Abstract
Pyrrole–imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition.
Highlights
Many DNA-binding small molecules can challenge a cell’s ability to accurately replicate its DNA
These results demonstrate that polyamides are capable of imposing replication stress and can activate both a non-canonical Chk1independent ATR-checkpoint response and the Fanconi anemia/breast cancer (FA/BRCA) pathway, resulting in S-phase delay
Hairpin pyrrole– imidazole (Py–Im) polyamides 1 and 2 were designed to target the androgen response element (ARE) (5 -GGTACANNNTGTTCT-3 [26]) and antagonize gene expression changes driven by the androgen receptor (AR) in the prostate cancer cell line, LNCaP (Figure 1A and B) [16,27]
Summary
Many DNA-binding small molecules can challenge a cell’s ability to accurately replicate its DNA. The master regulators of the DDR are ATR and ATM, two PI3 protein kinase family members, which respond to stalled replication forks and DNA breaks. ATR and ATM phosphorylate many substrates to stabilize the DNA replication fork and activate cell cycle checkpoints. During S-phase, ATR is recruited to sites of stalled replication by replication protein A (RPA)-bound single-stranded DNA (ssDNA) in the presence of DNA damage. ATR is activated by a complex of many proteins and phosphorylates a number of targets, among which Chk, a cell cycle checkpoint kinase, is best understood [3,4]. ATM is recruited to sites of double-stranded breaks (DSBs) by the Mre11–Rad50–NBS1 complex, where it can phosphorylate Chk, another cell cycle checkpoint kinase, and the histone variant H2AX [5]. We have studied the checkpoint response activated by DNA minor groove binding pyrrole– imidazole (Py–Im) polyamides to discover what response polyamides elicit
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