Centrosome Hyperamplification and Chromosomal Instability in Bladder Cancer

European Urology

European Urology 43 (2003) 505–515

Centrosome Hyperamplification and Chromosomal Instability in Bladder Cancer K. Kawamuraa,*, M. Moriyamaa, N. Shibaa, M. Ozakib, T. Tanakaa, T. Nojimac, K. Fujikawa-Yamamotod, R. Ikedaa, K. Suzukia a

Department of Urology, Kanazawa Medical University, 1-1 Daigaku Uchinada, Ishikawa, 920-0293, Japan Department of Human Genetics, Kanazawa Medical University, Ishikawa, Japan c Department of Pathology and Laboratory Medicine, Kanazawa Medical University, Ishikawa, Japan d Division of Basic Science, Kanazawa Medical University, Ishikawa, Japan b

Accepted 27 January 2003

Abstract Objective: Chromosomal instability (CIN) is a common feature of malignant tumors. Centrosome hyperamplification (CH) occurs frequently in human cancers, and may be a contributing factor in CIN. In this study, we investigated the relationship between CH and CIN in bladder cancer. Methods: Clinical samples obtained by transurethral resection from 22 patients with bladder cancer were examined (histological grade G1, 5 cases; G2, 6 cases; G3, 11 cases). CH was evaluated by immunohistochemistry using antipericentrin antibody. CIN was evaluated by fluorescence in situ hybridization (FISH). FISH probes for pericentromeric regions of chromosomes 3, 7, and 17 were hybridized to touch preparations of nuclei from frozen tissues. We also analyzed the centrosome replication cycle of bladder cancer by laser scanning cytometry (LSC). Results: Of the 22 cases examined, 18 (81.8%) had centrosome hyperamplification: CH 0, 4 cases (18.1%); CH I, 5 cases (22.7%); CH II, 5 cases (22.7%); CH III, 8 cases (36.4%). The grade of CH was directly proportional to the histological grade ( p ¼ 0:03, w2 test). LSC analysis showed that the centrosome replication cycle was well regulated in pathologically low-grade bladder cancer, which did not have chromosomal instability. In contrast, we found marked variability of centrosomes in pathologically high-grade bladder cancer, which had chromosomal instability. CH and CIN were both detected in pathologically high-grade tumors. The grade of CH was directly proportional to the CIN grade ( p ¼ 0:0079, w2 test). Conclusion: The results of the present study suggest that CH may be involved in CIN in bladder cancer. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Bladder cancer; Centrosome hyperamplification; Chromosomal instability; Pericentrin; FISH

1. Introduction The centrosome is composed of a pair of centrioles (core components of the centrosome in animal cells) and surrounding amorphous pericentriolar material.

Abbreviations: CH, centrosome hyperamplification; CIN, chromosomal instability; PBS, phosphate-buffered saline; NP-40, nonidet P-40; TBS, Tris-buffered saline; DAPI, 40 -diamidino 2-phenylindole; FISH, fluorescence in situ hybridization; LSC, laser scanning cytometry * Corresponding author. Tel. þ81-76-286-2211; Fax: þ81-76-286-5516. E-mail address: [email protected] (K. Kawamura).

The centrosome, a major microtubule-organizing center of animal cells, directs formation of bipolar mitotic spindles, which is essential for accurate chromosome segregation to daughter cells [1–3]. The hypothesis that the centrosome plays a key role in carcinogenesis was initially proposed by Boveri, who stated that loss of cellular polarity and increased chromosomal segregation errors characteristic of cancer cells result from defects in centrosome function [4]. The centrosome duplication cycle is a highly regulated process, and abrogation of its regulatory mechanisms results in uncontrolled amplification of centrosomes. Centrosome

0302-2838/03/$ – see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0302-2838(03)00056-3

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hyperamplification (CH) leads to formation of multipolar spindles and unequal segregation of chromosomes to daughter cells [5,6]. The variability in chromosome number observed in tumor cells has recently been termed chromosomal instability (CIN) [7]. Bladder cancer progression is accompanied by increased chromosomal instability and aneuploidy [8]. In this study, we investigated CH in bladder cancer, and assessed the relationship between CH and CIN. 2. Materials and methods 2.1. Tissue samples The samples came from 22 patients (17 men and 6 women) who underwent transurethral bladder biopsy for bladder cancer between December 2001, and June 2002. Mean age at diagnosis was 67 years (range, 52–83 years). Normal bladder epithelium was obtained in 3 cases. Specimens that could not be prepared for examination immediately after extraction were frozen in liquid nitrogen and stored at 80 8C until used. Sufficient material for histopathological diagnosis was collected. All tumors were pathologically evaluated by hematoxylin and eosin (H&E) staining. The histological grade of cancer was diagnosed by one of the authors (T.N.). Tumor grade was assessed according to the World Health Organization (WHO) criteria [9]. The tumors were classified according to the TNM system [10]. 2.2. Indirect immunofluorescence Touch preparations were generated by gently touching each bladder cancer biopsy specimen to a dry microscope slide. This brief contact allowed an adequate number of single tumor cells to adhere to the slide surface. Slides were then air dried at room temperature for 30 minutes. The specimen was fixed in 100% ethanol for 10 minutes at room temperature.

Cells were examined for centrosome amplification by immunostaining for pericentrin, a major component of the pericentrial material of the centrosome [11]. The clinical samples were washed with PBS and permeabilized with 1% nonidet P-40 (NP-40) in PBS for 5 minutes at room temperature. Cells were first incubated with blocking solution (10% normal goat serum in PBS) for 1 hour at room temperature, and then probed with anti-pericentrin polyclonal antibody for 1 hour at 37 8C. Negative controls were produced by omitting the primary antibody. The antibody–antigen complexes were detected by incubating cells with Alexa 488 goat anti-rabbit IgG antibody (Molecular Probes, The Netherlands) for 1 hour at room temperature. Each sample was washed three times with Trisbuffered saline (TBS) after each incubation, and then counterstained with 40 -diamidino 2-phenylindole (DAPI). The degree of CH in the clinical samples was graded as follows [12,13]: CH 0, no detectable amplification; CH I, amplification of centrosomes (n  3) in <5% of the cells; CH II, amplification in 5%, <20% of the cells; CH III, amplification in 20% of the cells. 2.3. Fluorescence in situ hybridization (FISH) Fluorescence in situ hybridization with centromeric probes is a sensitive technique that can detect aneusomy as numeric alterations of specific chromosomes [14]. To assay for CIN in tumor cells, we examined chromosomes in individual cells by FISH, using probes for chromosomes 3, 7 and 17. FISH probes to pericentromeric regions of chromosomes 3 (CEP3), 7 (CEP7) and 17 (CEP17) (Vysis) were hybridized to touch preparations of nuclei from frozen tissues according to published methods [14]. Probes were labeled with SpectrumOrange (CEP3), SpectrumGreen (CEP7), SpectrumAqua (CEP17) for simultaneous analysis. DNA was counterstained with DAPI. For each sample, 100 nuclei were scored for the number of signals for each of the three probes per nucleus. The modal signal number of each chromosome (M) was determined [7]. The fraction of cells with chromosome numbers that differed from the mode (variant fraction: F) was calculated for each chromosome [7]. The average variant fraction (F-AVG) for all three chromosomes (3, 7 and 17) was calculated as follows: F-AVG ¼ ðF3 þ F7 þ F17Þ=3, where F3, F7 and F17 are the variant fractions of chromosomes 3, 7 and 17, respectively. The degree of CIN was

Fig. 1. Centrosomes in normal bladder epithelium. Touch preparations were immunostained with anti-pericentrin antibody, and then stained with DAPI for visualization of nuclei. (a) The cell contained 1 centrosome juxtaposed to the nucleus. (b) The cell contained 2 centrosomes juxtaposed to the nucleus. Magnification: 600.

Table 1 Centrosome hyperamplification, chromosomal instability, DNA ploidy and pathological grade of bladder cancer

Chromosome 3

Chromosome 7

Chromosome 17

Average

Chromosome 3 Chromosome 7

Chromosome 17 Average DNA ploidy

Grade

Stage

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

2 2 2 4 2 4 4 3 4 5 5 2 4 4 5 4 7 7 4 4 7 16

2 2 2 4 3 4 3 4 4 4 4 2 4 4 7 4 10 7 3 4 8 10

2 2 1 4 2 4 2 4 5 3 4 2 3 3 5 4 8 5 5 4 4 11

2.0 2.0 1.7 4.0 2.3 4.0 3.0 3.7 4.3 4.0 4.3 2.0 3.7 3.7 5.7 4.0 8.3 6.3 4.0 4.0 6.3 12.3

2 7 1 15 0 27 21 39 11 39 61 44 21 42 50 16 49 43 24 38 66 75

8 28 34 21 9 37 18 48 25 30 35 42 24 25 42 37 53 29 56 37 41 64

G1 G1 G1 G1 G1 G2 G3 G3 G3 G2 G2 G2 G3 G3 G2 G3 G3 G3 G3 G2 G3 G3

pTa pTa pTa pTa pTa pTa pT1 pT1 pT1 pTa pT1 pTa pT2 pTa pT1 pT2 pT2 pT1 pT2 pT1 pT1 pT1

CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH

0 0 0 0 I I I I I II II II II II III III III III III III III III

0.0 0.0 0.0 0.0 2.1 2.4 2.7 3.2 4.8 5.4 5.4 8.3 11.8 16.4 20.5 23.8 23.9 24.2 26.4 30.4 43.0 46.9

CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN CIN

0 0 0 0 0 1 0 2 0 1 2 2 1 1 2 1 2 2 1 1 2 2

Mode (M)

Variant fraction (F)

2 1 3 17 11 24 19 45 18 29 28 44 18 25 43 51 61 49 25 28 54 57

Cytometry

4 12 13 18 7 29 19 44 18 33 41 43 21 31 45 35 54 40 35 34 54 65

Diploid Diploid Diploid Diploid Diploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid

Pathology K. Kawamura et al. / European Urology 43 (2003) 505–515

Case Centrosome FISH no. CH grade n > 3 (%) CIN grade

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graded as follows: CIN 0, the percent of cells with average nonmodal signal numbers (F-AVG) in <20% of the cells; CIN 1, F-AVG in 20%, <40% of the cells; CIN 2, F-AVG in 40% of the cells. 2.4. Laser scanning cytometry (LSC) We analyzed the centrosome replication cycle of bladder cancer by using LSC to measure DNA content and examine the centrosome. The cytometry procedure is described in detail elsewhere [15– 17]. Touch preparations were washed with PBS and permeabilized with 1% NP-40 in PBS for 5 minutes at room temperature. Cells were first incubated with blocking solution (10% normal goat serum in PBS) for 1 hour at room temperature and then incubated with anti-pericentrin polyclonal antibody [11] for 1 hour at 37 8C. The antibody–antigen complexes were detected by incubating cells with Alexa 488 goat anti-rabbit IgG antibody (Molecular Probes, The Netherlands) for 1 hour at room temperature. The cell nucleus was stained with propidium iodide (PI) solution (25 mg/ml, Sigma Chemical Co., St. Louis, MO, USA) containing 1 mg/ml RNase (Sigma), and DNA ploidy was analyzed using a laser scanning cytometer (LSC-01, Olympus; Tokyo, Japan). An excitation wavelength of 488 nm was used, and emission of the fluorochromes PI

and Alexa was measured using standard long-pass (570 nm) or bandpass (530 nm) filters, respectively. At least 5000 cells were measured per slide. For each cell, we measured the DNA content and examined the Alexa 488 fluorescence profile. The samples were photographed using a digital camera (DP-11, Olympus) mounted on the LSC microscope. 2.5. Statistic analysis The w2 test and linear regression analysis were used for statistical analysis. A p-value of less than 0.05 was considered to indicate statistical significance.

3. Results 3.1. Centrosome hyperamplification in clinical samples In the normal bladder epithelium (3 samples), centrosome number of n ¼ 1 was observed in 71% of the cells, n ¼ 2 in 29% and n  3 in 0%. Representative

Fig. 2. Examples of centrosome staining and FISH analysis of bladder cancer specimens. Cells were immunostained with anti-pericentrin antibody. Antibody–antigen complexes were detected with Alexa 488-conjugated anti-rabbit IgG antibody. Cells were also stained with DAPI for visualization of DNA. FISH probes to pericentromeric regions of chromosomes 3 (CEP3), 7 (CEP7) and 17 (CEP17) (Vysis) were hybridized to touch preparations of nuclei from frozen tissues. Case 2: G1 pTa, CH 0 (n  3 ratio, 0%), CIN 0 (F-AVG ¼ 12%). Case 22: G3 pT1, CH III (n  3 ratio, 65.9%), CIN 2 (F-AVG ¼ 65%). (a) Centrosome staining: touch preparations were immunostained with anti-pericentrin antibody, and then stained with DAPI for visualization of nuclei (magnification: 400). (b) FISH analysis: FISH probes to pericentromeric regions of chromosomes 3, 7 and 17 were hybridized to touch preparations of nuclei. Probes were labeled with Spectrum Orange (CEP3), Spectrum Green (CEP7) and Spectrum Aqua (CEP17) for simultaneous analysis (magnification: 600). (c) Distribution of centrosome number. (d) FISH. (e) Laser scanning cytometry: DNA ploidy analysis.

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Fig. 2. (Continued ).

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Table 2 Relationships among centrosome hyperamplification, pathological grade, and chromosomal instability Pathology

CH CH CH CH

0 I II III

p

Chromosomal instability

G1

G2

G3

CI 0

CI 2

CI 3

4 1 0 0

2 0 1 3

0 3 2 7

4 3 0 0

0 1 3 3

0 1 2 5

0.03

p

0.0079

immunostaining patterns of centrosomes in normal bladder epithelium are shown in Fig. 1. Table 1 shows data of CH, CIN, DNA ploidy, and pathological grade for each case. We detected centrosome hyperamplification in 18 out of the 22 (81.8%) bladder cancer cases: CH 0, 4 cases (18.1%); CH I, 5 cases (22.7%); CH II, 5 cases (22.7%); CH III, 8 cases (36.4%). There was a significant positive correlation between CH and histological grade ( p ¼ 0:03, w2 test) (Table 2). 3.2. Chromosomal instability in bladder cancer We found marked variability in chromosome copy number among individual cells. Fig. 2 shows centrosome number per cell and chromosome number per cell for 2 representative cases: case 2 (pathologically lowgrade) and case 22 (pathologically high-grade). In case 2 (G1 pTa, CH 0), there were only 2 copies of each chromosome in most cells. In contrast, in case 22 (pT1G3, CH II), there was clearly a highly degree of variability in number of chromosomes per cell. Fig. 2e shows DNA ploidy data obtained by LSC in cases 2 and 22. There was a significant positive correlation between centrosome hyperamplification and chromosomal instability (p ¼ 0:0079, w2 test) (Table 2). Fig. 3 shows the relationship between CIN (Fig. 3a) and CH (Fig. 3b) for each individual sample, revealing a clear pattern of positive association between CIN and CH. The linear regression findings of positive correlation between CIN and CH for individual chromosomes are as follows: chromosome 3, r ¼ 0:667, p ¼ 0:0006; chromosome 7, r ¼ 0:708, p ¼ 0:0002; chromosome 17, r ¼ 0:620, p ¼ 0:0021 (Fig. 3c). 3.3. Analysis of centrosome replication cycle using LSC Fig. 4a shows the centrosome replication state during the cell cycle of case 2 (G1 pTa, CH 0, CIN 0). Each daughter cell (postmitotic cell) receives only 1 centrosome after mitotic division. Over 98% of G1 cells contained 1 centrosome juxtaposed to the nucleus. S-phase cells contained 2 centrosomes juxtaposed to

the nucleus. Duplication begins near the G1/S boundary, and is completed in G2. In this tumor, the centrosome replication cycle is well regulated. Fig. 4b shows the centrosome replication state during the cell cycle of case 16 (G3 pT2, CH III, CIN 1). A diploid clone was detected in this tumor. CH was observed in 12% to 13% of the diploid clone cells. In contrast, CH was observed in 25% to 67% of aneuploid clone cells. Fig. 4c shows the centrosome replication state during the cell cycle of case 22 (G3 pT1, CH III, CIN 2). Only an aneuploid clone was detected in this tumor. CH was observed in 43% to 88% of aneuploid clone cells. Abnormal centrosomes juxtaposed to the nucleus were detected in 43% of postmitotic cells. CH was observed in 88% of the G2-phase cells and 78% of the M-phase cells.

4. Discussion CIN is a major characteristic of cancer cells. It facilitates carcinogenesis by increasing the probability of mutations responsible for malignant phenotypes. In most cases, CIN results from defective mitosis, including unequal distribution of chromosomes to daughter cells and failure to undergo cytokinesis, both of which can generate aneuploid cells [2,3,5,6]. CH, which leads to formation of aberrant mitotic spindles, is generally considered to be a major cause of CIN in human cancers [2,3,5,6]. Recently, there have been many reports on the role of CH in human cancers [12,13,18–24], including cancers of the breast [12,13,18,19,23], brain [20,21], lung [20], bile duct [22], colon [20], and prostate [24]. Lingle et al. reported abnormal centrosome features including structural defects, absence of centrioles, elevated levels of pericentrin staining, supernumerary structures, and increased microtubule nucleation [23]. Similarly, in the present study, we detected supernumerary structures in bladder cancer. Centrosomes in normal bladder epithelium were of consistent size, shape and number. Tarapore et al. proposed that there are 2 major regulatory mechanisms in centrosome duplication, one involving correct timing of initiation, and the other involving prevention of reduplication of centrosomes once they have duplicated. Another important aspect of the centrosome duplication cycle is that it always occurs in precise coordination with DNA replication. Loss of centrosome duplication cycle regulation or uncoupling of the centrosome duplication cycle from the DNA replication cycle leads to abnormal amplification of centrosomes, which in turn profoundly

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Fig. 3. Analysis of chromosomal instability and centrosome hyperamplification. (a) Plot of variant fraction (F) for each sample. For each chromosome tested, the modal chromosome number (M) was determined, and the fraction of cells with chromosome numbers that differed from the mode (variant fraction: F) is shown. (b) Plot of centrosome hyperamplification (n  3) for each sample. (c) Relationship between centrosome hyperamplification and chromosomal instability for each chromosome (F3, F7 and F17 are the variant fractions of chromosomes 3, 7 and 17, respectively): chromosome 3, F3 ¼ 18:661 þ 0:985  CH, r ¼ 0:667, p ¼ 0:0006; chromosome 7, F7 ¼ 18:355 þ 0:877  CH, r ¼ 0:708, p ¼ 0:0002; chromosome 17, F17 ¼ 26:722 þ 0:576  CH, r ¼ 0:620, p ¼ 0:0021.

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affects mitotic fidelity [25]. Lingle et al. proposed a model in which centrosome defects alter the normal assembly, organization and function of mitotic spindles, leading to missegregation of chromosomes. Such events could result in gains and losses of chromosomes that, together with the growth selection pressure that tumors experience, provide a mechanism by which cells could accumulate tumor-promoting genes (activated oncogenes) and lose normal copies of tumor suppressor genes [23]. The p53 gene, located at 17p13.1, is the most frequently altered gene in human cancers [26]. In urothelial cancer, the p53 gene has frequently been found to be inactivated. Mutations of p53 are generally regarded as a contributing factor in bladder cancer progression [27,28]. The p53 tumor suppressor protein has been

shown to be involved in regulation of centrosome duplication, and loss or mutational inactivation of p53 results in centrosome hyperamplification [5,6]. Other genes are also involved in centrosome duplication, including cyclin E [13], p21 [12,13], BRCA1 [29] and Gadd45 [30]. In the present study, centrosome hyperamplification (CH) occurred in histopathologically malignant tumors, and in the future, it will be necessary to investigate the relationship between CH and p53 mutations in bladder cancer as well as the genes involved in centrosome replication. The present study was conducted using touch preparations, but the disadvantage of these preparations was that not all cells obtained were cancer cells, and some fibroblasts as well as lymphocytes were present. We recently reported that the morphology of tumor

Fig. 4. Centrosome replication cycle and cell cycle. Bladder cancer cells were stained with anti-pericentrin antibody (Alexa 488, green) and PI (red), as described in Section 2. Original magnification: 600. (a) Case 2, G1 pTa, CH 0 (n  3 ratio, 0%), CIN 0 (F-AVG ¼ 12%). The centrosome replication cycle was well regulated in this tumor. (b) Case 16, G3 pT2, CH III (n  3 ratio, 23.8%), CIN 1 (F-AVG ¼ 35%). Diploid clone was detected in this tumor. CH was observed in 12% to 13% of diploid clone cells and 25% to 67% of aneuploid clone cells. (c) Case 22, G3 pT1, CH III (n  3 ratio, 46.9%), CIN 2 (F-AVG ¼ 65%). Only an aneuploid clone was detected in this tumor. CH was observed in 43% to 88% of aneuploid clone cells.

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cells at each cell cycle could be examined by laser scanning cytometry using touch preparations of bladder cancer [16]. In the present study, it was possible to prepare single cell preparations using touch preparations, and to observe centrosomes juxtaposed to the nucleus without affecting cell morphology. Mainguene et al. reported that image cytometry on touch preparations of bladder biopsies is a simple and reliable procedure for assessing DNA ploidy in urothelial carcinomas, thus providing great sensitivity for detecting small aneuploid peaks and multiploid tumors [31]. Urothelial cancer progression is accompanied by increased chromosomal instability and aneuploidy [8,32,33]. Cytogenic studies reveal frequent alterations in a variety of chromosomes, including chromosomes 1, 3, 7, 9, 11, and 17 [33–35]. Homozygous deletion of the p16 gene at 9p21 is one of most common alterations in urothelial cancer and occurs early in papillary urothelial cancer development [36,37]. Pycha et al. reported that patients (primary non-invasive bladder

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cancer: pTa) with recurrent and progressive disease had a high incidence of trisomy 7 and 17 [34]. Sarosdy et al. performed multi-target fluorescent in situ hybridization using urine samples collected from patients with transitional cell carcinoma. According to their study, the sensitivity of the Multi-target FISH assay was superior to that of cytology in detecting recurrent transitional cell carcinoma using chromosomes 3, 7, 17 and band 9p21 [35]. The present study investigated the relationship between centrosome hyperamplification and chromosomes 3, 7 and 17, which are thought to be involved in urothelial cancer progression. In this study, CH and CIN were both detected in pathologically high-grade tumors. CH may be the main contributing factor in CIN, and may be involved in tumor development and progression in bladder cancer. Although Boveri named the centrosome (based on the hypothesis that it is the dynamic center of the cell) nearly 100 years ago [4], molecular analysis of centrosome amplification has started only recently, and much remains unknown about this organelle. Identification

Fig. 4. (Continued ).

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Fig. 4. (Continued ).

of mechanisms underlying replication of the centrosome should help clarify the mechanism of chromosomal instability in bladder cancer, and enable us to develop cancer therapeutics that target centrosome replication.

Acknowledgements We are grateful to Chizue Domiki for her excellent technical assistance. This study was supported in part by a Grant from the Ministry of Education, Japan.

References [1] Brinkley BR. Microtubule organizing centers. Annu Rev Cell Biol 1985;1:145–72. [2] Andersen SS. Molecular characteristics of centrosome. Int Rev Cytol 1999;187:51–109. [3] Lange BM, Faragher AJ, March P, Gull K. Centriole duplication and maturation in animal cells. Curr Top Dev Biol 2000;49:235–49. [4] Boveri T. The origin of malignant tumors. Baltimore: Williams & Wilkins; 1929.

[5] Fukasawa K, Choi T, Kuriyama R, Rulong S, Vande Woude GF. Abnormal centrosome amplification in the absence of p53. Science 1996;271:1744–7. [6] Fukasawa K, Wiener F, Vande Woude GF, Mai S. Genomic instability and apoptosis are frequent in p53 deficient mice. Oncogene 1997;15:1295–302. [7] Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in colorectal cancer cancers. Nature 1997;386:623–7.

K. Kawamura et al. / European Urology 43 (2003) 505–515 [8] Zhao J, Richter J, Wagner U, Roth B, Schraml P, Zellweger TR, et al. Chromosomal imbalances in non invasive papillary bladder neoplasms (pTa). Cancer Res 1999;59:4658–61. [9] Mostofi F, Sobin L, Torloni H. Histological typing of urinary bladder tumors. Geneva: World Health Organization; 1973. [10] Sobin LH, Wittekind C. UICC TNM classification of malignant tumors, 6th ed. Revised. New York: Wiley; 2002. p. 199–202. [11] Doxsey SJ, Stein P, Evance L, Calarco P, Kirschner M. Pericentrin, a highly conserved protein of centrosomes involved in microtubule organization. Cell 1994;76:639–50. [12] Carroll PE, Okuda M, Horn FH, Biddinger P, Stambrook PJ, Gleich LL, et al. Centrosome hyperamplification in human cancer: chromosome instability induced by p53 mutation and/or Mdm2 overexpression. Oncogene 1999;18:1935–44. [13] Mussman JG, Horn HF, Carroll PE, Okuda M, Tarapore P, Donehower LA, et al. Synergistic induction of centrosome hyperamplification by loss of p53 and cyclin E overexpression. Oncogene 2000;19:1635–46. [14] Halling CH, King W, Sokolova IA, Meyer RG, Burkhardt HM, Halling AC, et al. A comparison of cytology and fluorescence in situ hybridization for the detection of urothelial carcinoma. J Urol 2000;164:1768–75. [15] Kawamura K, Ikeda R, Suzuki K. The relationships among DNA ploidy type determined by laser scanning cytometry: The overexpression of p53 protein and the numerical aberrations of chromosome 7 in bladder cancer. Acta Urol Japonica 2000;46: 377–88. [16] Kawamura K, Tanaka T, Ikeda R, Fujikawa-Yamamoto K, Suzuki K. DNA ploidy analysis of urinary tract epithelial tumors by laser scanning cytometry. Anal Quant Cytol Histol 2000;22:26–30. [17] Kawamura K, Kobayashi Y, Tanaka T, Ikeda R, Fujikawa-Yamamoto K, Suzuki K. Intranuclear localization of proliferative cell nuclear antigen during the cell cycle in renal cell carcinoma. Anal Quant Cytol Histol 2000;22:26–30. [18] Lingle WL, Lutz WH, Ingle JN, Maihle NJ, Salisbury JL. Centrosome hypertrophy in human breast tumors: Implications for genomic stability and cell polarity. Cell Biol 1998;95:2950–5. [19] Lingle WL, Salisbury JL. Altered centrosome structure is associated with abnormal mitoses in human breast tumors. Am J Pathol 1999; 155:1941–51. [20] Phihan GA, Purohit A, Wallace J, Knecht H, Woda B, Quesenberry P, et al. Centrosome defects and genetic instability in malignant tumors. Cancer Res 1998;58:3974–85. [21] Weber RG, Bridger JM, Benner A, Weisenberger D, Ehemann V, Reifenberger G, et al. Centrosome amplification as a possible mechanism for numerical chromosome aberrations in cerebral primitive neuroectodermal tumors with TP53 mutations. Cytogenet Cell Genet 1998;83:266–8. [22] Kuo KK, Sato N, Mizumoto K, Maehara N, Yonematsu H, Ker CG, et al. Centrosome abnormalities in human carcinomas of the

[23]

[24]

[25] [26] [27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

515

gallbladder and intrahepatic and extrahepatic bile ducts. Hepatology 2000;31:59–64. Lingle WL, Barret SL, Negron CV, D’Assoro AB, Boeneman KB, Liu W, et al. Centrosome amplification drives instability in breast tumor development. Proc Natl Acad Sci USA 2002;99:1978–83. Pihan GA, Purohit A, Wallace J, Malhotra R, Liotta L, Doxsey SJ. Centrosome defects can account for cellular and genetic changes that characterize prostate cancer progression. Cancer Res 2001;61: 2212–9. Tarapore P, Fukasawa K. p53 mutation and mitotic infidelity. Cancer Invest 2000;18:148–55. Harris CC, Hollstein M. Clinical implication of the p53 tumorsuppressor gene. N J Med 1993;329:1318–27. Fujimoto K, Yamada Y, Okajima E, Kakizoe T, Sasaki H, Sugimura T, et al. Frequent association of p53 gene mutation in invasive bladder cancer. Cancer Res 1992;52:1393–8. Sidransky D, Escenbach AV, Tsai YC, Jones P, Summerhayes I, Marshall F, et al. Identification of p53 gene mutation in bladder cancers and urine samples. Science 1991;252:706–9. Xu X, Weaver Z, Linkle SP, Li C, Gotay J, Wang X-W, et al. Centrosome amplification and a defective G2–M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoformdeficient cells. Mol Cell 1999;3:389–95. Hollander MC, Sheikh MS, Bulavin DV, Lundgren K, AugeriHenmueller L, Shehee R, et al. Genomic instability in Gadd45adeficient mice. Nat Genet 1999;23:176–84. Mainguene C, Choquenet C, Deplano C, Gavelli A, Clement N, Vanzo E, et al. DNA ploidy by image cytometry in urothelial carcinomas. Comparison of touch imprints and paraffin-embedded biopsies from 31 patients. Anal Quant Cytol Histol 1997;19:437–42. Sao JL, Eatable Y, Marcela ID, Hajyan K, Jones PA, Shibata D. Bladder cancer genotype stability during clinical progression. Gene Chromosome Cancer 2000;29:26–32. Yu DS, Chen HI, Chang SY. Chromosomal aberrations in transitional cell carcinoma: its correlation with tumor behavior. Urol Int 2002; 69:129–35. Pycha A, Mian C, Haitel A, Hofbauer J, Wiener H, Marberger M. Fluorescence in situ hybridization identifies more aggressive types of primarily noninvasive (stage pTa) bladder cancer. J Urol 1997;157: 2116–9. Sarosdy MF, Schellhammer P, Bokinsky G, Kahn P, Chao R, Yore L, et al. Clinical evaluation of a multi-target fluorescent in situ hybridization assay for detection of bladder cancer. J Urol 2002; 168:1950–4. Spruck III CH, Ohneseit PF, Gonzalez-Zulueta M, Esrig D, Miyao N, Tsai YC, et al. Two molecular pathways to transitional cell carcinoma of the bladder. Cancer Res 1994;54:784–8. Tsai YC, Nichols PW, Hiti AL, Williams Z, Skinner DG, Jones PA. Allelic losses of chromosomes 9, 11, and 17 in human bladder cancer. Cancer Res 1990;50:44–7.