RESUMEN
Platelet-activating factor (PAF) is a potent phospholipid modulator of inflammation that has diverse physiological and pathological functions. Previously, we demonstrated that PAF has an essential role in ultraviolet (UV)-induced immunosuppression and reduces the repair of damaged DNA, suggesting that UV-induced PAF is contributing to skin cancer initiation by inducing immune suppression and also affecting a proper DNA damage response. The exact role of PAF in modulating cell proliferation, differentiation or transformation is unclear. Here, we investigated the mechanism(s) by which PAF affects the cell cycle and impairs early DNA damage response. PAF arrests proliferation in transformed and nontransformed human mast cells by reducing the expression of cyclin-B1 and promoting the expression of p21. PAF-treated cells show a dose-dependent cell cycle arrest mainly at G2-M, and a decrease in the DNA damage response elements MCPH1/BRIT-1 and ataxia telangiectasia and rad related (ATR). In addition, PAF disrupts the localization of p-ataxia telangiectasia mutated (p-ATM), and phosphorylated-ataxia telangiectasia and rad related (p-ATR) at the site of DNA damage. Whereas the potent effect on cell cycle arrest may imply a tumor suppressor activity for PAF, the impairment of proper DNA damage response might implicate PAF as a tumor promoter. The outcome of these diverse effects may be dependent on specific cues in the microenvironment.
Asunto(s)
Daño del ADN/efectos de los fármacos , Mastocitos/efectos de los fármacos , Mastocitos/fisiología , Factor de Activación Plaquetaria/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/fisiología , Línea Celular , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Humanos , Mastocitos/citología , Mastocitos/metabolismo , Fosforilación , Factor de Activación Plaquetaria/análogos & derivadosRESUMEN
The Lewis(x) (Le(x)) trisaccharide (CD15) linked to proteins and glycolipids is highly expressed on the surface of normal human polymorphonuclear neutrophils (PMN) and several human neoplasias, such as breast and gastrointestinal carcinomas and chronic myeloid leukemias. FC-2.15 is an IgM murine mAb that specifically recognizes Le(x) and has been previously shown to mediate the in vitro lysis of Le(x)(+) cells by human complement. In a phase I clinical trial of FC-2.15, a temporary neutropenia was the main toxicity, and antitumor responses were observed. In order to characterize FC-2.15 further and determine the physiological relevance of Le(x) binding, the reactivity of FC-2.15 on PMN was investigated under several conditions. Flow cytometry revealed a strong reactivity of FC-2.15 with almost 100% of PMN, and Scatchard analysis demonstrated an affinity constant of 5.14 x 10(9) M(-1) and 1.11 x 10(6) antigen sites/cell. In vitro, the binding of Le(x) epitopes by FC-2.15 induced PMN homotypic aggregation, only 28.4 +/- 4.1% remaining as single cells. When PMN and the Le(x)(+) MCF-7 breast cancer cells were co-incubated, FC-2.15 induced heterotypic aggregation. In 51Cr-release assays employing human complement, FC-2.15 lysed 93.4 +/- 7.9% of PMN and 87.8 +/- 10.7% of MCF-7 cells. However, when the effect of FC-2.15 was tested in ex vivo circulating blood, no lytic activity against PMN was detected, whereas MCF-7 cells were still lysed. Blood smears demonstrated that FC-2.15 induced PMN agglutination and heterotypic aggregates when MCF-7 cells were present. A pretreatment of PMN with colchicine impaired PMN agglutination both in vitro (single PMN = 81.15 +/- 4.35%) and in ex vivo circulating blood. In the latter condition, FC-2.15-lytic activity was restored, suggesting that PMN homotypic aggregation by FC-2.15, but not lysis, is dependent on microtubule integrity and that PMN agglutination hinders their lysis. Moreover, when 51Cr-release assays were performed following agglutination, FC-2.15 cytotoxicity was restricted to isolated PMN. It is suggested that crosslinking of Le(x) epitopes by FC-2.15 induces PMN to form homotypic aggregates. It is suggested that the neutropenia observed in FC-2.15-treated patients would be due to PMN agglutination and margination, rather than lysis. In addition, FC-2.15 appears to be able to lyse Le(x)(+) tumor cells in circulation.
Asunto(s)
Aglutinación , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Neoplasias de la Mama/inmunología , Antígeno Lewis X/inmunología , Neutrófilos/fisiología , Animales , Agregación Celular , Femenino , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos BALB C , Neutropenia/etiología , Células Tumorales CultivadasRESUMEN
IIB-BR-G is an undifferentiated, highly heterogeneous, hormone receptor negative human breast cancer cell line previously established in our laboratory from a patient's primary tumor. An in vitro growing cell line (IIB-BR-G) and a xenotransplanted tumor growing in nude mice (IIB-BR-G(NUDE)) were derived. To further characterize these systems, immunocytochemical analysis was performed for differentiation antigens (PEM 200 kDa, CEA, NCA 90 kDa), blood-group related antigens (Le(x), sTn), oncogenes and tumor suppressor gene products (Her-2/neu protein, p53), metastasis-related cathepsin D and CD63/5.01 Ag, and the chemokine monocyte chemotactic protein 1 (MCP-1). Expression of markers was heterogeneous in these different systems. Previously reported karyotypic analysis has shown extensive chromosomal alterations including double min. Searching for oncogene amplification, we detected augmented copy number of c-myc and c-fos, the last one with two rearranged fragments. No amplification was found for c-erbB-2 in the cell line or in IIB-BR-G(NUDE), although this oncogene was amplified in the patient's primary tumor DNA. The differences observed between the patient's tumor, the cell line and the IIB-BR-G(NUDE) tumors are probably due to clonal expansion of cell variants not present in the original tumor. Electron microscopy of IIB-BR-G growing cells revealed epithelial characteristics with abundant dense granules, presumably secretory, distributed all over the cytoplasm and great nuclear pleomorphism. In vitro, IIB-BR-G cells showed a significant number of invading cells by Matrigel assay. After nearly 40 sequential subcutaneous passages of the original xenograft through nude mice, 80% of recipients developed spontaneous metastases, primarily to the lung and lymph nodes. Since this experimental model allowed to analyze changes produced in cancer cells from the primary tumor during adaptation to in vitro and in vivo growth, our results provide novel insights on the behaviour of hormone independent metastatic breast cancer.
Asunto(s)
Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Antígenos de Neoplasias/biosíntesis , Neoplasias de la Mama , Carcinoma Ductal de Mama , Femenino , Amplificación de Genes , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica , Receptores de Estrógenos/genética , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
FC-2.15 is a murine IgM monoclonal antibody that recognizes breast and colon human carcinomas, chronic myeloid leukemias, Sternberg cells of Hodgkin's lymphoma and some normal cells, such as peripheral polymorphonuclear granulocytes. It has been previously demonstrated that FC-2.15 recognizes the carbohydrate moiety of different glycoproteins. FC-2.15 is able to mediate the in vitro lysis of Ag-2.15+ cells by human complement. In a phase I clinical trial, FC-2.15 induced antitumor responses and reversible neutropenia was its main toxicity. In this work, analysis of epitope specificity has demonstrated that FC-2.15 specifically recognizes terminally exposed Lewis(x) trisaccharide but not sialyl-Lewis(x), Lewis(a), trifucosylated Lewis(y), blood-group antigens A and B, globo H and gangliosides. In polymorphonuclear granulocytes (PMN), myeloid leukemic cells and colon carcinoma T84 cells, Lewis(x) was found to be almost exclusively N-linked to the protein core, whereas in breast carcinoma MCF-7 cells, Lewis(x) appeared to be mostly O-linked. Treatment with neuraminidase increased detection by FC-2.15 in normal PMN, myeloid leukemia cells and T84 cells but not in MCF-7 cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Neoplasias de la Mama/inmunología , Antígeno Lewis X/inmunología , Animales , Especificidad de Anticuerpos , Línea Celular , Mapeo Epitopo , Haptenos , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Acquisition of invasive/metastatic potential is a key event in tumor progression. Cell surface glycoproteins and their respective matrix ligands have been implicated in this process. Recent evidence reveals that the secreted glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is highly expressed in different malignant tissues. The present study reports that the suppression of SPARC expression by human melanoma cells using a SPARC antisense expression vector results in a significant decrease in the in vitro adhesive and invasive capacities of tumor cells, completely abolishing their in vivo tumorigenicity. This is the first evidence that SPARC plays a key role in human melanoma invasive-metastatic phenotype development.
Asunto(s)
Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Melanoma Experimental/patología , Melanoma/patología , Oligonucleótidos Antisentido/genética , Osteonectina/genética , Animales , Adhesión Celular/genética , División Celular/genética , Regulación hacia Abajo , Humanos , Melanoma/genética , Melanoma Experimental/genética , Ratones , Transfección , Células Tumorales CultivadasRESUMEN
SPARC (secreted protein acidic and rich in cysteine) is an extracellular protein associated with tissues exhibiting high rates of cell proliferation and matrix remodeling. The current work shows that the human melanoma cell lines IIB-MEL-LES, IIB-MEL-IAN, and IIB-MEL-J and different human metastatic melanomas expressed high levels of SPARC mRNA and protein. By western blot analysis we detected a single secreted 42-kDa band in human diploid fibroblasts-conditioned medium and a 45- to 40-kDa doublet in the three melanoma cell lines and all the metastatic melanomas tested. Part of the melanoma samples and cell lines showed an additional doublet of 36-34 kDa. SPARC mRNA was expressed by the three established cell lines, 14 metastatic melanoma samples, and tumors raised in nude mice, and no spliced variants were found. The heterogeneous pattern of SPARC secreted by human melanoma cells is the result of post-translational glycosylation and a specific extracellular leupeptin-inhibitable cleavage. Unlike human fibroblasts, melanoma cells did not overexpress SPARC on addition of TGF-beta. Immunohistochemical analysis showed that SPARC was strongly expressed in 100% of primary melanomas (7 of 7) and metastatic melanomas (29 of 29), moderately expressed in most of the positive dysplastic nevi (13 of 14), and only weakly expressed in nevocellular nevi (4 of 25). Normal melanocytes did not express SPARC. The data suggest that the expression of SPARC is associated with the neoplastic progression of human melanoma.
Asunto(s)
Melanoma/patología , Osteonectina/biosíntesis , Transformación Celular Neoplásica , Regulación de la Expresión Génica/efectos de los fármacos , Glicosilación , Humanos , Inmunohistoquímica , Linfotoxina-alfa/farmacología , Melanoma/química , Melanoma/secundario , Metástasis de la Neoplasia/genética , Osteonectina/genética , ARN Mensajero/análisis , Células Tumorales Cultivadas/químicaRESUMEN
The incidence of melanoma is increasing rapidly, and in many cases the primary tumor is excised after metastatic spreading. In 80% of the cases, the first metastatic site is in regional lymph nodes (AJCC Stage III). After excision of these nodes, the patient is clinically disease-free, but the chances of recurrency vary between 40-80%. Thirty patients with stage III melanoma were treated in a non-randomized Phase II adjuvant trial with a vaccine consisting of a mixture of three allogeneic cell lines: IIB-MEL-J, IIB-MEL-LES and IIB-MEL-IAN (5 x 10(6) cells each). The cells were irradiated (5,000 cGy) and BCG was used as nonspecific stimulant. Before each vaccination (72 hr) the patients received cyclophosphamide (300 mg/sqm). The untreated control group was composed of 24 Stage III melanoma patients. Vaccination started within 60 days after surgery, and patients received 4 vaccinations, one every 21 days and then 1 every two months during the 1st year; 1 every three months during the 2nd year, and 1 every 6 months during the 3rd, 4th and 5th years. The treated group was composed by 19 men (63.3%) and 11 women (36.7%); average age: 47.6 +/- 14.1 years (range: 16-70 yr). The control group was composed by 18 men (75%) and 6 women (25%); average age 49.8 +/- 14.2 yr (range: 26-73 yr). The median disease free survival (DFS) calculated according to Kaplan-Meier was 7.0 months in the control group vs 20.0 months in the treated group (p < 0.001). The results of this clinical trial suggest that treatment with allogeneic cell vaccines increases DFS in stage III melanoma patients.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Melanoma/mortalidad , Melanoma/terapia , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/terapia , Adulto , Anciano , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Melanoma/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Cutáneas/patologíaRESUMEN
Cytokine gene transfer to tumor cells has been demonstrated to induce tumor rejection in different murine models. However, controversial results were presented for different cytokines. In order to study the antitumorigenic activity that has been proposed for IL-6, the poorly immunogenic melanoma B16 and the colon adenocarcinoma CT26-murine cell lines, were transduced with recombinant retrovirus expressing rat IL-6. In vivo studies showed that IL-6-producing-B 16 cells inoculated s.c. in syngeneic mice, exhibited reduced tumorigenicity compared to vector-transduced B 16 cells. The histology of growing IL-6-producing tumors showed a "pseudo-nodular" pattern which correlated with a strong inhibition of the in vitro invasive capacity of these cells. IL-6-producing-B 16 cells did not develop tumors in athymic nude mice suggesting that the antitumor effect is not mediated by a normal host-T- and B-cell response. In contrast, IL-6-producing CT26 cells grew as tumors in syngeneic mice with a faster growth rate than parental and vector-transduced cells, in accordance with an increased in vitro growth kinetics. These results indicate that IL-6 expression by tumor cells demonstrate different effects depending on the tumor cell model.
Asunto(s)
Interleucina-6/genética , Células Tumorales Cultivadas/inmunología , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , Linfocitos B/inmunología , División Celular , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Expresión Génica , Ingeniería Genética , Cinética , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Invasividad Neoplásica/genética , Invasividad Neoplásica/inmunología , Invasividad Neoplásica/patología , Trasplante de Neoplasias , Ratas , Linfocitos T/inmunología , Transducción Genética , Trasplante Isogénico , Células Tumorales Cultivadas/patologíaRESUMEN
The causes of decreased immune competence in melanoma patients as well as in other cancer patients are incompletely understood. The identification of the factor(s) responsible for this behaviour remains elusive. The present report demonstrates that an immunosuppressive activity (ISA), manifested in vitro as an inhibition of proliferation of human peripheral blood lymphocytes (PBL) induced both by phytohemagglutinin (PHA) or the cytokine IL-2, was exhibited by serum-free conditioned medium (SFCM) from the human melanoma cell line IIB-MEL-J, as well as by two other melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, established in our laboratory. The ISA was found to be exerted by a protein, which co-eluted with serum albumin in anionic exchange Mono-Q, gel filtration chromatography and Blue Sepharose columns. It showed a molecular weight (Mw) of 14 kDa when separated from albumin traces by means of a Sephacryl S 200 column. It is not recognized by a pan-specific anti-transforming growth factor-beta (TGF-beta) antibody as determined by Western blots assays performed on the SFCM. The immunosuppressive factor (ISF) is secreted by IIB-MEL-J cell line in soluble from and in very scarce amounts, non-detectable by polyacrylamide gel electrophoresis (PAGE). This characteristic difficults its obtention in adequate quantities to sequentiation. Since this inhibitory factor may have a role in protecting melanoma tumors from attack by the host immune system, preparative isolation will be attempted. But we consider these results only as preliminary one's.
Asunto(s)
Inmunosupresores/aislamiento & purificación , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Humanos , Tolerancia Inmunológica , Inmunosupresores/química , Inmunosupresores/farmacología , Técnicas In Vitro , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Prueba de Cultivo Mixto de Linfocitos , Melanoma/química , Peso Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/aislamiento & purificación , Fitohemaglutininas/farmacología , Solubilidad , Células Tumorales CultivadasRESUMEN
Previous studies from our laboratory have demonstrated that human melanoma cell lines and tumors expressed high levels of the extracellular protein SPARC. In order to demonstrate its role in human melanoma progression, IIB-MEL-LES human melanoma cells were transfected with SPARC full length c-DNA in the antisense orientation. In vivo studies demonstrated that all the control mice injected with parental cells developed tumors, while none of the mice injected with cells obtained from three different clones with diminished levels of SPARC expression, developed tumors. These studies suggest that SPARC may play a key role in human melanoma progression.
Asunto(s)
Melanoma/patología , Osteonectina/fisiología , Animales , Northern Blotting , Western Blotting , Células Clonales , ADN sin Sentido/genética , Humanos , Masculino , Melanoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Osteonectina/metabolismo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD3 and GD2 gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10, 20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.
Asunto(s)
Inmunohistoquímica , Melanoma Amelanótico/genética , Melanoma Amelanótico/patología , Células Tumorales Cultivadas , Adulto , Animales , Secuencia de Bases , Enzimas de Restricción del ADN , ADN de Neoplasias/análisis , ADN de Neoplasias/química , Gangliósidos/análisis , Antígenos HLA-DR/análisis , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Inmunofenotipificación , Cariotipificación , Masculino , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Trasplante de Neoplasias , Proteínas S100/análisis , Vimentina/análisisRESUMEN
High levels of cytosolic cathepsin D expression have been associated with poor prognosis in breast cancer node-negative patients. In this work, we provide evidence that three cell lines established from human metastatic melanomas--IIB-MEL-J, IIB-MEL-LES, and IIB-MEL-IAN--express high levels of procathepsin D mRNA. IIB-MEL-J cells secreted into the conditioned media about 30% of the newly synthesized protein, which was active at acidic pH. Melanoma tumors arising in nude mice after injection of the three different cell lines expressed high levels of procathepsin D mRNA. Moreover, 13 human metastatic melanomas expressed variable levels of procathepsin D mRNA. To study the possible association between cathepsin D expression and melanoma development, samples corresponding to 10 primary tumors, 11 metastatic melanomas, 10 dysplastic nevi, 27 nevocellular nevi, and normal melanocytes were studied by immunohistochemistry for cathepsin D-specific staining. We found that cathepsin D was expressed in all of the dysplastic nevi and primary and metastatic melanomas tested but in only 18% of nevocellular nevi (five of 27), whereas normal melanocytes showed no cathepsin D expression. The overall data indicate that cathepsin D is expressed at a high level by melanoma cells, and because of its expression in preneoplastic lesions, it may be associated with melanoma development.
Asunto(s)
Catepsina D/análisis , Síndrome del Nevo Displásico/metabolismo , Melanoma/secundario , Catepsina D/genética , Catepsina D/metabolismo , Medios de Cultivo Condicionados , Expresión Génica , Humanos , Inmunohistoquímica , Melanoma/química , Melanoma/genética , ARN Mensajero/análisis , Células Tumorales CultivadasRESUMEN
FC-2.15 is an IgM monoclonal antibody (MAb) obtained by immunizing Balb/c mice with tumor epithelial cells from a human undifferentiated primary breast carcinoma. FC-2.15 reacts with 93.9% (31/33) of human breast primary tumors, independently of their histology and hormone receptor content. Moreover, FC-2.15 reacts with 79.6 +/- 13.8% (mean +/- SD) of total breast malignant tumor cells and with 88.7 +/- 9.9% of proliferating tumor cells. It recognizes other neoplasia such as colon cancer, squamous carcinoma and melanoma. Among the normal tissues examined, strong cross-reactivity was found with kidney proximal convolute tubules, bone marrow myeloid progeny, peripheral granulocytes and large bowel epithelium. Through Western blots, FC-2.15 recognizes three major bands of Mr 160 kDa, 130 kDa and 115 kDa in membrane extracts of MCF-7 cells grown in nude mice and of human breast carcinoma and three major bands of 250 kDa, 185 kDa and 105 kDa in membrane extracts of peripheral granulocytes. This MAb mediates complement- cytotoxicity against malignant cells, reducing the clonogenic capacity of breast primary tumor cells and MCF-7 cells to 35.6 +/- 41.2% and 11.7 +/- 4.8% of control values respectively, whereas that of normal bone marrow cells is not affected (104.7 +/- 17.4%).
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Neoplasias de la Mama/inmunología , Neoplasias/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/aislamiento & purificación , Especificidad de Anticuerpos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Biomarcadores de Tumor/inmunología , Purgación de la Médula Ósea , Neoplasias de la Mama/terapia , Citotoxicidad Inmunológica , Femenino , Humanos , Inmunización , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Peso Molecular , Células Tumorales Cultivadas/inmunología , Ensayo de Tumor de Célula MadreRESUMEN
The establishment of a new human breast cancer cell line (IIB-BR-G) was successful after a previous growth of the cells isolated from a breast primary tumor in a female nude mouse. The IIB-BR-G cell line and the primary tumor do not express estrogen or progesterone receptors. Vimentin and keratin expression were found in the cell line and in the nude mouse tumor. This cell line displays high morphological heterogeneity with atypical multinucleated megacells, and it is capable of anchorage-independent growth and tumor formation in nude mice. The cytogenetic analysis confirmed its human origin and revealed multiple marker chromosomes and extensive chromosomal alterations including rearrangements, gains, losses, isochromosomes, and double minutes (DMs).
Asunto(s)
Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/patología , Menopausia , Receptores de Superficie Celular/análisis , Animales , Neoplasias de la Mama/química , Neoplasias de la Mama/genética , Carcinoma Intraductal no Infiltrante/química , Carcinoma Intraductal no Infiltrante/genética , Diferenciación Celular/fisiología , División Celular/fisiología , Medios de Cultivo , Femenino , Humanos , Inmunohistoquímica , Cariotipificación , Cinética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Trasplante de Neoplasias/patología , Células Tumorales CultivadasRESUMEN
We compared the morphology, clonogenic ability, Percoll gradient distribution, estrogen receptor proteins, and interactions with mesenchymal cells in MCF-7 breast tumor cells grown in medium containing fetal calf serum and insulin (FCS-I) or in a defined medium with insulin (ID) as the only growth factor. In the absence of serum and at densities below 5000-8000 cells/cm2, MCF-7 cells required epidermal growth factor, insulin, and thrombin. When cells reached a density of 23,000-26,000 cells/cm2, only insulin was necessary for optimal growth. In ID medium cells showed an enlarged Golgi apparatus and marked plasma membrane modifications, suggesting increased secretory activity. Moreover there was an increase in the release of protein products to the culture medium and a time-dependent ability of these cells to form macrocolonies in soft agar. On the contrary, cells in FCS-I showed no Golgi complex and few plasma membrane modifications. In both culture media tight junctions, desmosomes, and tonofilaments were present. We investigated the effect of conditioned media from MCF-7 cells growing in FCS-I or ID on the growth of primary rat vaginal fibroblasts. The growth of these mesenchymal cells was stimulated by FCS-I medium and inhibited by ID medium. By contrast, the embryonic fibroblast (preadipocyte) line CHEF/18 was also stimulated by FCS-I for the first 48 h, but thereafter ceased growth and acquired lipid droplets and a differentiated morphology. With ID medium, CHEF/18 cells were only partially inhibited with no changes in morphology. The Percoll gradient profiles of ID cells showed the same six fractions of increasing density as recently described. However, there was a progressive increase in subpopulations with higher growth rates and a decrease in the relative amount of the most differentiated cells. A unique feature of the growth analysis of MCF-7 cells in the absence of serum is the increased expression of the estradiol receptor gene. These studies show that the growth and differentiated properties of tumor cells can depend upon the cellular environment and offer a model system in which to further study this modulation.
Asunto(s)
Neoplasias de la Mama/patología , Transformación Celular Neoplásica/efectos de los fármacos , Receptores de Estradiol/fisiología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/ultraestructura , Medios de Cultivo/análisis , Medios de Cultivo/farmacología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/ultraestructura , Epitelio/metabolismo , Epitelio/patología , Epitelio/ultraestructura , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Inmunohistoquímica , Insulina/análisis , Insulina/farmacología , Metionina/metabolismo , Microscopía Electrónica , Proteínas/metabolismo , Ratas , Albúmina Sérica Bovina/análisis , Albúmina Sérica Bovina/farmacología , Radioisótopos de Azufre , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Células Tumorales Cultivadas/ultraestructuraRESUMEN
IIB-MEL-J is a highly heterogeneous newly established human melanoma cell line. The addition of 3 mM L-tyrosine to the culture medium produced (1) a great decrease in the cell growth rate, (2) a loss of the anchor-age-independent growth capacity, and (3) a change in the morphology of the cells to a fibroblastoid aspect. Coincident with these changes, an increase in subpopulations I and II and a decrease in subpopulations III and IV took place. In view of this evidence we consider that the cells have differentiated. The melanin production was not increased by the L-tyrosine treatment, suggesting that differentiation and melanin expression are not strictly correlated.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias Pulmonares/patología , Melanoma/patología , Tirosina/farmacología , Adhesión Celular/efectos de los fármacos , Separación Celular , Centrifugación por Gradiente de Densidad , Humanos , Neoplasias Pulmonares/metabolismo , Melaninas/metabolismo , Melanoma/metabolismo , Persona de Mediana Edad , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
The MCF-7 breast carcinoma cell line can be separated by Percoll density gradient centrifugation into several subpopulations, A to F, one of which (E) has been previously suggested to be highly enriched in stem cells. The anchorage-independent growth of the different fractions and its sensitivity to estradiol (E2) and tamoxifen (TAM) was assayed. The anchorage-independent growth capacity of the different fractions was E greater than A greater than B greater than D greater than C,F. The E fraction had the highest clonogenic index (6.62 +/- 1.18) and was unaffected by E2 or TAM. The karyotypic analysis of the E fraction revealed features similar to those of the unfractionated cell line. It is suggested that the high growth rate of fraction E is due to an enrichment in stem cells and not to the existence of a different clone.
Asunto(s)
Neoplasias de la Mama/patología , Estradiol/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Tamoxifeno/farmacología , Neoplasias de la Mama/genética , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Marcadores Genéticos , Humanos , Cariotipificación , Células Madre Neoplásicas/citologíaRESUMEN
In human breast cancer the proliferating cells appear to differ from those containing estrogen receptors (ER) as shown by studies on isolated cellular subpopulations. In this paper the in vitro effect of 17-beta-estradiol on cell proliferation in 30 primary breast tumors was studied. The effect of several estradiol concentrations was assayed, and the influence of diethylstilbestrol, tamoxifen, and nafoxidine was also tested. The response to these compounds was measured through the thymidine labeling index (TLI). When exposed to 10(-9) mol/l and 10(-8) mol/l estradiol, 14 of 19 ER-positive tumors and six of 11 ER-negative tumors were induced to further proliferate. The TLI increase over the control was 219% (P less than 0.05) at 10(-9) mol/l E2 and 258% (P less than 0.05) at 10(-8) mol/l E2 for ER-positive tumors, and 233% (0.1 less than P less than 0.2) at 10(-9) mol/l E2 and 321% (0.1 less than P less than 0.2) at 10(-8) mol/l E2 for ER-negative tumors. The addition of diethylstilbestrol and antiestrogens in vitro inhibited, to varying degrees, the estradiol-induced increase in the TLI irrespective of the ER-status. The response to E2 was correlated with the expression of the ras p21 protein and carcinoembryonic antigen. It was found that the ras p21 protein is preferentially expressed in ER-negative tumors, the opposite being true for carcinoembryonic antigen. The ras p21 protein is preferentially expressed in those ER-positive tumors that do not respond to estradiol with an increase in the TLI.
Asunto(s)
Neoplasias de la Mama/patología , Estradiol/farmacología , Neoplasias de la Mama/análisis , Antígeno Carcinoembrionario/análisis , División Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN de Neoplasias/análisis , Dietilestilbestrol/farmacología , Humanos , Nafoxidina/farmacología , Proteínas de Neoplasias/análisis , Neoplasias Hormono-Dependientes/análisis , Neoplasias Hormono-Dependientes/patología , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas p21(ras) , Receptores de Estrógenos/análisis , Estimulación Química , Tamoxifeno/farmacología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Exponentially growing MCF7 human breast cancer cells were separated in Percoll gradients into six different fractions of increasing density (A to F). These fractions could be subcultured and were found to contain different cellular subpopulations as defined by the following criteria: ability to generate other cellular subpopulations; growth rate; DNA synthesis; and expression of estrogen receptors, ras oncogene-encoded protein p21, and carcinoembryonic antigen. One of the minor fractions (E), which contained about 5% of the total cell number, appeared to contain the stem cells, on the basis of the following criteria: (i) its ability to reproduce the other cellular subpopulations, (ii) its high rate of growth and DNA synthesis, and (iii) the inability of the other subpopulations to generate it. The most differentiated subpopulation appeared to be the densest one (F), since it was the slowest growing and appeared to be the end point of the other subpopulations.
Asunto(s)
Neoplasias de la Mama/patología , Separación Celular/métodos , Modelos Biológicos , Neoplasias Hormono-Dependientes/patología , Células Tumorales Cultivadas/clasificación , Antígeno Carcinoembrionario/análisis , Centrifugación por Gradiente de Densidad , Inhibición de Contacto , Replicación del ADN , Femenino , Humanos , Proteínas de Neoplasias/análisis , Povidona , Receptores de Estrógenos/análisis , Dióxido de Silicio , Células Tumorales Cultivadas/análisis , Células Tumorales Cultivadas/patologíaRESUMEN
The combined effect of methotrexate (MTX) with dipyridamole, an inhibitor of nucleoside transport, was studied in ascitic Sarcoma 180 cells. It was determined that 10 microM MTX inhibits by greater than 90% deoxy[3H]uridine incorporation into DNA and that this MTX concentration inhibits DNA synthesis as revealed by deoxy[3H]cytidine but not [3H]thymidine incorporation into DNA. Exogenous thymidine (greater than or equal to 1 microM) in the cell culture medium enhances DNA synthesis in nontreated cells and fully restores it in MTX-treated cells, whereas hypoxanthine has no appreciable effect on DNA synthesis. Dipyridamole inhibits deoxy[3H]cytidine and [3H]thymidine uptake by these cells (IC50 = 0.2 and 3 microM, respectively) and blocks the increase in TTP pool produced by 1 microM thymidine in MTX-treated cells (23.1 +/- 4.7 pmol per 1 X 10(6) cells vs. 80.4 +/- 18.9 pmol per 1 X 10(6) cells). Dipyridamole at 10 microM enhances [3H]MTX accumulation by Sarcoma 180 cells and diminishes the efflux of the drug in previously loaded cells. It is suggested that the combination of inhibitors of the de novo pathway for pyrimidine biosynthesis, such as MTX, with inhibitors of the salvage pathway, such as dipyridamole, may increase the cytotoxic activity of MTX alone.