Combining deep RNA sequencing methodology with quantitative proteomics, Wiita and co-workers have investigated pathways in the final stages of cytotoxic chemotherapy-induced apoptosis.1 Their paper shows a myeloma cancer cell’s integrated survival response to in vitro treatment with a chemotherapeutic agent.
Most cytotoxic chemotherapeutic drugs induce apoptosis. Although the mechanisms involved are well known at the molecular level,2 the corresponding cellular survival response — in terms of protein transcription and translation — has not been studied in depth. In this paper, Wiita et al. reveal the global cellular response to a chemotherapeutic agent.
The researchers treated clonal myeloma cell line MM1.S with the protease inhibitor bortezomib, currently used for first-line clinical treatment for the disease. They harvested cells at different time points following treatment; material was collected to enable deep sequencing of mRNA (mRNA-seq), ribosome profiling, quantitative proteomics, and measurement of caspase and non-caspase enzymatic degradation events.
The team used ribosome profiling combined with mRNA-seq to compare proteome transcription with translation (ribosome occupancy) activity. They separated gene data into five clusters, using pathway analysis to identify intracellular activity affected by these changes:
- Transcription and translation increased (Upreg)
- Transcription and translation decreased (Downreg)
- No change in activity (Stable)
- No change in transcription, but translation increased (TE Up)
- No change in transcription, but translation decreased (TE Down)
Upreg pathways included protein ubiquitination and other post-translational modifications; Downreg included cell proliferation pathways; Stable pathways included those involved in apoptosis; TE Up pathways involved the response to unfolded proteins; TE Down pathways were those involved in ribosomal protein maintenance.
The researchers examined changes in the cell proteome using a quantitative proteomics approach, isobaric tag for relative and absolute quantitation (iTRAQ), followed by tandem mass spectrometry (MS). Cells were harvested at the defined time points, lysed and underwent trypsin digestion before addition of iTRAQ labeling reagent. Wiita et al. used two MS systems, including an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific), to analyze the combined iTRAQ-labeled samples. They identified the peptides using the human SwissProt database for reference.
When compared with proteins identified from the ribosome profiling, Wiita and co-authors found close agreement with the MS data. Although most proteins showed no change in abundance, the researchers did find that 12 out of the 2,686 proteins identified with iTRAQ increased following bortezomib treatment. They then used a label-free selected reaction monitoring (SRM) method to follow the biological time course of protein events in the MM1.S cells and to confirm the data obtained from iTRAQ labeling. Results correlated well (R = 0.8) and confirmed that only a small number of proteins increase with bortezomib-induced apoptosis.
Combining their data, Wiita et al. hypothesized that blocking the upregulated factors identified might overcome an early response or survival strategy by the cell to avoid apoptosis. Through pathway analysis they identified transcription factors — including HSF1, which is inhibited by KRIBB113 — as potential therapeutic targets. Addition of small-molecule inhibitor KRIBB11 accelerated the effect of bortezomib, causing significant cell death at an earlier time point.
Although transcription and translation is apparently upregulated for some genes following treatment with bortezomib, cellular protein abundance is relatively unchanged. Using total cellular RNA quantification and estimating total protein copy number per cell using label-free intensity-based absolute quantification (iBAQ) on an LTQ Orbitrap Velos mass spectrometer, the researchers developed a model to show that the lack of change is explained by RNA degradation offsetting the upregulation.
Another consideration was that protein degradation is enhanced in the treated cells. Wiita and co-workers developed a specific quantitative SRM assay to monitor caspase and non-caspase proteolytic cleavage products chronologically with induction of apoptosis. They found that proteolysis was neither increased in, nor targeted to, proteins upregulated by bortezomib treatment. Furthermore, bortezomib inhibits proteasomal activity,4 and the proteasomes themselves are subject to caspase destruction.5
In summary, the researchers consider that this integrated approach to investigating a cell’s response to cytotoxic chemotherapy has implications for developing and refining future treatments. Investigating early responses in RNA transcription and protein translation could potentially identify therapeutic pathways that further drive cells into apoptosis.
1. Wiita, A.P., et al. (2013) “Global cellular response to chemotherapy-induced apoptosis,” eLife, doi: 10.7554/eLife.01236.
2. Spencer, S.L., and Sorger, P.K. (2011) “Measuring and modeling apoptosis in single cells,” Cell, 144 (pp. 926–39), doi: 10.1016/j.cell.2011.03.002.
3. Yoon, Y.J., et al. (2011) “KRIBB11 inhibits HSP70 synthesis through inhibition of heat shock factor 1 function by impairing the recruitment of positive transcription elongation factor b to the hsp70 promoter,” Journal of Biological Chemistry, 286 (pp. 1737–47), doi: 10.1074/jbc.M110.179440.
4. Berkers, C.R., et al. (2005) “Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib,” Nature Methods, 2 (pp. 357–62), doi: 10.1038/nmeth759.
5. Gray, D.C., Mahrus, S., and Wells, J.A. (2010) “Activation of specific apoptotic caspases with an engineered small-molecule activated protease,” Cell, 142 (pp. 637–46), doi: 10.1016/j.cell.2010.07.014.
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