Flow-cytometric analysis of mixed cell populations using intermediate filament antibodies

Ramaekers, Frans C.S.; Beck, H. L. M.; Vooijs, G. P.; Herman, Chester J.

doi:10.1016/0014-4827(84)90467-1

Cited by 36 publications

(9 citation statements)

References 10 publications

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“…The decreases in cytokeratin polypeptides in ex vivo tumor cells can not be explained by masking of epitopes or as being a result of immunohistochemical artifacts, which establishes that cytokeratin levels indeed decrease during tumor transformation of breast epithelium. Cytokeratins are used as markers to identify breast carcinoma cells in various situations such as flow cytometry analyses of DNA ploidy [19,201, and detection of epithelial tumor cells that have metastasized to secondary sites such as the bone marrow [21, 221. Decrease or loss of cytokeratins in carcinomas can potentially be a problem in these types of studies, since the most malignant cells may escape detection.…”

Section: Discussionmentioning

confidence: 99%

Analysis of polypeptide expression in benign and malignant human breast lesions

et al. 1997

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Results of two-dimensional electrophoresis (2-DE) analyses of human breast carcinoma are described. Tumor cells were extracted and purified from breast carcinomas with different proliferative indeces and degrees of genomic stability. Cells purified from fibroadenoma tissue served as controls for benign cells. The following results were observed: (i) Analysis of samples from different areas of the same tumor showed a high degree of similarity in the pattern of polypeptide expression. Similarly, analysis of two tumors and their metastases revealed similar 2-DE profiles. (ii) In contrast, large variations were observed between different lesions with comparable histological characteristics. Larger differences in polypeptide expression were observed between potentially highly malignant carcinomas compared to comparisons of less malignant lesions. These differences were in the same order of magnitude as those observed comparing a breast carcinoma to a lung carcinoma. (iii) The levels of all cytokeratin forms resolved (CK7, CK8, CK15, and CK18) were significantly lower in carcinomas compared to fibroadenomas. (iv) The levels of high molecular weight tropomyosins (1-3) were lower in carcinomas compared to fibroadenomas. The expression of tropomyosin-1 was found to be 1.7-fold higher in primary tumors with metastatic spread to axillar lymph nodes compared to primary tumors with no evidence of metastasis (p < 0.05). (v) The expression of proliferating cell nuclear antigen (PCNA) and some members of the stress protein family (pHSP60, HSP90, and calreticulin) were higher in carcinomas. We conclude that malignant progression of breast carcinomas results in large heterogeneity in polypeptide expression between different tumors, but that some common themes such as decreased expression of cytokeratin and tropomyosin polypeptides can be discerned.

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Section: Discussionmentioning

confidence: 99%

Analysis of polypeptide expression in benign and malignant human breast lesions

et al. 1997

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“…The cell line was derived from a human squamous cell carcinoma of the tongue (Easty et al, 1981) For cytokeratin analysis ethanol fixed cells were labeled (30 min) with K-40, which is a broadly cross-reacting polyclonal antibody for cytokeratins as described elsewhere (Ramaekers et al, 1984;Bijman et al, 1986) or with 4 different monoclonal antibodies i.e., RCK-102, RCK-105, RKSE-60, and RCK-106, analysing cytokeratins 5+8, 7, 10, and 18 respectively (Ramaekers et al, 1983). For classification see Moll et al (1982).…”

Section: Chemicalsmentioning

confidence: 99%

Modulation of placental alkaline phosphatase activity and cytokeratins in human HN-1 cells by butyrate, retinoic acid, catecholamines and histamine

Bijman

Wagener²,

Rennes³

et al. 1987

Br J Cancer

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Summary The effects of butyrate and retinoic acid in combination with catecholamines or histamine on the HN-1 human head and neck squamous carcinoma cell line were investigated analysing cell proliferation, placental alkaline phosphatase (PLAP) activity, and relative cytokeratin content.Butyrate inhibited cell proliferation in agar, whereas retinoic acid induced a small inhibitory effect. Butyrate enhanced PLAP activity in a time related manner in contrast to retinoic acid, which had no significant effect. However, retinoic acid inhibited the efficacy of butyrate to induce PLAP activity. A synergistic enhancement of PLAP activity was demonstrated after treatment of butyrate pretreated cells with catecholamines or histamine. The f,-adrenergic antagonist propranolol partly inhibited the aforementioned enhancement of PLAP activity, whereas the x-adrenergic antagonist phentolamine further enhanced PLAP activity. Indirect labeling of keratins with a polyclonal antibody showed that cytokeratin content was enhanced by butyrate but not by retinoic acid. Further analysis of cytokeratin content using four monoclonal antibodies showed that labeling of cytokeratins (5+8) As is shown in numerous reports, butyrate, retinoic acid, interferon, glucocorticoids, polar solvents, and certain chemotherapeutic agents can modulate terminal differentiation (Augeron & Laboisse, 1984;Burke, 1986;Jetten, 1984;Reiss et al., 1985;Sartorelli, 1985), accompanied by inhibition of proliferation (Pierce & Wallace, 1971;Prasad & Sinha, 1976), morphological changes (Abe & Kufe, 1984;Kamech et al., 1986), and an increase in the production of specific antigens and enzymes (Reese & Politano, 1981;Morita et al., 1982;Abe & Kufe, 1984;Reese et al., 1985), such as CEA and alkaline phosphatase. These effects were in most cases reversible, although some authors claimed a more permanently affected cell progeny after treatment (Augeron & Laboisse, 1984;Reiss et al., 1985). It was indicated that for example butyrate and retinoic acid provided a trigger mechanism, which if properly administered to a cell would eventually lead to a terminally matured tumour cell.Recently it was shown that cAMP-elevating agents like catecholamines, potentiated the action of retinoic acid in eliciting normal epithelial cell differentiation (Schiff & Moore, 1985). Catecholamines have other intriguing effects on cells, including regulation of cell division (Kennedy et al., 1985;Tutton & Barkla, 1980), induction of alkaline phosphatase (Mary & Rao, 1981), and reduction of the total number of EGF receptors (Cruise et al., 1986). In the light of the increased number of EGF receptors on malignant squamous cells the aforementioned effect might be very promising (Cowley et al., 1986).The present study, using HN-1 squamous carcinoma cells, demonstrated that butyrate inhibited clonogenicity in soft agar, enhanced placental alkaline phosphatase (PLAP) activity, and increased relative cytokeratin content, in contrast to retinoic acid. Retinoic acid, however, inhibited the enhancement of PL...

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“…During in vitro aging, physiological cell parameters are modified such that there are: a) a gradual decline in proliferative capacity, a lengthened interphase interval, and a reduction of the number of cells actively synthesizing DNA; b) increased mean cell size and mass; c) a reduction in saturation density observed in monolayers; d) an increased number of binucleate/multinucleate cells or cells with lobed nuclei; and e) an increased number of lysosomes per cell (19).…”

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confidence: 99%

In vitro aging of articular chondrocytes identified by analysis of DNA and tubulin content and relationship to cell size and protein content

Ronot¹,

Gaillard‐Froger²,

Hainque³

et al. 1988

Cytometry

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In vitro senescence of chondrocytes, characterized by a decline in the proliferation rate during late passages, resulted from a rapid growth rate in early subcultures to a complete loss of division after seven to nine passages. One senescent-associated phenotypic change was the apparent increase in the density of cytoplasmic cytoskeletal proteins. We examined the relationship between tubulin content and growth (measured by DNA and total protein contents and cell volume), using flow cytometry, in the assessment of cytoskeleton analysis during in vitro aging. In contrast with previous microscopic observations of tubulin organization, flow eytometry revealed a tubulin content that was modulated as a function of protein content and/or cell volume.Key terms: Chondrocyte, in vitro aging, cytoskeleton, DNA, protein content, cell size A goal of flow cytometry in cell biology and cell culture is to explain the behavior of the cell in terms of the functional properties of its individual components. Some cellular components that have been studied successfully by flow cytometry include nucleic acids, proteins, and mitochondria, for which quantitation using fluorescent probes and/or monoclonal antibodies have been directly applicable (13,17,18). One major challenge for flow cytometry is to describe satisfactorily in vitro aging by analyzing cell morphology and structure and the related problems of cell growth, metabolism, and differentiation; all of these parameters are thought to depend on an intractably large number of specific interactions between cell compartments.During in vitro aging, physiological cell parameters are modified such that there are: a) a gradual decline in proliferative capacity, a lengthened interphase interval, and a reduction of the number of cells actively synthesizing DNA; b) increased mean cell size and mass; c) a reduction in saturation density observed in monolayers; d) an increased number of binucleate/multinucleate cells or cells with lobed nuclei; and e) an increased number of lysosomes per cell (19).These senescence-associated phenotypic changes have been correlated with a remarkable increase in the density and organization of the three cytoskeletal components, including actin-containing microfilaments, intermediate filaments, and microtubules in the enlarged cells (6, 22, 23).The cytoskeleton is an extensive, highly ordered structure with important functional roles in the determination of cell shape, cell and chromosome movements, disposition and movement of organelles, and cell division (2,4,5). Much of the current interest in the cytoskeleton has been triggered by technical developments, including high-voltage electron microscopy and deepetching and immunofluorescence microscopy (1). The controversial and confusing information regarding its structure and functions has stimulated research interest in the cytoskeleton, and details of its structure, functions, and organization have been related to in vitro senescence in several model systems.This report examines the senescence-associated phen...

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Flow-cytometric analysis of mixed cell populations using intermediate filament antibodies

Cited by 36 publications

References 10 publications

Analysis of polypeptide expression in benign and malignant human breast lesions

Analysis of polypeptide expression in benign and malignant human breast lesions

Modulation of placental alkaline phosphatase activity and cytokeratins in human HN-1 cells by butyrate, retinoic acid, catecholamines and histamine

In vitro aging of articular chondrocytes identified by analysis of DNA and tubulin content and relationship to cell size and protein content

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