Changes in immunocytochemical detectability of proteasome epitopes depending on cell growth and fixation conditions of lung cancer cell lines.
The localization of proteasome epitopes in the lung cancer cell lines NCI-H82, derived from a small cell lung cancer, and MR65, derived from a squamous cell lung carcinoma, was studied in relation to cell growth conditions. For this purpose the proteasome monoclonal antibodies MCP34 and MCP20 were applied to the cells growing under different nutritional conditions, resulting in different proliferative states. Using indirect immunofluorescence microscopy with brief fixation in methanol (5 sec, -20 degrees C) followed by three dips in acetone (5 sec at room temperature), it became obvious that the intracellular detectability of the proteasomes changes depending on the nutritional and proliferative status of the tumor cells. Two types of experiments were carried out: (1) cells were grown for two days at different cell densities, with an excess of culture medium, and (2) cells were seeded in a low cell density and monitored for 6 days without change of medium. In cells grown at low density, the proteasomes can be detected mainly in the nuclei, while the nucleoli are almost devoid of staining, and the cytoplasm is only slightly stained. In cells grown at high density, the staining pattern changes with a much less pronounced nuclear staining than in the cells at low density, while the cytoplasm remains slightly stained. In the nutrient depletion experiment similar changes were seen. In cells growing under favorable conditions (1 or 2 days in fresh medium) proteasomes are detected mainly in the nuclei, whereas when the medium becomes depleted of nutrients (4 or 5-day-old medium) the staining pattern changes to one with a much less pronounced nuclear staining. However, in immunofluorescence studies on cells grown under similar conditions but fixed in ethanol (-20 degrees C) for 15 min, the changes in proteasome localization pattern were not detected during medium depletion. Using this fixation protocol the proteasomes are detected mainly in the nuclei at all stages of the medium exhaustion experiment. These apparently contrasting results suggest that upon nutrient depletion the proteasome epitopes become less accessible to the antibodies used. Apparently, the epitopes can regain accessibility if an extended ethanol fixation is used. This hypothesis was confirmed by flow cytometry and immunoblotting experiments. In flow cytometry of ethanol-fixed cells the fluorescence intensity of only a minor part of the cell population decreases to some extent with medium depletion, but in the majority of the cells fluorescence remains at its initial level. The immunoblotting experiments show no quantitative changes in proteasome content of the tumor cells at the different growth conditions.
Machiels
B M
BM
Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands.
Henfling
M E
ME
Broers
J L
JL
Hendil
K B
KB
Ramaekers
F C
FC
D015153:Blotting, Western; D018288:Carcinoma, Small Cell / Q000276:immunology / Q000648:ultrastructure*; D002294:Carcinoma, Squamous Cell / Q000276:immunology / Q000648:ultrastructure*; D002448:Cell Adhesion; D002451:Cell Compartmentation; D002455:Cell Division; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000648:ultrastructure*; D003896:Desmosomes; D000939:Epitopes; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008175:Lung Neoplasms / Q000276:immunology / Q000648:ultrastructure*; D009097:Multienzyme Complexes / Q000276:immunology / Q000648:ultrastructure*; D046988:Proteasome Endopeptidase Complex; D016707:Tissue Fixation / Q000379:methods; D014407:Tumor Cells, Cultured
D000939:Epitopes; D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex