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1.
Chem Res Toxicol ; 32(9): 1733-1736, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31203605

RESUMO

Research in toxicology relies on in vitro models such as cell lines. These living models are prone to change and may be described in publications with insufficient information or quality control testing. This article sets out recommendations to improve the reliability of cell-based research.


Assuntos
Técnicas de Cultura de Células/normas , Linhagem Celular , Modelos Biológicos , Animais , Autenticação de Linhagem Celular , Humanos , Controle de Qualidade , Reprodutibilidade dos Testes , Toxicologia/métodos , Toxicologia/normas
2.
Int J Cancer ; 142(3): 561-572, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28940260

RESUMO

A variety of analytical approaches have indicated that melanoma cell line UCLA-SO-M14 (M14) and breast carcinoma cell line MDA-MB-435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross-contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA-MB-435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA-MB-435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA-MB-435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA-MB-435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research.


Assuntos
Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Melanoma/patologia , Neoplasias da Mama/genética , DNA de Neoplasias/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Melanoma/genética
3.
Mol Cancer ; 11: 64, 2012 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-22962849

RESUMO

BACKGROUND: Hepatocyte growth factor (HGF) induced c-Met activation is known as the main stimulus for hepatocyte proliferation and is essential for liver development and regeneration. Activation of HGF/c-Met signaling has been correlated with aggressive phenotype and poor prognosis in hepatocellular carcinoma (HCC). MUC1 is a transmembrane mucin, whose over-expression is reported in most cancers. Many of the oncogenic effects of MUC1 are believed to occur through the interaction of MUC1 with signaling molecules. To clarify the role of MUC1 in HGF/c-Met signaling, we determined whether MUC1 and c-Met interact cooperatively and what their role(s) is in hepatocarcinogenesis. RESULTS: MUC1 and c-Met over-expression levels were determined in highly motile and invasive, mesenchymal-like HCC cell lines, and in serial sections of cirrhotic and HCC tissues, and these levels were compared to those in normal liver tissues. Co-expression of both c-Met and MUC1 was found to be associated with the differentiation status of HCC. We further demonstrated an interaction between c-Met and MUC1 in HCC cells. HGF-induced c-Met phosphorylation decreased this interaction, and down-regulated MUC1 expression. Inhibition of c-Met activation restored HGF-mediated MUC1 down-regulation, and decreased the migratory and invasive abilities of HCC cells via inhibition of ß-catenin activation and c-Myc expression. In contrast, siRNA silencing of MUC1 increased HGF-induced c-Met activation and HGF-induced cell motility and invasion. CONCLUSIONS: These findings indicate that the crosstalk between MUC1 and c-Met in HCC could provide an advantage for invasion to HCC cells through the ß-catenin/c-Myc pathway. Thus, MUC1 and c-Met could serve as potential therapeutic targets in HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Hepáticas/metabolismo , Mucina-1/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Indóis/farmacologia , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/genética , Mucina-1/genética , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Sulfonamidas/farmacologia , beta Catenina/genética , beta Catenina/metabolismo
4.
Oncology ; 83(5): 264-72, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22992568

RESUMO

The association between vitamin D and thyroid cancer is unclear. It is unknown if CYP27A1 or CYP2R1 are present in normal thyroid or cancer cells and there is limited information regarding response to treatment with vitamin D. SV40 immortalized follicular cells (N-thy) and six thyroid cancer cell lines were treated with 10 µM vitamin D(3), 0.1 µM 1,25(OH)(2)D(3) or vehicle × 24 h. CYP27A1, CYP2R1, CYP27B1 and CYP24A1 mRNA were measured using quantitative real-time-PCR before and after treatment. Cell proliferation was also evaluated in TPC1 and C643 cells after treatment with D(3), 25(OH)D(3) and 1,25(OH)(2)D(3). Baseline CYP27A1 and CYP27B1 mRNA were present in all cells, CYP2R1 was higher and CYP24A1 mRNA was lower in cancer cell lines versus N-thy. TPC1 cells had increased CYP24A1 mRNA levels when treated with both D(3) (3.49, p < 0.001) and 1,25(OH)(2)D(3) (5.05, p < 0.001). C643 cells showed increased CYP24A1 mRNA expression when treated with 1,25(OH)(2)D(3) (5.36, p < 0.001). D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) all significantly decreased cell proliferation in TPC1 and C643 cells. Overall, both cancerous and N-thy cell lines express CYP27A1 and CYP2R1 in addition to CYP27B1, establishing the potential to metabolize D(3) to 1,25(OH)(2)D(3). Additionally, vitamin D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) all had an antiproliferative effect on two thyroid cancer cell lines.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/enzimologia , Vitamina D/metabolismo , Vitamina D/uso terapêutico , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Calcitriol/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Colecalciferol/uso terapêutico , Colestanotriol 26-Mono-Oxigenase/metabolismo , Colestanotriol 26-Mono-Oxigenase/uso terapêutico , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Família 2 do Citocromo P450 , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Esteroide Hidroxilases/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Vitamina D3 24-Hidroxilase
5.
Gynecol Oncol ; 127(1): 241-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22710073

RESUMO

OBJECTIVES: Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines. METHODS: Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed. RESULTS: Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively. CONCLUSIONS: Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.


Assuntos
Neoplasias do Endométrio/genética , Neoplasias Ovarianas/genética , Linhagem Celular Tumoral , Impressões Digitais de DNA/métodos , Neoplasias do Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Ovarianas/patologia
6.
SLAS Discov ; 26(10): 1268-1279, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34697958

RESUMO

Cell lines are essential models for biomedical research. However, they have a common and important problem that needs to be addressed. Cell lines can be misidentified, meaning that they no longer correspond to the donor from whom the cells were first obtained. This problem may arise due to cross-contamination: the accidental introduction of cells from another culture. The contaminant, which is often a rapidly dividing cell line, will overgrow and replace the original culture. The end result is a false cell line, also known as a misidentified or imposter cell line. False cell lines may come from an entirely different species, tissue, or cell type than the original donor. If undetected, false cell lines produce unreliable and irreproducible results that pollute the biomedical literature and threaten the development of reliable drug discovery and meaningful patient treatments.The goal of this study was to ascertain how widespread this problem is and how it affects the literature, as well as to estimate how much funding has been used to produce pools of scientific literature of questionable value. We focus on HEp-2 [HeLa] and Intestine 407 [HeLa], two false cell lines that are widely used in the scientific literature but were shown to be cross-contaminated in 1967. These two cell lines have been used in 8497 and 1397 published articles and extensively described as laryngeal cancer and normal intestine, respectively, rather than their true identity: the cervical cancer cell line HeLa. Discussed are tools, approaches, and resources that can address this issue-both retrospectively and prospectively.


Assuntos
Células HeLa/fisiologia , Intestinos/fisiologia , Pesquisa Biomédica/métodos , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Humanos , Estudos Prospectivos , Estudos Retrospectivos
8.
Mol Cancer Res ; 17(5): 1036-1048, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30733375

RESUMO

Cancer cell lines are critical models to study tumor progression and response to therapy. In 2008, we showed that approximately 50% of thyroid cancer cell lines were redundant or not of thyroid cancer origin. We therefore generated new authenticated thyroid cancer cell lines and patient-derived xenograft (PDX) models using in vitro and feeder cell approaches, and characterized these models in vitro and in vivo. We developed four thyroid cancer cell lines, two derived from 2 different patients with papillary thyroid cancer (PTC) pleural effusions, CUTC5, and CUTC48; one derived from a patient with anaplastic thyroid cancer (ATC), CUTC60; and one derived from a patient with follicular thyroid cancer (FTC), CUTC61. One PDX model (CUTC60-PDX) was also developed. Short tandem repeat (STR) genotyping showed that each cell line and PDX is unique and match the original patient tissue. The CUTC5 and CUTC60 cells harbor the BRAF (V600E) mutation, the CUTC48 cell line expresses the RET/PTC1 rearrangement, and the CUTC61 cells have the HRAS (Q61R) mutation. Moderate to high levels of PAX8 and variable levels of NKX2-1 were detected in each cell line and PDX. The CUTC5 and CUTC60 cell lines form tumors in orthotopic and flank xenograft mouse models. IMPLICATIONS: We have developed the second RET/PTC1-expressing PTC-derived cell line in existence, which is a major advance in studying RET signaling. We have further linked all cell lines to the originating patients, providing a set of novel, authenticated thyroid cancer cell lines and PDX models to study advanced thyroid cancer.


Assuntos
Adenocarcinoma Folicular/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Adenocarcinoma Folicular/genética , Idoso , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Mutação , Transplante de Neoplasias , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética
9.
Clin Cancer Res ; 25(10): 3141-3151, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30737244

RESUMO

PURPOSE: Thyroid cancer cell lines are valuable models but have been neglected in pancancer genomic studies. Moreover, their misidentification has been a significant problem. We aim to provide a validated dataset for thyroid cancer researchers. EXPERIMENTAL DESIGN: We performed next-generation sequencing (NGS) and analyzed the transcriptome of 60 authenticated thyroid cell lines and compared our findings with the known genomic defects in human thyroid cancers. RESULTS: Unsupervised transcriptomic analysis showed that 94% of thyroid cell lines clustered distinctly from other lineages. Thyroid cancer cell line mutations recapitulate those found in primary tumors (e.g., BRAF, RAS, or gene fusions). Mutations in the TERT promoter (83%) and TP53 (71%) were highly prevalent. There were frequent alterations in PTEN, PIK3CA, and of members of the SWI/SNF chromatin remodeling complex, mismatch repair, cell-cycle checkpoint, and histone methyl- and acetyltransferase functional groups. Copy number alterations (CNA) were more prevalent in cell lines derived from advanced versus differentiated cancers, as reported in primary tumors, although the precise CNAs were only partially recapitulated. Transcriptomic analysis showed that all cell lines were profoundly dedifferentiated, regardless of their derivation, making them good models for advanced disease. However, they maintained the BRAFV600E versus RAS-dependent consequences on MAPK transcriptional output, which correlated with differential sensitivity to MEK inhibitors. Paired primary tumor-cell line samples showed high concordance of mutations. Complete loss of p53 function in TP53 heterozygous tumors was the most prominent event selected during in vitro immortalization. CONCLUSIONS: This cell line resource will help inform future preclinical studies exploring tumor-specific dependencies.


Assuntos
Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Telomerase/genética , Neoplasias da Glândula Tireoide/genética , Proteína Supressora de Tumor p53/genética , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias da Glândula Tireoide/classificação , Neoplasias da Glândula Tireoide/patologia , Estudos de Validação como Assunto
10.
J Clin Endocrinol Metab ; 93(11): 4331-41, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18713817

RESUMO

CONTEXT: Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated. OBJECTIVE: We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis. RESULTS: Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines. CONCLUSIONS: Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.


Assuntos
Técnicas de Cultura de Células/normas , DNA de Neoplasias/genética , Perfilação da Expressão Gênica , Mutação , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Neoplasias da Glândula Tireoide/genética , Adenocarcinoma Folicular/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Feminino , Humanos , Masculino , Melanoma/genética , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Neoplasias da Glândula Tireoide/classificação
11.
Cancer Res ; 77(11): 2784-2788, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28455420

RESUMO

Continuous cell lines are widely used, but can result in invalid, irreproducible research data. Cell line misidentification is a common problem that can be detected by authentication testing; however, misidentified cell lines continue to be used in publications. Here we explore the impact of one misidentified cell line, KB (HeLa), on the scientific literature. We identified 574 articles between 2000 and 2014 that provided an incorrect attribution for KB, in accordance with its false identity as oral epidermoid carcinoma, but only 57 articles that provided a correct attribution for KB, as HeLa or cervical adenocarcinoma. Statistical analysis of 57 correct and 171 incorrect articles showed that the number of citations to these articles increased over time. Content analysis of 200 citing articles showed there was a tendency to describe the cell line in accordance with the description in the cited paper. Analysis of journal impact factor showed no significant difference between correct and incorrect groups. Articles using KB or citing that usage were most frequently published in the subject areas of pharmacology, pharmacy, oncology, and medicinal chemistry. These findings are important for science policy and support the need for journals to require authentication testing as a condition of publication. Cancer Res; 77(11); 2784-8. ©2017 AACR.


Assuntos
Pesquisa Biomédica/métodos , Linhagem Celular Tumoral , Células HeLa , Humanos
14.
Cancers (Basel) ; 5(2): 334-56, 2013 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-24216980

RESUMO

The epithelial to mesenchymal transition (EMT) enables epithelial cells with a migratory mesenchymal phenotype. It is activated in cancer cells and is involved in invasion, metastasis and stem-like properties. ZEB1, an E-box binding transcription factor, is a major suppressor of epithelial genes in lung cancer. In the present study, we show that in H358 non-small cell lung cancer cells, ZEB1 downregulates EpCAM (coding for an epithelial cell adhesion molecule), ESRP1 (epithelial splicing regulatory protein), ST14 (a membrane associated serine protease involved in HGF processing) and RAB25 (a small G-protein) by direct binding to these genes. Following ZEB1 induction, acetylation of histone H4 and histone H3 on lysine 9 (H3K9) and 27 (H3K27) was decreased on ZEB1 binding sites on these genes as demonstrated by chromatin immunoprecipitation. Of note, decreased H3K27 acetylation could be also detected by western blot and immunocytochemistry in ZEB1 induced cells. In lung cancers, H3K27 acetylation level was higher in the tumor compartment than in the corresponding stroma where ZEB1 was more often expressed. Since HDAC and DNA methylation inhibitors increased expression of ZEB1 target genes, targeting these epigenetic modifications would be expected to reduce metastasis.

15.
Mol Cancer Ther ; 12(12): 2675-84, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24121103

RESUMO

Inhibition of Wee1 is emerging as a novel therapeutic strategy for cancer, and some data suggest that cells with dysfunctional p53 are more sensitive to Wee1 inhibition combined with conventional chemotherapy than those with functional p53. We and others found that Wee1 inhibition sensitizes leukemia cells to cytarabine. Thus, we sought to determine whether chemosensitization by Wee1 inhibition is dependent on p53 dysfunction and whether combining Wee1 inhibition is tolerable and effective in vivo. Synergistic inhibition of proliferation with a Wee1 inhibitor in clinical development, MK1775, and cytarabine was observed in all acute myelogenous leukemia (AML) cell lines tested, regardless of p53 functionality. Mechanistic studies indicate that inhibition of Wee1 abrogates the S-phase checkpoint and augments apoptosis induced by cytarabine. In AML and lung cancer cell lines, genetic disruption of p53 did not alter the cells' enhanced sensitivity to antimetabolites with Wee1 inhibition. Finally, mice with AML were treated with cytarabine and/or MK1775. The combination of MK1775 and cytarabine was well tolerated in mice and enhanced the antileukemia effects of cytarabine, including survival. Thus, inhibition of Wee1 sensitizes hematologic and solid tumor cell lines to antimetabolite chemotherapeutics, whether p53 is functional or not, suggesting that the use of p53 mutation as a predictive biomarker for response to Wee1 inhibition may be restricted to certain cancers and/or chemotherapeutics. These data provide preclinical justification for testing MK1775 and cytarabine in patients with leukemia.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Camundongos , Pirimidinonas , Ensaios Antitumorais Modelo de Xenoenxerto
16.
PLoS One ; 8(7): e68815, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23874775

RESUMO

The purpose of this study was to explore the role of transcription factor Ets1 in estrogen receptor α (ERα)-positive breast cancer progression. We expressed human Ets1 or empty vector in four human ERα-positive breast cancer cell lines and observed increased colony formation. Further examination of cellular responses in stable Ets1-expressing MCF7 clones displayed increased proliferation, migration, and invasion. Ets1-expressing MCF7 tumors grown in the mammary fat pads of nude mice exhibited increased rates of tumor growth (7.36±2.47 mm(3)/day) compared to control MCF7 tumors (2.52±1.70 mm(3)/day), but maintained their dependence on estradiol for tumor growth. Proliferation marker Ki-67 staining was not different between control and Ets1-expressing tumors, but Ets1-expressing tumors exhibited large necrotic centers and elevated apoptotic staining. Ets1 was shown to cooperate with ERα and the p160 nuclear receptor coactivator (NCOA/SRC) family to increase activation of a consensus estrogen response element luciferase reporter construct. Ets1-expressing MCF7 cells also exhibited elevated expression of the ERα target genes, progesterone receptor and trefoil factor 1. Using GST-pulldown assays, Ets1 formed stable complexes containing both ERα and p160 nuclear receptor coactivators. Taken together, these data suggest that the Ets1-dependent estradiol sensitization of breast cancer cells and tumors may be partially due to the ability of Ets1 to cooperate with ERα and nuclear receptor coactivators to stimulate transcriptional activity of estrogen-dependent genes.


Assuntos
Neoplasias da Mama/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Expressão Gênica , Humanos , Antígeno Ki-67/metabolismo , Células MCF-7 , Camundongos , Complexos Multiproteicos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ligação Proteica , Proteína Proto-Oncogênica c-ets-1/genética , Elementos de Resposta , Carga Tumoral/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco
17.
Cancer Res ; 73(23): 7111-21, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24121493

RESUMO

The epithelial-mesenchymal transition (EMT) and its reversal, mesenchymal-epithelial transition (MET), are fundamental processes involved in tumor cell invasion and metastasis. SEMA3F is a secreted semaphorin and tumor suppressor downregulated by TGF-ß1 and ZEB1-induced EMT. Here, we report that neuropilin (NRP)-2, the high-affinity receptor for SEMA3F and a coreceptor for certain growth factors, is upregulated during TGF-ß1-driven EMT in lung cancer cells. Mechanistically, NRP2 upregulation was TßRI dependent and SMAD independent, occurring mainly at a posttranscriptional level involving increased association of mRNA with polyribosomes. Extracellular signal-regulated kinase (ERK) and AKT inhibition blocked NRP2 upregulation, whereas RNA interference-mediated attenuation of ZEB1 reduced steady-state NRP2 levels. In addition, NRP2 attenuation inhibited TGF-ß1-driven morphologic transformation, migration/invasion, ERK activation, growth suppression, and changes in gene expression. In a mouse xenograft model of lung cancer, NRP2 attenuation also inhibited locally invasive features of the tumor and reversed TGF-ß1-mediated growth inhibition. In support of these results, human lung cancer specimens with the highest NRP2 expression were predominantly E-cadherin negative. Furthermore, the presence of NRP2 staining strengthened the association of E-cadherin loss with high-grade tumors. Together, our results demonstrate that NRP2 contributes significantly to TGF-ß1-induced EMT in lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/genética , Neuropilina-2/genética , Fator de Crescimento Transformador beta1/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Células Tumorais Cultivadas , Regulação para Cima
18.
Endocr Relat Cancer ; 19(4): 575-88, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22696507

RESUMO

The functional significance of decreased RAP1GAP protein expression in human tumors is unclear. To identify targets of RAP1GAP downregulation in the thyroid gland, RAP1 and RAP2 protein expression in human thyroid cells and in primary thyroid tumors were analyzed. RAP1GAP and RAP2 were co-expressed in normal thyroid follicular cells. Intriguingly, RAP1 was not detected in normal thyroid cells, although it was detected in papillary thyroid carcinomas, which also expressed RAP2. Both RAP proteins were detected at the membrane in papillary thyroid tumors, suggesting that they are activated when RAP1GAP is downregulated. To explore the functional significance of RAP1GAP depletion, RAP1GAP was transiently expressed at the lowest level that is sufficient to block endogenous RAP2 activity in papillary and anaplastic thyroid carcinoma cell lines. RAP1GAP impaired the ability of cells to spread and migrate on collagen. Although RAP1GAP had no effect on protein tyrosine phosphorylation in growing cells, RAP1GAP impaired phosphorylation of focal adhesion kinase and paxillin at sites phosphorylated by SRC in cells acutely plated on collagen. SRC activity was increased in suspended cells, where it was inhibited by RAP1GAP. Inhibition of SRC kinase activity impaired cell spreading and motility. These findings identify SRC as a target of RAP1GAP depletion and suggest that the downregulation of RAP1GAP in thyroid tumors enhances SRC-dependent signals that regulate cellular architecture and motility.


Assuntos
Carcinoma Papilar/patologia , Movimento Celular , Citoesqueleto/fisiologia , Proteínas Ativadoras de GTPase/fisiologia , Neoplasias da Glândula Tireoide/patologia , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Regulação para Baixo/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Transfecção , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismo
19.
Endocr Relat Cancer ; 18(3): 301-10, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21367844

RESUMO

Increases in Rap activity have been associated with tumor progression. Although activating mutations in Rap have not been described, downregulation of Rap1GAP is frequent in human tumors including thyroid carcinomas. In this study, we explored whether endogenous Rap1GAP expression could be restored to thyroid tumor cells. The effects of deacetylase inhibitors and a demethylating agent, individually and in combination, were examined in four differentiated and six anaplastic thyroid carcinoma (ATC) cell lines. Treatment with the structurally distinct histone deacetylase (HDAC) inhibitors, sodium butyrate and trichostatin A, increased Rap1GAP expression in all the differentiated thyroid carcinoma cell lines and in four of the six ATC cell lines. The demethylating agent, 5-aza-deoxycytidine, restored Rap1GAP expression in one anaplastic cell line and enhanced the effects of HDAC inhibitors in a second anaplastic cell line. Western blotting indicated that Rap2 was highly expressed in human thyroid cancer cells. Importantly, treatment with HDAC inhibitors impaired Rap2 activity in both differentiated and anaplastic tumor cell lines. The mechanism through which Rap activity is repressed appears to entail effects on the expression of multiple Rap regulators, including RapGEFs and RapGAPs. These results suggest that HDAC inhibitors may provide a tractable approach to impair Rap activity in human tumor cells.


Assuntos
Butiratos/farmacologia , Carcinoma/patologia , Proteínas Ativadoras de GTPase/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Proteínas de Neoplasias/biossíntese , Neoplasias da Glândula Tireoide/patologia , Proteínas rap de Ligação ao GTP/antagonistas & inibidores , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidores , Adenocarcinoma Folicular/metabolismo , Adenocarcinoma Folicular/patologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Carcinoma/metabolismo , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Metilação de DNA/efeitos dos fármacos , Decitabina , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Proteínas Ativadoras de GTPase/genética , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/farmacologia , Neoplasias da Glândula Tireoide/metabolismo , Regulação para Cima/efeitos dos fármacos
20.
Cancer Chemother Pharmacol ; 67(1): 83-92, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20306194

RESUMO

PURPOSE: Osteosarcoma (OS) remains an incurable and ultimately fatal disease in many patients, and novel forms of therapy are needed. Improved models of OS that more closely mimic human disease would provide more robust information regarding the utility of novel therapies. Spontaneous OS in dogs may provide such a model. Pharmacologic inhibition of histone deacetylase (HDAC) enzymes has a variety of anti-tumor effects but may demonstrate the most utility when utilized in combination with standard cytotoxic therapies. We sought to determine the in vitro and in vivo effects of the HDAC inhibitor valproic acid (VPA) on doxorubicin (DOX) sensitivity in canine and human OS. METHODS: We evaluated the in vitro anti-proliferative and apoptotic effects of VPA/DOX combination treatment, alterations in histone acetylation and nuclear DOX accumulation resulting from VPA treatment, and the in vivo efficacy of combination therapy in a xenograft model. RESULTS: Treatment of canine and human OS cell lines with clinically achievable VPA concentrations resulted in increased histone acetylation but modest anti-proliferative effects. Pre-incubation with VPA followed by doxorubicin (DOX) resulted in significant growth inhibition and potentiation of apoptosis, associated with a dose-dependent increase in nuclear DOX accumulation. The combination of VPA and DOX was superior to either monotherapy in a canine OS xenograft model. CONCLUSION: These results demonstrate a rationale for the addition of HDAC inhibitors to current protocols for the treatment of OS and illustrate the similarities in response to HDAC inhibitors between human and canine OS, lending further credibility to the canine OS model.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Osteossarcoma/tratamento farmacológico , Ácido Valproico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Masculino , Camundongos , Camundongos Nus , Osteossarcoma/patologia , Especificidade da Espécie , Ácido Valproico/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
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