RESUMO
Wnt signalling has been implicated as a driver of tumour cell metastasis, but less is known about which branches of Wnt signalling are involved and when they act in the metastatic cascade. Here, using a unique intravital imaging platform and fluorescent reporters, we visualised ß-catenin/TCF-dependent and ATF2-dependent signalling activities during human cancer cell invasion, intravasation and metastatic lesion formation in the chick embryo host. We found that cancer cells readily shifted between states of low and high canonical Wnt activity. Cancer cells that displayed low Wnt canonical activity showed higher invasion and intravasation potential in primary tumours and in metastatic lesions. In contrast, cancer cells showing low ATF2-dependent activity were significantly less invasive both at the front of primary tumours and in metastatic lesions. Simultaneous visualisation of both these reporters using a double-reporter cell line confirmed their complementary activities in primary tumours and metastatic lesions. These findings might inform the development of therapies that target different branches of Wnt signalling at specific stages of metastasis.
Assuntos
Neoplasias , beta Catenina , Animais , Embrião de Galinha , Humanos , beta Catenina/metabolismo , Via de Sinalização Wnt , Neoplasias/genética , Linhagem Celular Tumoral , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismoRESUMO
Precise prognosis is crucial for selection of adjuvant therapy in breast cancer. Molecular subtyping is increasingly used to complement immunohistochemical and pathological classification and to predict recurrence. This study compares both outcomes in a clinical setting. Molecular subtyping (MammaPrint®, TargetPrint®, and BluePrint®) and pathological classification data were compared in a cohort of 143 breast cancer patients. High risk clinical factors were defined by a value of the proliferation factor Ki67 equal or higher than 14% and/or high histological grade. The results from molecular classification were considered as reference. Core needle biopsies were found to be comparable to surgery samples for molecular classification. Discrepancies were found between molecular and pathological subtyping of the samples, including misclassification of HER2-positive tumors and the identification of a significant percentage of genomic high risk T1N0 tumors. In addition, 20% of clinical low-risk tumors showed genomic high risk, while clinical high-risk samples included 42% of cases with genomic low risk. According to pathological subtyping, a considerable number of breast cancer patients would not receive the appropriate systemic therapy. Our findings support the need to determine the molecular subtype of invasive breast tumors to improve breast cancer management.
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Tissue culture has evolved considerably over the last few years, including cell culture in three dimensions, organoids, cocultures of different cell types and the use of diverse types of matrices in an attempt to mimic conditions that more closely resemble those found in the original tissue or organ. In this chapter, we describe how patient-derived breast tissue can be cultured on sponges for several days, maintaining their original architecture and with the capacity to respond to treatments. This protocol facilitates the study of the tissue responses without the need for extensive tissue manipulation, cell digestion or use of a biomaterial as scaffold, while maintaining the stroma and extracellular matrix organization. This method has the potential to improve preclinical testing by contributing to provide more accurate data reflecting cell-cell and cell-matrix interactions, tumor microenvironment, drug effects or stem cell function in normal- and pathophysiology of the breast.
Assuntos
Neoplasias da Mama , Organoides , Neoplasias da Mama/patologia , Técnicas de Cultura de Células/métodos , Feminino , Humanos , Organoides/metabolismo , Células-Tronco , Microambiente TumoralRESUMO
Excessive estrogen exposure is connected with increased risk of breast cancer and has been shown to promote epithelial-mesenchymal-transition. Malignant cancer cells accumulate changes in cell mechanical and biochemical properties, often leading to cell softening. In this work we have employed atomic force microscopy to probe the influence of estrogen on the viscoelastic properties of MCF-7 breast cancer cells cultured either in normal or hormone free-medium. Estrogen led to a significant softening of the cells in all studied cases, while growing cells in hormone free medium led to an increase in the studied elastic and viscoelastic moduli. In addition, fluorescence microscopy shows that E-cadherin distribution is changed in cells when culturing them under estrogenic conditions. Furthermore, cell-cell contacts seemed to be weakened. These results were supported by AFM imaging showing changes in surfaces roughness, cell-cell contacts and cell height as result of estrogen treatment. This study therefore provides further evidence for the role of estrogen signaling in breast cancer.
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The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at lysine 4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus of the five ING proteins. ING5 facilitates histone H3 acetylation by the HBO1 complex, and also H4 acetylation by the MOZ/MORF complex. We show that ING5 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING5, which contains the nuclear localization sequence, is flexible and disordered, but it binds dsDNA with micromolar affinity. NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule, and they bind H3K4me3 in the same way as the isolated PHD. We have observed that ING5 can form heterodimers with the highly homologous ING4, and that two of three primary tumor-associated mutants in the N-terminal domain strongly destabilize the coiled-coil structure. They also affect cell proliferation and cell cycle phase distribution, suggesting a driver role in cancer progression.
Assuntos
Histonas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Histonas/química , Humanos , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Alinhamento de Sequência , Fatores de Transcrição/química , Proteínas Supressoras de Tumor/químicaRESUMO
The SRC-family kinase LYN is highly expressed in triple-negative/basal-like breast cancer (TNBC) and in the cell of origin of these tumors, c-KIT-positive luminal progenitors. Here, we demonstrate LYN is a downstream effector of c-KIT in normal mammary cells and protective of apoptosis upon genotoxic stress. LYN activity is modulated by PIN1, a prolyl isomerase, and in BRCA1 mutant TNBC PIN1 upregulation activates LYN independently of c-KIT. Furthermore, the full-length LYN splice isoform (as opposed to the Δaa25-45 variant) drives migration and invasion of aggressive TNBC cells, while the ratio of splice variants is informative for breast cancer-specific survival across all breast cancers. Thus, dual mechanisms-uncoupling from upstream signals and splice isoform ratios-drive the activity of LYN in aggressive breast cancers.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Quinases da Família src/metabolismo , Adolescente , Adulto , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Isoenzimas/metabolismo , Camundongos , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-kit/metabolismo , Splicing de RNA/genética , Análise de Sobrevida , Regulação para Cima , Adulto Jovem , Quinases da Família src/genéticaRESUMO
Breast cancer is the most frequently diagnosed cancer in women and the second most common cancer overall, with nearly 1.7 million new cases worldwide every year. Breast cancer patients need accurate tools for early diagnosis and to improve treatment. Biomarkers are increasingly used to describe and evaluate tumours for prognosis, to facilitate and predict response to therapy and to evaluate residual tumor, post-treatment. Here, we evaluate different methods to separate Diaminobenzidine (DAB) from Hematoxylin and Eosin (H&E) staining for Wnt-1, a potential cytoplasmic breast cancer biomarker. A method comprising clustering and Color deconvolution allowed us to recognize and quantify Wnt-1 levels accurately at pixel levels. Experimental validation was conducted using a set of 12,288 blocks of m × n pixels without overlap, extracted from a Tissue Microarray (TMA) composed of 192 tissue cores. Intraclass Correlations (ICC) among evaluators of the data of 0.634 , 0.791 , 0.551 and 0.63 for each Allred class and an average ICC of 0.752 among evaluators and automatic classification were obtained. Furthermore, this method received an average rating of 4.26 out of 5 in the Wnt-1 segmentation process from the evaluators.
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Fluorescent-activated cell sorting (FACS) represents one of the key techniques that have been used to isolate and characterize stem cells, including cells from the mammary gland. A combination of approaches, including recognition of cell surface antigens and different cellular activities, has facilitated the identification of stem cells from the healthy mammary gland and from breast tumors. In this chapter we describe the protocol to use FACS to separate breast cancer stem cells, but most of the general principles discussed could be applied to sort other types of cells.
Assuntos
Neoplasias da Mama/metabolismo , Citometria de Fluxo , Células-Tronco Neoplásicas/metabolismo , Animais , Antígenos de Superfície/metabolismo , Neoplasias da Mama/patologia , Separação Celular/métodos , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem/métodosRESUMO
The side population (SP) assay has been utilized as a method for isolation and characterization of normal and cancer stem cells from a variety of tissues. However, the SP phenotype may not be a common property of all stem cells. This chapter reviews the principle and potential pitfalls of the SP assay with an emphasis on mammary gland SP cell analysis.
Assuntos
Células-Tronco Neoplásicas/metabolismo , Células da Side Population/metabolismo , Células-Tronco/metabolismo , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem/métodos , Células da Side Population/citologiaRESUMO
Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo and radiotherapy. Here, we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifenresistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely, ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2expressing cells, and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure, and also in the primary tumours of these patients, compared to those of responders. Together, these results suggest that development of tamoxifen resistance is driven by Sox2dependent activation of Wnt signalling in cancer stem/progenitor cells.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Fatores de Transcrição SOXB1/metabolismo , Tamoxifeno/uso terapêutico , Animais , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Retículo Endoplasmático/metabolismo , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Interferência de RNA , Recidiva , Fatores de Transcrição SOXB1/antagonistas & inibidores , Fatores de Transcrição SOXB1/genética , Análise de Sobrevida , Tamoxifeno/farmacologia , Transplante Heterólogo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Homeobox 9 (HOXB9), a nontransforming transcription factor overexpressed in breast cancer, alters tumor cell fate and promotes tumor progression and metastasis. Here we show that HOXB9 confers resistance to ionizing radiation by promoting DNA damage response. In nonirradiated cells, HOXB9 induces spontaneous DNA damage, phosphorylated histone 2AX and p53 binding protein 1 foci, and increases baseline ataxia telangiectasia mutated (ATM) phosphorylation. Upon ionizing radiation, ATM is hyperactivated in HOXB9-expressing cells during the early stages of the double-stranded DNA break (DSB) response, accelerating accumulation of phosphorylated histone 2AX, mediator of DNA-damage checkpoint 1, and p53 binding protein 1, at DSBs and enhances DSB repair. The effect of HOXB9 on the response to ionizing radiation requires the baseline ATM activity before irradiation and epithelial-to-mesenchymal transition induced by TGF-ß, a HOXB9 transcriptional target. Our results reveal the impact of a HOXB9-TGF-ß-ATM axis on checkpoint activation and DNA repair, suggesting that TGF-ß may be a key factor that links tumor microenvironment, tumor cell fate, DNA damage response, and radioresistance in a subset of HOXB9-overexpressing breast tumors.
Assuntos
Dano ao DNA , Transição Epitelial-Mesenquimal , Proteínas de Homeodomínio/metabolismo , Tolerância a Radiação , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos da radiação , Feminino , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
There is increasing evidence that breast cancers contain tumor-initiating cells with stem cell properties. The importance of estrogen in the development of the mammary gland and in breast cancer is well known, but the influence of estrogen on the stem cell population has not been assessed. We show that estrogen reduces the proportion of stem cells in the normal human mammary gland and in breast cancer cells. The embryonic stem cell genes NANOG, OCT4, and SOX2 are expressed in normal breast stem cells and at higher levels in breast tumor cells and their expression decreases upon differentiation. Overexpression of each stem cell gene reduces estrogen receptor (ER) expression, and increases the number of stem cells and their capacity for invasion, properties associated with tumorigenesis and poor prognosis. These results indicate that estrogen reduces the size of the human breast stem cell pool and may provide an explanation for the better prognosis of ER-positive tumors.
Assuntos
Mama/citologia , Mama/efeitos dos fármacos , Estrogênios/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Adulto , Antineoplásicos Hormonais/farmacologia , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Glândulas Mamárias Humanas/efeitos dos fármacos , Pessoa de Meia-Idade , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Esferoides Celulares/efeitos dos fármacos , Tamoxifeno/farmacologia , Adulto JovemRESUMO
The mechanisms underlying tumoral secretion of signaling molecules into the microenvironment, which modulates tumor cell fate, angiogenesis, invasion, and metastasis, are not well understood. Aberrant expression of transcription factors, which has been implicated in the tumorigenesis of several types of cancers, may provide a mechanism that induces the expression of growth and angiogenic factors in tumors, leading to their local increase in the tumor microenvironment, favoring tumor progression. In this report, we demonstrate that the transcription factor HOXB9 is overexpressed in breast carcinoma, where elevated expression correlates with high tumor grade. HOXB9 induces the expression of several angiogenic factors (VEGF, bFGF, IL-8, and ANGPTL-2), as well as ErbB (amphiregulin, epiregulin, and neuregulins) and TGF-ss, which activate their respective pathways, leading to increased cell motility and acquisition of mesenchymal phenotypes. In vivo, HOXB9 promotes the formation of large, well-vascularized tumors that metastasize to the lung. Thus, deregulated expression of HOXB9 contributes to breast cancer progression and lung metastasis by inducing several growth factors that alter tumor-specific cell fates and the tumor stromal microenvironment.
Assuntos
Neoplasias da Mama/genética , Proteínas de Homeodomínio/genética , Neoplasias Pulmonares/secundário , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/patologia , Humanos , Neovascularização PatológicaRESUMO
The retinoblastoma tumor suppressor protein (Rb) affects gene transcription both negatively and positively and through this regulates distinct cellular responses. Although cell cycle regulation requires gene repression, Rb's ability to promote differentiation and part of its antiproliferative activity appears to rely on the activation of gene transcription. We present evidence here that the RET finger protein (RFP)/tripartite motif protein 27 (TRIM 27) inhibits gene transcription activation by Rb but does not affect gene repression. RFP binds to Rb and prevents the degradation of the EID-1 inhibitor of histone acetylation and differentiation. Furthermore, ablation of RFP in U2OS osteosarcoma cells augments a transcriptional program indicative of lineage-specific differentiation in response to Rb. These findings provide precedent for a regulatory pathway that uncouples different Rb-dependent activities and thus silences specific cellular responses to Rb in a selective way.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteína do Retinoblastoma/metabolismo , Transcrição Gênica , Proteínas E1A de Adenovirus/genética , Western Blotting , Proteínas de Ciclo Celular/antagonistas & inibidores , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Luciferases/metabolismo , Modelos Biológicos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma , Testes de Precipitina , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras , Proteína do Retinoblastoma/genética , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Técnicas do Sistema de Duplo-HíbridoRESUMO
Mice carrying the bovine papillomavirus type I genome develop dermal fibrosarcomas in a multiple step process characterized by distinctive proliferative stages. Chromosomal aberrations are identified early in this tumorigenic pathway, however, the mechanism that originates them is unknown. Using a functional assay, we investigated the status of the mitotic spindle cell cycle checkpoint (MSCCC) that regulates the metaphase to anaphase transition, in cells representing different stages of fibrosarcoma progression. Loss of MSCCC activity was apparent in mild fibromatosis and completely abolished in aggressive fibromatosis and fibrosarcoma lesions. This altered MSCCC status was confirmed biochemically by deregulated expression of Cks1 protein and unscheduled cyclin B metabolism. Immunoprecipitation and sequencing analyses indicated that mutation of p53 was not required for the abrogation of the MSCCC. These results demonstrate that loss of mitotic spindle checkpoint activity predisposes to chromosomal instability at early stages of fibrosarcoma development. To our knowledge, these studies constitute the first report of a transition in MSCCC activity in a tumorigenesis model.
Assuntos
Aberrações Cromossômicas , Dano ao DNA/genética , Fibrossarcoma/genética , Genes cdc/fisiologia , Predisposição Genética para Doença/genética , Mitose/genética , Mutação/genética , Fuso Acromático/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/genética , Ciclina B/genética , Ciclina B/metabolismo , Progressão da Doença , Fibroma/genética , Camundongos , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Glucocorticoids influence many physiological processes, and in particular apoptosis, often with opposite effects depending on the cell type examined. We found that during fibrosarcoma development there is a strong increase in apoptosis at the tumor stage, which is repressed by dexamethasone to levels observed in normal fibroblasts. The anti-apoptotic Bcl-2 family protein Bcl-x(L) is induced by dexamethasone at the transcriptional level at all stages of fibrosarcoma development. The ligand-activated glucocorticoid receptor (GR) activates the Bcl-x promoter in transient transfection experiments, and GR binds to specific Bcl-x promoter sequences in vitro and in vivo. Furthermore, a GR antagonist abolishes this effect, indicating that Bcl-x(L) induction is mediated by GR. Importantly, exogenous Bcl-x(L) inhibits apoptosis and caspase-3 activity in fibrosarcoma cells to levels found in dexamethasone-treated fibrosarcoma cells. We conclude that Bcl-x(L) is a key target mediating the anti-apoptotic effects of glucocorticoids during fibrosarcoma development. These observations provide further understanding of the molecular basis of glucocorticoid regulation of cell death during tumorigenesis.
Assuntos
Apoptose , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Regulação Neoplásica da Expressão Gênica , Glucocorticoides/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ativação Transcricional , Northern Blotting , Caspases/metabolismo , Morte Celular , Cromatina/metabolismo , DNA/metabolismo , Dexametasona/farmacologia , Progressão da Doença , Humanos , Immunoblotting , Modelos Genéticos , Neoplasias/patologia , Plasmídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteína bcl-XRESUMO
Glucocorticoids inhibit proliferation of many cell types, but the relationship between the glucocorticoid receptor (GR) and the proteins regulating cell cycle progression is not fully understood. We previously found that during fibrosarcoma (FS) progression, GR displays only modest transcriptional activity in the preneoplastic stages, whereas it is highly active in FS cells. Now, we report that glucocorticoids reduce proliferation throughout FS development. The cyclin-dependent kinase inhibitor p16(INK4a) is frequently absent in many cancers, including FSs. We observed that p16(INK4a) protein expression is lost at the tumor stage of FS progression. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restores p16(INK4a) expression and reverts the phenotype of FS cells to low GR transcriptional activity, similar to that of the p16(INK4a)-expressing preneoplastic stages. Importantly, exogenous p16(INK4a) introduced by cotransfection is sufficient to reduce GR activity in FS cells, without affecting GR activity in p16-positive aggressive fibromatosis cells. Furthermore, GR transcriptional activity is elevated in mouse embryo fibroblasts derived from INK4a(-/-) mice compared with those derived from WT mice, implying that the difference in p16(INK4a) expression is sufficient to modulate GR activity. These results suggest a relationship between steroid hormone receptor activity and cell cycle inhibition, whereby absence of p16(INK4a) protein leads to higher GR transactivation activity and reduced cell sensitivity to dexamethasone. This observation might have important implications for current cancer therapies.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Fibrossarcoma/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metilação de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Dexametasona/farmacologia , Fibrossarcoma/etiologia , Fibrossarcoma/genética , Genes p16 , Camundongos , Lesões Pré-Cancerosas/etiologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Receptores de Glucocorticoides/genética , Ativação Transcricional , Células Tumorais CultivadasRESUMO
BACKGROUND: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown. METHODS: Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP. RESULTS: Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2-0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures. CONCLUSION: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology.