The XPF-ERCC1 endonuclease and homologous recombination contribute to the repair of minor groove DNA interstrand crosslinks in mammalian cells produced by the pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136

Abstract

SJG-136, a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, is a highly efficient interstrand crosslinking agent that reacts with guanine bases in a 5′-GATC-3′ sequence in the DNA minor groove. SJG-136 crosslinks form rapidly and persist compared to those produced by conventional crosslinking agents such as nitrogen mustard, melphalan or cisplatin which bind in the DNA major groove. A panel of Chinese hamster ovary (CHO) cells with defined defects in specific DNA repair pathways were exposed to the bi-functional agents SJG-136 and melphalan, and to their mono-functional analogues mmy-SJG and mono-functional melphalan. SJG-136 was >100 times more cytotoxic than melphalan, and the bi-functional agents were much more cytotoxic than their respective mono-functional analogues. Cellular sensitivity of both SJG-136 and melphalan was dependent on the XPF-ERCC1 heterodimer, and homologous recombination repair factors XRCC2 and XRCC3. The relative level of sensitivity of these repair mutant cell lines to SJG-136 was, however, significantly less than with major groove crosslinking agents. In contrast to melphalan, there was no clear correlation between sensitivity to SJG-136 and crosslink unhooking capacity measured using a modified comet assay. Furthermore, repair of SJG-136 crosslinks did not involve the formation of DNA double-strand breaks. SJG-136 cytotoxicity is likely to result from the poor recognition of DNA damage by repair proteins resulting in the slow repair of both mono-adducts and more importantly crosslinks in the minor groove.

Fludarabine-mediated suppression of the excision repair enzyme ERCC1 contributes to the cytotoxic synergy with the DNA minor groove crosslinking agent SJG-136 (NSC 694501) in chronic lymphocytic leukaemia cells

“…We also performed time-course experiments using both the comet assay and the Annexin V assay to establish the kinetics of the DNA crosslinking and apoptosis induction in primary CLL cells. In keeping with previous findings (Clingen et al, 2005), SJG-136 rapidly induced DNA crosslinks and these were maximal after 24 h exposure (data not shown). Apoptosis was also induced in a time-dependent manner with clear evidence of enhanced apoptotic cell killing in the cultures exposed to the combination of SJG-136 and fludarabine ( Figure 1D).…”

“…Importantly, this appears to be more marked in CLL cells when compared to normal lymphocytes (Moufarij et al, 2006). In addition, Clingen et al (2005) demonstrated that sensitivity to SJG-136 was dependent to some extent on ERCC1 expression in CHO cells. To determine whether ERCC1 was induced by SJG-136 in primary CLL cells, we performed real-time RT-PCR on RNA extracted from CLL cells exposed to SJG-136, fludarabine and both drugs in combination.…”

“…It has previously been demonstrated that fludarabine can inhibit DNA repair mechanisms and thereby synergise with cisplatin in the human chronic myeloid leukaemic K562 cell line (Yang et al, 1995;Li et al, 1997). More recently, Clingen et al (2005) reported that the XPF-ERCC1 endonuclease contributes to the repair of SJG-136-induced minor groove DNA ICL in Chinese hamster ovary (CHO) cells. Therefore, we hypothesised that fludarabine may suppress ERCC1 transcription and thereby enhance the cytotoxic effects of SJG-136.…”

“…This inhibition of ICL repair was associated with the inhibition of the nucleotide excision repair enzyme, ERCC1. In addition, a recent report clearly demonstrated a role for ERCC1 in the repair of SJG-136-induced DNA ICLs (Clingen et al, 2005). Therefore, this present study was designed to investigate whether fludarabinemediated inhibition of ERCC1-mediated DNA repair could positively impact on SJG-136 ICL patterns and enhance the resulting cytotoxicity of SJG-136 in CLL cells.…”

The use of molecular dynamics simulations to evaluate the DNA sequence-selectivity of G–A cross-linking PBD–duocarmycin dimers

“…The cytotoxicity of these molecules is thought to be directly related to the formation of interstrand cross-links (in this instance at Pu-GATC-Py sites [22,23]), and members of the PBD dimer class are now being used as payloads for antibody-drug conjugates (ADCs) [24,25]. Evidence is accumulating to suggest additional mechanisms of action including the formation of intrastrand cross-links [9,10,26] and mono-adducts, the inhibition of transcription factor binding [27,28] and the inhibition of enzymes such as endonucleases [29,30] and RNA polymerase [31].…”