Fig 1. PD 0332991 synchronization enhanced cytotoxic killing of HL-60 promyelocytic leukemia cells by Ara-C.

A. Human purified CD34⁺ cell from AML patient were cultured with mitomycin treated HS-5 human stromal cells. In left pannel primary AML cells co-treated with PD 0332991 (0, 0.5 and 1 µM) and Ara-C (0, 1 and 5 µM) for 24 hours and cell viability was determined by trypan blue staining. In right panel the same AML cells were treated with PD 0332991 (0, 0.5 and 1 µM) for 24 h to induce G1 arrest and then cultured in fresh medium for 12 h to allow re-entry into S-phase before the addition of Ara-C (0, 1 and 5 µM). Cell viability was determined by trypan blue staining after Ara-C 24 hours treatment. B. Schematic diagram of the protocol for cell cycle synchronization by reversible PD 0332991 arrest in G1 and subsequent cytotoxic killing by Ara-C upon release into S phase. C. HL-60 cells were cultured in 0.5 µM PD 0332991 for 24 hours, then released into fresh media and harvested at the indicated time points. BrdU was added at each time point indicated for 30 min, followed by propidium iodide (PI) staining and FACS analysis of BrdU uptake (left panel) and DNA content (right panel). Numbers indicate the percentages of BrdU-positive S phase cells. D. HL-60 cells were arrested by 0.5 µM PD 0332991 and released into fresh medium. Ara-C (50 nM) was added at 8 hr after PD 0332991 removal, and apoptosis induction measured by loss of mitochondrial membrane potential using the Mito Tracker Red CMXRos assay (Invitrogen). The time course of PD 0332991 arrest, release and Ara-C administration were indicated on the right. Numbers indicate the percentages of MT-negative cells. E. Inhibition of CDK4/6-dependent Rb phosphorylation on serine 807/811 (pRb) as an indication of induction of early G1 arrest 24 hours post PD 0332991. Release of PD 0332991 arrest and S phase entry was marked by re-phosphorylation Rb. pRb and overall Rb levels were detected by Immunoblotting at the indicated time points by the anti-phospho-Rb and anti-Rb antibodies (Cell Signaling). β-actin was also measured as the loading control. F. HL-60 cells were arrested in early G1 by PD 0332991 for 24 hrs and released synchronously into S-phase for 8 hours. Ara-C was added at the indicated doses for 48 hours, and cell viability was determined by trypan blue staining. These data are representative of 3 independent experiments; error bars indicate standard deviation. * Statistical significance, P < 0.05.