Role of p21WAF1 in the cellular response to UV

UV or g irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21WAF1 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21WAF1 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21WAF1 function.     

UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair

p53-mediated increase in cyclin-dependent kinase inhibitor p21(WAF1) protein is thought to be the major mediator of cell cycle arrest after DNA damage. Previously p21 protein levels have been reported to increase or to decrease after UV irradiation. We show that p21 protein is degraded after irradiation of a variety of cell types with low but not high doses of UV. Cell cycle arrest occurs despite p21 degradation via Tyr(15) inhibitory phosphorylation of cdk2 and differs from the classical p21-dependent checkpoint elicited by ionizing radiation. In contrast to the basal turnover of p21, degradation of p21 switches to ubiquitin/Skp2-dependent proteasome pathway following UV irradiation. ATR activation after UV irradiation is essential for signaling p21 degradation. Finally, UV-induced p21 degradation is essential for optimal DNA repair. These results provide novel insight into regulation of p21 protein and its role in the cellular response to DNA damage.     

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21(Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase delta. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.     

Regulation of Nucleotide Excision Repair by Nuclear Lamin B1

The nuclear lamins play important roles in the structural organization and function of the metazoan cell nucleus. Recent studies on B-type lamins identified a requirement for lamin B1 (LB1) in the regulation of cell proliferation in normal diploid cells. In order to further investigate the function of LB1 in proliferation, we disrupted its normal expression in U-2 OS human osteosarcoma and other tumor cell lines. Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis. The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation. Several proteins involved in the detection and repair of UV damage by the nucleotide excision repair (NER) pathway are down-regulated in LB1 silenced cells including DDB1, CSB and PCNA. We propose that LB1 regulates the DNA damage response to UV irradiation by modulating the expression of specific genes and activating persistent DNA damage signaling. Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.     

Binding sequence-dependent regulation of the human proliferating cell nuclear antigen promoter by p53

Exposure of a lung epithelial cell line to ionizing radiation (IR) arrests cell cycle progression through 48 h post-exposure. Coincidentally, IR differentially activates expression of the cell cycle inhibitor, p21/WAF1, and the DNA replication protein, proliferating cell nuclear antigen (PCNA). p21/WAF1 mRNA levels remain elevated through 48 h post-exposure to IR, while PCNA mRNA levels increase transiently at early times. Since p21/WAF1 inhibits DNA replication by directly binding PCNA, the relative levels of the two proteins can determine cell cycle progression. The PCNA p53-binding site displayed reduced p53 binding affinity in vitro relative to the distal p21/WAF1 p53-binding site. Substitution of the p21/WAF1 site for the resident p53-binding site in the PCNA promoter altered the responses to increasing amounts of p53 or IR in transient expression assays. The p21/WAF1 p53-binding site sustained activation of the chimeric PCNA promoter under conditions (high p53 levels or high dose IR) that the PCNA p53-binding site did not. Binding site-specific regulation by wild-type p53 was not observed with mutant p53 harboring a serine to alanine change at amino acid 46. Limited activation of the PCNA promoter by p53 and its modified forms would restrict the amount of PCNA made available for DNA repair.     

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.     

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.     

人衰老成纤维细胞经紫外线损伤后的DNA修复和细胞周期调控

 以体外培养的不同代龄的人胚肺二倍体成纤维细胞（２ Ｂ Ｓ）为对象,紫外线诱导 Ｄ Ｎ Ａ 损伤后,观察细胞形态、增殖特性、细胞周期、 Ｄ Ｎ Ａ 修复变化等细胞应答以及 ｇａｄｄ１５３、ｐ２１ Ｗ Ａ Ｆ１／ Ｃ Ｉ Ｐ１／ Ｓ Ｄ Ｉ１、ｐ５３ 等基因的转录水平的表达变化．结果显示：紫外线诱导 Ｄ Ｎ Ａ 损伤后,衰老（＞ ５５ 代）２ Ｂ Ｓ细胞形态及增殖能力的改变不如年轻细胞（＜ ３０ 代）显著;不同代龄的细胞损伤后均出现 Ｇ１ 期阻滞现象,年轻细胞 Ｇ１ 期阻滞率明显高于衰老细胞（ Ｐ＜ ００５）;衰老细胞总的修复能力较年轻细胞明显下降（ Ｐ＜ ００１）;同时,ｇａｄｄ１５３、ｐ２１、ｐ５３ 等的可诱导性均低于年轻 ２ Ｂ Ｓ细胞．由此,分别在细胞水平与基因水平反映了衰老细胞经紫外线照射损伤后的细胞应答变化与修复机能减退的关系．     

Checkpoint arrest signaling in response to UV damage is independent of nucleotide excision repair in Saccharomyces cerevisiae

The recognition of DNA double-stranded breaks or single-stranded DNA gaps as a precondition for cell cycle checkpoint arrest has been well established. However, how bulky base damage such as UV-induced pyrimidine dimers elicits a checkpoint response has remained elusive. Nucleotide excision repair represents the main pathway for UV dimer removal that results in strand interruptions. However, we demonstrate here that Rad53p hyperphosphorylation, an early event of checkpoint signaling in Saccharomyces cerevisiae, is independent of nucleotide excision repair (NER), even if replication as a source of secondary DNA damage is excluded. Thus, our data hint at primary base damage or at UV damage (primary or secondary) that does not need to be processed by NER as the relevant substrate of damage-sensing checkpoint proteins.     

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.     

Kruppel-like factor 4 mediates p53-dependent G1/S cell cycle arrest in response to DNA damage

The tumor suppressor p53 is required for the maintenance of genomic integrity following DNA damage. One mechanism by which p53 functions is to induce a block in the transition between the G(1) and S phase of the cell cycle. Previous studies indicate that the Krüppel-like factor 4 (KLF4) gene is activated following DNA damage and that such activation depends on p53. In addition, enforced expression of KLF4 causes G(1)/S arrest. The present study examines the requirement of KLF4 in mediating the p53-dependent cell cycle arrest process in response to DNA damage. We show that the G(1) population of a colon cancer cell line, HCT116, that is null for the p53 alleles (-/-) was abolished following gamma irradiation compared with cells with wild-type p53 (+/+). Conditional expression of KLF4 in irradiated HCT116 p53-/- cells restored the G(1) cell population to a level similar to that seen in irradiated HCT116 p53+/+ cells. Conversely, treatment of HCT116 p53+/+ cells with small interfering RNA (siRNA) specific for KLF4 significantly reduced the number of cells in the G(1) phase following gamma irradiation compared with the untreated control or those treated with a nonspecific siRNA. In each case the increase or decrease in KLF4 level because of conditional induction or siRNA inhibition, respectively, was accompanied by an increase or decrease in the level of p21(WAF1/CIP1). Results of our study indicate that KLF4 is an essential mediator of p53 in controlling G(1)/S progression of the cell cycle following DNA damage.