Download PDF
Horm Metab Res 2010; 42(3): 165-172
DOI: 10.1055/s-0029-1243190
Original Basic

© Georg Thieme Verlag KG Stuttgart · New York

Insulin-like Growth Factor Binding Protein-3 Induces G1 Cell Cycle Arrest with Inhibition of Cyclin-dependent Kinase 2 and 4 in MCF-7 Human Breast Cancer Cells

H.-S. Kim1 , W. J. Lee1 , S. W. Lee1 , H.-W. Chae1 , D. H. Kim1 , Y. Oh2
  • 1Department of Pediatrics, Yonsei University, Seoul, Korea
  • 2Department of Pathology, School of Medicine, Virginia Commonwealth University, Virginia, USA
Further Information

Publication History

received 06.04.2009

accepted 03.11.2009

Publication Date:
03 December 2009 (online)

Also available at
    Permissions and Reprints

Abstract

Insulin-like growth factor binding protein (IGFBP)-3 has been shown to potently inhibit proliferation of various cell types in an insulin-like growth factor (IGF)-independent manner. We have previously shown that IGFBP-3 induces apoptosis in an IGF-independent manner through the activation of caspases involved in a death receptor-mediated pathway in MCF-7 human breast cancer cells. In the present study, we present further evidence that IGFBP-3 inhibits cell proliferation through the induction of cell cycle arrest in the same cell line. Induction of IGFBP-3 in MCF-7 cells inhibited cell proliferation whereas presence of small interfering RNA against IGFBP-3 abolished cell inhibitory effect of IGFBP-3, suggesting that the observed growth inhibition is specific. Flow cytometry analysis showed that induced expression of IGFBP-3 led to an arrest of the cell cycle in G1-S phase. Western immunoblot analysis showed a significant decrease in the levels of the cell cycle-regulated proteins such as cyclin D1, cyclin D3, cyclin E, cyclin A, cyclin-dependent kinase (CDK) 2, CDK4, retinoblastoma protein (pRB), and phosph-pRB, suggesting a possible mechanism for cell cycle arrest by IGFBP-3. Northern blot analysis and real-time quantitative PCR demonstrated a significant decrease in gene expression of cyclin D1. Additional phosphorylation assay showed that IGFBP-3 decreased the phosphorylation activity of CDK2 and CDK4. These results show that cellular production of IGFBP-3 leads to G1 cell cycle arrest with inhibition of CDK2 and CDK4. Taken together, IGFBP-3 exerts its growth inhibitory action through not only induction of apoptosis but also the G1 cell cycle arrest in human breast cancer cells.

Key words

insulin-like growth factor-binding protein-3 - cell cycle arrest - breast cancer - cyclin D1 - CDK2 - CDK4

References

  • 1 Jones JI, Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions.  Endocr Rev. 1995;  16 3-34
    Google Scholar
  • 2 Kim H-S, Nagalla SR, Oh Y, Wilson E, Roberts Jr CT, Rosenfeld RG. Identification of a connective tissue growth factor as a member of the IGFBP superfamily.  Proc Natl Acad Sci USA. 1997;  94 12981-12986
    Google Scholar
  • 3 Hwa V, Oh Y, Rosenfeld RG. The insulin-like growth factor-binding protein (IGFBP)superfamily.  Endocr Rev. 1999;  20 761-787
    Google Scholar
  • 4 Firth SM, Baxter RC. Cellular actions of the insulin-like growth factor binding proteins.  Endocr Rev. 2002;  23 824-854
    Google Scholar
  • 5 Blat C, Delbe J, Villaudy J, Chatelain G, Golde A, Harel L. Inhibitory diffuse factor 45 bifunctional activity. As a cell growth inhibitor and as an insulin-like growth factor I-binding protein.  J Biol Chem. 1989;  264 12449-12454
    Google Scholar
  • 6 Liu L, Delbe J, Blat C, Zapf J, Harel L. Insulin-like growth factor binding protein-3 (IGFBP-3), an inhibitor of serum growth factors other than IGF-I and -II.  J Cell Physiol. 1992;  153 15-21
    Google Scholar
  • 7 Cohn P, Lamson G, Okajima T, Rosenfeld RG. Transfection of the human insulin-like growth factor binding protein-3 gene into Balb/c fibroblasts inhibits cellular growth.  Mol Endocrinol. 1993;  7 380-386
    Google Scholar
  • 8 Oh Y, Muller HL, Lamson G, Rosenfeld RG. Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578 T human breast cancer cells. Cell surface binding and growth inhibition.  J Biol Chem. 1993;  268 14964-14971
    Google Scholar
  • 9 Valentinis B, Bhala A, DeAngelis T, Baserga R, Cohen P. The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene.  Mol Endocrinol. 1995;  9 361-367
    Google Scholar
  • 10 Hong J, Zhang G, Dong F, Rechler MM. Insulin-like growth factor (IGF)-binding protein-3 mutants that do not bind IGF-I or IGF-II stimulate apoptosis in human prostate cancer cells.  J Biol Chem. 2002;  277 10489-10497
    Google Scholar
  • 11 Ali O, Cohen P, Lee K-W. Epidemiology and biology of insulin-like growth factor binding protein-3 (IGFBP-3) as an anti-cancer molecule.  Horm Metab Res. 2003;  35 726-733
    Google Scholar
  • 12 Rajah R, Valentinis B, Cohen P. Insulin-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-β1 on programmed cell death through a p53- and IGF-independent mechanism.  J Biol Chem. 1997;  272 12181-12188
    Google Scholar
  • 13 Nickerson T, Huynh H, Pollak M. Insulin-like growth factor binding protein-3 induces apoptosis in MCF-7 breast cancer cells.  Biochem Biophys Res Commun. 1997;  237 690-693
    Google Scholar
  • 14 Gill ZP, Perks CM, Newcomb PV, Holly JMP. Insulin-like growth factor-binding protein-3 (IGFBP-3) predisposes breast cancer cells to programmed cell death in a non-IGF-dependent manner.  J Biol Chem. 1997;  272 25602-25607
    Google Scholar
  • 15 Butt AJ, Firth SM, King MA, Baxter RC. Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells.  J Biol Chem. 2000;  275 39174-39181
    Google Scholar
  • 16 Kim H-S, Ingermann AR, Tsubaki J, Twigg SM, Walker GE, Oh Y. Insulin-like growth factor-binding protein 3 induces caspase-dependent apoptosis through a death receptor-mediated pathway in MCF-7 human breast cancer cells.  Cancer Res. 2004;  64 2229-2237
    Google Scholar
  • 17 Velez-Yanguas MC, Kalebic T, Maggi M, Kappel CC, Letterio J, Uskokovic M, Helman LJ. 1 alpha, 25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24–5531) modulation of insulin-like growth factor-binding protein-3 and induction of differentiation and growth arrest in a human osteosarcoma cell line.  J Clin Endocrinol Metab. 1996;  81 93-99
    Google Scholar
  • 18 Boyle BJ, Zhao XY, Cohen P, Feldman D. Insulin-like growth factor binding protein-3 mediates 1α,25-dihydroxyvitamin D3 growth inhibition in the LNCaP prostate cancer cell line through the cyclin-dependent kinase inhibitory protein, p21/WAF1.  J Urol. 2001;  165 1319-1324
    Google Scholar
  • 19 Firth SM, Fanayan S, Benn D, Baxter RC. Development of resistance to insulin-like growth factor binding protein-3 in transfected T47D breast cancer cells.  Biochem Biophys Res Commun. 1998;  246 325-329
    Google Scholar
  • 20 Butt AJ, Fraley KA, Firth SM, Baxter RC. IGF-binding protein-3-induced growth inhibition and apoptosis do not require cell surface binding and nuclear translocation in human breast cancer cells.  2002;  143 2693-2699
    Google Scholar
  • 21 Sherr CJ. Cancer cell cycles.  Science. 1996;  274 1672-1677
    Google Scholar
  • 22 Morgan DO. Cyclin-dependent kinases:engines, clocks, and microprocessors.  Annu Rev Cell Dev Biol. 1997;  13 261-291
    Google Scholar
  • 23 Johnson DG, Walker CL. Cyclins and cell cycle checkpoints.  Ann Rev Pharmacol Toxicol. 1999;  39 295-312
    Google Scholar
  • 24 Reed SI. Control of G1/S transition.  Cancer Surv. 1997;  29 7-23
    Google Scholar
  • 25 Weinberg RA. The retinoblastoma protein and cell cycle control.  Cell. 1995;  81 323-330
    Google Scholar
  • 26 Dyson N. The regulation of E2F by pRB-family proteins.  Genes Dev. 1998;  12 2245-2262
    Google Scholar
  • 27 Sakaguchi K, Yanagishita M, Takeuchi Y, Aurbach GD. Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.  J Biol Chem. 1991;  266 7270-7278
    Google Scholar
  • 28 Yu B, Lane ME, Pestell RG, Albanese C, Wadler S. Downregulation of cyclin D1 alters cdk 4- and cdk 2-specific phosphorylation of retinoblastoma protein.  Mol Cell Biol Res Commun. 2000;  3 352-359
    Google Scholar
  • 29 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delata Delta C(T)) Method.  Methods. 2001;  25 402-408
    Google Scholar
  • 30 Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells.  Cancer Res. 1996;  56 2973-2978
    Google Scholar
  • 31 Sherr CJ. Mammalian G1 cyclins.  Cell. 1993;  73 1059-1065
    Google Scholar
  • 32 Sweeney KJ, Musgrove EA, Watts CK, Sutherland RL. Cyclins and breast cancer.  Cancer Treat Res. 1996;  83 141-170
    Google Scholar
  • 33 Alenzi FQB. Links between apoptosis, proliferation and the cell cycle.  Br J Biomed Science. 2004;  61 99-102
    Google Scholar
  • 34 Quelle DE, Ashmun RA, Shurtleff SA, Kato JY, Bar-Sagi D, Roussel MF, Sherr CJ. Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts.  Genes Dev. 1993;  7 1559-1571
    Google Scholar
  • 35 Resnitzky D, Gossen M, Bujard H, Reed SI. Acceleration of the G1/S phase transition by expression of cyclin D1 and E with an inducible system.  Mol Cell Biol. 1994;  14 1669-1679
    Google Scholar
  • 36 Imoto M, Doki Y, Jian W, Han EK, Weinstein IB. Effects of cyclin D1 overexpression on G1 progression-related events.  Exp Cell Res. 1997;  236 173-180
    Google Scholar
  • 37 Tam SW, Theodoras AM, Shay JW, Draetta GF, Pagano M. Differential expression and regulation of Cyclin D1 protein in normal and tumor human cells:association with Cdk4 is required for Cyclin D1 function in G1 progression.  Oncogene. 1994;  9 2663-2674
    Google Scholar
  • 38 Dulic V, Lees E, Reed SI. Association of human cyclin E with a periodic G1-S phase protein kinase.  Science. 1992;  257 1958-1961
    Google Scholar
  • 39 Lovec H, Sewing A, Lucibello FC, Muller R, Moroy T. Oncogenic activity of cyclin D1 revealed through corporation with Ha-ras; link between cell cycle control and malignant transformation.  Oncogene. 1994;  9 323-326
    Google Scholar
  • 40 Hinds PW, Dowdy SF, Eaton EN, Arnold A, Weinberg RA. Function of a human cyclin gene as an oncogene.  Proc Natl Acad Sci U S A. 1994;  91 709-713
    Google Scholar
  • 41 Wang TC, Cardiff RD, Zukerberg L, Lees E, Arnold A, Schmidt EV. Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice.  Nature. 1994;  369 669-671
    Google Scholar
  • 42 Buckley MF, Sweeney KJ, Hamilton JA, Sini RL, Manning DL, Nicholson RI, deFazio A, Watts CK, Musgrove EA, Sutherland RL. Expression and amplification of cyclin genes in human breast cancer.  Oncogene. 1993;  8 2127-2133
    Google Scholar
  • 43 Bartkova J, Lukas J, Muller H, Strauss M, Gusterson B, Bartek J. Abnormal patterns of D-type cyclin expression and G1 regulation in human head and neck cancer.  Cancer Res. 1995;  55 949-956
    Google Scholar
  • 44 Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4.  Nature. 1993;  366 704-707
    Google Scholar
  • 45 El-Diery WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzier KW, Vogelstein B. WAF1, a potential mediators of p53 tumor suppression.  Cell. 1993;  75 817-825
    Google Scholar
  • 46 Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-cdk protein kinase activity, is related to p21.  Cell. 1994;  78 67-74
    Google Scholar
  • 47 Helin K. Regulation of cell proliferation by the E2F transcription factors.  Curr Opin Genet Dev. 1998;  8 28-35
    Google Scholar
  • 48 Nevins JR. Toward an understanding of the functional complexity of the E2F and retinoblastoma families.  Cell Growth Differ. 1998;  9 585-593
    Google Scholar

Correspondence

H.-S. KimMD, PhD 

Department of Pediatrics Gangnam Severance Hospital

Yonsei University 712 Earnju-Ro

Gangnam-Gu

Seoul 135-720

Korea

Phone: +82/2/2019 3355

Fax: +82/2/3461 9473

Email: kimho@yuhs.ac

      >