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Journal of Histochemistry and Cytochemistry, Vol. 47, 139-150, February 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

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

Cynthia A. Schandla, Shuli Lia, Gian G. Rea, Weimin Fana, and Mark C. Willinghama
a Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina

Correspondence to: Mark C. Willingham, Dept. of Pathology, Wake Forest U. School of Medicine, Medical Center Boulevard, Winston–Salem, NC 27157-1072.

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. (J Histochem Cytochem 47:139–149, 1999)

Key Words: bcl-2, chromosomes, mitosis, phosphorylation, nucleus, apoptosis, immunocytochemistry


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