Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and guinea pig antikeratin antisera.

Asch, Bonnie B.; Burstein, Nelson A.; Vidrich, Alda; Sun, Tung‐Tien

doi:10.1073/pnas.78.9.5643

Cited by 59 publications

(25 citation statements)

References 24 publications

(25 reference statements)

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“…Therefore, it also will be useful in defining the role and relationship of the various cytokeratins during myoepithelial and luminal epithelial development and differentiation. Precedence for such myoepithelium-specific keratins has been established in the mammary gland of rodents (12,13). However, the antibodies used in these studies do not crossreact with their human counterparts; and thus, they cannot be applied toward investigations of the human myoepithelium.…”

Section: Discussionmentioning

confidence: 99%

Monoclonal antibody that defines human myoepithelium.

Dairkee¹,

Blayney²,

Smith³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.Myoepithelial cells are derived from ectoderm and are known to exhibit both epithelial and mesenchymal characteristics (1, 2). They are situated between acinar or ductal luminal cells and the basal lamina in a number of secretory glands such as breast, lacrimal, eccrine, and apocrine sweat glands and various salivary glands (3).We report here on a monoclonal antibody directed toward a cytoskeletal keratin that is unique to the myoepithelial or "basal" layer of epithelial cells in human mammary ducts, sweat glands, and salivary glands. Using polyacrylamide gel electrophoresis and immunoblotting, we have found the apparent molecular weight of this basal epithelium-specific keratin to be 51,000 daltons. The antibody is generally nonreactive with luminal epithelium, stroma, muscle, fat, and vasculature. Therefore, it appears to be a useful tool in studying the role of the myoepithelium in the normal development of the breast, during lactation and involution, and in malignancy and metastasis.Recently, considerable advances have been made in tumor diagnosis by immunocytochemical typing of intermediate filaments. For example, carcinomas are characterized by antibodies to cytokeratins; sarcomas of muscle cells, by anti-desmin; nonmuscle sarcomas, by anti-vimentin; and gliomas, by anti-glial fibrillary acidic protein (for review, see ref. 4). The myoepithelium-specific monoclonal antibody described in this report has allowed us to define and characterize subclasses of human mammary carcinomas that would not be possible to distinguish by conventional techniques. The potential application of this antibody in the prognosis of human mammary carcinoma is discussed. MATERIALS AND METHODSImmunization. BALB/c mice raised under pathogen-free conditions were injected via subcutaneous or intravenous routes for 5 weeks at weekly intervals with whole-cell preparations of a secondary culture of an infiltrating ductal breast carcinoma. For each inoculation, 106 cells, which had been cultured as previously described (5), were prepared by scraping the monolayer with a rubber policeman into phosphate-buffered saline. Splenocytes from immunized animals were fused with SP2/0 mouse myeloma cells in the presence of polyethylene glycol (Mr 4000; Merck) by the procedure of Kohler and Milstein (6). Monocl...

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Section: Discussionmentioning

confidence: 99%

Monoclonal antibody that defines human myoepithelium.

Dairkee¹,

Blayney²,

Smith³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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“…I. Note that the 65-67,000-dalton keratin was detected only in the in vivo epidermis (lane I) but not in any cultured epithelial cells (lanes [2][3][4][5][6][7][8][9][10], and that the 58/56,000-dalton keratins were detected in all stratified squamous cells (lanes 2-6). The arrowhead on the right denotes the 66,000-dalton protein previously found in preparations of various types of intermediate filaments (43,64).…”

Section: A E 3 a N T I B O D Ymentioning

confidence: 99%

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies.

Nelson¹,

Sun²

1983

The Journal of Cell Biology

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338

169

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The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000-and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500-and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500-and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000-and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000-and 58,000-dalton keratin classes. These findings suggest that the 50,000-and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000-and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes.The keratins are a family of water-insoluble proteins that form tonofilaments (a class of intermediate-sized filament), which are present in almost all vertebrate epithelia (13,16,17,59,61,62). Since keratins are usually not detectable in nonepithelial tissues including fibroblasts, muscles, and nerves, immunohistochemical staining of keratins facilitates the detection and identification of epithelial cells, both in tissue section and in culture (1, 2, 7, 8, 24, 35, 37, 46-48, 54, 55, 69).Biochemical data indicate that the polypeptide composition of keratin filaments varies depending on epithelial cell type (12,14,15,18,21,26,39), cellular growth environment (3,12,20,22,23,32,53,60), histological differentiation stage (6, 9, 2 l, 49, 5 l, 56, 66, 67, 70), and embryonic development period (4, 11). It is therefore not surprising that many keratin species have been described in the literature. To study the biological significance of keratin heterogeneity, we have previously prepared three monoclonal antikeratin antibodies (designated A...

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“…Recently, antibodies to major components of the cytoskeleton, the intermediate-sized filaments (1), have been successfully used to distinguish different classes of tumors by nonmorphological criteria. For example, epithelia-derived tumor cells, including carcinomas, contain cytokeratin filaments whereas mesenchymally derived tumors such as sarcomas contain only filaments of the vimentin type (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). This immunocytochemical staining procedure has turned out to be valuable in clinical diagnosis (e.g., refs.…”

mentioning

confidence: 99%

“…Antibodies to determinants of a certain subgroup ofcytokeratins therefore will not react-with cells lacking these determinants in their specific type of cytokeratin filaments. This could give rise to "false negative" findings (5,7,10,13,15,18). Therefore, we have looked for a compositionally less variable protein common to epithelial cells that might serve as a conservative marker for epithelium-derived tumors.…”

mentioning

confidence: 99%

Immunocytochemical identification of epithelium-derived human tumors with antibodies to desmosomal plaque proteins.

Franke¹,

Moll²,

Mueller³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Epithelial cells contain desmosomes, special intercellular junctions providing sites of membrane attachment for intermediate-sized filaments of the cytokeratin type (tonofilaments). Such sites of anchorage of tonofilaments appear as dense plaques on the cytoplasmic side ofthe desmosomal membrane. We have isolated desmosome-enriched fractions from bovine snout epidermis and-tongue mucosa and have characterized the major protein associated with the desmosomal plaque. This protein occurs in equimolar amounts oftwo polypeptides OfMr 250,000 (desmoplakin I) and Mr 215,000 (desmoplakin II) which are chemically and immunologically related. Antibodies raised against desmoplakins allow the identification and localization of this protein in epithelial cells grown-in tissues or in vitro and show crossreaction in species as diverse as man, mouse, and chicken. Using immunolocalization at the light and electron microscope levels, we show that these antibodies bind specifically to desmosomal plaques. Antibodies to desmoplakins have been used successfully for detection of desmosomal proteins in a broad variety of epitheliumderived human tumors, including primary carcinomas and their metastases, irrespective of the morphology of the specific tumor. Nonepithelial tumors examined have been negative. We propose to use antibodies to desmoplakins and to cytokeratins in pathological diagnosis as two independent markers for the positive immunocytochemical identification and classification of epithelium derived tumors.

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Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and guinea pig antikeratin antisera.

Cited by 59 publications

References 24 publications

Monoclonal antibody that defines human myoepithelium.

Monoclonal antibody that defines human myoepithelium.

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies.

Immunocytochemical identification of epithelium-derived human tumors with antibodies to desmosomal plaque proteins.

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