RESUMEN
BACKGROUND: The majority of patients diagnosed with advanced epithelial ovarian carcinoma (EOC) relapse with resistant disease, and there are no biomarkers that possess clinical utility to identify or monitor these patients. This study aimed to identify secreted proteins ('secretome') collected from human EOC cell lines that differ in their inherent platinum sensitivity. METHODS: Secreted proteins collected from conditioned medium from ovarian cancer cell lines that vary in their sensitivity to cisplatin were digested with trypsin and analysed by liquid chromatography-tandem mass spectrometry for peptide identification. RESULTS: Of the 1688 proteins identified, 16 possessed significant differential abundances (P<0.05) between the platinum-resistant and -sensitive cell lines. A number of these were verified by immunoblot, including COL11A1, which was also found to be associated with worse progression-free survival (PFS; N=723) and overall survival (OS; N=1183) as assessed from publicly available transcript expression data from ovarian cancer tumour specimens. CONCLUSION: Secretome proteomics of EOC cells resulted in the identification of a novel candidate biomarker, COL11A1. The expression level of COL11A1 correlates to worse PFS and OS, and is predicted to reside in peripheral circulation making this an attractive candidate for validation in sera as a biomarker of cisplatin resistance and poor outcome.
Asunto(s)
Biomarcadores de Tumor/sangre , Colágeno Tipo XI/sangre , Proteínas de Neoplasias/sangre , Neoplasias Glandulares y Epiteliales/sangre , Neoplasias Ováricas/sangre , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Cisplatino/farmacología , Colágeno Tipo XI/biosíntesis , Colágeno Tipo XI/genética , Medios de Cultivo , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Femenino , Expresión Génica , Humanos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Proteómica , Tasa de SupervivenciaRESUMEN
BACKGROUND: Lobular endocervical glandular hyperplasia (LEGH) is a rare lesion of the uterine cervix. It has been proposed that LEGH may represent a precursor lesion to a group of mucinous adenocarcinoma with gastric phenotype (GA) that is independent of high-risk human papillomavirus (H-HPV) infection. Carbonic anhydrase-IX (CA-IX) is highly expressed in conventional glandular lesions (CGLs). However, expression of CA-IX in LEGH or GA has not been studied. METHODS: In all, 12 CGLs, 7 LEGHs, 6 LEGHs with coexisting adenocarcinoma in situ (AIS, 3) and GA (3) were identified from Japanese women with a cytological diagnosis of atypical glandular cells of undetermined significance. Immunostaining was used to detect CA-IX and p16(INK)4(a) (hereafter termed p16) protein expression in the tissues and CA-IX protein expression in the Papanicolaou smears (PSs). Polymerase chain reaction was used to detect H-HPV DNA in liquid-based cytology. RESULTS: Out of 12 (83%) CGLs, 10 were positive with H-HPV and high levels of CA-IX expression were seen in all (100%) cases. P16 protein expression was observed in 11 out of 12 (92%) cases. None of the LEGHs, LEGHs with AIS or GA were positive for H-HPV and only 8 out of 13 (62%) showed focal weak (1+) p16 expression. In contrast, all cases (100%) exhibited strong CA-IX protein expression. CONCLUSION: Our study suggests that there are different molecular mechanisms of carcinogenesis resulting in CGLs vs LEGHs associated with AIS or GA. There is also a possible link between LEGHs and GAs. Furthermore, CA-IX expression may serve as a useful biomarker for the detection of GAs in the absence of H-HPV infection.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Anhidrasas Carbónicas/metabolismo , Hiperplasia/patología , Neoplasias Glandulares y Epiteliales/patología , Infecciones por Papillomavirus/patología , Neoplasias del Cuello Uterino/patología , Adenocarcinoma Mucinoso/enzimología , Adenocarcinoma Mucinoso/patología , Adenocarcinoma Mucinoso/virología , Adulto , Anciano , Anciano de 80 o más Años , Anhidrasa Carbónica IX , Carcinoma Lobular/enzimología , Carcinoma Lobular/patología , Carcinoma Lobular/virología , ADN Viral/genética , Femenino , Humanos , Hiperplasia/enzimología , Hiperplasia/virología , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/enzimología , Neoplasias Glandulares y Epiteliales/virología , Papillomaviridae/genética , Infecciones por Papillomavirus/enzimología , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa , Pronóstico , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/patología , Neoplasias Gástricas/virología , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/virologíaRESUMEN
BACKGROUND: Breast cancer 1, early onset (BRCA1) is a tumour-suppressor gene associated with familial epithelial ovarian cancer (EOC). Reduced BRCA1 expression is associated with enhanced sensitivity to platinum-based chemotherapy. We sought to examine the prognostic relevance of BRCA1 expression in EOC patients treated with intraperitoneal platinum/taxane. METHODS: The GOG-172 was a phase III, multi-institutional randomised trial of intravenous paclitaxel and cisplatin (IV therapy) vs intravenous paclitaxel, intraperitoneal cisplatin plus paclitaxel (IP therapy) in patients with optimally resected stage III EOC. The BRCA1 expression was assessed with immunohistochemistry (IHC) staining blinded to clinical outcome in archival tumour specimens. Slides with îº10% staining were defined as aberrant and >10% as normal. Correlations between BRCA1 expression and progression-free survival (PFS) and overall survival (OS) were analysed using Kaplan-Meier method and Cox regression analysis. RESULTS: Of the 393 patients, 189 tumours had aberrant expression, and 204 had normal BRCA1 expression. There was an interaction between BRCA1 expression and route of administration on OS (P=0.014) but not PFS (P=0.054). In tumours with normal BRCA1 expression, the median OS was 58 months for IP group vs 50 months for IV group (P=0.818). In tumours with aberrant BRCA1 expression, the median OS was 84 vs 47 months in the IP vs IV group, respectively (P=0.0002). Aberrant BRCA1 expression was an independent prognostic factor for better survival in women randomised to IP therapy (hazard ratio (HR)=0.67, 95% confidence interval (CI)=0.47-0.97, P=0.032). Similar survival was observed in the IV and IP patients with normal BRCA1 expression. Multivariate but not univariate modelling demonstrated that IV patients with aberrant vs normal BRCA1 expression had worse survival. CONCLUSION: Decreased BRCA1 expression is associated with a 36-month survival improvement in patients with EOC treated with IP chemotherapy. Although these results merit validation in future studies, the results suggest that decreased BRCA1 expression predicts for improved response to cisplatin-based IP chemotherapy with cisplatin and paclitaxel.
Asunto(s)
Adenocarcinoma de Células Claras/mortalidad , Adenocarcinoma Mucinoso/mortalidad , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteína BRCA1/metabolismo , Cistadenocarcinoma Seroso/mortalidad , Neoplasias Endometriales/mortalidad , Neoplasias Ováricas/mortalidad , Adenocarcinoma de Células Claras/tratamiento farmacológico , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma Mucinoso/tratamiento farmacológico , Adenocarcinoma Mucinoso/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Cisplatino/administración & dosificación , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/metabolismo , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Inyecciones Intraperitoneales , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Paclitaxel/administración & dosificación , Pronóstico , Tasa de SupervivenciaRESUMEN
BACKGROUND: High-risk human papillomavirus (H-HPV) infection is linked to cervical neoplasia but its role in detecting cervical glandular lesions (GLs) is unclear. Carbonic anhydrase IX (CA-IX) is a hypoxic biomarker that is highly expressed in neoplastic cervical GLs. The diagnostic utility of these biomarkers was evaluated by the Gynecologic Oncology Group in Japanese women with a cytological diagnosis of atypical glandular cells. METHODS: Immunostaining was used to detect CA-IX in a conventional Pap smear. Immunoreactivity of CA-IX was interpreted by a panel of pathologists blinded to the histological diagnosis. Polymerase chain reaction was used to detect H-HPV in a liquid-based cytology specimen. RESULTS: Significant cervical lesions (SCLs), defined as cervical intraepithelial neoplasia (CIN2, CIN3), adenocarcinoma in situ or invasive carcinoma, were observed in 37/88 (42%) of women. CA-IX testing alone (n=88) had a sensitivity of 89, 100 or 73% for SCLs, GLs or significant squamous lesions (SLs), respectively, with a false negative rate (FNR) of 14%. Testing for H-HPV (n=84) had a sensitivity of 65, 53 or 80% for SCLs, GLs or SLs, respectively, with a FNR of 22%. The combination of CA-IX and H-HPV testing had a sensitivity of 97, 100 or 93% for SCLs, GLs or SLs, respectively, with a FNR of 5%. Among eight H-HPV-negative GLs, six (75%) had a diagnosis of lobular endocervical glandular hyperplasia (LEGH). CONCLUSION: The combination of CA-IX and HPV testing improved the diagnostic accuracy. The low rate of H-HPV positivity in the GLs was associated with coexisting LEGH independent of H-HPV.
Asunto(s)
Alphapapillomavirus/patogenicidad , Anhidrasas Carbónicas/metabolismo , Displasia del Cuello del Útero/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Alphapapillomavirus/genética , Femenino , Genotipo , Humanos , Japón , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Displasia del Cuello del Útero/enzimología , Displasia del Cuello del Útero/virologíaRESUMEN
Polypyrimidine tract-binding protein (PTB) is an RNA-binding protein with multiple functions in the regulation of RNA processing and IRES-mediated translation. We report here overexpression of PTB in a majority of epithelial ovarian tumors revealed by immunoblotting and tissue microarray (TMA) staining. By western blotting, we found that PTB was overexpressed in 17 out of 19 ovarian tumor specimens compared to their matched-normal tissues. By TMA staining, we found PTB expression in 38 out of 44 ovarian cancer cases but only in two out of nine normal adjacent tissues. PTB is also overexpressed in SV40 large T-antigen immortalized ovarian epithelial cells compared to normal human ovarian epithelial cells. Using doxycycline-inducible small interfering RNA technology, we found that knockdown of PTB expression in the ovarian tumor cell line A2780 substantially impaired tumor cell proliferation, anchorage-independent growth and in vitro invasiveness. These results suggest that overexpression of PTB is an important component of the multistep process of tumorigenesis, and might be required for the development and maintenance of epithelial ovarian tumors. Moreover, because of its novel role in tumor cell growth and invasiveness, shown here for the first time, PTB may be a novel therapeutic target in the treatment of ovarian cancer.
Asunto(s)
Neoplasias Ováricas/patología , Proteína de Unión al Tracto de Polipirimidina/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Inmunohistoquímica , Invasividad Neoplásica , Neoplasias Ováricas/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Interferencia de ARN , Análisis de Matrices TisularesRESUMEN
Epidermal growth factor (EGF) is a multifunctional regulator of mammary epithelial cells (MEC) that transduces its signals through the EGF receptor (EGFR). To clarify the role of the EGFR in the mammary gland, EGFR expression, localization and function were examined during different developmental stages in rats. Immunoblot analysis demonstrated high levels of EGFR during puberty, pregnancy and involution as well as at sexual maturity, and low levels throughout lactation. An immunohistochemical assay was used to show that EGFR was distinctly expressed in a variety of cell types throughout mammary glands from virgin rats and rats during pregnancy and involution, and was down-regulated in all cell types throughout lactation. To examine the relationship between EGFR expression and function, primary MEC were cultured under conditions that induced physiologically relevant growth, morphogenesis and lactogenesis. Cultured MEC expressed an in vivo-like profile of EGFR. EGFR was high in immature MEC, down-regulated in functionally differentiated MEC, and then up-regulated in terminally differentiated and apoptotic MEC. An inhibitor of the tyrosine kinase domain of EGFR was used to demonstrate that EGFR signaling was required for growth and differentiation of immature MEC, and for survival of terminally differentiated MEC, but not for maintaining functional differentiation.
Asunto(s)
Receptores ErbB/fisiología , Regulación del Desarrollo de la Expresión Génica , Glándulas Mamarias Animales/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adipocitos/metabolismo , Animales , Apoptosis , Diferenciación Celular , División Celular , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Femenino , Fibroblastos/metabolismo , Humanos , Lactancia , Glándulas Mamarias Animales/citología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Morfogénesis , Organoides/metabolismo , Embarazo , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Maduración SexualRESUMEN
The effect of tumor necrosis factor-alpha (TNF alpha) on the growth and differentiation of normal rat mammary epithelial cells was evaluated using a model system in which cells were grown within a reconstituted basement membrane under defined serum-free medium conditions. TNF alpha (5-10,000 U/ml) stimulated mammary epithelial cell proliferation both under conditions previously considered optimal for their growth as well as in medium deficient in epidermal growth factor (EGF). Moreover, TNF alpha could completely substitute for EGF for cell proliferation. Under optimal conditions, TNF alpha had no effect on morphological differentiation, but in medium either lacking or deficient in EGF or when suboptimal reconstituted basement membrane was used, TNF alpha (5-100 U/ml) had a marked stimulatory effect on lobular and ductal morphogenesis. The effect of TNF alpha on functional differentiation, as assessed by casein production, was more complex. In optimal lactogenic medium, TNF alpha (10-10,000 U/ml) inhibited casein production. In the absence of EGF, however, the effect of TNF alpha appeared to follow a bell-shaped curve. Thus, omission of EGF per se resulted in a marked suppression of casein production, possibly secondary to an inhibition of morphological development. At low concentrations (approximately 5 U/ml), TNF alpha stimulated casein production in parallel to its stimulation of morphological differentiation, although not to the same extent as in medium containing optimal levels of EGF. However, once maximal stimulation of morphogenesis had been achieved, further increasing the TNF alpha concentration from 5 to 100 U/ml resulted in a concentration-dependent inhibition of casein production. This suggests that TNF alpha may have a direct inhibitory effect on casein gene expression. In summary, this is the first study to report that the multifunctional cytokine TNF alpha is a potential regulator of the growth and development of the mammary gland.
Asunto(s)
Glándulas Mamarias Animales/citología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Membrana Basal/fisiología , Caseínas/análisis , Caseínas/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Cinética , Glándulas Mamarias Animales/metabolismo , Ratas , Ratas EndogámicasRESUMEN
The interaction of the retinoid RE80 with the lactogenic and mammogenic regulators of mammary gland development was investigated using a mammary epithelial cell (MEC) primary culture model in which cells from young virgin rats were cultured within a reconstituted basement membrane using defined serum-free medium. RE80 (10(-10) M) was able to substitute completely for epidermal growth factor and partially for hydrocortisone in stimulating both morphological and functional (casein accumulation) differentiation of the MEC. In contrast, the requirement of PRL for both differentiation processes was absolute. Furthermore, RE80 was found to abrogate the inhibitory effect of progesterone on casein accumulation and to act as an antiprogestin in terms of morphological effects. Under optimal medium conditions, RE80 also inhibited cell proliferation. This inhibition did not require epidermal growth factor, hydrocortisone, progesterone, or PRL, but, unexpectedly, was enhanced in medium deficient in or lacking hydrocortisone. Additionally, RE80 induced the death of differentiated MEC, an effect that was found to require hydrocortisone. These results suggest that retinoids may modulate transcription of the casein gene family, either directly by activation of the binding of retinoic acid receptors to the casein promoter or indirectly by modulation of the effects of other hormones. The antiproliferative effect of retinoid may also be direct or indirect by virtue of down-regulation of the receptors for one of the mitogenic hormones, possibly progesterone.
Asunto(s)
Benzoatos/farmacología , Diferenciación Celular/efectos de los fármacos , Hormonas/farmacología , Glándulas Mamarias Animales/citología , Retinoides/farmacología , Tetrahidronaftalenos/farmacología , Animales , Muerte Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Hidrocortisona/farmacología , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Progesterona/farmacología , Prolactina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Our laboratory has shown that tumor necrosis factor-alpha (TNF alpha) can regulate normal mammary epithelial cell (MEC) growth, morphogenesis, and, under certain circumstances, functional differentiation in a manner similar to epidermal growth factor (EGF). As TNF alpha has been shown to up-regulate EGF receptor (EGFR) expression and function in other systems, the present studies were undertaken to determine whether TNF alpha action in MEC was indirect through stimulation of the EGFR. An inhibitor of EGFR tyrosine kinase activity, PD158780, failed to block proliferation induced by 40 ng/ml TNF alpha and only partially inhibited growth in response to 2 ng/ml TNF alpha. PD158780 was also unable to suppress the extensive morphological development induced by either TNF alpha concentration. In contrast, the effects of TNF alpha and PD158780 on functional differentiation (i.e. casein accumulation) were time dependent. When measured on day 7 after 48 h of treatment, casein accumulation was unaffected by either concentration of TNF alpha or by PD158780. When assessed on day 21 after 16 days of treatment, however, casein levels were decreased by 40 ng/ml TNF alpha and increased by PD158780. Significantly, this PD158780-induced increase in casein was not observed in MEC that had been treated with both PD158780 and TNF alpha. These results thus suggest that EGFR tyrosine kinase activity is not necessary for TNF alpha action in normal MEC.
Asunto(s)
Receptores ErbB/fisiología , Glándulas Mamarias Animales/citología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Epiteliales , Receptores ErbB/antagonistas & inhibidores , Femenino , Pirimidinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The ability of retinoids to modulate the proliferation as well as the morphological and functional differentiation of normal mammary epithelial cells isolated from pubescent female virgin rats was evaluated in serum-free primary culture. The retinobenzoic acid derivative RE80, present continuously or for only a limited time in culture, inhibited proliferation with an IC50 of less than 10(-10) M. In contrast, all-trans-retinoic acid (RA) inhibited proliferation with an IC50 of approximately 10(-8) M. In addition to effects on proliferation, RE80 and RA stimulated end bud colonies to differentiate to lobular alveolar colonies, inhibited alveolar budding, and suppressed the outgrowth of squamous colonies. Both retinoids also markedly stimulated functional differentiation, as assessed by accumulation of the major milk protein casein, and stimulated the synthesis of a approximately 73- to 74-kilodalton protein identified as a member of the transferrin family. Moreover, both retinoids stimulated cell death in the differentiated cell population. RE80 was approximately 100-fold more potent than RA for all of these effects. These data suggest that several mechanisms may contribute to the chemopreventive and/or therapeutic efficacy of retinoids in breast cancer, including inhibition of proliferation, stimulation of cell death, and/or induction of differentiation.
Asunto(s)
Benzoatos/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Glándulas Mamarias Animales/citología , Retinoides/farmacología , Tetrahidronaftalenos/farmacología , Tretinoina/farmacología , Animales , Western Blotting , Caseínas/metabolismo , Muerte Celular/efectos de los fármacos , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Studies were undertaken to examine the natural role of ErbB2, ErbB3, and ErbB4 during the development of normal rat mammary epithelial cells (MECs) in vivo and in vitro. Immunohistochemical analysis demonstrated that mammary gland terminal end buds expressed abundant ErbB2 and ErbB4 but limited ErbB3 in pubescent rats, whereas luminal epithelial cells in nulliparous rats expressed ErbB2, ErbB3, and/or ErbB4. During pregnancy, ductal epithelial cells and stromal cells expressed abundant ErbB3 but limited ErbB2. Although ErbB2 and ErbB3 were downregulated throughout lactation, both receptors were re-expressed during involution. In contrast, ErbB4 was downregulated throughout pregnancy, lactation, and involution. Immunoblotting and immunoprecipitation studies confirmed the developmental expression of ErbB2 and ErbB3 in the mammary gland and the co-localization of distinct ErbB receptors in the mammary gland of nulliparous rats. In agreement with our in vivo findings, primary culture studies demonstrated that ErbB2 and ErbB3 were expressed in functionally immature, terminally differentiated and apoptotic MECs, and downregulated in functionally differentiated MECs. ErbB receptor signaling was required for epithelial cell growth, functional differentiation, and morphogenesis of immature MECs, and the survival of terminally differentiated MECs. Finally, ErbB4 expression did not interfere with functional differentiation and apoptosis of normal MECs.
Asunto(s)
Células Epiteliales/fisiología , Receptores ErbB/aislamiento & purificación , Glándulas Mamarias Animales/fisiología , Receptor ErbB-2/aislamiento & purificación , Receptor ErbB-3/aislamiento & purificación , Reproducción/fisiología , Animales , Apoptosis , Diferenciación Celular , Técnicas de Cultivo , Dimerización , Células Epiteliales/citología , Receptores ErbB/metabolismo , Femenino , Lactancia/fisiología , Glándulas Mamarias Animales/citología , Embarazo , Unión Proteica , Ratas , Ratas Sprague-Dawley , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Receptor ErbB-4 , Distribución TisularRESUMEN
Stromal-epithelial interactions play a profound role in regulating normal and tumor development in the mammary gland. The molecular details of these events, however, are incompletely understood. A novel serum-free transwell coculture system was developed to study the natural paracrine interactions between mammary epithelial cells (MEC) and mammary fibroblasts (MFC) isolated from normal rats during puberty. The MEC were cultured within a reconstituted basement membrane (RBM) in transwell inserts with or without MFC in the lower well. The presence of MFC stimulated epithelial cell growth, induced alveolar morphogenesis, and enhanced casein accumulation, a marker of the functional differentiation of MEC, but did not induce ductal morphogenesis. Potent mitogenic, morphogenic, and lactogenic effects were observed when the MFC were cultured either on plastic or within a layer of RBM. Although most MFC maintained on plastic died after 1 wk in serum-free medium, fibroblast survival was enhanced significantly when the MFC were cultured within the RBM. Taken together, this in vitro model effectively reconstitutes a physiologically relevant three-dimensional microenvironment for MEC and MFC, and seems ideal for studying the locally derived factors that regulate the developmental fate of the epithelial and fibroblast compartments of the mammary gland.
Asunto(s)
Mama/citología , Diferenciación Celular , Células Epiteliales/citología , Fibroblastos/citología , Animales , División Celular , Técnicas de Cocultivo , Medio de Cultivo Libre de Suero , Femenino , Morfogénesis , Ratas , Ratas Sprague-DawleyRESUMEN
Our previous study revealed that two splicing factors, polypyrimidine tract-binding protein (PTB) and SRp20, were upregulated in epithelial ovarian cancer (EOC) and knockdown of PTB expression inhibited ovarian tumor cell growth and transformation properties. In this report, we show that knockdown of SRp20 expression in ovarian cancer cells also causes substantial inhibition of tumor cell growth and colony formation in soft agar and the extent of such inhibition appeared to correlate with the extent of suppression of SRp20. Massive knockdown of SRp20 expression triggered remarkable apoptosis in these cells. These results suggest that overexpression of SRp20 is required for ovarian tumor cell growth and survival. Immunohistochemical staining for PTB and SRp20 of two specialized tissue microarrays, one containing benign ovarian tumors, borderline/low malignant potential (LMP) ovarian tumors as well as invasive EOC and the other containing invasive EOC ranging from stage I to stage IV disease, reveals that PTB and SRp20 are both expressed differentially between benign tumors and invasive EOC, and between borderline/LMP tumors and invasive EOC. There were more all-negative or mixed staining cases (at least two evaluable section cores per case) in benign tumors than in invasive EOC, whereas there were more all-positive staining cases in invasive EOC than in the other two disease classifications. Among invasive EOC, the majority of cases were stained all positive for both PTB and SRp20, and there were no significant differences in average staining or frequency of positive cancer cells between any of the tumor stages. Therefore, the expression of PTB and SRp20 is associated with malignancy of ovarian tumors but not with stage of invasive EOC.
Asunto(s)
Técnicas de Silenciamiento del Gen , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Proteínas de Unión al ARN/fisiología , Secuencia de Bases , División Celular/genética , Cartilla de ADN , Femenino , Humanos , Inmunohistoquímica , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Interferencia de ARN , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-ArgininaRESUMEN
Model systems have been developed to investigate the complex and coordinated regulation of mammary gland development and transformation. Primary cultures, using newly isolated cells or tissue, are optimal for such studies since, in comparison to immortalized cell lines, the normal signal transduction pathways are presumed to be intact. Three such models are described, including whole organ culture, mammary epithelial cell (MEC) organoids, and MEC-stromal cocultures. Studies using whole-organ culture have the advantage that the normal glandular architecture remains intact, the MEC can undergo lobuloalveolar development and express milk proteins in a hormone dependent manner, and, following hormonal withdrawal, undergo involution. Moreover, transformation of the MEC is readily accomplished. Culture of isolated MEC organoids within an EHS-derived reconstituted basement membrane permits extensive proliferation, branching end bud and alveolar morphogenesis, and accumulation of milk protein and lipid in a physiologically relevant hormone- and growth factor-dependent manner. This model can thus be utilized to investigate the mechanism by which various modulators exert their direct effects on the epithelium. Finally, in view of compelling evidence for stromal-epithelial interactions during normal mammary gland development, and potentially also during the development of malignancy, models in which MEC can be cocultured with enriched populations of stroma offer considerable potential as a tool to understand the nature and mechanisms of the interactions that occur during the various developmental states, and how such interactions may go awry during carcinogenesis.
Asunto(s)
Mama/citología , Comunicación Celular , Técnicas de Cultivo de Célula/métodos , Glándulas Mamarias Animales/citología , Animales , Mama/patología , Femenino , Humanos , Glándulas Mamarias Animales/patologíaRESUMEN
We have recently described a primary culture system which allows for extensive proliferation and functional differentiation of immature mammary epithelial cells. Herein, these findings are extended to demonstrate that a distinct pattern of ductal and alveolar morphogenesis can be induced within the mammary organoids isolated from virgin female rats and cultured within an Engelbreth-Holm-Swarm sarcoma-derived reconstituted basement membrane under defined serum-free conditions. The lobular and multilobular organoids that emerged resemble the alveoli of the mammary gland in gross form, multicellular architecture, and cytologic and functional differentiation, while the ductal organoids expressed characteristics typical of mammary gland ducts in vivo. The epithelial cells within the alveolar- and duct-like organoids displayed the capability of secreting two morphologically distinct milk products, casein and lipid, into the luminal compartment. The expression of histiotypic morphogenesis and mammary-specific functional differentiation by the cultured mammary organoids proceeded in the absence of a morphologically distinct basal lamina. We illustrate that development highly reminiscent of that which naturally occurs in the mammary gland in vivo can be induced and supported in vitro under defined serum-free conditions. In addition, the methodologies are available to simultaneously monitor mammary organoid morphogenesis, growth, and functional differentiation. This system should serve as a unique model in which the regulation of branching morphogenesis, development, gene expression, and transformation can be examined.
Asunto(s)
Glándulas Mamarias Animales/ultraestructura , Organoides/ultraestructura , Animales , Membrana Basal/fisiología , Membrana Basal/ultraestructura , Diferenciación Celular/fisiología , Células Cultivadas , Células Epiteliales , Epitelio/fisiología , Epitelio/ultraestructura , Femenino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/fisiología , Microscopía Electrónica , Morfogénesis/fisiología , Organoides/fisiología , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
The mammary gland is a dynamic tissue that undergoes epithelial expansion and invasion during puberty and cycles of branching and lobular morphogenesis, secretory differentiation, and regression during pregnancy, lactation, and involution. The alteration in the mammary gland epithelium during its postnatal differentiation is accompanied by changes in the multiple stromal cell types present in this complex tissue. The postnatal plasticity of the epithelium, endothelium, and stromal cells of the mammary gland may contribute to its susceptibility to carcinogenesis. The purpose of this review is to assist researchers in recognizing histological changes in the epithelium and stroma of the rat mammary gland throughout development.
Asunto(s)
Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratas/anatomía & histología , Envejecimiento , Animales , Células Epiteliales/citología , Femenino , Lactancia , Glándulas Mamarias Animales/embriología , Morfogénesis , Embarazo , Maduración SexualRESUMEN
The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on proliferation and differentiation of normal mammary epithelial cells from 50-day-old virgin rats was investigated using a model system that allows for full morphological and functional development of the cells. In this model, mammary epithelial cells are grown within a reconstituted basement membrane in a defined serum-free medium. PMA at a concentration of 10(-6) M effected translocation of protein kinase C from cytosol to membrane. At the same concentration, it stimulated cell proliferation both in the presence and absence of EGF, and this stimulation was observed even when PMA exposure was limited to 15 min at the time of each media change. In contrast to the effect on proliferation, PMA at concentrations of 10(-7) and 10(-6) M inhibited functional differentiation as assessed by casein accumulation. Phorbol 12,13-dibutyrate at 10(-6) M also stimulated proliferation and inhibited casein accumulation and was more effective than PMA in both cases. In contrast, the nonactive tumor promoter 4-alpha PMA had no effect on either proliferation or differentiation. One of the most striking effects of PMA was its ability to stimulate an atypical ductal morphogenesis, as manifested by the formation of intricate web-like colonies, and to inhibit the development of the well-differentiated alveolar-like multilobular colonies. PMA was also shown to completely suppress the growth of the squamous-like colonies that develop when EGF is absent or deficient. These effects of phorbol esters in mammary epithelial cells to stimulate proliferation, inhibit functional differentiation, and stimulate the development of ductal colonies are consistent with the suggestion that the signal transduction pathways evoked by PMA could act to stimulate the growth of initiated cells or render normal cells more sensitive to carcinogen.
Asunto(s)
Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Animales , Caseínas/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Glándulas Mamarias Animales/metabolismo , Forbol 12,13-Dibutirato/farmacología , Proteína Quinasa C/metabolismo , Ratas , Valores de ReferenciaRESUMEN
Mammary epithelial organoids (MEO), isolated from pubescent rats, were cultured within a reconstituted basement membrane in transwell inserts, in the presence or absence of mature mammary adipocytes in the lower well. This system allowed for free medium exchange between the two compartments, without direct cell-to-cell contact. When cultured in serum-free medium supplemented with insulin, prolactin, hydrocortisone, progesterone, and various epidermal growth factor (EGF) concentrations, mammary adipocytes did not affect epithelial cell growth, but enhanced epithelial differentiation. Casein and lipid accumulations were monitored as indicators of functional differentiation of MEO. Mammary adipocytes significantly enhanced casein and lipid accumulation within the MEO, independently of EGF concentration. Furthermore, adipocytes induced MEO to preferentially undergo alveolar morphogenesis, inhibited squamous outgrowth, and increased lumen size. These findings demonstrate that morphological and functional differentiation of mammary epithelial cells is profoundly enhanced by the adipose stroma and that these effects are mediated by diffusible paracrine factors. This new model can be exploited in future studies to define the mechanisms whereby hormones and growth factors regulate mammary gland development and carcinogenesis. Moreover, it could complement in vivo reconstitution/transplantation studies, which are currently employed to evaluate the role of specific gene deletions in the regulation of mammary development.
Asunto(s)
Adipocitos/fisiología , Glándulas Mamarias Animales/fisiología , Adipocitos/citología , Animales , Diferenciación Celular , División Celular , Células Cultivadas , Células Epiteliales/citología , Femenino , Macrófagos Alveolares/citología , Glándulas Mamarias Animales/citología , Morfogénesis , Ratas , Ratas Sprague-Dawley , Células MadreRESUMEN
It has previously been shown that mammary stromal cells possess the ability to maintain a fibroblast-like phenotype or differentiate in vitro into mature adipocytes in a hormone-dependent manner. This paper reports that rat mammary stromal cells can also differentiate into capillary-like structures in vitro when cultured on a reconstituted basement membrane (RBM). The differentiation potential of mammary stromal cells was compared with that of human umbilical vein endothelial cells (HUVEC) and 3T3-L1 preadipocytes. When cultured on plastic, mammary stromal cells, 3T3-L1 and HUVEC maintained a fibroblast-like phenotype. Mammary stromal cells and 3T3-L1, but not HUVEC, differentiated into mature adipocytes when cultured in adipogenic medium. When plated on reconstituted basement membrane, all three cell types began to migrate and organize themselves into an interconnected capillary network. By 18-20 h, mammary stromal cells organized into complex, highly branched capillary-like tubules whereas 3T3-L1 cells and HUVEC formed more simple structures. Cross-sectional analysis demonstrated the presence of an internal lumen. Mammary stromal cells were unique in their ability to progressively develop into a three-dimensional, highly branched network invading the RBM surface. The network formation was enhanced by the presence of vascular endothelial growth factor (VEGF) and was inhibited by the anti-angiogenic drug suramin. Western blotting analysis demonstrated the presence of the endothelial-specific marker flk-1, as well as the presence of the tight-junction-associated protein ZO-1. Mammary stromal cell differentiation into capillary structures was not a terminal state, since these cells were still able to differentiate into adipocytes when exposed to adipogenic medium. These findings suggest that mammary stromal cells differentiate into fibroblasts, adipocytes or vascular structures in a hormone- and substatum-dependent manner, and may explain the dramatic changes in stromal composition during both normal mammary gland development and tumorigenesis.
Asunto(s)
Adipocitos/citología , Endotelio Vascular/citología , Matriz Extracelular/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Células 3T3/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Membrana Celular , Células Cultivadas , Medios de Cultivo , Factores de Crecimiento Endotelial/farmacología , Femenino , Fibroblastos/citología , Hormonas/metabolismo , Humanos , Metabolismo de los Lípidos , Linfocinas/farmacología , Glándulas Mamarias Animales/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Fenotipo , Fosfoproteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Suramina/farmacología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Proteína de la Zonula Occludens-1RESUMEN
The epithelial cell-specific effects of prolactin and epidermal growth factor (EGF) on the development of normal rat mammary epithelial cells (MEC) were evaluated using a three dimensional primary culture model developed in our laboratory. Non-milk-producing MEC were isolated as spherical end bud-like mammary epithelial organoids (MEO) from pubescent virgin female rats. The cultured MEO developed into elaborate multilobular and lobuloductal alveolar organoids composed of cytologically and functionally differentiated MEC. Prolactin (0.01-10 micrograms/ml) and EGF (1-100 ng/ml) were each required for induction of cell growth, extensive alveolar, as well as multilobular branching morphogenesis, and casein accumulation. MEO cultured without prolactin for 14 days remained sensitive to the mitogenic, morphogenic, and lactogenic effects of prolactin upon subsequent exposure. Similarly, cells cultured in the absence of EGF remained sensitive to the mitogenic and lactogenic effects of EGF, but were less responsive to its morphogenic effects when it was added on day 14 of a 21-day culture period. If exposure to prolactin was terminated after the first week, the magnitude of the mitogenic and lactogenic effects, but not the morphogenic response was decreased. Removal of EGF on day 7 also reduced the mitogenic response, but did not have any effect on the magnitude of the lactogenic or morphogenic responses. These studies demonstrate that physiologically relevant development of normal MEC can be induced in culture and that this model system can be used to study the mechanisms by which prolactin and EGF regulate the complex developmental pathways operative in the mammary gland.