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

Abstract

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 significant role in this process. NuMA resides in the nucleus during interphase and becomes transiently associated with mitotic centrosomes after multiple steps 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 localization. Here we report on nuclear organization of NuMA during the cell cycle in estrogen responsive MCF-7 breast cancer cells and in androgen responsive 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 asso-ciated with distinct material corresponding to a speckled or punctate immunofluorescence appearance in the nucleus while MPM-2 is uniformly dispersed in the nucleus. At prophase NuMA disperses in the cytoplasm and associates with microtubules while MPM-2 is uniformly distributed in the cytoplasm. During metaphase or anaphase anti-NuMA labeling is associated with spindle fibers. During telophase NuMA relocates to electron-dense areas around chromatin and finally to the reconstituted nuclei. These results demonstrate NuMA organization in MCF-7 and LNCaP cells in the log phase of cell culture growth.

Remodeling of Centrosomes in Intraspecies and Interspecies Nuclear Transfer Porcine Embryos

“…19 Inaccurate NuMA regulation and atypical NuMA distribution will result in abnormal mitosis 56 and may result in an inability to support normal embryonic development. We have studied NuMA in somatic cells, 57 pig oocytes 18 and intraspecies and interspecies nuclear transfer embryos and have shown that NuMA is contributed to the reconstructed oocyte by the donor cell nucleus. 17 We do not yet know the mechanisms by which NuMA becomes translocated to the spindle poles in cloned embryos.…”

The mammalian centrosome and its functional significance

“…As nuclear matrix protein NuMA is involved in DNA replication and transcription. It displays distinct staining patterns in the nucleus (Merdes and Cleveland 1998 ; Zeng 2000 ; Gobert et al 2001 ) and stains heavily in mitosis when using anti-NuMA antibodies (Saredi et al 1997 ; Merdes et al 2000 ; Gobert et al 2001 ; Zhong et al 2005 ; Liu et al 2006 ). Inaccurate NuMA regulation and atypical NuMA distribution will result in abnormal mitosis as has been reported for cancer cells (Saunders et al 2000 ).…”

Cellular expression, localization and interactions of the product of the human MOST-1 gene associated with breast and prostate cancers

“…Several proteins which are misfolded or overexpressed have been described, especially in neurodegenerative disorders and many types of cancers. Examples include cystatin C in susceptible neurons in Alzheimer's disease (11), NuMA which is an important protein for the formation of mitotic centrosomes (12), and CHMP1 which is a nuclear matrix protein affecting chromatin structure and cell cycle progression (13). Overexpression of the NABC1 protein in cancer cells results in detergent-stable homodimers that leads to punctate localization in cultured cells (14).…”