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16. Layher, S. and J. E. Cleaver (1996) Expression levels of the XPA gene in human and mouse cells. Mutat. Rex (In press)

17. Cleaver, J. E., G. H. Thomas, J. E. Trosko and J. H. Lett (1972) Excision repair (dimer excision, strand breakage and repair rep- lication) in primary culture of eukaryotic (bovine) cells. Exp. Cell Res. 74. 67-80.

Signal Transduction Mechanisms in P hotocarcinogenesis

Alice P. Pentland

Departments of Medicine and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO, USA

Exposure to UV light B (UVB, 290-320 nm) produces in- flammation acutely, whereas chronic exposure can cause tu- mor formation (1). The generation of DNA photoproducts has been shown to initiate tumorigenesis (2), but the mech- anisms by which UVB also promotes tumor growth are un- clear. It is likely that these mechanisms are related to the signalling events that occur acutely in response to tissue ir- radiation. Recently, numerous advances have been made in the understanding of these cellular responses. The majority of these studies have been done using UVC wavelengths (200-290 nm). These wavelengths are convenient for study because they are readily available in most laboratories. How- ever, these wavelengths do not penetrate the ozone layer in the atmosphere ( 1 ) and so are not responsible for human photocarcinogenesis; this is a major flaw in studies per- formed using this approach. Instead, wavelengths between

c c Mitogenesla, Streaa responses

Differentlation (growth Inhibition)

Figure 4. Proposed signalling pathway for transcriptional activation initiated by irradiation with UVC. Growth factor receptors for EGF (epidermal growth factor), IL-I (interleukin 1) and TNF (tumor ne- crosis factor) have been implicated. The signalling molecules pic- tured in association with the EGF receptor have been documented to he necessary for signalling by this receptor in response to its ligand, EGF, but have not been directly linked to its activation by UVC. Ras and the kinases pictured downstream from Ras have all been shown to participate.

W AA

z PGE,

Figure 5. Proposed pathway for UVB-initiated signal transduction. The EGF receptor is known to be activated, and increased synthesis and phosphorylation of cPLA, (cytosolic phospholipase A2) have been documented. The MAP kinase and v-src are implicated by indirect evidence.

290 and 320 nm (termed UVB radiation) are clearly linked to UV irradiation-induced inflammation and carcinogenesis, particularly squamous cell and basal cell carcinoma (l,2). Further studies extending the work performed using UVC light sources are now being done using UVB irradiation.

Signal transduction mechanisms elicited by UVC radiation

In studies conducted by Devary et ul. (3), the signalling ini- tiated by UVC was examined by dissecting the mechanisms leading to activation of the transcription factor c-jun. These studies clearly implicated tyrosine kinases in the signalling pathway and suggested that active oxygen species activate v-src at the membrane. These studies demonstrated through the use of dominant negative mutants of v-src, Ha-ras and raf- 1 as well as selective tyrosine kinase inhibitors that early activation of src kinases acted via Ha-ras and raf- 1 to initiate c-jun phosphorylation within 15 min of irradiation. Gluta- thione supplementation by treatment of the cells with N- acetylcysteine attenuated the induction, suggesting oxidative stress as a key mechanism in the activation process.

These observations were extended to demonstrate that many of the initial events stimulated by irradiation parallel the activation of mitogenesis pathways. In studies of UVC signalling by Sachsenmaier et ul. (4), the UVC response ap- peared to be mediated viu basic fibroblast growth factor (bFGF), interleukin (1L)- 1 and epidermal growth factor re- ceptors. Activation of the pre-existing transcription factors c-fos, c-jun and TCFElk-I, as well as protein synthesis- independent transcriptional activation of c-fos and c-jun were studied. When cellular growth factor receptors were inhibited by prior down-regulation, the UVC response was inhibited. This observation was confirmed through the use of suramin to block receptor activation and by expression of dominant negative epidermal growth factor (EGF)-receptor mutants. Down modulation was specific to the receptor type studied. It appears that the initiation of a growthhepair sig- nalling pathway is integral to the UVC response (4). In sep- arate studies, a highly UVC-inducible kinase has been iden- tified as JNK-1, also termed SAPK (stress activated protein

380 Hasan Mukhtar eta/.

kinase), which is capable of robust phosphorylation of c-jun (Fig. 4) (5,6).

In addition to activation of MAPK signalling pathways, NF-KB is activated by UVC exposure as well (7). In a series of elegant experiments, NF-KB activation was found to occur in the cytosol, without involvement of the nucleus, confirm- ing the importance of extranuclear events in the UVC re- sponse. Cytoplasts, which were prepared to eliminate the nucleus, were found to be fully capable of NF-KB induction. Irradiated cytoplasts could readily activate a NF-KB-depen- dent human immunodeficiency virus (H1V)-luciferase con- struct (7). Collectively, these experiments demonstrate that cytoplasmic signalling pathways are necessary for early ac- tivation events after UVC irradiation.

Signal transduction mechanisms elicited by UVB radiation

Some of the observations that have been made regarding the signalling pathways initiated by UVC exposure have been confirmed in cells that have been exposed to UVB irradia- tion. The activation of NF-KB by UVB has been demonstrat- ed in human epidermoid carcinoma cells (8). In experiments studying the up-regulation of IL-6 expression after UVB ex- posure, it was demonstrated that cytosolic extracts free of nuclei were capable of activating NF-KB. This activation was modulated significantly by scavenging of oxygen free radi- cals (8).

One of the most apparent changes after acute UVB irra- diation is the appearance of redness in irradiated skin. At early time points (<24 h), much of this redness is due to the synthesis of prostaglandins (PG) ( 1 ) . The key enzyme me- diating the increase in the UV-induced synthesis of PGE, in UVB irradiation injury has recently been demonstrated to be cytosolic phospholipase A2 (cPLA,) (9). Studies examining the UVB-signalling mechanisms responsible for up-regulat- ing the expression of cPLA, have shown that it may be ac- tivated via an EGF-receptor mediated pathway (lo), a mech- anism that parallels the extensive observations of kinase ac- tivation by UVC (Fig. 5). Analysis of the mechanism of this increase in activity revealed that both increased synthesis and activation of CPLA, occurred ( 1 1 ). These increases were modulated by the redox status of the cell. If cellular gluta- thione stores were supplemented by the addition of N-acetyl- cysteine, the induction of cPLA, by UVB irradiation was attenuated.

The mechanism by which these acute signalling changes in response to irradiation are related to the ultimate devel- opment of tumors is less clear. The evidence indicating that oxidative stress underlies some aspect of the signalling ini- tiated by irradiation may be one common link by which acute irradiation ultimately causes tumors. Further work is needed to understand the linkage of early UVB-induced ac- tivation events with the ultimate formation of skin cancer.

References

1. Hawk, J. L. M. and J. A. Parrish (1983) Responses of normal skin to ultraviolet radiation. In Photoimmunology (Edited by J. Parrish, M. Kripke and W. Morrison), pp. 219-260. Plenum Medical Books, New York.

2. Ananthaswamy, H. N. and W. E. Pierceall (1990) Molecular

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mechanisms of ultraviolet radiation carcinogenesis. Photochem. Photobiol. 52, 11 19-1 136. Devary, Y., R. A. Gottlieb, T. Smeal and M. Karin (1992) The mammalian ultraviolet response is triggered by activation of src tyrosine kinases. Cell 71, 1081-1091. Sachsenmaier, C., A. Radler-Pohl, R. Zinck, A. Nordheim, P. Herrlich and H. J. Rahmsdorf (1994) Involvement of growth factor receptors in the mammalian UVC response. Cell 78, 963- 972. Derijard, B., M. Hibi, L H . Wu, T. Barrett, B. Su, T. Deng, M. Karin and R. J. Davis (1994) JNKl: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c- Jun activation domain. Cell 76, 1025-1037. Yan, M., T. Dai, J. Deak, J. Kyriakis, L. Zon, J. Woodgett and D. Templeton (1 994) Activation of stress activated protein ki- nase by MEKKl phosphorylation of its activator SEKl. Nature 372, 798-800. Devary, Y., C. Rosette, J. DiDinato and M. Karin (1993) NF- kB activation by ultraviolet light not dependent on a nuclear signal. Science 261, 1442-1445. Simon, M., Y. Aragane, A. Schwarz, T. Luger and T. Schwarz (1994) UVB light induces nuclear factor B (NF-kB) activity independently from chromosomal DNA damage in cell-free cy- tosolic extracts J . Invest. Dermafol. 102, 422427. Gresham, A,, J. Masferrer, X. Chen, S . Leal-Khouri and A. P. Pentland (1996) Increased synthesis of the high molecular weight cytosolic phospholipase A, mediates early UV-induced prostaglandin E, in human skin. Am. J. Physiol. (In press) Miller, C. C., P. Hale and A. P. Pentland (1994) Ultraviolet B injury increases prostaglandin synthesis through a tyrosine ki- nase-dependent pathway. Evidence for UVB-induced epidermal growth factor receptor activation. J. Bid. Chem. 269, 3529- 3533. Chen, X., A. Gresham and A. P. Pentland (1995) Oxidative stress mediates synthesis and phosphorylation of cytosolic phos- pholipase A, after UVB injury. Biochim. Biophys. Acta. (In press)

Oxidant, Antioxidant Status and Photocarcinogenesis: The Role of Gene Activation

Rex M. Tyrrell

Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland

Oxidative processes have been implicated in many disease processes including cancer. Experimental data derived from a variety of biological systems are consistent with the current notion that the oxidative component of UVB radiation may contribute to skin carcinogenesis. Dietary antioxidants can re- duce UV-induced skin cancer in rodents (1) and this process can also be suppressed by topically applied carotenoids (2). Deficiency of selenium, which inhibits the activity of the an- tioxidant selenium-dependent glutathione peroxidase enzyme, accelerates development of UVB-induced tumors (3). Re- cently, an antioxidant polyphenolic fraction isolated from green tea has also been shown to protect hairless mice against UVB radiation-induced carcinogenesis (4). Although such data clearly implicate UV-induced oxidative reactions in UV carcinogenesis, the nature of the critical changes and the stage of the carcinogenesis process at which they occur is not yet