RESUMEN
Mipp is a kelch-related, placental-specific gene that is ectopically expressed in many BALB/c mouse mammary carcinomas of various etiologies. The Kelch family encompasses proteins that are emerging as key links between microfilaments and a variety of cellular structures and functions. Mouse mammary tumors express two mipp transcripts (2.2 and 5.6 kb). We cloned the 2.2 kb mipp mRNA and analysed the product of its 1.7 kb ORF. The 584 residue MIPP protein has an N-terminal BTB domain and six C-terminal tandem Kelch repeats. Despite expression of two mipp RNAs, only a single MIPP protein is expressed in mammary tumors. MIPP protein binds to microfilaments in vitro and co-immunoprecipitates with actin. MIPP co-localized with concanavalin A at the endoplasmic reticulum, suggesting that MIPP might mediate interactions between microtubules and actin filaments. Because MIPP expression is widespread in mouse mammary tumors, it might contribute to tumorigenesis. Although MIPP had little effect on the growth rate of human breast cell lines following transfection, it greatly reduced the formation of duct-like structures on reconstituted basement membrane. Our results suggest that MIPP could contribute to malignant progression in the mouse mammary epithelial cells by perverting their response to cues from the extracellular matrix.
Asunto(s)
Actinas/metabolismo , Neoplasias Mamarias Animales/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/genética , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Clonación Molecular , Concanavalina A/farmacología , ADN Complementario/metabolismo , Retículo Endoplásmico/metabolismo , Matriz Extracelular/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Monoéster Fosfórico Hidrolasas/química , Pruebas de Precipitina , Estructura Terciaria de Proteína , Transfección , Células Tumorales CultivadasRESUMEN
The production of heritable changes in gene expression is the driving force in the development and progression of breast cancer. Such changes can result from mutations or from epigenetic events such as hypermethylation of DNA and hypoacetylation of histones. Histone acetylation and DNA methylation are major determinants of chromatin structure, and chromatin structure is a primary regulator of gene transcription. Cancer cells frequently contain both mutated genes and genes with altered expression due to one or more epigenetic mechanisms. This review describes the epigenetic changes that disrupt normal chromatin architecture and modify the expression of key genes in breast cancer cells. The structural integrity of the latter genes is usually intact, but their expression has been substantially altered due to methylation in their promoter region or deacetylation of histones that interact with their promoter region or both mechanisms. Genes affected by epigenetic changes in breast cancers include HoxA5, p21WAF, gelsolin, BRCA1, BRCA2, E-cadherin, steroid hormone receptors, and retinoic acid receptor II. Because these epigenetic modifications are usually reversible by treatment with certain drugs, they represent vulnerabilities in the cancer cell that can be exploited as novel targets for new prevention and therapeutic strategies.
Asunto(s)
Neoplasias de la Mama/genética , Cromatina/genética , Regulación Neoplásica de la Expresión Génica , Metilación de ADN , Femenino , Genoma , Inhibidores de Histona Desacetilasas , Humanos , Regiones Promotoras GenéticasRESUMEN
The actin cytoskeleton underlies several normal cellular functions and is deranged during carcinogenesis. Gelsolin, a multifunctional actin-binding protein, is downregulated in several types of tumors and its abnormal expression is one of the most common defects noted in invasive breast carcinoma (ICA). This study utilizes immunohistochemistry to examine the expression of gelsolin in 95 ICA, 59 ductal carcinoma in situ (DCIS) and 36 benign lesions, including 17 atypical ductal hyperplasia (ADH). Cytoplasmic staining was scored as positive, reduced or negative. Gelsolin expression was then correlated with patient's age, tumor size, histologic grade and lymph node status. All unremarkable breast biopsies, 88% of ADH, 44% of DCIS and 28% of ICA were positive for gelsolin. This represents a significant difference among the groups (p = < 0.0001) and the trend towards reduced gelsolin with the progression to ICA is significantly linear (p = < 0.0001). For invasive carcinoma, patients older than 44 years were significantly more likely to have decreased expression of gelsolin than patients 44 years old and younger (p = 0.007). Bivariate analysis showed no correlation of gelsolin expression with lymph node status (p = 0.62), tumor size (p = 0.10), histologic grade (p = 0.42), estrogen receptor status (p = 1.0) or other clinicopathologic parameters. In clinical follow-up, there were 18 breast tumor related deaths within a median follow-up time of 4.2 years. Survival analysis indicated that the level of gelsolin expression may be associated with survival (p = 0.06). In summary, the frequency of gelsolin deficiency increases significantly with progression from ADH to DCIS to ICA. Additionally, gelsolin expression may be an independent marker of prognosis.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Carcinoma Intraductal no Infiltrante/fisiopatología , Carcinoma/fisiopatología , Transformación Celular Neoplásica , Gelsolina/biosíntesis , Invasividad Neoplásica , Adulto , Anciano , Anciano de 80 o más Años , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Estudios de Seguimiento , Gelsolina/farmacología , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Lesiones Precancerosas , Pronóstico , Receptores de Estrógenos/análisis , Análisis de SupervivenciaRESUMEN
Gelsolin is an actin-binding/severing protein expressed in intracellular and secreted forms. It is a major regulator of the form and function of the actin cytoskeleton in most all cells. Here we demonstrate that female mice with a targeted deletion of the gelsolin gene (Gsn-/-) have defects in mammary gland morphogenesis. Two distinct defects were identified in the gelsolin-null mammary gland. First, the mammary anlage from Gsn-/- mice failed to elongate at the onset of puberty and remained rudimentary until approximately 9 weeks of age, early block (Gsn-/-(EB)). Second, after the mammary epithelium had filled the mammary fat pad, a complete lack of terminal branching, or late block, was observed (Gsn-/-(LB)). The Gsn-/-(EB) was seen in 70% of Gsn-/- mice and appeared to be dependent on a modifier gene(s) in addition to the loss of gelsolin. Gsn-/-(LB) was observed in all Gsn-/- mice. Terminal end buds (TEBs) were not evident in the mammary anlage from Gsn-/-(EB) mice until approximately 9 weeks of age. Cellular proliferation in the terminal ductal regions of Gsn-/-(EB) females was detected by bromodeoxyuridine incorporation, but was less than that found in the TEBs of age-matched controls. In mice deficient for gelsolin, mammary gland architecture was unaltered at the histological level. Lobuloalveolar development was delayed in response to pregnancy in mammary glands of Gsn-/- mice but was otherwise normal. Lactation and involution in the gelsolin-null animals were similar to those of wild-type mice. Transplantation of epithelium devoid of gelsolin into a wild-type (GsnWT) mammary fat pad resulted in proper arborization of the ductal tree. Transplantation of GsnWT epithelium into the Gsn-/- fat pad recapitulated the lack of terminal branching seen in Gsn-/- females. These results indicate that gelsolin is required in the mammary stroma for proper ductal morphogenesis. Our results provide the first evidence of an actin regulatory protein affecting mammary ductal growth through stromal-epithelial communication.
Asunto(s)
Gelsolina/metabolismo , Glándulas Mamarias Animales/embriología , Morfogénesis/fisiología , Tejido Adiposo/embriología , Animales , Cruzamientos Genéticos , Inducción Embrionaria , Epitelio/embriología , Epitelio/trasplante , Femenino , Gelsolina/deficiencia , Gelsolina/genética , Heterocigoto , Masculino , Glándulas Mamarias Animales/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Embarazo , Transducción de Señal , Células del Estroma/fisiología , Células del Estroma/trasplanteRESUMEN
Expression of gelsolin, an actin filament regulatory protein, in human breast ductal carcinoma in situ (DCIS) was analyzed by immunohistochemistry using a monoclonal antibody. Formalin-fixed paraffin-embedded tissues from 59 pure DCIS specimens and 33 DCIS specimens with associated invasive components were evaluated for gelsolin reactivity and compared to eight normal breast cases and 76 invasive breast cancers. The proportion of cases exhibiting negative/low expression of gelsolin in the epithelium was as follows -- normal, 0%; pure DCIS, 56%; DCIS associated with invasion, 58% in the DCIS component and 66% in the invasive component; invasive carcinoma, 70%. These data demonstrate that down-regulation of gelsolin expression in breast epithelium frequently parallels progression to malignancy. Testing gelsolin expression (normal vs. negative/low levels) in the DCIS lesions for associations with patient age or any of the following histopathologic parameters revealed no significant (95% probability level) correlations -- tumor size; pathologic (Van Nuys system) grade; nuclear grade; necrosis; presence of histologic calcifications; presence or type of adjacent benign lesions; architectural histologic pattern; and mammographic extent. Gelsolin loss was more commonly associated with mammographic soft tissue lesions as compared to calcified lesions (P = 0.009). A positive trend of borderline significance (P = 0.06) found in the DCIS with invasion group was a correlation between down-regulated gelsolin expression in the DCIS component and size (< versus > or = 15 mm) of the invasive tumor. In conclusion, reduced gelsolin protein is detectable in at least half of breast lesions which have progressed to DCIS. The trend between increasing gelsolin loss and malignant progression from normal epithelium to DCIS to invasive breast cancer (P < 0.0001) suggests additional investigation is needed to determine the potential of altered gelsolin expression as a marker for prognosis and for therapeutic interventions in breast cancer.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/genética , Carcinoma in Situ/genética , Carcinoma Ductal de Mama/genética , Gelsolina/biosíntesis , Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica/genética , Proteínas de Neoplasias/biosíntesis , Adulto , Anciano , Biomarcadores de Tumor/genética , Enfermedades de la Mama/genética , Enfermedades de la Mama/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma in Situ/clasificación , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Carcinoma Ductal de Mama/clasificación , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Progresión de la Enfermedad , Femenino , Gelsolina/genética , Humanos , Persona de Mediana Edad , Proteínas de Neoplasias/genéticaRESUMEN
Decreased gelsolin and increased cyclin D1 are among the most common defects found in human and rodent breast cancers. Our purpose was to determine the frequency of concurrence of these 2 alterations in this malignancy. Our results demonstrate that gelsolin protein and mRNA were significantly reduced in 80-100% of rodent mammary carcinomas that developed spontaneously, following oncogene introduction, or after treatment with viral, chemical or hormonal agents. The reduction in gelsolin most likely occurs during the transition from preneoplasia to carcinoma because hyperplasias had normal levels of gelsolin whereas microtumors had reduced expression. Southern analysis revealed no major mutations in the gelsolin gene of tumors with low expression. Cyclin D1 mRNA was increased in 50-100% of these rodent mammary tumors, although the cyclin D1 gene was not amplified. By nuclear runon assay, downregulation of gelsolin in both human and mouse mammary cancer cells involved diminished transcription and, conversely, human breast cancer cells expressing high levels of cyclin D1 had increased initiation of cyclin D1 transcription compared with cyclin D1 low expressors. Thus, alteration in the rate of transcription appears to be an important factor underlying the dysfunction of these genes. According to our data, concurrent deregulation of gelsolin and cyclin D1 is highly prevalent among breast cancers of humans and rodents, with both defects present in 89% of the neoplasms analyzed in this study. In fact, most tumors in every rodent model of mammary tumorigenesis examined had the 2 alterations.
Asunto(s)
Neoplasias de la Mama/genética , Ciclina D1/genética , Gelsolina/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Experimentales/genética , 9,10-Dimetil-1,2-benzantraceno , Animales , Antígenos Transformadores de Poliomavirus/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Regulación de la Expresión Génica , Humanos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Virus del Tumor Mamario del Ratón , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Embarazo , Biosíntesis de Proteínas , ARN Mensajero/genética , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Gelsolin is a multifunctional, actin-binding protein that is greatly decreased in many transformed cell lines and tumor tissues, including breast cancers. Downregulation of gelsolin RNA occurs in most breast cancers of rats, mice, and humans, but gross mutations of the gelsolin gene have not been found. Here we demonstrate by PCR and RT-PCR analysis that there are no point mutations in putative regulatory regions or the entire coding region of the cytoplasmic isoform of the gelsolin gene in human breast cancer cells (BCC). To determine if epigenetic modification is involved in downregulating gelsolin expression in MDA-MB-231 (MDA231), MCF7, and T47D BCC, we have used Southern blot analysis, 5-azacytidine (5aza) treatment, and trichostatin A (TSA) treatment. Southern blot analysis performed on genomic DNA demonstrated altered CpG methylation within intron 1 in DNA from all BCC compared to normal, mortal human mammary epithelial cells (HMEC). Treatment of the BCC with 5aza converted the DNA restriction pattern to that seen in untreated HMEC genomic DNA and caused modest increases in gelsolin RNA and protein. Incubation with TSA, an inhibitor of histone deacetylase, induced a dramatic upregulation of gelsolin RNA and protein levels which preceded apoptotic death of all BCC within 48-60 h. Our data support a role for epigenetic changes in chromatin structure leading to downregulation of gelsolin expression in human breast cancer. To our knowledge, this is the first example of a tumor suppressor gene downregulated in human breast cancer by changes in histone acetylation.
Asunto(s)
Gelsolina/biosíntesis , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Proteínas de Neoplasias/biosíntesis , Procesamiento Proteico-Postraduccional , Acetilación , Apoptosis , Azacitidina/farmacología , Southern Blotting , Mama/citología , Ciclo Celular , Células Cultivadas , Islas de CpG , Metilación de ADN , Análisis Mutacional de ADN , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Gelsolina/genética , Inhibidores de Histona Desacetilasas , Humanos , Ácidos Hidroxámicos/farmacología , Intrones/genética , Proteínas de Neoplasias/genética , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/química , Gelsolina/análisis , Neoplasias Mamarias Experimentales/química , Actinas/análisis , Animales , Mama/química , Gelsolina/genética , Humanos , Glándulas Mamarias Animales/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Fosfolipasa D/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Genes de Partícula A Intracisternal , Neoplasias Mamarias Experimentales/genética , Placenta/fisiología , Proteínas Gestacionales/genética , Animales , Secuencia de Bases , Northern Blotting , Femenino , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Especificidad de Órganos , Embarazo , Proteínas Gestacionales/biosíntesis , Secuencias Repetitivas de Ácidos Nucleicos , RetroelementosRESUMEN
Much of our knowledge about the intricate pathways and molecular mechanisms involved in the conversion of a normal mammary epithelial cell to malignancy derives from studies on mammary tumorigenesis induced by the retrovirus mouse mammary tumor virus. In addition, three DNA tumor viruses, simian virus 40, polyomavirus, and human papillomavirus, have been instrumental in dissecting the series of steps comprising neoplastic progression of mammary epithelium, particularly with cultured human breast cells. Endogenous transposons are analogous bioagents receiving increased attention recently. At least 10% of the cell genome consists of transposable elements, a growing number of which have been implicated in mutagenizing DNA in a variety of human tissues and disorders. Research efforts have therefore intensified to determine if endogenous elements such as retrotransposons participate in the development of breast cancer in animals and humans.
Asunto(s)
Neoplasias de la Mama/patología , Neoplasias de la Mama/virología , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/virología , Virus Oncogénicos , Retroelementos , Animales , Transformación Celular Neoplásica , Transformación Celular Viral , Femenino , Humanos , RatonesRESUMEN
Human LINE-1 (L1Hs) retrotransposons can act as insertional mutagens and are expressed in a variety of tumors, including breast cancer. The purpose of the present study was to examine expression of the p40 protein encoded by the first open reading frame of a L1Hs element in normal human breast tissue of patients without malignant breast disease and in nontumor breast tissue adjacent to cancer and to compare it to expression in breast carcinomas. An antiserum specific for the L1Hs p40 protein was used to analyze its expression in 5 reduction mammoplasties, 16 primary breast cancers, 1 lymph node metastasis and 13 non-malignant breast tissues adjacent to matched primaries by western blotting and/or immunocytochemistry. The immunoreactive band observed on westerns consistently had a M(r) of approximately 46 kDa. Westerns detected some p40 protein expression in all malignant and nonmalignant tissues examined, although 4 of 5 reduction mammoplasties had very low or trace levels as compared with tumors. Nonmalignant breast tissues adjacent to cancers showed significant western band reactivity, and all 15 tumors were positive. Immunocytochemistry revealed staining specificity of the antibody for epithelial cells. Of 12 invasive carcinomas examined, 100% were positive for the p40 protein, whereas one reduction with benign proliferative disease was very weakly reactive, two histologically normal reductions were negative, and 4 of 6 nonmalignant tissues adjacent to cancers were negative. Our data indicated that expression of the L1Hs p40 protein was often elevated in tumor cells of human breast cancers compared to epithelium of normal mammary gland.
Asunto(s)
Neoplasias de la Mama/genética , Mama/metabolismo , Carcinoma/genética , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/biosíntesis , Regulación de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Retroelementos/genética , Adulto , Anciano , Western Blotting , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , Persona de Mediana Edad , Mutagénesis Insercional , Proteínas de Neoplasias/genética , Teratocarcinoma/patologíaRESUMEN
Endogenous murine leukemia virus-related elements (MLVEs) are often overexpressed in primary mammary carcinomas of BALB/c mice. We therefore searched for mutations associated with MLVEs and found amplified sequences of the ecotropic MLVE in hormonally and chemically induced mammary neoplasms. Restriction fragment length polymorphism (RFLP) analysis revealed DNA rearrangements consistent with 1-10 or more new copies of the ecotropic MLVE in the genome of these tumors. This is the first evidence of mutations involving an endogenous retrovirus other than mouse mammary tumor virus in mouse mammary carcinomas.
Asunto(s)
Virus de la Leucemia Murina/genética , Neoplasias Mamarias Experimentales/virología , Mutación , 9,10-Dimetil-1,2-benzantraceno , Animales , Carcinógenos , Cocarcinogénesis , ADN de Neoplasias/genética , ADN Viral/genética , Femenino , Amplificación de Genes , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/virología , Neoplasias Mamarias Experimentales/inducido químicamente , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Neoplasias Hormono-Dependientes/virología , Polimorfismo de Longitud del Fragmento de Restricción , EmbarazoRESUMEN
Amplified expression of the endogenous retrotransposons, intracisternal A particles (IAPs) and murine leukemia virus-related elements (MLVEs), along with decreased expression of VL30 elements frequently occurs during mouse mammary tumorigenesis. We have now analyzed the expression of these retroelements during the normal developmental and differentiation cycle of the mammary gland as found in virgin, pregnant, lactating, and postlactation adult female BALB/c mice. Retrotransposon expression was either unchanged or decreased during the progressive stages of the cycle compared to virgin tissue. Likewise, growth of mammary epithelial cells in primary culture had little or no effect on expression of IAPs, MLVEs and VL30 sequences. Thus, the dramatic changes involving these retrotransposons in many mouse mammary tumors appear unrelated to any normal state.
Asunto(s)
Elementos Transponibles de ADN/genética , Expresión Génica/genética , Glándulas Mamarias Animales/crecimiento & desarrollo , Animales , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , ADN Complementario , Células Epiteliales , Femenino , Genes de Partícula A Intracisternal , Genes Virales/genética , Lactancia , Glándulas Mamarias Animales/citología , Ratones , Ratones Endogámicos BALB C , Embarazo , ARN/análisis , Retroviridae/genéticaRESUMEN
Of the several families of endogenous retrovirus-like elements present in the mouse genome, only mouse mammary tumor virus has been analyzed for its role in mammary carcinogenesis. Very little is known about the expression and activities of other retro-elements in normal and malignant mammary epithelium. We have begun investigating the possible involvement of the 3 retrotransposons, intracisternal A particles (IAPs), murine-leukemia-virus-related (MuLVr) elements, and VL30 sequences, in neoplastic progression of the mammary gland in BALB/c mice. The purpose of the present study was to determine which of these elements was active in primary mammary carcinomas induced by chemical, hormonal and viral agents. Each of these cancers had aberrant expression of at least one of the latter retrovirus-like components. IAP and/or MuLVr sequences were over-expressed 3 to 100-fold in most of the tumors as compared with normal mammary tissue, whereas VL30 expression was markedly decreased by 5- to 35-fold in almost all of the neoplasms. Our results thus demonstrate that substantial changes in the expression of one or more of these 3 families of endogenous retrotransposons are triggered during mouse mammary tumorigenesis, regardless of etiology. Direct involvement of IAPs and MuLVr elements in neoplastic progression by transposition and insertional mutagenesis in the genome of several hematopoietic cell types has already been demonstrated. Their elevated expression in many mammary carcinomas suggests that these retrotransposons may also be potential participants in some pathways of mouse mammary carcinogenesis.
Asunto(s)
Elementos Transponibles de ADN/fisiología , Neoplasias Mamarias Experimentales/genética , ARN Neoplásico/análisis , 9,10-Dimetil-1,2-benzantraceno , Animales , Northern Blotting , Femenino , Virus de la Leucemia Murina/genética , Glándulas Mamarias Animales , Neoplasias Mamarias Experimentales/etiología , Ratones , Ratones Endogámicos BALB CRESUMEN
Keratin polypeptides obtained from mouse mammary epithelial cells (MMEC) were found to be modified by covalent attachment of lipids. MMEC in primary culture were incubated in 3H-palmitate and treated with 1.5M KCl/1% Triton X-100 to obtain a cytoskeletal (CS) fraction containing primarily keratin and actin filaments. After exhaustive extraction to remove labeled lipids, the CS proteins were separated by gel electrophoresis, and the labeled 46 kD (K18) and 55 kD (K8) keratin polypeptides were excised, subjected to acid hydrolysis and the chloroform-soluble products were resolved on thin layer chromatography. For both keratins, covalently bound lipid included major peaks which co-chromatographed with fatty acid standards. Also, unlabeled lipid resolving with fatty acid standards was found covalently bound to both keratins. The results are discussed in terms of keratin-lipid-membrane interactions.
Asunto(s)
Queratinas/metabolismo , Metabolismo de los Lípidos , Glándulas Mamarias Animales/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Cromatografía en Capa Delgada , Proteínas del Citoesqueleto/metabolismo , Electroforesis en Gel de Poliacrilamida , Epitelio/metabolismo , Femenino , Glándulas Mamarias Animales/citología , Ratones , Ratones Endogámicos BALB C , Palmitatos/metabolismo , EmbarazoRESUMEN
Cytoskeletal proteins obtained from mouse mammary epithelial cells (MMEC) were found to be modified by covalent attachment of lipids. Primary cultures of MMEC were incubated in the presence of 3H-palmitate for 4 h. A cytoskeletal (CS) fraction was prepared by treatment of the cells with 1.5M KCl and 1% Triton X-100. The residual material, consisting primarily of keratin and actin filaments was exhaustively (10-20 rounds, including sonications) extracted with chloroform/methanol to remove non-covalently bound labeled lipids. The CS protein was then acid-hydrolyzed and the chloroform-soluble products subjected to thin layer chromatography (TLC). Two-thirds of the covalently bound radiolabel appeared as a very hydrophobic peak on a TLC system optimized for separation of neutral lipids. Ten percent separated into 4-5 peaks on a polar lipid TLC system. A small amount of label was traced to fatty acid-like components. Autoradiography of two-dimensional gels indicated that all the CS proteins resolvable by Coomassie blue staining were also radiolabeled. The results are discussed in terms of CS-lipid-membrane interactions.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Glándulas Mamarias Animales/metabolismo , Lípidos de la Membrana/metabolismo , Palmitatos/metabolismo , Animales , Células Cultivadas , Cromatografía en Capa Delgada , Proteínas del Citoesqueleto/química , Femenino , Ratones , Ratones Endogámicos BALB C , Palmitatos/química , Unión Proteica , TritioRESUMEN
Growth of normal and malignant mouse mammary epithelial cells (MMEC) on a biomatrix of substrate-attached material from 3T3-L1 preadipocytes was evaluated to devise culture conditions that are suitable for transformation studies but do not involve embedding cells in a gel. The biomatrix was prepared as described by Levine and Stockdale, and serum-free medium contained bovine serum albumin, insulin, progesterone, prolactin, and linoleic acid. Each cell type produced a distinctive pattern of colony architecture in this culture system. Cells from virgin mice (vMMEC) usually formed elaborate, three-dimensional structures resembling ducts and alveoli; cells from pregnant mice (pMMEC) grew as flat monolayers; and tumor cells grew in multilayered clusters. Cell growth was monitored by an assay for succinate dehydrogenase. Similar growth rates were found through Day 8 in cultures of vMMEC and D2 carcinoma cells. Growth of vMMEC slowed thereafter, whereas tumor cells typically continued growing through Day 14 to 18. Increase in cell number during 18 days in culture was 3-, 7-, 9-, and 11-fold for cells from pregnant and virgin mice, BALB/cfC3H and D2 carcinomas, respectively. The percent cells in S phase on Day 2 of culture was 9% for pMMEC, 4 to 11% for BALB/cfC3H tumor cells, 20% for vMMEC, and 24% for D2 tumor cells. Thus, this culture system promotes extended growth of MMEC and offers several advantages over embedding cells in a collagen gel. It may therefore be applicable to in vitro transformation studies with MMEC.
Asunto(s)
Glándulas Mamarias Animales/citología , Neoplasias Mamarias Experimentales/patología , Tejido Adiposo/citología , Animales , Membrana Basal/citología , Adhesión Celular , División Celular , Células Cultivadas , Células Epiteliales , Epitelio/metabolismo , Matriz Extracelular/fisiología , Proteínas de la Matriz Extracelular/fisiología , Queratinas/metabolismo , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Fase S , Factores de TiempoRESUMEN
Evidence presented previously indicated that shape changes in circulating cancer cells within the microvasculature may lead to rapid, lethal rupture of the cell surface membranes, thereby contributing to the inefficiency of this phase of hematogenous metastasis. As the cytoskeleton is known to regulate cell shape and, in at least some cases, to be attached to the surface membrane, we have determined whether or not it plays a role in inhibiting lethal, deformation-associated, mechanically induced surface membrane trauma. Thus, Ehrlich ascites tumor (EAT) cells were treated with different cytoskeleton-perturbing agents, and their susceptibility to filtration trauma on passage through Nuclepore membranes was determined. In contrast to the involvement of the cytoskeleton in nonlethal cell deformation, agents which disrupt intracellular networks of microtubules, microfilaments and intermediate filaments had little or no direct effect on EAT cell susceptibility to rapid, mechanically induced, lethal trauma.