The involvement of Timeless, Tipin, Claspin, ATR and Chk1 in SCC was investigated by using siRNA to deplete hTERT-expressing normal human fibroblasts (NHF) of these proteins and examining metaphase preparations for aberrant pairing between sister chromatids.
At 48 h after introduction of siRNAs, levels of targeted protein were reduced by ≥ 95% compared to NHF1-hTERT that were electroporated with the non-targeting control (NTC) siRNA (). Similar to previous reports using cancer cell lines, depletion of Timeless reduced the stability of Tipin and vice versa.3,6,9,14
When NHF1-hTERT were electroporated with siRNAs targeting Chk1, Timeless, Tipin or Claspin, the targeted protein was reduced, but the levels of the other three proteins also decreased by 40–50%. These proteins may regulate each other's expression, and indeed, Chk1 has been shown to regulate the stability of Claspin.32
However, the abundance of these proteins is higher during S phase in normal human fibroblasts and other cell lines.4,11,12,33,34
Equivalent amounts of protein were loaded per well for SDS-PAGE, but the S-phase fraction in NHF1-hTERT cultures depleted of Chk1, Timeless, Tipin or Claspin was reduced (data not shown), which could account for the lower levels of these proteins when any one of them was targeted by siRNA. It remains to be formally examined whether Chk1 and Tim-Tipin regulate each other's stability.
Figure 1 Depletion of checkpoint/RFPC proteins attenuates UVC-induced phosphorylation of Chk1 in NHF1-hTERT. Forty-eight h after electroporation with siRNAs, NHF1-hTERT were exposed to 0 or 2.5 J/m2 UVC. Cells were harvested 1 hour after exposure. (A) A representative (more ...)
To demonstrate functional depletion of checkpoint proteins, ATR-dependent phosphorylation of Chk1 at S345 was examined one hour after exposure to 2.5 J/m2
(). It has been shown that inhibition of DNA replication following exposures to low fluences of UVC results from active signaling as opposed to passive obstruction of replication forks.35
Depletion of ATR reduced UVC-induced activation of Chk1 similar to depletion of Chk1 itself. Targeting of Timeless, Tipin, or Claspin attenuated UVC-induced activation of Chk1, consistent with previous reports in cancer cell lines that these proteins mediate ATR-dependent activation of Chk1 in response to DNA-damaging agents.3,6–8
Despite ≥ 95% depletion of mediator proteins, the UVC-induced P-Chk1 S345 could not be fully attenuated, on average, by targeting of Timeless, Tipin or Claspin or by co-targeting of Timeless and Claspin (), perhaps reflecting the contribution of other mediators to ATR-dependent intra-S checkpoint signaling.2
Giemsa-stained metaphase spreads were prepared to examine whether NHF1-hTERT that were depleted of checkpoint proteins would exhibit defects in SCC. At metaphase, sister chromatids were fully paired in cells electroporated with NTC siRNA (). Timeless-depleted cells, however, exhibited striking defects in SCC (). A range of defects was observed, from partial separation of sister chromatids to complete premature anaphase. In metaphases exhibiting partial separation of sister chromatids, some chromatids were still normally paired whereas others showed loss of both arm and centromeric cohesion. Fluorescence in situ hybridization (FISH) probes to centromere 9 and the CDKN2A locus were used to confirm that what was observed by Giemsa staining was, indeed, loss of centromeric cohesion ( and D). Chromosome 9 centromeric sequences and CDKN2A loci were detected with green or red fluorescent probes, respectively. Chromosome 9 centromeres were fully cohered in 100% of metaphases from NHF1-hTERT electroporated with NTC siRNA. In contrast, the fluorescent signals of the centromeres of chromosome 9 were separated in 38% of metaphases from Timeless-depleted cells.
Figure 2 Metaphases from Timeless-depleted NHF1-hTERT display defective sister chromatid cohesion. (A) Metaphase from NHF1-hTERT 48 h after introduction of NTC siRNA. (B) Metaphases from Timeless-depleted NHF1-hTERT depicting range of discohesion phenotypes from (more ...)
Defective SCC was observed in 27 ± 5.4% of Giemsa-stained metaphases prepared from NHF1-hTERT depleted of Timeless, representing a 100-fold increase over the control level of 0.23 ± 0.17% (). However, Tipin siRNA did not produce the same effect on SCC as Timeless siRNA despite the dependency of the two proteins on each other for their levels of expression. Only 2 ± 2% of metaphases exhibited defective SCC when Tipin was targeted by siRNA. Similar to depletion of Tipin, 4 ± 1.5% of metaphases exhibited defective SCC when cells were depleted of Claspin. Although the percentage of metaphases with defective SCC was not as dramatic with depletion of Tipin or Claspin compared to depletion of Timeless, loss of Tipin or Claspin produced statistically significant ~10–20-fold increases in defective SCC compared to the control, respectively. Co-targeting of Timeless and Tipin produced defective SCC in 24 ± 2.5% of metaphases, a result that was not different from targeting of Timeless alone. Additionally, 22 ± 5.3% of metaphases showed defective SCC with co-targeting of Timeless and Claspin. NHF1-hTERT that were depleted of ATR or Chk1 exhibited 1 ± 1% or 0.7 ± 0.7% of metaphases with defective SCC, respectively; these results were not statistically different from the control. The percent of metaphases exhibiting partial discohesion versus complete premature anaphase for each knockdown is reported in Supplemental Figure 1
Figure 3 Metaphases from checkpoint/RFPC-depleted NHF1-hTERT display defective sister chromatid cohesion. NHF1-hTERT metaphases were prepared 48 h after electroporation with siRNAs. Error bars represent standard error of the mean (S.E.M.) from 25–50 determinations (more ...)
One possibility for the 10-fold difference in defective SCC when targeting either Timeless (27%) or Tipin (2%) by siRNA could have been off-target effects of the Timeless siRNA. Tim-05 or Tim-06 siRNAs (designated by their Dharmacon catalog numbers) were equally effective at knocking down Timeless and reducing Tipin. Depletion of Timeless by either siRNA was accompanied by attenuation of UVC-induced Chk1 phosphorylation at S345 in NHF1-hTERT (). Importantly, 37 ± 4.8% of metaphases from cells that were electroporated with Tim-06 siRNA exhibited defects in SCC (), similar to the degree of defective SCC observed with Tim-05 siRNA ( and FISH result).
Figure 4 Defective sister chromatid cohesion in Timeless-depleted NHF1-hTERT is not an off-target effect of Timeless siRNA. (A) Comparison of Timeless depletion by two different siRNAs (Tim-05 or Tim-06). Forty-eight h after electroporation of siRNAs, cells were (more ...)
Lastly, to determine whether disruption of SCC by depletion of Timeless was a stereotypic response in normal human diploid fibroblasts, the analysis was repeated in a second line, NHF10-hTERT (). The depletion of targeted proteins and the attenuation of UVC-induced P-Chk1 S345 in NHF10-hTERT were similar to that observed in NHF1-hTERT (data not shown). Defective SCC was observed in 56 ± 2.8% of Giemsa-stained metaphases prepared from NHF10-hTERT depleted of Timeless, representing a 100-fold increase over the control level of 0.45 ± 0.18%. When accounting for the higher baseline of discohesion in NHF10-hTERT, all knockdowns (except for ATR) showed results that were highly similar to those obtained from NHF1-hTERT (Sup. Fig. 2
Figure 5 Metaphases from checkpoint/RFPC-depleted NHF10-hTERT display defective sister chromatid cohesion. NHF10-hTERT metaphases were prepared 48 h after electroporation with siRNAs. Error bars represent standard error of the mean (S.E.M.) from 25–50 (more ...)