Doses Of UV-C Radiation Research Articles

Mcl–1 Promotes Drug Resistance in Pancreatic Cancer Cells

Due to the lack of effective therapy, pancreatic adenocarcinoma is unalteredly causing every fifth cancer–related death in the western world. On the cellular level, resistance to conventional chemotherapy is reflected by a pronounced apoptosis resistance of pancreatic cancer cells. In search of stimuli overcoming apoptosis resistance in pancreatic cancer, we found that UV-irradiation rapidly induced apoptosis in all of 11 pancreatic cancer cell lines (n=11) tested. UV-induced apoptosis occurred rapidly, was dose-dependent and did not require ongoing protein synthesis. Activation of Bax and release of cytochrom c occurred independently of caspase-activity. On the mitochondrial level, rapid elimination of Mcl–1 occurred upstream of Bax-activation. Blocking Mcl–1-degradation by inhibition of the proteasome, completely abolished UV-induced activation of Bax, activation of effector caspases and subsequent induction of cell death. However, elimination of Mcl–1 by siRNA-mediated knockdown was not sufficient to induce apoptosis, and knockdown of Mcl–1 did not affect tumor cell growth in vitro. In contrast, knockdown of Mcl–1 strongly sensitized pancreatic cancer cells to very low doses of UVC-irradiation. Most strinkingly, knockdown of Mcl–1 sensitized pancreatic cancer cells to various cytotoxic stimuli. These results point to a major role of Mcl–1 as an inhibitor of apoptosis in response to various stimuli in pancreatic cancer cells. Our findings suggest that elimination of Mcl–1 may extremely facilitate the induction of apoptosis in response to cytotoxic drugs. Thus, pharmacological targeting of Mcl–1 may be an attractive strategy to overcome drug resistance in pancreatic cancer.

Characterization of UVC-induced DNA damage in bloodstains: forensic implications.

The ability to detect DNA polymorphisms using molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role within the criminal justice system, but one of the limiting factors with the technology is that DNA isolated from biological stains recovered from the crime scene is sometimes so damaged as to be intractable to analysis. Potential remedies for damaged DNA are likely to be dependent upon the precise nature of the DNA damage present in any particular sample but, unfortunately, current knowledge of the biochemical nature, and the extent, of such DNA damage in dried biological stains is rudimentary. As a model for DNA damage assessment in biological stains recovered from crime scenes, we have subjected human bloodstains and naked DNA in the hydrated and dehydrated states to varying doses of UVC radiation. It was possible to damage the DNA sufficiently in a bloodstain to cause a standard autosomal short tandem repeat (STR) profile to be lost. However, a detailed analysis of the process, based upon assays developed to detect bipyrimidine photoproducts (BPPPs), single- and double-strand breaks, and DNA-DNA crosslinks, produced some unexpected findings. Contrary to the situation with living tissues or cells in culture, the predominant UVC-induced damage to DNA in bloodstains appears not to be pyrimidine dimers. Although some evidence for the presence of BPPPs and DNA crosslinks was obtained, the major form of UVC damage causing genetic profile loss appeared to be single-strand breaks. It was not possible, however, to preclude the possibility that a combination of damage types was responsible for the profile loss observed. We demonstrate here that a significant measure of protection against UVC-mediated genetic profile loss in dried biological stain material is afforded by the dehydrated state of the DNA and, to a lesser extent, the DNA cellular milieu.

Functional genomics of UV radiation responses in human cells

The gene expression responses of MCF-7, a p53 wild-type (wt) human cell line, were monitored by cDNA microarray hybridization after exposure to different wavelengths of UV irradiation. Equitoxic doses of UVA, UVB, and UVC radiation were used to reduce survival to 37%. The effects of suramin, a signal pathway inhibitor, on the gene expression responses to the three UV wavelengths were also compared in this model system. UVB radiation triggered the broadest gene expression responses, and 172 genes were found to be consistently responsive in at least two-thirds of independent UVB experiments. These UVB radiation-responsive genes encode proteins with diverse cellular roles including cell cycle control, DNA repair, signaling, transcription, protein synthesis, protein degradation, and RNA metabolism. The set of UVB-responsive genes included most of the genes responding to an equitoxic dose of UVC radiation, plus additional genes that were not strongly triggered by UVC radiation. There was also some overlap with genes responding to an equitoxic dose of UVA radiation, although responses to this lower energy UV radiation were overall weaker. Signaling through growth factor receptors and other cytokine receptors was shown to have a major role in mediating UV radiation stress responses, as suramin, which inhibits such receptors, attenuated responses to UV radiation in nearly all the cases. Inhibition by suramin was greater for UVC than for UVB irradiation. This probably reflects the more prominent role in UVB damage response of signaling by reactive oxygen species, which would not be affected by suramin. Our results with suramin demonstrate the power of cDNA microarray hybridization to illuminate the global effects of a pharmacologic inhibitor on cell signaling.

Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses.

DNA damage caused by UV radiation initiates cellular recovery mechanisms, which involve activation of DNA damage response pathways, cell cycle arrest and apoptosis. To assess cellular transcriptional responses to UVC-induced DNA damage we compared time course responses of human skin fibroblasts to low and high doses of UVC radiation known to induce a transient cellular replicative arrest or apoptosis, respectively. UVC radiation elicited >3-fold changes in 460 out of 12,000 transcripts and 89% of these represented downregulated transcripts. Only 5% of the regulated genes were common to both low and high doses of radiation. Cells inflicted with a low dose of UVC exhibited transcription profiles demonstrating transient regulation followed by recovery, whereas the responses were persistent after the high dose. A detailed clustering analysis and functional classification of the targets implied regulation of biologically divergent responses and suggested involvement of transcriptional and translational machinery, inflammatory, anti-proliferative and anti-angiogenic responses. The data support the notion that UVC radiation induces prominent, dose-dependent downregulation of transcription. However, the data strongly suggest that transcriptional repression is also target gene selective. Furthermore, the results demonstrate that dose-dependent induction of cell cycle arrest and apoptosis by UVC radiation are transcriptionally highly distinct responses.

UV-C radiation as a novel technique for keeping quality of fresh processed ‘Lollo Rosso’ lettuce

Fresh processed ‘Lollo Rosso’ lettuce ( Lactuca sativa) was exposed to different doses (0.4, 0.81, 2.44, 4.07, and 8.14 kJ/m 2) of ultraviolet-C (254 nm, UV-C) radiation, and stored up to 9 and 10 days at 5 °C. While minimal processing did not induce significant increases in respiration rate, UV-C radiation did. Consequently, gas composition within bags containing radiated and control lettuce pieces changed according with the increase in respiration rate. All UV-C radiation doses decreased growth of psychrotrophic bacteria, coliform, and yeast, but only significant differences were found when the highest level was applied. UV-C radiation seemed to stimulate the growth of lactic acid bacteria, probably due to reduced growth of competitive flora. After 7 days of storage the highest dose resulted in increased tissue brightness, and browning was reduced when 2.44, 4.07, and 8.14 kJ/m 2 were applied. UV-C radiation at an appropriate dose could reduce microbial loads without adversely affecting sensorial quality of ‘Lollo Rosso’ lettuce.

Use of a combined enzymatic digestion/ESI mass spectrometry assay to study the effect of TATA-binding protein on photoproduct formation in a TATA box.

Recently, it was reported that TATA-binding protein (TBP) enhances (6-4) photoproduct formation in a TATA box under UVC irradiation [Aboussekhra and Thoma (1999) EMBO J. 18, 433-443]. The conclusions of that study were based on an indirect enzymatic assay that was not specific for (6-4) photoproducts. Herein we report the use of a recently developed coupled enzymatic digestion/mass spectrometry assay [Wang et al. (1999) Chem. Res. Toxicol. 12, 1077-1082] to identify unambiguously and quantify the photoproducts formed in a TATA box-containing dodecamer duplex sequence in the presence or absence of TBP binding. Exposure of the adenovirus major late promoter TATA box to a high dose of UVC irradiation in the absence of the C-terminal domain of yeast TBP leads to predominant formation of the cis-syn dimer within the T(4) tract, whereas exposure in the presence of TBP leads to almost exclusive formation of the (6-4) photoproduct. In contrast, the (6-4) product is not detected at high doses of UVB irradiation in the absence of TBP but is detected in the presence of TBP, although the cis-syn product predominates. When the products of UVB irradiation were subsequently exposed to a high dose of UVC irradiation in the presence of TBP, the (6-4) photoproduct again becomes nearly the exclusive photoproduct, indicating that the cis-syn dimer is being reversed to TT by UVC light. Both cis-syn and (6-4) photoproducts are formed in approximately equal amounts upon irradiation with small doses of UVC in the presence of TBP, but the fraction of (6-4) photoproduct increases with dose. Through the use of a TATA box containing a site-specifically deuterated thymine, it was found that (6-4) photoproducts formed most selectively at the second and third positions of the T(4) tract upon either UVB or UVC irradiation in the presence of TBP. By using the same substrate, it was found that UVC-induced TA formation was inhibited by TBP binding and that TA formation was greatest at the 5' end of the TATA sequence.

Inactivation of infectious salmon anaemia virus, viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus in water using UVC irradiation.

The UVC irradiation doses necessary for a 99.9% (3-log) inactivation of 3 different fish pathogenic viruses diluted in freshwater/seawater and wastewater from a fish processing plant were determined. The results showed that both infectious salmon anaemia virus (ISAV) and viral haemorrhagic septicaemia virus (VHSV) were very sensitive to UVC irradiation, showing a 3-log reduction of infectivity in freshwater of 33 +/- 3.5 and 7.9 +/- 1.5 J m(-2), respectively, while that of infectious pancreatic necrosis virus (IPNV) was substantially higher, 1188 +/- 57 J m(-2). Using ISAV as a model, a comparison of the effect of UVC irradiation on virus isolation versus reverse transcription polymerase chain reaction (RT-PCR) showed that considerably higher UVC doses, depending on the length of the amplified product, were necessary to abolish RT-PCR detection of viral RNA.

The consequence of p53 overexpression for liver tumor development and the response of transformed murine hepatocytes to genotoxic agents.

To analyse the effect of p53 on liver tumor development, we generated transgenic mice overexpressing wild-type p53 in the liver and crossed them with transgenic mice in which the expression of the SV40 large T antigen (TAg) induces hepatic tumors. Remarkably, whereas preneoplastic TAg liver exhibited anisocaryosis and anisocytosis, TAg/p53 liver never presented any dysplastic cells. Moreover, whereas expression of p53 did not affect hepatic development, its constitutive expression in tumorigenic livers resulted in a significantly enhanced apoptosis once nodules had appeared. In contrast, p53 overexpression did not modify the elevated proliferation of TAg-transformed hepatocytes and had no effect on hepatocarcinoma progression. In vitro analysis of primary hepatocytes exposed to various genotoxic agents showed that p53 failed to sensitize normal or TAg-transformed hepatocytes to apoptosis, except when high doses of doxorubicin, UV-B and UV-C radiation were used. Our results confirmed that the hepatocyte cell type is very resistant to genotoxic agents and showed that constitutive expression of p53 failed to improve their responsiveness. In addition, our results showed that suppression of dysplastic cells, probably by restoring normal cytokinesis and karyokinesis, and enhancement of apoptosis by means of p53 overexpression were insufficient to counteract or delay the TAg-induced liver tumoral progression. Oncogene (2000) 19, 3498 - 3507

Chronic lymphocytic leukemia lymphocytes lack the capacity to repair UVC-induced lesions

Cells from chronic lymphocytic leukemia (CLL) patients and from healthy individuals were irradiated with UVC and incubated for varying periods of time. The number of single strand breaks and alkali-labile sites was determined by comet analysis. Unirradiated CLL and healthy cells exhibited no significant numbers of single strand breaks. The extent of DNA damage was found to increase with dose for both healthy and CLL cells. However, the CLL cells had much more extensive DNA fragmentation than healthy cells at each dose. Deoxyribonucleoside supplemented medium inhibited comet formation in both cell types. Thymidine alone produced the same effect. In healthy cells, repair of lesions was complete after 4 h of incubation as indicated by the absence of comet formation. The CLL cells exhibited no significant repair even after 48 h. CLL lymphocytes are killed by very low doses of UVC radiation. The results reported here suggest that this hypersensitivity results from the inability of CLL cells to repair UVC-induced DNA damage and a contributing factor is the low amounts of intracellular deoxyribonucleosides.

Light and death: photons and apoptosis.

Phototherapies like photodynamic therapy (PDT), UVA1, UVB, and PUVA treat skin diseases. These phototherapies work because they alter cytokine profiles, change immune cytotoxicity in the skin, and directly kill diseased cells by apoptosis. Apoptosis is a term that only describes the morphologic changes a cell undergoes during this mode of cell death. The terms "immediate", "intermediate", and "delayed" apoptosis segregate the different apoptotic mechanisms into three kinetic categories, whereas the terms preprogrammed cell death (pre-PCD) and programmed cell death (PCD) describe the underlying mechanisms. Immediate apoptosis (T< or =0.5 h post-exposure) is triggered by singlet-oxygen damage that opens the mitochondrial megachannel, which can be mediated by PDT or UVA1 radiation. It is a pre-PCD mechanism of apoptosis, i.e., protein synthesis is not required post-insult, because all the necessary components are constitutively synthesized and only need to be activated. Intermediate apoptosis (T< or =4 h>0.5 h) is initiated by receptor cross-linking on the plasma membrane, which can be achieved using high doses of UVB or UVC radiation. It is also a pre-PCD mechanism. Delayed apoptosis (T>4 h) is induced by DNA damage that can be caused by X-rays, PUVA, UVC, UVB, UVA, and PDT. It is a PCD mechanism of apoptosis, i.e., protein synthesis is required post-insult. These three apoptotic mechanisms each access one of two "points-of-no-return" located on the mitochondrial membrane, which activate different, but not mutually exclusive, final pathways of apoptosis. This review discusses the latest findings on these apoptotic mechanisms and their implications in phototherapies.

The Martian and extraterrestrial UV radiation environment—1. Biological and closed-loop ecosystem considerations

The Martian surface is exposed to both UVC radiation (<280 nm) and higher doses of UVB (280–315 nm) compared to the surface of the Earth. Terrestrial organisms have not evolved to cope with such high levels of UVC and UVB and thus any attempts to introduce organisms to Mars, particularly in closed-loop life support systems that use ambient sunlight, must address this problem. Here we examine the UV radiation environment of Mars with respect to biological systems. Action spectra and UV surface fluxes are used to estimate the UV stress that both DNA and chloroplasts would experience. From this vantage point it is possible to consider appropriate measures to address the problem of the Martian UV environment for future long term human exploration and settlement strategies. Some prospects for improving the UV tolerance of organisms are also discussed. Existing artificial ecosystems such as Biosphere 2 can provide some insights into design strategies pertient to high UV environments. Some prospects for improving the UV tolerance of organisms are also discussed. The data also have implications for the establishment of closed-loop ecosystems using natural sunlight on the lunar surface and elsewhere in the Solar System.

Biological Effects of High Ultraviolet Radiation on Early Earth—a Theoretical Evaluation

The surface of early Earth was exposed to both UVC radiation (<280nm) and higher doses of UVB (280–315 nm) compared with the surface of present day Earth. The degree to which this radiation environment acted as a selection pressure on organisms and biological systems has rarely been theoretically examined with respect to the biologically effective irradiances that ancient organisms would receive. Here action spectra for DNA inactivation and isolated chloroplast inhibition are used to estimate biologically effective irradiances on archean Earth. Comparisons are made with present day Earth. The theoretical estimations on the UV radiation screening required to protect DNA on archean Earth compare well with field and laboratory observations on protective strategies found in present day microbial communities. They suggest that many physical and biological methods may have been effective and would have allowed for the radiation of life even under the high UV radiation regimes of archean Earth. Such strategies would also have provided effective reduction of photoinhibition by UV radiation. The data also suggest that the UV regime on the surface of Mars is not a life limiting factorper se, although other environmental factors such as desiccation and low temperatures may contribute towards the apparent lack of the surface biota.

Signal transduction through atypical PKCs, but not the EGF receptor, is necessary for UVC-induced AP-1 activation in immortal murine cells.

The exposure of mammalian cells to ultraviolet (u.v.) irradiation leads to activation of transcription factors, such as AP-1 and NFkappaB. It is postulated that the EGF receptor but not protein kinase C (PKC) is the major membrane mediator in UVC-induced signal transduction. We demonstrate here that the antisense oligonucleotides of PKC zeta and the dominant negative mutant of PKC lambda/iota as well as dominant negative PKC zeta markedly blocked UVC-induced AP-1 activity. In contrast, UVC-induced AP-1 activity in cells devoid of the EGF receptor (B82), is not significantly different from that of the stable transfectants with a kinase-deficient EGF receptor (B82M721), or wild-type EGF receptor (B82L). This was found at all UVC irradiation doses and time courses studied, while high levels of EGF-induced AP-1 activity were observed in B82L cells but not in B82 cells. This evidence strongly suggests that atypical PKCs, but not the EGF receptor, is necessary for UVC-induced AP-1 activation in JB6 and B82 cells.

Drosophila melanogaster larvae detect low doses of UVC radiation as manifested by a writhing response

Exposure of insects and higher orders of animals to UVC radiation has been shown to result in toxicity with a delayed expression. We have observed an immediate writhing response in slowly wandering third instar larvae of Drosophila melanogaster exposed to UVC radiation at doses that were not lethal. UVB and UVA radiation had no effect. Mutants for the visual and sensory systems appeared to respond normally, but CO2 anesthesia resulted in reversible inhibition, ebony and silver mutants, which affect different pathways in catecholamine metabolism, showed an absent to reduced response. Using UVC lasers, we were able to demonstrate a response in different regions of the larval body. Furthermore, a 193 nm laser that penetrates only 2–5 μm was able to induce a response but unable to kill the larvae. These results suggest a photochemical reaction occurs in the cuticle which produces free radicals that stimulate the nerves and muscle which are present immediately below the epidermis. Possible targets for the UVC radiation are catecholic compounds secreted and processed into the cuticle of third instar larvae just prior to pupariation whose primary function is to crosslink the protein and carbohydrate components. © 1996 Wiley-Liss, Inc.

Drosophila melanogaster larvae detect low doses of UVC radiation as manifested by a writhing response.

Exposure of insects and higher orders of animals to UVC radiation has been shown to result in toxicity with a delayed expression. We have observed an immediate writhing response in slowly wandering third instar larvae of Drosophila melanogaster exposed to UVC radiation at doses that were not lethal. UVB and UVA radiation had no effect. Mutants for the visual and sensory systems appeared to respond normally, but CO(2) anesthesia resulted in reversible inhibition. ebony and silver mutants, which affect different pathways in catecholamine metabolism, showed an absent to reduced response. Using UVC lasers, we were able to demonstrate a response in different regions of the larval body. Furthermore, a 193 nm laser that penetrates only 2-5 micron(s) was able to induce a response but unable to kill the larvae. These results suggest a photochemical reaction occurs in the cuticle which produces free radicals that stimulate the nerves and muscle which are present immediately below the epidermis. Possible targets for the UVC radiation are catecholic compounds secreted and processed into the cuticle of third instar larvae just to pupariation whose primary function is to crosslink the protein and carbohydrate components.

DNA damage and repair after low doses of UV-C radiation; comparable rates of repair in rodent and human cells.

The UV endonuclease assay for cyclobutane pyrimidine dimers in the DNA of UV-C-irradiated mammalian cells has been modified by replacing alkaline sucrose sedimentation with alkaline unwinding and hydroxylapatite chromatography to determine the number of DNA breaks introduced by the endonuclease. Dimers induced by doses as low as 0.25 Jm-2 can be detected and the assay has been used to examine the capacity of human, hamster and mouse cells to remove damage inflicted by sublethal doses of UV-C. In addition, incision activity has been measured by incubating cells with DNA synthesis inhibitors after irradiation with UV-C. In rodent and human cells, given a dose of UV-C of 1 Jm-2 about half of the endonuclease-sensitive sites are lost in 5-6 h. The incision capacity of these cells corresponds well with the extent of removal of dimers. Thus, although rodent cells are normally considered to be relatively deficient in nucleotide excision repair, we find that rodent and human cells have comparable capacities to deal with low levels of UV-C-induced damage.

Flow cytometric analysis of DNA sensitivity to nuclease S1 in UVC-irradiated human fibroblasts

The sensitivity of human fibroblast DNA towards the activity of nuclease S1 was investigated in situ after irradiation of the cells with UVC light. DNA digestion was assessed using flow cytometry by staining the nuclease-resistant DNA fraction with the specific fluorochrome propidium iodide (PI). The results showed a non-linear dependence of DNA digestion on the UVC irradiation dose between 0 and 20 J m −2. About 70% of the UVC-induced DNA sensitivity to nuclease S1 was lost after 30 min of repair. These results suggest that flow cytometry may be useful for assessing the heterogeneity of cell response to UV damage.

In vitro reduction of peroxidation in UVC‐irradiated cell cultures by concurrent exposure with Bowman‐Birk protease inhibitor

Bowman-Birk protease inhibitor (BBI), extracted from soybean flour, has been shown to protect cells from damage after UVC irradiation. BBI also reduces the number of transformed foci following exposure to the same doses of UVC irradiation. Proteins have been reported to act as antioxidants by their sheer intracellular bulk. In this study we assessed BBI's ability to attenuate UVC-irradiation-induced peroxidation. Balb c/3T3 cells, clone A31, subcloned by this laboratory were used. Treatment groups consisted of cells suspended in Hanks' Balanced Salt Solution with and without 100 micrograms/ml of a crude soybean extract containing the Bowman-Birk protease inhibitor. Cells were exposed to irradiation in suspension by using GE (G8T5) germicidal lamps which deliver predominantly 254 nm light at 0.385 J/m2/s as measured at the cell surface. Aliquots of UVC-exposed cells were homogenized in HBSS in the presence and absence of 100 micrograms/ml BBI with a motor-driven Teflon pestle and assayed for peroxidation. The results showed significant (P less than 0.0001) protection afforded by BBI against peroxidation at 1.16 (65%), 1.93 (60%, and 2.70 (48%) J/m2 of UVC irradiation. Transformation was reduced to 0.33 foci per dish at all levels of radiation exposure.

The Time Course of UVB and UVC Erythema

The time course of ultraviolet erythema was measured using six different exposure doses of UVC and UVB radiation in each of eight adult subjects. The intensity of erythema was measured by reflectance spectrophotometry at 4, 8, 24, 36, and 48 h after irradiation. In five subjects there was no significant difference between the form of the UVB and UVC erythema time-course. In three subjects a significant difference was observed, but this was random rather than systematic between subjects. The results do not confirm the previously reported major differences in time course between the two qualities of radiation.

Effects of short-wave ultraviolet radiation (UV-C) on delayed hypersensitivity in the guinea-pig.

Guinea-pigs, previously sensitized to dinitrochlorobenzene (DNCB), were exposed to varying doses of UV-C radiation (254 nm) on one of the flanks for a period of 15 days. They were then patch-tested with DNCB immediately after UV treatment. The responses to DNCB were diminished both in the irradiated and non-irradiated skin compared with control animals which had not received UV-C radiation. This effect was dose dependent and could only be demonstrated for a limited period of time. Guinea-pig exposed to UV-C radiation did not show a decreased response to sodium lauryl sulphate, which is a potent irritant. The influence of UV-C on delayed hypersensitivity therefore seems to be an immunological effect.