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1.
J Exp Clin Cancer Res ; 39(1): 69, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312295

RESUMO

BACKGROUND: Liquid biopsy (LB) in early-stage, non-metastatic colorectal cancer (CRC) must be sensitive enough to detect extremely low circulating tumor DNA (ctDNA) levels. This challenge has been seldom and non-systematically investigated. METHODS: Next generation sequencing (NGS) and digital PCR (dPCR) were combined to test tumor DNAs (tDNAs) and paired ctDNAs collected at surgery from 39 patients, 12 of whom were also monitored during the immediate post-surgery follow up. Patients treated for metastatic disease (n = 14) were included as controls. RESULTS: NGS and dPCR concordantly (100% agreement) called at least one single nucleotide variant (SNV) in 34 tDNAs, estimated differences in allelic frequencies being negligible (±1.4%). However, despite dPCR testing, SNVs were only detectable in 15/34 (44.1%) ctDNAs from patients at surgery, as opposed to 14/14 (100%) metastatic patients. This was likely due to striking differences (average 10 times, up to 500) in ctDNA levels between groups. NGS revealed blood-only SNVs, suggesting spatial heterogeneity since pre-surgery disease stages, and raising the combined NGS/dPCR sensitivity to 58.8%. ctDNA levels at surgery correlated with neither tumor size, stage, grade, or nodal status, nor with variant abundance in paired tDNA. LB sensitivity reached 63.6% when ctDNA was combined with CEA. Finally, persistence and absence of ctDNA on the first conventional (month 3) post-surgery follow-up were associated with fast relapse and a disease-free status in 3 and 7 patients, respectively. CONCLUSIONS: A simple clinical NGS/dPCR/CEA combination effectively addresses the LB challenge in a fraction of non-metastatic CRC patients.


Assuntos
DNA Tumoral Circulante/metabolismo , Neoplasias Colorretais/cirurgia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biópsia Líquida/métodos , Estudos Transversais , Feminino , Humanos , Masculino
3.
Hum Reprod ; 29(12): 2802-13, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25336713

RESUMO

STUDY QUESTION: Can next-generation sequencing (NGS) techniques be used reliably for comprehensive aneuploidy screening of human embryos from patients undergoing IVF treatments, with the purpose of identifying and selecting chromosomally normal embryos for transfer? SUMMARY ANSWER: Extensive application of NGS in clinical preimplantation genetic screening (PGS) cycles demonstrates that this methodology is reliable, allowing identification and transfer of euploid embryos resulting in ongoing pregnancies. WHAT IS KNOWN ALREADY: The effectiveness of PGS is dependent upon the biology of the early embryo and the limitations of the technology. Fluorescence in situ hybridization, used to test for a few chromosomes, has largely been superseded by microarray techniques that test all 24 chromosomes. Array comparative genomic hybridization (array-CGH) has been demonstrated to be an accurate PGS method and has become the de facto gold standard, but new techniques, such as NGS, continue to emerge. STUDY DESIGN, SIZE, DURATION: The study consisted of a prospective trial involving a double blind parallel evaluation, with both NGS and array-CGH techniques, of 192 blastocysts obtained from 55 consecutive clinical PGS cycles undertaken during the period of September to October 2013. Consistency of NGS-based aneuploidy detection was assessed by matching the results obtained with array-CGH-based diagnoses. Primary outcome measure was accuracy of the chromosomal analysis; secondary outcome measures were clinical outcomes. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Fifty-five patients (median age 39.3 years, range 32-46) undergoing PGS were enrolled in the study. All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on Day 5 of development or Day 6/7 for slower growing embryos. The method involved whole genome amplification followed by both NGS and array-CGH. The MiSeq control software, real-time analysis and reporter performed on-board primary and secondary bioinformatics analysis. Copy number variation analysis was accomplished with BlueFuse Multi software. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 192 blastocysts were blindly evaluated with the NGS-based protocol. Paired comparison between NGS and array-CGH from individual embryos showed concordant results in 191/192 (99.5%) of the blastocysts tested. In total 4608 chromosomes were assessed, 211 (4.6%) of which carried a copy number imbalance. NGS specificity for aneuploidy calling (consistency of chromosome copy number assignment) was 99.98% (4333/4334; 95% confidence interval [95% CI]: 99.87-100) with a sensitivity of 100% (211/211, 95% CI: 99.25-100). Despite one discordant result, NGS specificity and sensitivity for aneuploid embryo calling (24-chromosome diagnosis consistency) were both 100% since the discordant sample presented several other aneuploidies. Clinical application of the NGS-based approach revealed 74/192 (38.5%) euploid blastocysts. Following transfer of 50 embryos in 47 women, 34 women had positive hCG levels: 30 pregnancies continued, confirmed by at least one fetal sac and heart beat (63.8% clinical pregnancy rate/embryo transfer), 3 were biochemical and 1 miscarried. A total of 32 embryos implanted and led to the presence of a fetal sac (64.0% implantation rate). All pregnancies went to term resulting in the birth of 31 healthy babies. LIMITATION, REASON FOR CAUTION: Although clinical results reported high pregnancy outcomes following transfer of screened embryos, further data and broad-based clinical application are required to better define the role of NGS in PGS. Before recommending widespread application, a randomized controlled trial confirming its clinical effectiveness is advisable. WIDER IMPLICATION OF THE FINDING: This is the first study reporting extensive application of NGS-based comprehensive aneuploidy screening on embryos at blastocyst stage in a clinical setting versus array-CGH as test of reference. NGS has demonstrated a reliable methodology, with the potential to improve chromosomal diagnosis on embryos especially in terms of high-throughput, automation and ability to detect aneuploidy. NGS methodology may represent a valuable alternative to the other comprehensive aneuploidy screening techniques currently available. STUDY FUNDING/COMPETING INTERESTS: No external funding was sought for this study. Drs F.K. and C.-E.M. are full-time employees of Illumina, Inc., which provided NGS library and sequencing reagents for the study. All other authors have no conflicts to declare. TRIAL REGISTRATION NUMBER: Not applicable.


Assuntos
Aneuploidia , Diagnóstico Pré-Implantação/métodos , Análise de Sequência de DNA/métodos , Adulto , Método Duplo-Cego , Técnicas de Cultura Embrionária , Transferência Embrionária , Feminino , Genoma Humano , Humanos , Pessoa de Meia-Idade , Sensibilidade e Especificidade
4.
Fertil Steril ; 101(5): 1375-82, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24613537

RESUMO

OBJECTIVE: To validate a next-generation sequencing (NGS)-based method for 24-chromosome aneuploidy screening and to investigate its applicability to preimplantation genetic screening (PGS). DESIGN: Retrospective blinded study. SETTING: Reference laboratory. PATIENT(S): Karyotypically defined chromosomally abnormal single cells and whole-genome amplification (WGA) products, previously analyzed by array comparative genomic hybridization (array-CGH), selected from 68 clinical PGS cycles with embryos biopsied at cleavage stage. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Consistency of NGS-based diagnosis of aneuploidy compared with either conventional karyotyping of single cells or array-CGH diagnoses of single blastomeres. RESULT(S): Eighteen single cells and 190 WGA products from single blastomeres, were blindly evaluated with the NGS-based protocol. In total, 4,992 chromosomes were assessed, 402 of which carried a copy number imbalance. NGS specificity for aneuploidy call (consistency of chromosome copy number assignment) was 99.98% (95% confidence interval [CI] 99.88%-100%) with a sensitivity of 100% (95% CI 99.08%-100%). NGS specificity for aneuploid embryo call (24-chromosome diagnosis consistency) was 100% (95% CI 94.59%-100%) with a sensitivity of 100% (95% CI 97.39%-100%). CONCLUSION(S): This is the first study reporting extensive preclinical validation and accuracy assessment of NGS-based comprehensive aneuploidy screening on single cells. Given the high level of consistency with an established methodology, such as array-CGH, NGS has demonstrated a robust high-throughput methodology ready for clinical application in reproductive medicine, with potential advantages of reduced costs and enhanced precision.


Assuntos
Aneuploidia , Hibridização de Ácido Nucleico , Diagnóstico Pré-Natal/normas , Diagnóstico Pré-Natal/tendências , Adulto , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/normas , Sequenciamento de Nucleotídeos em Larga Escala/tendências , Humanos , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico/normas , Técnicas de Amplificação de Ácido Nucleico/tendências , Hibridização de Ácido Nucleico/métodos , Gravidez , Ensaios Clínicos Controlados Aleatórios como Assunto/normas , Ensaios Clínicos Controlados Aleatórios como Assunto/tendências , Estudos Retrospectivos , Método Simples-Cego
5.
J Cell Physiol ; 207(1): 232-7, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16331689

RESUMO

With the aim to find novel partners of human Cyclin T2a, we performed a two-hybrid screening in yeast using the full-length cDNA of this cyclin as bait, and a human heart cDNA library as preys source. Upon several interesting genes selected, our attention has been focused on the cDNA coding for PKNalpha, a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. Co-immunoprecipitation and in vitro pull-down assays independently confirmed the interaction between the two proteins. Luciferase assays, performed on NIH3T3 cell extracts after transfection with a MyoD-responsive promoter, pointed out that PKNalpha was able to enhance MyoD-dependent transcription, and that this effect was further increased when cyclin T2a was co-overexpressed. Finally, overexpression of both Cyclin T2a and PKNalpha in C2C12 cells strongly enhanced the expression of myogenic differentiation markers, such as Myogenin and Myosin Heavy Chain, during starvation-induced differentiation. Taken together, our data strengthen the hypothesis that Cyclin T2a plays a role in muscle differentiation, and propose PKNalpha as a novel partner of Cyclin T2a in this process.


Assuntos
Diferenciação Celular , Ciclinas/metabolismo , Células Musculares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Sítios de Ligação/genética , Ligação Competitiva , Linhagem Celular , Ciclina T , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Ciclinas/genética , DNA Complementar/genética , Expressão Gênica/genética , Humanos , Camundongos , Células Musculares/citologia , Mutação , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Células NIH 3T3 , Plasmídeos/genética , Ligação Proteica , Proteína Quinase C , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido
6.
J Anat ; 205(5): 405-12, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15575889

RESUMO

Cyclin D1 is a key cell-cycle regulatory protein required for the cell to progress through G1 to S phase. We have shown by Western blot analysis that cyclin D1 has a wide distribution in adult mouse tissues, with its level of expression being tissue-dependent. Immunohistochemistry has also shown that cyclin D1 may be present in the cytoplasm, in the nucleus or in both these cell compartments: cytoplasmic staining was observed in both proliferating cells (e.g. kidney, intestine, stomach and salivary gland) and in the non-dividing cells (the mature neurons of adult brain), while nuclear staining was seen in the neurons of the embryonic nervous system. Immunoelectron microscopy results indicate that, in tissues where cyclin D1 is present in both compartments (e.g. intestinal enterocytes), it may move via nuclear pores from the nucleus to the cytoplasm, and vice versa. The findings as a whole suggest that cyclin D1 may play multiple roles within specific tissues, probably by interacting with different substrates, and that its transit between nuclear and cytoplasmic compartments may help maintain cell homeostasis.


Assuntos
Núcleo Celular/química , Ciclina D1/análise , Citoplasma/química , Células Epiteliais/química , Animais , Transporte Biológico , Encéfalo , Ciclo Celular , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Intestinos/química , Túbulos Renais/química , Pulmão/química , Camundongos , Microscopia Imunoeletrônica , Neurônios/química , Glândulas Salivares/química , Estômago/química
7.
Biol Cell ; 96(4): 303-11, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15145534

RESUMO

Signaling through the Notch cell surface receptors is a highly conserved mechanism of cell fate specification. Notch signaling regulates proliferation, differentiation and cell death. In vertebrates, putative gene duplication has originated four Notch genes, Notch-1, -2, -3 and -4. They have been implicated in neurogenesis, hematopoiesis, T-cell development, vasculogenesis and brain cortical growth. We have investigated Notch-1 distribution in normal human tissues by immunohistochemistry and immunoblot. We detected widespread expression of Notch-1 cytoplasmatic staining, with different tissue distributions in the different organs examined. In particular, high expression of Notch-1 was detected in the intermediate suprabasal layers, but not in the dead cells at the extreme periphery of stratified epithelia. Moreover, a low/intermediate level of Notch-1 was observed in lymphocytes in several peripheral lymphoid tissues; in particular the germinal centers of lymph nodes showed the most abundant number of positive cells, which appeared to be centroblasts/immunoblasts based on nuclear morphology. Notch-1 participates in keratinocytes differentiation. We showed by Western blot analysis that Notch-1 level was clearly increased in HaCaT cells after Ca(++) addition and remained substantially elevated until late differentiation stages. These results suggest that Notch-1 may function in numerous cell types in processes beyond cell fate determination, such as neuronal plasticity, muscle hypertrophy, liver regeneration, and germinal center lymphopoiesis during the immune response.


Assuntos
Queratinócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/metabolismo , Western Blotting , Diferenciação Celular , Linhagem Celular Transformada , Feminino , Humanos , Imuno-Histoquímica , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Masculino , Especificidade de Órgãos , Receptor Notch1 , Transdução de Sinais , Pele/citologia , Pele/metabolismo , Distribuição Tecidual
8.
J Cell Physiol ; 199(1): 134-9, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14978742

RESUMO

The adenoviral E1A proteins have been implicated in promotion of proliferation and transformation, inhibition of differentiation, induction of apoptosis, regulation of transcription, and suppression of tumor growth. The ability of E1A to override the fundamental controls of host cells is based on its ability to physically interact with several cellular proteins. We recently characterized RACK1 as a new E1A-interacting protein. In this report, we show that the extreme N-terminal region of E1A, spanning from aminoacids 1-36, and the conserved WD regions of RACK1 are responsible for this interaction. We also demonstrate that E1A and RACK1 colocalize at the perinuclear membrane in the cells. Furthermore, we provide evidence that E1A is able to antagonize the inhibitory effects of RACK1 on Src activity. These results suggest that RACK1 signaling pathway may be a functional target of E1A, contributing to E1A oncogenic effect in the host cells.


Assuntos
Proteínas E1A de Adenovirus/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Camundongos , Células NIH 3T3 , Testes de Precipitina , Ligação Proteica , Receptores de Quinase C Ativada , Transfecção , Quinases da Família src/metabolismo
9.
Biochem J ; 369(Pt 3): 477-84, 2003 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-12371907

RESUMO

Thyroid hormone receptors (TRs) and members of the myocyte enhancer factor 2 (MEF2) family are involved in the regulation of muscle-specific gene expression during myogenesis. Physical interaction between these two factors is required to synergistically activate gene transcription. p300/cAMP-response-element-binding-protein ('CREB')-binding protein (CBP) interacting with transcription factors is able to increase their activity on target gene promoters. We investigated the role of p300 in regulating the TR-MEF2A complex. To this end, we mapped the regions of these proteins involved in physical interactions and we evaluated the expression of a chloramphenicol acetyltransferase (CAT) reporter gene in U2OS cells under control of the alpha-myosin heavy chain promoter containing the thyroid hormone response element (TRE). Our results suggested a role of p300/CBP in mediating the transactivation effects of the TR-retenoid X receptor (RxR)-MEF2A complex. Our findings showed that the same C-terminal portion of p300 binds the N-terminal domains of both TR and MEF2A, and our in vivo studies demonstrated that TR, MEF2A and p300 form a ternary complex. Moreover, by the use of CAT assays, we demonstrated that adenovirus E1A inhibits activation of transcription by TR-RxR-MEF2A-p300 but not by TR-RxR-MEF2A. Our data suggested that p300 can bind and modulate the activity of TR-RxR-MEF2A at TRE. In addition, it is speculated that p300 might modulate the activity of the TR-RxR-MEF2A complex by recruiting a hypothetical endogenous inhibitor which may act like adenovirus E1A.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células COS , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Proteínas de Domínio MADS , Fatores de Transcrição MEF2 , Fatores de Regulação Miogênica , Proteínas Nucleares/genética , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Receptores X de Retinoides , Transativadores/genética , Fatores de Transcrição/genética
10.
Cell Tissue Res ; 309(3): 381-6, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12195294

RESUMO

Although the structural and pharmacological properties of endothelin (ET) receptors have been studied, little is known concerning their physiological significance, even if each subtype is supposed to have a distinct physiological action. Thus, to further elucidate the physiological function of this receptor, we examined the presence and distribution of the endothelin-B receptor (ET(B)) subtype in tissues of the lizard Podarcis sicula, using immunoblotting and immunohistochemistry. Immunoblotting indicated that, although the ET(B) receptor appears to be ubiquitous, it is present at different levels in the tissues examined. Furthermore, immunohistochemistry showed that this receptor is very abundant in endothelial cells of all tissues, suggesting that there is an ET(B)-mediated autocrine system of endothelin, which plays an important role in the regulation of endothelial cell function. On the other hand, the presence of ET(B) immunoreactivity also in endocrine systems such as adrenal and thyroid glands suggests an involvement also in the paracrine system of these organs.


Assuntos
Receptores de Endotelina/análise , Glândulas Suprarrenais/citologia , Animais , Sistema Cardiovascular/citologia , Tecido Conjuntivo , Sistema Digestório/citologia , Endotélio Vascular/citologia , Feminino , Immunoblotting , Imuno-Histoquímica , Rim/citologia , Lagartos , Masculino , Neurônios/citologia , Receptor de Endotelina B , Pele/citologia , Glândula Tireoide/citologia
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