Ultraviolet radiation-induced interleukin 6 release in HeLa cells is mediated via membrane events in a DNA damage-independent way

Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damage and activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 degrees C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominant over nuclear effects. UV-induced IL-6 release is mediated via NFkappaB since the NFkappaB inhibitor MG132 or transfection of cells with a super-repressor form of the NFkappaB inhibitor IkappaB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage.     

DNA Replication Arrest in Response to Genotoxic Stress Provokes Early Activation of Stress-Activated Protein Kinases (SAPK/JNK)

The impact of DNA damage-induced replication blockage for early activation of stress kinases [stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)] is largely unknown. Here, we show that induction of dual phosphorylation of SAPK/JNK by the DNA polymerase inhibitor aphidicolin was not ameliorated by additional exposure to ultraviolet (UV) light, indicating that overlapping mechanisms participate in signaling to SAPK/JNK triggered by both agents. UV-induced DNA replication blockage, cyclobutane pyrimidine dimer formation and DNA strand break induction coincided with SAPK/JNK phosphorylation at early (≤ 30 min) but not late (≥ 2 h) time points after exposure. Genotoxin-stimulated SAPK/JNK activation was attenuated in nonproliferating cells, indicating that S phase-dependent mechanisms are involved in signaling to SAPK/JNK. Correspondingly, UV-induced phosphorylation of SAPK/JNK was higher in S-phase cells as compared with G₁-phase cells. Activation of SAPK/JNK by genotoxins was below detection limit in nonproliferating human peripheral blood lymphocytes, whereas peripheral blood lymphocytes stimulated to proliferation displayed clear SAPK/JNK activation. UV-induced phosphorylation of SAPK/JNK was attenuated in XPC-defective cells, ameliorated in BRCA2 mutated cells and not changed in cells lacking ATM, DNA-PK, CSB, XPA, p53, ERCC1 or PARP as compared with the corresponding wild types. Based on these data, we suggest that DNA replication blockage caused by genotoxin-induced DNA damage contributes to early activation of SAPK/JNK.     

Histone H2AX Phosphorylation after Cell Irradiation with UV-B: Relationship to Cell Cycle Phase and Induction of Apoptosis

Damage to DNA that engenders double-strand breaks (DSBs) triggers phosphorylation of histone H2AX on Ser-139. Expression of phosphorylated H2AX (_H2AX) can be revealed immunocytochemically; the intensity of ?H2AX immunofluorescence (IF) measured by cytometry was reported to correlate with the frequency of DSBs induced by X-ray radiation or by DNA damaging antitumor drugs. The aim of the present study was to measure expression of ?H2AX following exposure of HeLa and HL-60 cells to a wide range of doses of UV-B light (6.1 J/m²-3.45 kJ/m²) and using multiparameter flow and laser scanning cytometry (LSC) to correlate DNA damage with cell cycle phase and induction of apoptosis. In both cell lines, the highest degree of H2AX phosphorylation induced by UV was seen in S-phase cells, particularly during early portion of S. In cells that did not replicate DNA (G₁, G₂ and M) the degree of H2AX phosphorylation was markedly lower than that in S-phase cells, and was strongly UV dose-dependent. Furthermore, the level of UV-induced γH2AX in G₁, G₂ and M was much higher in HeLa- than in HL-60-cells. Apoptotic cells become apparent >2h after exposure to UV and exhibited nearly an order of magnitude higher intensity of γH2AX IF than that initially induced by UV; predominantly S-phase cells underwent apoptosis. While the suppression of DNA replication aphidicolin prevented the induction of H2AX phosphorylation by UV in most S phase cells, it had no effect on a small cohort of cells that appeared to be entering S-phase, that expressed very high levels of γH2AX. Furthermore, aphidicolin itself induced γH2AX in early-S phase cells. The induction of γH2AX by UV was inhibited, but the incidence of apoptosis increased, by 5 mM caffeine, a known inhibitor of PI-3-related kinases. The data are consistent with the notion that H2AX phosphorylation observed throughout S phase reflects formation of DSBs due to the collision of replication forks with the UV-induced primary DNA lesions. Induction of γH2AX in GG₁, GG₂ and M is likely a response to the primary DSBs generated during UV exposure and/or DNA repair. It is unclear why the latter process was more pronounced in HeLa than in HL-60 cells.     

Apoptosis induced by disruption of the actin cytoskeleton is mediated via activation of CD95 (Fas/APO-1)

Activation of the death receptor CD95 by its ligand or by UV radiation is associated with receptor clustering. The mechanism underlying this clustering is mostly unclear. Here we show that although disruption of the actin cytoskeleton by cytochalasin B (CyB) itself induces moderate apoptosis, it enhances apoptosis in HeLa cells induced either by UV radiation or an agonistic anti-CD95 antibody. CyB augments UV-induced apoptosis independently of UV-mediated DNA damage, since induction of DNA repair by exogenous DNA repair enzymes did not alter its enhancing effect. Inhibition of caspase-8, the most upstream caspase in CD95 signaling, blocked the apoptotic effect of CyB and the enhancing effect on UV- and CD95-induced apoptosis. Confocal laser scanning microscopy revealed that (i) CyB induces CD95 clustering, (ii) enhances UV-induced CD95 clustering, and (iii) CD95 clusters colocalize with disrupted actin filaments, suggesting a link between receptor clustering and actin rearrangement. Disruption of CD95 signaling by a dominant negative mutant of the signaling protein FADD protected from CyB-induced apoptosis and prevented the UV-enhancing effect. Accordingly, both the apoptotic and the enhancing effect of CyB was reduced in epidermal cells obtained from CD95 deficient mice (lpr) when compared to wild-type mice. These data suggest that disruption of the cytoskeleton causes apoptosis via activation of CD95 and enhances UV-induced apoptosis, possibly via aiding receptor clustering.     

Photoreversion of ultraviolet induced apoptosis in Rat Kangaroo cells

Rat kangaroo(Potorous tridactylus) cells efficiently repair 254 nm ultraviolet light (UV) induced cyclobutane pyrlmidine dimers (CPDs) through photoreactivation, leading to an enhancement of survival when cells are exposed to photoreactivation light (PRL) immediately after UV-irradiation. This work presents evidence that at least part of the UV-irradiated cells die through apoptosis, as demonstrated by DNA fragmentation and chromatin condensation. The induction of this kind of cell death can be reversed through photoreactivation immediately after irradiation, indicating that CPDs are essential signals for the initiation of apoptosis by UV-irradiation. Exposure to PRL 24 h after UV-irradiation does not reverse the induction of apoptosis, implying that the cells are committed to die at this time after irradiation. Inhibition of DNA synthesis during this period of time following UV-irradiation, and before exposure to PRL, does not avoid apoptosis. Since similar results were obtained in G_o confluent and G₁/S synchronized cells, the signals for the UV-induced apoptosis do not seem to be related to a specific phase of cell cycle. Nevertheless, by adding 3-aminobenzamide (3AB—an inhibitor of poly(ADP-ribose) polymerase) in the cell medium after UV-irradiation, apoptosis endpoints were partially reversed if cells are exposed to PRL 24 h later. This result strongly indicates that poly(ADP-ribose) is an intermediary signal for UV-induced apoptosis in mammalian cells.     

Different cell cycle mechanisms between UV-induced and X-ray-induced apoptosis in WiDr colorectal carcinoma cells

Although DNA-damaging agents such as ultraviolet (UV) and X-ray can induce apoptosis, the difference in the apoptotic mechanism is not clearly understood. In the present study, we investigated the effects of these two genotoxic agents on the induction of DNA damage and subsequent apoptotic cell death from the viewpoint of cell cycle regulation by using WiDr cells. Transient G1 arrest was observed after UV exposure, whereas G2 but not G1 arrest was induced after X-ray irradiation. UV-exposure could induce G1 arrest in both mutant-type (mt-p53) and wild-type p53 (wt-p53) cells, but obvious G1 arrest was not observed in the cells lacking in p53 expression. An increase in the DNA fragmentation was observed at S phase in UV-irradiated cells and at G2 phase in X-irradiated cells, respectively. UV-irradiated cells showed an increase production of p53 protein and accumulation of p21 protein. On the contrary, both p53 and p21 proteins remained at a low level in X-irradiated cells. Treatment with aphidicolin, an S phase blocking agent, prolonged cell cycle arrest and reduced the rate of apoptotic cell death in both UV-irradiated and X-irradiated cells. From these results, it is suggested that UV-induced apoptosis occurs mainly at S phase and is regulated by increased production of p53 and p21 proteins, while X-ray-induced apoptosis occurs after G2 blockade and may be independent of p53.     

Positioning atypical protein kinase C isoforms in the UV-induced apoptotic signaling cascade.

Recent studies have documented the involvement of the atypical protein kinase C (aPKC) isoforms in important cellular functions such as cell proliferation and survival. Exposure of cells to a genotoxic stimulus that induces apoptosis, such as UV irradiation, leads to a profound inhibition of the atypical PKC activity in vivo. In this study, we addressed the relationship between this phenomenon and different proteins involved in the apoptotic response. We show that (i) the inhibition of the aPKC activity precedes UV-induced apoptosis; (ii) UV-induced aPKC inhibition and apoptosis are independent of p53; (iii) Bcl-2 proteins are potent modulators of aPKC activity; and (iv) the aPKCs are located upstream of the interleukin-converting enzyme-like protease system, which is required for the induction of apoptosis by both Par-4 (a selective aPKC inhibitor) and UV irradiation. We also demonstrate here that inhibition of aPKC activity leads to a decrease in mitogen-activated protein (MAP) kinase activity and simultaneously an increase in p38 activity. Both effects are critical for the induction of apoptosis in response to Par-4 expression and UV irradiation. Collectively, these results clarify the position of the aPKCs in the UV-induced apoptotic pathway and strongly suggest that MAP kinases play a role in this signaling cascade.     

IL-18 reduces ultraviolet radiation-induced DNA damage and thereby affects photoimmunosuppression

UV-induced DNA damage has been recognized as the major molecular trigger for photoimmunosuppression. IL-12 prevents UV-induced immunosuppression via its recently discovered capacity to reduce DNA damage presumably via induction of DNA repair. Because IL-18 shares some biological activities with IL-12 we studied the effect of IL-18 on UV-induced DNA damage and immunosuppression. IL-18 reduced UV-induced apoptosis of keratinocytes and supported long-term cell survival on UV exposure. Injection of IL-18 into mice that were exposed to UV radiation significantly lowered the number of apoptotic keratinocytes. Accordingly, radiation immunohistochemistry revealed reduced amounts of DNA damage in epidermal cells upon injection of IL-18. These effects were not observed in DNA repair-deficient (XpaKO) mice, indicating that IL-18 like IL-12 reduces DNA damage via DNA repair. UV-mediated suppression of the induction of contact hypersensitivity, which is known to be primarily triggered by DNA damage, was prevented upon injection of IL-18 before UV exposure in wild-type but not in XpaKO mice. In contrast to IL-12, IL-18 was not able either in wild-type or in XpaKO mice to break UV-induced immunotolerance that is mediated via regulatory T cells rather than in a DNA damage-dependent fashion. This result indicates that IL-12 is still unique in its capacity to restore immune responses because of its effect on regulatory T cells. Together, these data identify IL-18 as a further cytokine that exhibits the capacity to affect DNA repair. Though being primarily a proinflammatory cytokine through this capacity, IL-18 can also foster an immune response that is suppressed by UV radiation.     

PUVA-induced apoptosis involves mitochondrial dysfunction caused by the opening of the permeability transition pore

Damaged DNA-binding activity comprises two major protein components, DDB1 and DDB2, which are implicated in the repair of ultraviolet (UV) radiation-induced DNA damage. The possible role of DDB2 as a determinant of cellular sensitivity to UV was investigated. The abundance of DDB2 in UV-resistant HeLa cell lines was increased compared with that in the parental UV-sensitive cells. Stable transfection of the resistant cells with DDB2 antisense cDNA resulted in marked depletion of DDB2 protein and restored cellular sensitivity to UV-induced apoptosis. Whereas the extent of UV-induced activation of apoptosis executioners, including DNA fragmentation factor, and caspase-3 were reduced in the UV-resistant cells compared with those apparent in the sensitive cells, depletion of DDB2 from the resistant cells restored the normal activation patterns for these proteins. In contrast, overexpressing DDB2 in DDB2-depleted cells with recombinant adenovirus, which carries ddb2 cDNA, markedly inhibited the extent of UV-induced activation of DNA fragmentation factor, and caspase-3. Interestingly, a mutated form of DDB2, which is defective in interacting with DDB1 and binding to UV-damaged DNA, also markedly inhibited the activation of apoptosis executioners. These results indicate that DDB2 is a modulator of UV-induced apoptosis, and that UV resistance can be overcome by inhibition of DDB2. The findings also suggest that modulation of UV-induced apoptosis by DDB2 may be independent of DNA repair.     

Ultraviolet Light Induces Apoptosis via Direct Activation of CD95 (Fas/APO-1) Independently of Its Ligand CD95L

Induction of apoptosis in keratinocytes by UV light is a critical event in photocarcinogenesis. Although p53 is of importance in this process, evidence exists that other pathways play a role as well. Therefore, we studied whether the apoptosis-related surface molecule CD95 (Fas/APO-1) is involved. The human keratinocyte cell line HaCaT expresses CD95 and undergoes apoptosis after treatment with UV light or with the ligand of CD95 (CD95L). Incubation with a neutralizing CD95 antibody completely prevented CD95L-induced apoptosis but not UV-induced apoptosis, initially suggesting that the CD95 pathway may not be involved. However, the protease CPP32, a downstream molecule of the CD95 pathway, was activated in UV-exposed HaCaT cells, and UV-induced apoptosis was blocked by the ICE protease inhibitor zVAD, implying that at least similar downstream events are involved in CD95- and UV-induced apoptosis. Activation of CD95 results in recruitment of the Fas-associated protein with death domain (FADD) that activates ICE proteases. Immunoprecipitation of UV-exposed HaCaT cells revealed that UV light also induces recruitment of FADD to CD95. Since neutralizing anti-CD95 antibodies failed to prevent UV-induced apoptosis, this suggested that UV light directly activates CD95 independently of the ligand CD95L. Confocal laser scanning microscopy showed that UV light induced clustering of CD95 in the same fashion as CD95L. Prevention of UV-induced CD95 clustering by irradiating cells at 10°C was associated with a significantly reduced death rate. Together, these data indicate that UV light directly stimulates CD95 and thereby activates the CD95 pathway to induce apoptosis independently of the natural ligand CD95L. These findings further support the concept that UV light can affect targets at the plasma membrane, thereby even inducing apoptosis.     

Role of Gab1 in UV-induced c-Jun NH2-terminal kinase activation and cell apoptosis

Exposure of mammalian cells to UV irradiation leads to activation of the c-Jun NH(2)-terminal protein kinase (JNK) pathway, which is associated with cell apoptosis. However, the molecular mechanism for JNK activation by UV exposure is not fully understood. We show here an essential role of a multisubstrate adapter, Gab1, in this signaling cascade. Gab1-deficient mouse fibroblast cells were defective in induction of JNK activity by UV exposure or heat shock, and this defect was rescued by reintroduction of Gab1 into Gab1(-/-) cells. Consistently, Gab1(-/-) cells displayed reduced caspase 3 induction and apoptotic cell death in response to UV irradiation. Gab1 was constitutively complexed with JNK and became tyrosine phosphorylated in UV-irradiated cells. Genetic and pharmaceutical analyses suggest the involvement of c-Met and the Src family tyrosine kinases in mediating UV-induced Gab1 phosphorylation as well as JNK activation. In aggregate, these observations identify a new function of Gab1 in the response of mammalian cells to UV light.     

Role of G1 phase in the UV-induced apoptosis of EL-4 mouse lymphoma cells

Although UV is known to induce apoptotic cell death to various animal cells, relationship between cell cycle and UV-induced apoptosis is still unclear. In this study, we investigated the role of G1 phase in UV-induced apoptosis by using EL-4 mouse lymphoma cells which have wild type p53. After 500 J/m UV irradiation, an increase of apoptotic fraction was accompanied by cell cycle accumulation in the G1 phase. Apoptotic fraction after UV-exposure was remarkably augmented by treatment with 2-AP, a G1 checkpoint inhibitor. In contrast, aphidicolin, an inhibitor of DNA polymerase , suppressed the rate of apoptotic fraction.These results suggest that mandatory cell cycle progression from G1 to S leaves the damaged DNA unrepaired and may increase the apoptotic fraction. To investigate the precise mechanism in the G1 phase, UV was exposed to the G1-synchronized cells and apoptotic fraction was serially observed. Synchronized EL-4 cells passed through the G1 phase in 8 h. Within the G1 phase, late-G1 cells (6 h after M) were more sensitive to UV-induced apoptosis than early-G1 cells (2 h after M) (49.7 ± 9.0% vs. 41.5 ± 8.5%, p < 0.05). In HL-60 cells, lacking in p53 expression, such a difference was not observed. Western blot analysis revealed that expression of p53 in synchronized EL-4 cells was increasingly enhanced during G1 phase. After UV-exposure, p53 expression gradually decreased in early-G1 cells, but it was kept at almost the same level in late-G1 cells. In addition, bcl-2 expression in early-G1 cells showed a more rapid and larger increase than that in late-G1 cells. These results suggest that susceptibility of the G1 cells to UV-induced apoptosis depends on their position within the G1 phase, and late-G1 is more sensitive than early-G1. Sensitivity to UV-induced apoptosis is closely related to the expression level of p53 and bcl-2 proteins. Early-G1 cells may be able to take enough time to repair damaged DNA until they reach the G1 checkpoint compared to the late-G1 cells.     

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.     

Inhibition of polymerases-alpha and -beta completely blocks DNA repair induced by UV irradiation in cultured mouse neuronal cells

The effects of hydroxyurea, aphidicolin and dideoxythymidine on UV-induced DNA repair of mouse neuronal granular cells were studied. Aphidicolin, which is considered a specific inhibitor of polymerase-alpha, decreased spontaneous DNA synthesis by 93% and totally suppressed DNA repair. Dideoxythymidine, an inhibitor of polymerase-beta, was more potent in decreasing scheduled DNA synthesis than aphidicolin, and also completely blocked the UV-induced DNA repair. Hydroxyurea, a specific inhibitor of ribonucleotide reductase, inhibited scheduled DNA synthesis, but unscheduled DNA synthesis after UV irradiation was always well detectable. Our data suggest that in neuronal cells from 5 to 10 days old mice both polymerases-alpha and -beta are required for both DNA synthesis and repair. These two enzymes may act jointly in filling up the gaps along the DNA molecule and elongating the DNA chain.     

DNA polymerase delta mediates excision repair in growing cells damaged with ultraviolet radiation

In confluent, stationary phase cells, an aphidicolin-sensitive DNA polymerase mediates UV-induced excision repair, but the situation in growing cells is still controversial. The sensitivity of repair synthesis to aphidicolin, an inhibitor of DNA polymerases alpha and delta, was determined in growth phase and confluent normal human fibroblasts (AG1518) using several techniques. Repair synthesis in confluent cells was always inhibited by aphidicolin, no matter which measurement technique was used. However, the inhibition of repair synthesis in growth-phase cells by aphidicolin was only detectable when techniques unaffected by changes in nucleotide metabolism were used. We conclude that UV-induced repair synthesis in growing cells is actually aphidicolin sensitive, but that this inhibition can be obscured by changes in nucleotide metabolism. Employing butylphenyl-deoxyguanosine triphosphate, a potent inhibitor of polymerase alpha and a weak inhibitor of delta, we have obtained evidence that polymerase delta is responsible for repair synthesis in growth-phase cells following UV irradiation.     

Cell line dependent involvement of ceramide in ultraviolet light-induced apoptosis

Ultraviolet light (UV) activates an acid sphingomyelinase (ASMase) pathway, which hydrolyzes sphingomyeline to ceramide. Ceramide has been found to be a second messenger, which activates the c-jun N-terminal kinase (JNK) that is required for apoptotic cell death. However, the role of ceramide in UV-induced JNK activation and apoptosis remains controversial. In this study, we examined the correlation among ceramide production, JNK activation and cell apoptosis after UV-irradiation in three cell lines: 293 (kidney), Jurkat (lymphocytes) and MCF-7 (breast) were used in this study. The ceramide production was analyzed using the diacylglycerol kinase assay method. The JNK activation was measured by Western blot analysis using an antibody specifically recognizing phosphorylated JNK. Cell apoptosis was determined by morphological change or flow cytometry. Our data show that UV-irradiation induces ceramide production in both 293 and Jurkat cells. Inhibition of ceramide production by desipramine (25–50 M) reduced UV-induced JNK activation in both 293 and Jurkat cells; and protects 293 cells from UV-induced apoptosis. However, inhibition of ceramide production does not prevent Jurkat cells from UV-induced apoptosis. In addition, our data demonstrates that UV-irradiation induces JNK activation and apoptosis of MCF-7 cells without production of detectable amounts of ceramide after UV-irradiation. These results suggest that UV-induced JNK activation and apoptosis can be mediated through a ceramide dependent or an independent pathway.     

UV-Induced Apoptosis in Resistant HeLa Cells

Recently, apoptosis (genetically programmed cell death) induced by UV hasbeen documented in some cell culture models. However, the significance ofapoptosis in UV-induced cytotoxicity and resistance is uncertain. In thisstudy, we investigated the induction of apoptosis in HeLa cells and itsrole in acquired UV-resistance. The membrane receptor Fas was induced toassembly, and its immediate downstream target, caspase-8, was induced byUV in a dose- and time-dependent manner. Caspase-10, another possiblecandidate for forming the death-inducing signaling complex with Fas, wasalso activated in a dose- and time-dependent manner. There was significantactivation of caspase 9, 3 and 2 by UV. The apoptotic pathways appeared tobe normal in acquired UV-resistant HeLa cells. In addition, there was a UVdose-dependent induction of chromatin condensation in both parental andUV-resistant cells. However, resistant cells displayed significant reductionin chromatin condensation at lower doses. Inhibition of caspase-3 activation byspecific inhibitor significantly reduced the chromatin condensation in bothcell types, and unexpectedly, the difference between the two cell lines wascompletely eradicated, suggesting that the caspase-3 pathway plays asignificant role in reducing apoptosis in resistant cells. The resultsindicate that UV induces apoptosis by direct activation of apoptoticproteins in HeLa and resistant cells. Although resistant cells displayedpartial inhibition of UV-induced apoptosis through the caspase-3 pathway,there was no consistent difference in the activation of this and relatedcaspase-9 caspases compared to parental HeLa cells.