Quantitative protein network monitoring in response to DNA damage.
Conventional molecular biology techniques have identified a large number of cell signaling pathways; however, the importance of these pathways often varies, depending on factors such as treatment type, dose, time after treatment, and cell type. Here, we describe a technique using "reverse-phase" protein lysate microarrays (RPAs) to acquire multiple dimensions of information on protein dynamics in response to DNA damage. Whole-cell lysates from three cellular stress treatments (IR, UV, and ADR) were collected at four doses per treatment, and each, in turn, at 10 time points, resulting in a single-slide RPA consisting of 10,240 features, including replicates. The dynamic molecular profile of 18 unique protein species was compared to phenotypic fate by FACS analysis for corresponding stress conditions. Our initial quantitative results in this new platform confirmed that (1) there is clear stress dose-response effect in p53 protein and (2) a comparison of the rates of increase of p21 and Cyclin D3/p53-Ser15 in response to DNA damage may be associated with the pattern of DNA content. This method, offering a quantitative time-course monitoring of protein expression levels, can provide an experimental reference for developing mathematical models of cell signaling dynamics. Although the present study focuses on the DNA damage-repair pathway, the technique is generally useful to the study of protein signaling.
Nishizuka
Satoshi
S
Molecular Therapeutics Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. nishizus@mail.nih.gov
Ramalingam
Sundhar
S
Spurrier
Brett
B
Washburn
Frank L
FL
Krishna
Ramya
R
Honkanen
Peter
P
Young
Lynn
L
Tsutomu
Shimura
S
Steeg
Patricia S
PS
Austin
John
J
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology; D045325:HCT116 Cells; D006801:Humans; D040081:Protein Array Analysis; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D018797:Cell Cycle Proteins; D011506:Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases