Circadian clock, carcinogenesis, chronochemotherapy connections. 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. Overall design: 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.

生物钟（circadian clock）可调控小鼠中近50%的蛋白编码基因的表达，在人类中大概率亦是如此。因此，生物钟紊乱被认为会引发包括癌症在内的严重病理效应。然而，针对因夜班或轮班工作导致生物钟紊乱人群的流行病学研究，所得结果相互矛盾，无法就生物钟紊乱是否会提升乳腺、卵巢、前列腺或结直肠癌的发病率达成科学共识。同样，携带生物钟紊乱突变的基因工程小鼠，其自发或辐射诱导的癌症发病率并未高于野生型对照。由于包括细胞周期与细胞分裂在内的诸多细胞功能，至少部分由分子钟组分（CLOCK、BMAL1、CRYs、PERs）调控，因此学界也曾推测，化疗的适时施用可提升化疗药物的疗效并减轻其毒副作用。但目前时辰化疗（chronochemotherapy）的实证尝试并未取得有益效果。

本研究利用携带或不携带人类肿瘤异种移植物的小鼠，以单核苷酸分辨率、并结合生物钟时间维度，在全基因组范围内定位了抗癌药物顺铂（cisplatin）处理后DNA损伤与修复的位点。数据表明，此类基于机制的研究或可提供设计合理时辰化疗方案所需的信息。

实验整体设计：
患者肿瘤样本采集遵循杜克大学机构审查委员会（IRB）批准的方案（Pr000089222），所有参与本研究的患者均已签署书面知情同意书。患者来源异种移植模型（Patient Derived Xenografts, PDX）的构建参照我们既往研究（Somarelli等, MCT 2020）中的方法。简言之，将切除的肿瘤用磷酸盐缓冲液（PBS）洗涤并剪切成小于2mm的小块。为获得单细胞悬液，使用组织解离试剂盒（gentleMACS解离仪）进一步解离肿瘤组织块。随后，将150 μL浓度为150 mg/ml的均质化肿瘤组织悬液注射至8~10周龄JAX NOD.CB17-PrkdcSCID-J小鼠的侧翼（该小鼠购自杜克大学啮齿类动物遗传与繁育中心）。每日观察小鼠，待首次出现肿瘤迹象后，每隔一日使用数显游标卡尺测量肿瘤体积。当肿瘤直径达到1cm×1cm时（即第一代PDX），将其取材并传代以构建第二代PDX。第三代PDX被视为模型稳定。所有动物实验操作均符合杜克大学机构动物护理与使用委员会的相关规定。

我们利用本实验室开发的切除修复测序（Excision Repair-sequencing, XR-seq）技术，研究了小鼠体内培养的人类PDX以及小鼠肝肿瘤中的顺铂修复情况。XR-seq可在核苷酸分辨率下全基因组定位修复位点。在注射顺铂2小时后，通过二氧化碳窒息处死小鼠，取出肝脏与肾脏，用预冷PBS充分洗涤后，使用特氟龙均质器以15次往复将组织在5 mL预冷PBS中均质化，直至柱塞可自由滑动。将均质化的组织转移至50 mL离心管中，以2500 rpm离心4分钟（离心机型号CL2，货号004260F；赛默飞世尔科技），弃去上清，沉淀物用预冷PBS洗涤三次以去除脂肪物质。将沉淀物重悬于5 mL预冷缓冲液A [25 mM Hepes（pH 7.9）、100 mM KCl、12 mM MgCl₂、0.5 mM EDTA、2 mM DTT、12.5%甘油、0.5% Nonidet P-40]，每肝组织或双肾脏对应此体系，冰浴孵育10分钟。将重悬的细胞转移至预冷的杜恩匀浆器中，使用紧配柱塞冰浴裂解60次。随后以14000 rpm、4℃离心30分钟沉淀染色质组分（离心机型号5418，货号022620304；艾本德）。收集上清用于第一轮免疫沉淀：使用抗TFIIH p89抗体（G-10）与抗p62抗体（H-10；圣克鲁兹生物技术公司）。随后对DNA进行第二轮免疫沉淀，使用抗顺铂抗体。切除产物经氰化钠（NaCN）处理以去除铂（Pt）后进行PCR扩增。后续步骤包括寡核苷酸与接头的使用，均参照我们既往报道的XR-seq实验流程。