RESUMO
The Genetic Association Information Network (GAIN) Data Access Committee was established in June 2007 to provide prompt and fair access to data from six genome-wide association studies through the database of Genotypes and Phenotypes (dbGaP). Of 945 project requests received through 2011, 749 (79%) have been approved; median receipt-to-approval time decreased from 14 days in 2007 to 8 days in 2011. Over half (54%) of the proposed research uses were for GAIN-specific phenotypes; other uses were for method development (26%) and adding controls to other studies (17%). Eight data-management incidents, defined as compromises of any of the data-use conditions, occurred among nine approved users; most were procedural violations, and none violated participant confidentiality. Over 5 years of experience with GAIN data access has demonstrated substantial use of GAIN data by investigators from academic, nonprofit, and for-profit institutions with relatively few and contained policy violations. The availability of GAIN data has allowed for advances in both the understanding of the genetic underpinnings of mental-health disorders, diabetes, and psoriasis and the development and refinement of statistical methods for identifying genetic and environmental factors related to complex common diseases.
Assuntos
Acesso à Informação , Pesquisa Biomédica , Bases de Dados Factuais , Estudo de Associação Genômica Ampla , Serviços de Informação , Humanos , PesquisadoresRESUMO
The Human Microbiome Project (HMP), funded as an initiative of the NIH Roadmap for Biomedical Research (http://nihroadmap.nih.gov), is a multi-component community resource. The goals of the HMP are: (1) to take advantage of new, high-throughput technologies to characterize the human microbiome more fully by studying samples from multiple body sites from each of at least 250 "normal" volunteers; (2) to determine whether there are associations between changes in the microbiome and health/disease by studying several different medical conditions; and (3) to provide both a standardized data resource and new technological approaches to enable such studies to be undertaken broadly in the scientific community. The ethical, legal, and social implications of such research are being systematically studied as well. The ultimate objective of the HMP is to demonstrate that there are opportunities to improve human health through monitoring or manipulation of the human microbiome. The history and implementation of this new program are described here.
Assuntos
Bactérias , Trato Gastrointestinal/microbiologia , Metagenoma/genética , Boca/microbiologia , National Institutes of Health (U.S.) , Pele/microbiologia , Vagina/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Feminino , Humanos , Programas Nacionais de Saúde , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Estados UnidosRESUMO
Mutations in two branch-point sequences (BPS) in intron 3 of the XPC DNA repair gene affect pre-mRNA splicing in association with xeroderma pigmentosum (XP) with many skin cancers (XP101TMA) or no skin cancer (XP72TMA), respectively. To investigate the mechanism of these abnormalities we now report that transfection of minigenes with these mutations revealed abnormal XPC pre-mRNA splicing that mimicked pre-mRNA splicing in the patients' cells. DNA oligonucleotide-directed RNase H digestion demonstrated that mutations in these BPS disrupt U2 snRNP-BPS interaction. XP101TMA cells had no detectable XPC protein but XP72TMA had 29% of normal levels. A small amount of XPC protein was detected at sites of localized ultraviolet (UV)-damaged DNA in XP72TMA cells which then recruited other nucleotide excision repair (NER) proteins. In contrast, XP101TMA cells had no detectable recruitment of XPC or other NER proteins. Post-UV survival and photoproduct assays revealed greater reduction in DNA repair in XP101TMA cells than in XP72TMA. Thus mutations in XPC BPS resulted in disruption of U2 snRNP-BPS interaction leading to abnormal pre-mRNA splicing and reduced XPC protein. At the cellular level these changes were associated with features of reduced DNA repair including diminished NER protein recruitment, reduced post-UV survival and impaired photoproduct removal.
Assuntos
Proteínas de Ligação a DNA/genética , Mutação/genética , Precursores de RNA/genética , Splicing de RNA/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Xeroderma Pigmentoso/genética , Sequência de Bases , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , DNA/genética , DNA/metabolismo , Dano ao DNA , Reparo do DNA/efeitos da radiação , Éxons/genética , Genoma Humano/genética , Humanos , Dados de Sequência Molecular , Fenótipo , Ligação Proteica/efeitos da radiação , Dímeros de Pirimidina/metabolismo , Splicing de RNA/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Raios Ultravioleta , Xeroderma Pigmentoso/patologiaRESUMO
Two unrelated xeroderma pigmentosum (XP) patients, with and without neurological abnormalities, respectively, had identical defects in the XPC DNA nucleotide excision repair (NER) gene. Patient XP21BE, a 27-year-old woman, had developmental delay and early onset of sensorineural hearing loss. In contrast, patient XP329BE, a 13-year-old boy, had a normal neurological examination. Both patients had marked lentiginous hyperpigmentation and multiple skin cancers at an early age. Their cultured fibroblasts showed similar hypersensitivity to killing by UV and reduced repair of DNA photoproducts. Cells from both patients had a homozygous c.2T>G mutation in the XPC gene which changed the ATG initiation codon to arginine (AGG). Both had low levels of XPC message and no detectable XPC protein on Western blotting. There was no functional XPC activity in both as revealed by the failure of localization of XPC and other NER proteins at the sites of UV-induced DNA damage in a sensitive in vivo immunofluorescence assay. XPC cDNA containing the initiation codon mutation was functionally inactive in a post-UV host cell reactivation (HCR) assay. Microsatellite markers flanking the XPC gene showed only a small region of identity ( approximately 30kBP), indicating that the patients were not closely related. Thus, the initiation codon mutation resulted in DNA repair deficiency in cells from both patients and greatly increased cancer susceptibility. The neurological abnormalities in patient XP21BE may be related to close consanguinity and simultaneous inheritance of other recessive genes or other gene modifying effects rather than the influence of XPC gene itself.
Assuntos
Códon de Iniciação/metabolismo , Proteínas de Ligação a DNA/genética , Mutação , Doenças do Sistema Nervoso/genética , Xeroderma Pigmentoso/genética , Adolescente , Adulto , Linhagem Celular , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Repetições de Microssatélites , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Raios Ultravioleta , Xeroderma Pigmentoso/complicações , Xeroderma Pigmentoso/metabolismoRESUMO
The viral early-to-late switch of papillomavirus infection is tightly linked to keratinocyte differentiation and is mediated in part by alternative mRNA splicing. Here, we report that SRp20, a cellular splicing factor, controls the early-to-late switch via interactions with A/C-rich RNA elements. An A/C-rich SE4 element regulates the selection of a bovine papillomavirus type 1 (BPV-1) late-specific splice site, and binding of SRp20 to SE4 suppresses this selection. Expression of late BPV-1 L1 or human papillomavirus (HPV) L1, the major capsid protein, inversely correlates with SRp20 levels in the terminally differentiated keratinocytes. In HPV type 16, a similar SRp20-interacting element also controls the viral early-to-late switch. Keratinocytes in raft cultures, which support L1 expression, make considerably less SRp20 than keratinocytes in monolayer cultures, which do not support L1 expression. Conversely, abundant SRp20 in cancer cells or undifferentiated keratinocytes is important for the expression of the viral early E6 and E7 by promoting the expression of cellular transcription factor SP1 for transactivation of viral early promoters.
Assuntos
Papillomavirus Bovino 1/fisiologia , Regulação Viral da Expressão Gênica , Papillomavirus Humano 16/fisiologia , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Linhagem Celular Tumoral , Humanos , Queratinócitos/virologia , Sítios de Splice de RNA , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Fatores de Processamento de Serina-ArgininaRESUMO
Defects in the xeroderma pigmentosum type B (XPB) gene (ERCC3), a DNA helicase involved in nucleotide excision repair (NER) and an essential subunit of the basal transcription factor, TFIIH, have been described in only three families. We report three new XPB families: one has two sisters with relatively mild xeroderma pigmentosum (XP) symptoms not previously associated with XPB mutations and two have severe XP/Cockayne syndrome (CS) complex symptoms. All XP-B cells had reduced NER and post-ultraviolet (UV) cell viability. Surprisingly, cells from the milder XP sisters had the same missense mutation (c.296T>C, p.F99S) that was previously reported in two mild XP/CS complex brothers. These cells had higher levels of XPB protein than the severely affected XP/CS complex patients. An XPB expression vector with the p.F99S mutation partially complemented the NER defect in XP-B cells. The three severely affected XP/CS complex families all have the same splice acceptor site mutation (c.2218-6C>A, p.Q739insX42) in one allele. This resulted in alteration of 41 amino acids at the C terminus, producing partial NER complementation. This limited number of mutations probably reflects the very restricted range of alterations of this vital protein that are compatible with life. We found new mutations in the second allele yielding markedly truncated proteins in all five XP or XP/CS complex families: c.1273C>T, p.R425X; c.471+1G>A, p.K157insTSDSX; c.807-808delTT, p.F270X; c.1421-1422insA, p.D474EfsX475; and c.1633C>T, p.Q545X. The remarkable phenotypic heterogeneity of XPB is associated with partially active missense mutations in milder patients while severe XP/CS complex patients have nonsense mutations in both alleles with low levels of altered XPB proteins.
Assuntos
Síndrome de Cockayne/complicações , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Xeroderma Pigmentoso/complicações , Xeroderma Pigmentoso/genética , Anormalidades Múltiplas/genética , Adulto , Processamento Alternativo , Carcinoma Basocelular/genética , Sobrevivência Celular/efeitos da radiação , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Doenças Desmielinizantes/genética , Neoplasias Oculares/genética , Feminino , Expressão Gênica , Genoma Humano , Heterozigoto , Humanos , Masculino , Melanoma/genética , Pessoa de Meia-Idade , Mutação , Núcleo Familiar , Fenótipo , Transtornos de Fotossensibilidade/genética , Polimorfismo Genético , RNA/biossíntese , Raios Ultravioleta/efeitos adversosRESUMO
Papillomaviruses are a group of small non-enveloped DNA tumor viruses whose infection usually causes benign epithelial lesions (warts). Certain types of HPVs, such as HPV-16, HPV-18, and HPV-31, have been recognized as causative agents of cervical cancer and anal cancer and their infections, which arise via sexual transmission, are associated with more than 95% of cervical cancer. Papillomaviruses infect keratinocytes in the basal layer of stratified squamous epithelia and replicate in the nucleus of infected keratinocytes in a differentiation-dependent manner. Viral gene expression in infected cells depends on cell differentiation and is tightly regulated at the transcriptional and post-transcriptional levels. A noteworthy feature of all papillomavirus transcripts is that they are transcribed as a bicistronic or polycistronic form containing two or more ORFs and are polyadenylated at either an early or late poly(A) site. In the past ten years, remarkable progress has been made in understanding how this complex viral gene expression is regulated at the level of transcription (such as via DNA methylation) and particularly post-transcription (including RNA splicing, polyadenylation, and translation). Current knowledge of papillomavirus mRNA structure and RNA processing has provided some clues on how to control viral oncogene expression. However, we still have little knowledge about which mRNAs are used to translate each viral protein. Continuing research on post-transcriptional regulation of papillomavirus infection will remain as a future focus to provide more insights into papillomavirus-host interactions, the virus life-cycle, and viral oncogenesis.
Assuntos
Metilação de DNA , Regulação Viral da Expressão Gênica , Papillomaviridae/genética , Genoma , Infecções por Papillomavirus/fisiopatologia , Interferência de RNA , Splicing de RNA , Transcrição GênicaRESUMO
Promising molecular techniques may allow for testing of novel and complex hypotheses such as defining gene expression profiles in specific cells, tumors, or their microenvironments. For most large cancer epidemiologic and population-based studies, however, application of such promising techniques may not be possible owing to constraints of specimen preservation from paraffin-embedded tissues. Alternative methods would ideally preserve tissue morphology and not degrade DNA or RNA. We conducted a comparison of snap-freezing (freezing with liquid nitrogen), ethanol-fixation with low melt polyester wax embedding, and RNAlater-preservation techniques to determine which method was optimal for subsequent assessment of gene expression changes in cervical cancer. From each of 15 women with cancer and 30 without, we procured 3 pieces of cervical tissue and compared snap-freezing, ethanol-fixation, and RNAlater-preservation techniques. Despite slight loss in morphologic quality from snap-frozen cervix tissues, RNA quality was equivalent to or better than RNAlater-preserved tissues and significantly exceeded that from ethanol-fixed/polyester wax embedded tissue. In conclusion, despite the moderate logistical constraints in set-up that required either liquid nitrogen or dry ice on-site for snap-freezing tissue, the ease of downstream processing and consistent high quality RNA made it preferable to the other 2 methods.
Assuntos
Colo do Útero/química , RNA Neoplásico/normas , Manejo de Espécimes/normas , Fixação de Tecidos/normas , Neoplasias do Colo do Útero/diagnóstico , Criopreservação , Etanol/química , Feminino , Humanos , RNA Neoplásico/análise , RNA Neoplásico/isolamento & purificação , Manejo de Espécimes/métodos , Inclusão do Tecido , Fixação de Tecidos/métodos , Ceras/químicaRESUMO
XPC DNA repair gene mutations result in the cancer-prone disorder xeroderma pigmentosum. The XPC gene spans 33 kb and has 16 exons (82-882 bp) and 15 introns (0.08-5.4 kb). A 1.6 kb intron was found within exon 5. Sensitive real- time quantitative reverse transcription-polymerase chain reaction methods were developed to measure full-length XPC mRNA (the predominant form) and isoforms that skipped exons 4, 7 or 12. Exon 7 was skipped in approximately 0.07% of XPC mRNAs, consistent with the high information content of the exon 7 splice acceptor and donor sites (12.3 and 10.4 bits). In contrast, exon 4 was skipped in approximately 0.7% of the XPC mRNAs, consistent with the low information content of the exon 4 splice acceptor (-0.1 bits). A new common C/A single nucleotide polymorphism in the XPC intron 11 splice acceptor site (58% C in 97 normals) decreased its information content from 7.5 to 5.1 bits. Fibroblasts homozygous for A/A had significantly higher levels (approximately 2.6-fold) of the XPC mRNA isoform that skipped exon 12 than those homozygous for C/C. This abnormally spliced XPC mRNA isoform has diminished DNA repair function and may contribute to cancer susceptibility.
Assuntos
Processamento Alternativo/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Polimorfismo de Nucleotídeo Único/genética , Sítios de Splice de RNA/genética , Éxons/genética , Fibroblastos , Frequência do Gene/genética , Haplótipos/genética , Humanos , Íntrons/genética , Desequilíbrio de Ligação/genética , Dados de Sequência Molecular , Neoplasias/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Isoformas de Proteínas/genética , Deleção de SequênciaRESUMO
Keratinocytes undergo a finite number of divisions in culture before senescing. The high-risk human papillomavirus (HPV) E6 and E7 oncoproteins prevent keratinocyte senescence and extend life span by interacting with p53 and pRb, respectively, and also by transcriptionally activating the human telomerase reverse transcriptase (hTERT) gene, which encodes the catalytic subunit of telomerase. We correlated telomerase activity, which was measured by a highly sensitive and quantitative real-time quantitative-PCR-based telomeric repeat amplification protocol assay, with telomere length and the expression of hTERT, p16(INK4a), and HPV-16 E6 and E7 in keratinocytes grown under two culture conditions. Primary human foreskin keratinocytes (HFKs) cultured in keratinocyte serum-free medium on plastic senesced at approximately 13 population doublings (PDs). Senescence was accompanied by a dramatic increase in p16(INK4A) levels, a marked decrease in telomerase, and only a slight decrease in telomere length. In contrast, HFKs grown in F medium on 3T3 fibroblast feeders maintained elevated telomerase and lower levels of p16(INK4A) for 60 PDs before senescing approximately 81 PDs. E7 was shown to act synergistically with E6 to super induce telomerase expression in a feeder environment-dependent manner. Culture of both HFKs and HFK/16E6E7 cells in the feeder environment significantly increased the number of doublings that these cells could undergo without a significant reduction in telomere length. Finally, transfer of either HFKs or HFK/16E6E7 cells from plastic to the feeder fibroblast culture system significantly induced telomerase activity. This induction in telomerase was fully reversible and largely attributable to the medium. Our results suggest that the influence of keratinocyte culture conditions on the expression of telomerase and p16(INK4A) and on telomere maintenance is responsible, at least partially, for the differences in proliferative capacity, senescence, and HPV-keratinocyte interactions seen in the two culture systems.
Assuntos
Queratinócitos/citologia , Queratinócitos/enzimologia , Proteínas Oncogênicas Virais/genética , Proteínas Repressoras , Telomerase/biossíntese , Animais , Células Cultivadas , Senescência Celular/fisiologia , Meios de Cultura , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Proteínas de Ligação a DNA , Indução Enzimática , Humanos , Queratinócitos/fisiologia , Camundongos , Proteínas Oncogênicas Virais/biossíntese , Proteínas E7 de Papillomavirus , Telômero/genética , Telômero/metabolismo , Transdução GenéticaRESUMO
Among tumor sites, cervical cancer offers an ideal model for investigating differences in gene expression associated with transitions from normal to precancer and invasion to cancer. To evaluate the validity of assessing gene expression in cervical tissues acquired in a clinical setting, we investigated whether standard procedures, namely the application of acetic acid and/or Lugol's iodine, employed for the visualization of colposcopically directed biopsies, altered patterns in oligonucleotide (oligo) arrays. We compared microarray profiles from six women, each with three adjacent tissue samples removed from benign hysterectomy specimens and treated as follows: immediately frozen, acetic acid application only, acetic acid, and Lugol's iodine. Of the 22,464 original spots on the microarray, 4,850 spots were expressed at detectable levels for further evaluation upon data normalization and filtration. For each spot, the difference between topical applications was computed, and P values were calculated using a bivariate T2 test. Upon adjustment for multiple comparisons using both the Holm's and Hochberg's procedures as well as the False Discovery Rate (Benjamini-Hochberg and Benjamini-Yeuketili [BY]), we failed to identify genes differentially expressed and conclude that standard precolposcopic procedures do not substantially affect the overall gene expression patterns in the normal cervix.
Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/normas , Colo do Útero/metabolismo , Colposcopia , Perfilação da Expressão Gênica , Neoplasias do Colo do Útero/diagnóstico , Artefatos , Biópsia , Colo do Útero/patologia , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias do Colo do Útero/patologiaRESUMO
Xeroderma pigmentosum-variant (XP-V) patients have sun sensitivity and increased skin cancer risk. Their cells have normal nucleotide excision repair, but have defects in the POLH gene encoding an error-prone polymerase, DNA polymerase eta (pol eta). To survey the molecular basis of XP-V worldwide, we measured pol eta protein in skin fibroblasts from putative XP-V patients (aged 8-66 years) from 10 families in North America, Turkey, Israel, Germany, and Korea. Pol eta was undetectable in cells from patients in eight families, whereas two showed faint bands. DNA sequencing identified 10 different POLH mutations. There were two splicing, one nonsense, five frameshift (3 deletion and 2 insertion), and two missense mutations. Nine of these mutations involved the catalytic domain. Although affected siblings had similar clinical features, the relation between the clinical features and the mutations was not clear. POLH mRNA levels were normal or reduced by 50% in three cell strains with undetectable levels of pol eta protein, indicating that nonsense-mediated message decay was limited. We found a wide spectrum of mutations in the POLH gene among XP-V patients in different countries, suggesting that many of these mutations arose independently.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Mutação/genética , Xeroderma Pigmentoso/genética , Adolescente , Adulto , Idoso , Ásia , Criança , Códon sem Sentido/genética , DNA Polimerase Dirigida por DNA/metabolismo , Europa (Continente) , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Mutação da Fase de Leitura/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , América do Norte , Linhagem , RNA Mensageiro/metabolismo , Xeroderma Pigmentoso/etnologia , Xeroderma Pigmentoso/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) is a proangiogenic factor upregulated in many tumors. The alternative splicing of VEGF mRNA renders 3 major isoforms of 121, 165 and 189 amino-acids in humans (1 less amino-acid for each mouse VEGF isoform). We have designed isoform specific real time QRT-PCR assays to quantitate VEGF transcripts in mouse and human normal and malignant prostates. In the human normal prostate, VEGF(165) was the predominant isoform (62.8% +/- 5.2%), followed by VEGF(121) (22.5% +/- 6.3%) and VEGF(189) (p < 0.001) (14.6% +/- 2.1%). Prostate tumors showed a significant increase in the percentage of VEGF(121) and decreases in VEGF(165) (p < 0.01) and VEGF(189) (p < 0.05). However, the amount of total VEGF mRNA was similar between normal and malignant prostates. VEGF(164) was the transcript with the highest expression in the mouse normal prostate. Unlike human prostate cancer, tumors from TRAMP mice demonstrated a significant increase in total VEGF mRNA levels and in each of the VEGF isoforms, without changes in the relative isoform ratios. Morpholino phosphorodiamide antisense oligonucleotide technology was used to increase the relative amount of VEGF(121) while proportionally decreasing VEGF(165) and VEGF(189) levels in human prostate cell lines, through the modification of alternative splicing, without changing transcription levels and total amount of VEGF. The increase in the VEGF(121)/VEGF(165-189) ratio in PC3 cells resulted in a dramatic increase in prostate tumor angiogenesis in vivo. Our results underscore the importance of VEGF(121) in human prostate carcinoma and demonstrate that the relative expression of the different VEGF isoforms has an impact on prostate carcinogenesis.
Assuntos
Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Processamento Alternativo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Morfolinas/administração & dosagem , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/genética , Neoplasias da Próstata/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
HPV16 DNA is often integrated in cancers, disrupting the E1 or E2 genes. E2 can repress the E6/E7 promoter, but other models have been proposed to explain why integration promotes malignant progression. E1 and E2 are required for viral replication, and so genetic analysis of their role in transcriptional regulation is complex. Therefore, we developed an extrachromosomal vector containing HPV16 to undertake a genetic analysis of the E1 and E2 genes. We demonstrate that the E2 protein is primarily a transcriptional repressor when expressed from the virus. Furthermore, repression requires both the transactivation function of E2 and specific binding of E2 to the LCR. We find no evidence that the E1 protein directly modulates HPV16 gene expression. However, certain E1 mutations modulated transcription indirectly by altering splicing of E2 mRNA species. These data provide important insight into which E1 and E2 functions are optimal targets for anti-viral therapies.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Sequência de Bases , DNA Viral , Herpesvirus Humano 4 , Dados de Sequência Molecular , Mutagênese Insercional , Fases de Leitura Aberta , RNA Viral , Ativação Transcricional , Replicação ViralRESUMO
Despite recent emerging evidence suggesting that cancer stem cells subsist in a variety of tumors, it is not yet fully elucidated whether postnatal stem cells are directly involved in tumorigenesis. We used murine bone marrow-derived mesenchymal stem cells (BMMSCs) as a model to test a hypothesis that tumorigenesis may originate from spontaneous mutation of stem cells. In this study, we demonstrated that murine BMMSCs, after numerous passages, obtained unlimited population doublings and proceeded to a malignant transformation state, resulting in fibrosarcoma formation in vivo. Transformed BMMSCs colonized to multiple organs when delivered systemically through the tail vein. Fibrosarcoma cells formed by transformed BMMSCs contained cancer progenitors, which were capable of generating colony clusters in vitro and fibrosarcoma in vivo by the second administration. The mechanism by which BMMSCs transformed to malignant cells was associated with accumulated chromosomal abnormalities, gradual elevation in telomerase activity, and increased c-myc expression. Moreover, BMMSCs and their transformed counterpart, fibrosarcoma-forming cells, demonstrated different sensitivity to anti-cancer drugs. BMMSCs/fibrosarcoma transformation system may provide an ideal system to elucidate the mechanism of how stem cells become cancer cells and to screen anti-sarcoma drugs.
Assuntos
Transformação Celular Neoplásica/genética , Instabilidade Cromossômica , Células-Tronco Hematopoéticas/patologia , Células-Tronco Mesenquimais/patologia , Animais , Aberrações Cromossômicas , Ensaios de Seleção de Medicamentos Antitumorais , Fibrossarcoma/etiologia , Fibrossarcoma/genética , Fibrossarcoma/patologia , Transplante de Células-Tronco Hematopoéticas , Hibridização in Situ Fluorescente , Técnicas In Vitro , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Nus , Mutação , Ensaio Tumoral de Célula-TroncoRESUMO
Xeroderma pigmentosum group C (XP-C) is a rare autosomal recessive disorder. Patients with two mutant alleles of the XPC DNA repair gene have sun sensitivity and a 1000-fold increase in skin cancers. Clinically normal parents of XP-C patients have one mutant allele and one normal allele. As a step toward evaluating cancer risk in these XPC heterozygotes we characterized cells from 16 XP families. We identified 15 causative mutations (5 frameshift, 6 nonsense and 4 splicing) in the XPC gene in cells from 16 XP probands. All had premature termination codons (PTC) and absence of normal XPC protein on western blotting. The cell lines from 26 parents were heterozygous for the same mutations. We employed a real-time quantitative reverse transcriptase-PCR assay as a rapid and sensitive method to measure XPC mRNA levels. The mean XPC mRNA levels in the cell lines from the XP-C probands were 24% (P<10(-7)) of that in 10 normal controls. This reduced XPC mRNA level in cells from XP-C patients was caused by the PTC that induces nonsense-mediated mRNA decay. The mean XPC mRNA levels in cell lines from the heterozygous XP-C carriers were intermediate (59%, P=10(-4)) between the values for the XP patients and the normal controls. This study demonstrates reduced XPC mRNA levels in XP-C patients and heterozygotes. Thus, XPC mRNA levels may be evaluated as a marker of cancer susceptibility in carriers of mutations in the XPC gene.
Assuntos
Códon sem Sentido/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Mutação/genética , RNA Mensageiro/genética , Xeroderma Pigmentoso/genética , Adolescente , Adulto , Western Blotting , Criança , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Heterozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Pais , Reação em Cadeia da Polimerase , Sítios de Splice de RNA , RNA Mensageiro/metabolismo , Xeroderma Pigmentoso/metabolismoRESUMO
Most human cancer cells display increased telomerase activity that appears to be critical for continued cell proliferation and tumor formation. The E6 protein of malignancy-associated human papillomaviruses increases cellular telomerase in primary human keratinocytes at least partly via transcriptional activation of the telomerase catalytic subunit, hTERT. In the present study, we investigated whether E6AP, a ubiquitin ligase well known for binding and mediating some of the activities of the E6 oncoprotein, participated in the transactivation of the hTERT promoter. Our results demonstrate that E6 mutants that fail to bind E6AP are also defective for increasing telomerase activity and transactivating the hTERT promoter. More importantly, E6AP knock-out mouse cells and small interfering RNA techniques demonstrated that E6AP was required for hTERT promoter transactivation in both mouse and human cells. Neither E6 nor E6AP bound to the hTERT promoter or activated the promoter in the absence of the partner protein. With all transactivation-competent E6 proteins, induction of the hTERT promoter was dependent upon E box elements in the core promoter. It appears, therefore, that E6-mediated activation of the hTERT promoter requires a complex of E6-E6AP to engage the hTERT promoter and that activation is dependent upon Myc binding sites in the promoter. The recruitment of a cellular ubiquitin ligase to the hTERT promoter during E6-mediated transcriptional activation suggests a role for the local ubiquitination (and potential degradation) of promoter-associated regulatory proteins, including the Myc protein.
Assuntos
Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/genética , Proteínas Repressoras/metabolismo , Telomerase/metabolismo , Ativação Transcricional , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/fisiologia , Acetilcisteína/metabolismo , Animais , Sítios de Ligação , Western Blotting , Domínio Catalítico , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA , Fibroblastos/metabolismo , Vetores Genéticos/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Queratinócitos/metabolismo , Luciferases/metabolismo , Camundongos , Camundongos Knockout , Mutação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção , Ubiquitina/metabolismoRESUMO
Previous studies have suggested that most papillomaviruses enter the host cell via clathrin-dependent receptor-mediated endocytosis but have not addressed later steps in viral entry. To examine these events, we followed the localization of L2 and packaged DNA after entry of infectious virions or L1/L2 pseudovirions. Confocal microscopic analyses of HeLa cells showed a time-dependent uncoating of capsids in cytoplasmic vesicles and the accumulation of both L2 and viral DNA at distinct nuclear domains identified as nuclear domain 10 (ND10). Both L2 and the pseudogenome had a punctate distribution and localized to ND10 in promyelocytic leukemia protein (PML)-expressing cells, whereas L2 had a diffuse nuclear distribution in PML-/- cells. The number of pseudovirus-infected cells was an order of magnitude higher in the PML+ cells compared with the PML-/- cells, and viral genome transcription after infection with authentic bovine papillomavirus virions was similarly elevated in PML+ cells. The results identify a role for PML in the enhancement of viral infectivity in the early part of the life cycle. We propose a model in which L2 chaperones the viral genome to ND10 to efficiently initiate viral transcription.
Assuntos
Papillomavirus Bovino 1/fisiologia , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares/biossíntese , Infecções por Papillomavirus/metabolismo , Fatores de Transcrição/biossíntese , Animais , Papillomavirus Bovino 1/crescimento & desenvolvimento , Papillomavirus Bovino 1/patogenicidade , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Bovinos , Núcleo Celular/metabolismo , Núcleo Celular/virologia , DNA Viral/genética , DNA Viral/metabolismo , Expressão Gênica , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , Pseudogenes/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética , Proteínas Supressoras de Tumor , Dedos de ZincoRESUMO
Equine infectious anemia virus (EIAV), a lentivirus distantly related to HIV-1, encodes regulatory proteins, EIAV Tat (ETat) and Rev (ERev), from a four-exon mRNA. Exon 3 of the tat/rev mRNA contains a 30-nucleotide purine-rich element (PRE) which binds both ERev and SF2/ASF, a member of the SR family of RNA splicing factors. To better understand the role of this element in the regulation of EIAV pre-mRNA splicing, we quantified the effects of mutation or deletion of the PRE on exon 3 splicing in vitro and on alternative splicing in vivo. We also determined the branch point elements upstream of exons 3 and 4. In vitro splicing of exon 3 to exon 4 was not affected by mutation of the PRE, and addition of purified SR proteins enhanced splicing independently of the PRE. In vitro splicing of exon 2 to exon 3 was dependent on the PRE; under conditions of excess SR proteins, either the PRE or the 5' splice site of exon 3 was sufficient to activate splicing. We applied isoform-specific primers in real-time RT-PCR reactions to quantitatively analyze alternative splicing in cells transfected with rev-minus EIAV provirus constructs. In the context of provirus with wild-type exon 3, greater than 80% of the viral mRNAs were multiply spliced, and of these, less than 1% excluded exon 3. Deletion of the PRE resulted in a decrease in the relative amount of multiply spliced mRNA to about 40% of the total and approximately 39% of the viral mRNA excluded exon 3. Ectopic expression of ERev caused a decrease in the relative amount of multiply spliced mRNA to approximately 50% of the total and increased mRNAs that excluded exon 3 to about 4%. Over-expression of SF2/ASF in cells transfected with wild-type provirus constructs inhibited splicing but did not significantly alter exon 3 skipping.
Assuntos
Processamento Alternativo , Regulação Viral da Expressão Gênica , Vírus da Anemia Infecciosa Equina/genética , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Éxons , Produtos do Gene rev/genética , Produtos do Gene rev/metabolismo , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Purinas/química , Precursores de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Fatores de Processamento de Serina-ArgininaRESUMO
The human papillomavirus type 16 (HPV-16) E5 protein is an 83-amino-acid, hydrophobic polypeptide that has been localized to intracellular membranes when overexpressed in COS-1 cells. While the HPV-16 E5 protein appears to modulate endosomal pH and signal transduction pathways, genetic analysis of its biological activities has been hampered by low (usually nondetectable) levels of expression in stable cell lines. Sequence analysis of the native HPV-16 E5 gene revealed that infrequent-use codons are used for 33 of its 83 amino acids and, in an effort to optimize E5 expression, we converted these codons to those more common in mammalian genes. The modified gene, 16E5*, generated protein levels that were six- to ninefold higher than those of wild-type HPV-16 E5, whereas the levels of mRNA were unchanged. 16E5* protein was detectable in keratinocytes by immunoblotting, immunoprecipitation, and immunofluorescence techniques and formed disulfide-dependent dimers and higher-order oligomers. Unlike the bovine papillomavirus E5 protein, which is present in the Golgi, 16E5* was localized primarily to the endoplasmic reticulum and its expression reduced the in vitro life span of keratinocytes.