Quantitative and qualitative immunofluorescence studies of neoplastic cells transfected with a construct encoding p53-EGFP.
The p53 protein is a major regulator of cell cycle progression and apoptosis. We used a p53-enhanced green fluorescent protein (EGFP) construct for transfections into human breast cancer (MCF-7) cells. Cells expressing p53-EGFP showed an increased apoptotic index compared to cells transfected with EGFP alone. Interestingly, apoptotic cells showed localization of p53-EGFP to both nuclei and cytoplasm, whereas non-apoptotic cells usually only showed nuclear localization of p53-EGFP. This result is in agreement with the hypothesis that p53 induces apoptosis by interaction with both nuclear and cytoplasmic targets. Transfected p53-deficient osteosarcoma cells were used for immunofluorescence quantitation. The intensity of immunofluorescence for either p53 or EGFP showed excellent linear correlation to the EGFP autofluorescence, proving that measurements of immunofluorescence intensities can be used for determining endogenous protein levels.
Mortensen
K
K
Division of Cell Biology, Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
Larsson
L I
LI
D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins