title: IGFBP-rP1 induces p21 expression through a p53-independent pathway, leading to cellular senescence of MCF-7 breast cancer cells.
abstract: OBJECTIVE
  Insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1), a member of the IGFBP super family, was identified as a potent tumor suppressor in several carcinomas. IGFBP-rP1 was down-regulated in primary breast cancer tissues and several breast cancer cell lines but overexpressed in senescent human mammary epithelial cells (HMECs), suggesting that IGFBP-rP1 might be a tumor suppressor in breast cancer and the tumor suppressor role of IGFBP-rP1 might be associated with cellular senescence. The aim of the study was to observe the effect of IGFBP-rP1 on cellular senescence and the molecular events mediating this biological effect in MCF-7 breast cancer cells.
  
  
  METHODS
  DNA fragment-encoding IGFBP-rP1 was cloned in-frame N-terminally to EGFP gene to generate IGFBP-rP1-EGFP fusion protein expression plasmid (pEGFP-IGFBP-rP1). The plasmid pEGFP-IGFBP-rP1 was then transfected into MCF-7 cells, and the proliferation, cell cycle distribution, cellular senescence, and cell cycle-related protein expression of MCF-7 cells were examined by trypan blue exclusion, flow cytometry, senescence-associated galactosidase (SA-β-gal) staining, and Western blot analysis, respectively. Two shRNA plasmid vectors against p21 or p53 gene were constructed and stably transfected into the MCF-7 cells to determine the involvement of p21 or p53 in cellular senescence induced by IGFBP-rP1.
  
  
  RESULTS
  Transfection of IGFBP-rP1 or addition of condition medium (CM) from IGFBP-rP1-transfected cells in MCF-7 cells caused induction of a variety of senescent phenotypes, such as decrease in cell proliferation, increase in G0/G1 cell cycle arrest cells, change in cell morphology, and increase in senescence-associated galactosidase (SA-β-gal) activity. IGFBP-rP1-induced growth arrest is associated with enhanced expression of the cyclin-dependent kinase inhibitor p21 and dephosphorylation of the retinoblastoma protein (pRB). Cell proliferation block and cellular senescence induction in response to IGFBP-rP1 were partially reversed by p21 knockdown in MCF-7 cells. Knockdown of p53 in MCF-7 cells did not influence the growth inhibition, cellular senescence, and p21 expression of the cells in response to IGFBP-rP1 transfection.
  
  
  CONCLUSIONS
  Results from this study suggest that cellular senescence induced by IGFBP-rP1 is mediated at least in part by p21 enhanced expression, which regulated through the p53-independent pathway. IGFBP-rP1 might be one of the key molecules that trigger cellular senescence in breast cancer. Restoration of IGFBP-rP1 function might have therapeutic significance in breast cancer.
authors.affiliation: Experimental Center of Molecular Medicine, Luohe Medical College, No 148 University Road, 462002 Luohe, Henan Province, China.
authors.forename: Shuguang
authors.initials: S
authors.lastname: Zuo
authors.forename: Chang
authors.initials: C
authors.lastname: Liu
authors.forename: Jianguo
authors.initials: J
authors.lastname: Wang
authors.forename: Fuqing
authors.initials: F
authors.lastname: Wang
authors.forename: Wanling
authors.initials: W
authors.lastname: Xu
authors.forename: Shao
authors.initials: S
authors.lastname: Cui
authors.forename: Lei
authors.initials: L
authors.lastname: Yuan
authors.forename: Xudong
authors.initials: X
authors.lastname: Chen
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Fan
authors.forename: Mingchen
authors.initials: M
authors.lastname: Cui
authors.forename: Guohua
authors.initials: G
authors.lastname: Song
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D059447:Cell Cycle Checkpoints / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D048708:Cell Growth Processes / Q000502:physiology; D045744:Cell Line, Tumor; D016922:Cellular Senescence / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000378:metabolism; D005260:Female; D016193:G1 Phase / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques / Q000379:methods; D016147:Genes, Tumor Suppressor; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016192:Resting Phase, Cell Cycle; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection / Q000379:methods; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018969:Insulin-Like Growth Factor Binding Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C102529:insulin-like growth factor binding protein-related protein 1