Seckel syndrome exhibits cellular features demonstrating defects in the ATR-signalling pathway

Abstract

To date, the only reported genetic defect identified in the developmental disorder, Seckel syndrome, is a mutation in ataxia telangiectasia and Rad3-related protein (ATR). Seckel syndrome is clinically and genetically heterogeneous and whether defects in ATR significantly contribute to Seckel syndrome is unclear. Firstly, we characterize ATR-Seckel cells for their response to DNA damage. ATR-Seckel cells display impaired phosphorylation of ATR-dependent substrates, impaired G2/M checkpoint arrest and elevated micronucleus (MN) formation following exposure to UV and agents that cause replication stalling. We describe a novel phenotype, designated nuclear fragmentation (NF), that occurs following replication arrest. Finally, we report that ATR-Seckel cells have an endogenously increased number of centrosomes in mitotic cells demonstrating a novel role for ATR in regulating centrosome stability. We exploit these phenotypes to examine cell lines derived from additional unrelated Seckel syndrome patients. We show that impaired phosphorylation of ATR-dependent substrates is a common but not invariant feature of Seckel syndrome cell lines. In contrast, all cell lines displayed defective G2/M arrest, increased levels of NF and MN formation following exposure to agents that cause replication stalling. All the Seckel syndrome cell lines examined showed increased endogenous centrosome numbers. Though ATR cDNA can complement the defects in ATR-Seckel cells, it failed to complement any of the additional cell lines. We conclude that Seckel syndrome represents a further damage response disorder that is uniquely associated with defects in the ATR-signalling pathway resulting in failed checkpoint arrest following exposure to replication fork stalling.

Human cancer cells require ATR for cell cycle progression following exposure to ionizing radiation

“…DLD-ATR-Seckel cells that had been treated with UV 30 min before labeling showed a markedly reduced BrdU-positive fraction as compared with wildtype DLD1 (Figure 5d). This suggests that ATR is essential for S-phase recovery following exposure to UV, consistent with earlier studies (Osborn et al, 2002;Alderton et al, 2004). IR treatment, however, caused decreased BrdU uptake in both cell lines, consistent with the observed decrease in thymidine uptake (Figure 5a).…”

“…Analysis of ATR transcripts by reverse transcription (RT)-PCR showed the appearance of a major amplified product with a size (295 bp) that was consistent with skipping of exon 9 in DLD-ATR-HET and DLD-ATR-Seckel cells (Figure 1c Immunoblotting revealed a decreased amount of ATR protein in DLD-ATR-HET cells, whereas ATR protein was undetectable in DLD-ATR-Seckel cells (Figure 1d). A significant phenotype previously observed in cells from ATR-Seckel patients, and directly attributed to ATR deficiency, is a marked sensitivity to ultraviolet radiation (UV) (O'Driscoll et al, 2003;Alderton et al, 2004). As measured by clonogenic survival, the DLD-ATR-Seckel cells were approximately 10-fold more sensitive to UV than wild-type DLD1 over a range of doses (Figure 2a), thereby providing functional confirmation of ATR deficiency in this engineered cell line.…”

Ataxia-telangiectasia and Rad3-related and DNA-dependent protein kinase cooperate in G2 checkpoint activation by the DNA strand-breaking nucleoside analogue 2′-C-cyano-2′-deoxy-1-β-d-arabino-pentofuranosylcytosine

“…2D). Similarly, when the Seckel cell line GM09703 (32), which retains f50% of ATR protein level compared with GM02188, was treated with 40 or 60 Amol/L of CNDAC followed by colcemid, cells exhibited mitotic scorings of 9% or 14%, respectively, relative to that of colcemid alone (P < 0.01). In contrast, another cell line, GM018367, which is derived from a Seckel syndrome patient with a splicing mutation (2101A!G) in the ATR gene (33) and has little residual expression of ATR, presented a mitotic index of 26% in response to colcemid alone.…”

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

“…It is thought that ATR-ATRIP, as part of the DNA replication machinery, associated with RPA, monitors single stranded DNA damage during the progression of DNA replication fork elongation (Niida and Nakanishi, 2006;Shechter et al, 2004b). In addition, ATR functions in both S and M phases include regulating late DNA replication origin firing, replication fork elongation, restarting stalled replication forks and involvement in centrosome stability (Shechter et al, 2004a;Cha and Kleckner, 2002;Sorensen et al, 2004;Friedel et al, 2009;Alderton et al, 2004;Smith et al, 1998).…”

“…Seckel syndrome is clinically characterised by dwarfism, abnormal brain development and microcephaly (Majewski and Goecke, 1982). Seckel cells show higher sensitivity to replication fork stalling agents and illustrate defects in downstream ATR DNA damage responses exemplified by impaired H2AX, Rad17, Nbs1 P53, and Chk1 phosphorylation (O'Driscoll et al, 2003;Alderton et al, 2004). In addition, Seckel cells exposed to UV were unable to execute the G2/M checkpoint (the G2/M checkpoint is described in section 1.4.3) (Alderton et al, 2004).…”