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Volume 43, Issue 5, Pages 505-515 (May 2003) Centrosome Hyperamplification and Chromosomal Instability in Bladder Cancer K. Kawamura, M. Moriyama, N. Shiba, M. Ozaki, T. Tanaka, T. Nojima, K. Fujikawa-Yamamoto, R. Ikeda, K. Suzuki European Urology Volume 43, Issue 5, Pages 505-515 (May 2003) DOI: 10.1016/S0302-2838(03)00056-3

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×. European Urology 2003 43, 505-515DOI: (10.1016/S0302-2838(03)00056-3)

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=12%). Case 22: G3 pT1, CH III (n≥3 ratio, 65.9%), CIN 2 (F-AVG=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. European Urology 2003 43, 505-515DOI: (10.1016/S0302-2838(03)00056-3)

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=12%). Case 22: G3 pT1, CH III (n≥3 ratio, 65.9%), CIN 2 (F-AVG=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. European Urology 2003 43, 505-515DOI: (10.1016/S0302-2838(03)00056-3)

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=18.661+0.985×CH, r=0.667, p=0.0006; chromosome 7, F7=18.355+0.877×CH, r=0.708, p=0.0002; chromosome 17, F17=26.722+0.576×CH, r=0.620, p=0.0021. European Urology 2003 43, 505-515DOI: (10.1016/S0302-2838(03)00056-3)

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=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=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=65%). Only an aneuploid clone was detected in this tumor. CH was observed in 43% to 88% of aneuploid clone cells. European Urology 2003 43, 505-515DOI: (10.1016/S0302-2838(03)00056-3)

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=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=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=65%). Only an aneuploid clone was detected in this tumor. CH was observed in 43% to 88% of aneuploid clone cells. European Urology 2003 43, 505-515DOI: (10.1016/S0302-2838(03)00056-3)

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=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=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=65%). Only an aneuploid clone was detected in this tumor. CH was observed in 43% to 88% of aneuploid clone cells. European Urology 2003 43, 505-515DOI: (10.1016/S0302-2838(03)00056-3)