Mitotic chromosomal bcl-2: I. Stable expression throughout the cell cycle and association with isolated chromosomes

Bcl-2 is present in a cytoplasmic distribution in cells that express high l evels of this oncoprotein. In contrast, using immunocytochemistry in cells expressing low levels of bcl-2, such as KB human carcinoma cells, we and ot hers have shown that bcl-2 is present on the surface of mitotic chromosomes . However, monoclonal antibodies reactive with an epitope representing amin o 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 immu nocytochemistry. Immunofluorescence studies in several other human cell lin es showed the same mitotic distribution of bcl-2. Other studies using flow cytometry also showed selective mitotic phase detection of bcl-2. A compari son 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. Wi th RNase protection and Western blot analyses, cells synchronized at variou s 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 apparen t molecular weight as that seen in comparison with authentic bcl-2 overexpr essed in the cytoplasm. The retention of bcl-2 on chromosomes in unfixed, p ermeabilized 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 molec ular weight, and was phosphorylated in the same manner as that seen in whol e-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 b cl-2 is somehow modified or masked in interphase.