes and DNA methylation profile of tumour suppressor genes between FA and normal samples. FA samples exhibited decreased expression levels of genes involved in epigenetic regulation and hypomethylation in the promoter regions of tumour suppressor genes. Treatment of FA cells with histone deacetylase inhibitor Vorinostat increased the expression of DNM3Tβ and reduced the levels of CIITA and HDAC9, PAK1, USP16, all involved in different aspects of epigenetic and immune regulation. Given the ability of Vorinostat to modulate epigenetic genes in FA patients, we investigated its functional effects on the FA phenotype. This was assessed by incubating FA cells with Vorinostat and quantifying chromosomal breaks induced by DNA cross-linking agents. Treatment of FA cells with Vorinostat resulted in a significant reduction of aberrant cells (81% on average). Our results suggest that epigenetic mechanisms may play a role in oncogenesis in FA. Epigenetic agents may be helpful in improving the phenotype of FA patients, potentially reducing tumour incidence in this population.     

Status of Bone Mineral Density in Patients Selected for Cardiac Transplantation

ObjectiveTo determine the prevalence and correlates of low bone mineral density (BMD) in ambulatory outpatients with end-stage heart failure who were awaiting cardiac transplantation.MethodsFifty-five cardiac transplant candidates with end-stage heart failure were enrolled in this study. Bone mineral density at the lumbar spine and proximal femur was determined by dual-energy x-ray absorptiometry. Laboratory studies included serum alkaline phosphatase, calcium, intact parathyroid hormone, and 25-hydroxyvitamin D.ResultsThe mean proximal femur and lumbar spine Z scores were 0.3 ± 1.1 and 0.3 ± 1.5, respectively. The mean BMD was not lower than that of the age- and sex- matched reference population. Z scores were less than -1 in 23% at the lumbar spine and 15% at the proximal femoral neck. On the basis of T scores, osteopenia (T scores between -1 and -2.5) was present in 24% (confidence interval, 13% to 35%) of patients at the lumbar spine and in 20% (confidence interval, 10% to 30%) at the proximal femur; osteoporosis (T scores of less than -2.5) was present in 4% of the study population. Half of the patients in this study sample had elevated intact parathyroid hormone levels, and a third of the patients had low 25-hydroxyvitamin D levels.ConclusionLumbar spine and hip BMD measurements were not significantly low relative to age and sex in ambulatory patients with heart failure awaiting cardiac transplantation. (Endocr Pract. 2008;14:704-712)     

Alterations of Mitochondrial DNA in CEM Cells Selected for Resistance Toward DDC Toxicity

2 ′,3 ′-dideoxycytidine (ddC) is a nucleoside analog that has been shown to produce a delayed toxicity which may be due to the depletion of mitochondrial DNA (mtDNA). In order to gain further understanding of the events involved in mitochondrial toxicity, two different CEM cell lines were selected for resistance to the delayed ddC toxicity.     

正常精子和圆头精子差异表达蛋白的分离与鉴定

圆头精子症是一种表现为顶体缺失的男性不育症.为了研究正常精子和圆头精子的蛋白质组成差异,用固相pH梯度双向凝胶电泳和质谱分析等蛋白质组学方法,分离了30份正常和3份圆头精子标本的蛋白质.对其中16个在正常精子中有高丰度表达而在圆头精子中缺失,和1个在圆头精子中表达明显下调的蛋白质点,以及在圆头精子中存在而在正常精子中缺失的蛋白质簇W和点X,进行了肽质指纹分析和蛋白质鉴定,获得8个点的肽质量指纹图,经MS-Fit软件搜索SWISS-PROT数据库来鉴定其身分.发现其中3个点与高尔基体相关、2个点与蛋白酶体相关、2个点为锌指蛋白,对其功能和与圆头精子形成的可能关系进行了初步探讨.     

Epigenetic Alterations in the Brain Associated with HIV-1 Infection and Methamphetamine Dependence

HIV involvement of the CNS continues to be a significant problem despite successful use of combination antiretroviral therapy (cART). Drugs of abuse can act in concert with HIV proteins to damage glia and neurons, worsening the neurotoxicity caused by HIV alone. Methamphetamine (METH) is a highly addictive psychostimulant drug, abuse of which has reached epidemic proportions and is associated with high-risk sexual behavior, increased HIV transmission, and development of drug resistance. HIV infection and METH dependence can have synergistic pathological effects, with preferential involvement of frontostriatal circuits. At the molecular level, epigenetic alterations have been reported for both HIV-1 infection and drug abuse, but the neuropathological pathways triggered by their combined effects are less known. We investigated epigenetic changes in the brain associated with HIV and METH. We analyzed postmortem frontal cortex tissue from 27 HIV seropositive individuals, 13 of which had a history of METH dependence, in comparison to 14 cases who never used METH. We detected changes in the expression of DNMT1, at mRNA and protein levels, that resulted in the increase of global DNA methylation. Genome-wide profiling of DNA methylation in a subset of cases, showed differential methylation on genes related to neurodegeneration; dopamine metabolism and transport; and oxidative phosphorylation. We provide evidence for the synergy of HIV and METH dependence on the patterns of DNA methylation on the host brain, which results in a distinctive landscape for the comorbid condition. Importantly, we identified new epigenetic targets that might aid in understanding the aggravated neurodegenerative, cognitive, motor and behavioral symptoms observed in persons living with HIV and addictions.     

Magnetic-activated Cell Sorting before Cryopreservation Preserves Mitochondrial Integrity in Human Spermatozoa

Superparamagnetic annexin-V conjugated microbeads are able to eliminate spermatozoa with externalized phosphatidylserine, a membrane feature of apoptotic cells as well as spermatozoa with deteriorated plasma membrane. Our objective was to evaluate the effects of annexin-V Magnetic-Activated Cell Sorting (MACS) in cryopreservation–thawing protocols and on integrity of sperm mitochondrial transmembrane potential and mitochondrial integrity survival rate (MSR). Mature spermatozoa of 10 healthy donors were prepared by density gradient centrifugation and divided into 2 aliquots afterwards. The first one was subjected to annexin-V MACS followed by cryopreservation and thawing, while the second was cryopreserved–thawed without MACS to serve as control. Annexin-negative sperm separated by MACS showed significantly higher levels of intact mitochondria following cryopreservation–thawing (45.4±8.6%) compared to sperm that were not separated (15.8±4.6%, p<0.01). Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize cryopreservation–thawing outcome. MACS using annexin-V microbeads enhances the percentage of spermatozoa with intact transmembrane mitochondrial potential and mitochondrial integrity survival rates following cryopreservation.     

VLDL双向选择肉鸡群SSR指纹分析

对初步进行VLDL双向选择的三个世代高、低脂群肉鸡进行SSR指纹分析,评定各基因座基因频率的变化,进而判断SSR标记与肉鸡肥度性状VLDL的相关关系,为低脂肉鸡的早期选育奠定基础。5个微卫星引物一个未扩增出产物,其余4个引物扩增出14个微卫星位点。各位点在高、低脂系中的基因频率经卡方检验,一世代有一个基因座的基因频率差异显著（P<0.05）;二世代两个基因座差异显著;三世代共检测到4个基因座差异显著。 Abstract:SSR fingerprints were analyzed in three generations of fat line (FL) and lean line (LL) of broiler chickens.Changes in gene frequencies of every locus were evaluated.Thus the relationship between SSR markers and VLDL (a trait representing fat mass of broiler),which is the basis for early selection of LL broiler,was examined.Fourteen microsatellite locus were successfully amplified with 4 of 5 primers used.The results of χ2 test for the gene frequencies of every locus show that one locus was significantly different in generation 1(P<0.05),two in generation 2 and 4 in generation 3.     

Epigenetic Alterations at Genomic Loci Modified by Gene Targeting in Arabidopsis thaliana

Gene Targeting (GT) is the integration of an introduced vector into a specific chromosomal site, via homologous recombination. It is considered an effective tool for precise genome editing, with far-reaching implications in biological research and biotechnology, and is widely used in mice, with the potential of becoming routine in many species. Nevertheless, the epigenetic status of the targeted allele remains largely unexplored. Using GT-modified lines of the model plant Arabidopsis thaliana, we show that the DNA methylation profile of the targeted locus is changed following GT. This effect is non-directional as methylation can be either completely lost, maintained with minor alterations or show instability in the generations subsequent to GT. As DNA methylation is known to be involved in several cellular processes, GT-related alterations may result in unexpected or even unnoticed perturbations. Our analysis shows that GT may be used as a new tool for generating epialleles, for example, to study the role of gene body methylation. In addition, the analysis of DNA methylation at the targeted locus may be utilized to investigate the mechanism of GT, many aspects of which are still unknown.