RESUMO
The phylogeography of wild boars (WB) and domestic pigs (Sus scrofa) has contributed important insights into where and when domestication occurred. The geographic distribution of two core haplotypes (E1a and E1c) of the main European phylogenetic clade suggests that Central Europe was an early domestication centre, although the complexity of the pattern does not exclude the possibility that multiple domestication events occurred in different regions. To investigate the relationships among WB and domestic pig breeds in Iberia, a fragment of the mitochondrial DNA control region from a large sample (n=409) of WB and local pig breeds was co-analysed with published sequences from other European populations. The Iberian sample revealed a high frequency of a sub-cluster (E1c) of the European haplogroup E1 in 77% of total Iberian samples, 96% of WB, 90% of Alentejano (Portugal) and 87% of Iberian breed pigs (Spain; Black Hairy, Black Hairless and Red varieties). Low genetic distance (F'(ST) = 0.105) was observed between Alentejano (Portugal) and Iberian breed pigs (Spain). Alentejano and Iberian breed pigs showed low genetic distances to both Iberian and Central European WB (average F'(ST) =0.345 and 0.215, respectively). This pattern suggests that early pig husbandry in the Iberian Peninsula did not solely rely on imported Central European stock, but also included the recruitment of local WB.
Assuntos
Fluxo Gênico , Sus scrofa/genética , Suínos/genética , Animais , DNA Mitocondrial/genética , Variação Genética , Haplótipos , PortugalRESUMO
The sequence of heat shock-induced perturbations in protein synthesis and cytoskeletal organization was investigated in primary cultures of mouse mammary epithelial cells (MMEC). Exposure of the cells to 45 degrees C for 15 min caused a marked inhibition of protein synthesis through 2 h after heart. Resumption of protein synthesis began by 4 h, was complete by 8 h, and was accompanied by induction of four major heat shock proteins (HSPs) of 68, 70, 89, and 110 kD. Fluorescent cytochemistry studies indicated that heat shock elicited a reversible change in the organization of keratin filaments (KFs) and actin filaments but had a negligible effect on microtubules. Changes in the organization of KFs progressed gradually with maximal retraction and collapse into the perinuclear zone occurring at 1-2 h after heat followed by restoration to the fully extended state at 8 h. In contrast, actin filaments disappeared immediately after heat treatment and then rapidly returned within 30-60 min to their original appearance. The translocation of many organelles first into and then away from the juxtanuclear area along with the disruption and reformation of polyribosomes were concurrent with the sequential changes in distribution of KFs. The recovery of the arrangement of KFs coincided with but was independent of the resumption of protein synthesis and induction of HSPs. Thermotolerance could be induced in protein synthesis and KFs, but not in actin filaments, by a conditioning heat treatment. Neither protein synthesis nor induction of HSPs was necessary for the acquisition of thermotolerance in the KFs. The results are compatible with the possibility that protein synthesis may depend on the integrity of the KF network in MMEC. Heat shock thus can efficiently disarrange the KF system in a large population of epithelial cells, thereby facilitating studies on the functions of this cytoskeletal component.
Assuntos
Citoesqueleto/ultraestrutura , Proteínas de Choque Térmico/biossíntese , Temperatura Alta , Queratinas , Biossíntese de Proteínas , Citoesqueleto de Actina/ultraestrutura , Actinas , Animais , Núcleo Celular/ultraestrutura , Células Cultivadas , Colchicina/farmacologia , Cicloeximida/farmacologia , Citocalasina D , Citocalasinas/farmacologia , Citoesqueleto/efeitos dos fármacos , Imunofluorescência , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Organelas/fisiologia , Organelas/ultraestruturaRESUMO
The concanavalin A (Con A)-induced agglutinability of normal, preneoplastic, and neoplastic mouse mammary epithelial cells was examined. Cells freshly dissociated from normal mammary glands, hyperplastic alveolar nodules, or primary mammary adenocarcinomas by collagenase digestion in the presence of bovine serum albumin were strongly agglutinated by low concentrations of Con A. After short-term culture in vitro, however, cells from all three types of tissue were only weakly agglutinated by Con A, as measured by both suspension and hemadsorption assays. By comparison, cells of three established mammary tumor culture lines agglutinated strongly in the presence of the lectin. Treatment of the normal, preneoplastic, and neoplastic mammary cells in primary cultures with either trypsin or collagenase had little or no effect on their agglutinability, whereas hyaluronidase significantly increased their reactivity. Studies with fluorescein-tagged Con A indicated that all three cell types were capable of binding the lectin. The results were consistent with previous evidence suggesting that neoplastic transformation of mouse mammary epithelial cells is not manifested in vitro by several of the alterations in growth patterns, intercellular interactions, and surface properties that usually accompany transformation of fibroblastic cells.
Assuntos
Concanavalina A/farmacologia , Neoplasias Mamárias Experimentais/patologia , Lesões Pré-Cancerosas/patologia , Animais , Células Cultivadas , Feminino , Testes de Hemaglutinação , Hialuronoglucosaminidase/farmacologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Receptores de Concanavalina ARESUMO
The phenotypic heterogeneity of epithelial cells during hematogenous metastasis from primary mammary carcinomas of BALB/cfC3H mice was examined. Antisera to keratins were used in immunofluorescence assays to identify epithelial and myoepithelial cells. Both cell types were located in frozen sections of not only the primary lesions but also pulmonary metastases. Tumor cells disseminating to the lungs were recovered from the bloodstream of tumor-bearing mice with the use of a quantitative density gradient centrifugation procedure. Carcinoma cells were detected in the circulation of 62% of mice at the time of assay, but all tumors examined had released cells into the circulation at some point in their history. Epithelial cells and myoepithelial cells were detected simultaneously in 21% of mice, whereas other mice had only epithelial cells (16%) or myoepithelial cells (25%) in their circulation at the time of assay. Single cells and multicellular emboli of each cell type were disseminated, together with similar numbers of heterogeneous emboli containing both epithelial and myoepithelial cells. The evidence shows that the presence of both mammary epithelial and myoepithelial cells in pulmonary metastases from mouse mammary carcinomas can be accounted for by their hematogenous delivery as differentiated cells.
Assuntos
Neoplasias Mamárias Experimentais/patologia , Células Neoplásicas Circulantes , Animais , Epitélio/patologia , Feminino , Queratinas/análise , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , FenótipoRESUMO
Cell lines were established from 2 primary hepatocellular carcinomas (HC's) and 3 sarcomas produced in Syrian golden hamsters inoculated as newborns with chicken embryo lethal orphan (CELO) virus. Cell lines from 2 sarcomas (COT, CMT) and 1 HC (CEHEP) produced CELO virus-specific T-antigen. The antigen was not detected in cells of the third sarcoma line (RCT) until they had undergone more than 34 passages in vitro. Although 5-10% of cells in the second HC line (CILT/2) contained T-antigen during early passages, it was not demonstrable after the fifth subculture. Nevertheless, cells of both HC lines possessed CELO virus tumor-specific transplantation antigen. All 5 cell lines also contained hamster type R particles, and both HC lines had type C and intracytoplasmic type A particles. The percentage of carcinoma cells producing type R particles increased during cultivation in vitro, whereas the number of cells with type A particles decreased. Treatment with dibutyryl cyclic AMP and theophylline enhanced the number of cells producing type C particles in 1 HC line and type R particles in 2 sarcoma lines.
Assuntos
Infecções por Adenoviridae/etiologia , Adenoviridae/isolamento & purificação , Aviadenovirus/isolamento & purificação , Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Sarcoma Experimental/etiologia , Adolescente , Animais , Antígenos Virais , Aviadenovirus/imunologia , Bucladesina/farmacologia , Carcinoma Hepatocelular/ultraestrutura , Linhagem Celular , Cricetinae , Antígenos de Histocompatibilidade , Humanos , Corpos de Inclusão Viral , Neoplasias Hepáticas/ultraestrutura , Mesocricetus , Neoplasias Experimentais/etiologia , Replicação Viral/efeitos dos fármacosRESUMO
Normal, preneoplastic, and neoplastic primary cultures of mouse mammary epithelial cells were distinguishable on the basis of water proton nuclear magnetic resonance (NMR) relaxation times--i.e., spin-lattice relaxation time (T1) and spin-spin relaxation time (T2). T1 values were 916 +/- 24 msec for normal cells, 1,029 +/- 24 msec for preneoplastic cells, and 1,155 +/- 42 msec for neoplastic cells. This method of distinction between normal and neoplastic cells (P less than 0.001) and normal and preneoplastic cells (P less than 0.005) supported previous findings in whole tissues. NMR relaxation times resulted in better distinction between these cell populations than any other technique except direct histology. The T1 and T2 values of water protons in cells grown in primary culture were higher than those of established mouse mammary cancer cell lines. The differences in T1 and T2 did not correlate with cellular hydration. The data suggested a basic difference in water-macromolecular surface interactions among normal, preneoplastic, and neoplastic cells.
Assuntos
Água Corporal/metabolismo , Espectroscopia de Ressonância Magnética , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Animais , Células Cultivadas , Epitélio/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Fatores de TempoRESUMO
In order to develop new probes for investigating cellular alterations accompanying neoplastic progression of mouse mammary epithelium, rat monoclonal antibodies were prepared against cytoskeletal extracts of cultured normal and malignant mouse mammary epithelial cells. One of these antibodies, VG10, reacted in immunofluorescent staining with the majority of mouse mammary epithelial cells from hyperplastic alveolar nodule outgrowth lines of hormonal, chemical, and viral etiologies and the primary adenocarcinomas arising spontaneously from the nodules. Moreover, most mammary carcinomas developing in BALB/cfC3H, BALB/cV, and 7,12-dimethylbenzanthracene-treated BALB/c mice also had cells recognized by VG10. In contrast, normal mouse mammary epithelial cells from virgin and pregnant mice were unreactive. Immunoelectron microscopy identified the reactive component in the abnormal cells as intracisternal A-particles (IAPs), endogenous retrovirions with the ability to transpose in the mouse genome. Western blotting experiments established that the VG10 antibody is specific for the Mr 73,000 gag protein of IAPs. The results indicate that expression of IAPs is a common occurrence in the evolution of malignancy in mammary epithelium of BALB/c mice and may be induced during the transformation of normal cells to the preneoplastic stage. Expression of IAPs is therefore a candidate marker for a step in neoplastic progression in this system and has the potential for contributing to the carcinogenic process.
Assuntos
Anticorpos Monoclonais , Elementos de DNA Transponíveis , Expressão Gênica , Genes de Partícula A Intracisternal , Neoplasias Mamárias Experimentais/genética , Proto-Oncogenes , 9,10-Dimetil-1,2-benzantraceno , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Células Epiteliais , Feminino , Imunofluorescência , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Valores de ReferênciaRESUMO
Three monoclonal anti-keratin antibodies, AE1, AE3, and AE4, were used to compare the expression of keratins in normal, preneoplastic, and malignant mouse mammary epithelial cells growing in primary culture. In indirect immunofluorescence, AE1 did not stain normal cells but did stain a minority of preneoplastic and carcinoma cells. AE3 reacted with a subpopulation of epithelial cells in both the normal and abnormal cultures, except for certain cultures from one type of tumor wherein all of the epithelial cells were reactive. AE4 decorated an elaborate keratin filament network in all cultured mammary epithelial cells, regardless of neoplastic state. In double-label immunofluorescence, a guinea pig anti-keratin antiserum, which reacts preferentially with myoepithelial cells, exhibited coincident staining with AE1 in the tumor cultures and AE3 in the normal and most tumor cultures, indicating that the cells recognized by the antibodies in these populations were myoepithelial. Immunoblot experiments with cytoskeletal polypeptides extracted from the normal and tumor cells demonstrated that the set of keratins recognized by each monoclonal antibody was essentially the same in all of the cells except for a Mr 40,000 component that was present in normal cells but either absent or diminished in the cancer cells. Thus, while normal cells had Mr 40,000 and 50,000 keratins recognized by AE1, the epitope detected by this antibody was apparently concealed or "masked" in situ. AE3 reacted in immunoblots with a major keratin group (Mr 54,000-55,000) and a minor keratin (Mr 57,000), while AE4 reacted only with the Mr 54,000-55,000 keratin species. Because immunofluorescence with AE4 showed that the Mr 54,000-55,000 keratin group was present in all mammary epithelial cells, the AE3-reactive epitope must be masked in the majority of normal and tumor cells. The data therefore showed that epitopes on three major keratins, the Mr 40,000, 50,000, and 54,000-55,000 group, were "masked" in normal cells, whereas in tumor cells "masking" involved primarily the Mr 54,000-55,000 keratin. Attempts to "unmask" the epitopes recognized by AE1 in normal cells or to increase the number of cells reactive with AE3 in the normal and tumor cultures failed. Thus, certain cultured preneoplastic and neoplastic mammary cells with a myoepithelial phenotype have an altered organization of keratins that is manifested by a keratin antigenic determinant which is visible by immunocytochemistry in the abnormal cells but not in normal mouse mammary cells. This is the first demonstration that the immunoreactivity of keratins can be modified during neoplastic progression of epithelial cells.
Assuntos
Antígenos de Neoplasias/análise , Queratinas/imunologia , Glândulas Mamárias Animais/imunologia , Neoplasias Mamárias Experimentais/imunologia , Lesões Pré-Cancerosas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Epitélio/imunologia , Epitélio/metabolismo , Epitopos , Feminino , Imunofluorescência , Ponto Isoelétrico , Queratinas/metabolismo , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Peso Molecular , Lesões Pré-Cancerosas/metabolismoRESUMO
The keratins and other cytoskeletal proteins expressed by normal, preneoplastic, and malignant mammary tissues in BALB/c mice and by cells in primary cultures established from these tissues were analyzed and compared. The preneoplastic lesions were hyperplastic alveolar nodules (HAN) derived originally from mice treated by hormonal stimulation (D2), exposed to a chemical carcinogen (C4), or spontaneously expressing mouse mammary tumor virus (CV2) and maintained by serial transplantation. All tumors were mammary adenocarcinomas which developed as primary neoplasms from the HAN outgrowth lines. Cytoskeletal extracts were prepared from the tissues and cultured cells and subjected to two-dimensional polyacrylamide gel electrophoresis. Comparison of the major polypeptides in the normal and abnormal tissue extracts revealed considerable similarities in the cytoskeletal profiles. Three basic and seven acidic polypeptides ranging in molecular weight from 40,000 to 90,000 were regularly identified. However, notable differences were also found. A Mr 55,000 keratin (IEF 55) was prominent in one HAN, the D2, and all tumor tissues but not in normal gland. Likewise, a Mr 46,000 polypeptide (IEF 46), which has been tentatively identified previously as a keratin, was absent in normal epithelium but present in all abnormal tissues except the C4 and CV2 HAN. A Mr 58,000 polypeptide (NEPHGE 58) was not detected in normal gland or the C4 lesions but was found in all other abnormal tissues. The overall pattern of polypeptides in cytoskeletal extracts from normal and abnormal mammary cells in primary culture resembled that of the corresponding tissue but also had important differences. In all cell cultures, IEF 46 and IEF 55 were major species, while the larger and more basic components were markedly reduced. A Mr 56,000 polypeptide (NEPHGE 56) was detected only in C4 HAN and C4 and CV2 tumor cells. Trace or small, variable amounts of a Mr 57,000 basic keratin (NEPHGE 57) were present in normal and D2 tissues and cultured cells. NEPHGE 57 was dramatically increased in C4 and CV2 tissues and cultured cells and may be related to expression of squamous metaplasia and keratinization which are characteristic of these lesions. Although production of IEF 46 and IEF 55 may be associated with neoplastic progression of mammary epithelium, particularly in vivo, the association is not exclusive since normal cells express these polypeptides when grown in primary culture. In addition, correlations between altered keratin expression and the mode of induction of the mammary lesions were not obvious.
Assuntos
Adenocarcinoma/análise , Queratinas/análise , Glândulas Mamárias Animais/análise , Neoplasias Mamárias Experimentais/análise , Animais , Citoesqueleto/análise , Eletroforese em Gel de Poliacrilamida , Feminino , Hiperplasia , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Peso MolecularRESUMO
Previous studies have demonstrated that normal and malignant mouse mammary cells are indistinguishable in many surface-related properties that often denote transformation of other cell types such as fibroblasts. In the present investigation, the interactions of normal, dysplastic, and malignant mammary epithelial cells with fibronectin in tissues and cultures were examined by indirect immunofluorescence. Cells lining the lumina of ducts and alveoli in normal and dysplastic mouse and human mammary tissues abutted a layer of fibronectin along their basal surfaces that included the region of the basement membrane and the underlying stroma. Moreover, double staining for keratin and fibronectin revealed that myoepithelial cells were surrounded by the matrix protein. In contrast, tumor cells in adenocarcinomas and ductal carcinomas were not directly associated with fibronectin. The accumulation of fibronectin in primary cultures prepared from mouse mammary tissues paralleled the distribution seen in vivo. A matrix of fibronectin formed beneath normal and preneoplastic mammary cells within 4 to 6 days after plating, whereas tumor cells were negative, regardless of the age or density of the culture. This correlation with in vivo results did not extend to cells of established mammary tumor culture lines which readily accumulated pericellular networks of fibronectin. Addition of exogenous fibronectin to primary cultures enhanced formation of a basal matrix by normal cells but had no effect on the negative status of the tumor cells. The results indicate that mammary tumor cells in tissues and in primary cultures have an altered capacity to interact with fibronectin. However, this alteration is not necessarily expressed by established mammary tumor cell lines.
Assuntos
Neoplasias da Mama/metabolismo , Fibronectinas/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Feminino , Imunofluorescência , Histocitoquímica , Humanos , Camundongos , Fotomicrografia , GravidezRESUMO
Epithelial cell cultures of normal mammary gland, preneoplastic hyperplastic nodule outgrowth lines, primary tumors, and transplanted tumors, all derived from BALB/c mice, were examined for their response to cytochalasin B to determine if cells of primary mammary tumors multinucleated and if preneoplastic hyperplastic alveolar nodule cells responded differently than cells of primary tumors. Established tumorigenic and nontumorigenic cell lines were also examined as positive and negative controls. The standard assay conditions were optimized at 1 microgram CB per ml for 48 hr. The results, expressed as the mean percentage of cells exhibiting three or more nuclei per cell were: normal mammary cells, 5%; preneoplastic mammary cells, 4%; primary mammary tumor cells, 36%; transplanted mammary tumors, 70%; tumorigenic established cell lines, 80%; and nontumorigenic established cell lines, 5%. The frequency of tumor cells exhibiting multinucleation increased with serial transplantation in vivo and with serial passage in vitro. The results demonstrate that neoplastic cells within a primary tumor exhibit uncontrolled nuclear division and that uncontrolled nuclear division is a distinguishing characteristic between preneoplastic and neoplastic mammary cells.
Assuntos
Núcleo Celular/efeitos dos fármacos , Citocalasina B/farmacologia , Neoplasias Mamárias Experimentais/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Animais , Linhagem Celular , Núcleo Celular/ultraestrutura , Feminino , Técnicas In Vitro , Neoplasias Mamárias Experimentais/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Lesões Pré-Cancerosas/ultraestruturaRESUMO
The distribution and organization of microtubules (MT's) and actin-containing microfilaments (MF's) were examined in epithelial cells of primary cultures established from normal, preneoplastic, and neoplastic mouse mammary tissues and in cells of three clonal culture lines derived from murine mammary adenocarcinomas. No consistent differences in the cytoskeletal components were found among cell populations of the primary cultures as revealed either by indirect immunofluorescence using antibodies to tubulin and actin or by electron microscopy. Overall, the majority of cells in the three types of primary cultures possessed elaborate complexes of MT's and actin filaments after fluorescent staining with the appropriate antibodies, and abundant MT's and MF's were found in the cells at the ultrastructural level. Similar patterns of MT's and MF's were observed in cells of two of the clonal mammary tumor lines. Cells of the third line, however, exhibited intricate networks of MT's but had a reduction in actin cables detectable by the immunofluorescence procedure. Moreover, MF's were difficult to locate by electron microscopy. The results suggest that the lesion(s) in growth control in the neoplastic mammary cells may not involve any gross alterations in MT's or MF's.
Assuntos
Citoplasma/ultraestrutura , Citoesqueleto/ultraestrutura , Glândulas Mamárias Animais/ultraestrutura , Neoplasias Mamárias Experimentais/ultraestrutura , Microtúbulos/ultraestrutura , Lesões Pré-Cancerosas/ultraestrutura , Actinas/metabolismo , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Epitélio/ultraestrutura , Feminino , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Lesões Pré-Cancerosas/metabolismoRESUMO
Previous electrophoretic analysis has indicated that keratins 13 and 16 (K13 and K16) are not present in normal human breast epithelium, but K16 is expressed in some ductal carcinomas (Moll et al., Cell, 31: 11-24, 1982). K16 may thus represent a marker for identifying a subset of ductal carcinomas and for distinguishing such tumor cells from normal cells. To explore this possibility further, we have used the monoclonal antibody Ks8.12, reportedly specific for K13 and K16 (Huszar et al., Differentiation, 31: 141-153, 1986), to examine keratin expression in human breast tissues. In immunocytochemistry the antibody reacted with epithelial cells of all normal samples, hyperplasias, and fibroadenomas. The staining was moderate to strong and always heterogeneous, involving most but not all luminal cells of ducts and acini. Myoepithelial cells were never stained. Of twenty-one ductal carcinomas examined, 90% gave a very weak or no reaction. The remaining 10% of cancers exhibited moderate to strong staining in about 50% of tumor cells. Cytoskeletal polypeptides extracted from the tissues and separated by polyacrylamide gel electrophoresis were analyzed by Western blotting to identify the polypeptides recognized by Ks8.12. In samples from normal tissue and a fibroadenoma, the antibody recognized a major Mr 48,000 component, a size appropriate for K16. In extracts from a hyperplasia and two of four carcinomas, the antibody detected a Mr 54,000 polypeptide, as well as a Mr 48,000 band, properties consistent with K13 and K16, respectively. Our results provide the first evidence indicating that K16 is present in normal as well as abnormal human breast epithelium. In addition, the data suggest that K13 may be expressed in some benign lesions and ductal carcinomas of the breast. Accordingly, Ks8.12 may prove to be useful for subclassifying ductal carcinomas and for discriminating between normal and certain benign and malignant disorders of human mammary epithelium.
Assuntos
Anticorpos Monoclonais , Doenças Mamárias/metabolismo , Neoplasias da Mama/metabolismo , Mama/metabolismo , Queratinas/genética , Western Blotting , Epitélio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Queratinas/biossíntese , Peso MolecularRESUMO
Mammary tumorigenesis was surveyed in retired breeding females in four sublines of the C3H strain: in standard milk-transmitted early oncogenic mouse mammary tumor virus (MMTV)-infected C3H/He and C3H/Ki mice, and in standard milk-transmitted early oncogenic MMTV free C3Hf/He and C3Hf/Ki mice. All of 58 C3H/Ki mice and 98% (306 of 309) of the C3H/He mice developed palpable mammary tumors at average ages of 276 and 284 days, respectively. Thirty-one % (47 of 152) of the C3Hf/Ki mice and 77% (168 of 218) of the C3Hf/He mice developed palpable mammary tumors at average ages of 798 and 757 days, respectively. The mammary tumors removed from C3H/He and C3H/Ki mice were all adenocarcinomas of epithelial origin, and all contained MMTV. The mammary tumors removed from C3Hf/He and C3Hf/Ki mice were either adenocarcinomas or sarcomas. The carcinomas were of epithelial origin and all expressed the late oncogenic endogenous MMTV. The sarcomas were of histiocyte or fibrocyte origin and contained neither virus particles nor MMTV antigenic markers. It is concluded that exogenous standard milk-transmitted oncogenic MMTV oncogenesis in C3H mice is not modified by host genetic factors. In contrast, late oncogenic endogenous MMTV oncogenesis is influenced both by host genetic control of the expression of the late oncogenic MMTV provirus and by the location of the proviral genes in the germline DNA.
Assuntos
Neoplasias Mamárias Experimentais/etiologia , Vírus do Tumor Mamário do Camundongo/isolamento & purificação , Adenocarcinoma/etiologia , Adenocarcinoma/patologia , Animais , Modelos Animais de Doenças , Feminino , Genes Virais , Queratinas/análise , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Endogâmicos C3H , Sarcoma/etiologia , Sarcoma/patologia , Vimentina/análiseRESUMO
The effects of selenium on three mammary epithelial cell lines (YN-4, WAZ-2t, and CL-S1) grown in vitro were examined by immunocytochemical and transmission electron microscopy technique. The primary effect of selenium at the ultrastructural level was the appearance of electron-dense inclusions within the mitochondrial matrix. The mitochondrial inclusions were seen in all three cell lines, although most readily induced in YN-4 cells, the cell line which is most sensitive to selenium-mediated growth inhibition. Selenium at 5 x 10(-8) and 5 x 10(-6) M did not alter cytoplasmic microtubules or intermediate filament networks, as determined by immunocytochemical staining. Immunocytochemical staining for cytoplasmic filaments and microtubules, and transmission electron microscopy observations, supported the contention that cells from all three cell lines were epithelial in origin, since they contained abundant desmosomes and were uniformly positive for keratin intermediate filaments. Whereas line YN-4 was negative for vimentin intermediate filaments, a minority (5 to 24%) of the cells in lines CL-S1 and WAZ-2t stained positively. In addition, the tumorigenicity of these three cell lines was assessed by in vitro growth assays and in vivo transplantation assays. Cell lines YN-4 and WAZ-2t, but not line CL-S1, were tumorigenic in syngeneic mice. All tumors were mammary adenocarcinomas. Cytochalasin B-induced multinucleation assay and growth as multicellular spheroides correlated positively with in vivo tumorigenicity, whereas saturation density and growth in low Ca2+ medium were not correlated with tumorigenicity. It is speculated that one of the early effects of selenium-mediated growth inhibition may be a modulation of mitochondrial function.
Assuntos
Glândulas Mamárias Animais/efeitos dos fármacos , Selênio/farmacologia , Animais , Linhagem Celular , Citoesqueleto/ultraestrutura , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/ultraestrutura , Imunofluorescência , Corpos de Inclusão/ultraestrutura , Queratinas/metabolismo , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/ultraestrutura , Camundongos , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Mitocôndrias/ultraestruturaRESUMO
We previously demonstrated that epithelial cells growing in primary cultures derived from normal and neoplastic mouse mammary tissues are indistinguishable on the basis of morphology, surface topography, agglutinability by concanavalin A, or of number, cytoplasmic location, or organization of microtubules and actin-containing microfilaments. In the present study, the modulation and interrelationships of these features were investigated using the drugs colchicine and cytochalasin B and the enzymes trypsin, collagenase, and hyaluronidase. Untreated cells of both types were only weakly agglutinable by concanavalin A. Pretreatment of the cells with colchicine, cytochalasin B, or hyaluronidase increased cytoagglutination by the lectin dramatically, whereas neither trypsin nor collagenase significantly altered reactivity of the cells. Binding studies utilizing fluorescein-tagged concanavalin A indicated that the enhanced agglutinability did not correlate with any particular distribution of lectin receptors on the cells. At the doses used, colchicine and cytocholasin B induced disruption of microtubules and microfilament networks, respectively, but no effects on either type of cytoskeletal element were observed after hyaluronidase, trypsin, or collagenase. The increased agglutinability was not associated with any specific surface conformation since scanning electron microscopy revealed that untreated cells and cells exposed to colchicine, hyaluronidase, and collagenase were flat and possessed variable numbers of small microvilli. In contrast, following incubation with cytochalasin B or trypsin, the cells underwent retraction and rounding, and developed complex surface structures such as blebs, ruffles, and filopodia. Thus, no single factor appeared responsible for determining the agglutinable or nonagglutinable state of the mammary cells. Morover, no differences were detected in the surface responses of the normal and malignant cells under any of the conditions used.
Assuntos
Neoplasias Mamárias Experimentais/ultraestrutura , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Células Cultivadas , Colchicina/farmacologia , Concanavalina A/farmacologia , Citocalasina B/farmacologia , Feminino , Hemadsorção , Hialuronoglucosaminidase/farmacologia , Camundongos , Colagenase Microbiana/farmacologia , Microscopia Eletrônica de Varredura , Receptores de Concanavalina A/metabolismo , Propriedades de Superfície , Tripsina/farmacologiaRESUMO
125I-radiolabeled guinea pig fibrinogen was used to measure the influx (20 min) and accumulation (18 h) of fibrinogen/fibrin in three transplantable carcinomas (Lewis lung, TA3/St mammary, and MOT ovarian) growing in the subcutaneous space of syngeneic mice. Fibrinogen influx and, to an even greater extent, fibrin accumulation were substantially increased in all three tumors, as compared with normal control tissues. A significantly larger fraction of tumor-associated than control tissue radioactivity was insoluble in 3 M urea, a property of cross-linked fibrin. Positive identification of cross-linked fibrin was made by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of tumor extracts. Tumor fibrin deposits were localized by immunoperoxidase staining of tissue sections. Fibrin accumulation was also significantly increased in premalignant hyperplastic alveolar nodules that had been transplanted to cleared mammary fat pads, as compared with normal mammary tissue, and was further increased in primary mammary carcinomas that arose from hyperplastic alveolar nodules. These findings generalize to the mouse the principles that tumor vessels are hyperpermeable to plasma proteins and that fibrin accumulates in transplantable and primary tumors. Further, they demonstrate that tumor fibrin is cross-linked and therefore analogous to the fibrin deposited in thrombi, wounds, and cellular immunity.
Assuntos
Carcinoma/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Lesões Pré-Cancerosas/metabolismo , Animais , Carcinoma/irrigação sanguínea , Eletroforese em Gel de Poliacrilamida , Técnicas Imunoenzimáticas , Neoplasias Mamárias Experimentais/irrigação sanguínea , Camundongos , Transplante de Neoplasias , Lesões Pré-Cancerosas/irrigação sanguíneaRESUMO
Mammary epithelial cells from 4-month-old virgin BALB/c mice were cultured inside collagen gels in the following serum-free media: Dulbecco's modified Eagle's medium:Hams's F-12 (1:1) supplemented with: insulin (10 micrograms/ml), bovine serum albumin (5 mg/ml), and epidermal growth factor (5 ng/ml); insulin, bovine serum albumin, progesterone (0.05 microgram/ml), and prolactin (1 microgram/ml); insulin, bovine serum albumin, progesterone, prolactin, and linoleic acid (10 micrograms/ml). Cells proliferated in all these media. The cells were treated with 0.01 micrograms/ml of 7,12-dimethylbenz(a)anthracene or 100 micrograms/ml of N-nitroso-N-methylurea on day 3 of culture and, subsequently, at 1-week intervals for 3-6 weeks. Tetradecanoylphorbol acetate (0.1 micrograms/ml) was added to selected cultures. The cultures were maintained for up to 9 weeks; the cells were then removed from the collagen gels, placed in monolayer culture for 2 days, and removed from monolayer culture, and 5 X 10(5) cells were transplanted to each of the gland-free mammary fat pads of 3-week-old female mice. Approximately 10 weeks after transplantation, the transplanted mammary fat pads were examined for outgrowths. Cells that were not treated with carcinogen and cultured for up to 9 weeks in different serum-free media and transplanted to the gland-free mammary fat pad produced only ductal outgrowths similar in morphology to the ducts of the virgin host's mammary glands. Six treatments with 7,12-dimethylbenz(a)anthracene, of cells grown in the presence of epidermal growth factor, induced 31% spindle cell tumors, 17% ductal hyperplasias, and 5% lobuloalveolar hyperplasias. Cells that were grown in epidermal growth factor and treated three times with N-nitroso-N-methylurea produced 23% ductal hyperplasias and 17% lobuloalveolar hyperplasias. Cells grown in the presence of progesterone and prolactin and treated three times with 7,12-dimethylbenz(a)anthracene produced up to 23% lobuloalveolar hyperplasias and 12% ductal hyperplasias. Three treatments with N-nitroso-N-methylurea of cells grown in progesterone- and prolactin-containing media produced a maximum of 50% lobuloalveolar hyperplasias and 33% ductal hyperplasias. The lobuloalveolar hyperplasias have the characteristics of the precancerous hyperplastic alveolar nodules found in mouse mammary tumorigenesis. The in vitro carcinogen-induced lobuloalveolar hyperplasias were transplantable, maintained their lobuloalveolar morphology in virgin hosts, and produced carcinomas.
Assuntos
Transformação Celular Neoplásica , Colágeno/farmacologia , Glândulas Mamárias Animais/patologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Fenômenos Fisiológicos Sanguíneos , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Epitélio/patologia , Feminino , Géis , Hiperplasia , Glândulas Mamárias Animais/transplante , Neoplasias Mamárias Experimentais/patologia , Metilnitrosoureia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Down-regulation of gelsolin, an actin-binding protein, is frequently found in several types of transformed cells and tumors. The present study demonstrates that gelsolin protein and RNA were absent or markedly reduced in human breast cancer cell lines relative to "normal" mortal human mammary epithelial cells and benign, immortalized cell lines. Moreover, actin filaments were usually attenuated coincident with the reduction in gelsolin. Gelsolin was also missing or greatly decreased in 70% of 30 human sporadic, invasive breast carcinomas examined by immunocytochemistry and in 100% of virally induced mouse and chemically induced rat mammary carcinomas evaluated by Northern analysis. Southern analysis revealed no major mutations in the gelsolin gene of human breast cancer cells. Our results show that partial or total loss of gelsolin expression is common to the majority of breast cancers of diverse etiologies in three animal species and point to gelsolin as a candidate suppressor of breast cancer.
Assuntos
Neoplasias da Mama/química , Gelsolina/análise , Neoplasias Mamárias Experimentais/química , Actinas/análise , Animais , Mama/química , Gelsolina/genética , Humanos , Glândulas Mamárias Animais/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Fosfolipase D/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Células Tumorais CultivadasRESUMO
A placenta-specific gene, MIPP, is transcriptionally regulated in BALB/c mice by a solo long terminal repeat (LTR) of an intracisternal A-particle (IAP), an endogenous retrotransposon. Expression of IAPs, which is also promoted by LTR sequences, is a frequent aberration in many mouse mammary tumors of BALB/c mice. Given that these retroelements and the placental gene have a common promoter, we hypothesized that the tumors also express the gene. Northern blot analysis and RT-PCR revealed high expression of the placenta-specific gene in BALB/c mouse mammary preneoplasias and carcinomas of diverse etiologies, but not in normal mammary gland from virgin, pregnant and lactating mice. The preneoplasias and tumors expressed two transcripts, one of which is apparently unique to the mammary lesions. The other transcript is the same as one expressed in placenta that is not promoted by the IAP LTR. Despite the parallel expression of the placental gene and IAPs in the mammary tissues, RT-PCR showed that LTR sequences are absent from tumor-associated MIPP transcripts. Southern analysis revealed no gross mutations of the MIPP gene in mammary preneoplasias and tumors. The ectopic expression of the placenta-specific gene in BALB/c mouse mammary preneoplasias and carcinomas raises the possibility that it acts as an oncogene.