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 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.
Schandl
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CA
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina, USA.
Li
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Fan
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Willingham
M C
MC
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002453:Cell Cycle; D002478:Cells, Cultured; D002875:Chromosomes / Q000378:metabolism*; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008938:Mitosis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger