Figure 1.
Clock phenotypes of colon cancer cell lines.
(A–G) Cells were lentivirally transduced with a Bmal1-luciferase construct and bioluminescence was measured over 6 days. Given are detrended time series (black) and the best corresponding cosine fit (blue). Cell line names marked in green represent the test cell line (U2OS); blue - strong oscillators (SW480, HCT116); red - weak oscillators (HT29, LIM1215, RKO, CaCo2). (H) Plotted are the mRNA levels relative to Bmal1 of seven clock genes for the colon cancer cells lines. Note that the bars represent ratios to Bmal1.
Table 1.
Circadian properties vary within the different cell lines.
Figure 2.
Microarray analysis reveals clock-related gene expression signature.
(A) Shown is the heatmap generated with the list of 45 discriminative genes. Heatmaps were generated using Pearson distance function and ward clustering. Colour bar (left) indicates the expression levels for genes in the array, from green (low expression) to red (high expression). Colour bar (top) indicates class membership. Blue indicates strong oscillator, red indicates weak oscillator, and green indicates test sample (U2OS). Genes are ordered by profile similarity. (B) A comprehensive regulatory network of the mammalian circadian clock. We used a curated text mining approach to search for interactions between our genes of interest. The network represents 108 novel interactions, supported by 132 PubMed references. In the core of the network (orange circles) the main components of the canonical feedback loops are shown. The outer shell of the network (grey circles) shows the clock-regulated genes and proteins feeding back to the core components and thereby potentially influencing the oscillations. Red lines represent inhibitory interactions; green lines, activating interactions; grey lines, other kinds of interactions. A large format of the network is provided as Figure S2. (C, D) Networks of circadian and cancer regulation for the discriminative genes. All networks were generated using the GeneView software (expected rate of false positive interactions, 10%) with additional data retrieved from the STRING database. (C) Network of interaction correlating circadian clock genes (all genes in (B)) and discriminative genes. A large format of the network is provided as Figure S3. (D) Network of interaction correlating discriminative genes and cancer genes. A large format of the network is provided as Figure S4. Cancer-related genes are represented in orange, clock genes in blue, discriminative genes in green and common genes in violet (e.g. TNFα). Grey lines represent text-mined interactions, blue lines interactions from the STRING database, and yellow lines common interactions. A list of colon specific interactions (by filtering for colon cancer related terms) is given in Table S2.
Table 2.
List of week/strong oscillator cell line discriminative genes.
Table 3.
Selected cancer-related genes, core-clock genes and clock-related genes.
Figure 3.
Differential circadian phenotypes of human keratinocytes and their Ras-transformed variants.
Bmal1-Luciferase bioluminescence was recorded for 5 days after dexamethasone synchronization (A). Representative results from five independent experiments are shown (HaCaT II4 n = 3). Normal human keratinocytes display an average period of 23.4±0.4 hours with a peak phase at 16.4±0.2 hours after of synchronization (B, C). HRAS transformed HaCaT A5RT3 show a significantly longer period (HaCaT A5RT3 24.9±0.2 hours p<0.05, Student's t-test) and a significant phase delay of approximately 1.5 hours (p<0.05, Student's t-test). An significantly earlier phase of 0.24 and 0.85 hours was observed in HaCaT I7 and HaCaT II4 cell lines, compared to normal keratinocytes (HaCaT II4, p<0.05, Student's t-test). (D) Phase delay of H-Ras-transformed human keratinocytes upon temperature entrainment. HaCaT and HaCaT A5RT3 were entrained with temperature cycles for 4 days consisting of 10 hours 37°C and 10 hours 33°C with 2-hour ramps and subsequently released to constant 37°C. Bioluminescence of the Bmal1-driven luciferase reporter was recorded for at least 8 days. Shown are representative detrended data from two independent experiments.
Figure 4.
Induction of Ras expression in rat fibroblasts.
IR2 and IR4 rat fibroblasts (1×105 cells) were plated in 35 mm dishes 24 hours previous to measurement, and treated with either 20 mM IPTG for Ras induction (red line), or with sterile H2O (blue line) immediately after synchronization with dexamethasone (A) for IR2 and (D) for IR4, or for 48 hours previous to real-time rhythmicity measurement (B) for IR2 and (E) for IR4. 0.5×105 IR2 (C) and IR4 (F) cells were plated in 35 mm dishes and treated with either 20 mM IPTG for Ras induction (red line), or with sterile H2O (blue line) for 72 hours previous to synchronization and bioluminescence was monitored for several days (A and B: 6 counts/h in LumiCycle; C: 12 counts/h in TopCount). Shown are representative detrended data from three independent experiments.
Figure 5.
Effect of KRAS induction in HKe3 and HKe3 clone 8 cell lines.
Shown are the results from 3 independent experiments. (A) HKe3 have a period of 25.3±0.59 hours (p<0.05, Student's t-test), (B) HKe3 cells treated with mifepristone show a small period increase (26.1±0.68 hours p<0.05, Student's t-test). (C) HKe3 clone8 present a period of 25.1±0.3 hours (p<0.05, Student's t-test). (D) HKe3 clone8 cells treated with mifepristone show a large period increase (37.79±0.96 hours p<0.05, Student's t-test). (E) Western Blot analysis for RAS and phosphorylated ERK in HKe3 and HKe3 clone8 cells following K-Ras induction by mifepristone. Extracts where prepared from untreated cells (−) or 48 hours after addition of mifepristone (+). Levels of RAS and phosphorylated ERK were analysed. GAPDH was used as loading control.
Figure 6.
Differential gene expression of core clock genes in normal- and H-Ras-transformed human keratinocytes.
(A) HaCaT and H-Ras transformed HaCaT A5RT3 cells were synchronized with dexamethasone and after 24 hours harvested in regular 3-hour intervals Cry1 and Per2 gene expression magnitudes (relative to Gapdh) in HaCaT and HaCaT A5RT3 cells were significantly different (p<0.001, Mann-Whitney U-test). Shown are means and SEM of three independent experiments. (B) Shown are in silico expression profiles for Per and Cry obtained from simulations with our model over approximately 6 days. The blue curve represents the wild type (WT) non-perturbed situation (τ = 23 hours) and the red line represents the result of a single parameter perturbation (τ = 23.7 hours).
Figure 7.
In silico perturbation of BMAL-mediated transcription reproduces HaCaT vs A5RT3 phenotype.
(A) HaCaT and HRAS transformed HaCaT A5RT3 cells were synchronized with dexamethasone and after 24 hours harvested in regular 3-hour intervals. Shown are representative results from three independent experiments. Normal human keratinocytes shows an average free-running period of 22.9±0.2 hours (p<0.05, Student's t-test), and a peak at 15.9±0.4 hours (p<0.05, Student's t-test) after synchronization. H-Ras transformed HaCaT A5RT3, present a wide period (HaCaT A5RT3 24.5±0.1 hours p<0.05, Student's t-test) and a peak at 16.9±0.05 h (p<0.05, Student's t-test). (B) Shown are in silico expression profiles for Bmal1 obtained from simulations with our model over approximately 7 days. The blue curve represents the wild type (WT) non-perturbed situation (τ = 23 hours) and the red line represents the result of a perturbation in BMAL-mediated transcription (τ = 24.1 hours).
Figure 8.
Ras transformation affects the circadian clock via MAPK signalling.
(A) Shown are in silico-generated gene expression oscillations with wild type (WT) in blue, RAS overexpressing in red (i.e., reducing the BMAL1 mediated-transcription by 60% (ktt = 0.4) compared to WT) and reducing RAS activity (i.e., increasing BMAL1 mediated-transcription by 60% (ktt = 1.6)) in green. (B) Western Blot analysis of RAS protein expression for the HaCaT cell lines and its derivatives (HaCaTI7, HaCaTII4, A5RT3), shows clear RAS overexpression measured in A5RT3. (C) Phosphorylated ERK (pERK) is shown for HaCaT cells under different conditions: non-treated cells, (−), cells treated with DMSO (D), cells treated with U0126 (U). HaCaT cells were treated with a MEK inhibitor (UO126 20 µmol) after synchronization with dexamethasone and circadian activity of Bmal1-luciferase reporter was measured over 8 days. (D) Shown are the results from 3 independent experiments. Non-treated (NT) HaCaT have a period of 22.9±0.2 hours (p<0.05, Student's t-test), HaCaT A5RT3 present a larger period of 24.5±0.1 hours (p<0.05, Student's t-test). Cells treated with UO126 (20 µM) show a period decrease (22.2±0.1 hours, p<0.05, Student's t-test) and a phase advance.