Jeffrey L. Salisbury

Single-walled carbon nanotube-induced mitotic disruption

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 μg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 μg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Centrosome amplification drives chromosomal instability in breast tumor development

Earlier studies of invasive breast tumors have shown that 60–80% are aneuploid and ≈80% exhibit amplified centrosomes. In this study, we investigated the relationship of centrosome amplification with aneuploidy, chromosomal instability, p53 mutation, and loss of differentiation in human breast tumors. Twenty invasive breast tumors and seven normal breast tissues were analyzed by fluorescence in situ hybridization with centromeric probes to chromosomes 3, 7, and 17. We analyzed these tumors for both aneuploidy and unstable karyotypes as determined by chromosomal instability. The results were then tested for correlation with three measures of centrosome amplification: centrosome size, centrosome number, and centrosome microtubule nucleation capacity. Centrosome size and centrosome number both showed a positive, significant, linear correlation with aneuploidy and chromosomal instability. Microtubule nucleation capacity showed no such correlation, but did correlate significantly with loss of tissue differentiation. Centrosome amplification was detected in in situ ductal carcinomas, suggesting that centrosome amplification is an early event in these lesions. Centrosome amplification and chromosomal instability occurred independently of p53 mutation, whereas p53 mutation was associated with a significant increase in centrosome microtubule nucleation capacity. Together, these results demonstrate that independent aspects of centrosome amplification correlate with chromosomal instability and loss of tissue differentiation and may be involved in tumor development and progression. These results further suggest that aspects of centrosome amplification may have clinical diagnostic and/or prognostic value and that the centrosome may be a potential target for cancer therapy.

Centrosome amplification and the development of cancer.

Recent studies have implicated centrosome amplification in the origin of chromosomal instability during tumor development. Theodor Boveri first suggested this notion nearly a century ago (Boveri, 1914). While the role of centrosome amplification in the origin of malignant tumors remains a controversial issue, several emerging lines of evidence suggest that cell cycle pathways converge on the centrosome and implicate this organelle in the control of cell cycle progression in addition to its function as a microtubule organizing center. Here we review the relationship between cell cycle regulation and centrosome amplification in the development of cancer. The centrosome influences cell structure through the nucleation and organization of cytoplasmic microtubules (Kirschner and Mitchison, 1986). Interphase cells contain a single centrosome that is typically located near the nucleus and contains of a pair of centrioles that anchor the recruitment of pericentriolar material including the microtubule nucleating protein g-tubulin (Bobinnec et al., 1998). Centrioles are small organelles (*200 nm diameter and 400 nm in length) consisting of a cylindrical array of nine triplet microtubules (Dutcher, 2001a,b). The centrosome is duplicated once, and only once, during a normal cell cycle to give rise to two centrosomes that function as the spindle poles of the dividing cell (Kellogg, 1989). This process is most clearly illustrated by duplication of the centrioles themselves. In early G1 phase of the cell cycle the two centrioles are typically oriented in a characteristic orthogonal arrangement. As cells pass the G1 restriction point and commit to DNA replication and subsequent cell division, the two centrioles separate a short distance from one another and nascent procentrioles form at the proximal end and orthogonal to each pre-existing centriole (Adams and Kilmartin, 2000; Wheatley, 1982). During G2/M phase centrosome duplication is completed and each new centrosome (i.e. mitotic spindle pole) contains one old and one new centriole. The presence of only two centrosomes in the cell as it enters mitosis ensures the equal segregation of sister chromatids to each daughter cell. Mitotic spindle poles also play a role in determining the position and orientation of the cleavage furrow and in exit from cytokinesis (Khodjakov and Rieder, 2001; Piel et al., 2001).

Altered Centrosome Structure Is Associated with Abnormal Mitoses in Human Breast Tumors

Centrosomes are the major microtubule organizing center in mammalian cells and establish the spindle poles during mitosis. Centrosome defects have been implicated in disease and tumor progression and have been associated with nullizygosity of the p53 tumor suppressor gene. In the present ultrastructural analysis of 31 human breast tumors, we found that centrosomes of most tumors had significant alterations compared to centrosomes of normal breast tissue. These alterations in included 1) supernumerary centrioles, 2) excess pericentriolar material, 3) disrupted centriole barrel structure, 4) unincorporated microtubule complexes, 5) centrioles of unusual length, 6) centrioles functioning as ciliary basal bodies, and 7) mispositioned centrosomes. These alterations are associated with changes in cell polarity, changes in cell and tissue differentiation, and chromosome missegregation through multipolar mitoses. Significantly, the presence of excess pericentriolar material was associated with the highest frequency of abnormal mitoses. Centrosome abnormalities may confer a mutator phenotype to tumors, occasionally yielding cells with a selective advantage that emerge and thrive, thus leading the tumor to a more aggressive state.

Induction of aneuploidy by single-walled carbon nanotubes

Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single‐walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus. Environ. Mol. Mutagen., 2009. Published 2009 Wiley‐Liss, Inc.

Centrosomes and cancer

The centrosome functions as the major microtubule organizing center (MTOC) of the cell and as such it determines the number, polarity, and organization of interphase and mitotic microtubules. Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division are all dependent on the normal function of the centrosome and on its orderly duplication, once and only once, in each cell cycle. Malignant tumor cells show characteristic defects in cell and tissue architecture and in chromosome number that can be attributed to inappropriate centrosome behavior during tumor progression. In this review, we will summarize recent observations linking centrosome defects to disruption of normal cell and tissue organization and to chromosomal instability found in malignant tumors.

Estrogen mediates Aurora-A overexpression, centrosome amplification, chromosomal instability, and breast cancer in female ACI rats

Estrogens play a crucial role in the causation and development of sporadic human breast cancer (BC). Chromosomal instability (CIN) is a defining trait of early human ductal carcinoma in situ (DCIS) and is believed to precipitate breast oncogenesis. We reported earlier that 100% of female ACI (August/Copenhagen/Irish) rats treated with essentially physiological serum levels of 17β-estradiol lead to mammary gland tumors with histopathologic, cellular, molecular, and ploidy changes remarkably similar to those seen in human DCIS and invasive sporadic ductal BC. Aurora-A (Aur-A), a centrosome kinase, and centrosome amplification have been implicated in the origin of aneuploidy via CIN. After 4 mo of estradiol treatment, levels of Aur-A and centrosomal proteins, γ-tubulin and centrin, rose significantly in female ACI rat mammary glands and remained elevated in mammary tumors at 5–6 mo of estrogen treatment. Centrosome amplification was initially detected at 3 mo of treatment in focal dysplasias, before DCIS. At 5–6 mo, 90% of the mammary tumor centrosomes were amplified. Comparative genomic hybridization revealed nonrandom amplified chromosome regions in seven chromosomes with a frequency of 55–82% in 11 primary tumors each from individual rats. Thus, we report that estrogen is causally linked via estrogen receptor α to Aur-A overexpression, centrosome amplification, CIN, and aneuploidy leading to BC in susceptible mammary gland cells.

Proteins Required for Centrosome Clustering in Cancer Cells

Identified in an RNA interference screen, proteins that prevent spindle multipolarity in human cancer cells may prove to be promising drug targets. Releasing Tension Could Help Tame Tumor Cells Tumor cells do some things very well—proliferate, for example—but do other things very badly. Instead of orderly, bipolar cytoskeletal machinery that neatly pulls the chromosomes apart during cell division, tumor cells are often multipolar, a state caused by extra copies of the microtubule organizer, the centrosome. These extra organizing centers form multipolar spindle apparatuses, the scaffolds on which cell division takes place, thus producing abnormal division and cell death. Some cells successfully correct this defect by bundling the extra spindles along with the primary ones, and these cells go on to divide properly and proliferate, becoming thriving tumors. Leber and colleagues have taken advantage of this organizational weakness of tumor cells and have identified the proteins these cells use to bundle their spindles next to the primary ones. These proteins are prime targets for new chemotherapeutic drugs, which would prevent corrective bundling, sentencing the cells to abnormal cell division and death. The authors used a small interfering RNA screen to eliminate the expression of 21,000 genes, one by one, in a squamous cell carcinoma cell line. By finding which cells had more than the usual two spindle poles during mitosis—indicative of extra centrosomes—and then verifying these in a second screen, the authors identified 82 genes that encode proteins that the cancer cells use to inhibit damaging multiple centrosomes. Among these proteins are many involved in attaching chromosomes to the cytoskeletal scaffold: those in the chromosomal passenger complex, a regulator of chromosome-microtubule interactions and spindle tension at the point where the spindle attaches; members of the Ndc80 complex, a contact point for chromosome-microtubule attachment; and CENPT, part of a DNA binding complex at the attachment point. Another particularly interesting protein picked up by the screen is CEP164, which is found at the ends of centriole appendages, which are microtubule anchors at the centrosome. By looking for commonalities among these proteins, the authors hypothesized that spindle tension might be a key characteristic required for effective corrective spindle bundling. They tested this idea by measuring the distance between the chromosomes, confirming a central role for tension in this homeostatic mechanism, by which cancer cells compensate for extra centrosomes. Therefore, interfering with the function of any of the proteins found in the screen could, in principle, eliminate this tension and prevent bundling, leading to tumor cell death. These potential drug targets that the cell uses to bypass normal protective mechanisms against cancer are new weapons that can be added to the arsenal of anticancer drug developers. Current cancer chemotherapies are limited by the lack of tumor-specific targets, which would allow for selective eradication of malignant cells without affecting healthy tissues. In contrast to normal cells, most tumor cells contain multiple centrosomes, which tend to cause the formation of multipolar mitotic spindles, chromosome segregation defects, and cell death. Nevertheless, many cancer cells divide successfully because they can cluster multiple centrosomes into two spindle poles. Inhibition of this centrosomal clustering, with consequent induction of multipolar spindles and subsequent cell death, would specifically target cancer cells and overcome one limitation of current cancer treatments. We have performed a genome-wide RNA interference screen to identify proteins involved in the prevention of spindle multipolarity in human cancer cells with supernumerary centrosomes. The chromosomal passenger complex, Ndc80 microtubule-kinetochore attachment complex, sister chromatid cohesion, and microtubule formation via the augmin complex were identified as necessary for centrosomal clustering. We show that spindle tension is required to cluster multiple centrosomes into a bipolar spindle array in tumor cells with extra centrosomes. These findings may explain the specificity of drugs that interfere with spindle tension for cancer cells and provide entry points for the development of therapeutics.

Phosphorylation of centrin during the cell cycle and its role in centriole separation preceding centrosome duplication

Once during each cell cycle, mitotic spindle poles arise by separation of newly duplicated centrosomes. We report here the involvement of phosphorylation of the centrosomal protein centrin in this process. We show that centrin is phosphorylated at serine residue 170 during the G2/M phase of the cell cycle. Indirect immunofluorescence staining of HeLa cells using a phosphocentrin-specific antibody reveals intense labeling of mitotic spindle poles during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosomes. Cultured cells undergo a dramatic increase in centrin phosphorylation following the experimental elevation of PKA activity, suggesting that this kinase can phosphorylate centrinin vivo. Surprisingly, elevated PKA activity also resulted intense phosphocentrin antibody labeling of interphase centrosomes and in the concurrent movement of individual centrioles apart from one another. Taken together, these results suggest that centrin phosphorylation signals the separation of centrosomes at prophase and implicates centrin phosphorylation in centriole separation that normally precedes centrosome duplication.

Ontogeny of Phex/PHEX Protein Expression in Mouse Embryo and Subcellular Localization in Osteoblasts†

PHEX, a phosphate‐regulating gene with homologies to endopeptidases on the X chromosome, is mutated in X‐linked hypophosphatemia (XLH) in humans and mice (Hyp). Although recent observations indicate that Phex protein is expressed primarily in bone and may play an important role in osteoblast function and bone mineralization, the pattern of the Phex protein expression in the developing skeleton and its subcellular localization in osteoblasts remain unknown. We examined the ontogeny of the Phex protein in the developing mouse embryo and its subcellular localization in osteoblasts using a specific antibody to the protein. Immunohistochemical staining of mouse embryos revealed expression of Phex in osteogenic precursors in developing vertebral bodies and developing long bones on day 16 postcoitum (pc) and thereafter. Calvaria from day 18 pc mice showed Phex epitopes in osteoblasts. No Phex immunoreactivity was detected in lung, heart, hepatocytes, kidney, intestine, skeletal muscle, or adipose tissue of mouse embryos. Interestingly, embryonic mouse skin showed moderate amounts of Phex immunostaining. In postnatal mice, Phex expression was observed in osteoblasts and osteocytes. Moderate expression of Phex was seen in odontoblasts and slight immunoreactivity was observed in ameloblasts. Confocal microscopy revealed the presence of immunoreactive PHEX protein in the Golgi apparatus and endoplasmic reticulum of osteoblasts from normal mice and in osteoblasts from Hyp mice transduced with a human PHEX viral expression vector. PHEX protein was not detected in untransduced Hyp osteoblasts. These data indicate that Phex protein is expressed in osteoblasts and osteocytes during the embryonic and postnatal periods and that within bone, Phex may be a unique marker for cells of the osteoblast/osteocyte lineage.