Long-Term Kinetic Analysis of the Effect of the Circadian Clock and Transcription on Repair of Cisplatin-DNA Adducts in Mouse Liver

Cisplatin is the most commonly used chemotherapeutic drug for treating solid tumors. However, its toxicity and the innate or acquired resistance of cancer cells to the drug limit its usefulness. Cisplatin kills cells by forming cisplatin-DNA adducts, most commonly in the form of Pt-d(GpG) diadduct. We recently showed that repair of this adduct within 2 hours following injection is controlled by two circadian programs: (1) The circadian clock controls transcription of 2000 genes in mouse liver and thereby controls repair of the transcribed strand of these genes via transcription coupled repair in a rhythmic fashion unique to each gene's phase of transcription. (2) The basal excision repair activity itself is controlled by the circadian clock with a single phase at which the repair of the non-transcribed strand and the rest of the genome take place. Here, we have followed the repair kinetic for long periods genome-wide both globally and at single nucleotide resolution by the Excision Repair-sequencing (XR-seq) method to gain further insight into cisplatin DNA damage and repair with the aim of improving the therapeutic index of the drug. Our data show that transcription-driven repair is complete after two days, while completion of basal repair of nontranscribed strands, silent genes and intergenic regions requires weeks. In rhythmically controlled genes, oscillations of transcribed repair are observed up to 2 days post-injection, and in both constitutively transcribed and rhythmic genes, we see a trend in template strand repair with time from the 5'to 3' end. These findings add to the complexity of circadian- and transcription-dependent and -independent control of repair exhibited by this model organism in response to cisplatin. Overall design: To follow the Pt-d(GpG) repair pattern over a 10-day period, 59 mice were injected with cisplatin (10mg/kg) at ZT20 (3:00AM) on day 1, and for the first two days afterwards 3 mice were sacrificed at 4 hr intervals, their livers were harvested, and Pt-d(GpG) repair was quantified by slot blot and XR-seq. For the remaining mice, six to eight per time point were sacrificed at 4, 8 and 10 days after cisplatin injection and were processed likewise. An additional 12 mice were injected with cisplatin on day 1, as above, and livers were harvested for slot blot analysis at 5 day intervals from day 15 to day 70. Insufficient material was available for XR-seq analysis of samples obtained later than 10 days after injection.