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 fro m a squamous cell lung carcinoma, was studied in relation to cell grow th conditions, For this purpose the proteasome monoclonal antibodies M CP34 and MCP20 were applied to the cells growing under different nutri tional 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 ro om temperature), it became obvious that the intracellular detectabilit y of the proteasomes changes depending on the nutritional and prolifer ative status of the tumor cells. Two types of experiments were carried out: (I) cells were grown for two days at different cell densities, w ith an excess of culture medium, and (2) cells were seeded in a low ce ll density and monitored for 6 days without change of medium. In cells grown at low density the proteasomes can he detected mainly in the nu clei, while the nucleoli are almost devoid of staining, and the cytopl asm is only slightly stained. In cells grown at high density; the stai ning pattern changes with a much less pronounced nuclear staining than in the cells at low density, while the cytoplasm remains slightly sta ined. In the nutrient depletion experiment similar changes were seen. In cells growing under favorable conditions (1 or 2 days in fresh medi um) proteasomes are detected mainly in the nuclei, whereas when the me dium becomes depleted of nutrients (4 or 5-day-old medium) the stainin g pattern changes to one Kith a much less pronounced nuclear staining. However, in immunofluorescence studies on cells grown under similar c onditions but fixed in ethanol (-20 degrees C) for 15 min, the changes in proteasome localization pattern were not detected during medium de pletion. Using this fixation protocol the proteasomes are detected mai nly in the nuclei at all stages of the medium exhaustion experiment. T hese apparently contrasting results suggest that upon nutrient depleti on the proteasome epitopes become less accessible to the antibodies us ed. Apparently, the epitopes can regain accessibility if an extended e thanol fixation is used. This hypothesis was confirmed by flow cytomet ry and immunoblotting experiments. In nom cytometry of ethanol-fixed c ells the fluorescence intensity of only a minor part of the cell popul ation decreases to some extent with medium depletion, but in the major ity of the cells fluorescence remains at its initial level. The immuno blotting experiments show no quantitative changes in proteasome conten t of the tumor cells at the different growth conditions.