Mitotic Chromosomal Bcl-2: II. Localization to Interphase Nuclei

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Abstract
We have previously shown, by immunofluorescence of fixed cells, that bcl-2 is found only in mitotic chromosomes in KB cultured human tumor cells expressing low levels of this oncoprotein. However, other studies showed that bcl-2 did not change its levels during the cell cycle when analyzed using Western blots. In this study we analyzed the distribution of bcl-2 during interphase, the point at which it is undetectable by immunofluorescence, using biochemical extraction, immunoprecipitation, and cell fractionation with Western blots. Interestingly, when carefully examined by immunofluorescence in fixed cells, the earliest point in the cell cycle showing bcl-2 localization was early G2, in which bcl-2 could be found within the intact nucleus. In spite of showing no immunofluorescence reaction in fixed interphase cells, immunoprecipitation of gentle detergent extracts showed that bcl-2 from interphase cells reacted readily with the antibody used (#124) after extraction. However, immunoprecipitation using anti-bcl-2 followed by Western blots using anti-Bax showed that, unlike overexpressing cells, this bcl-2 was not complexed with Bax. Classical cell fractionation methods were used to separate nuclei from cytosol and cell membranes. Surprisingly, these experiments clearly showed that essentially all of the bcl-2 in interphase KB cells was present in the nucleus. Therefore, the lack of reaction in fixed cells with anti-bcl-2 antibody reflects either a masking or a conformational change of the reactive epitope in bcl-2 present within the nucleus. By correlation, this change may be related to the phosphorylation of bcl-2 that occurs just before mitosis. The nature of this novel yet highly conserved nuclear form of bcl-2 and the understanding of its function will require further study.