Monthly Archives: April 2008

Commentary: Colonization of titles

You have probably noticed that a high fraction of scientific papers have colons in their titles. Several people have written humorous commentaries on this. Although these authors clearly see the use of colons as a growing trend, they did not present hard evidence for the increase in the usage of colons in the titles of scientific publications.

Out of curiosity, I thus wrote a small script to count the fraction of papers in Medline that have colons in their titles for each of the past 25 years. The result is shown in the plot below (note that the y-axis does not start at zero):

The conclusion is very clear: the fraction of titles with colons has increased linearly from 15% to 24% over the past 20 years. One could object that this effect may be explained by the increase in apologies (which often have a title “Retraction: …”) or by the NAR special issues on databases and web servers (which contain hundreds papers with titles such as “YADB: yet another database”). However, these add up to less than 2% of the papers with colonized titles and are thus insufficient to explain the observed 9% increase.

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Analysis: Cell-cycle expression of cancer genes

I have long used a data integration approach to obtain a global picture of eukaryotic cell-cycle regulation. The cell cycle is a popular research topic in part because of its importance for cancer research. I thus recently compared microarray expression data on the human cell cycle to genes with mutations that have been causally implicated in various forms of cancer.

From the Cancer Genome Project website, I downloaded a list of 353 human genes that are implicated in cancer. Using the identifier mapping file from STRING, I was able to automatically map 338 of these genes to the set of human genes from Ensembl that I used in earlier cell-cycle studies. 295 of the 338 genes were present on the microarrays used in the cell-cycle expression study by Whitfield et al. (2002). However, only 23 of these are among the 600 periodically expressed genes identified in the reanalysis by Jensen et al. (2006). The many numbers are illustrated in the diagram below:

By random chance, 295*600/12097 = 15 of the 295 genes would be expected to be periodically expressed, and the enrichment is thus only a bit over 1.5 fold. Although this enrichment is statistically significantly (P < 3%, Fisher’s exact test), the correlation is clearly not strong enough to allow prediction of novel cancer genes.

My step was to look at the evolutionary conservation of the 23 periodically expressed cancer genes. Only 12 of them belong to an orthologous group. Half of them do thus not appear to have orthologs in budding yeast, fission yeast, or Arabidopsis thaliana. Only three periodically expressed cancer genes have orthologs in all of these organisms. One of these genes is periodically expressed onlt in human, one in human and fission yeast, and one in all four organisms (a histone subunit).

In summary, it seems that one cannot say much about cancer based on cell-cycle mRNA expression data. This is perhaps not surprising considering that the transcriptional regulation does not seem to vary much between cancer cells and normal cells.

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Analysis: Cancer or not, cell-cycle expression stays the same

The groups of Ziv Bar-Joseph and Itamar Simon recently published a paper in PNAS on a new microarray study of the cell cycle of primary human fibroblasts:

Genome-wide transcriptional analysis of the human cell cycle identifies genes differentially regulated in normal and cancer cells

Characterization of the transcriptional regulatory network of the normal cell cycle is essential for understanding the perturbations that lead to cancer. However, the complete set of cycling genes in primary cells has not yet been identified. Here, we report the results of genome-wide expression profiling experiments on synchronized primary human foreskin fibroblasts across the cell cycle. Using a combined experimental and computational approach to deconvolve measured expression values into ‘‘single-cell’’ expression profiles, we were able to overcome the limitations inherent in synchronizing nontransformed mammalian cells. This allowed us to identify 480 periodically expressed genes in primary human foreskin fibroblasts. Analysis of the reconstructed primary cell profiles and comparison with published expression datasets from synchronized transformed cells reveals a large number of genes that cycle exclusively in primary cells. This conclusion was supported by both bioinformatic analysis and experiments performed on other cell types. We suggest that this approach will help pinpoint genetic elements contributing to normal cell growth and cellular transformation.

In contrast to the earlier study by Whitfield et al. (2002), which was performed on HeLa cells, Ziv Bar-Joseph et al. worked on non-transformed fibroblasts. The dataset thus offers a first global view of the differences between the cell cycle of normal human cells and that of cancer cells.

To compare their list of cell-cycle-regulated human genes to the one the I have used so far, I mapped their 480 genes to Ensembl using the mapping file from the STRING database. This resulted in a list of 410 genes, that is 70 genes could not be mapped by the automatic procedure. Whereas this is far from a perfect mapping, it is sufficient to judge the quality of the list.

The plots below show the fraction of a benchmark set that is identified as function of the number of genes that is proposed to be periodically expressed during the cell cycle. In each plot, I compare the results for the list of 410 obtained from the new study by Bar-Joseph et al., the analysis by Whitfield et al., and the reanalysis of the latter dataset by Jensen et al. (2006) (available from Cyclebase.org). To make the comparison as fair as possible, I only considered the subset of genes that were present in both microarray designs. The first plot uses as benchmark a set of 63 genes that have been identified as periodically expressed in targeted small-scale studies:

Three sets of cell-cycle-regulated human genes compared to benchmark set B1

I also benchmarked the three gene lists against a second benchmark set, which consists of predicted target genes of E2F cell-cycle transcription factors:

Three sets of cell-cycle-regulated human genes compared to benchmark set B2

Both benchmarks suggest that the three lists are of very comparable quality, but that the list by Whitfield and coworkers is much more inclusive than the one from Bar-Joseph and coworkers. In other words, the former list has better sensitivity whereas the latter has better specificity. This is consistent with the results presented by Bar-Joseph et al., who conclude that their list is more reliable than the previously published list. However, this is probably not due to better quality of the raw expression data, since reanalysis of the data by Whitfield et al. yielded a list with almost identical sensitivity and specificity (that is the red curve is very close to the blue cross in both plots).

Although the two lists of periodically expressed are of comparable quality, they may still contain very different sets of genes. I therefore decided to compare the list of genes that are periodically expressed in the time course on primary fibroblasts and in each of the four time courses on HeLa cells. To make this comparison as easy as possible, I selected the top-364 cycling genes from each of the four HeLa time courses based on the reanalysis by Jensen et al. (2006). The ten Venn diagrams below show all pairwise comparisons of the five lists of 364 genes each:

The average overlap between the list by Bar-Joseph et al. and an experiment from Whitfield et al. is 114 genes. By comparison, the average overlap between the top-364 lists from two individual experiments from Whitfield et al. is 123 genes. Although the overlap may seem low, I thus believe that it is due to the poor reproducibility between microarray time courses rather than due to genuine differences between primary fibroblasts and HeLa cells as suggested by Bar-Joseph and colleagues.

Although cancer cells have to circumvent the regulatory mechanisms that would normally prevent cell proliferation, the cell cycle itself appears to function the same way as in normal cells. In other words, the difference does not lie in the “engine” but in the “brakes”, which have been sabotaged in cancer cells.

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Commentary: Viewing the cell cycle in a new light

Atsushi Miyawaki’s lab from RIKEN has recently published a Cell paper that describes a novel approach for how to monitor cell-cycle progression of individual cells:

Visualizing spatiotemporal dynamics of multicellular cell-cycle progression

The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

The clever idea was to fuse a red- and a green-emitting fluorescent protein to Cdt1 and Geminin, respectively. Cdt1 is ubiquitinated by SCFSkp2 at the onset of S phase, which causes it to be rapidly degraded by the proteasome, whereas Geminin is targeted for proteolytic degradation by APCCdh1 in late M phase. By fluorescent labeling of two proteins, Miyawaki and colleagues managed to make mouse cells that become increasingly red during G1 phase, yellow around the G1/S transition, and increasingly green through S, G2, and M phase. It is thus possible to monitor the cell-cycle states of individual cells with a microscope.

The movie below follows a few HeLa cells for 3-4 cell cycles:

The authors also show how their construct can be used for imaging the cell-cycle state of the cells in a slice of a mouse brain or a mouse embryo. I expect that this will become an indispensable tool for unraveling the links between cell-cycle control and developmental processes.

For more details, I strongly recommend that you read Jake Young’s post at Pure Pedantry.

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