Table 1
Taqman assays used for qRT-PCR gene expression analysis.
| Gene | Taqman Assay | Interrogated sequence (RefSeq) | Amplicon length |
|---|---|---|---|
| Per2 | Hs00256143_m1 | NM_022817.2 | 121 |
| BMAL1/ARNTL | Hs00154147_m1 | NM_001030272.2 | 112 |
| CRY1 | Hs00172734_m1 | NM_004075.4 | 84 |
| CRY2 | Hs00323654_m1 | NM_001127457.2 | 75 |
| CLOCK | Hs00231857_m1 | NM_001267843.1 | 88 |
| REV-ERBa/NR1D1 | Hs00253876_m1 | NM_021724.4 | 60 |
| PPARa | Hs00947536_m1 | NM_001001928.2 | 62 |
| SIRT1 | Hs01009006_m1 | NM_001142498.1 | 91 |
| c-myc | Hs00153408_m1 | NM_002467.4 | 107 |
Figure 1
Temperature change is a more suitable method for synchronization of human PBMC populations than using 10% or 50% FBS in cell culture. PBMC’s were synchronized either by culturing in 10% FBS for 12 hours followed by sample collection (A) or by culturing in 50% FBS (B) or by culturing cells at the reduced temperature of 32°C for 12 hours followed by 12 hours of culture at 37°C for a further 12 hours before sample collection (C). In each experiment, RNA samples were harvested from cell pellets from three different donors at 6hr intervals over a period of 42hrs for qRT-PCR analysis BMAL1 and Per2 expression over this period. ΔΔCt analysis was performed in SDS 5.4 (Applied Biosystems). GAPDH was used as a reference control for all analyses. Average expression across the three donor samples for each synchronization method tested is shown in boxes.
Figure 2
Core and ancillary circadian genes are down-regulated in PBMCs derived from patients diagnosed with either AML or ALL and show further changes in expression at the end of treatment and during disease relapse. RNA samples from PBMC cells were harvested directly from patients newly diagnosed with AML or ALL, towards the end of treatment for both AML or ALL and during disease relapse for AML patients. The core clock genes including Per2, BMAL1, Cry1, Cry2 and clock; and the ancillary clock genes Rev-ERBa and PPARa were examined by qRT-PCR. Arbitrary copy numbers were calculated, normalized to GAPDH and calibrated relative to control samples (control samples were given a value of 1). Mean mRNA levels are plotted relative to control samples (mean ± SD). Statistical differences were determined by student’s t-test assuming unequal variances. Statistical significance at *p < 0.05, **p < 0.01 and ***p < 0.001.
Figure 3
Core and ancillary circadian genes are down-regulated in PBMCs derived from patients diagnosed with either CML or CLL and show further changes in expression at the end of treatment. RNA samples from PBMC cells were harvested directly from patients newly diagnosed with AML or ALL, towards the end of treatment for both AML or ALL and during disease relapse for AML patients. The core clock genes including Per2, BMAL1, Cry1, Cry2 and Clock and the ancillary clock genes Rev-ERBa and PPARa were examined by qRT-PCR and copy numbers were plotted relative to control samples (mean ± SD) following normalization by GAPDH. Statistical differences were determined by student’s t-test assuming unequal variances. Statistical significance at *p < 0.05, **p < 0.01 and ***p < 0.001.
Figure 4
Circadian oscillations are lost in PBMCs derived from patients diagnosed with CML. PBMCs derived from newly diagnosed AML, ALL, CML and CLL patients were synchronized using the temperature change method. Following this RNA samples were harvested from the cells every 6hrs over a period of 30hrs and circadian gene expression analyzed by qRT-PCR. Analysis was performed for the following category of genes; Core clock (Per2/BMAL1, Cry2/clock), ancillary clock (Rev-ERBa/PPARa) and modifier (SIRT1/c-myc). PBMCs from each patient category (n = 5) were pooled together before synchronization. Raw Ct values were converted to arbitrary copy numbers and normalized to copy numbers for GAPDH.
Figure 5
Treatment with the Sirt1 inhibitor EX527 rescues the oscillation of BMAL1 in CML patients and regulates oscillation of core circadian genes in CLL patients. PBMCs derived from newly diagnosed AML, ALL, CML and CLL patients were first synchronized and then treated with the SIRT1 inhibitor EX527 (30uM). RNA samples were then harvested every 6hrs after treatment over a period of 30hrs and gene expression analyzed by qRT-PCR for core clock genes (A) (BMAL1/Per2) and (B) ancillary clock genes (Rev-ERBa/PPARa). PBMCs from each patient category (n = 5) were pooled together before synchronization.
Figure 6
Treatment with the Sirt1 inhibitor EX527 leads to altered responses in both the early and late-response circadian clock and modifier genes across the different sub-types of leukemia patients. PBMCs derived from newly diagnosed AML, ALL, CML and CLL patients were first synchronized and then treated with the SIRT1 inhibitor EX527 (30uM). RNA samples were then harvested every 6hrs after treatment over a period of 30hrs and gene expression analyzed by RT-qPCR for core clock genes (BMAL1/Per2) and ancillary clock genes (Rev-Erba/PPARa). PBMCs from each patient category (n = 5) were pooled together before synchronization. (A) Sirt1 inhibition by EX527 in control samples over the 30h test period. (B) Circadian clock gene expression in the first 6h of treatment with EX527 (early response), (C) Circadian clock gene expression after 18h of treatment with EX527 (late-response), (D) Expression of clock modifier genes Sirt1 and c-myc after 6h of treatment with EX527 and (E) Expression of clock modifier genes Sirt1 and c-myc after 18h treatment with EX527.