Abstract
BackgroundCancer cells are characterized by a deregulated cell cycle that facilitates abnormal proliferation by allowing cells to by-pass tightly regulated molecular checkpoints such as the G1/S restriction point. To facilitate early diagnosis and the identification of new drug targets, current research efforts focus on studies that could lead to the development of protein panels that collectively can improve the effectiveness of our response to the detection of a life-threatening disease.MethodsEstrogen-responsive MCF-7 cells were cultured and arrested by serum deprivation in the G1-stage of the cell cycle, and fractionated into nuclear and cytoplasmic fractions. The protein extracts were trypsinized and analyzed by liquid chromatography - mass spectrometry (MS), and the data were interpreted with the Thermo Electron Bioworks software. Biological characterization of the data, selection of cancer markers, and identification of protein interaction networks was accomplished with a combination of bioinformatics tools provided by GoMiner, DAVID and STRING.ResultsThe objective of this work was to explore via MS proteomic profiling technologies and bioinformatics data mining whether randomly identified cancer markers can be associated with the G1-stage of the cell cycle, i.e., the stage in which cancer cells differ most from normal cells, and whether any functional networks can be identified between these markers and placed in the broader context of cell regulatory pathways. The study enabled the identification of over 2000 proteins and 153 cancer markers, and revealed for the first time that the G1-stage of the cell cycle is not only a rich source of cancer markers, but also a host to an intricate network of functional relationships within the majority of these markers. Three major clusters of interacting proteins emerged: (a) signaling, (b) DNA repair, and (c) oxidative phosphorylation.ConclusionsThe identification of cancer marker regulatory components that act not alone, but within networks, represents an invaluable resource for elucidating the moxlecular mechanisms that govern the uncontrolled proliferation of cancer cells, as well as for catalyzing the development of protein panels with biomarker and drug target potential, screening tests with improved sensitivity and specificity, and novel cancer therapies aimed at pursuing multiple drug targets.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-710) contains supplementary material, which is available to authorized users.
Highlights
Cancer cells are characterized by a deregulated cell cycle that facilitates abnormal proliferation by allowing cells to by-pass tightly regulated molecular checkpoints such as the G1/S restriction point
To increase the number of identifiable proteins and generate a comprehensive map of the biological processes that unfold in the G1-stage of the cell cycle, the MCF-7 cells were separated into nuclear and cytoplasmic fractions
Three biological replicates were prepared to enable a confident selection of identifiable proteins, and five LC-mass spectrometry (MS)/MS technical replicates were performed to maximize the number of identifiable proteins and the number of spectral counts per protein [7,8]
Summary
Cancer cells are characterized by a deregulated cell cycle that facilitates abnormal proliferation by allowing cells to by-pass tightly regulated molecular checkpoints such as the G1/S restriction point. DNA damage checkpoints at the G1/ S and G2/M transition boundaries, and spindle checkpoints during the M-phase, have been recognized These checkpoints allow either for DNA repair, or correct chromosome alignment on the mitotic spindle, respectively, before the steps of the cell cycle can proceed. Key to its regulation is the phosphorylation of the retinoblastoma protein (pRb or RB1) by active cyclin D-CDK4/6 and cyclin E-CDK2 complexes in early and late G1, respectively, an event that results in the release of E2F transcription factors that signal the cell to continue into the S-phase, replicate and proliferate. As the mechanisms used by cancer cells to escape the Rrestriction point continue to remain unclear, the objective of this study was to use mass spectrometry technologies to generate a comprehensive map of proteins that are expressed in the critical G1-stage of the cell cycle in a representative model system of ER + breast cancer such as MCF-7, and make use of bioinformatics tools to explore: (a) whether cancer marker proteins reported by previous studies (rather unrelated) can be associated with this stage of the cell cycle, (b) whether a particular subcellular localization is characteristic for these proteins, and (c) whether these markers form regulatory networks that promote cell proliferation and can be placed in a broader context of cancer-relevant functional roles to advance a panel with biomarker and drug target potential
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