Real Time PCR analysis shows accumulation of mRNA for a. GCLc and b. GCLm in both the NCTC and HaCaT cell lines after exposure to SUL (5μM). Both the.
Identification and Characterization of Chemopreventive Mechanisms in Human Skin. T Devling, L McLellan, CD Lindsay * and J Hayes. Biomedical Research Centre, University of Dundee Medical School, Ninewells Hospital, Dundee, UK. * Biomedical Sciences Department, Dstl Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
Abstract Here we have investigated the response to and effects of chemical inducing agents on the detoxification enzyme systems present in two human skin cell lines (HaCaT and NCTC 2544) with the aim of developing a protective strategy against cytotoxic insult. Our results showed an increased expression of antioxidant and detoxification enzyme systems Further to this, the results in one of the two cells lines (HaCaT) correlated with results demonstrated in a primary human skin cell model, establishing the use of one of these cell lines as a suitable model for skin. Further investigation showed that cells pretreated with the chemical inducing agent sulforaphane (SUL) had an enhanced antioxidant status including up regulation of enzymatic and non protein thiol antioxidant capacity. Pretreated cells were able to respond more favourably to antioxidant depletion. Further experiments with the pro-oxidant menadione showed enhanced survival in SUL pretreated cells. However subsequent experiments with the potent alkylating agent melphalan demonstrated the inability of pre-treated cells to withstand cytotoxic insult even though they showed increased antioxidant capacity.
Introduction The stress response in mammalian cells is characterised by the modulation of gene expression during and after the exposure to cytotoxic insult. Increasing levels of oxidative stress, chemical stress, UV light and pathogenic organisms are known to affect regulation and expression of cytoprotective genes in stressed cells.(1) One such effector of this response is the Nrf2 transcription factor which, acting through the Antioxidant Response Element (ARE), can regulate genes encoding a battery of enzymes serving antioxidant or detoxicant functions within the cell.(2) The control of this battery is of particular interest in regard to protection from cytotoxic insult. Human keratinocytes are regularly exposed to the aforementioned insults. As a consequence they possess a number of systems aimed at dealing effectively with this stress. Accordingly a number of these systems have been shown to respond to activators of the Nrf2 pathway in mammalian cells.(3) The modulation of these pathways which include the antioxidant enzyme NAD(P)H Quinone Oxido Reductase 1 (NQO1) and the glutathione (GSH) synthesising Glutamate Cysteine Ligase enzyme (GCL),(4,5) may offer subsequent protection from exposure to cytotoxic agents. Here we investigate the detoxification systems present in two human keratinocyte cell lines (HaCaT and NCTC 2544) and their response to chemical inducing agents in comparison with a primary cell line (NHEK). We then attempt to produce a chemopreventive strategy to the potent alkylating agent melphalan through the modulation of these enzyme systems.
Results Identification of an ARE-Driven Response in HaCaT cells, a human keratinocyte cell line.
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Figure 1a. Cells transiently transfected with the mNQO1 promoter region display both basal and SUL (5 μM) induced activity. Mutation of the ARE sequence abolishes this activity. Figure 1b. RT-PCR analysis demonstrates the HaCaT cell line possesses mRNA encoding the Nrf1, Nrf2 and small Maf protein. a.
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Figures 2a and 2b. Real Time PCR analysis shows accumulation of mRNA for a. GCLc and b. GCLm in both the NCTC and HaCaT cell lines after exposure to SUL (5μM). Both the timing and extent of accumulation reflect the results seen at the protein levels and highlight the difference between the two cell lines. Figure 2c. Real Time PCR comparison of NHEK and HaCaT cell gene expression in response to SUL (5μM). Similarities exist between the HaCaT and NHEK enzyme expression in both basal and inducible conditions. Western blot analysis of NHEK cells after exposure to SUL (5μM) for 24 hours (not shown) demonstrated induction of both antioxidant and detoxicant enzymes. FI represents fold increase in product expression. a.
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Figure 3a. Levels of intracellular GSH are raised in HaCaT cells pre-treated with SUL. Subsequent exposure to 0.5mM diethyl maleate for 2 hours is shown to have less of an initial effect on the depletion of cellular GSH. Recovery from this initial depletion is also more extensive in pre-treated cells. Figure 3b. Pretreatment of HaCaT cells with SUL (5μM) significantly enhances cell survival after exposure to the prooxidant menadione. a. 35
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Figure 4. Effect of the alkylating agent melphalan. The toxicity profile of melphalan is unaltered in cells pre-treated with SUL using MTT toxicity assay after 48 hours exposure. These results indicate no protective effect was gained from pre-treatment of cells with SUL in these conditions
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Discussion/Conclusions •
Human keratinocyte cell lines possess the factors necessary and demonstrate the ability to activate an ARE driven reporter construct in response to chemical treatment. As well as an inducible role for the ARE there appears to be basal activity through this element.
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This activity is linked to the up-regulation of detoxification enzymes, at both the protein and mRNA level, whose encoding genes contain known or suspected AREs.
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Comparison of the two cell lines with a primary keratinocyte cell model demonstrates the ARE response of the HaCaT cell line closely resembles the primary human keratinocyte cell system justifying its preferential use over the NCTC 2544 cell line and its general application as a model cell system.
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Cells pre-treated with SUL show a 1.8 fold increase in the levels of GSH and a greater rate of recovery to GSH depletion. This is suspected, in part, to be due to increased levels of GCLm + GCLc, the subunits of the GCL heterodimer involved in the coordinated GSH synthesis pathway.
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HaCaT cells pre-treated with SUL show an enhanced survival profile when exposed to the pro-oxidant menadione, a reactive quinone. The contribution of an increased level of GCL and NQO1 are of further interest.
•
Cell survival is not enhanced after pre-treatment with SUL and subsequent exposure to the potent alkylating agent melphalan. Although Mel has been shown to react with GSH it is also known to spontaneously bind to both DNA and protein within the cell hence limiting cellular function and leading to cell death. Although up regulation of the GSH synthesising enzymes occurs there is no alteration in cellular GST status, in this instance the increased antioxidant capacity may not be utilised by the cell through a GST catalysed reaction. It should also be noted that the skin may possess enhanced detoxification systems as a consequence of its increased exposure to environmental
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stressing agents hence any intervention here may be seen as trying to increase the capacity of an already activated system. •
It remains to be seen whether SUL or other chemopreventative agents can offer greater levels of protection against other agents such as UV light or benzo[a]pyrene.
References 1. Hayes, JD, McMahon, M (2001). Cancer Lett. 174, 103-13. 2. McMahon, M, Itoh, K, Yamamoto, M, et al., (2001). Cancer Res. 61, 3299-307. 3. Zhang, Y, Talalay, P (1998). Cancer Res. 58, 4632-9. 4. Wild, AC, Moinova, HR, Mulcahy, RT (1999). J. Biol. Chem. 274, 33627-36. 5. Bonnesen, C, Eggleston, IM, Hayes, JD (2001). Cancer Res. 61, 6120-30.
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