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DNA aneuploidy and centrosome amplification in canine tumor cell lines
Tissue and Cell 61 (2019) 67–71
Contents lists available at ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
DNA aneuploidy and centrosome amplification in canine tumor cell lines a
b
a
T
b,c
Yoshifumi Endo , Manabu Watanabe , Nozomi Miyajima-Magara , Maki Igarashi , ⁎ Manabu Mochizukia, Ryohei Nishimuraa, Sumio Suganob, Nobuo Sasakia, Takayuki Nakagawaa, a
Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan c Biochemistry Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo, Tokyo 104-0045, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Centrosome amplification DNA aneuploidy Canine tumor cell line
DNA aneuploidy, the altered DNA content of a cell, is a common feature of canine tumors. However, it is unclear whether aneuploid DNA in canine tumor cells show centrosome amplification (CA), which contributes to numerical and structural chromosome aberrations that result in DNA aneuploidy. Here, we evaluated whether DNA aneuploidy and CA occur concurrently in canine tumor cell lines. Centrosome numbers were evaluated in 18 canine tumor cell lines by immunocytochemistry with anti-γ-tubulin antibody, and DNA content was evaluated by flow cytometry using propidium iodide. A total of 15 cell lines showed DNA aneuploidy, and CA was observed in 5 of these 15 cell lines. Together, our results suggest that DNA aneuploidy in canine tumor cells might be explained at least in part by CA. In addition, cell lines with CA may be useful tools to examine the detailed relationship between CA and DNA aneuploidy and the molecular mechanism of CA in canine tumor.
1. Introduction DNA aneuploidy, characterized by altered nuclear DNA content, is a common feature of human cancer. This abnormality results from numerical and structural chromosomal aberrations and is an inevitable result of chromosome instability, which plays an important role in carcinogenesis (Danielsen et al., 2016). DNA aneuploidy is also found in several canine tumors (Ayl et al., 1992; Clemo et al., 1994; Fox et al., 1990; Hellmen et al., 1993), but the underlying mechanisms are not fully elucidated. The centrosome is the major microtubule-organizing center in animal cells and plays a key role in accurate chromosome segregation during mitosis. Impaired regulation of the numerical integrity of centrosomes can result in centrosome amplification (CA), which is the presence of more than two centrosomes in a cell. CA leads to aberrant mitotic spindles with multiple (> 2) spindle poles and unequal segregation of chromosomes to daughter cells, resulting in DNA aneuploidy (Fukasawa, 2005; Pihan, 2013). CA is found in several human cancers and is associated with chromosome instability and DNA aneuploidy (Ghadimi et al., 2000; Pihan et al., 1998). Two studies have described CA in canine tumors (Kaneko et al., 2005; Setoguchi et al., 2001), revealing CA in two osteosarcoma (OSA)
cell lines and in some canine tumors using immunocytochemistry and immunohistochemistry with anti γ-tubulin antibody. However, DNA aneuploidy was not concurrently evaluated with CA. There are also very few reports of canine tumor cell lines showing CA. Therefore, in this study, we characterized four major canine tumor cell lines for both DNA aneuploidy and CA to gain insights into the mechanisms of DNA aneuploidy in canine tumors. 2. Material and methods 2.1. Cells This study analyzed 18 cell lines: 6 mammary gland tumor (MGT) cell lines (CHMp, CHMm, CIPp, CIPm, CNMp, and CNMm), 3 OSA cell lines (POS, HOS, and OOS), 6 melanoma cell lines (CMeC1, CMeC2, KMeC, LMeC, CMM1, and CMM2), and 3 mast cell tumor (MCT) cell lines (CoMS, VI-MC, and CM-MC). The origin of these cell lines was indicated in the supplementary material 1. Cells were maintained as described previously (Hong et al., 1998; Inoue et al., 2004; Ishiguro et al., 2001; Kadosawa et al., 1994; Ohashi et al., 2002; Takahashi et al., 2001; Uyama et al., 2006).
⁎ Corresponding author at: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail address: [email protected] (T. Nakagawa).
https://doi.org/10.1016/j.tice.2019.09.003 Received 10 July 2019; Received in revised form 3 September 2019; Accepted 13 September 2019 Available online 19 September 2019 0040-8166/ © 2019 Elsevier Ltd. All rights reserved.
Tissue and Cell 61 (2019) 67–71
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2.2. Immunocytochemistry
Table 1 DNA ploidy and centrosome amplification of canine tumor cell lines. Tumors
Cell Lines
DNA index
DNA ploidy
Frequency of CA(%)
Normal Melanoma
PBMC CMeC1 CMeC2
1.0 2.42 ± 0.022 1.82 ± 0.011
Diploid Aneuploid Aneuploid (multiploid)
3.0 2.7 5.0
Aneuploid Aneuploid Aneuploid (multiploid)
8.0 3.7 2.0
Near-diploid Aneuploid Aneuploid Aneuploid Near-diploid Near-diploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid
2.7 30.7 3.0 16.3 3.7 2.3 4.0 3.3 4.0 6.7 20.3 41.3 20.0
clone1 clone2
KMeC LMeC CMM1
clone1 clone2
Osteosarcoma
MGT
MCT
CMM2 POS HOS OOS CHM-p CHM-m CIP-p CIP-m CNM-p CNM-m CoMS VI-MC CM-MC
2.37 2.05 1.64 1.89
± ± ± ±
0.019 0.014 0.011 0.011
2.85 ± 0.029 1.06 ± 0.004 1.73 ± 0.005 1.29 ± 0.005 1.83 ± 0.075 1.08 ± 0.007 1.09 ± 0.009 2.03 ± 0.023 2.04 ± 0.021 1.12 ± 0.012 2.04 ± 0.021 1.49 ± 0.003 1.53 ± 0.014 1.42 ± 0.015
All cells except MCT cells were cultured on chamber slides (Lab-Tek II; Thermo Fisher Scientific; IL, USA) to sub-confluent growth, washed with phosphate buffer solution (PBS), and fixed in ice-cold methanol for 5 min at −20 °C. MCT cells and peripheral blood mononuclear cells (PBMCs) were washed with PBS, centrifuged on poly-L-lysine-coated slides (Matsunami; Osaka, Japan) for 5 min at 470 × g at 25 °C, and fixed in the same manner. Cells were then washed with PBS and permeabilized with 1% NP-40 in PBS for 5 min. Cells were first incubated with blocking solution (10% normal goat serum in PBS) for 1 h, then probed with mouse anti-γ-tubulin monoclonal antibody (1:1000), (GTU-88; Sigma-Aldrich; MO, USA) for 1 h. The antigen-antibody complex was detected after incubation for 1 h at room temperature with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen; CA, USA). Samples were washed three times with PBS for 30 min after incubation and nuclei were counterstained with propidium iodide (Sigma-Aldrich). Slide were mounted with mounting medium (Invitrogen) and visualized under a confocal laser scanning microscopy (LSM700; Zeiss; Oberkochen, Germany). Number of centrosomes per cell was counted for 300 cells in 3 different fields of view. Mean values were calculated, and multiple statistical comparisons were made by analysis of variance (ANOVA) using Microsoft Excel for Windows. Statistically significant differences in the number of centrosomes per cell were estimated using Bonferroni’s multiple comparison test. P < 0.05 was considered statistically significant.
*
*
* * *
* P < 0.05 compared with the value of PBMCs.
Fig. 1. Representative results of flow cytometric analysis of DNA ploidy. a) Peripheral blood mononuclear cells (PBMCs) derived from a healthy dog as an internal standard (IS). b) Near-diploid cell line CMM2 (DNA index 〈DI〉 = 1.06). Aneuploid cell lines c) CM-MC (DI = 1.42) and d) POS (DI = 1.73). 68
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Fig. 2. Centrosome amplification in canine tumor cell lines. Immunostaining of centrosomes in peripheral blood mononuclear cells (PBMCs, A) of a clinically healthy dog and representative cell lines of each canine tumors, the MCT cell line VI-MCs (B), the OSA cell line POS (C), the MGT cell line CHM-p (D), the melanoma cell line CMeC1 (E). For example, Panels A show immunostained centrosomes, and panels A´ show the images of nucleus counterstained with PI DNA dye in the same microscopic fields. Panels A˝ show the merged image. Panel A, D and E show the centrosome of PBMCs and cell lines without CA that is visible as a tiny, discrete dot signal close to the nucleus. Panel B and C show cell lines with CA, which contain over three centrosomes. Bar =10 μm. Arrows show centrosomes.
content was measured using a BD FACScan flow cytometer (Becton Dickinson; CA, USA) configured with a 488-nm argon ion laser. PBMCs from healthy beagles were used as an internal DNA ploidy standard. PBMCs were isolated by gradient centrifugation and stained with propidium iodide as described for tumor cells. A total of 10,000 events per sample were obtained, and each measurement was repeated three times.
2.3. Flow cytometry Cells were harvested by trypsinization, washed once with PBS, and fixed in 70% ethanol for 30 min. Fixed cells were briefly pelleted, resuspended in PBS, and treated with 100 U/mL RNase A (QIAGEN; Hilden, Germany) for 30 min at 37 °C. Propidium iodide (50 μg/mL; Sigma-Aldrich) was added to the cell suspension prior to analysis. DNA 69
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specimens of both tumors (Kaneko et al., 2005; Setoguchi et al., 2001). Abnormal centrosomes were detected in some of the most common human cancers, and in some cancers, CA was found at the earliest stage of cancer development (Chan, 2011; Pihan et al., 2003). A recent report demonstrated the oncogenic potential of centrosome amplification in mammals (Levine et al., 2017). Therefore, it has been suggested that DNA aneuploidy caused by CA may be involved in the tumorigenesis and malignant transformation of both tumors. In conclusion, our study is the first report on CA in canine tumor cell lines with DNA aneuploidy. Our results favor the hypothesis that CA contributes to chromosome instability and, ultimately, to DNA aneuploidy in a canine tumor, especially OSA and MCT. However, a limitation of this study is that it only evaluated cell lines. Therefore, further investigation including cytogenetic analysis of cell lines and tissue specimens is needed to confirm the relationship between CA and DNA aneuploidy in canine tumors (OSA and MCT).
DNA histograms were analyzed using Flowjo software (TreeStar software; CA, USA). The median coefficient of variation (CV) of the G1 peaks of the PBMC standards was 2.0%. The DNA index (DI) was calculated by determining the ratio of the DNA content of the aneuploid peak to the DNA content of the diploid peak in the DNA histogram. The DNA aneuploid population had a DI > 1.1 N and the near-diploid population had 1.0 < DI < 1.1. DNA multiploidy was indicated by two or more distinct aneuploid G0/G1 peaks. 3. Results Table 1 shows the DI values in all canine malignant tumor cell lines. Of the 18 cell lines, 15 had a DI > 1.1 N, indicating the presence of DNA aneuploidy (Fig. 1). One melanoma (KMeC) and three MGT (CIPp, CIPm, and CNMm) cell lines showed approximately tetraploid DNA content, which is characterized by twice the cellular DNA content of normal cells. Near-diploid DNA content was observed in three cell lines (CMM2, CHMp, and CHMm) (Fig. 1). Fig. 2 displays the immunostaining for γ-tubulin in PBMCs which used as controls and representative cell lines of each tumors. In PBMCs, CHM-p (MGT) and CMeC1 (melanoma), the centrosome was recognized as a tiny, discrete dot signal always adjacent to the nucleus, whereas VIMCs (MCT) and POS (osteosarcoma) had more than three dot signals, indicating CA. Among the 15 DNA aneuploid cell lines, the percentages of cells with CA were significantly higher in 5 cell lines (all MCT and two of the three OSA cell lines) compared with those in PBMCs (P < 0.05; Table 1). Of the MCT cell lines with CA, 41% were VI-MCs, 20% were CoMS, and 20% were CM-MCs; of the OSA cell lines with CA, 31% were POS and 16% were OOS. In the remaining aneuploid cell lines and near-diploid cell lines, the percentages of cells with CA was very low, with no statistical difference compared to those in PBMCs.
Formatting of funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest None. Acknowledgement We would like to thank Editage (www.editage.com) for English language editing. References
4. Discussion Ayl, R.D., Couto, C.G., Hammer, A.S., Weisbrode, S., Ericson, J.G., Mathes, L., 1992. Correlation of DNA ploidy to tumor histologic grade, clinical variables, and survival in dogs with mast cell tumors. Vet. Pathol. 29, 386–390. Chan, J.Y., 2011. A clinical overview of centrosome amplification in human cancers. Int. J. Biol. Sci. 7, 1122–1144. Clemo, F.A., DeNicola, D.B., Carlton, W.W., Morrison, W.B., Walker, E., 1994. Flow cytometric DNA ploidy analysis in canine transitional cell carcinoma of urinary bladders. Vet. Pathol. 31, 207–215. Danielsen, H.E., Pradhan, M., Novelli, M., 2016. Revisiting tumour aneuploidy - the place of ploidy assessment in the molecular era. Nat. Rev. Clin. Oncol. 13, 291–304. Fox, M.H., Armstrong, L.W., Withrow, S.J., Powers, B.E., LaRue, S.M., Straw, R.C., Gillette, E.L., 1990. Comparison of DNA aneuploidy of primary and metastatic spontaneous canine osteosarcomas. Cancer Res. 50, 6176–6178. Fukasawa, K., 2005. Centrosome amplification, chromosome instability and cancer development. Cancer Lett. 230, 6–19. Ghadimi, B.M., Sackett, D.L., Difilippantonio, M.J., Schrock, E., Neumann, T., Jauho, A., Auer, G., Ried, T., 2000. Centrosome amplification and instability occurs exclusively in aneuploid, but not in diploid colorectal cancer cell lines, and correlates with numerical chromosomal aberrations. Genes Chromosomes Cancer 27, 183–190. Hellmen, E., Bergstrom, R., Holmberg, L., Spangberg, I.B., Hansson, K., Lindgren, A., 1993. Prognostic factors in canine mammary tumors: a multivariate study of 202 consecutive cases. Vet. Pathol. 30, 20–27. Hong, S.H., Kadosawa, T., Mochizuki, M., Matsunaga, S., Nishimura, R., Sasaki, N., 1998. Establishment and characterization of two cell lines derived from canine spontaneous osteosarcoma. J. Vet. Med. Sci. 60, 757–760. Inoue, K., Ohashi, E., Kadosawa, T., Hong, S.H., Matsunaga, S., Mochizuki, M., Nishimura, R., Sasaki, N., 2004. Establishment and characterization of four canine melanoma cell lines. J. Vet. Med. Sci. 66, 1437–1440. Ishiguro, T., Kadosawa, T., Mori, K., Takagi, S., Okumura, M., Fujinaga, T., 2001. Establishment and characterization of a new canine mast cell tumor cell line. J. Vet. Med. Sci. 63, 1031–1034. Kadosawa, T., Nozaki, K., Sasaki, N., Takeuchi, A., 1994. Establishment and characterization of a new cell line from a canine osteosarcoma. J. Vet. Med. Sci. 56, 1167–1169. Kaneko, N., Okuda, M., Toyama, N., Oikawa, T., Watanabe, M., Kanaya, N., Yazawa, M., Hasegawa, K., Morimoto, M., Hayashi, T., Une, S., Nakaichi, M., Taura, Y., Tsujimoto, H., Inokuma, H., 2005. Detection of centrosome amplification as a surrogate marker of dysfunction in the p53 pathway -p53 gene mutation or MDM2 overexpression. Vet. Comp. Oncol. 3, 203–210. Kops, G.J., Weaver, B.A., Cleveland, D.W., 2005. On the road to cancer: aneuploidy and the mitotic checkpoint. Nat. Rev. Cancer 5, 773–785. Levine, M.S., Bakker, B., Boeckx, B., Moyett, J., Lu, J., Vitre, B., Spierings, D.C., Lansdorp,
In the present study, we found DNA aneuploidy in a majority of canine tumor cell lines, with some cell lines revealing significantly increased centrosome numbers. To our knowledge, this is the first report confirming CA in DNA aneuploid canine tumor cell lines. DNA aneuploidy results from numerical and structural chromosome aberrations. CA participates in promoting unequal chromosome segregation and chromosome breakage by formation of multipolar spindle and merotelic chromosome attachment (Pihan, 2013). Therefore, our results might provide further evidence for the possible connection between DNA aneuploidy and CA in canine tumor cells. We found 10 DNA aneuploid canine tumor cell lines in which CA did not frequently occur. Of these 10 cell lines, 4 revealed approximately tetraploid content. In previous reports, some of these cell lines showed numerical chromosome abnormalities (Inoue et al., 2004), which are caused not only by CA, but also by impaired spindle assembly checkpoint (SAC) response (Kops et al., 2005). The SAC is a signaling cascade that arrests the cell cycle in mitosis, even when a single chromosome is not properly attached to the microtubule and correctly aligned at the metaphase plate (Musacchio and Salmon, 2007). Thus, the SAC monitors the accurate segregation of sister chromatids into the dividing daughter cells during mitosis. If the SAC is defective, mitotic slippage in mitotic cells leads to progression to the next cell cycle without chromosomal segregation or cell division, resulting in tetraploidy (Tanaka et al., 2018). Therefore, impaired SAC or other unknown mechanisms might partly contribute to DNA aneuploidy in canine tumor cells; however, further research is needed. CA was frequently observed in DNA aneuploid cells of OSA and MCT cell lines, although in few cells. Previous reports have shown DNA aneuploidy in both tumors, particularly in MCT; DNA aneuploidy has been associated with the pathological grade of tumors and survival time (Ayl et al., 1992; Fox et al., 1990). Moreover, although the number of specimens examined is very small, CA has been observed in clinical 70
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chromosome instability occur together in pre-invasive carcinomas. Cancer Res. 63, 1398–1404. Setoguchi, A., Okuda, M., Nishida, E., Yazawa, M., Ishizaka, T., Hong, S.H., Hisasue, M., Nishimura, R., Sasaki, N., Yoshikawa, Y., Masuda, K., Ohno, K., Tsujimoto, H., 2001. Results of hyperamplification of centrosomes in naturally developing tumors of dogs. Am. J. Vet. Res. 62, 1134–1141. Takahashi, T., Kitani, S., Nagase, M., Mochizuki, M., Nishimura, R., Morita, Y., Sasaki, N., 2001. IgG-mediated histamine release from canine mastocytoma-derived cells. Int. Arch. Allergy Immunol. 125, 228–235. Tanaka, K., Goto, H., Nishimura, Y., Kasahara, K., Mizoguchi, A., Inagaki, M., 2018. Tetraploidy in cancer and its possible link to aging. Cancer Sci. 109, 2632–2640. Uyama, R., Nakagawa, T., Hong, S.H., Mochizuki, M., Nishimura, R., Sasaki, N., 2006. Establishment of four pairs of canine mammary tumor cell lines derived from primary and metastatic origin and their E-cadherin expression. Vet. Comp. Oncol. 4, 104–113.
P.M., Cleveland, D.W., Lambrechts, D., Foijer, F., Holland, A.J., 2017. Centrosome amplification is sufficient to promote spontaneous tumorigenesis in mammals. Dev. Cell 40, 313–322. Musacchio, A., Salmon, E.D., 2007. The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8, 379–393. Ohashi, E., Inoue, K., Kagechika, H., Hong, S.H., Takayuki, Nakagawa, Takahashi, T., Mochizuki, M., Nishimura, R., Sasaki, N., 2002. Effect of natural and synthetic retinoids on the proliferation and differentiation of three canine melanoma cell lines. J. Vet. Med. Sci. 64, 169–172. Pihan, G.A., 2013. Centrosome dysfunction contributes to chromosome instability, chromoanagenesis, and genome reprograming in cancer. Front. Oncol. 3, 277. Pihan, G.A., Purohit, A., Wallace, J., Knecht, H., Woda, B., Quesenberry, P., Doxsey, S.J., 1998. Centrosome defects and genetic instability in malignant tumors. Cancer Res. 58, 3974–3985. Pihan, G.A., Wallace, J., Zhou, Y., Doxsey, S.J., 2003. Centrosome abnormalities and
71
Contents lists available at ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
DNA aneuploidy and centrosome amplification in canine tumor cell lines a
b
a
T
b,c
Yoshifumi Endo , Manabu Watanabe , Nozomi Miyajima-Magara , Maki Igarashi , ⁎ Manabu Mochizukia, Ryohei Nishimuraa, Sumio Suganob, Nobuo Sasakia, Takayuki Nakagawaa, a
Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan c Biochemistry Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo, Tokyo 104-0045, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Centrosome amplification DNA aneuploidy Canine tumor cell line
DNA aneuploidy, the altered DNA content of a cell, is a common feature of canine tumors. However, it is unclear whether aneuploid DNA in canine tumor cells show centrosome amplification (CA), which contributes to numerical and structural chromosome aberrations that result in DNA aneuploidy. Here, we evaluated whether DNA aneuploidy and CA occur concurrently in canine tumor cell lines. Centrosome numbers were evaluated in 18 canine tumor cell lines by immunocytochemistry with anti-γ-tubulin antibody, and DNA content was evaluated by flow cytometry using propidium iodide. A total of 15 cell lines showed DNA aneuploidy, and CA was observed in 5 of these 15 cell lines. Together, our results suggest that DNA aneuploidy in canine tumor cells might be explained at least in part by CA. In addition, cell lines with CA may be useful tools to examine the detailed relationship between CA and DNA aneuploidy and the molecular mechanism of CA in canine tumor.
1. Introduction DNA aneuploidy, characterized by altered nuclear DNA content, is a common feature of human cancer. This abnormality results from numerical and structural chromosomal aberrations and is an inevitable result of chromosome instability, which plays an important role in carcinogenesis (Danielsen et al., 2016). DNA aneuploidy is also found in several canine tumors (Ayl et al., 1992; Clemo et al., 1994; Fox et al., 1990; Hellmen et al., 1993), but the underlying mechanisms are not fully elucidated. The centrosome is the major microtubule-organizing center in animal cells and plays a key role in accurate chromosome segregation during mitosis. Impaired regulation of the numerical integrity of centrosomes can result in centrosome amplification (CA), which is the presence of more than two centrosomes in a cell. CA leads to aberrant mitotic spindles with multiple (> 2) spindle poles and unequal segregation of chromosomes to daughter cells, resulting in DNA aneuploidy (Fukasawa, 2005; Pihan, 2013). CA is found in several human cancers and is associated with chromosome instability and DNA aneuploidy (Ghadimi et al., 2000; Pihan et al., 1998). Two studies have described CA in canine tumors (Kaneko et al., 2005; Setoguchi et al., 2001), revealing CA in two osteosarcoma (OSA)
cell lines and in some canine tumors using immunocytochemistry and immunohistochemistry with anti γ-tubulin antibody. However, DNA aneuploidy was not concurrently evaluated with CA. There are also very few reports of canine tumor cell lines showing CA. Therefore, in this study, we characterized four major canine tumor cell lines for both DNA aneuploidy and CA to gain insights into the mechanisms of DNA aneuploidy in canine tumors. 2. Material and methods 2.1. Cells This study analyzed 18 cell lines: 6 mammary gland tumor (MGT) cell lines (CHMp, CHMm, CIPp, CIPm, CNMp, and CNMm), 3 OSA cell lines (POS, HOS, and OOS), 6 melanoma cell lines (CMeC1, CMeC2, KMeC, LMeC, CMM1, and CMM2), and 3 mast cell tumor (MCT) cell lines (CoMS, VI-MC, and CM-MC). The origin of these cell lines was indicated in the supplementary material 1. Cells were maintained as described previously (Hong et al., 1998; Inoue et al., 2004; Ishiguro et al., 2001; Kadosawa et al., 1994; Ohashi et al., 2002; Takahashi et al., 2001; Uyama et al., 2006).
⁎ Corresponding author at: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail address: [email protected] (T. Nakagawa).
https://doi.org/10.1016/j.tice.2019.09.003 Received 10 July 2019; Received in revised form 3 September 2019; Accepted 13 September 2019 Available online 19 September 2019 0040-8166/ © 2019 Elsevier Ltd. All rights reserved.
Tissue and Cell 61 (2019) 67–71
Y. Endo, et al.
2.2. Immunocytochemistry
Table 1 DNA ploidy and centrosome amplification of canine tumor cell lines. Tumors
Cell Lines
DNA index
DNA ploidy
Frequency of CA(%)
Normal Melanoma
PBMC CMeC1 CMeC2
1.0 2.42 ± 0.022 1.82 ± 0.011
Diploid Aneuploid Aneuploid (multiploid)
3.0 2.7 5.0
Aneuploid Aneuploid Aneuploid (multiploid)
8.0 3.7 2.0
Near-diploid Aneuploid Aneuploid Aneuploid Near-diploid Near-diploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid
2.7 30.7 3.0 16.3 3.7 2.3 4.0 3.3 4.0 6.7 20.3 41.3 20.0
clone1 clone2
KMeC LMeC CMM1
clone1 clone2
Osteosarcoma
MGT
MCT
CMM2 POS HOS OOS CHM-p CHM-m CIP-p CIP-m CNM-p CNM-m CoMS VI-MC CM-MC
2.37 2.05 1.64 1.89
± ± ± ±
0.019 0.014 0.011 0.011
2.85 ± 0.029 1.06 ± 0.004 1.73 ± 0.005 1.29 ± 0.005 1.83 ± 0.075 1.08 ± 0.007 1.09 ± 0.009 2.03 ± 0.023 2.04 ± 0.021 1.12 ± 0.012 2.04 ± 0.021 1.49 ± 0.003 1.53 ± 0.014 1.42 ± 0.015
All cells except MCT cells were cultured on chamber slides (Lab-Tek II; Thermo Fisher Scientific; IL, USA) to sub-confluent growth, washed with phosphate buffer solution (PBS), and fixed in ice-cold methanol for 5 min at −20 °C. MCT cells and peripheral blood mononuclear cells (PBMCs) were washed with PBS, centrifuged on poly-L-lysine-coated slides (Matsunami; Osaka, Japan) for 5 min at 470 × g at 25 °C, and fixed in the same manner. Cells were then washed with PBS and permeabilized with 1% NP-40 in PBS for 5 min. Cells were first incubated with blocking solution (10% normal goat serum in PBS) for 1 h, then probed with mouse anti-γ-tubulin monoclonal antibody (1:1000), (GTU-88; Sigma-Aldrich; MO, USA) for 1 h. The antigen-antibody complex was detected after incubation for 1 h at room temperature with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen; CA, USA). Samples were washed three times with PBS for 30 min after incubation and nuclei were counterstained with propidium iodide (Sigma-Aldrich). Slide were mounted with mounting medium (Invitrogen) and visualized under a confocal laser scanning microscopy (LSM700; Zeiss; Oberkochen, Germany). Number of centrosomes per cell was counted for 300 cells in 3 different fields of view. Mean values were calculated, and multiple statistical comparisons were made by analysis of variance (ANOVA) using Microsoft Excel for Windows. Statistically significant differences in the number of centrosomes per cell were estimated using Bonferroni’s multiple comparison test. P < 0.05 was considered statistically significant.
*
*
* * *
* P < 0.05 compared with the value of PBMCs.
Fig. 1. Representative results of flow cytometric analysis of DNA ploidy. a) Peripheral blood mononuclear cells (PBMCs) derived from a healthy dog as an internal standard (IS). b) Near-diploid cell line CMM2 (DNA index 〈DI〉 = 1.06). Aneuploid cell lines c) CM-MC (DI = 1.42) and d) POS (DI = 1.73). 68
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Fig. 2. Centrosome amplification in canine tumor cell lines. Immunostaining of centrosomes in peripheral blood mononuclear cells (PBMCs, A) of a clinically healthy dog and representative cell lines of each canine tumors, the MCT cell line VI-MCs (B), the OSA cell line POS (C), the MGT cell line CHM-p (D), the melanoma cell line CMeC1 (E). For example, Panels A show immunostained centrosomes, and panels A´ show the images of nucleus counterstained with PI DNA dye in the same microscopic fields. Panels A˝ show the merged image. Panel A, D and E show the centrosome of PBMCs and cell lines without CA that is visible as a tiny, discrete dot signal close to the nucleus. Panel B and C show cell lines with CA, which contain over three centrosomes. Bar =10 μm. Arrows show centrosomes.
content was measured using a BD FACScan flow cytometer (Becton Dickinson; CA, USA) configured with a 488-nm argon ion laser. PBMCs from healthy beagles were used as an internal DNA ploidy standard. PBMCs were isolated by gradient centrifugation and stained with propidium iodide as described for tumor cells. A total of 10,000 events per sample were obtained, and each measurement was repeated three times.
2.3. Flow cytometry Cells were harvested by trypsinization, washed once with PBS, and fixed in 70% ethanol for 30 min. Fixed cells were briefly pelleted, resuspended in PBS, and treated with 100 U/mL RNase A (QIAGEN; Hilden, Germany) for 30 min at 37 °C. Propidium iodide (50 μg/mL; Sigma-Aldrich) was added to the cell suspension prior to analysis. DNA 69
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specimens of both tumors (Kaneko et al., 2005; Setoguchi et al., 2001). Abnormal centrosomes were detected in some of the most common human cancers, and in some cancers, CA was found at the earliest stage of cancer development (Chan, 2011; Pihan et al., 2003). A recent report demonstrated the oncogenic potential of centrosome amplification in mammals (Levine et al., 2017). Therefore, it has been suggested that DNA aneuploidy caused by CA may be involved in the tumorigenesis and malignant transformation of both tumors. In conclusion, our study is the first report on CA in canine tumor cell lines with DNA aneuploidy. Our results favor the hypothesis that CA contributes to chromosome instability and, ultimately, to DNA aneuploidy in a canine tumor, especially OSA and MCT. However, a limitation of this study is that it only evaluated cell lines. Therefore, further investigation including cytogenetic analysis of cell lines and tissue specimens is needed to confirm the relationship between CA and DNA aneuploidy in canine tumors (OSA and MCT).
DNA histograms were analyzed using Flowjo software (TreeStar software; CA, USA). The median coefficient of variation (CV) of the G1 peaks of the PBMC standards was 2.0%. The DNA index (DI) was calculated by determining the ratio of the DNA content of the aneuploid peak to the DNA content of the diploid peak in the DNA histogram. The DNA aneuploid population had a DI > 1.1 N and the near-diploid population had 1.0 < DI < 1.1. DNA multiploidy was indicated by two or more distinct aneuploid G0/G1 peaks. 3. Results Table 1 shows the DI values in all canine malignant tumor cell lines. Of the 18 cell lines, 15 had a DI > 1.1 N, indicating the presence of DNA aneuploidy (Fig. 1). One melanoma (KMeC) and three MGT (CIPp, CIPm, and CNMm) cell lines showed approximately tetraploid DNA content, which is characterized by twice the cellular DNA content of normal cells. Near-diploid DNA content was observed in three cell lines (CMM2, CHMp, and CHMm) (Fig. 1). Fig. 2 displays the immunostaining for γ-tubulin in PBMCs which used as controls and representative cell lines of each tumors. In PBMCs, CHM-p (MGT) and CMeC1 (melanoma), the centrosome was recognized as a tiny, discrete dot signal always adjacent to the nucleus, whereas VIMCs (MCT) and POS (osteosarcoma) had more than three dot signals, indicating CA. Among the 15 DNA aneuploid cell lines, the percentages of cells with CA were significantly higher in 5 cell lines (all MCT and two of the three OSA cell lines) compared with those in PBMCs (P < 0.05; Table 1). Of the MCT cell lines with CA, 41% were VI-MCs, 20% were CoMS, and 20% were CM-MCs; of the OSA cell lines with CA, 31% were POS and 16% were OOS. In the remaining aneuploid cell lines and near-diploid cell lines, the percentages of cells with CA was very low, with no statistical difference compared to those in PBMCs.
Formatting of funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest None. Acknowledgement We would like to thank Editage (www.editage.com) for English language editing. References
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In the present study, we found DNA aneuploidy in a majority of canine tumor cell lines, with some cell lines revealing significantly increased centrosome numbers. To our knowledge, this is the first report confirming CA in DNA aneuploid canine tumor cell lines. DNA aneuploidy results from numerical and structural chromosome aberrations. CA participates in promoting unequal chromosome segregation and chromosome breakage by formation of multipolar spindle and merotelic chromosome attachment (Pihan, 2013). Therefore, our results might provide further evidence for the possible connection between DNA aneuploidy and CA in canine tumor cells. We found 10 DNA aneuploid canine tumor cell lines in which CA did not frequently occur. Of these 10 cell lines, 4 revealed approximately tetraploid content. In previous reports, some of these cell lines showed numerical chromosome abnormalities (Inoue et al., 2004), which are caused not only by CA, but also by impaired spindle assembly checkpoint (SAC) response (Kops et al., 2005). The SAC is a signaling cascade that arrests the cell cycle in mitosis, even when a single chromosome is not properly attached to the microtubule and correctly aligned at the metaphase plate (Musacchio and Salmon, 2007). Thus, the SAC monitors the accurate segregation of sister chromatids into the dividing daughter cells during mitosis. If the SAC is defective, mitotic slippage in mitotic cells leads to progression to the next cell cycle without chromosomal segregation or cell division, resulting in tetraploidy (Tanaka et al., 2018). Therefore, impaired SAC or other unknown mechanisms might partly contribute to DNA aneuploidy in canine tumor cells; however, further research is needed. CA was frequently observed in DNA aneuploid cells of OSA and MCT cell lines, although in few cells. Previous reports have shown DNA aneuploidy in both tumors, particularly in MCT; DNA aneuploidy has been associated with the pathological grade of tumors and survival time (Ayl et al., 1992; Fox et al., 1990). Moreover, although the number of specimens examined is very small, CA has been observed in clinical 70
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P.M., Cleveland, D.W., Lambrechts, D., Foijer, F., Holland, A.J., 2017. Centrosome amplification is sufficient to promote spontaneous tumorigenesis in mammals. Dev. Cell 40, 313–322. Musacchio, A., Salmon, E.D., 2007. The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8, 379–393. Ohashi, E., Inoue, K., Kagechika, H., Hong, S.H., Takayuki, Nakagawa, Takahashi, T., Mochizuki, M., Nishimura, R., Sasaki, N., 2002. Effect of natural and synthetic retinoids on the proliferation and differentiation of three canine melanoma cell lines. J. Vet. Med. Sci. 64, 169–172. Pihan, G.A., 2013. Centrosome dysfunction contributes to chromosome instability, chromoanagenesis, and genome reprograming in cancer. Front. Oncol. 3, 277. Pihan, G.A., Purohit, A., Wallace, J., Knecht, H., Woda, B., Quesenberry, P., Doxsey, S.J., 1998. Centrosome defects and genetic instability in malignant tumors. Cancer Res. 58, 3974–3985. Pihan, G.A., Wallace, J., Zhou, Y., Doxsey, S.J., 2003. Centrosome abnormalities and
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