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Link to original content: https://pubmed.ncbi.nlm.nih.gov/10393932
Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB radiation - PubMed Skip to main page content
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. 1999 Jul 6;96(14):7974-9.
doi: 10.1073/pnas.96.14.7974.

Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB radiation

D Kulms  1 , B Pöppelmann, D Yarosh, T A Luger, J Krutmann, T Schwarz
Affiliations

Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB radiation

D Kulms et al. Proc Natl Acad Sci U S A. .

Abstract

UVB-induced DNA damage is a crucial event in UVB-mediated apoptosis. On the other hand, UVB directly activates death receptors on the cell surface including CD95, implying that UVB-induced apoptosis can be initiated at the cell membrane through death receptor clustering. This study was performed to measure the relative contribution of nuclear and membrane effects in UVB-induced apoptosis of the human epithelial cell line HeLa. UVB-mediated DNA damage can be reduced by treating cells with liposomes containing the repair enzyme photolyase followed by exposure to photoreactivating light. Addition of photolyase followed by photoreactivation after UVB reduced the apoptosis rate significantly, whereas empty liposomes had no effect. Likewise, photoreactivating treatment did not affect apoptosis induced by the ligand of CD95, CD95L. UVB exposure at 4 degrees C, which prevents CD95 clustering, also reduced the apoptosis rate, but to a lesser extent. When cells were exposed to UVB at 4 degrees C and treated with photolyase plus photoreactivating light, UVB-induced apoptosis was almost completely prevented. Inhibition of caspase-3, a downstream protease in the CD95 signaling pathway, blocked both CD95L and UVB-induced apoptosis, whereas blockage of caspase-8, the most proximal caspase, inhibited CD95L-mediated apoptosis completely, but UVB-induced apoptosis only partially. Although according to these data nuclear effects seem to be slightly more effective in mediating UVB-induced apoptosis than membrane events, both are necessary for the complete apoptotic response. Thus, this study shows that nuclear and membrane effects are not mutually exclusive and that both components contribute independently to a complete response to UVB.

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Figures

Figure 1
Figure 1
(a) Apoptosis is reduced on UVB exposure at low temperature. HeLa cells were UVB-irradiated (400 J/m2) at either 37°C or after being kept at 4°C for 10, 20, or 30 min. After irradiation, cells were kept on ice for 5 min and then incubated at 37°C for 16 hr. Control cells were not UVB-exposed but were incubated at 4°C for 30 min or kept at 37°C. (b) Cells were incubated with different concentrations of cis-platin either at 37°C or kept for 30 min before and 5 min after cis-platin stimulation. Apoptosis was evaluated 16 hr later with a cell death ELISA. Rate of apoptosis is reflected by the enrichment of nucleosomes in the cytoplasm shown on the y axis (mean ± SD of triplicate samples). Data presented show one representative of two independently performed experiments.
Figure 2
Figure 2
Enhancement of DNA repair reduces UVB-induced apoptosis. After UVB irradiation, increasing amounts of Photosomes were applied to the medium and cells were incubated at 37°C for 1 hr. Subsequently, photoreactivation was carried out by irradiating cells with PRL. For control purposes, cells were either left untreated, irradiated with PRL or UVB alone, or with UVB and PRL in the absence of Photosomes. In addition, cells were incubated with empty liposomes (lip) followed by exposure to PRL. After 16 hr at 37°C, apoptosis was evaluated with a cell death ELISA. Enrichment factor was used as a parameter of apoptosis and is given as the mean ± SD of three independently performed experiments.
Figure 3
Figure 3
Photoreactivation reduces UVB-induced DNA damage. Cells were left untreated (lane 1) or were exposed either to UVB (lane 2), to PRL (lane 3), or to UVB followed by PRL (lane 4). In lanes 5 and 6, UVB-exposed cells were treated with Photosomes (lane 5) or empty liposomes (lane 6) for 1 hr followed by exposure to PRL. Immediately after photoreactivation, genomic DNA was extracted and subjected to Southwestern dot-blot analysis by using an Ab against thymine–thymine CPD.
Figure 4
Figure 4
Enhancement of DNA repair by photoreactivation reduces caspase-3 activation and PARP cleavage. Cells were left untreated (lane 1) or exposed either to UVB (lane 2), to PRL (lane 3), or to UVB followed by PRL (lane 4). In lanes 5 and 6, UVB-exposed cells were treated with Photosomes (lane 5) or empty liposomes (lane 6) for 1 hr followed by exposure to PRL. After 16 hr, proteins were subjected to Western blot analysis with a caspase-3 Ab (a) or a PARP Ab (b). Equal loading was monitored with an Ab directed against α-tubulin.
Figure 5
Figure 5
Photoreactivation does not affect CD95-mediated apoptosis. Cells were exposed to UVB, CD95L or to an agonistic CD95-Ab. Subsequently, cells were treated with Photosomes or liposomes followed by PRL. Apoptosis was evaluated 16 hr later by using a cell death ELISA. Rate of apoptosis is reflected by the enrichment of nucleosomes in the cytoplasm, shown on the y axis (mean ± SD of triplicate samples). Data presented show one representative of two independently performed experiments.
Figure 6
Figure 6
Enhancement of DNA repair and prevention of surface receptor aggregation most effectively block UVB-induced apoptosis. HeLa cells were UVB exposed at 37°C (open bars) or at 4°C (filled bars). Subsequently, cells were treated with Photosomes or liposomes followed by PRL, and 16 hr later, apoptosis was evaluated by using a cell death ELISA. Enrichment factor was used as a parameter of apoptosis and is given as the mean ± SD of three independently performed experiments.
Figure 7
Figure 7
Caspase inhibitors suppress CD95- and UVB-mediated apoptosis differentially. Cells were treated with one of the caspase inhibitors z-VAD, z-DEVD, or z-IETD (20 μM each). Cells were UVB-irradiated or incubated with CD95L and an agonistic CD95-Ab, respectively, 1 hr later. Control cells were stimulated with the inhibitors only. Apoptosis was evaluated 16 hr later by using a cell death ELISA. Enrichment factor was used as a parameter of apoptosis and is given as the mean ± SD of three independently performed experiments.
Figure 8
Figure 8
Proposed signaling model for UVB-induced apoptosis.
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