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, 105 (3), 955-60

Genome-wide Transcriptional Analysis of the Human Cell Cycle Identifies Genes Differentially Regulated in Normal and Cancer Cells

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Genome-wide Transcriptional Analysis of the Human Cell Cycle Identifies Genes Differentially Regulated in Normal and Cancer Cells

Ziv Bar-Joseph et al. Proc Natl Acad Sci U S A.

Abstract

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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Cell cycle synchronization. The cell cycle distribution of cells synchronized by (a) serum starvation (one cycle) or (b) thymidine block (two cycles) was monitored by FACS. The number of cells (arbitrary units) is plotted against DNA content for time points after release. The percentage of cells in G1, S, and G2 stages of the cell cycle at each time point is shown.
Fig. 2.
Fig. 2.
Data deconvolution. (a) Due to loss of synchronization, cells (gray dots) are distributed around the actual time (t). Using a synchronization loss model, this distribution can be determined. The actual measurement at time t is an average of the expression values of the gene (black dot) in all cells and is thus not an accurate representation of the single-cell expression value for this gene at time t. Using deconvolution on data from multiple time points, we can recover the underlying expression pattern for gene i (ui). (b) A diagram depicting the percentage of cells entering mitosis at each time point after release from the thymidine block as determined by time-lapse cinematography (gray) and as predicted by the synchronization loss model from the FACS data (black). Note the high correlation between the two distributions (R = 0.76, ANOVA P < 10−4). (c) Expression profile of the BIRC5 gene as measured by microarray analysis of the thymidine block experiment. Raw data (gray triangles) and deconvolved data (black squares).
Fig. 3.
Fig. 3.
Characterization of three cycling gene groups. (a) Venn diagram depicting the overlap between cycling genes identified in Whitfield's (16) and our datasets. The numbers in each section of the diagram reflect the number of genes in each group (see SI Table 5 for a list of all of the cycling genes). (b) Expression data for each group of cycling genes (log ratio to unsynchronized culture) is represented by color, using a heat map, where red indicates induced expression and green indicates repressed expression. For the HeLa cells, we used data published in ref. of the Thy-Thy 3 (T-T3) and Thy-Noc (T-N) experiments. For the primary FF cells, we used the deconvolved expression data for the serum starvation (SS) and thymidine block (T) experiments. The genes within a group have been ordered (vertically) according to their assigned cell cycle stage, which is indicated on the right of the heat maps. Enrichment analysis of each group's members for functional cell cycle GO categories is represented by rectangles next to that group (for the full analysis, see SI Table 2). The length of a rectangle depicts the percentage of cycling genes that fall into the category (2.7–27%), and the color depicts the significance of the enrichment. The significance levels (in parentheses) were corrected for multiple hypotheses (see Materials and Methods) and are indicated by three levels of gray color for P < 0.05, <0.005 and <0.001. White rectangles indicate no enrichment (P > 0.05).
Fig. 4.
Fig. 4.
Expression in normal cells. The average gene expression level for each of the three groups in proliferating and arrested cells is shown. The asterisks designate cases with a significant difference (P < 0.05; paired t test) between proliferating and arrested cells. (a and b) Data of proliferating (blue) and senescent (red) IMR-90 cells from an oncogene-induced senescence experiment (20) (a) and a senescence bypass experiment (21) (b). (c) Data of endometrium tissue (42) in the proliferative (blue) and senescent early, middle, and late secretory (red, yellow, and black, respectively) phases. (d) Average expression levels in a variety of normal tissues (22) for genes in the common (orange), primary FF (yellow) and HeLa (blue) groups. The asterisks designate a significant difference (P < 0.05; t test) between the expression of the genes within a group and the expression of all genes measured in that tissue. Note the different expression pattern of quiescent and proliferating tissues. Similar results were obtained by using an additional dataset (SI Fig. 8).
Fig. 5.
Fig. 5.
Expression in cancer tissues. (a) The average gene expression level is diagrammed for the common and primary FF groups in normal arrested IMR-90 primary fibroblasts (red, the same data as in Fig. 4b) and fibrosarcoma (26) (green). Significant (P < 0.0007; paired t test) difference between the normal and the cancer samples was observed only for the common group. (b) Expression data from a variety of tissues and cell lines (27) were averaged according to three categories—normal quiescent samples (such as lung, liver, and heart), normal proliferating samples (such as testis, thymus, and bone marrow), and cancer samples. The average expression of genes in the common (orange) and primary FF (yellow) groups is shown. Note that a significant difference (P < 0.0002; t test) between the expression behaviors of the two groups is observed only when considering cancer cells. (c and d) RNA levels of genes from the primary FF (c) and common (d) groups were measured in normal primary endothelial cells, HUVECs (Pr), and a fibrosarcoma cancer cell line, HT1080 (Ca), by semiquantitative RT-PCR at several cell cycle stages (SI Appendix). The level of expression at G1/S and G2/M for each gene is presented relative to its expression at G1 after normalizing for GAPDH. The averages and the standard deviations (error bars) of duplicate measurements are shown. The predicted peak of expression of each gene is G1/S for RBL1, C1ORF73, and CYCLINE1; G2 for FYN; G2/M for PER2, WHSC1, and C17ORF41; and M for CYCLINB1.

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