, Volume 71, Issue 1, pp 219–229 | Cite as

Nuclear co-expression of p21 and p27 induced effective cell-cycle arrest in T24 cells treated with BCG

  • Sumiko WatanabeEmail author
  • Shota Yamaguchi
  • Naoto Fujii
  • Natsuki Eguchi
  • Hitoshi Katsuta
  • Setsuo Sugishima
  • Tsuyoshi Iwasaka
  • Tsunehisa Kaku
Original Article


A proposed mechanism underlying the effect of bacillus Calmette–Guérin (BCG) treatment for bladder cancer cells is as follows: BCG-induced crosslinking of cell-surface receptors results in the activation of signaling cascades, including cell-cycle regulators. However, the clinical significance of cell-cycle regulators such as p21 and p27 is controversial. Here we investigated the relationship between BCG exposure and p21 and p27. We used confocal laser microscopy to examine the expression levels of pKi67, p21 and p27 in T24 cells (derived from human urothelial carcinoma) exposed six times to BCG. We performed dual immunofluorescence staining methods for p21 and p27 and observed the localization of nuclear and cytoplasm expressions. We investigated the priority of p27 over p21 regarding nuclear expression by using p27 Stealth RNAi™ (p27-siRNA). With 2-h BCG exposure, the nuclear-expression level of p21 and p27 was highest, while pKi67 was lowest. The percentage of double nuclear-expression of p21 and p27 in BCG cells was significantly higher than that in control cells during the 1st to 6th exposure (P < 0.05), and the expression of pKi67 showed the opposite of this pattern. Approximately 10% of the nuclear p21 was independent of p27, whereas the cytoplasmic p21 was dependent on p27. Our results suggested that the nuclear co-expression of p21 and p27 caused effective cell-cycle arrest, and thus the evaluation of the nuclear co-expression of p21 and p27 might help determine the effectiveness of BCG treatment.


BCG Cell-cycle arrest T24 cells Nuclear co-expression p27-siRNA 



This study was supported by JSPS KAKENHI Grant Nos. #JP25460459 and #JP17K08744.

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interest.


  1. Babjuk M, Böhle A, Burger M, Compérat E, Kaasinen E, Palou J, Rouprêt M, van Rhijn BWG, Shariat S, Sylvester R, Zigeuner R (2015) Guidelines on Non-muscle-invasive Bladder Cancer (Ta, T1 and CIS). Accessed 8 Apr 2017
  2. Besson A, Gurian-West M, Schmidt A, Hall A, Roberts JM (2004) p27Kip1 modulates cell migration through the regulation of RhoA activation. Genes Dev 18:862–876CrossRefGoogle Scholar
  3. Björklund MA, Vaahtomeri K, Peltonen K, Viollet B, Mäkelä TP, Band AM, Laiho M (2010) Non-CDK-bound p27(p27NCDK) is a marker for cell stress and is regulated through the AKT/PKB and AMPK-kinase pathways. Exp Cell Res 316:762–774CrossRefGoogle Scholar
  4. Blagosklonny MV (2002) Are p27 and p21 cytoplasmic oncoproteins? Cell Cycle 1:391–393CrossRefGoogle Scholar
  5. Chen F, Zhang G, Iwamoto Y, See WA (2005) BCG directly induces cell cycle arrest in human transitional carcinoma cell lines as a consequence of integrin cross-linking. BMC Urol 5:8. CrossRefGoogle Scholar
  6. Cooper MJ, Haluschak JJ, Johnson D, Schwartz S, Morrison LJ, Lippa M, Hatzivassiliou G, Tan J (1994) p53 mutations in bladder carcinoma cell lines. Oncol Res 6:569–579Google Scholar
  7. Cormio L, Tolve I, Annese P, Saracino A, Zamparese R, Sanguedolce F, Bufo P, Battaglia M, Selvaggi FP, Carrieri G (2009) Altered p53 and pRb expression is predictive of response to BCG treatment in T1G3 bladder cancer. Anticancer Res 29:4201–4204Google Scholar
  8. Hall MC, Chang SS, Dalbagni G, Pruthi RS, Seigne JD, Skinner EC, Wolf JS Jr, Schellhammer PF (2007) The Bladder Cancer Clinical Guideline Update Panel: guideline for the management of nonmuscle invasive bladder cancer (stages Ta, T1, and Tis): 2007 update. J Urol 178:2314–2330CrossRefGoogle Scholar
  9. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805–816CrossRefGoogle Scholar
  10. Herr HW, Milan TN, Dalbagni G (2015) BCG-refractory vs. BCG-relapsing non-muscle-invasive bladder cancer: a prospective cohort outcomes study. Urol Oncol 33:108.e1–108.e4CrossRefGoogle Scholar
  11. Kawasaki H, Taira K, Morris KV (2005) siRNA induced transcriptional gene silencing in mammalian cells. Cell Cycle 4:442–448CrossRefGoogle Scholar
  12. Kim YJ, Ha YS, Kim SK, Yoon HY, Lym MS, Kim MJ, Moon SK, Choi YH, Kim WJ (2010) Gene signatures for the prediction of response to Bacillus Calmette–Guérin immunotherapy in primary pT1 bladder cancers. Clin Cancer Res 16:2131–2137CrossRefGoogle Scholar
  13. Kiselyov A, Bunimovich-Mendrazitsky S, Startsev V (2015) Treatment of non-muscle invasive bladder cancer with Bacillus Calmette–Guérin (BCG): biological markers and simulation studies. BBA Clin 4:27–34CrossRefGoogle Scholar
  14. Kossatz U, Malek NP (2007) p27: tumor suppressor and oncogene …? Cell Res 17:832–833CrossRefGoogle Scholar
  15. Larrea MD, Hong F, Wander SA, da Silva TG, Helfman D, Lannigan D, Smith JA, Slingerland JM (2009) RSK1 drives p27Kip1 phosphorylation at T198 to promote RhoA inhibition and increase cell motility. Proc Natl Acad Sci USA 106:9268–9273CrossRefGoogle Scholar
  16. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, Slingerland JM (2002) PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 8:1153–1160CrossRefGoogle Scholar
  17. MacCallum DE, Hall PA (2000) The biochemical characterization of the DNA binding activity of pKi67. J Pathol 191:286–298CrossRefGoogle Scholar
  18. McAllister SS, Becker-Hapak M, Pintucci G, Pagano M, Dowdy SF (2003) Novel p27kip1 C-terminal scatter domain mediates Rac-dependent cell migration independent of cell cycle arrest functions. Mol Cell Biol 23:216–228CrossRefGoogle Scholar
  19. Orlando S, Gallastegui E, Besson A, Abril G, Aligué R, Pujol MJ, Bachs O (2015) p27Kip1 and p21Cip1 collaborate in the regulation of transcription by recruiting cyclin-Cdk complexes on the promoters of target genes. Nucleic Acids Res 43:6860–6873CrossRefGoogle Scholar
  20. Park J, Song C, Shin E, Hong JH, Kim CS, Ahn H (2013) Do molecular biomarkers have prognostic value in primary T1G3 bladder cancer treated with bacillus Calmette–Guérin intravesical therapy? Urol Oncol Semin Orig Investig 31:849–856CrossRefGoogle Scholar
  21. Philipp-Staheli J, Kim KH, Payne SR, Gurley KE, Liggitt D, Longton G, Kemp CJ (2002) Pathway-specific tumor suppression. Reduction of p27 accelerates gastrointestinal tumorigenesis in Apc mutant mice, but not in Smad3 mutant mice. Cancer Cell 1:355–368CrossRefGoogle Scholar
  22. Saitoh H, Mori K, Kudoh S, Itoh H, Takahashi N, Suzuki T (2002) BCG effects on telomerase activity in bladder cancer cell lines. Int J Clin Oncol 7:165–170CrossRefGoogle Scholar
  23. Sato M, Yanai H, Morito T, Oda W, Shin-no Y, Yamadori I, Tshushima T, Yoshino T (2011) Association between the expression pattern of p16, pRb and p53 and the response to intravesical bacillus Calmette–Guérin therapy in patients with urothelial carcinoma in situ of the urinary bladder. Pathol Int 61:456–460CrossRefGoogle Scholar
  24. See WA, Zhang G, Chen F, Cao Y (2010) p21 expression by human urothelial carcinoma cells modulates the phenotypic response to BCG. Urol Oncol 28:526–533CrossRefGoogle Scholar
  25. Shariat SF, Blenz C, Godoy G, Fradet Y, Ashfraq R, Karakiewicz PI, Isbarn H, Jeldres C, Rigaud J, Sagalowsky AI (2009) Predictive value of combined immunohistochemical markers in patients with pT1 urothelial carcinoma at radical cystectomy. J Urol 182:78–84CrossRefGoogle Scholar
  26. Stefano VD, Giacca M, Capogrossi MC, Crescenzi M, Martelli F (2011) Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle. JBC 286:8644–8654CrossRefGoogle Scholar
  27. Toyoshima H, Hunter T (1994) p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78:67–74CrossRefGoogle Scholar
  28. van Rhijn BW, Vis AN, van der Kwast TH, Kirkels WJ, Radvanyi F, Ooms EC, Chopin DK, Boevé ER, Jöbsis AC, Zwarthoff EC (2003) Molecular grading of urothelial cell carcinoma with fibroblast growth factor receptor 3 and MIB-1 is superior to pathologic grade for the prediction of clinical outcome. J Clin Oncol 21:1912–1921CrossRefGoogle Scholar
  29. van Rhijn BW, Liu L, Vis AN, Bostrom PJ, Zuiverloon TCM, Fleshner NE, Aa MNM, Alkhateeb SS, Bangma CH, Jewett MAS, Zwarthoff EC, Bapat B, van der Kwast TH, Zlotta AR (2012) Prognostic value of molecular markers, substage and European Organisation for the Research and Treatment of Cancer risk scores in primary T1 bladder cancer. BJU Int 110:1169–1176CrossRefGoogle Scholar
  30. Witjes JA (2006) Management of BCG failures in superficial bladder cancer: a review. Eur Urol 49:790–797CrossRefGoogle Scholar
  31. Wu FY, Wang SE, Sanders ME (2006) Reduction of cytosolic p27(Kip1) inhibits cancer cell motility, survival, and tumorigenicity. Cancer Res 66:2162–2172CrossRefGoogle Scholar
  32. Yoon MK, Mitrea DM, Ou L, Kriwacki RW (2012) Cell cycle regulation by the intrinsically disordered proteins p21 and p27. Biochem Soc Trans 40:981–988CrossRefGoogle Scholar
  33. Yu DS, Wu CL, Ping SY, Keng C, Shen KH (2015) Bacille Calmette–Guérin can induce cellular apoptosis of urothelial cancer directly through toll-like receptor 7 activation. Kaohsiung J Med Sci 31:391–397CrossRefGoogle Scholar
  34. Zhang G, Chen F, Cao Y, See WA (2007) Bacillus Calmette–Guérin induces p21 expression in human transitional carcinoma cell lines via an immediate early, p53 independent pathway. Urol Oncol 25:221–227CrossRefGoogle Scholar
  35. Zhou BP, Liao Y, Xia W, Spohn B, Lee MH, Hung MC (2001) Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in Her-2/neu-overexpressing cells. Nat Cell Biol 3:245–252CrossRefGoogle Scholar
  36. Zlotta AR, Noel JC, Fayt I, Drowart A, Van Vooren JP, Huygen K, Simon J, Schulman CC (1999) Correlation and prognostic significance of p53, p21WAF1/CAP1 and Ki-67 expression in patients with superficial bladder tumors treated with Bacillus Calmette–Guérin intravesical therapy. J Urol 161:792–798CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Sumiko Watanabe
    • 1
    Email author
  • Shota Yamaguchi
    • 2
  • Naoto Fujii
    • 2
  • Natsuki Eguchi
    • 2
  • Hitoshi Katsuta
    • 1
  • Setsuo Sugishima
    • 1
  • Tsuyoshi Iwasaka
    • 3
  • Tsunehisa Kaku
    • 1
  1. 1.Department of Health Sciences, Faculty of Medical SciencesKyushu UniversityFukuoka CityJapan
  2. 2.Department of Health Sciences, Graduate School of Medical SciencesKyushu UniversityFukuoka CityJapan
  3. 3.Department of Obstetrics and GynecologyTakagi HospitalOkawa CityJapan

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