Circadian clock, carcinogenesis, chronochemotherapy connections

The circadian clock controls the expression of nearly 50% of protein coding genes in mice, and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens. Patient’s tumor samples were collected under Duke Institutional Review Board (IRB) approved protocol (Pr000089222) and written informed consent were obtained from all patients who participated in the study. The generation of PDX was performed as described in our previous studies (Somarelli et al, MCT 2020). Briefly, the resected tumor was washed with phosphate buffered saline (PBS) and minced to small fragments (<2mm). To produce single cell suspension, tumor fragments were further dissociated with tissue dissociation kit (gentleMACS Dissociator). Subsequently, 150 µL of homogenized tumor tissue suspension (150 mg/ml concentration) was injected into the flank of 8-10-week-old JAX NOD.CB17-PrkdcSCID-J mice (obtained from the Duke University Rodent Genetic and Breeding Core). Mice were observed daily and after first sign of tumor appearance, tumor size was checked every other day by digital caliper Vernier. Once the tumor size reached a size of 1cm X 1cm in diameters (1st generation), it was harvested, and passaged to make 2nd generation PDX. PDX were deemed stable after the 3rd generation. All animal procedures were according to Duke University Institutional Animal Care and Use Committee. We use the Excision Repair-sequencing (XR-seq) method developed in our lab to study cisplatin repair in both human Patient Derived Xenografts (PDXs) cultivated in mice and in mouse liver tumors. XR-seq maps repair sites genome-wide at nucleotide resolution. Two hours after injecting cisplatin, the mice were sacrificed by carbon dioxide exposure, the liver and kidneys were removed and washed extensively with cold PBS, and then homogenized in 5 mL ice-cold PBS using 15 strokes of a Teflon homogenizer, at which point the plunger moved freely. The homogenized tissues were transferred into 50-mL tubes and pelleted by centrifugation in a centrifuge (Model CL2, cat. no. 004260F; Thermo Fisher) at 2,500 rpm for 4 min, the supernatant was discarded, and the pellets were washed three times to remove fatty material. The pellets were suspended in 5 mL ice-cold Buffer A [25 mM Hepes (pH 7.9)], 100 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 2 mM DTT, 12.5% glycerol, 0.5% Nonidet P-40)/per liver or two kidneys and incubated for 10 min on ice. Resuspended cells were transferred to an ice-cold Dounce homogenizer and lysed on ice with 60 strokes using a tight plunger. The chromatin fraction was then pelleted by centrifugation for 30 min at 14,000 rpm at 4 °C in a centrifuge (Model 5418, cat. no. 022620304; Eppendorf). The supernatants were harvested for the first immunoprecipitation [anti-TFIIH, p89 antibody (G-10) and p62 antibody (H-10); Santa Cruz Biotechnology]. The DNAs were subjected to a second round of immunoprecipitation with anti-cisplatin antibody. Excision products were treated with NaCN to remove Pt before PCR. Remaining steps, including oligonucleotides and adaptors were according to the previously described XR-seq procedure.