Mitotic Chromosomal Bcl-2: I. Stable Expression Throughout the Cell Cycle and Association with Isolated Chromosomes

Abstract

Bcl-2 is present in a cytoplasmic distribution in cells that express high levels of this oncoprotein. In contrast, using immunocytochemistry in cells expressing low levels of bcl-2, such as KB human carcinoma cells, we and others have shown that bcl-2 is present on the surface of mitotic chromosomes. However, monoclonal antibodies reactive with an epitope representing amino acids 41-54 of the bcl-2 sequence did not detect bcl-2 in other phases of the cell cycle. This study extended those earlier findings to determine if bcl-2 was expressed as a cyclin or if this pattern was an artifact of immunocytochemistry. Immunofluorescence studies in several other human cell lines showed the same mitotic distribution of bcl-2. Other studies using flow cytometry also showed selective mitotic phase detection of bcl-2. A comparison of available commercial antibodies showed that, in spite of reactivity with denatured bcl-2 on Western blots, clear reactivity with bcl-2 in fixed cells was found only with those reactive with the (a.a. 41-54) epitope. With RNase protection and Western blot analyses, cells synchronized at various stages of the cell cycle showed constant levels of bcl-2 mRNA and protein. Analysis of bcl-2 using Western blots showed a band with the same apparent molecular weight as that seen in comparison with authentic bcl-2 overexpressed in the cytoplasm. The retention of bcl-2 on chromosomes in unfixed, permeabilized preparations was influenced by protease treatment, phosphate, and pH. Studies using isolated chromosomes showed that much of the bcl-2 in these cells was attached to chromosomes in mitosis, had the expected molecular weight, and was phosphorylated in the same manner as that seen in whole-cell extracts. These results show that bcl-2 is not a cyclin and that the bcl-2 localized on chromosomes is the same molecule seen by immunoblotting. These results suggest that the reactive (a.a. 41-54) epitope present in bcl-2 is somehow modified or masked in interphase.