Cisplatin is one of the most commonly used anticancer drugs. It kills cancer cells by damaging their DNA, and hence cellular DNA repair capacity is an important determinant of its efficacy. Here, we investigated the repair of cisplatin-induced DNA damage in mouse liver and testis tissue extracts prepared at regular intervals over the course of a day. We find that the XPA protein, which plays an essential role in repair of cisplatin damage by nucleotide excision repair, exhibits circadian oscillation in the liver but not in testis. Consequently, removal of cisplatin adducts in liver extracts, but not in testis extracts, exhibits a circadian pattern with zenith at ∼5 pm and nadir at ∼5 am. Furthermore, we find that the circadian oscillation of XPA is achieved both by regulation of transcription by the core circadian clock proteins including cryptochrome and by regulation at the posttranslational level by the HERC2 ubiquitin ligase. These findings may be used as a guide for timing of cisplatin chemotherapy.

The circadian clock regulates the daily rhythms in the physiology and behavior of many organisms, including mice and humans. These cyclical changes at molecular and macroscopic levels affect the organism's response to environmental stimuli such as light and food intake and the toxicity and efficacy of chemo- and radiotherapeutic agents. In this work, we investigated the circadian behavior of the nucleotide excision repair capacity in the mouse cerebrum to gain some insight into the optimal circadian time for favorable therapeutic response with minimal side effects in cancer treatment with chemotherapeutic drugs that produce bulky adducts in DNA. We find that nucleotide excision repair activity in the mouse cortex is highest in the afternoon/evening hours and is at its lowest in the night/early morning hours. The circadian oscillation of the repair capacity is caused at least in part by the circadian oscillation of the xeroderma pigmentosum A DNA damage recognition protein.

Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.

Purpose: Previous study identified E2F1 as a key mediator of non–muscle-invasive bladder cancer (NMIBC) progression. The aim of this study was to identify the E2F1-related genes associated with poor prognosis and aggressive characteristics of bladder cancer. Experimental Design: Microarray analysis was performed to find E2F1-related genes associated with tumor progression and aggressiveness in the gene expression data from 165 primary patients with bladder cancer. The biologic activity of E2F1-related genes in tumor progression and aggressiveness was confirmed with experimental assays using bladder cancer cells and tumor xenograft assay. Results: The expression of E2F1 was significantly associated with EZH2 and SUZ12. The overexpression of E2F1, EZH2, and SUZ12 enhanced cancer progression including cell colony formation, migration, and invasiveness. Knockdown of these genes reduced motility, blocked invasion, and decreased tumor size in vivo. E2F1 bound the proximal EZH2 and SUZ12 promoter to activate transcription, suggesting that E2F1 and its downstream effectors, EZH2 and SUZ12, could be important mediators for the cancer progression. In addition, we confirmed an association between these genes and aggressive characteristics. Interestingly, the treatment of anticancer drugs to the cells overexpressing E2F1, EZH2, and SUZ12 induced the expression of CD44, KLF4, OCT4, and ABCG2 known as cancer stem cell (CSC)–related genes. Conclusions: The link between E2F1, EZH2, and/or SUZ12 revealed that E2f1 directly regulates transcription of the EZH2 and SUZ12 genes. The signature of E2F1–EZH2–SUZ12 shows a predictive value for prognosis in bladder tumors and the E2F1–EZH2–SUZ12–driven transcriptional events may regulate the cancer aggressiveness and chemo-resistance, which may provide opportunity for development of new treatment modalities. Clin Cancer Res; 21(23); 5391–403. ©2015 AACR.

The XPA (Xeroderma pigmentosum A) protein is one of the six core factors of the human nucleotide excision repair system. In this study we show that XPA is a rate-limiting factor in all human cell lines tested, including a normal human fibroblast cell line. The level of XPA is controlled at the transcriptional level by the molecular circadian clock and at the post-translational level by a HECT domain family E3 ubiquitin ligase called HERC2. Stabilization of XPA by downregulation of HERC2 moderately enhances excision repair activity. Conversely, downregulation of XPA by siRNA reduces excision repair activity in proportion to the level of XPA. Ubiquitination and proteolysis of XPA are inhibited by DNA damage that promotes tight association of the protein with chromatin and its dissociation from the HERC2 E3 ligase. Finally, in agreement with a recent report, we find that XPA is post-translationally modified by acetylation. However, contrary to the previous claim, we find that in mouse liver only a small fraction of XPA is acetylated and that downregulation of SIRT1 deacetylase in two human cell lines does not affect the overall repair rate. Collectively, the data reveal that XPA is a limiting factor in excision repair and that its level is coordinately regulated by the circadian clock, the ubiquitin–proteasome system and DNA damage.