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1.
Proc Natl Acad Sci U S A ; 112(27): 8403-8, 2015 Jul 07.
Article in English | MEDLINE | ID: mdl-26080435

ABSTRACT

Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.


Subject(s)
Antigens, Neoplasm/genetics , Introns/genetics , Neoplasm Proteins/genetics , Prostatic Neoplasms/genetics , RNA, Long Noncoding/genetics , Tumor Suppressor Proteins/genetics , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Animals , Antigens, Neoplasm/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , HEK293 Cells , HeLa Cells , Humans , Immunoblotting , MCF-7 Cells , Male , Mice, SCID , Molecular Sequence Data , Neoplasm Proteins/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Binding , RNA Interference , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Long Noncoding/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor Assays/methods
2.
Circ Cardiovasc Genet ; 7(3): 335-43, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24951659

ABSTRACT

BACKGROUND: Genome-wide association studies have identified thousands of genetic variants that influence a variety of diseases and health-related quantitative traits. However, the causal variants underlying the majority of genetic associations remain unknown. Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium Targeted Sequencing Study aims to follow up genome-wide association study signals and identify novel associations of the allelic spectrum of identified variants with cardiovascular-related traits. METHODS AND RESULTS: The study included 4231 participants from 3 CHARGE cohorts: the Atherosclerosis Risk in Communities Study, the Cardiovascular Health Study, and the Framingham Heart Study. We used a case-cohort design in which we selected both a random sample of participants and participants with extreme phenotypes for each of 14 traits. We sequenced and analyzed 77 genomic loci, which had previously been associated with ≥1 of 14 phenotypes. A total of 52 736 variants were characterized by sequencing and passed our stringent quality control criteria. For common variants (minor allele frequency ≥1%), we performed unweighted regression analyses to obtain P values for associations and weighted regression analyses to obtain effect estimates that accounted for the sampling design. For rare variants, we applied 2 approaches: collapsed aggregate statistics and joint analysis of variants using the sequence kernel association test. CONCLUSIONS: We sequenced 77 genomic loci in participants from 3 cohorts. We established a set of filters to identify high-quality variants and implemented statistical and bioinformatics strategies to analyze the sequence data and identify potentially functional variants within genome-wide association study loci.


Subject(s)
Aging/genetics , Genome-Wide Association Study , Heart Diseases/genetics , Adult , Aged , Aged, 80 and over , Cohort Studies , Female , Genetic Variation , Genomics , Heart Diseases/epidemiology , Humans , Male , Middle Aged , Polymorphism, Single Nucleotide , Research Design , Sequence Analysis, DNA
3.
BMC Genomics ; 15: 86, 2014 Jan 30.
Article in English | MEDLINE | ID: mdl-24479613

ABSTRACT

BACKGROUND: The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. RESULTS: Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. CONCLUSIONS: Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.


Subject(s)
Bees/genetics , Genes, Insect , Animals , Base Composition , Databases, Genetic , Interspersed Repetitive Sequences/genetics , Molecular Sequence Annotation , Open Reading Frames/genetics , Peptides/analysis , Sequence Analysis, RNA , Sequence Homology, Amino Acid
4.
PLoS Genet ; 9(4): e1003443, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23593035

ABSTRACT

We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD.


Subject(s)
Child Development Disorders, Pervasive/genetics , Exome , Genome-Wide Association Study , Case-Control Studies , Child , Child Development Disorders, Pervasive/physiopathology , Genetic Predisposition to Disease , Genetic Variation , Humans , Population Control , Sequence Analysis, DNA , Software
5.
Neuron ; 77(2): 235-42, 2013 Jan 23.
Article in English | MEDLINE | ID: mdl-23352160

ABSTRACT

To characterize the role of rare complete human knockouts in autism spectrum disorders (ASDs), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a 2-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤ 5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudoautosomal regions on the X chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together, these results provide compelling evidence that rare autosomal and X chromosome complete gene knockouts are important inherited risk factors for ASD.


Subject(s)
Child Development Disorders, Pervasive/diagnosis , Child Development Disorders, Pervasive/genetics , Demography/methods , Gene Deletion , Loss of Heterozygosity/genetics , Case-Control Studies , Child Development Disorders, Pervasive/epidemiology , Child, Preschool , Chromosomes, Human, X/genetics , Female , Genetic Variation/genetics , Homozygote , Humans , Linkage Disequilibrium/genetics , Male , Risk Factors
6.
Nature ; 485(7397): 242-5, 2012 Apr 04.
Article in English | MEDLINE | ID: mdl-22495311

ABSTRACT

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.


Subject(s)
Autistic Disorder/genetics , DNA-Binding Proteins/genetics , Exons/genetics , Genetic Predisposition to Disease/genetics , Mutation/genetics , Transcription Factors/genetics , Case-Control Studies , Exome/genetics , Family Health , Humans , Models, Genetic , Multifactorial Inheritance/genetics , Phenotype , Poisson Distribution , Protein Interaction Maps
7.
Nature ; 482(7384): 173-8, 2012 Feb 08.
Article in English | MEDLINE | ID: mdl-22318601

ABSTRACT

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.


Subject(s)
Drosophila melanogaster/genetics , Genome-Wide Association Study , Genomics , Quantitative Trait Loci/genetics , Alleles , Animals , Centromere/genetics , Chromosomes, Insect/genetics , Genotype , Phenotype , Polymorphism, Single Nucleotide/genetics , Selection, Genetic/genetics , Starvation/genetics , Telomere/genetics , X Chromosome/genetics
8.
Nat Genet ; 44(2): 165-9, 2011 Dec 25.
Article in English | MEDLINE | ID: mdl-22197930

ABSTRACT

We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.


Subject(s)
MAP Kinase Kinase Kinase 5/genetics , MAP Kinase Kinase Kinases/genetics , Melanoma/genetics , Mutation , Skin Neoplasms/genetics , Antineoplastic Agents/pharmacology , Base Sequence , Cell Line, Tumor , Dacarbazine/analogs & derivatives , Dacarbazine/pharmacology , Exome , Humans , Loss of Heterozygosity , Melanoma/drug therapy , Melanoma/secondary , Molecular Sequence Data , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Skin Neoplasms/drug therapy , Skin Neoplasms/pathology , Temozolomide , Tumor Cells, Cultured
9.
Am J Med Genet A ; 155A(9): 2071-7, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21834044

ABSTRACT

Polymicrogyria is a disorder of neuronal development resulting in structurally abnormal cerebral hemispheres characterized by over-folding and abnormal lamination of the cerebral cortex. Polymicrogyria is frequently associated with severe neurologic deficits including intellectual disability, motor problems, and epilepsy. There are acquired and genetic causes of polymicrogyria, but most patients with a presumed genetic etiology lack a specific diagnosis. Here we report using whole-exome sequencing to identify compound heterozygous mutations in the WD repeat domain 62 (WDR62) gene as the cause of recurrent polymicrogyria in a sibling pair. Sanger sequencing confirmed that the siblings both inherited 1-bp (maternal allele) and 2-bp (paternal allele) frameshift deletions, which predict premature truncation of WDR62, a protein that has a role in early cortical development. The probands are from a non-consanguineous family of Northern European descent, suggesting that autosomal recessive PMG due to compound heterozygous mutation of WDR62 might be a relatively common cause of PMG in the population. Further studies to identify mutation frequency in the population are needed.


Subject(s)
Abnormalities, Multiple/genetics , Exome , Malformations of Cortical Development/genetics , Nerve Tissue Proteins/genetics , Adult , Base Sequence , Cell Cycle Proteins , Child , Craniofacial Abnormalities/genetics , Female , Frameshift Mutation , Genetic Testing , Heterozygote , High-Throughput Nucleotide Sequencing , Humans , Magnetic Resonance Imaging , Male , Mutation , Sequence Analysis, DNA , Sequence Deletion , Siblings
10.
Genome Biol ; 12(7): R68, 2011 Jul 25.
Article in English | MEDLINE | ID: mdl-21787409

ABSTRACT

BACKGROUND: Enrichment of loci by DNA hybridization-capture, followed by high-throughput sequencing, is an important tool in modern genetics. Currently, the most common targets for enrichment are the protein coding exons represented by the consensus coding DNA sequence (CCDS). The CCDS, however, excludes many actual or computationally predicted coding exons present in other databases, such as RefSeq and Vega, and non-coding functional elements such as untranslated and regulatory regions. The number of variants per base pair (variant density) and our ability to interrogate regions outside of the CCDS regions is consequently less well understood. RESULTS: We examine capture sequence data from outside of the CCDS regions and find that extremes of GC content that are present in different subregions of the genome can reduce the local capture sequence coverage to less than 50% relative to the CCDS. This effect is due to biases inherent in both the Illumina and SOLiD sequencing platforms that are exacerbated by the capture process. Interestingly, for two subregion types, microRNA and predicted exons, the capture process yields higher than expected coverage when compared to whole genome sequencing. Lastly, we examine the variation present in non-CCDS regions and find that predicted exons, as well as exonic regions specific to RefSeq and Vega, show much higher variant densities than the CCDS. CONCLUSIONS: We show that regions outside of the CCDS perform less efficiently in capture sequence experiments. Further, we show that the variant density in computationally predicted exons is more than 2.5-times higher than that observed in the CCDS.


Subject(s)
Consensus Sequence , Exome , Exons , Open Reading Frames/genetics , Sequence Analysis, DNA , Alleles , Computational Biology , Gene Frequency , Genome, Human , Humans , Introns , Mutation Rate , Polymorphism, Single Nucleotide
11.
Sci Transl Med ; 3(87): 87re3, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21677200

ABSTRACT

Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)-responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins.


Subject(s)
Dystonic Disorders , Genome, Human , Patient Care , Sequence Analysis, DNA , Adolescent , Decision Making , Dystonic Disorders/drug therapy , Dystonic Disorders/genetics , Female , Humans , Levodopa/therapeutic use , Male , Pedigree , Treatment Outcome , Twins, Dizygotic/genetics
12.
Nat Commun ; 1: 131, 2010 Nov 30.
Article in English | MEDLINE | ID: mdl-21119644

ABSTRACT

Accurately determining the distribution of rare variants is an important goal of human genetics, but resequencing of a sample large enough for this purpose has been unfeasible until now. Here, we applied Sanger sequencing of genomic PCR amplicons to resequence the diabetes-associated genes KCNJ11 and HHEX in 13,715 people (10,422 European Americans and 3,293 African Americans) and validated amplicons potentially harbouring rare variants using 454 pyrosequencing. We observed far more variation (expected variant-site count ∼578) than would have been predicted on the basis of earlier surveys, which could only capture the distribution of common variants. By comparison with earlier estimates based on common variants, our model shows a clear genetic signal of accelerating population growth, suggesting that humanity harbours a myriad of rare, deleterious variants, and that disease risk and the burden of disease in contemporary populations may be heavily influenced by the distribution of rare variants.

13.
Genome Biol ; 11(6): R62, 2010.
Article in English | MEDLINE | ID: mdl-20565776

ABSTRACT

We have developed a solution-based method for targeted DNA capture-sequencing that is directed to the complete human exome. Using this approach allows the discovery of greater than 95% of all expected heterozygous singe base variants, requires as little as 3 Gbp of raw sequence data and constitutes an effective tool for identifying rare coding alleles in large scale genomic studies.


Subject(s)
Base Pairing/genetics , Databases, Nucleic Acid , Exons/genetics , Sequence Analysis, DNA/methods , Gene Library , Haplotypes/genetics , Humans , Polymorphism, Single Nucleotide/genetics , Reproducibility of Results , Sequence Alignment , Solutions
14.
Biochem Pharmacol ; 77(7): 1139-50, 2009 Apr 01.
Article in English | MEDLINE | ID: mdl-19185564

ABSTRACT

Although the primary response to Adriamycin (doxorubicin) in p53 mutant MDA-MB231 and p53 null MCF-7/E6 breast tumor cells is apoptotic cell death, the residual surviving population appears to be in a state of senescence, based on cell morphology, beta galactosidase staining, induction of p21(waf1/cip1) and down regulation of cdc2/cdk1. Suppression of apoptosis in MDA-MB231 and MCF-7/E6 cells treated with Adriamycin using the broad spectrum caspase inhibitor, zvad-Fmk, results in substantial induction of autophagy. Overall sensitivity to Adriamycin, measured by clonogenic survival, is not altered in the cells undergoing autophagy, consistent with autophagy contributing to cell death in response to Adriamycin. The free radical scavengers, glutathione and N-acetyl cysteine attenuate the accelerated senescence response to Adriamycin in MCF-7 cells as well as in MDA-MB231 and MCF-7/E6 cells, but protect primarily the MCF-7 cells, indicating that reactive oxygen is unlikely to be directly responsible for Adriamycin toxicity in breast tumor cells. Expression of caspase 3 or induced expression of c-myc in MCF-7 cells fails to abrogate accelerated senescence induced by Adriamycin. Taken together, these studies suggest that accelerated senescence induced by Adriamycin is similar in cells with wild type p53 and in cells lacking functional p53 with regard to the upregulation of p21(waf1/cip1), down regulation of cdc2 and the involvement of reactive oxygen species. Furthermore, accelerated senescence, autophagy and apoptosis all appear to be effective in suppressing self-renewal capacity in breast tumor cells exposed to Adriamycin.


Subject(s)
Apoptosis/physiology , Autophagy/physiology , Breast Neoplasms/metabolism , Cellular Senescence/physiology , Doxorubicin/pharmacology , Reactive Oxygen Species/metabolism , Apoptosis/drug effects , Autophagy/drug effects , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cellular Senescence/drug effects , Humans
15.
Biochem Pharmacol ; 74(7): 981-91, 2007 Oct 01.
Article in English | MEDLINE | ID: mdl-17692290

ABSTRACT

JG-03-14, a substituted pyrrole that inhibits microtubule polymerization, was screened against MCF-7 (p53 wild type), MDA-MB231 (p53 mutant), MCF-7/caspase 3 and MCF-7/ADR (multidrug resistant) breast tumor cell lines. Cell viability and growth inhibition were assessed by the crystal violet dye assay. Apoptosis was evaluated by the TUNEL assay, cell cycle distribution by flow cytometry, autophagy by acridine orange staining of vesicle formation, and senescence based on beta-galactosidase staining and cell morphology. Our studies indicate that exposure to JG-03-14, at a concentration of 500 nM, induces time-dependent cell death in the MCF-7 and MDA-MB231 cell lines. In MCF-7 cells, a residual surviving cell population was found to be senescent; in contrast, there was no surviving senescent population in treated MDA-MB231 cells. No proliferative recovery was detected over a period of 15 days post-treatment in either cell line. Both the TUNEL assay and FLOW cytometry indicated a relatively limited degree of apoptosis (<10%) in response to drug treatment in MCF-7 cells with more extensive apoptosis (but <20%) in MDA-MB231 cells; acidic vacuole formation indicative of autophagic cell death was relatively extensive in both MCF-7 and MDA-MB231 cells. In addition, JG-03-14 induced the formation of a large hyperdiploid cell population in MDA-MB231 cells. JG-03-14 also demonstrated pronounced anti-proliferative activity in MCF-7/caspase 3 cells and in the MCF-7/ADR cell line. The observation that JG-03-14 promotes autophagic cell death and also retains activity in tumor cells expressing the multidrug resistance pump indicates that novel microtubule poisons of the substituted pyrroles class may hold promise in the treatment of breast cancer.


Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Polyploidy , Pyrroles/pharmacology , Aging/drug effects , Caspase 3/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Chromosome Aberrations , Drug Resistance, Neoplasm , Humans , Microtubules/drug effects , Molecular Structure , Pyrroles/chemistry , Time Factors
16.
Breast Cancer Res Treat ; 103(3): 283-91, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17260099

ABSTRACT

TSLC1 and DAL-1 are tumor suppressor genes involved in cell adhesion. In this study, we examined the expression and methylation pattern of these genes in breast cancer cell lines and primary breast carcinomas. TSLC1 expression was lost in 5 of 8 (63%) and DAL-1 expression was lost in 6 of 8 (75%) breast cancer cell lines, respectively. Downregulation of TSLC1 expression was observed in 43 of 50 (86%) and of DAL-1 expression in 26 of 55 (47%) primary breast carcinomas. TSLC1 methylation was found in 4 of 8 (50%) and DAL-1 methylation was observed in 6 of 8 (75%) breast cancer cell lines, respectively. Of 95 primary breast carcinomas 46 (48%) were TSLC1 methylated and 26 (27%) were DAL-1 methylated. Twenty of 43 (47%) and 10 of 26 (38%) primary breast cancer samples which showed downregulation of TSLC1 and DAL-1 expression were unmethylated for these genes. Re-expression of TSLC1 and DAL-1 was observed after treatment of BT-20 cells with 5-aza-2'-deoxycytidine and TSA. Samples from patients with grade 3 tumors were more frequently TSLC1 and TSLC1 and/or DAL-1 methylated than samples from patients with grade 1 and 2 tumors (P = 0.032, P = 0.023). Moreover, TSLC1 methylation correlated with loss of both ER and PgR staining (P = 0.011, P = 0.02). Our findings suggest that TSLC1 and DAL-1 are involved in the pathogenesis of breast cancer and are frequently inactivated by methylation.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Immunoglobulins/biosynthesis , Immunoglobulins/genetics , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Tumor Suppressor Proteins/biosynthesis , Tumor Suppressor Proteins/genetics , Azacitidine/analogs & derivatives , Azacitidine/chemistry , Carcinoma/metabolism , Cell Adhesion , Cell Adhesion Molecule-1 , Cell Adhesion Molecules , Cell Line, Tumor , DNA Methylation , Decitabine , Epigenesis, Genetic , Female , Humans , Microfilament Proteins , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sulfites/chemistry
17.
Mol Cancer Ther ; 5(11): 2786-97, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17121925

ABSTRACT

1,25-Dihydroxyvitamin D(3) and vitamin D(3) analogues, such as EB 1089, potentiate the response to ionizing radiation in breast tumor cells. The current studies address the basis for this interaction by evaluating DNA damage and repair, the effect of interference with reactive oxygen generation, the involvement of p53 and caspase-3, signaling through c-myc, as well as the induction of senescence and multiple modes of cell death. EB 1089 failed to increase the extent of radiation-induced DNA damage or to attenuate the rate of DNA repair. The reactive oxygen scavengers N-acetyl-l-cysteine and reduced glutathione failed to protect the cells from the promotion of cell death by EB 1089 and radiation. Whereas MCF-7 cells expressing caspase-3 showed significant apoptosis with radiation alone as well as with EB 1089 followed by radiation, EB 1089 maintained its ability to confer susceptibility to radiation-induced cell killing, in large part by interference with proliferative recovery. In contrast, in breast tumor cells lacking p53, where radiation promoted extensive apoptosis and the cells failed to recover after radiation treatment, EB 1089 failed to influence the effect of radiation. EB 1089 treatment interfered with radiation-induced suppression of c-myc; however, induction of c-myc did not prevent senescence by radiation alone or radiation-induced cell death promoted by EB 1089. EB 1089 did not increase the extent of micronucleation, indicative of mitotic catastrophe, induced by radiation alone. However, EB 1089 did promote extensive autophagic cell death in the irradiated cells. Taken together, these studies suggest that the effect of EB 1089 treatment on the radiation response is related in part to enhanced promotion of autophagic cell death and in part to interference with the proliferative recovery that occurs with radiation alone in p53 wild-type breast tumor cells.


Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Autophagy/radiation effects , Breast Neoplasms/metabolism , Calcitriol/analogs & derivatives , Radiation Tolerance , Breast Neoplasms/pathology , Calcitriol/pharmacology , Caspase 3/metabolism , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Cellular Senescence/radiation effects , DNA Damage/drug effects , DNA Damage/radiation effects , DNA Repair/drug effects , DNA Repair/radiation effects , Female , Free Radicals/metabolism , Humans , Reactive Oxygen Species/metabolism , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism
18.
Mol Cancer ; 5: 4, 2006 Jan 18.
Article in English | MEDLINE | ID: mdl-16420693

ABSTRACT

BACKGROUND: DAL-1 (Differentially Expressed in Adenocarcinoma of the Lung)/4.1B is a member of the protein 4.1 superfamily that has been shown to suppress growth in lung, breast and brain tumor cells. In the case of the caspase-3 deficient MCF-7 breast cancer cells, this growth suppression has been shown to be partially mediated by the induction of apoptosis. However the exact mechanism of action of DAL-1/4.1B is unknown. Recently, protein arginine N-methyltransferase 3 (PRMT3) was identified as a DAL-1/4.1B interacting protein. Protein arginine methyltransferases (PRMTs) posttranslationally methylate the arginine residues of proteins, a modification which has been implicated in the regulation of multiple cellular processes including nuclear-cytoplasmic transport, signal transduction, and transcription. RESULTS: To investigate the role of protein methylation in cell death induced by DAL-1/4.1B, DAL-1/4.1B-inducible MCF-7 cells were examined for apoptosis and caspase activation in the absence and presence of the protein methylation inhibitor adenosine dialdehyde (AdOX). Flow cytometry analysis revealed that apoptosis was primarily associated with the activation of caspase 8, and inhibition of this activation blocked the ability of DAL-1/4.1B to induce cell death. CONCLUSION: These results suggest that protein methylation cooperates with DAL-1/4.1B-associated caspase 8-specific activation to induce apoptosis in breast cancer cells.


Subject(s)
Apoptosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Membrane Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Blotting, Western , Breast Neoplasms/enzymology , Caspase 3/metabolism , Caspase 8/metabolism , Cell Line, Tumor , Enzyme Activation , Humans , Methylation , Microfilament Proteins
19.
Oncogene ; 24(43): 6502-15, 2005 Sep 29.
Article in English | MEDLINE | ID: mdl-16007173

ABSTRACT

4.1B is a member of the protein 4.1 superfamily of proteins that link transmembrane proteins to the actin cytoskeleton. The 4.1B gene localizes to chromosome 18p11.3, which undergoes loss of heterozygosity in mammary tumors. Here, we examine the expression of 4.1B in murine mammary epithelium and find that 4.1B is dramatically upregulated in mammary epithelial cells during pregnancy when there is extensive cell proliferation. In contrast, 4.1B is not expressed in virgin, lactating, or involuting mammary epithelium. To examine the consequence of 4.1B loss on mammary epithelial cell proliferation, we analysed mammary glands in 4.1B-null mice. 4.1B loss results in a significant increase in mammary epithelial cell proliferation during pregnancy, but has no effect on mammary epithelial cell proliferation, in virgin or involuting mice. Furthermore, we show that 4.1B inhibits the proliferation of mammary epithelial cell lines by inducing a G1 cell cycle arrest, characterized by decreased cyclin A expression and reduced Rb phosphorylation, and accompanied by reduced erbB2 phosphorylation. This cell cycle arrest does not involve alterations in the activities of MAPK, JNK, or Akt. Collectively, our findings demonstrate that 4.1B regulates mammary epithelial cell proliferation during pregnancy and suggest that its loss may influence mammary carcinoma pathogenesis in multiparous women.


Subject(s)
Mammary Glands, Animal/physiology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation , DNA-Binding Proteins/metabolism , Epithelial Cells/cytology , Female , G1 Phase/genetics , Gene Expression Regulation, Developmental , Humans , Lactation , Mammary Glands, Animal/cytology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Microfilament Proteins , Phosphorylation , Pregnancy , Pregnancy, Animal/genetics , Receptor, ErbB-2/metabolism , Signal Transduction , Transcription Factors/metabolism , Tumor Cells, Cultured
20.
Biochem Biophys Res Commun ; 329(2): 522-30, 2005 Apr 08.
Article in English | MEDLINE | ID: mdl-15737618

ABSTRACT

We previously identified DAL-1/4.1B as a growth suppression protein involved in the pathogenesis of lung, breast, and meningioma tumors. Using yeast two-hybrid interaction cloning, protein arginine N-methyltransferase 3 (PRMT3) was originally identified as a DAL-1/4.1B-interacting protein. PRMTs catalyze the sequential transfer of methyl groups from S-adeonsyl-l-methionine to the guanidino nitrogens of arginine residues in proteins, the effect of which can include regulation of signal transduction, transcription regulation, and RNA transport, suggesting that modulating this event may have far-reaching impact. In this study, we assessed the impact of DAL-1/4.1B binding on the activity of another family member, PRMT5, both in vitro and in cells. In contrast to PRMT3, DAL-1/4.1B was found to mediate PRMT5 by either inhibiting (Sm proteins) or enhancing (myelin basic protein) protein methylation. We propose that this interaction between a tumor suppressor and a post-translational methylation enzyme is of biological importance in controlling tumorigenesis.


Subject(s)
Breast Neoplasms/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Methyltransferases/chemistry , Protein Methyltransferases/metabolism , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolism , Binding Sites , Cell Line , Enzyme Activation , Genes, Tumor Suppressor , Humans , Kidney/metabolism , Microfilament Proteins , Protein Binding , Protein-Arginine N-Methyltransferases , Substrate Specificity
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