articles
Nitric oxide upregulates expression of DNA-PKcs to protect cells from DNAdamaging anti-tumour agents Weiming Xu*, Lizhi Liu*, Graeme C. M. Smith†‡ and lan G. Charles*§ *The Wolfson Institute for Biomedical Research, The Cruciform Building, University College London, Gower Street, London WC1E 6BT, UK †Wellcome Trust/CRC Institute and Department of Zoology, Cambridge University, Tennis Court Road, Cambridge CB2 1QR, UK ‡Present address: KuDOS Pharmaceuticals Ltd. Cambridge, CB4 4WG, UK §e-mail:
[email protected]
Nitric-oxide synthase (NOS) activity has been detected in many human tumours, although its function is unclear. Here we show that exposure of cells to nitric oxide (NO) results in a 4–5-fold increase in expression of the DNA-dependent protein-kinase catalytic subunit (DNA-PKcs), one of the key enzymes involved in repairing double-stranded DNA breaks. This NO-mediated increase in enzymatically active DNA-PK not only protects cells from the toxic effects of NO, but also provides crossprotection against clinically important DNA-damaging agents, such as X-ray radiation, adriamycin, bleomycin and cisplatin. The NO-mediated increase in DNA-PKcs described here demonstrates the presence of a new and highly effective NO-mediated mechanism for DNA repair.
Results Construction of an NO-inducible cell line. To control precisely the generation of NO in a cell, we used an ecdysone-inducible mammalian expression system13. We cloned a 4-kilobase (kb) fragment of the complementary DNA encoding human inducible nitric oxide synthase (iNOS)14 into the vector pIND, to form pIND–hiNOSf. This construct was transfected into the human fetal kidney cell line EcR293, which contains the heterodimeric receptor for retinoid X (RXR) and the ecdysone receptor (EcR). Following transfection and double selection using G418 (selection for pINO–hiNOSf) and zeocin (selection for pVg–RXR) for 14 days, 20 stable transfectant cell lines were established. Of these, six could be induced to generate NO at various concentrations after treatment with 10 µM muristerone A for 24 h. We selected one of the transfectants, EcR293 clone
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11, for further study. We used the radioactive citrulline assay15 to determine NOS activity after addition of muristerone A. Treatment of EcR293 clone-11 cells with 1 µM and 10 µM muristerone A resulted in values for NOS activity of 6 ± 2 pmol min–1 mg–1 and 33 ± 4 pmol min–1 mg–1, respectively (Fig. 1a). NOS activity was abolished by addition of the NOS inhibitor N-iminoethyl-L-ornithine (LNIO; 20 µM). We used A549 human lung carcinoma cells treated with cytokines as a control, and observed a NOS activity of 20 ± 5 pmol min–1 mg–1. As the concentration of muristerone A was increased, levels of both iNOS messenger RNA and iNOS protein also increased (Fig. 1b). investiNO mediates an increase in DNA-PKcs expression. To gate the effect of NO on gene expression, we used a strategy involving differential hybridization of mRNA-derived probes
NOS activity (pmol min –1 mg –1)
he free-radical gas NO, generated by the NOS family of enzymes, is a pleiotropic signalling molecule that has been identified as a mediator for a wide range of physiological and pathophysiological events1,2. NOS activity has been observed in human tumour cell lines3,4 and cells from tumour biopsies5–7. However, the precise function(s) of NO in tumour biology remains unclear, and several lines of research have indicated that NO may have dual effects that are apparently contradictory. For example, low concentrations of NO are thought to upregulate p53 and promote both tumour growth and neovascularisation, whereas higher concentrations have been shown to have anti-tumour properties8–10. The dual functions of NO in tumour biology may be partly explained by the dose-dependency of interactions of NO, and related reactive nitrogen-oxide species, with DNA. These interactions can result in both single and double-strand breaks11. We are interested in the role of NO in cancer, and particularly in how NO can alter the pattern of gene expression in tumour cells. To address this question, we have constructed a cell line, regulated by muristerone A, that is capable of generating NO at levels found clinically in cells from biopsies of human tumours. We used this cell line in differential-array hybridization experiments to investigate the effect of NO on gene expression. Here we show that NO can upregulate transcription of the gene encoding DNA-PKcs12. Increased transcription of the DNA-PKcs gene correlates with an increase in enzyme activity, and provides functional protection from DNAdamaging agents.
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Figure 1 Generation of NO by EcR293 clone-11 cells. a, NOS activity in EcR293 and EcR293 clone-11 cells grown in the absence (–) or presence (indicated concentration) of muristerone A, together with L-NIO where indicated. A549 human lung carcinoma cells induced by a cytokine cocktail mixture (CCM) were used as a control. NOS activity was determined by the conversion of L-arginine to L-citrulline; values are means ± s.d. from three separate assays. Northern (upper panels) and western (lower panels) blots for the indicated species in cells grown in the absence (–) or presence (indicated concentration) of muristerone A.
NATURE CELL BIOLOGY | VOL 2 | JUNE 2000 | www.nature.com/ncb © 2000 Macmillan Magazines Ltd
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with normalized cDNA arrays. Two different populations of mRNA were isolated, one from EcR293 clone-11 cells, and the other from the same cells generating NO after treatment with 10 µM muristerone A for 24 h. cDNA probes, prepared from mRNA isolated from these cells, were radioactively labelled and used to hybridize with a normalized human cDNA expression array containing 588 genes. (Atlas human DNA expression array, Clontech). Analysis of the resulting differential-hybridization pattern showed that the signal for DNA-PKcs was increased in cells expressing NO. To confirm that these changes in hybridization signal on the filter array correspond to changes in mRNA abundance, we carried out Northern blots, using a probe for DNAPKcs (Fig. 2)16. We extracted polyA+ mRNA from untreated EcR293 clone-11 cells and from cells expressing NO after treatment with 1 µM or 10 µM muristerone A (Fig. 2, upper panel). To control for differences in RNA loading, the intensity of the DNAPKcs signal was calculated as a percentage, ± s.d., of the β-actin signal, and increased by a factor of 1.2 ± 0.5 or 2.4 ± 0.4 in the presence of 1 µM or 10 µM muristerone A, respectively (Fig. 2a, lower panel). This increase was reduced (Fig. 2b) by addition of the NOS inhibitor L-NIO (10 µM muristerone A in the presence of 20 µM L-NIO; decrease by a factor of 1.2 ± 0.1). The level of DNA-PKcs mRNA in the parental cell line EcR293 remained unchanged after treatment with 10 µM muristerone A. Experiments using shorter induction times (4 h or 12 h) also failed to produce any significant increase in levels of DNA-PKcs mRNA (data not shown). To determine whether this increase in the level of DNA-PKcs mRNA corresponds to an increase in DNA-PKcs protein, we carried out western blots (Fig. 3a). We used SDS–polyacrylamide-gel electrophoresis (SDS–PAGE) to separate whole-cell extracts of protein from untreated cells, and protein from cells generating NO after treatment with 1 µM, 5 µM or 10 µM muristerone A, or 10 µM muristerone A in the presence of 20 µM L-NIO. Western blotting with a specific polyclonal antibody against DNA-PKcs17 showed an
Figure 3 Expression of DNA-PKcs. a, Western blots obtained from whole-cell extracts. Cells were grown in the absence (–) or presence (indicated concentration plus that of L-NIO where applicable) of muristerone A. After blotting against DNAPKcs, the filter was stripped and reprobed with an antibody against Ku-80 as a control for equal loading. b, Western blots obtained from nuclear lysates. Cells were grown in the absence (–) or presence (indicated concentration) of muristerone A.
Radiolabel incorporation (c.p.m.)
Figure 2 Expression of DNA-PKcs mRNA. a, Upper and middle panels, northern blots against polyA+ mRNA for the indicated proteins in cells grown in the absence (–) or presence (indicated concentration) of muristerone A. Lower panel, quantified levels of DNA-PKcs mRNA in the blot, relative to those of β-actin. b, Upper and middle panels, northern blots against polyA+mRNA for the indicated proteins in cells grown in the absence (–) or presence (indicated concentration plus that of L-NIO where applicable) of muristerone A. Lower panel, quantified levels of DNA-PKcs in the blot, relative to those of β-actin. Values in the graphs are means ± s.d. from three separate experiments.
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Figure 4 DNA-PK pulldown peptide assay. a, Enzyme activity of DNA-PK in EcR293 clone-11 cells in the absence (–) or presence (indicated concentration plus that of L-NIO where applicable) of muristerone A. Radiolabelled peptides derived from wild-type (WT) and mutant (M) versions of p53 were used as substrates. b, DNA-PK activity in nuclear extracts from cells grown in the absence (–) or presence (indicated concentration plus that of L-NIO where applicable) of muristerone A. Values are means ± s.d. from three separate experiments.
increase in the level of DNA-PKcs protein, correlating with the increase in NO concentration. The largest increase in the level of DNA-PKcs was seen after treatment with 10 µM muristerone A for 24 h. To ensure that these differences in levels of DNA-PKcs were not artefacts of the whole-cell preparation, we carried out similar experiments using protein prepared from nuclear extracts. Western blotting of these samples (Fig. 3b) supported our data from wholecell extracts. We carried out measurements of DNA-PK activity using the DNA-PK ‘pulldown’ peptide assay as described18. Increases in DNA-PK activity by factors of 1.8 and 3.5 were observed in extracts from cells generating NO after treatment with 1 µM and 10 µM
© 2000 Macmillan Magazines Ltd NATURE CELL BIOLOGY | VOL 2 | JUNE 2000 | www.nature.com/ncb
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Figure 6 NO-mediated increase in Sp1 binding. Electrophoretic mobility-shift assay (EMSA) showing binding of Sp1 and OCT-1 consensus oligonucleotides to DNA. Lane 1, control with no cell extract; lane 2, control HeLa nuclear extract; lane 3, untreated control EcR293 cells; lane 4, EcR293 cells treated with 900 µM SIN-1 for 16 h; lane 5, untreated EcR293 clone-11 cells; lane 6, EcR293 clone-11 cells treated with 10 µM muristerone A for 16 h; lane 7, control HeLa nuclear extract treated with a 100-fold excess of unlabelled Sp1 oligonucleotide. Lane 8, control HeLa nuclear extract treated with labelled OCT1 probe and a 100-fold excess of unlabelled OCT1 oligonucleotide, lane 9, control HeLa nuclear extract treated with labelled OCT1 probe; lane 10, untreated control EcR293 cells; lane 11, EcR293 cells treated with 900 µM SIN-1 for 16 h in the presence of labelled OCT1 probe.
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Figure 5 NO-dependent increase in DNA-PKcs promoter activity. a, Upper panel, the DNA-PKcs promoter region of the promoter–reporter construct p507, showing putative binding sites for Sp1, E2F, an overlapping WT-1/EGR-1/EGR-2 motif and a potential initiator consensus element (Inr)19. Lower panel, dual luciferase assays of EcR293 clone-11 cells exposed to the indicated concentrations of muristerone A (plus L-NIO where indicated) or SIN-1. Cells were co-transfected with the DNA-PKcs promoter–reporter plasmid and the internal control plasmid pRL–tk, expressing Renilla luciferase under the control of the tk promoter. Relative firefly luciferase activity (DNAPKcs promoter) was calculated by dividing the firefly reading by the Renilla luciferase value. Values are means ± s.d. from six separate experiments. **denotes P
