GFP-centrin as a marker for centriole dynamics in the human breast cancer cell line MCF-7.
Centrosome duplication plays an important role in genomic stability through bipolar spindle formation and equal chromosome segregation during mitosis. Defects in centrosome duplication and centrosome amplification correlate with aggressive tumors and aneuploidy. Cyclin-dependent cell cycle regulators play a key role in signaling centrosome duplication and the tumor suppressor genes p53, BRCA1 and BRCA2 are suspected to function at mitotic checkpoints that monitor centrosome duplication. The relationship between loss of hormone dependence in breast cancer, and signaling of centrosome duplication in tumor progression is not known. We have developed a MCF-7 cell line expressing GFP-centrin that allows direct visualization of centriole duplication during the cell cycle in living cells. GFP-centrin is expressed and selectively incorporated into the structure of both centrioles making them clearly visible in living cells. Our studies demonstrate three important aspects of recombinant GFP-centrin incorporation into centrioles. 1) GFP-centrin transfected cells grow normally in culture and show no adverse effect associated with GFP-centrin expression; 2) newly duplicated centrioles incorporate centrin during their genesis; and 3) GFP-centrin incorporation into centrioles does not grossly affect cell cycle progression, or centrosome function.
D'Assoro
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AB
Tumor Biology Program, Mayo Clinic Foundation, Rochester, MN 55905, USA.
Stivala
F
F
Barrett
S
S
Ferrigno
G
G
Salisbury
J L
JL
D000782:Aneuploidy; D014408:Biomarkers, Tumor / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002453:Cell Cycle / Q000235:genetics*; D002502:Centrioles / Q000235:genetics*; D005260:Female; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics*; D008954:Models, Biological; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; C424436:Cetn2 protein, mouse; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins