Fig 1.
Chemical structure of cytidine and RX-3117.
Fig 2.
UCK1 and UCK2 mRNA levels 72 hr after siRNA transfection.
(A) Relative UCK1 and UCK2 mRNA levels after transfection in A549 cells. (B) Relative UCK1 and UCK2 mRNA levels after transfection in SW1573 cells. Values are means ± SEM from 2–3 separate experiments. The gene expression in control cells was set at 100% for each separate experiment in order to calculate the relative gene expression in siRNA treated cells.
Fig 3.
Effect of UCK1 and UCK2 down regulation on RX-3117 cytotoxicity in A549 (A) and SW1573 (B) cells. Values are means ± SEM (n = 6). Values were normalized to 100% for untreated cells. As controls we also used cells transfected with control scrambled siRNA (data not shown). Drugs were added 24 hr after transfection. Cells were exposed to drugs for 48 hr. ECyd was used as a positive control. The protective effect of siUCK2 was significant in both A549 (p = 0.004) and SW1573 cells (p = 0.003). Significance is marked with an asterisk (*).
Fig 4.
Validation of UCK2 down regulation and UCK2 expression in normal and cancer tissues.
Silencing of UCK2 measured by western blotting; expression in cells treated with siRNA against UCK1 and UCK2 was compared to no siRNA (siNeg) (A). For immunocytochemistry; silence of UCK2 by siRNA was compared to untreated control in A59 and SW1573 (B). Examples of normal placenta from two women stained with the UCK2 antibody (C), normal liver from two patients (D) and liver metastases from separate human colorectal cancers (E).
Fig 5.
Regression analysis of UCK1/UCK2 expression and enzyme activity for [2-14C]-uridine and [3H]-RX-3117 in the cell line panel (A and B) and xenograft cells (C and D). In both panels UCK2 mRNA expression correlated significantly (p<0.001) with UCK enzyme activity measured with both [2-14C]-uridine (r = 0.828 for the cell lines and r = 0.878 for the xenograft cells) and [3H]-RX-3117 (r = 0.803 for the cell lines and r = 0.915 for the xenograft cells). UCK1 mRNA expression did not correlate with enzyme activity with either substrate (r = -0.721 and -0.585, respectively for the cell lines and r = -0.119 and r = -0.056 respectively for the xenograft cells). The cell lines in panel A and B shown in increasing UCK activity are: U937, A549, CCRF-CEM, SW1573, AG6000 and A2780. In panel C and D the sequence is LS174T, BT474, RT112, Raji B, SW780, PANC-1 and C-33A. The r-coefficient is given in the legend along for each gene. Significant correlations are marked with an asterisk (*).
Fig 6.
Correlation between the UCK2 mRNA expression of the cell lines in panels A and B (Fig 5) with phosphorylation and the sensitivity to RX-3117. The cell lines starting from highest UCK2 expression and sensitivity are: A2780, U937, A549, SW1573 and CCRF-CEM. (A) Correlation between sensitivity to RX-3117 and mRNA expression of UCK2. (B) RX-3117 phosphorylation in panel cells. RX-3117MP formation after 120 minutes correlated with UCK expression. (C) Protein expression of UCK1 and UCK2; the two left lanes are positive controls for UCK1 and UCK2 for which high levels of purified protein was used, which shows some cross-reactivity. Equal amounts of protein were put on the gels.
Fig 7.
Rescue of cell cycle effects and proliferation inhibition of cell lines upon treatment with RX-3117 conditioned with Uridine or Cytidine.
(A) The cell cycle distribution was monitored 36 hours after exposure to 33.3 μM RX-3117 or 33.3 μM RX-3117 with 100 μM of uridine or cytidine in A549 and SW1573 cells. (B) Proliferation of A549 and SW1573 cell lines treated with 33.3 μM of RX-3117 during 24 hours or with RX-3117 plus 100 μM of uridine/cytidine.