Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

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266

Skin cancer is the most common form of cancer in the United States. The main cause of this cancer is DNA damage induced by the UV component of sunlight. In humans and mice, UV damage is removed by the nucleotide excision repair system. Here, we report that a rate-limiting subunit of excision repair, the xeroderma pigmentosum group A (XPA) protein, and the excision repair rate exhibit daily rhythmicity in mouse skin, with a minimum in the morning and a maximum in the afternoon/evening. In parallel with the rhythmicity of repair rate, we find that mice exposed to UV radiation (UVR) at 4:00 AM display a decreased latency and about a fivefold increased multiplicity of skin cancer (invasive squamous cell carcinoma) than mice exposed to UVR at 4:00 PM. We conclude that time of day of exposure to UVR is a contributing factor to its carcinogenicity in mice, and possibly in humans.circadian clock | cryptochrome | sunbathing | tanning salons S kin cancer is the most common form of cancer in the United States. With over 1.3 million new cases each year, it constitutes nearly 40% of all diagnosed cancers (1). Moreover, because of changes in lifestyle and the environment, the incidence of skin cancer is steadily increasing (2). The main causative agent of skin cancer is the UV component of sunlight. UV radiation (UVR) produces two major lesions in DNA, the cyclobutane pyrimidine dimer (CPD) and the (6-4) photoproduct [(6-4) PP], both of which are mutagenic and carcinogenic in animal model systems and are thought to be the primary cause of skin cancer in humans (3-7).In mice and humans, nucleotide excision repair is the sole repair system for removing CPDs and (6-4) PPs from DNA. As a consequence, humans with hereditary mutations in excision repair genes suffer from xeroderma pigmentosum, a syndrome characterized by a nearly 5,000-fold increase in skin cancer in sunlight-exposed areas of the afflicted individuals (8). Excision repair involves photoproduct removal by dual incisions bracketing the lesion, removal of the damage in the form of a 24-to 32-nt-long oligomer, filling in the resulting single-stranded gap, and sealing by ligase (9). The dual incision is carried out by six excision repair factors: RPA, xeroderma pigmentosum group A (XPA), XPC, TFIIH, XPG, and XPF-ERCC1 (10). Recently, in a study that analyzed liver and brain tissues from mice, it was found that XPA, a critical protein involved in damage recognition and a rate-limiting factor in excision repair, is controlled by the core molecular circadian clock (11, 12). As a consequence, excision repair activity exhibited circadian rhythmicity in these organs, increasing during the day to reach a maximum at 4-6:00 PM and decreasing during the night to a minimum at 4-6:00 AM.Here we analyzed the expression pattern of XPA and excision repair activity in mouse skin. We found that protein and repair activity exhibit a circadian rhythm similar to that found in the liver and brain. To determine whether this rhythmicity affected UV-induced skin cancer development we exposed a...

Section: Resultssupporting

confidence: 92%

“…In vivo repair of CPD and (6-4) PP was measured as reported previously (20,22). Mouse skin was collected at various time points after UVB treatment and immediately flash frozen to an index card and skin patches were stored at −80°C until processing.…”

Section: Methodsmentioning

confidence: 99%

Control of skin cancer by the circadian rhythm

Gaddameedhi

Selby²,

Kaufmann

et al. 2011

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205

266

“…UV irradiation was performed as described with the indicated dose (12). After incubation at 37°C for the indicated time, cells were washed and collected in cold PBS.…”

Section: Methodsmentioning

confidence: 99%

“…To determine which genomic regions were repaired rapidly after damage and which were repaired more slowly, we conducted a time course of CPD and (6-4)PP repair in normal human skin fibroblasts (NHF1). The bulk repair kinetics of these two types of damage is known to be quite different (4,12,13). CPDs, which do not distort the DNA helix as strongly and escape recognition by the general repair machinery, are recognized primarily in a transcription-coupled manner.…”

Section: Identification Of Sites Of Cpd Damage Repaired Early and Latmentioning

confidence: 99%

Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis

Adar

Lieb

et al. 2016

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160

226

We recently developed a high-resolution genome-wide assay for mapping DNA excision repair named eXcision Repair-sequencing (XR-seq) and have now used XR-seq to determine which regions of the genome are subject to repair very soon after UV exposure and which regions are repaired later. Over a time course, we measured repair of the UV-induced damage of cyclobutane pyrimidine dimers (CPDs) (at 1, 4, 8, 16, 24, and 48 h) and (6-4)pyrimidinepyrimidone photoproducts [(6-4)PPs] (at 5 and 20 min and 1, 2, and 4 h) in normal human skin fibroblasts. Each type of damage has distinct repair kinetics. The (6-4)PPs are detected as early as 5 min after UV treatment, with the bulk of repair completed by 4 h. Repair of CPDs, which we previously showed is intimately coupled to transcription, is slower and in certain regions persists even 2 d after UV irradiation. We compared our results to the Encyclopedia of DNA Elements data regarding histone modifications, chromatin state, and transcription. For both damage types, and for both transcription-coupled and general excision repair, the earliest repair occurred preferentially in active and open chromatin states. Conversely, repair in regions classified as "heterochromatic" and "repressed" was relatively low at early time points, with repair persisting into the late time points. Damage that remains during DNA replication increases the risk for mutagenesis. Indeed, laterepaired regions are associated with a higher level of cancer-linked mutations. In summary, we show that XR-seq is a powerful approach for studying relationships among chromatin state, DNA repair, genome stability, mutagenesis, and carcinogenesis.DNA repair | DNA damage | chromatin | transcription | mutation D NA damage blocks transcription and replication and compromises the integrity of the genome. Bulky adducts in DNA, the focus of this work, are caused by a variety of genotoxic agents including UV radiation in sunlight. In human cells, this damage is removed exclusively by the nucleotide excision repair mechanism (1-3). In nucleotide excision repair, a single-stranded oligomer that contains the bulky adduct is excised by dual incisions bracketing the lesion. The resulting gap is filled in by DNA polymerases, and the newly synthesized repair patch is then sealed by DNA ligase. In general excision repair, damage recognition is achieved by the repair factors xeroderma pigmentosum (XP) complementation group C (XPC) together with replication protein A (RPA) and XPA (4-6). In DNA that is being actively transcribed, damage recognition can also be achieved by a stalled RNA polymerase II, which, with the aid of the Cockayne Syndrome B (CSB) protein, accelerates the recruitment of the excision repair factors. This recognition process leads to transcription-coupled repair (7,8). The subsequent dual-incision reaction is carried out by the core excision repair factors RPA, XPA, transcription factor II H (TFIIH), XPG, and XPF-ERCC1, releasing an excised oligomer that is 24-32 nucleotides (nt) in length (6, 9).The mechanism of DNA...

“…The clonogenic survival assay was done as described previously (32,33) with some modifications. Cells were seeded at low density to ensure the formation of 200 colonies per six-well plate mRNA Measurement by Quantitative PCR.…”

Section: Methodsmentioning

confidence: 99%

Regulation of apoptosis by the circadian clock through NF-κB signaling

Lee

Sancar

2011

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In mice and humans the circadian rhythm of many biochemical reactions, physiology, and behavior is generated by a transcriptionaltranslation feedback loop (TTFL) made up of the so-called core clock genes/proteins. The circadian system interfaces with most signaling pathways including those involved in cell proliferation and inflammation. Cryptochrome (CRY) is a core clock protein that plays an essential role in the repressive arm of the TTFL. It was recently reported that mutation of CRY in p53-null mice delayed the onset of cancer. It was therefore suggested that CRY mutation may activate p53-independent apoptosis pathways, which eliminate premalignant and malignant cells and thus delay overt tumor formation. Here we show that CRY mutation sensitizes p53 mutant and oncogenically transformed cells to tumor necrosis factor α (TNFα)-initiated apoptosis by interfacing with the NF-κB signaling pathway through the GSK3β kinase and alleviating prosurvival NF-κB signaling. These findings provide a mechanistic foundation for the delayed onset of tumorigenesis in clock-disrupted p53 mutant mice and suggest unique therapeutic strategies for treating cancers associated with p53 mutation. hepatocellular carcinoma | inflammatory cytokine | extrinsic apoptotic pathway T he circadian rhythm in mammalian organisms is generated by a molecular clock comprising four gene/protein groups (1-5): The CLOCK (NPAS2) and BMAL1 transactivators and the cryptochrome 1 and 2 (CRY1/2) and the period 1 and 2 (PER1/ 2) repressors. CLOCK (or NPAS2) and BMAL1 make heterodimers, which act on the promoters of CRY1/2 and PER1/2 and activate their transcription. The CRY and PER proteins in turn, after a time delay of ill-defined mechanism, inhibit the CLOCK-BMAL1 transactivator and hence their own transcription to close the TTFL. This core TTFL directly or indirectly affects most cellular functions, and as a result, clock disruption by core clock gene mutation is expected to have serious repercussions at the cellular and organismal levels.We recently reported that mice of the p53 −/− Cry1 −/− Cry −/− (hereafter p53 KO Cry DKO ) genotype exhibited delayed onset of spontaneous cancer relative to p53 −/− (p53 KO ) mice (6). When analyzed for their response to genotoxic stress, it was found that p53 KO Cry DKO cells were more sensitive to UV-induced apoptosis than p53 KO cells even though they are identical in terms of DNA repair and DNA damage checkpoint functions (6). Therefore, we ascribed the reduced clonogenic survival of p53 KO Cry DKO cells upon UV irradiation, relative to p53 KO cells, to enhanced p53-independent apoptosis as a consequence of CRY mutation. We also suggested that the delayed onset of cancer and prolonged lifespan of mice of p53 KO Cry DKO genotype may have been caused by enhanced apoptosis of oncogenically transformed cells before giving rise to macroscopic tumors (6). In line with these findings we recently determined that p53 KO Cry DKO tumors are more responsive to oxaliplatin than p53 KO tumors (7). However, UV and oxaliplatin...