Immunofluorescence of γ-tubulin in tumor centrosomes. (A) In this overview of a breast tumor cryosection, γ-tubulin appears as many large spots scattered randomly throughout the tumor, similar to the distribution of centrin in Fig. 1E. Double labeling (B–D) of tumor centrosomes by using antibodies to centrin (B, red) and γ-tubulin (C, green) show virtual coincidence of signal (D, yellow). (Bar = 20 μm.)

Inappropriate phosphorylation of breast tumor centrosomes. (A) Specificity of αHPC antibodies for phosphocentrin. Western blot analysis of phosphorylated and nonphosphorylated bacterially expressed recombinant centrin (A, gel lanes a′ and b′) and autoradiography of the same gel lanes (A, gel lanes a and b). Comparison of the Western blot analysis (A, gel lane a′) by using mAb 20H5 that reacts with centrin regardless of phosphorylation status and the corresponding autoradiogram (A, gel lane a) demonstrates that the slower migrating centrin band is phosphorylated. Western blot analysis (A, gel lane b′) by using αHPC antibody to detect phosphorylated centrin demonstrates reactivity only with the slower migrating form of centrin and no reaction with the nonphosphorylated form. Analysis of whole cell extracts demonstrates that centrin is phosphorylated in breast tumor tissue (A, compare gel lanes c and d). (B) A section of a normal breast duct labeled with αHPC antibody demonstrates only a background level of staining in normal epithelial cells. (C) A section of a breast adenocarcinoma labeled with αHPC antibody demonstrates that centrosomes stain intensely, indicating that centrin is phosphorylated in tumor cells. (D–E) Immunofluorescence of the normal breast epithelial cell line MCF10A stained with αHPC antibody for phosphocentrin and propidium iodide for DNA. The spindle poles of a mitotic cell react strongly with αHPC antibody (D), whereas interphase cells show no labeling of their centrosomes (E). Tumor centrosome and mitotic MCF10A cell staining with αHPC are completely eliminated with a fivefold excess of competing phosphopeptide (data not shown). (Bar = 20 μm.)

Centrosome number and size in normal breast duct epithelia (A–C) and adenocarcinoma cells (D–F). Hematoxylin and eosin-stained paraffin sections of a normal human breast duct (A) and a breast adenocarcinoma (D). (B) Confocal image stack of a normal breast duct stained for centrioles with anticentrin mAb 20H5 (fluorescein isothiocyanate secondary antibody) and for nuclear DNA with propidium iodide. Approximately 20 pairs of centrioles are located apical to the nuclei of epithelial cells that line this normal duct. (C) Binary processed image showing the volume of centrin labeling for the same normal epithelial image stack shown in B, from which a portion of the data in Table 1 was derived. (E) Confocal image stack of a breast adenocarcinoma stained as above. Many large centrin-staining spots mark the location of abnormal centrosomes in the tumor tissue. (F) Binary processed image showing the volume of centrin labeling for the same tumor image stack shown in E, from which a portion of the data in Table 1 was derived. (Bar = 20 μm.)

Normal and tumor centrosome ultrastructure. Electron micrographs of thin sections of normal epithelial cell centrosomes (A–B) and centrosomes of tumor cells (C–E). (A) A normal epithelial cell centrosome illustrating the orientation of the pair of centriole cylinders and sparse pericentriolar material surrounding them. (B) A centrosome of another normal breast epithelial cell illustrating two centriole profiles and sparse pericentriolar material. The three tumor cell centrosomes illustrated in C–E show large accumulations of densely staining pericentriolar material with numerous centrioles and procentrioles. (E) A section of a tumor centrosome that includes five centriole and procentriole profiles (arrowheads). (Bar = 0.5 μm.)

Tumor centrosomes nucleate more microtubules than do normal epithelial cell centrosomes. (A) A section of normal breast tissue after incubation with Xenopus egg extract and staining for microtubules. (D) A section of a breast tumor illustrating nucleation of large numbers of microtubules by many individual adenocarcinoma cell centrosomes. Touch preparations were analyzed for the purpose of making accurate counts of microtubules nucleated by normal breast epithelial cells (B–C) and by tumor cells (E–F). Normal breast epithelial cells nucleate <45 microtubules that originate from a single, distinctly focused MTOC (B–C). In contrast, tumor cells nucleate many more microtubules that originate from multiple large MTOCs (E–F). Microtubules are stained with antitubulin antibodies, and nuclei are stained by using propidium iodide. Data from 50 normal and 50 tumor cells are summarized in Table 1C. (Bar = 20 μm.)