Skip to main content
SpringerLink
Log in

Non-nuclear localization of Ki-67 in human colorectal cancer cells grown as multicellular layers

  • Research Article
  • Published:
Archives of Pharmacal Research Aims and scope Submit manuscript

Abstract

Multicellular layers (MCL) of cancer cells is an in vitro 3-dimensional (3D) model that mimics avascular microregions of human solid tumors and has been shown to be useful in pharmacokinetic–pharmacodynamic studies of anticancer agents. We investigated whether Ki-67, which is widely used as a proliferation marker, can be used to evaluate changes in proliferative fractions following drug exposure in MCL of HT-29 human colorectal cancer cells. Ki-67 expression was monitored and compared between cancer cells cultured as monolayers or MCL. Drug distribution and Ki-67 expression were evaluated within MCL following exposure to doxorubicin and paclitaxel. Ki-67 expression was observed in the nuclei of proliferating cells in monolayers, tumor xenograft, and multicellular spheroids. In MCL, however, Ki-67 expression was detected in the membrane/cytoplasm as well as in the nucleus throughout MCL. Neither the location nor the level of expression changed following drug exposure, whereas drug-induced apoptosis increased. Our data show that membranous/cytoplasmic Ki-67 may not be valid as a marker for the proliferative activity of cells grown as MCL. Studies for non-nuclear localization and its mechanism in 3D in vitro models of other cell lines are warranted.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Al-Abd, A.M., J.H. Lee, S.Y. Kim, N. Kun, and H.J. Kuh. 2008. Novel application of multicellular layers culture for in situ evaluation of cytotoxicity and penetration of paclitaxel. Cancer Science 99: 423–431.

    Article  PubMed  CAS  Google Scholar 

  • Campbell-Thompson, M., I.J. Lynch, and B. Bhardwaj. 2001. Expression of estrogen receptor (ER) subtypes and ERbeta isoforms in colon cancer. Cancer Research 61: 632–640.

    PubMed  CAS  Google Scholar 

  • Clairwood, M., A. LaChance, and M. Murphy. 2011. Cytoplasmic immunoreactivity for Ki67 in an invasive amelanotic melanoma–foe or faux pas? Journal of Cutaneous Pathology 38: 318–319.

    Article  PubMed  Google Scholar 

  • Cowan, D.S., K.O. Hicks, and W.R. Wilson. 1996. Multicellular membranes as an in vitro model for extravascular diffusion in tumours. British Journal of Cancer 27: S28–S31.

    CAS  Google Scholar 

  • du Manoir, S., P. Guillaud, E. Camus, D. Seigneurin, and G. Brugal. 1991. Ki-67 labeling in postmitotic cells defines different Ki-67 pathways within the 2c compartment. Cytometry 12: 455–463.

    Article  PubMed  Google Scholar 

  • Elliott, N.T., and F. Yuan. 2011. A review of three-dimensional in vitro tissue models for drug discovery and transport studies. Journal of Pharmaceutical Sciences 100: 59–74.

    Article  PubMed  CAS  Google Scholar 

  • Faratian, D., A. Munro, C. Twelves, and J.M. Bartlett. 2009. Membranous and cytoplasmic staining of Ki67 is associated with HER2 and ER status in invasive breast carcinoma. Histopathology 54: 254–257.

    Article  PubMed  Google Scholar 

  • Friedrich, J., W. Eder, J. Castaneda, M. Doss, E. Huber, R. Ebner, and L.A. Kunz-Schughart. 2007. A reliable tool to determine cell viability in complex 3-d culture: The acid phosphatase assay. Journal of Biomolecular Screening 12: 925–937.

    Article  PubMed  CAS  Google Scholar 

  • Gerdes, J., H. Lemke, H. Baisch, H.H. Wacker, U. Schwab, and H. Stein. 1984. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. Journal of Immunology 133: 1710–1715.

    PubMed  CAS  Google Scholar 

  • Hattori, H. 2002. Sclerosing haemangioma of the lung is positive for MIB-1 in cell membrane and cytoplasmic staining pattern. Histopathology 40: 291–293.

    Article  PubMed  CAS  Google Scholar 

  • Hirokawa, M., and J.A. Carney. 2000. Cell membrane and cytoplasmic staining for MIB-1 in hyalinizing trabecular adenoma of the thyroid gland. The American Journal of Surgical Pathology 24: 575–578.

    Article  PubMed  CAS  Google Scholar 

  • Kee, N., S. Sivalingam, R. Boonstra, and J.M. Wojtowicz. 2002. The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. Journal of Neuroscience Methods 115: 97–105.

    Article  PubMed  CAS  Google Scholar 

  • Kyle, A.H., L.A. Huxham, J.H. Baker, H.E. Burston, and A.I. Minchinton. 2003. Tumor distribution of bromodeoxyuridine-labeled cells is strongly dose dependent. Cancer Research 63: 5707–5711.

    PubMed  CAS  Google Scholar 

  • Kyle, A.H., L.A. Huxham, A.S. Chiam, D.H. Sim, and A.I. Minchinton. 2004. Direct assessment of drug penetration into tissue using a novel application of three-dimensional cell culture. Cancer Research 64: 6304–6309.

    Article  PubMed  CAS  Google Scholar 

  • Leonardo, E., M. Volante, M. Barbareschi, and A. Cavazza. 2007. Dei Tos, A. P., Bussolati, G., and Papotti, M., Cell membrane reactivity of MIB-1 antibody to Ki67 in human tumors: Fact or artifact? Applied Immunohistochemistry Molecular Morphology 15: 220–223.

    Article  PubMed  CAS  Google Scholar 

  • MacCallum, D.E., and P.A. Hall. 1999. Biochemical characterization of pKi67 with the identification of a mitotic-specific form associated with hyperphosphorylation and altered DNA binding. Experimental Cell Research 252: 186–198.

    Article  PubMed  CAS  Google Scholar 

  • Mayot, D., M.C. Béné, C. Perrin, and G.C. Faure. 1993. Restricted expression of Ki-67 in cholesteatoma epithelium. Archives of Otolaryngology: Head and Neck Surgery 119: 656–658.

    Article  PubMed  CAS  Google Scholar 

  • Minchinton, A.I., and I.F. Tannock. 2006. Drug penetration in solid tumours. Nature Reviews Cancer 6: 583–592.

    Article  PubMed  CAS  Google Scholar 

  • Ortiz-Rey, J.A., A. Peteiro, and A. Fernández-Costas. 2009. Gómez de María, C., and Antón, I., Cell membrane and cytoplasmic immunoreactivity of MIB-1 in invasive breast carcinoma. Applied Immunohistochemistry Molecular Morphology 17: 563–564.

    Article  PubMed  CAS  Google Scholar 

  • Padrón, J.M., C.L. van der Wilt, K. Smid, E. Smitskamp-Wilms, H.H. Backus, P.E. Pizao, G. Giaccone, and G.J. Peters. 2000. The multilayered postconfluent cell culture as a model for drug screening. Critical Reviews in Oncology/Hematology 36: 141–157.

    Article  PubMed  Google Scholar 

  • Schmidt, M.H., R. Broll, H.P. Bruch, S. Finniss, O. Bögler, and M. Duchrow. 2004. Proliferation marker pKi-67 occurs in different isoforms with various cellular effects. Journal of Cellular Biochemistry 91: 1280–1292.

    Article  PubMed  CAS  Google Scholar 

  • Scholzen, T., and J. Gerdes. 2000. The Ki-67 protein: From the known and the unknown. Journal of Cellular Physiology 182: 311–322.

    Article  PubMed  CAS  Google Scholar 

  • Singh, B., R. Li, L. Xu, A. Poluri, S. Patel, A.R. Shaha, D. Pfister, E. Sherman, A. Goberdhan, R.M. Hoffman, and J. Shah. 2002. Prediction of survival in patients with head and neck cancer using the histoculture drug response assay. Head Neck 24: 437–442.

    Article  PubMed  Google Scholar 

  • Tashiro, T., M. Hirokawa, H. Harada, S. Yokoyama, and T. Sano. 2002. Cell membrane expression of MIB-1 in salivary gland pleomorphic adenoma. Histopathology 41: 559–561.

    Article  PubMed  CAS  Google Scholar 

  • Trédan, O., C.M. Galmarini, K. Patel, and I.F. Tannock. 2007. Drug resistance and the solid tumor microenvironment. Journal of the National Cancer Institute 99: 1441–1454.

    Article  PubMed  Google Scholar 

  • Urruticoechea, A., I.E. Smith, and M. Dowsett. 2005. Proliferation marker Ki-67 in early breast cancer. Journal of Clinical Oncology 23: 7212–7220.

    Article  PubMed  CAS  Google Scholar 

  • van Dierendonck, J.H., R. Keijzer, C.J. van de Velde, and C.J. Cornelisse. 1989. Nuclear distribution of the Ki-67 antigen during the cell cycle: Comparison with growth fraction in human breast cancer cells. Cancer Research 49: 2999–3006.

    PubMed  Google Scholar 

  • Walker, R.A., and R.S. Camplejohn. 1988. Comparison of monoclonal antibody Ki-67 reactivity with grade and DNA flow cytometry of breast carcinomas. British Journal of Cancer 57: 281–283.

    Article  PubMed  CAS  Google Scholar 

  • Wong, S.F., Y. da No, Y.Y. Choi, D.S. Kim, B.G. Chung, and S.H. Lee. 2011. Concave microwell based size-controllable hepatosphere as a three-dimensional liver tissue model. Biomaterials 32: 8087–8096.

    Article  PubMed  CAS  Google Scholar 

  • Xu, H., Y. Yu, D. Marciniak, A.K. Rishi, F.H. Sarkar, O. Kucuk, and A.P. Majumdar. 2005. Epidermal growth factor receptor (EGFR)-related protein inhibits multiple members of the EGFR family in colon and breast cancer cells. Molecular Cancer Therapeutics 4: 435–442.

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by Mid-career Researcher Program through NRF Grant funded by the MEST (No. 2012R1A2A2A01003361) and the Nano-Biotechnology Project (Regenomics, 2011-0007745) of the Korean Science and Engineering Foundation (KOSEF). Yu-Jin Kim was sponsored by the Ministry of Knowledge Economy (MKE) and Korea Industrial Technology Foundation (KOTEF) through the Human Resource Training Project for Strategic Technology

Author information

Authors and Affiliations

  1. Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-ku, Seoul, 137-701, South Korea

    Yu-Jin Kim & Hyo-Jeong Kuh

  2. Department of Biomedical Engineering, College of Health Science, Korea University, Jeongneung 3-dong, Seongbuk-gu, Seoul, 136-703, South Korea

    Sang-Hoon Lee

  3. Divison of Pharmaceutical Sciences, College of Pharmacy, Chung-Ang University, 221 Heuksuk-dong, Dongjak-gu, Seoul, 156-756, South Korea

    Jaehwi Lee

Authors
  1. Yu-Jin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sang-Hoon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jaehwi Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hyo-Jeong Kuh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hyo-Jeong Kuh.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, YJ., Lee, SH., Lee, J. et al. Non-nuclear localization of Ki-67 in human colorectal cancer cells grown as multicellular layers. Arch. Pharm. Res. 36, 634–640 (2013). https://doi.org/10.1007/s12272-013-0061-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12272-013-0061-2

Keywords

Search

Navigation