Global Investigation of p53-induced Apoptosis Through Quantitative Proteomic Profiling Using Comparative Amino Acid-coded Tagging

Abstract

p53-induced apoptosis plays a pivotal role in the suppression of tumorigenesis, and mutations in p53 have been found in more than 50% of human tumors. By comparing the proteome of a human colorectal cancer cell transfected with inducible p53 (DLD-1.p53) with that of the control DLD-1 cell line using amino acid-coded mass tagging (AACT)-assisted mass spectrometry, we have broadly identified proteins that are upregulated at the execution stage of the p53-mediated apoptosis. In cell culturing, the deuterium-labeled (heavy) amino acids were incorporated into the proteome of the induced DLD-1.p53 cells, whereas the DLD-1.vector cells were grown in the unlabeled medium. In high-throughput LC-ESI-MS/MS analyses, the AACT-containing peptides were paired with their unlabeled counterparts, and their relative spectral intensities, reflecting the differential protein expression, were quantified. In addition, our novel AACT-MS method utilized a number of different heavy amino acids as internal markers that significantly increased the peptide sequence coverage for both quantitation and identification purposes. As a result, we were able to identify differentially regulated protein isozymes that would be difficult to distinguish by ICAT-MS methods and to obtain a large dataset of the proteins with altered expression in the late stage of p53-induced apoptosis. The regulated proteins we identified are associated with several distinct functional categories: cell cycle arrest and p53 binding, protein chaperoning, plasma membrane dynamics, stress response, antioxidant enzymes, and anaerobic glycolysis. This result suggests that the p53-induced apoptosis involves the systematic activation of multiple pathways that are glycolysis-relevant, energy-dependent, oxidative stress-mediated, and possibly mediated through interorganelle crosstalks.

Differential antiproliferative mechanisms of novel derivative of benzimidazo[1,2-α]quinoline in colon cancer cells depending on their p53 status

“…Several reports dealing with identification of p53 targets in colon carcinoma cells by proteomic tools have given prominence to Annexin I in p53-mediated apoptosis (22,23); for example, proteomic analysis of mitomycin C -treated HCT 116 cells carrying wt-p53 revealed Annexin I in the group of up-regulated proteins containing p53-binding site (23). Our proteomic analysis revealed an elevated expression of this protein in treated HCT 116 cells as well.…”

Externalized Glycolytic Enzymes Are Novel, Conserved, and Early Biomarkers of Apoptosis

“…Still, our results are consistent with findings from other proteomic studies. For example, Gu et al (59) observed that several glycolytic enzymes (including EnoA and GAPDH) were up-regulated at late times of death (triggered by activation of p53), and Jin et al (45) found EnoA and GAPDH in microparticles recovered from human plasma, …”

TRPC-mediated actin-myosin contraction is critical for BBB disruption following hypoxic stress

“…Differential expression of proteins in cells exposed to hypoxia (1% O 2, 1, 3, or 6 h) was determined by mass spectrometry (MS) using the iTRAQ system of isotopic labeling for the identification and quantitation of proteins (20,21,68). In brief, 100-g protein mixtures from each treatment group (control, 1, 3, and 6 h hypoxia) were precipitated with acetone.…”