- Email: [email protected]
ULTRAVIOLET B IRRADIATION MODULATES SUSCEPTIBILITY TO TUMOUR NECROSIS FACTOR-α-INDUCED APOPTOSIS VIA INDUCTION OF DEATH RECEPTORS IN MURINE FIBROBLASTS
Cell Biology International 2001, Vol. 25, No. 12, 1221–1228 doi:10.1006/cbir.2001.0805, available online at http://www.idealibrary.com on
ULTRAVIOLET B IRRADIATION MODULATES SUSCEPTIBILITY TO TUMOUR NECROSIS FACTOR--INDUCED APOPTOSIS VIA INDUCTION OF DEATH RECEPTORS IN MURINE FIBROBLASTS HIROKAZU KIMURA1,2, HISANORI MINAKAMI3 and AKIRA SHOJI1 1
Department of Biological Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515; Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Gunma 371-0052; 3 Department of Reproductive and Developmental Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan 2
Received 8 July 2001; accepted 13 July 2001
Ultraviolet B (UVB) irradiation causes cell death by apoptosis in murine fibroblast cells. Tumor necrosis factor- (TNF-) is also a well known inducer of apoptosis, although the physiological significance of this activity is poorly understood. We investigated the effects of pretreatment with UVB (312 nm) on TNF--induced apoptosis in murine fibroblast cells. UVB enhanced susceptibility to cell death by TNF- in a dose-dependent manner. UVB but not TNF- induced the expression of TNF receptor type-1 (TNFR-1) and type-2 (TNFR-2) in a dose-dependent manner. Expression of Fas (CD95) and Fas-ligand (Fas-L), and significant DNA fragmentation were observed in the cells that died. These results suggest that UVB irradiation modulates susceptibility to TNF--induced apoptosis through the induction of TNFRs, Fas, and Fas-L in 2001 Academic Press murine fibroblasts. K: apoptosis; fibroblast; TNF-; TNF receptor; ultraviolet.
INTRODUCTION The cells on the surface of the body are bombarded with ultraviolet (UV) irradiation (Doll and Peto, 1981). UV irradiation causes sunburn, inflammation, tissue injury, and skin cancer (Doll and Peto, 1981; Madfu and Thomas, 1993). Oncogenes and protooncogenes such as c-myc, c-fos and c-jun are activated by UV irradiation; thus, it is a physical carcinogen (Doll and Peto, 1981; Angel and Karin, 1991; Nakafuku et al., 1992). Excessive UV irradiation also induces Fas (CD95), Fas-ligand (Fas-L) and cell death by apoptosis in murine fibroblast cells (Kimura et al., 1999, 2000). Thus, UV irradiation can have diverse effects on cells (Angel and Karin, 1991). These data indicate that cells will die if the damage is severe enough, whereas cells with only a mild injury may undergo To whom correspondence should be addressed: Dr Akira Shoji, Department of Biological Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan. Fax: +81-277-30-1443. E-mail: [email protected] 1065–6995/01/121221+08 $35.00/0
repair and thus survive (Kimura et al., 1999, 2000). It is generally believed that abnormal cells that can potentially lead to defective cellular offspring are programmed to die by apoptosis (Thompson, 1995; Jacobson et al., 1997; Kimura et al., 1999). UVinduced cell death by apoptosis may act to prevent the reproduction of abnormal cells. UV irradiation stimulates the production of tumor necrosis factor (TNF) in various cells, such as human keratinocytes, macrophages, and corneal stroma cells (Bazzoni et al., 1994; Kennedy et al., 1997; Kibital et al., 1998). TNF- is a proinflammatory cytokine and is now recognized as a critical component that orchestrates many different aspects of host inflammatory defences, including differentiation and proliferation, as well as the ability to induce cell death (Ware et al., 1996; Nagata, 1997; Sheikh et al., 1998; Kothney-Wilkins et al., 1999). These results suggest that TNF- is involved in UV-induced inflammation, cell death, and tissue injuries (Bazzoni et al., 1994; Kennedy et al., 1997; Kibitel et al., 1998; Sheikh et al., 1998; 2001 Academic Press
1222
Kothny-Wilkins et al., 1999). There are two types of TNF receptor (TNFR) (Tartaglia and Goeddel, 1992). TNF binds to TNFRs, and both receptors can transduce a death signal or an anti-death signal in cells (Martin and Cotter, 1991; Chinnaiyan et al., 1995; Liu et al., 1996; Duckett and Thompson, 1997; Nagata, 1997; Baker and Reddy, 1998; Sheikh et al., 1998; Ledgerwood et al., 1999). The type-1 and type-2 receptors also have different roles (Tartaglia et al., 1993; Rothe et al., 1995; Baker and Reddy, 1998; Sheikh et al., 1998; Ledgerwood et al., 1999). Recently, UV-exposed lung carcinoma cells have been shown to be easily killed by TNF- (Sheikh et al., 1998). However, the possible effects of UV on TNFRs is not exactly understood in this phenomenon. Fibroblasts are important cells in the skin and connective tissues, which may always be exposed to UV radiation (Kimura et al., 1999, 2000). We found that UVB modulated susceptibility to TNF--induced cell death through the induction of death receptors such as TNFRs, Fas, and Fas-L in murine fibroblasts (L-929 cells). We report here the effects of pretreatment with UVB on TNF--induced apoptosis in murine fibroblasts. MATERIALS AND METHODS Cells and cell culture Murine fibroblast L-929 cells (NTCC clone 929) were purchased from Dainippon Pharmaceutical Co. Ltd (Osaka, Japan) and grown in Eagle’s minimal essential medium (MEM) without phenol red (Nissui Pharmaceutical Co. Ltd, Tokyo), containing 10% fetal calf serum (FCS) at 37C in an atmosphere of 5% CO2 (Kimura et al., 1999, 2000). UVB exposure and addition of TNF- The L-929 cells were grown in 96-well microplates (Corning Co. Ltd, NY, U.S.A.) until confluent (3.1–3.3105/cm2). The culture medium was replaced with 50 l of MEM containing 2% fetal calf serum (FCS). The cells were then irradiated with UVB (312 nm, 60 to 960 mJ/cm2) using a UVB generator (Atto, DT-20 MP, Tokyo, Japan) (Kimura et al., 1999; Kimura et al., 2000). Just after the exposure to UVB radiation, 50 l of various concentrations of recombinant mouse TNF- (Strathmann Biotech., Hannover, Germany) was added. The concentration of TNF- ranged from 0 to 100 ng/ml. The cells were incubated for 17 h at 37C in an atmosphere of 5%
Cell Biology International, Vol. 25, No. 12, 2001
CO2. Because phenol red can cause a phototoxic reaction, we used MEM that did not contain this dye. Measurement of cell viability After 17 h incubation at 37C in an atmosphere of 5% CO2, cell viability was examined using the crystal violet method (Itoh et al., 1991). Dye uptake by cells was measured at 550 nm using a microplate autoreader (SJEIA-II, Sanko Junyaku Co. Ltd, Tokyo). Analysis of Fas, Fas-L, TNFR-1 and TNFR-2 expression by fluorescence flow cytometry The treated cells were washed in a culture flask (25 cm2) with phosphate-buffered saline (PBS) without calcium and magnesium. The cells were detached from the flask wall with a cell scraper, dispersed by gentle pipetting, and passed through a 45 m filter (Millipore, Bedford, MA, U.S.A.). An aliquot of 20 g/ml of anti-mouse rabbit polyclonal Fas IgG antibody, anti-mouse rabbit polyclonal Fas-L IgG antibody, anti-mouse goat polyclonal TNFR-1 IgG antibody, anti-mouse goat polyclonal TNFR-2 IgG antibody (mouse and rat reactive, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), or isotype-matched control antibody (anti-rabbit or anti-goat normal control IgG, Genzyme, Minneapolis, MN, U.S.A.) was added to a cell suspension of 4–6105/ml in PBS and incubated for 1 h at 4C. The cells were washed twice with PBS, then stained with 10 g/ml of anti-rabbit or anti-goat FITC-conjugated F(ab )2 fragment (Organon Tecnica, Durham, NC, U.S.A.) (Kimura et al., 1999). Fluorescence flow cytometric analysis was then performed (Epics XL System II, Coulter Co. Ltd, Tokyo). Analysis of DNA fragmentation After UVB pretreatment and incubation with TNF- for 17 h, the cells attached to a culture flask were washed with PBS. After washing, the cells were detached with a cell scraper, dispersed by gentle pipetting and collected by centrifugation (1500g, 15 min, 4C). DNA from these cells was purified according to a method previously reported (Laird et al., 1991). The purified DNA was electrophoresed on a 1.5% agarose gel and stained with ethidium bromide (Wako Pure Chemicals Co. Ltd, Tokyo).
Cell Biology International, Vol. 25, No. 12, 2001
1223
Fig. 1. Microscopic findings in cells treated with UVB alone and with UVB plus TNF-. L-929 murine fibroblasts were exposed to no UVB (a, control), 100 pg/ml of TNF- alone for 17 h (b), 480 mJ/cm2 of UVB alone for 17 h (c), or 100 pg/ml of TNF- for 17 h after 480 mJ/cm2 of UVB (d). The cells were visualized by light microscopy under 100 magnification. Bar indicates 100 m.
RESULTS Light microscopy findings in cells treated with UVB and/or TNF- Using light microscopy, we observed morphological changes in the cells caused by UVB and TNF- after 17 h incubation. Cells treated with 100 pg/ml of TNF- alone did not detach from the microplate walls (Fig. 1B). A small fraction of the cells treated with UVB (480 mJ/cm2) alone had detached from the plate wall (Fig. 1C). Morphological changes were also observed in these cells. A significant number of cells treated with UVB (480 mJ/cm2) and TNF- (100 pg/ml) had detached from the plate wall (Fig. 1D). Effects of various doses of UVB and TNF on cell viability We investigated the interrelationship between various doses of UVB and TNF- with respect to their
effect on cell viability after 17 h incubation (Fig. 2). In the control cells not exposed to UVB a higher concentration than 1 ng/ml of TNF- was required to induce cell death. A concentration of 1 ng/ml of TNF- killed approximately 50% of the cells, 70% of the cells, and 90% of the cells when the cells were pretreated with 120 mJ/cm2, 240 mJ/cm2, and 480 mJ/cm2 of UVB, respectively. Thus, the dose of TNF- required to kill the cells decreased as the pretreatment dose of UVB increased, suggesting that UVB modulated susceptibility to TNF--induced cell death. UVB-induced expression of TNFR-1 and TNFR-2 in L-929 cells The expression of TNFR-1 and TNFR-2 was examined in the control cells (exposed to neither UVB nor TNF-), cells treated with 100 pg/ml of TNF- alone, treated with 480 mJ/cm2 of UVB alone, or treated with both 100 pg/ml of TNF-
1224
Cell Biology International, Vol. 25, No. 12, 2001
TNFR-1 expression (A)
0 0.1
50
(E) Isotype
Count
75
TNFR-2 expression 100
Isotype
Count
100
Cell viability (%)
100
(A) (B) (C) (D) (E)
0 0.1
1000
1000 FITC
FITC 100
100 (B)
(F)
2
3
4
10 10 10 10 TNF-α concentration (pg/ml)
5
10
Fig. 2. Effects of various doses of UVB and TNF- on cell viability. Just after exposure to various doses of UVB (0–480 mJ/cm2), the various doses of TNF- were added to the culture medium. Cell viability was assessed by the crystal violet method after 17 h incubation. The vertical bar indicates the meanstandard error (SE) of triplicate experiments. (A) Non-irradiated cells; (B) cells treated with 60 mJ/cm2 of UVB; (C) cells treated with 120 mJ/cm2 of UVB; (D) cells treated with 240 mJ/cm2 of UVB; (E) cells treated with 480 mJ/cm2 of UVB.
Count
0 0.1
0 0.1
1000 FITC
100
100 (C)
(G) A
0 0.1
B
0 0.1
1000 FITC
UVB and TNF--induced expression of Fas and Fas-L in L-929 cells The expression of Fas and Fas-L was examined in the control cells (exposed to neither UVB nor TNF-), cells treated with 100 pg/ml of TNF- alone, treated with 480 mJ/cm2 of UVB alone, or treated with both 100 pg/ml of TNF- and 480 mJ/cm2 of UVB (Fig. 5). Fas was constitutively
1000 FITC
100
100 (H) Count
(D) Count
and 480 mJ/cm2 of UVB (Fig. 3). Both TNFR-1 and TNFR-2 were constitutively expressed (Fig. 3A and E). Expression of TNFR-1 or TNFR-2 (Fig. 3B and F) was not induced by the addition of 100 pg/ml of TNF- as compared with the control cells (Fig. 3A and E). The expression of TNFR-1 and TNFR-2 was markedly induced by UVB alone (Fig. 3C and G). The addition of TNF- to cells pretreated with UVB did not further induce the expression of TNFR-1 or TNFR-2 (Fig. 3D and H) as compared with UVB alone. We investigated the effects of the dose of UVB on the expression of TNFR-1 and TNFR-2. The relative area, defined as the B/A100 (the areas of A and B as shown in Fig. 3G), was calculated (Fig. 4). UVB increased TNFR-1 and TNFR-2 expression in a dose-dependent manner.
1000 FITC
Count
1
Count
0
Relative cell number
Count
25
0 0.1
1000 FITC
0 0.1
1000 FITC
Fluorescence intensity (log)
Fig. 3. UVB-induced expression of TNFR-1 and TNFR-2 in L-929 cells. Panels A, B, C, and D represent experiments for the detection of TNFR-1; panels E, F, G and H represent experiments for the detection of TNFR-2. (A and E) Untreated cells (exposed to neither UVB nor TNF-); (B and F) cells incubated with 100 pg/ml of TNF- alone for 17 h; (C and G) cells 17 h after UVB (480 mJ/cm2) exposure alone; (E and H) cells pretreated with UVB (480 mJ/cm2) and incubated with TNF- (100 pg/ml) for 17 h.
expressed in the control cells (Fig. 5A), and Fas-L was slightly expressed in the control cells (Fig. 5A and E). Expression of Fas or Fas-L (Fig. 5B and F) was not induced by the addition of 100 pg/ml of TNF- as compared with the control cells (Fig. 5A and E). The expression of Fas and Fas-L was slightly induced by UVB alone (Fig. 5C and G).
Cell Biology International, Vol. 25, No. 12, 2001
1225
Fas expression
FasL expression 100
100
TNFR-1 TNFR-2
Isotype
Isotype
(A)
(E)
30
Count
Count
Relative area (%)
40
0 0.1
20
0 0.1
1000
100
100
10
UVB dose (mJ/cm )
Fig. 4. Effects of the UVB dose on the expression of TNFR-1 and TNFR-2. The relative area was defined as 100B/A (areas of A and B are depicted in Fig. 3G, (A) area under the entire curve of all the cells, (B) area under the curve of cells expressing either TNFR-1 or TNFR-2). Detailed experimental conditions are described in the text. Vertical bar indicates the meanSE of triplicate experiments. L-929 cells were exposed to various doses of UVB (60–960 mJ/cm2). Following exposure, the cells were analysed for the expression of TNFR-1 and TNFR-2.
The addition of TNF- to cells pretreated with UVB markedly induced the expression of both Fas and Fas-L (Fig. 5D and H) as compared with UVB alone. DNA fragmentation in L-929 cells treated with UVB and/or TNF- We examined the serial changes of DNA fragmentation in adherent control cells (exposed to neither UVB nor TNF-) and in adherent cells treated with UVB alone, TNF- alone or both UVB and TNF-. No fragmentation was observed in the control cells (exposed to neither UVB nor TNF-), the cells treated with 60 mJ/cm2 or 480 mJ/cm2 of UVB alone, or the cells treated with 100 pg/ml of TNF- alone (Fig. 6, lanes 1 to 4). In contrast, the cells treated with 480 mJ/cm2 UVB plus 100 pg/ml of TNF- showed marked DNA fragmentation (Fig. 6, lane 7), strongly suggesting that the cell death induced by this treatment was due to apoptosis. DISCUSSION We have demonstrated that pretreatment with UVB markedly enhanced the susceptibility of
Count
1000
0 0.1
0 0.1
1000
1000 FITC
FITC 100
100 (C)
(G) Count
800 2
0 0.1
0 0.1
1000
1000 FITC
FITC 100
100 (D)
(H) Count
600
Count
400
Count
200
(F)
Count
(B)
Relative cell number
0
1000 FITC
FITC
0 0.1
1000 FITC
0 0.1
1000 FITC
Fluorescence intensity (log)
Fig. 5. UVB and TNF--induced expression of Fas and Fas-L in L-929 cells. (A, B, C, and D) Experiments for detection of Fas; (E, F, G, and H) experiments for detection of Fas-L. (A and E) Untreated cells (not exposed to UVB or TNF-); (B and F) cells incubated with 100 pg/ml of TNF- alone for 17 h; (C and G) cells 17 h after UVB (480 mJ/cm2) exposure alone; (E and H) cells pretreated with UVB (480 mJ/cm2) and incubated with TNF- (100 pg/ml) for 17 h.
murine fibroblasts to TNF--induced cell death. This may have been caused by promoting an increase in TNFRs after UVB irradiation. In addition, 100 pg/ml of TNF-, which in itself did not induce Fas or Fas-L in the absence of pretreatment with UVB, enhanced the expression of Fas and Fas-L when the cells were pretreated with UVB. More than 1 ng/ml of TNF- is required to
1226
Cell Biology International, Vol. 25, No. 12, 2001
DNA fragmentation M
1
2
3
4
5
6
7
Ori
1057 bp
Fig. 6. DNA fragmentation in L-929 cells treated with UVB and TNF-. L-929 cells were treated with various doses of UVB and TNF- either alone or in combination. The cells were lysed and DNA was prepared from the cells to determine the extent of DNA fragmentation after UVB pretreatment and incubation with TNF- for 17 h. M, marker (X174/Hinc II digest); lane 1, control cells (exposed to neither UVB nor TNF-); lane 2, cells treated with 60 mJ/cm2 of UVB alone; lane 3, cells treated with 480 mJ/cm2 of UVB alone; lane 4, cells treated with 100 pg/ml of TNF- alone; lane 5, cells treated with 5 ng/ml of TNF- alone; lane 6, cells treated with 50 ng/ml of TNF- alone; lane 7, cells treated with 480 mJ/cm2 of UVB plus 100 pg/ml of TNF-.
induce cell death in non-irradiated cells, while 50 pg/ml of TNF- is sufficient to cause cell death in cells pretreated with 480 mJ/cm2 of UVB. All these results suggest that UVB irradiation can modulate susceptibility to TNF--induced cell death through the induction of death receptors such as TNFRs, Fas and Fas-L in murine fibroblasts. These death receptors may have played a role in the observed cell death. Fas and Fas-L expression may precede apoptosis (Nagata, 1997; Kimura et al., 1999). The expression of Fas and Fas-L was markedly increased in cells treated with both 480 mJ/cm2 of UVB and 100 pg/ml of TNF- in the present study, and approximately 70% of the cells died over the subsequent 17 h. In addition, significant DNA fragmentation was observed in these cells (Figs. 6 & 7). These results suggested that the cell death seen in this study was due to apoptosis. There are two types of TNF receptor in some cells (Tartaglia et al., 1993; Baker and Reddy, 1998;
Ledgerwood et al., 1999). Numerous reports suggest that both TNFR-1 and TNFR-2 can transduce both an apoptotic signal and an anti-apoptotic signal (Martin and Cotter, 1991; Chinnaiyan et al., 1995; Liu et al., 1996; Duckett and Thompson, 1997; Nagata, 1997; Baker and Reddy, 1998; Sheikh et al., 1998; Ledgerwood et al., 1999). However, our results do not show whether UVB irradiation altered the nature of TNFRs or altered signal transduction at a site downstream of TNFRs. Induction of TNFRs was only examined by flow cytometry in this study. However, previous studies suggest that increased expression of TNFR genes and TNFR mRNAs are associated with induction of TNFRs (Jin et al., 2000). It is possible that expression of TNFR genes and TNFR mRNAs may have been increased in the cells treated with UVB and TNF-. This hypothesis remains to be studied. Excessive exposure to UV causes inflammation in skin tissue (Madfu and Thomas, 1993). UV irradiation also induces the production of TNF in various cell types such as human keratinocytes, macrophages, and corneal cells (Bazzoni et al., 1994; Kennedy et al., 1997; Kibitel et al., 1998). It is believed that injured cells are removed to prevent the reproduction of any abnormal cells (Thompson, 1995; Jacobson et al., 1997). Because TNF is a proinflammatory cytokine and a wellknown inducer of apoptosis, it is reasonable to postulate that TNF is involved in UV-induced skin injury and cell death. To our knowledge, there has been only one study that examined the interrelationship between UV irradiation, TNF, and TNFR with respect to cell death. In that study, Sheikh et al. (1998) found that UV enhanced the susceptibility of lung cancer cells to TNF-induced cell death. In this study, we found that UVB induced TNFRs. This may explain why UV pretreatment made the cells more vulnerable to TNF-. Increased expression of TNFRs, Fas, and Fas-L may be a likely explanation for an enhanced susceptibility to TNF- in cells treated with UV. The dose of UVB used in this study was 60–960 mJ/ cm2 at 312 nm. Based on the biological effects of UVB, a dose of 480 mJ/cm2 corresponds to approximately 4 h of sunbathing during the summer at sea level (Madfu and Thomas, 1993). Thus, our experimental conditions may be clinically relevant, for instance, to the effects of sunbathing. A large dose of UV itself kills cells (Hall et al., 1988; Martin and Cotter, 1991). However, mild UV irradiation can act as a physical carcinogen (Doll and Peto, 1981; Angel and Karin, 1991; Madfu et al., 1993; Thompson, 1995). This
Cell Biology International, Vol. 25, No. 12, 2001
may be due to the replication of abnormal cells in a population of mildly injured cells (Thompson, 1995; Jacobson et al., 1997). Therefore, mildly injured cells may need to be removed (Thompson, 1995; Jacobson et al., 1997). TNF- can induce cell death in a number of different sensitive cell types in vitro, such as blood cells, keratinocytes, and fibroblasts, although the biological significance of this activity is not well understood (Baker and Reddy, 1998; Ledgerwood et al., 1999). In this study, a clinically relevant dose of UVB made fibroblasts vulnerable to TNF-, perhaps through the induction of death-related molecules such as TNFRs, Fas, and Fas-L, thus leading to cell death. Because UV has been reported to stimulate TNF release from various cells (Bazzoni et al., 1994; Kennedy et al., 1997; Kibitel et al., 1998; Andersson et al., 1988), it is possible that both enhanced TNF release and increased expression of death receptors caused by UV exposure may work additively in inducing cell death after sunbathing. ACKNOWLEDGEMENTS We are most grateful to Ms Masae Ohbuchi (Gunma Prefectural Insitute of Public Health and Environmental Sciences) for her skilful assistance. REFERENCES A U, S B, A J, M G, 1988. Concomitant production of different lymphokines in activated T cells. Eur J Immunol 18: 2081–2084. A P, K M, 1991. The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta 1072: 129–157. B S-J, R E-P, 1998. Modulation of life and death by the TNF receptor superfamily. Oncogene 17: 3261–3270. B F, K V, S A, J C-V, B B, 1994. Analysis of tumor necrosis factor promotes responses to ultraviolet light. J Clin Invest 93: 56–62. C A-M, O’R K, T M, D V-M, 1995. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81: 505–512. D R, P R, 1981. The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst 66: 1191–1308. D C-S, T C-B, 1997. CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival. Genes Dev 11: 2810–2821. H E-J, A M, B J, B C, C S-B, F M, G C, H T, M J, O N, 1988. Basic radiobiology. Am J Clin Oncl 11: 220–252. I N, Y S, I A, Y M, M S, S M, H A, S Y, N S, 1991. The
1227
polypeptide encoded by cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66: 233–243. J M-D, W M, R M-C, 1997. Programmed cell death in animal development. Cell 88: 347–354. J L, R D-P, C T-D, P S-J, H C-W, P T-L, S R-G, 2000. Enhanced murine macrophage TNF receptor shedding by cytosine-guanine sequences in oligodeoxynucleotides. Enhanced murine macrophage TNF receptor shedding by cytosine-guanine sequences in oligodeoxy-nucleotides. J Immunol 165: 5153–5160. K M, K K-H, H B, B J, P S, M C, E H, R J-T, A C-A, A J-C, 1997. Ultraviolet irradiation induces the production of multiple cytokines by human corneal cells. Invest Ophthalmol Vis Sci 38: 2483–2491. K J, H V, A L, O’C A, S B-M, Y D, 1998. UV-DNA damage in mouse and human cells induces the expression of tumor necrosis factor alpha. Photochem Photobiol 67: 541–546. K H, M H, S I, O K, S A, 1999. Phorbol ester facilitates apoptosis in murine fibroblasts pretreated by mild ultraviolet radiation. J Biochem 126: 340–346. K H, M H, O K, S A, 2000. Cu-Zn superoxide dismutase inhibits lactate dehydrogenase release and protects against cell death in murine fibroblasts pretreated with ultraviolet radiation. Cell Biol Int 24: 459–465. K-W G, K D, L T-A, K M, S T, 1999. Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosisinducing ligand and CD95-induced apoptosis but not from ultraviolet radiation-induced apoptosis. J Biol Chem 274: 28916–28921. L P-W, Z A, L K, R M-A, J R, B A, 1991. Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19: 4293. L E-C, P J-S, B J-R, 1999. Recent advances in the molecular basis of TNF signal transduction. Lab Invest 79: 1041–1050. L Z-G, H H, G D-V, K M, 1996. Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell 87: 565–576. M A-P, T B-F, 1993. Preventive treatment of sunburn, dermatoheliosis, and skin cancer with sunprotective agents. In: Thomas B-F, Arthur Z-E, Klaus W, Irwin M-F, Frank A, eds. Dermatology in general medicine. New York, McGraw-Hill. 1689–1717. M S-J, C T-G, 1991. Ultraviolet B irradiation of human leukaemia HL-60 cells in vitro induces apoptosis. Int J Radiat Biol 59: 1001–1016. N S, 1997. Apoptosis by death factors. Cell 88: 355–365. N M, S T, K Y, 1992. Differentiation factors, including nerve growth factor, fibroblast growth factor, and interleukin-6, induce an accumulation of an active Ras. GTP complex in rat pheochromocytoma PC 12 cells. J Biol Chem 267: 19448–19454. O N-K, C A, P V-C, F P-A, K J-D, G J-R, S E-M, T L-D, U S-R, K M-C, 1997. Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem 272: 16351–16357.
1228
R M, P M-G, H W-J, A T-M, G D-V, 1995. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83: 1243–1252. S M-S, A M-J, H Y, F A-J, 1998. Ultraviolet-irradiation-induced apoptosis is mediated via ligand independent activation of tumor necrosis factor receptor 1. Oncogene 17: 2555–2563. T L-A, G D-V, 1992. Two TNF receptors. Immunol Today 13: 151–153.
Cell Biology International, Vol. 25, No. 12, 2001
T L-A, A T-M, W G-H, G D-V, 1993. A novel domain within the 55 kd TNF receptor signals cell death. Cell 74: 845–853. T C-B, 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456–1462. W C-F, VA S, VA T-L, 1996. Apoptosis mediated by the TNF-related cytokine and receptor families. J Cell Biochem 60: 47–55.
ULTRAVIOLET B IRRADIATION MODULATES SUSCEPTIBILITY TO TUMOUR NECROSIS FACTOR--INDUCED APOPTOSIS VIA INDUCTION OF DEATH RECEPTORS IN MURINE FIBROBLASTS HIROKAZU KIMURA1,2, HISANORI MINAKAMI3 and AKIRA SHOJI1 1
Department of Biological Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515; Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Gunma 371-0052; 3 Department of Reproductive and Developmental Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan 2
Received 8 July 2001; accepted 13 July 2001
Ultraviolet B (UVB) irradiation causes cell death by apoptosis in murine fibroblast cells. Tumor necrosis factor- (TNF-) is also a well known inducer of apoptosis, although the physiological significance of this activity is poorly understood. We investigated the effects of pretreatment with UVB (312 nm) on TNF--induced apoptosis in murine fibroblast cells. UVB enhanced susceptibility to cell death by TNF- in a dose-dependent manner. UVB but not TNF- induced the expression of TNF receptor type-1 (TNFR-1) and type-2 (TNFR-2) in a dose-dependent manner. Expression of Fas (CD95) and Fas-ligand (Fas-L), and significant DNA fragmentation were observed in the cells that died. These results suggest that UVB irradiation modulates susceptibility to TNF--induced apoptosis through the induction of TNFRs, Fas, and Fas-L in 2001 Academic Press murine fibroblasts. K: apoptosis; fibroblast; TNF-; TNF receptor; ultraviolet.
INTRODUCTION The cells on the surface of the body are bombarded with ultraviolet (UV) irradiation (Doll and Peto, 1981). UV irradiation causes sunburn, inflammation, tissue injury, and skin cancer (Doll and Peto, 1981; Madfu and Thomas, 1993). Oncogenes and protooncogenes such as c-myc, c-fos and c-jun are activated by UV irradiation; thus, it is a physical carcinogen (Doll and Peto, 1981; Angel and Karin, 1991; Nakafuku et al., 1992). Excessive UV irradiation also induces Fas (CD95), Fas-ligand (Fas-L) and cell death by apoptosis in murine fibroblast cells (Kimura et al., 1999, 2000). Thus, UV irradiation can have diverse effects on cells (Angel and Karin, 1991). These data indicate that cells will die if the damage is severe enough, whereas cells with only a mild injury may undergo To whom correspondence should be addressed: Dr Akira Shoji, Department of Biological Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan. Fax: +81-277-30-1443. E-mail: [email protected] 1065–6995/01/121221+08 $35.00/0
repair and thus survive (Kimura et al., 1999, 2000). It is generally believed that abnormal cells that can potentially lead to defective cellular offspring are programmed to die by apoptosis (Thompson, 1995; Jacobson et al., 1997; Kimura et al., 1999). UVinduced cell death by apoptosis may act to prevent the reproduction of abnormal cells. UV irradiation stimulates the production of tumor necrosis factor (TNF) in various cells, such as human keratinocytes, macrophages, and corneal stroma cells (Bazzoni et al., 1994; Kennedy et al., 1997; Kibital et al., 1998). TNF- is a proinflammatory cytokine and is now recognized as a critical component that orchestrates many different aspects of host inflammatory defences, including differentiation and proliferation, as well as the ability to induce cell death (Ware et al., 1996; Nagata, 1997; Sheikh et al., 1998; Kothney-Wilkins et al., 1999). These results suggest that TNF- is involved in UV-induced inflammation, cell death, and tissue injuries (Bazzoni et al., 1994; Kennedy et al., 1997; Kibitel et al., 1998; Sheikh et al., 1998; 2001 Academic Press
1222
Kothny-Wilkins et al., 1999). There are two types of TNF receptor (TNFR) (Tartaglia and Goeddel, 1992). TNF binds to TNFRs, and both receptors can transduce a death signal or an anti-death signal in cells (Martin and Cotter, 1991; Chinnaiyan et al., 1995; Liu et al., 1996; Duckett and Thompson, 1997; Nagata, 1997; Baker and Reddy, 1998; Sheikh et al., 1998; Ledgerwood et al., 1999). The type-1 and type-2 receptors also have different roles (Tartaglia et al., 1993; Rothe et al., 1995; Baker and Reddy, 1998; Sheikh et al., 1998; Ledgerwood et al., 1999). Recently, UV-exposed lung carcinoma cells have been shown to be easily killed by TNF- (Sheikh et al., 1998). However, the possible effects of UV on TNFRs is not exactly understood in this phenomenon. Fibroblasts are important cells in the skin and connective tissues, which may always be exposed to UV radiation (Kimura et al., 1999, 2000). We found that UVB modulated susceptibility to TNF--induced cell death through the induction of death receptors such as TNFRs, Fas, and Fas-L in murine fibroblasts (L-929 cells). We report here the effects of pretreatment with UVB on TNF--induced apoptosis in murine fibroblasts. MATERIALS AND METHODS Cells and cell culture Murine fibroblast L-929 cells (NTCC clone 929) were purchased from Dainippon Pharmaceutical Co. Ltd (Osaka, Japan) and grown in Eagle’s minimal essential medium (MEM) without phenol red (Nissui Pharmaceutical Co. Ltd, Tokyo), containing 10% fetal calf serum (FCS) at 37C in an atmosphere of 5% CO2 (Kimura et al., 1999, 2000). UVB exposure and addition of TNF- The L-929 cells were grown in 96-well microplates (Corning Co. Ltd, NY, U.S.A.) until confluent (3.1–3.3105/cm2). The culture medium was replaced with 50 l of MEM containing 2% fetal calf serum (FCS). The cells were then irradiated with UVB (312 nm, 60 to 960 mJ/cm2) using a UVB generator (Atto, DT-20 MP, Tokyo, Japan) (Kimura et al., 1999; Kimura et al., 2000). Just after the exposure to UVB radiation, 50 l of various concentrations of recombinant mouse TNF- (Strathmann Biotech., Hannover, Germany) was added. The concentration of TNF- ranged from 0 to 100 ng/ml. The cells were incubated for 17 h at 37C in an atmosphere of 5%
Cell Biology International, Vol. 25, No. 12, 2001
CO2. Because phenol red can cause a phototoxic reaction, we used MEM that did not contain this dye. Measurement of cell viability After 17 h incubation at 37C in an atmosphere of 5% CO2, cell viability was examined using the crystal violet method (Itoh et al., 1991). Dye uptake by cells was measured at 550 nm using a microplate autoreader (SJEIA-II, Sanko Junyaku Co. Ltd, Tokyo). Analysis of Fas, Fas-L, TNFR-1 and TNFR-2 expression by fluorescence flow cytometry The treated cells were washed in a culture flask (25 cm2) with phosphate-buffered saline (PBS) without calcium and magnesium. The cells were detached from the flask wall with a cell scraper, dispersed by gentle pipetting, and passed through a 45 m filter (Millipore, Bedford, MA, U.S.A.). An aliquot of 20 g/ml of anti-mouse rabbit polyclonal Fas IgG antibody, anti-mouse rabbit polyclonal Fas-L IgG antibody, anti-mouse goat polyclonal TNFR-1 IgG antibody, anti-mouse goat polyclonal TNFR-2 IgG antibody (mouse and rat reactive, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), or isotype-matched control antibody (anti-rabbit or anti-goat normal control IgG, Genzyme, Minneapolis, MN, U.S.A.) was added to a cell suspension of 4–6105/ml in PBS and incubated for 1 h at 4C. The cells were washed twice with PBS, then stained with 10 g/ml of anti-rabbit or anti-goat FITC-conjugated F(ab )2 fragment (Organon Tecnica, Durham, NC, U.S.A.) (Kimura et al., 1999). Fluorescence flow cytometric analysis was then performed (Epics XL System II, Coulter Co. Ltd, Tokyo). Analysis of DNA fragmentation After UVB pretreatment and incubation with TNF- for 17 h, the cells attached to a culture flask were washed with PBS. After washing, the cells were detached with a cell scraper, dispersed by gentle pipetting and collected by centrifugation (1500g, 15 min, 4C). DNA from these cells was purified according to a method previously reported (Laird et al., 1991). The purified DNA was electrophoresed on a 1.5% agarose gel and stained with ethidium bromide (Wako Pure Chemicals Co. Ltd, Tokyo).
Cell Biology International, Vol. 25, No. 12, 2001
1223
Fig. 1. Microscopic findings in cells treated with UVB alone and with UVB plus TNF-. L-929 murine fibroblasts were exposed to no UVB (a, control), 100 pg/ml of TNF- alone for 17 h (b), 480 mJ/cm2 of UVB alone for 17 h (c), or 100 pg/ml of TNF- for 17 h after 480 mJ/cm2 of UVB (d). The cells were visualized by light microscopy under 100 magnification. Bar indicates 100 m.
RESULTS Light microscopy findings in cells treated with UVB and/or TNF- Using light microscopy, we observed morphological changes in the cells caused by UVB and TNF- after 17 h incubation. Cells treated with 100 pg/ml of TNF- alone did not detach from the microplate walls (Fig. 1B). A small fraction of the cells treated with UVB (480 mJ/cm2) alone had detached from the plate wall (Fig. 1C). Morphological changes were also observed in these cells. A significant number of cells treated with UVB (480 mJ/cm2) and TNF- (100 pg/ml) had detached from the plate wall (Fig. 1D). Effects of various doses of UVB and TNF on cell viability We investigated the interrelationship between various doses of UVB and TNF- with respect to their
effect on cell viability after 17 h incubation (Fig. 2). In the control cells not exposed to UVB a higher concentration than 1 ng/ml of TNF- was required to induce cell death. A concentration of 1 ng/ml of TNF- killed approximately 50% of the cells, 70% of the cells, and 90% of the cells when the cells were pretreated with 120 mJ/cm2, 240 mJ/cm2, and 480 mJ/cm2 of UVB, respectively. Thus, the dose of TNF- required to kill the cells decreased as the pretreatment dose of UVB increased, suggesting that UVB modulated susceptibility to TNF--induced cell death. UVB-induced expression of TNFR-1 and TNFR-2 in L-929 cells The expression of TNFR-1 and TNFR-2 was examined in the control cells (exposed to neither UVB nor TNF-), cells treated with 100 pg/ml of TNF- alone, treated with 480 mJ/cm2 of UVB alone, or treated with both 100 pg/ml of TNF-
1224
Cell Biology International, Vol. 25, No. 12, 2001
TNFR-1 expression (A)
0 0.1
50
(E) Isotype
Count
75
TNFR-2 expression 100
Isotype
Count
100
Cell viability (%)
100
(A) (B) (C) (D) (E)
0 0.1
1000
1000 FITC
FITC 100
100 (B)
(F)
2
3
4
10 10 10 10 TNF-α concentration (pg/ml)
5
10
Fig. 2. Effects of various doses of UVB and TNF- on cell viability. Just after exposure to various doses of UVB (0–480 mJ/cm2), the various doses of TNF- were added to the culture medium. Cell viability was assessed by the crystal violet method after 17 h incubation. The vertical bar indicates the meanstandard error (SE) of triplicate experiments. (A) Non-irradiated cells; (B) cells treated with 60 mJ/cm2 of UVB; (C) cells treated with 120 mJ/cm2 of UVB; (D) cells treated with 240 mJ/cm2 of UVB; (E) cells treated with 480 mJ/cm2 of UVB.
Count
0 0.1
0 0.1
1000 FITC
100
100 (C)
(G) A
0 0.1
B
0 0.1
1000 FITC
UVB and TNF--induced expression of Fas and Fas-L in L-929 cells The expression of Fas and Fas-L was examined in the control cells (exposed to neither UVB nor TNF-), cells treated with 100 pg/ml of TNF- alone, treated with 480 mJ/cm2 of UVB alone, or treated with both 100 pg/ml of TNF- and 480 mJ/cm2 of UVB (Fig. 5). Fas was constitutively
1000 FITC
100
100 (H) Count
(D) Count
and 480 mJ/cm2 of UVB (Fig. 3). Both TNFR-1 and TNFR-2 were constitutively expressed (Fig. 3A and E). Expression of TNFR-1 or TNFR-2 (Fig. 3B and F) was not induced by the addition of 100 pg/ml of TNF- as compared with the control cells (Fig. 3A and E). The expression of TNFR-1 and TNFR-2 was markedly induced by UVB alone (Fig. 3C and G). The addition of TNF- to cells pretreated with UVB did not further induce the expression of TNFR-1 or TNFR-2 (Fig. 3D and H) as compared with UVB alone. We investigated the effects of the dose of UVB on the expression of TNFR-1 and TNFR-2. The relative area, defined as the B/A100 (the areas of A and B as shown in Fig. 3G), was calculated (Fig. 4). UVB increased TNFR-1 and TNFR-2 expression in a dose-dependent manner.
1000 FITC
Count
1
Count
0
Relative cell number
Count
25
0 0.1
1000 FITC
0 0.1
1000 FITC
Fluorescence intensity (log)
Fig. 3. UVB-induced expression of TNFR-1 and TNFR-2 in L-929 cells. Panels A, B, C, and D represent experiments for the detection of TNFR-1; panels E, F, G and H represent experiments for the detection of TNFR-2. (A and E) Untreated cells (exposed to neither UVB nor TNF-); (B and F) cells incubated with 100 pg/ml of TNF- alone for 17 h; (C and G) cells 17 h after UVB (480 mJ/cm2) exposure alone; (E and H) cells pretreated with UVB (480 mJ/cm2) and incubated with TNF- (100 pg/ml) for 17 h.
expressed in the control cells (Fig. 5A), and Fas-L was slightly expressed in the control cells (Fig. 5A and E). Expression of Fas or Fas-L (Fig. 5B and F) was not induced by the addition of 100 pg/ml of TNF- as compared with the control cells (Fig. 5A and E). The expression of Fas and Fas-L was slightly induced by UVB alone (Fig. 5C and G).
Cell Biology International, Vol. 25, No. 12, 2001
1225
Fas expression
FasL expression 100
100
TNFR-1 TNFR-2
Isotype
Isotype
(A)
(E)
30
Count
Count
Relative area (%)
40
0 0.1
20
0 0.1
1000
100
100
10
UVB dose (mJ/cm )
Fig. 4. Effects of the UVB dose on the expression of TNFR-1 and TNFR-2. The relative area was defined as 100B/A (areas of A and B are depicted in Fig. 3G, (A) area under the entire curve of all the cells, (B) area under the curve of cells expressing either TNFR-1 or TNFR-2). Detailed experimental conditions are described in the text. Vertical bar indicates the meanSE of triplicate experiments. L-929 cells were exposed to various doses of UVB (60–960 mJ/cm2). Following exposure, the cells were analysed for the expression of TNFR-1 and TNFR-2.
The addition of TNF- to cells pretreated with UVB markedly induced the expression of both Fas and Fas-L (Fig. 5D and H) as compared with UVB alone. DNA fragmentation in L-929 cells treated with UVB and/or TNF- We examined the serial changes of DNA fragmentation in adherent control cells (exposed to neither UVB nor TNF-) and in adherent cells treated with UVB alone, TNF- alone or both UVB and TNF-. No fragmentation was observed in the control cells (exposed to neither UVB nor TNF-), the cells treated with 60 mJ/cm2 or 480 mJ/cm2 of UVB alone, or the cells treated with 100 pg/ml of TNF- alone (Fig. 6, lanes 1 to 4). In contrast, the cells treated with 480 mJ/cm2 UVB plus 100 pg/ml of TNF- showed marked DNA fragmentation (Fig. 6, lane 7), strongly suggesting that the cell death induced by this treatment was due to apoptosis. DISCUSSION We have demonstrated that pretreatment with UVB markedly enhanced the susceptibility of
Count
1000
0 0.1
0 0.1
1000
1000 FITC
FITC 100
100 (C)
(G) Count
800 2
0 0.1
0 0.1
1000
1000 FITC
FITC 100
100 (D)
(H) Count
600
Count
400
Count
200
(F)
Count
(B)
Relative cell number
0
1000 FITC
FITC
0 0.1
1000 FITC
0 0.1
1000 FITC
Fluorescence intensity (log)
Fig. 5. UVB and TNF--induced expression of Fas and Fas-L in L-929 cells. (A, B, C, and D) Experiments for detection of Fas; (E, F, G, and H) experiments for detection of Fas-L. (A and E) Untreated cells (not exposed to UVB or TNF-); (B and F) cells incubated with 100 pg/ml of TNF- alone for 17 h; (C and G) cells 17 h after UVB (480 mJ/cm2) exposure alone; (E and H) cells pretreated with UVB (480 mJ/cm2) and incubated with TNF- (100 pg/ml) for 17 h.
murine fibroblasts to TNF--induced cell death. This may have been caused by promoting an increase in TNFRs after UVB irradiation. In addition, 100 pg/ml of TNF-, which in itself did not induce Fas or Fas-L in the absence of pretreatment with UVB, enhanced the expression of Fas and Fas-L when the cells were pretreated with UVB. More than 1 ng/ml of TNF- is required to
1226
Cell Biology International, Vol. 25, No. 12, 2001
DNA fragmentation M
1
2
3
4
5
6
7
Ori
1057 bp
Fig. 6. DNA fragmentation in L-929 cells treated with UVB and TNF-. L-929 cells were treated with various doses of UVB and TNF- either alone or in combination. The cells were lysed and DNA was prepared from the cells to determine the extent of DNA fragmentation after UVB pretreatment and incubation with TNF- for 17 h. M, marker (X174/Hinc II digest); lane 1, control cells (exposed to neither UVB nor TNF-); lane 2, cells treated with 60 mJ/cm2 of UVB alone; lane 3, cells treated with 480 mJ/cm2 of UVB alone; lane 4, cells treated with 100 pg/ml of TNF- alone; lane 5, cells treated with 5 ng/ml of TNF- alone; lane 6, cells treated with 50 ng/ml of TNF- alone; lane 7, cells treated with 480 mJ/cm2 of UVB plus 100 pg/ml of TNF-.
induce cell death in non-irradiated cells, while 50 pg/ml of TNF- is sufficient to cause cell death in cells pretreated with 480 mJ/cm2 of UVB. All these results suggest that UVB irradiation can modulate susceptibility to TNF--induced cell death through the induction of death receptors such as TNFRs, Fas and Fas-L in murine fibroblasts. These death receptors may have played a role in the observed cell death. Fas and Fas-L expression may precede apoptosis (Nagata, 1997; Kimura et al., 1999). The expression of Fas and Fas-L was markedly increased in cells treated with both 480 mJ/cm2 of UVB and 100 pg/ml of TNF- in the present study, and approximately 70% of the cells died over the subsequent 17 h. In addition, significant DNA fragmentation was observed in these cells (Figs. 6 & 7). These results suggested that the cell death seen in this study was due to apoptosis. There are two types of TNF receptor in some cells (Tartaglia et al., 1993; Baker and Reddy, 1998;
Ledgerwood et al., 1999). Numerous reports suggest that both TNFR-1 and TNFR-2 can transduce both an apoptotic signal and an anti-apoptotic signal (Martin and Cotter, 1991; Chinnaiyan et al., 1995; Liu et al., 1996; Duckett and Thompson, 1997; Nagata, 1997; Baker and Reddy, 1998; Sheikh et al., 1998; Ledgerwood et al., 1999). However, our results do not show whether UVB irradiation altered the nature of TNFRs or altered signal transduction at a site downstream of TNFRs. Induction of TNFRs was only examined by flow cytometry in this study. However, previous studies suggest that increased expression of TNFR genes and TNFR mRNAs are associated with induction of TNFRs (Jin et al., 2000). It is possible that expression of TNFR genes and TNFR mRNAs may have been increased in the cells treated with UVB and TNF-. This hypothesis remains to be studied. Excessive exposure to UV causes inflammation in skin tissue (Madfu and Thomas, 1993). UV irradiation also induces the production of TNF in various cell types such as human keratinocytes, macrophages, and corneal cells (Bazzoni et al., 1994; Kennedy et al., 1997; Kibitel et al., 1998). It is believed that injured cells are removed to prevent the reproduction of any abnormal cells (Thompson, 1995; Jacobson et al., 1997). Because TNF is a proinflammatory cytokine and a wellknown inducer of apoptosis, it is reasonable to postulate that TNF is involved in UV-induced skin injury and cell death. To our knowledge, there has been only one study that examined the interrelationship between UV irradiation, TNF, and TNFR with respect to cell death. In that study, Sheikh et al. (1998) found that UV enhanced the susceptibility of lung cancer cells to TNF-induced cell death. In this study, we found that UVB induced TNFRs. This may explain why UV pretreatment made the cells more vulnerable to TNF-. Increased expression of TNFRs, Fas, and Fas-L may be a likely explanation for an enhanced susceptibility to TNF- in cells treated with UV. The dose of UVB used in this study was 60–960 mJ/ cm2 at 312 nm. Based on the biological effects of UVB, a dose of 480 mJ/cm2 corresponds to approximately 4 h of sunbathing during the summer at sea level (Madfu and Thomas, 1993). Thus, our experimental conditions may be clinically relevant, for instance, to the effects of sunbathing. A large dose of UV itself kills cells (Hall et al., 1988; Martin and Cotter, 1991). However, mild UV irradiation can act as a physical carcinogen (Doll and Peto, 1981; Angel and Karin, 1991; Madfu et al., 1993; Thompson, 1995). This
Cell Biology International, Vol. 25, No. 12, 2001
may be due to the replication of abnormal cells in a population of mildly injured cells (Thompson, 1995; Jacobson et al., 1997). Therefore, mildly injured cells may need to be removed (Thompson, 1995; Jacobson et al., 1997). TNF- can induce cell death in a number of different sensitive cell types in vitro, such as blood cells, keratinocytes, and fibroblasts, although the biological significance of this activity is not well understood (Baker and Reddy, 1998; Ledgerwood et al., 1999). In this study, a clinically relevant dose of UVB made fibroblasts vulnerable to TNF-, perhaps through the induction of death-related molecules such as TNFRs, Fas, and Fas-L, thus leading to cell death. Because UV has been reported to stimulate TNF release from various cells (Bazzoni et al., 1994; Kennedy et al., 1997; Kibitel et al., 1998; Andersson et al., 1988), it is possible that both enhanced TNF release and increased expression of death receptors caused by UV exposure may work additively in inducing cell death after sunbathing. ACKNOWLEDGEMENTS We are most grateful to Ms Masae Ohbuchi (Gunma Prefectural Insitute of Public Health and Environmental Sciences) for her skilful assistance. REFERENCES A U, S B, A J, M G, 1988. Concomitant production of different lymphokines in activated T cells. Eur J Immunol 18: 2081–2084. A P, K M, 1991. The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta 1072: 129–157. B S-J, R E-P, 1998. Modulation of life and death by the TNF receptor superfamily. Oncogene 17: 3261–3270. B F, K V, S A, J C-V, B B, 1994. Analysis of tumor necrosis factor promotes responses to ultraviolet light. J Clin Invest 93: 56–62. C A-M, O’R K, T M, D V-M, 1995. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81: 505–512. D R, P R, 1981. The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst 66: 1191–1308. D C-S, T C-B, 1997. CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival. Genes Dev 11: 2810–2821. H E-J, A M, B J, B C, C S-B, F M, G C, H T, M J, O N, 1988. Basic radiobiology. Am J Clin Oncl 11: 220–252. I N, Y S, I A, Y M, M S, S M, H A, S Y, N S, 1991. The
1227
polypeptide encoded by cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66: 233–243. J M-D, W M, R M-C, 1997. Programmed cell death in animal development. Cell 88: 347–354. J L, R D-P, C T-D, P S-J, H C-W, P T-L, S R-G, 2000. Enhanced murine macrophage TNF receptor shedding by cytosine-guanine sequences in oligodeoxynucleotides. Enhanced murine macrophage TNF receptor shedding by cytosine-guanine sequences in oligodeoxy-nucleotides. J Immunol 165: 5153–5160. K M, K K-H, H B, B J, P S, M C, E H, R J-T, A C-A, A J-C, 1997. Ultraviolet irradiation induces the production of multiple cytokines by human corneal cells. Invest Ophthalmol Vis Sci 38: 2483–2491. K J, H V, A L, O’C A, S B-M, Y D, 1998. UV-DNA damage in mouse and human cells induces the expression of tumor necrosis factor alpha. Photochem Photobiol 67: 541–546. K H, M H, S I, O K, S A, 1999. Phorbol ester facilitates apoptosis in murine fibroblasts pretreated by mild ultraviolet radiation. J Biochem 126: 340–346. K H, M H, O K, S A, 2000. Cu-Zn superoxide dismutase inhibits lactate dehydrogenase release and protects against cell death in murine fibroblasts pretreated with ultraviolet radiation. Cell Biol Int 24: 459–465. K-W G, K D, L T-A, K M, S T, 1999. Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosisinducing ligand and CD95-induced apoptosis but not from ultraviolet radiation-induced apoptosis. J Biol Chem 274: 28916–28921. L P-W, Z A, L K, R M-A, J R, B A, 1991. Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19: 4293. L E-C, P J-S, B J-R, 1999. Recent advances in the molecular basis of TNF signal transduction. Lab Invest 79: 1041–1050. L Z-G, H H, G D-V, K M, 1996. Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell 87: 565–576. M A-P, T B-F, 1993. Preventive treatment of sunburn, dermatoheliosis, and skin cancer with sunprotective agents. In: Thomas B-F, Arthur Z-E, Klaus W, Irwin M-F, Frank A, eds. Dermatology in general medicine. New York, McGraw-Hill. 1689–1717. M S-J, C T-G, 1991. Ultraviolet B irradiation of human leukaemia HL-60 cells in vitro induces apoptosis. Int J Radiat Biol 59: 1001–1016. N S, 1997. Apoptosis by death factors. Cell 88: 355–365. N M, S T, K Y, 1992. Differentiation factors, including nerve growth factor, fibroblast growth factor, and interleukin-6, induce an accumulation of an active Ras. GTP complex in rat pheochromocytoma PC 12 cells. J Biol Chem 267: 19448–19454. O N-K, C A, P V-C, F P-A, K J-D, G J-R, S E-M, T L-D, U S-R, K M-C, 1997. Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem 272: 16351–16357.
1228
R M, P M-G, H W-J, A T-M, G D-V, 1995. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83: 1243–1252. S M-S, A M-J, H Y, F A-J, 1998. Ultraviolet-irradiation-induced apoptosis is mediated via ligand independent activation of tumor necrosis factor receptor 1. Oncogene 17: 2555–2563. T L-A, G D-V, 1992. Two TNF receptors. Immunol Today 13: 151–153.
Cell Biology International, Vol. 25, No. 12, 2001
T L-A, A T-M, W G-H, G D-V, 1993. A novel domain within the 55 kd TNF receptor signals cell death. Cell 74: 845–853. T C-B, 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456–1462. W C-F, VA S, VA T-L, 1996. Apoptosis mediated by the TNF-related cytokine and receptor families. J Cell Biochem 60: 47–55.