DNA polymerase eta, the product of the xeroderma pigmentosum variant gene and a target of p53, modulates the DNA damage checkpoint and p53 activation

DNA polymerase eta (PolH) is the product of the xeroderma pigmentosum variant (XPV) gene and a well-characterized Y-family DNA polymerase for translesion synthesis. Cells derived from XPV patients are unable to faithfully bypass UV photoproducts and DNA adducts and thus acquire genetic mutations. Here, we found that PolH can be up-regulated by DNA breaks induced by ionizing radiation or chemotherapeutic agents, and knockdown of PolH gives cells resistance to apoptosis induced by DNA breaks in multiple cell lines and cell types in a p53-dependent manner. To explore the underlying mechanism, we examined p53 activation upon DNA breaks and found that p53 activation is impaired in PolH knockdown cells and PolH-null primary fibroblasts. Importantly, reconstitution of PolH into PolH knockdown cells restores p53 activation. Moreover, we provide evidence that, upon DNA breaks, PolH is partially colocalized with phosphorylated ATM at gamma-H2AX foci and knockdown of PolH impairs ATM to phosphorylate Chk2 and p53. However, upon DNA damage by UV, PolH knockdown cells exhibit two opposing temporal responses: at the early stage, knockdown of PolH suppresses p53 activation and gives cells resistance to UV-induced apoptosis in a p53-dependent manner; at the late stage, knockdown of PolH suppresses DNA repair, leading to sustained activation of p53 and increased susceptibility to apoptosis in both a p53-dependent and a p53-independent manner. Taken together, we found that PolH has a novel role in the DNA damage checkpoint and that a p53 target can modulate the DNA damage response and subsequently regulate p53 activation.     

Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.     

p53-degradation by HPV-16 E6 preferentially affects the removal of cyclobutane pyrimidine dimers from non-transcribed strand and sensitizes mammary epithelial cells to UV-irradiation

Nucleotide excision repair (NER), the most versatile and ubiquitous mechanism for DNA repair, operates to remove many types of DNA base lesions. We have studied the role of p53 function in modulating the repair of DNA damage following UV irradiation in normal and p53-compromised human mammary epithelial cells (HMEC). The effect of UV-induced DNA damage on cellular cytotoxicity and apoptosis was determined in conjunction with global, gene- and strand-specific repair. Cytotoxicity studies, using clonogenic survival and MTT assays, showed that HPV-16 E6-expressing HMEC were more UV sensitive than p53-WT cell lines. High apoptotic index obtained with p53-compromised cells was in conformity to both the low clonogenic survival and the low cellular viability. No discernible differences in the formation of initial UV-induced cyclobutane pyrimidine dimers (CPD) were observed in the cell lines of varying p53 functional status. However, the extent and the rate of damage removal from genome overall were highest for p53-WT cells. Further examination of strand-specific repair in the p53 gene revealed that the removal of CPD in the non-transcribed strand (NTS) was slower in p53-compromised cells compared to the normal p53-WT cell lines. These results suggest that loss of p53 function, in the absence of other genetic alterations, decreased both overall amount of CPD repaired and their removal rate from the genome. Additionally, normal function of p53 is required for the repair of the NTS, but not of the transcribed strand (TS) in genomic DNA in human epithelial cells. Thus, failure of quantitative removal of CPD by global genomic repair (GGR), due to loss of p53 function, causes the enhanced UV sensitivity and increased damage-induced apoptosis via a p53-independent pathway. Nevertheless, recovery of cells from UV damage requires normal p53 function and efficient GGR.     

Dysregulation of lysophospholipid signaling by p53 in malignant cells and the tumor microenvironment

The TP53 gene has been widely studied for its roles in cell cycle control, maintaining genome stability, activating repair mechanisms upon DNA damage, and initiating apoptosis should repair mechanisms fail. Thus, it is not surprising that mutations of p53 are the most common genetic alterations found in human cancer. Emerging evidence indicates that dysregulation of lipid metabolism by p53 can have a profound impact not only on cancer cells but also cells of the tumor microenvironment (TME). In particular, intermediates of the sphingolipid and lysophospholipid pathways regulate many cellular responses common to p53 such as cell survival, migration, DNA damage repair and apoptosis. The majority of these cellular events become dysregulated in cancer as well as cell senescence. In this review, we will provide an account on the seminal contributions of Prof. Lina Obeid, who deciphered the crosstalk between p53 and the sphingolipid pathway particularly in modulating DNA damage repair and apoptosis in non-transformed as well as transformed cells. We will also provide insights on the integrative role of p53 with the lysophosphatidic acid (LPA) signaling pathway in cancer progression and TME regulation.     

The oncogenic phosphatase WIP1 negatively regulates nucleotide excision repair

Nucleotide excision repair (NER) is the only mechanism in humans to repair UV-induced DNA lesions such as pyrimidine (6-4) pyrimidone photoproducts and cyclobutane pyrimidine dimers (CPDs). In response to UV damage, the ataxia telangiectasia mutated and Rad3-related (ATR) kinase phosphorylates and activates several downstream effector proteins, such as p53 and XPA, to arrest cell cycle progression, stimulate DNA repair, or initiate apoptosis. However, following the completion of DNA repair, there must be active mechanisms that restore the cell to a prestressed homeostatic state. An important part of this recovery must include a process to reduce p53 and NER activity as well as to remove repair protein complexes from the DNA damage sites. Since activation of the damage response occurs in part through phosphorylation, phosphatases are obvious candidates as homeostatic regulators of the DNA damage and repair responses. Therefore, we investigated whether the serine/threonine wild-type p53-induced phosphatase 1 (WIP1/PPM1D) might regulate NER. WIP1 overexpression inhibits the kinetics of NER and CPD repair, whereas WIP1 depletion enhances NER kinetics and CPD repair. This NER suppression is dependent on WIP1 phosphatase activity, as phosphatase-dead WIP1 mutants failed to inhibit NER. Moreover, WIP1 suppresses the kinetics of UV-induced damage repair largely through effects on NER, as XPD-deficient cells are not further suppressed in repairing UV damage by overexpressed WIP1. Wip1 null mice quickly repair their CPD and undergo less UV-induced apoptosis than their wild-type counterparts. In vitro phosphatase assays identify XPA and XPC as two potential WIP1 targets in the NER pathway. Thus WIP1 may suppress NER kinetics by dephosphorylating and inactivating XPA and XPC and other NER proteins and regulators after UV-induced DNA damage is repaired.     

p38γ regulates UV-induced checkpoint signaling and repair of UV-induced DNA damage

In eukaryotic cells, DNA damage triggers activation of checkpoint signaling pathways that coordinate cell cycle arrest and repair of damaged DNA. These DNA damage responses serve to maintain genome stability and prevent accumulation of genetic mutations and development of cancer. The p38 MAPK was previously implicated in cellular responses to several types of DNA damage. However, the role of each of the four p38 isoforms and the mechanism for their involvement in DNA damage responses remained poorly understood. In this study, we demonstrate that p38γ, but not the other p38 isoforms, contributes to the survival of UV-treated cells. Deletion of p38γ sensitizes cells to UV exposure, accompanied by prolonged S phase cell cycle arrest and increased rate of apoptosis. Further investigation reveal that p38γ is essential for the optimal activation of the checkpoint signaling caused by UV, and for the efficient repair of UV-induced DNA damage. These findings have established a novel role of p38γ in UV-induced DNA damage responses, and suggested that p38γ contributes to the ability of cells to cope with UV exposure by regulating the checkpoint signaling pathways and the repair of damaged DNA.     

A Mouse Model Uncovers LKB1 as an UVB-Induced DNA Damage Sensor Mediating CDKN1A (p21WAF1/CIP1) Degradation

Exposure to ultraviolet (UV) radiation from sunlight accounts for 90% of the symptoms of premature skin aging and skin cancer. The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome and in a spectrum of epithelial cancers whose etiology suggests a cooperation with environmental insults. Here we analyzed the role of LKB1 in a UV-dependent mouse skin cancer model and show that LKB1 haploinsufficiency is enough to impede UVB-induced DNA damage repair, contributing to tumor development driven by aberrant growth factor signaling. We demonstrate that LKB1 and its downstream kinase NUAK1 bind to CDKN1A. In response to UVB irradiation, LKB1 together with NUAK1 phosphorylates CDKN1A regulating the DNA damage response. Upon UVB treatment, LKB1 or NUAK1 deficiency results in CDKN1A accumulation, impaired DNA repair and resistance to apoptosis. Importantly, analysis of human tumor samples suggests that LKB1 mutational status could be a prognostic risk factor for UV-induced skin cancer. Altogether, our results identify LKB1 as a DNA damage sensor protein regulating skin UV-induced DNA damage response.     

How Disruption of Cell Cycle Regulators Might Predispose to Sun-Induced Skin Cancer

The Ink4a/Arf ( CDKN2a) locus encodes two proteins that regulate distinct important tumor suppressor pathways represented by p53 and Rb. Loss of either p16^INK4a or p19^ARF was recently reported to reduce the ability of mouse cells to repair UV-induced DNA damage and to induce a UV-mutator phenotype. This observation was independent of cell cycle effects incurred by either p16INK4a and/or p19ARF loss, as it was demonstrable in unirradiated cells using UV-treated DNA. We suggest that this might explain why germ line mutations of INK4a/ARF predispose mainly to malignant melanoma, a UV-induced skin cancer, and provides a molecular explanation for the link between melanoma-genesis and impaired DNA repair. It also further demonstrates that regulation of cell cycle check points and DNA repair in response to genomic insults, such as ultraviolet irradiation are intricately interwoven processes. Differences in the apoptotic response to ultraviolet light between melanocytes and keratinocytes might explain why INK4a/ARF mutations predispose to malignant melanoma, but not to keratinocyte-derived skin cancers.     

Expression of tumor suppressor p53 facilitates DNA repair but not UV-induced G2/M arrest or apoptosis in Chinese hamster ovary CHO-K1 cells

Tumor suppressor p53 is an essential regulator in mammalian cellular responses to DNA damage including cell cycle arrest and apoptosis. Our study with Chinese hamster ovary CHO-K1 cells indicates that when p53 expression and its transactivation capacity was inhibited by siRNA, UVC-induced G2/M arrest or apoptosis were unaffected as revealed by flow cyotmetric analyses and other measurements. However, inhibition of p53 rendered the cells slower to repair UV-induced damages upon a plasmid as shown in host cell reactivation assay. Furthermore, the nuclear extract (NE) of p53 siRNA-treated cells was inactive to excise the UV-induced DNA adducts as analyzed by comet assay. Consistently, the immunodepletion of p53 also deprived the excision activity of the NE in the similar experiment. Thus, tumor suppressor p53 of CHO-K1 cells may facilitate removal of UV-induced DNA damages partly via its involvement in the repair mechanism.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation, and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.     

Epidermal malignant tumors: pathogenesis, influence of UV light and apoptosis

Basal cell carcinoma and squamous cell carcinoma, collectively termed non-melanoma skin cancers are the most common malignant tumors in humans. Basal cell carcinoma grows slowly and metastatic spread is very rare. Squamous cell carcinoma is characterized by infiltrative, destructive growth and metastasis. Long-term exposure of skin to UV light has a great impact on development of these epidermal malignancies. UV light induces cascade of events like well known DNA damage of keratinocytes as well as still completely undetermined influence on apoptotic process through expression of proapoptotic and antiapoptotic molecules. The major role in development of skin cancer is given to proapoptotic p53 molecule or tumor suppressor gene which mutation due to UV exposure leads to resistance of DNA-damaged cell to apoptosis. Other proapoptotic molecules such as Fas ligand (FasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are strongly expressed in basal cell carcinoma and squamous cell carcinoma that could be explained by the ability of tumor to escape the attack of immune system.     

Selective cell death of p53-insufficient cancer cells is induced by knockdown of the mRNA export molecule GANP

Cancer cells often contain p53 abnormalities that impair cell-cycle checkpoint progression and cause resistance to various anti-cancer treatments. DNA damage occurs at actively transcribed genes during G1-phase in yeast cells that have a deficient mRNA export capacity. Here, we show that germinal center-associated nuclear protein (GANP), a homologue of yeast Sac3 that is involved in mRNA export, is indispensable for ensuring the stability of human genomic DNA and that GANP knockdown causes apoptosis and necrosis of p53-insufficient cancer cells. Ganp small interfering RNA (siGanp)-induced DNA damage, accompanied by a decrease in the number of cells in S-phase, caused late apoptosis and necrosis in p53-insufficient cancer cells through both caspase-dependent and -independent mechanisms. siGanp effectively induced DNA damage leading to cell death in p53-insufficient cancer cells in vitro and protect the growth of cancer cells transplanted into immunocompromized mice, suggesting that siGanp has potential as a selective treatment for p53-insufficient cancer cells.     

Stepping up melanocytes to the challenge of UV exposure

Exposure to solar ultraviolet radiation (UV) is the main etiological factor for skin cancer, including melanoma. Cutaneous pigmentation, particularly eumelanin, afforded by melanocytes is the main photoprotective mechanism, as it prevents UV-induced DNA damage in the epidermis. Therefore, maintaining genomic stability of melanocytes is crucial for prevention of melanoma, as well as keratinocyte-derived basal and squamous cell carcinoma. A critical independent factor for preventing melanoma is DNA repair capacity. The response of melanocytes to UV is mediated mainly by a network of paracrine factors that not only activate melanogenesis, but also DNA repair, anti-oxidant, and survival pathways that are pivotal for maintenance of genomic stability and prevention of malignant transformation or apoptosis. However, little is known about the stress response of melanocytes to UV and the regulation of DNA repair pathways in melanocytes. Unraveling these mechanisms might lead to strategies to prevent melanoma, as well as non-melanoma skin cancer.