Cytokeratin 8 associates with the external leaflet of plasma membranes in tumour cells

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Abstract

We reported the identification of tumour-associated antigens from head and neck carcinomas, including cytokeratin 8 (CK8). These antigens were isolated based on the humoral immune response they elicit in vivo using the antibody-mediated identification of antigens technology. Unlike healthy squamous epithelium, tumour cells displayed CK8 at the plasma membrane. However, the actual presence of CK8 at the plasma membrane is still a matter of debate. Here, we have analyzed the expression of CK8 in detail using confocal laser scanning microscopy and circumstantiated its localization at the plasma membrane of carcinoma cells. Healthy human tissues were devoid of CK8 at the membrane, with the exception of hepatocytes. Moreover, membrane-associated CK8 molecules experienced a re-distribution throughout mitosis, which was associated with phosphorylation at serine 73. Phosphorylated CK8 redistributed into dense speckles and relocated to the plasma membrane upon cytokinesis. Thus, CK8 possesses genuine extracellular epitopes on tumour cells, which may represent valuable targets for therapy.

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Materials and methods

Cell lines and primary samples. PCI-1, PCI-13, HLAC-78, HLAC-79, ANT-1, 22-A, and FaDu are squamous cell carcinoma lines derived from head and neck carcinomas. SkBr3 and MCF-7 are breast carcinoma cell lines. HCT-8 is a colon carcinoma cell line. All cell lines were propagated at 37 °C in a 5% CO2-humidified incubator in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Biochrom AG, Berlin, Germany), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were

Cytokeratin 8 localizes to the plasma membrane of carcinoma cells

First, we analyzed the expression of CK8 on non-permeabilized carcinoma cell lines of different origin, i.e., head and neck (PCI-1, PCI-13, HLAC-78, HLAC-79, ANT-1, and FaDu, 22-A), breast (SkBr3, MCF-7), cervix (HeLa), colon carcinomas (HCT-8), and adenovirus-transformed human embryonic kidney cells (HEK293), which have an epithelial phenotype. CK8 was detected using an antibody recognizing the 12 most C-terminal amino acids in combination with a FITC-conjugated secondary antibody and measured

Discussion

Upon screening of primary carcinomas with autologous serum antibodies using a novel technology termed AMIDA, we have identified CK8 as a target for the humoral immune response in patients suffering from head and neck squamous cell carcinomas [17]. In this screen, CK8 was isolated from a membrane fraction of cells and was subsequently demonstrated to localize to the plasma membrane of primary carcinomas upon flow cytometry analysis. However, the localization of CK8 at the external leaflet of the

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    These authors contributed equally to this work.

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