Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells

We are pleased that the reviewers provided a positive assessment of our study, recognizing the value of extending our previous analysis of cell cycle variation in protein expression to include here measurements of protein levels in NB4 cells arrested at different stages of interphase, which we compare with data from elutriated NB4 cells at the same stages of interphase. We respond below to the specific comments provided by the reviewers, explaining how we have revised the manuscript to address these points. In addition to the points raised by the reviewers, we have also improved the revised manuscript by including an additional figure panel (Figure 4E) documenting the ratio of CDT1:geminin protein expression in cells treated with RO‐3306. This supports an expanded discussion of our proteomic characterization here of the effects of the CDK1 inhibitor RO‐3306 on arrested cells, which the reviewers highlighted as one of the most novel aspects of the study.

This study builds upon the authors' previous mass spectrometry analysis of the cell cycle in elutriated cells. In this study they have compared the changes in the proteome in cells treated by serum starvation, hydroxyurea and the Cdk1 inhibitor RO3306. The authors use these treatments to obtain cells in G0/G1, S and G2 phase. They find very significant differences in proteins enriched using these treatments compared to elutriation.

This follow up study contains some useful but perhaps not unexpected data. While the idea of comparing the proteome in drug-arrested cells to elutriation is certainly worthwhile, the choice of conditions used does not throw as much light on the drawbacks to cell synchronisation techniques as the authors suggest. For example, serum-starvation is not generally used to obtain G1 phase cells: most cell cycle researchers are aware that G0 phase cells are in a very different state from G1 cells, as shown by the classic studies by Anders Zetterberg. Thus, it is not particularly informative to compare serum starved cells to G1 phase cells; it would have been more useful to add back serum and analyse the cells after they had committed to another round of replication.

Similarly, hydroxyurea is more commonly used to induce DNA damage or replication stress. Most mammalian synchronisation regimes use a thymidine block and release protocol because cells recover from this more readily than from HU treatment.

We agree that serum starvation and hydroxyurea are both classic techniques for cell synchronization, and are aware there are several methods, such as thymidine block and release, that are now more commonly used in recent cell cycle studies. However, we feel it is important to note that the arrest treatments we examine in this study are still being used to synchronise cells in conjunction with cell cycle analyses, as can be seen by performing a PubMed search for the terms ‘hydroxyurea’ and ‘serum starvation’. We also note that these arrest treatments are featured in recent books (e.g., Methods in Molecular Biology) and review papers on synchronization methods. We have explicitly discussed in the revised manuscript the use of other synchronization methods not covered in this study and indicate our intention to address also the proteomic consequences of arrest and release procedures in future studies. However, given the previous and ongoing use of the classic arrest procedures in the cell cycle literature, we feel that it is useful and relevant to provide this analysis of how hydroxyurea and serum starvation affect protein expression at a global level.

Regarding the novelty of this study, while we are aware of previous literature on the physiological and biochemical effects of hydroxyurea and serum starvation, we believe our present study represents the first time that the global proteome response to these treatments has been examined quantitatively and compared with data from elutriated cells at the same stages of interphase. We feel this allows for an unbiased and novel analysis of the effects of these arrest treatments that can begin to unravel to what extent changes in protein expression reflect bona fide cell cycle regulation as opposed to effects of metabolic perturbation that do not occur during physiological progression of cells through interphase. Reassuringly, our unbiased proteomic analysis on arrested NB4 cells is highly consistent with previous findings (e.g., effects of serum starvation on metabolism and hydroxyurea on replication stress). We feel this validates the proteomic methodology used here and illustrates how it can be applied in future to characterize the effects of drugs and other treatments on cellular physiology. However, while our data for cells arrested with either serum starvation or hydroxyurea are in general consistent with previous studies, we note that an interesting new finding is that serum starvation also induces changes to chromatin modifiers and chromatin components. As the reviewers point out (below), changes in core histone levels were unexpected. This demonstrates how our unbiased proteomic approach can reveal an unanticipated, and previously undocumented, effect of serum starvation on cells.

The RO3306 data are more interesting because they give an insight into the role of Cdk1 in preventing re-replication.

We agree that the RO‐3306 data are particularly interesting and represent a timely component of this study. RO‐3306 is a recently developed Cdk1 inhibitor that is now being more widely used to synchronize cells in G2. We agree with the reviewers that our proteomic data on cells treated with RO‐3306 reveal an important role of Cdk1 in preventing re‐replication in G2. To expand and emphasize this point, we have included additional data in the revised manuscript that support this conclusion. Specifically, we have performed additional quantitative measurements of the Cdt1:geminin ratio, which has been shown to be critical for regulating re‐replication in G2. We show that this ratio is abnormally high in Cdk1‐inhibited G2 cells, compared with control, vehicle‐treated cells, consistent with RO‐3306‐induced re‐licensing. We have also increased the focus of the study towards the analysis of RO‐3306 by modifying the Abstract and expanding the relevant section of the Discussion.

Similar work has previously been done in yeast and using transcript profiling; this work should at least be cited.

We agree and have now included citations to several transcriptome profiling experiments in yeast and in mammalian cells.

It would have been interesting to go further and correlate the findings between the studies.

We agree in principle but plan to address this more comprehensively in future when we can compare the published transcript data with our protein datasets, including our planned future exploration also of arrest and release methods with increased proteomic depth.

It would also be interesting to know if the authors have performed GO analysis separately for the set of proteins that correlate well with the elutriation experiment and the ones that do not, to try to better identify those processes in which changes in protein abundance due to the arrest method are most affected.

GO analysis was performed separately for the set of proteins that correlate well between elutriation and the arrest datasets. The only significantly enriched functions that emerged were ‘mitosis’, ‘cell division’, etc. Similarly, GO analysis was performed on the proteins that do not correlate well. All these GO analysis results are shown in Figures 3 and 4, which illustrate the biological processes that are specifically modulated by each arrest method and were not detected to be changing in elutriated cells.

In a similar vein, a comparison to an asynchronous population should be included to determine the enriched proteins in each phase.

Proteome measurements were also made in asynchronous cells in this analysis. Fold changes and implementation of fold‐change cut-offs were based on pairwise comparisons between each treatment and an asynchronous population. Similarly, p‐value calculations include measurements made in an asynchronous population where available. The supplementary table that is provided in the revised manuscript provides separate columns that indicate whether a protein satisfies both fold change and p‐value cut-offs for each treatment.

The one experimental concern lies with the observation of histone upregulation in serum-starved cells. This goes against what has been reported on the very tight control of histone levels.

Although histone levels are tightly controlled in general, several recent papers, which we now reference in the revised manuscript, provide exciting evidence that histone levels can be modulated under different environmental contexts and biochemical treatments (Celona et al. PLoS Biol 2011; Feser et al. Mol Cell 2010; Karnavas et al. Frontiers Physiol 2014).

It is possible that this particular observation is the result of an overcompensation of total protein levels prior to mass spec analysis. Since G0/1 cells are likely much smaller and of lower protein content than S or G2 phase cells, it could be that the high abundance of histones in serum starved samples only reflects a higher number of cells. This issue should be addressed or commented upon as appropriate.

While we recognize that G0/G1 cells are smaller and therefore likely have lower protein content than either S, or G2 phase cells, we consider overcompensation is unlikely and further note that in any event this is not different for the arrested and elutriated cells that we are comparing. Thus, in elutriated cells, we observed by MS analysis that the levels of core histone proteins, as a proportion of total cellular protein content, do not significantly change across the cell cycle. In contrast, in arrested cells, which were processed for MS analysis in the same manner as elutriated cells, we observe a significant, reproducible change in core histone proteins.

In summary, the author should re-write their paper to de-emphasise the importance of their results for interpreting cell synchronisation techniques and put the results in context particularly with respect to the non-equivalence of G0 and G1 phases, and the DNA damage induced by hydroxyurea treatment.

We agree that the importance of our results does not need further embellishment. We had not intended to comment on all cell synchronization methods and have therefore re‐focused the Abstract and text accordingly. We hope that it is now apparent that we are only commenting here on our analysis of two classic methods of synchronization, namely serum starvation and hydroxyurea, and on a relatively new synchronization method using RO‐3306. Additionally, we have revised the Introduction to include a more detailed description of the literature on the physiological effects of hydroxyurea and serum starvation, which we hope will provide better context for readers to interpret the results of our unbiased, system‐wide analysis. We trust that these data will be useful to the community and seen as part of an ongoing project to apply state of the art quantitative proteomics methods to systematically characterize the global regulation of the proteome as cells progress through the cell cycle.