Salisbury, Jeffrey L.

Centrin is a calcium-binding centrosome/basal body–associated protein involved in duplication and segregation of these organelles in eukaryotes. We had shown that disruption of one of the centrin genes (centrin1) in Leishmania amastigotes resulted in failure of both basal body duplication and cytokinesis. Here, we undertook to define the role of centrin1 (TbCen1) in the duplication and segregation of basal body and its associated organelles kinetoplast and Golgi, as well as its role in cytokinesis of the procyclic form of Trypanosoma brucei by depleting its protein using RNA inhibition methodology. TbCen1-depleted cells showed significant reduction in growth compared with control cells. Morphological analysis of these cells showed they were large and pleomorphic with multiple detached flagella. Both immunofluorescence assays using organelle-specific antibodies and electron microscopic analysis showed that TbCen1-deficient cells contained multiple basal bodies, kinetoplasts, Golgi, and nuclei. These multiple organelles were, however, closely clustered together, indicating duplication without segregation in the absence of centrin. This failure in organelle segregation may be the likely cause of inhibition of cytokinesis, suggesting for the first time a new and unique role for centrin in the segregation of organelles without affecting their multiplication in the procyclic form of T. brucei.

Abstract Estrogen-induced Syrian hamster tumors in the kidney represent a useful model to gain insight into the role of estrogens in oncogenic processes. We provided evidence that early tumor foci in the kidney arise from interstitial ectopic uterine-like germinal stem cells, and that early tumor foci and well-established tumors are highly aneuploid (92–94%). The molecular mechanisms whereby estrogens mediate this process are unclear. Here, we report that estrogen treatment induced significant increases in Aurora A protein expression (8.7-fold), activity (2.6-fold), mRNA (6.0-fold), and Aurora B protein expression (4.6-fold) in tumors, compared with age-matched cholesterol-treated kidneys. Immunohistochemistry revealed that this increase in Aurora A and B protein expression was essentially confined to cells within early and large tumor foci at 3.5 and 6 months of estrogen treatment, respectively. Upon estrogen withdrawal or coadministration of tamoxifen for 10 days, a 78% to 79% and 81% to 64% reduction in Aurora A and B expression, respectively, were observed in primary tumors compared with tumors continuously exposed to estrogens. These data indicate that overexpressed Aurora A and B in these tumors are under estrogen control via estrogen receptor α. Aurora A coenriched with the centrosome fraction isolated from tumors in the kidney. Centrosome amplification (number and area/cell) was detected in early tumor foci and large tumors but not in adjacent uninvolved or age-matched control kidneys. Taken together, these data indicate that persistent overexpression of Aurora A and B is under estrogen control, and is coincident with centrosome amplification, chromosomal instability, and aneuploidy, and represent an important mechanism driving tumorigenesis. [Cancer Res 2007;67(7):2957–63]

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.

The centrosome plays an important role in maintenance of cell polarity and in progression through the cell cycle by determining the number, polarity, and organization of interphase and mitotic microtubules. By examining a set of 35 high grade human breast tumors, we show that centrosomes of adenocarcinoma cells generally display abnormal structure, aberrant protein phosphorylation, and increased microtubule nucleating capacity in comparison to centrosomes of normal breast epithelial and stromal tissues. These structural and functional centrosome defects have important implications for understanding the mechanisms by which genomic instability and loss of cell polarity develop in solid tumors.

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.

The avian erythroblastosis viral oncogene (v- erb B) encodes a receptor tyrosine kinase that possesses sarcomagenic and leukemogenic potential. We have expressed transforming and nontransforming mutants of v- erb B in fibroblasts to detect transformation-associated signal transduction events. Coimmunoprecipitation and affinity chromatography have been used to identify a transformation-associated, tyrosine phosphorylated, multiprotein complex. This complex consists of Src homologous collagen protein (Shc), growth factor receptor binding protein 2 (Grb2), son of sevenless (Sos), and a novel tyrosine phosphorylated form of the cytoskeletal regulatory protein caldesmon. Immunofluorescence localization studies further reveal that, in contrast to the distribution of caldesmon along actin stress fibers in normal fibroblasts, caldesmon colocalizes with Shc in plasma membrane blebs in transformed fibroblasts. This colocalization of caldesmon and Shc correlates with actin stress fiber disassembly and v- erb B-mediated transformation. The tyrosine phosphorylation of caldesmon, and its association with the Shc–Grb2–Sos signaling complex directly links tyrosine kinase oncogenic signaling events with cytoskeletal regulatory processes, and may define one mechanism regulating actin stress fiber disassembly in transformed cells.