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1.
Proc Natl Acad Sci U S A ; 109(8): 2724-9, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22003129

RESUMO

Breast cancers are comprised of molecularly distinct subtypes that may respond differently to pathway-targeted therapies now under development. Collections of breast cancer cell lines mirror many of the molecular subtypes and pathways found in tumors, suggesting that treatment of cell lines with candidate therapeutic compounds can guide identification of associations between molecular subtypes, pathways, and drug response. In a test of 77 therapeutic compounds, nearly all drugs showed differential responses across these cell lines, and approximately one third showed subtype-, pathway-, and/or genomic aberration-specific responses. These observations suggest mechanisms of response and resistance and may inform efforts to develop molecular assays that predict clinical response.


Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/classificação , Neoplasias da Mama/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Dosagem de Genes/genética , Humanos , Modelos Biológicos , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacos
2.
Cancer Cell ; 10(6): 515-27, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17157791

RESUMO

Recent studies suggest that thousands of genes may contribute to breast cancer pathophysiologies when deregulated by genomic or epigenomic events. Here, we describe a model "system" to appraise the functional contributions of these genes to breast cancer subsets. In general, the recurrent genomic and transcriptional characteristics of 51 breast cancer cell lines mirror those of 145 primary breast tumors, although some significant differences are documented. The cell lines that comprise the system also exhibit the substantial genomic, transcriptional, and biological heterogeneity found in primary tumors. We show, using Trastuzumab (Herceptin) monotherapy as an example, that the system can be used to identify molecular features that predict or indicate response to targeted therapies or other physiological perturbations.


Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Proteínas de Neoplasias/análise
3.
PLoS One ; 15(9): e0238594, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32911489

RESUMO

Intratumor Heterogeneity (ITH) is a functionally important property of tumor tissue and may be involved in drug resistance mechanisms. Although descriptions of ITH can be traced back to very early reports about cancer tissue, mechanistic investigations are still limited by the precision of analysis methods and access to relevant tissue sources. PDX models have provided a reproducible source of tissue with at least a partial representation of naturally occurring ITH. We investigated the properties of phenotypically distinct cell populations by Fluorescence activated cell sorting (FACS) tissue derived cells from multiple tumors from a triple negative breast cancer patient derived xenograft (PDX) model. We subsequently subjected each population to in depth gene expression analysis. Our findings suggest that process related gene expression changes (caused by tissue dissociation and FACS sorting) are restricted to Immediate Early Genes (IEGs). This allowed us to discover highly reproducible gene expression profiles of distinct cellular compartments identifiable by cell surface markers in this particular tumor model. Within the context of data from a previously published model our work suggests that gene expression profiles associated with hypoxia, stemness and drug resistance may reside in tumor subpopulations predictably growing in PDX models. This approach provides a novel opportunity for prospective mechanistic studies of ITH.


Assuntos
Regulação Neoplásica da Expressão Gênica , Transcriptoma , Neoplasias de Mama Triplo Negativas/genética , Animais , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Oncotarget ; 10(38): 3592-3604, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31217895

RESUMO

Barrett's esophagus (BE) is metaplasia of the squamous epithelium to a specialized columnar epithelium. BE progresses through low- and high-grade dysplasia before developing into esophageal adenocarcinoma. The BE microenvironment is not well defined. We compare 12 human clinical BE and adjacent normal squamous epithelium biopsies using single cell immunophenotyping by flow cytometry. A cassette of 19 epithelial and immune cell markers was used to detect differences between cellular compartments in normal and BE tissues. We found that the BE microenvironment has an immunological landscape distinct from adjacent normal epithelium. BE has an increased percentage of epithelial cells with a concomitant decrease in the percentage of immune cells, accompanied by a shift in the immune landscape from a predominantly T cell rich microenvironment in normal tissue to a B cell rich landscape in BE tissue. Hierarchical clustering separates BE and normal samples into two discrete groups based upon our 19-marker panel, but also reveals unexpected, shared phenotypes for three patients. Our results suggest that flow based single cell analysis may have the potential for revealing clinically relevant differences between BE and normal adjacent tissue, and that surface immunophenotypes could identify specific subpopulations from dysplastic tissue for further investigation.

6.
Sci Rep ; 8(1): 5035, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29568081

RESUMO

Comprehensive molecular analysis of rare circulating tumor cells (CTCs) and cell clusters is often hampered by low throughput and purity, as well as cell loss. To address this, we developed a fully integrated platform for flow cytometry-based isolation of CTCs and clusters from blood that can be combined with whole transcriptome analysis or targeted RNA transcript quantification. Downstream molecular signature can be linked to cell phenotype through index sorting. This newly developed platform utilizes in-line magnetic particle-based leukocyte depletion, and acoustic cell focusing and washing to achieve >98% reduction of blood cells and non-cellular debris, along with >1.5 log-fold enrichment of spiked tumor cells. We could also detect 1 spiked-in tumor cell in 1 million WBCs in 4/7 replicates. Importantly, the use of a large 200µm nozzle and low sheath pressure (3.5 psi) minimized shear forces, thereby maintaining cell viability and integrity while allowing for simultaneous recovery of single cells and clusters from blood. As proof of principle, we isolated and transcriptionally characterized 63 single CTCs from a genetically engineered pancreatic cancer mouse model (n = 12 mice) and, using index sorting, were able to identify distinct epithelial and mesenchymal sub-populations based on linked single cell protein and gene expression.


Assuntos
Células Neoplásicas Circulantes/metabolismo , Neoplasias Pancreáticas/patologia , Análise de Célula Única/métodos , Animais , Linhagem Celular Tumoral/transplante , Separação Celular/métodos , Modelos Animais de Doenças , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/métodos , Humanos , Procedimentos de Redução de Leucócitos/instrumentação , Procedimentos de Redução de Leucócitos/métodos , Biópsia Líquida/métodos , Imãs , Camundongos , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/genética
7.
Mol Cancer Res ; 15(4): 429-438, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28039356

RESUMO

Cancer tissue functions as an ecosystem of a diverse set of cells that interact in a complex tumor microenvironment. Genomic tools applied to biopsies in bulk fail to account for this tumor heterogeneity, whereas single-cell imaging methods limit the number of cells which can be assessed or are very resource intensive. The current study presents methods based on flow cytometric analysis and cell sorting using known cell surface markers (CXCR4/CD184, CD24, THY1/CD90) to identify and interrogate distinct groups of cells in triple-negative breast cancer clinical biopsy specimens from patient-derived xenograft (PDX) models. The results demonstrate that flow cytometric analysis allows a relevant subgrouping of cancer tissue and that sorting of these subgroups provides insights into cancer cell populations with unique, reproducible, and functionally divergent gene expression profiles. The discovery of a drug resistance signature implies that uncovering the functional interaction between these populations will lead to deeper understanding of cancer progression and drug response.Implications: PDX-derived human breast cancer tissue was investigated at the single-cell level, and cell subpopulations defined by surface markers were identified which suggest specific roles for distinct cellular compartments within a solid tumor. Mol Cancer Res; 15(4); 429-38. ©2016 AACR.


Assuntos
Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/métodos , Imunofenotipagem/métodos , Análise de Célula Única/métodos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Animais , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Transplante de Neoplasias , Fenótipo , Receptores CXCR4/metabolismo
8.
Cytometry B Clin Cytom ; 70(3): 107-14, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16498671

RESUMO

BACKGROUND: We previously reported a flow cytometry technique to monitor pharmacodynamic effects of the raf kinase inhibitor BAY 43-9006 based on the ability of phorbol ester (PMA) to phosphorylate extracellular-regulated kinase (ERK) in peripheral blood (Chow et al., Cytometry 2001;46:72-78). In this article, we describe its application to phase I trials of BAY 43-9006 in solid tumor and AML/MDS patients. METHODS: The previously described whole blood lysis method was used to monitor BAY 43-9006 effects on peripheral T-cells of solid tumor patients. A modified whole blood fixation protocol was developed for the AML/MDS trial, using the c-kit ligand stem cell factor (SCF) to activate ERK as an alternative to PMA, and incorporating immunophenotypic markers to identify leukemic blasts. RESULTS: At all dose levels of BAY 43-9006 used to treat solid tumor patients, ERK could be activated by PMA in peripheral T-cells and we were not able to show inhibition of raf kinase. A similar effect was seen in the lymphocytes of AML/MDS patients during treatment with BAY 43-9006. However, we found strong inhibition when ERK was activated via c-kit using SCF. Furthermore, normal donor CD34+ve stem cells were much more sensitive to BAY 43-9006 when ERK was activated by SCF, compared to PMA. CONCLUSIONS: These findings support the further development of flow cytometry applications to monitor signal transduction inhibitors during early phase clinical trials.


Assuntos
Benzenossulfonatos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Síndromes Mielodisplásicas/tratamento farmacológico , Neoplasias/tratamento farmacológico , Piridinas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/análise , Antígenos CD34/análise , Antígenos de Diferenciação Mielomonocítica/análise , Benzenossulfonatos/farmacologia , Antígenos CD13/análise , Complexo CD3/análise , Monitoramento de Medicamentos/métodos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo/métodos , Hemólise , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/metabolismo , Leucócitos Mononucleares/química , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/metabolismo , Neoplasias/sangue , Neoplasias/metabolismo , Niacinamida/análogos & derivados , Compostos de Fenilureia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Sorafenibe , Fator de Células-Tronco/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
9.
Proc Natl Acad Sci U S A ; 100(17): 9940-5, 2003 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-12909712

RESUMO

Genes subject to monoallelic expression are expressed from only one of the two alleles either selected at random (random monoallelic genes) or in a parent-of-origin specific manner (imprinted genes). Because high densities of long interspersed nuclear element (LINE)-1 transposon sequence have been implicated in X-inactivation, we asked whether monoallelically expressed autosomal genes are also flanked by high densities of LINE-1 sequence. A statistical analysis of repeat content in the regions surrounding monoallelically and biallelically expressed genes revealed that random monoallelic genes were flanked by significantly higher densities of LINE-1 sequence, evolutionarily more recent and less truncated LINE-1 elements, fewer CpG islands, and fewer base-pairs of short interspersed nuclear elements (SINEs) sequence than biallelically expressed genes. Random monoallelic and imprinted genes were pooled and subjected to a clustering analysis algorithm, which found two clusters on the basis of aforementioned sequence characteristics. Interestingly, these clusters did not follow the random monoallelic vs. imprinted classifications. We infer that chromosomal sequence context plays a role in monoallelic gene expression and may involve the recognition of long repeats or other features. The sequence characteristics that distinguished the high-LINE-1 category were used to identify more than 1,000 additional genes from the human and mouse genomes as candidate genes for monoallelic expression.


Assuntos
Alelos , Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , Algoritmos , Animais , Ilhas de CpG , Mecanismo Genético de Compensação de Dose , Feminino , Genoma , Genoma Humano , Humanos , Camundongos , Elementos Nucleotídeos Curtos e Dispersos
10.
Biochem Biophys Res Commun ; 308(4): 784-92, 2003 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-12927787

RESUMO

We report the cloning of the NKIAMRE gene located on human chromosome 5q31.1. It encodes a novel 52kDa Cdc2-related kinase with a 1.5kb open reading frame. Like MAP kinases, NKIAMRE contains a Thr-X-Tyr (TXY) motif in the activation loop domain. Similar to cdks, NKIAMRE contains the putative negative regulatory Ser14 and Tyr15 residues and the cyclin-binding motif, NKIAMRE, from which it derives its name. Human NKIAMRE has significant amino acid identity to related kinases in rat, mouse, Caenorhabditis elegans, and Drosophila, and is widely expressed in human tissues and cell lines. Confocal microscopy demonstrates that NKIAMRE localizes to the cytoplasm. NKIAMRE is activated by treatment of cells with phorbol 12-myristate 13-acetate. Mutation of the ATP-binding Lys-33 to arginine and the Thr-Glu-Tyr motif to Ala-Glu-Phe abolished its ability to phosphorylate myelin basic protein. NKIAMRE is a member of a conserved family of kinases with homology to both MAP kinases and cyclin-dependent kinases.


Assuntos
Proteína Quinase CDC2/química , Proteína Quinase CDC2/fisiologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arginina/química , Sequência de Bases , Northern Blotting , Western Blotting , Encéfalo/metabolismo , Células COS , Cromossomos Humanos Par 5 , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/metabolismo , Éxons , Biblioteca Gênica , Vetores Genéticos , Humanos , Lisina/química , Sistema de Sinalização das MAP Quinases , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteína Básica da Mielina/química , Miocárdio/metabolismo , Fases de Leitura Aberta , Fosforilação , Filogenia , Testes de Precipitina , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Serina/química , Acetato de Tetradecanoilforbol/química , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas
11.
Nature ; 420(6916): 696-700, 2002 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-12442173

RESUMO

In a variety of cells, the Ca2+ signalling process is mediated by the endoplasmic-reticulum-membrane-associated Ca2+ release channel, inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Being ubiquitous and present in organisms ranging from humans to Caenorhabditis elegans, InsP3R has a vital role in the control of cellular and physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, behaviour, memory and learning. Mouse type I InsP3R (InsP3R1), found in high abundance in cerebellar Purkinje cells, is a polypeptide with three major functionally distinct regions: the amino-terminal InsP3-binding region, the central modulatory region and the carboxy-terminal channel region. Here we present a 2.2-A crystal structure of the InsP3-binding core of mouse InsP3R1 in complex with InsP3. The asymmetric, boomerang-like structure consists of an N-terminal beta-trefoil domain and a C-terminal alpha-helical domain containing an 'armadillo repeat'-like fold. The cleft formed by the two domains exposes a cluster of arginine and lysine residues that coordinate the three phosphoryl groups of InsP3. Putative Ca2+-binding sites are identified in two separate locations within the InsP3-binding core.


Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Receptores de Inositol 1,4,5-Trifosfato , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
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