Identification of Fhit as a post-transcriptional effector of Thymidine Kinase 1 expression

Abstract

FHIT is a genome caretaker gene that is silenced in >50% of cancers. Loss of Fhit protein expression promotes accumulation of DNA damage, affects apoptosis and epithelial-mesenchymal transition, though molecular mechanisms underlying these alterations have not been fully elucidated. Initiation of genome instability directly follows Fhit loss and the associated reduced Thymidine Kinase 1 (TK1) protein expression. The effects on TK1 of Fhit knockdown and Fhit induction in the current study confirmed the role of Fhit in regulating TK1 expression. Changes in Fhit expression did not impact TK1 protein turnover or transcription from the TK1 promoter, nor steady-state levels of TK1 mRNA or turnover. Polysome profile analysis showed that up-regulated Fhit expression resulted in decreased TK1 RNA in non-translating messenger ribonucleoproteins and increased ribosome density on TK1 mRNA. Fhit does not bind RNA but its expression increased luciferase expression from a transgene bearing the TK1 5'-UTR. Fhit has been reported to act as a scavenger decapping enzyme, and a similar result with a mutant (H96) that binds but does not cleave nucleoside 5',5'-triphosphates suggests the impact on TK1 translation is due to its ability to modulate the intracellular level of cap-like molecules. Consistent with this, cells expressing Fhit mutants with reduced activity toward cap-like dinucleotides exhibit DNA damage resulting from TK1 deficiency, whereas cells expressing wild-type Fhit or the H96N mutant do not. The results have implications for the mechanism by which Fhit regulates TK1 mRNA, and more broadly, for its modulation of multiple functions as tumor suppressor/genome caretaker.

Keywords: 5′-UTR; Fhit; Thymidine kinase 1; Translational control.

Fig. 1. S-phase specific TK1 protein synthesis in Fhit-positive and negative cells

A. Immunoblot of Vinculin, TK1, and Fhit in 293 cells 72 h post transfection with siCtrl and siFHIT. B. Immunoblot of Vinculin, TK1, and Fhit in H1299 E1 and D1 induced cells. Cells were harvested 48 h after treatment with ponasterone A. C. Immunoblot of TK1 and GAPDH in siRNA transfected 293 cells treated for indicated times with 10 μM MG132 (left panel). Relative TK1 protein expression determined by densitometric analysis of the immunoblots (right panel). D. Immunoblot of TK1 and GAPDH in siRNA transfected 293 cells treated for indicated times with 100 μg/ml cycloheximide (CHX) (left panel). Protein lysates from siFHIT cells were loaded at 2:1 ratio compared to siCtrl lysates to more accurately measure rate of degradation. Relative TK1 protein expression determined by densitometric analysis of immunoblots (right panel). E. Immunoblot of TK1 and Cyclin A2 in siRNA transfected 293 cells synchronized by thymidine/nocodazole block and released for up to 24 h. Cells pass through S and G2 phases between 16-24 h after release as determined by Cyclin A2 expression. The initial ‘0’ lanes on the two blots show siFHIT and siCtrl lanes for direct comparison to the juxtaposed ‘0’ lanes for the siCtrl and siFHIT time-course immunoblots of TK1 and Cyclin A2 expression. Error bars indicate standard deviation.

Fig. 2. Fhit does not regulate the transcription or stability of TK1 mRNA

A. H1299 E1 and D1 induced cells were co-transfected with plasmids expressing firefly luciferase under control of the TK1 promoter and Renilla luciferase. Results are presented as the ratio of D1/E1 for relative light units of firefly luciferase normalized to Renilla luciferase and represent the mean ± standard deviation (n=3). B. RT-qPCR analysis of relative TK1 mRNA expression or RPLP0, an internal control, in H1299 E1 and D1 induced cells. mRNA levels, shown as fold change after induction of WT Fhit, were normalized to an internal luciferase control transcript. C. H1299 E1 and D1 induced cells were treated with DRB to block transcription. TK1 mRNA was determined by RT-qPCR and normalized to 18S rRNA and luciferase that was added as a recovery control. The insert is a Western blot of cytoplasmic extract from these cells probed with antibodies to Fhit and tubulin. While this is specific to C it is representative of Western blots done for A and B. The results in B and C represent the mean ± SEM from three independent experiments each performed in triplicate.

Fig. 3. Fhit increases polysome-bound TK1 mRNA

A. Cytoplasmic extracts from H1299 E1 and D1 induced cells were separated on a 10-50% sucrose gradient and fractions were monitored continuously at 254 nm. A representative gradient (of 3 independent gradients) is shown for each cell line and a representative Western blot of Fhit expression is shown in the inset. B. Even-numbered fractions were spiked with 1 ng of luciferase RNA as a control for sample recovery, and recovered RNA was analyzed by RT-qPCR for TK1 and luciferase. TK1 was normalized to luciferase and plotted as the percent of mRNA in each gradient fraction relative to input. Results shown are the means ± SEM of three independent experiments each performed in triplicate.

Fig. 4. Fhit mediates translational regulation through the TK1 5’-UTR

A. H1299 D1 and D1 induced cells were transfected with a plasmid expressing firefly luciferase with no added 5’-UTR (control, grey bars), or the same plasmid expressing firefly luciferase with the TK1 5’-UTR (TK1, black bars). Results were normalized to co-transfected Renilla luciferase. Fhit induction is shown in the Western blot at the top. B. The experiment in A was repeated using Fhit-negative H1299 E1 cells treated with or without ponasterone A. Data shown in A and B are the means ±SEM of three independent experiments (each measured in triplicate). The asterisk (*) indicates p <0.05 as determined by two-tailed Student T test.

Fig. 5. Translational regulation of TK1 is mediated by Fhit scavenger decapping activity

A. Parental H1299 cells were co-transfected with plasmids expressing firefly luciferase with the TK1 5’-UTR and Renilla luciferase, and either empty vector or plasmids expressing WT Fhit or the catalytically inactive H96N mutant. The impact of Fhit catalytic activity on expression driven by the TK1 5’-UTR was determined by dual luciferase assays. Data shown are the means ±SEM of three independent experiments (each measured in triplicate). The asterisk (*) indicates p <0.05 as determined by two-tailed Student T test. B. Extracts from DcpS positive HCT116 cells and H1299 cells used in this study were analyzed by Western blotting for DcpS and for Vinculin. C. H1299 E1 cells were transfected with control or DcpS siRNA and monitored by Western blotting for DcpS, Fhit, Vinculin and TK1.

Fig. 6. Assessment of DNA damage in cells expressing Fhit

Fhit genome caretaker function requires binding and cleavage of Fhit substrate to prevent DSBs. A. Neutral comet assay of H1299 cells ± 48 h ponasterone A treatment. Box plots of tail moments: D1, n = 292; D1 induced, n = 207; H96N induced, n = 185; Y114F induced, n = 152. Scatter plots of tail moments include data from 3 separate experiments. Statistical significance was determined using the wilcoxon test. B. Representative photos of neutral comet assay fields of H1299 cells 48 h after induction of WT or mutant Fhit proteins. C. Immunoblot assay showing levels of TK1 and Fhit protein expression in the H1299 cells examined in panels A and B. Densitometry of TK1 expression relative to Vinculin loading control.