Immunolocalization of NuMA and phosphorylated proteins during the cell cycle in human breast and prostate cancer cells as analyzed by immunofluorescence and postembedding immunoelectron microscopy

The formation of mitotic centrosomes is a complex process in which a number of cellular proteins translocate to mitotic poles and play a critical role in the organization of the mitotic apparatus. The 238-kDa nuclear mitotic apparatus protein NuMA is one of the important proteins that plays a signif icant role in this process. NuMA resides in the nucleus during interphase a nd becomes transiently associated with mitotic centrosomes after multiple s teps of phosphorylations. The role of NuMA in the interphase nucleus is not well known but it is clear that NuMA responds to external signals (such as hormones) that induce cell division, or heat shock that induces apoptosis. In order to determine the function of NuMA it is important to study its lo calization. Here we report on nuclear organization of NuMA during the cell cycle in estrogen responsive MCF-7 breast cancer cells and in androgen resp onsive LNCaP prostate cancer cells using immunoelectron microscopy, and on correlation to MPM-2 monoclonal phosphoprotein antibody. These results show that NuMA is present in speckled and punctate form associated with distinc t material corresponding to a speckled or punctate immunofluorescence appea rance in the nucleus while MPM-2 is uniformly dispersed in the nucleus. At prophase NuMA disperses in the cytoplasm and associates with microtubules w hile MPM-2 is uniformly distributed in the cytoplasm. During metaphase or a naphase anti-NuMA labeling is associated with spindle fibers. During teloph ase NuMA relocates to electron-dense areas around chromatin and finally to the reconstituted nuclei. These results demonstrate NuMA organization in MC F-7 and LNCaP cells in the log phase of cell culture growth.