Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins.
The advent of DNA microarray technology will likely have a major impact on the molecular classification and understanding of human cancer. Obtaining a global perspective of proteins expressed in cancer cells is considerably more challenging. Here we describe a microarray-based platform that can be used to measure protein levels and activities in a complex biological milieu such as a cellular lysate. Using a protein microarray made up of 1920 elements (146 distinct antibodies) we were able to monitor alterations of protein levels in LoVo colon carcinoma cells treated with ionizing radiation. The protein microarray approach revealed radiation-induced up-regulation of apoptotic regulators including p53, DNA fragmentation factor 40/caspase activated DNase, DNA fragmentation factor 45/inhibitor of caspase activated DNase, tumor necrosis factor-related apoptosis-inducing ligand, death receptor 5, decoy receptor 2, FLICE-like inhibitory protein, signal transducers and activators of transcription 1alpha, and uncoupling protein 2, among others. Consistent with this observation, an increased percentage of apoptosis was observed in irradiated LoVo cells. Interestingly, we also observed radiation-induced down-regulation of carcinoembryonic antigen, a prototypic cancer biomarker. Selected proteins assessed by microarray were validated by traditional immunoblotting. Taken together, our work suggests that protein/antibody microarrays will facilitate high-throughput proteomic studies of human cancer and carcinogenesis.
Sreekumar
A
A
Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Nyati
M K
MK
Varambally
S
S
Barrette
T R
TR
Ghosh
D
D
Lawrence
T S
TS
Chinnaiyan
A M
AM
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D051017:Apoptosis Regulatory Proteins; D002272:Carcinoembryonic Antigen / Q000096:biosynthesis / Q000235:genetics; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000096:biosynthesis / Q000235:genetics; D015536:Down-Regulation / Q000528:radiation effects; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects*; D006801:Humans; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D000075223:Poly-ADP-Ribose Binding Proteins; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D051017:Apoptosis Regulatory Proteins; D002272:Carcinoembryonic Antigen; D009363:Neoplasm Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins; D012333:RNA, Messenger; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; C505639:TNFRSF10B protein, human; D016159:Tumor Suppressor Protein p53; C109664:caspase-activated DNase inhibitor; C113696:DFFB protein, human; D003851:Deoxyribonucleases