Haem oxygenase-1 prevents cell death by regulating cellular iron

articles Haem oxygenase-1 prevents cell death by regulating cellular iron Christopher D. Ferris*†**, Samie R. Jaffrey*, Akira Sawa*, Masaaki Takahashi*, Stephen D. Brady*, Roxanne K. Barrow*, Steven A. Tysoe‡, Herman Wolosker*, David E. Barañano*, Sylvain Doré*, Kenneth D. Poss§ and Solomon H. Snyder*¶# *Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA †Department of Medicine (Division of Gastroenterology), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA ‡Department of Chemistry and Physics, Skidmore College, Saratoga Springs, New York 12866, USA §Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA ¶Departments of Pharmacology and Molecular Sciences, and Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA #e-mail: [email protected] **e-mail: [email protected] Haem oxygenase-1 (HO1) is a heat-shock protein that is induced by stressful stimuli. Here we demonstrate a cytoprotective role for HO1: cell death produced by serum deprivation, staurosporine or etoposide is markedly accentuated in cells from mice with a targeted deletion of the HO1 gene, and greatly reduced in cells that overexpress HO1. Iron efflux from cells is augmented by HO1 transfection and reduced in HO1-deficient fibroblasts. Iron accumulation in HO1-deficient cells explains their death: iron chelators protect HO1-deficient fibroblasts from cell death. Thus, cytoprotection by HO1 is attributable to its augmentation of iron efflux, reflecting a role for HO1 in modulating intracellular iron levels and regulating cell viability. he cellular response to stressful stimuli includes the induction of stress-response proteins, including heat-shock proteins (HSPs)1,2. Some stress-response proteins, such as superoxide dismutase, modulate intracellular levels of free radicals, while others, such as members of the Hsp70 and chaperonin families, protect intracellular proteins from denaturation3,4. Proteins other than stress-response proteins, such as heat-shock transcription factors, are also induced following exposure to stressful stimuli and are thought to regulate the expression of the key proteins that participate in cellular defense or repair processes5. One HSP, Hsp32, also known as haem oxygenase-1 (HO1), is induced following exposure to several stressful stimuli, including lipopolysaccharide, haem, phorbol esters, ultraviolet radiation, hydrogen peroxide, heavy metals and organic chemicals6–11. Increased levels of HO1 messenger RNA are observed within minutes of exposure of cells to stresses, and amounts of HO1 protein remain raised for hours after stress10,11. The rapid induction of HO1 following a wide variety of stressful stimuli indicates that it may participate in the cellular response to stress. Acting in concert with cytochrome P450 reductase (CPR) and biliverdin reductase12, HO1 converts haem into bilirubin, carbon monoxide and iron. Paradoxically, these three products are toxic. Thus, the role of HO1 in the cellular response to stress is poorly understood. Here we study the function of HO1 in the cellular response to stress. We find that expression of HO1, in the absence of other HSPs, is both necessary and sufficient to protect cells from toxicity elicited by serum deprivation, showing that HO1 has a protective function. The absence of HO1 leads to iron accumulation in liver tissue and increased cellular iron in cultured fibroblasts, whereas HO1 overexpression decreases cellular iron levels. Protection of cells by HO1 parallels a decrease in intracellular iron amounts, and HO1’s protection of cells is mimicked by iron chelation. Thus HO1 is cytoprotective, and its modulation of intracellular iron levels is a physiological mechanism for determining cell viability. T Results HO1 expression blocks serum-deprivation-induced cell death. We reasoned that, if HO1 activity is cytoprotective, HO1-deficient 152 (HO1−/−) cells should be more susceptible to stressful or toxic insults than are wild-type cells. We therefore subjected HO1−/− fibroblasts to serum deprivation, staurosporine treatment or etoposide treatment, three conditions that induce cellular stress. Serum deprivation is a widely used model of cellular stress that is associated with depletion of growth factors and nutrients, and may elicit cell death through oxidative stress and subsequent apoptosis13–15. We compared wild-type and HO1−/− cells following serum deprivation. To determine the extent of cell death, we studied both nuclear morphology and DNA-ladder formation (a characteristic of apoptotic cells). Before serum deprivation, wild-type and HO1−/− cells showed similar nuclear morphology (Fig. 1a) and a low rate of spontaneous apoptosis (3±2% for wild-type cells and 4±2% for HO1−/− cells, P>0.05) as detected by staining with Hoechst 33258. However, following serum deprivation, most nuclei from HO1−/− cells were small and condensed (89±9%), consistent with them having undergone apoptotic cell death, whereas nuclei from wild-type cells remained normal, with only 6±3% (P<0.01 compared with HO1−/− cells) showing condensed apoptotic nuclei (Fig. 1a,c). Moreover, distinct DNA-ladder formation was apparent in the HO1−/− cells, whereas wild-type cells showed no evidence of DNA fragmentation (Fig. 1b). In other experiments we treated wild-type and HO1−/− cells with staurosporine (200 nM) or etoposide (100 µM) for 4–24 h and monitored cell death by DNA fragmentation using agarose-gel electrophoresis. Large DNA fragments can be observed in some cells in the initial stages of apoptosis16. After 20 h of treatment with either staurosporine or etoposide, we observed large DNA fragments (>20 kilobases in length) in HO1−/− cells but not in wild-type cells (data not shown). These data indicate that HO1 is necessary to protect cells from apoptotic cell death, particularly in response to serum deprivation. Physiological stressors induce HO1 and several other HSPs. To mimic HO1 induction in the absence of the induction of other proteins, we generated cell lines showing stable overexpression of either CPR alone or HO1 and CPR together, as CPR co-expression is required to achieve maximal increases in HO1 enzymatic activity17 (data not shown). We then monitored cell death in response to serum deprivation. After 4 days of serum deprivation, CPR-293 © 1999 Macmillan Magazines Ltd NATURE CELL BIOLOGY | VOL 1 | JULY 1999 | cellbio.nature.com articles 75 50 cells exhibited substantial nuclear condensation and fragmentation (66±5% of cells, Fig. 2A). In contrast, HO1/CPR-293 cells had 6±2% apoptotic nuclei (P<0.01 compared with CPR-293 cells, Fig. 2A). Analysis of apoptosis by DNA laddering confirmed that HO1 transfection affords significant cytoprotection, as no DNA ladder was apparent in HO1/CPR-293 cells following serum deprivation (Fig. 2B). Thus, HO1 expression is sufficient to protect cells from serum-deprivation-induced apoptosis. HO1 regulates cellular iron. To identify a possible mechanism for the protective effect of HO1 induction, we studied HO1−/− mice. As previously observed18, iron accumulation as detected by Prussian blue histochemistry did not become evident in the livers of HO1−/− animals until the animals reached ~40 weeks of age (data not shown). To determine whether iron actually accumulates earlier than this, we measured total iron levels in the livers of 10-week old HO1−/− animals by atomic emission spectrometry (AES). At this stage, iron levels were significantly higher in the HO1−/− animals (11.9±0.4 pmol µg–1 protein, n=4) than in wild-type animals (6.9±0.2 pmol µg–1 protein, n=6, P<0.001). Thus, iron accumulates NATURE CELL BIOLOGY | VOL 1 | JULY 1999 | cellbio.nature.com eru 1 –/– ;–s HO HO Figure 1 Increased apoptosis in HO1−/− fibroblasts following serum deprivation. Primary cultures of fibroblasts from HO1−/− mice and their wild-type littermates were established as described in Methods. a, Condensed nuclear morphology in HO1−/− but not wild-type fibroblasts following serum deprivation. Primary fibroblast cultures (0.5 × 106 cells) were incubated in complete (DMEM/ 10% FCS) media or incomplete (DMEM-only) media for 20 h. Following this incubation, light microscopy of wild-type cells revealed grossly normal cellular morphology in the presence and absence of serum (FCS). Nuclear morphology was determined by fluorescence microscopy following staining with Hoechst 33258, a cell-permeable, fluorescent dye, as described in Methods. For both wild-type and HO1−/− cells, occasional condensed nuclei were observed in the presence of serum, consistent with spontaneous apoptosis in primary cell culture. The images shown m m eru WT ;–s eru WT ;+s m 25 eru – 1 –/– ;+s + m – – serum c 100 Apoptotic cells (%) HO1–/– WT serum + + serum – serum + serum b HO1–/– WT a are typical examples of many microscopic fields studied in at least three separate experiments. b, DNA-ladder formation in HO1−/− but not wild-type fibroblasts following serum deprivation. DNA-ladder formation in the presence (lanes 2, 4) or absence (lanes 3, 5) of serum was determined as described in Methods. After centrifugation to remove intact genomic DNA, fragmented DNA was visualized with ethidium bromide following electrophoresis on a 2% horizontal agarose gel. Distinct ladder formation is apparent only in HO1−/− cells after serum deprivation, consistent with significant apoptosis of these cells. For comparison, molecular size markers (123-base-pair ladder) are shown in lane 1. The bands seen in lane 5 have the expected molecular size, being multiples of about 180 base pairs. c, Percentage of apoptotic cells in wild-type and HO1−/− cultures in the presence and absence of serum. in the livers of young HO1−/− animals before it can be detected by Prussian blue staining, indicating that iron homeostasis is disrupted in these animals. To ascertain the cellular basis for iron accumulation in HO1−/− tissues, we studied the effect of HO1 overexpression on iron deposition in CPR-293 and HO1/CPR-293 cells. We could not reliably detect iron levels in these cells by AES; thus we labelled cells with 55 Fe to allow a sensitive determination of cellular iron. Iron uptake was performed in the presence of complete media containing transferrin and other physiological chelators of iron that prevent precipitation of ferric hydroxide complexes19 (see Methods). In HO1/ CPR-293 cells, 55Fe uptake was substantially lower than in CPR-293 cells from 20 to 90 min (Fig. 3a). In other experiments, using 55Fe– transferrin in phosphate-buffered saline (PBS), we observed levels of 55Fe uptake and differences between HO1-transfected and control cells (data not shown) that were similar to those observed in the presence of complete media. With prolonged labelling periods (48– 72 h), 55Fe levels were the same for HO1-transfected and control cells (data not shown). As the major storage site for cellular iron, © 1999 Macmillan Magazines Ltd 153 articles –serum +serum A B a b 93 -2 R CP 3 R 9 -2 CPR HEK-293 CP c 1/ HO d HO1/CPR HEK-293 1 2 Figure 2 Transfection of HO1 into HEK-293 cells protects against serumdeprivation-induced apoptosis. A, Fragmented and condensed nuclear morphology is seen in CPR-293 but not HO1/CPR-293 cells following serum deprivation. Using cell lines showing stable expression of either CPR (a, b) or CPR and HO1 (c, d), we evaluated the effect of HO1 on cell survival during serum deprivation (b, d). HEK-293 cells are significantly more resistant to serumdeprivation-induced cell death than are primary fibroblasts. Nuclear morphology was determined after 24, 48, 72, and 96 h of serum deprivation. Nuclear morphology was visualized using the fluorescent dye Hoechst 33258 (see Methods). Significant protection by HO1 was observed at 72 h (data not shown) and 96 h, when many of the cells expressing CPR alone were apoptotic. As shown, after 96 h of serum deprivation many CPR-293 cells (b) have fragmented and condensed nuclei (white arrows) whereas only a few HO1/CPR-293 cells (d, white arrow) appear apoptotic. The data shown are typical examples from many microscopic fields observed; this experiment was repeated, with the same results being obtained. B, DNA ladders form in CPR-293 but not HO1/CPR-293 cells following serum deprivation. Serum deprivation of cells stably expressing either CPR or HO1 plus CPR was done as in A. DNA-ladder formation was determined as described in Methods. Characteristic DNAladder formation is seen in CPR-293 cells but not in HO1/CPR-293 cells, consistent with protection from cell death by HO1 expression. ferritin concentrations reflect cellular iron levels20, and increase following HO1 induction in some cells21. To assess levels of stored iron in these cells, we monitored ferritin expression. Western blot analysis revealed equal levels of ferritin in CPR-293 and HO1/CPR-293 cells (data not shown), indicating that this iron pool is not regulated by HO1 in these cells. Thus, HO1 expression can reduce iron levels in non-ferritin-associated pools. HO1 promotes cellular iron efflux. To determine whether increased iron release, as well as reduced iron uptake, could account for the decreased iron levels in HO1/CPR-293 cells, we evaluated iron release by replacing the labelling media with fresh media without 55 Fe after washing the cells with PBS and 100 µM deferoxamine. The rate of 55Fe release following labelling of the cellular iron was increased twofold in HO1/CPR-293 cells compared with control cells (Fig. 3b). Treatment of these cells with the HO inhibitor tinprotoporphyrin IX (SnPPIX) reduced 55Fe release from HO1/CPR293 cells to a level below that observed in CPR-293 cells. The potency of SnPPIX in inhibiting iron release (half-maximal inhibitory concentration (IC50) = 7 µM) is similar to its potency in assays of HO enzymatic activity22. Thus, transfection of HO1 appears to shift the equilibrium of iron transport towards iron efflux, lowering cellular iron levels. The link between HO catalytic activity and alterations in 55Fe efflux indicates a role for HO1 in regulating the mobilization of cellular iron. As SnPPIX blocks iron release in HO1/CPR-293 cells to levels below those observed in CPR-293 cells, we wondered whether baseline HO activity could regulate basal 55Fe release from CPR-293 cells. We could not detect HO1 in CPR-293 cells, but substantial levels of HO2 were measurable. SnPPIX blocks 55Fe efflux from these cells (IC50=5 µM), indicating that HO2 activity may account for basal iron efflux in these cells. To study further the physiological role of HO1 in regulating iron flux, we used fibroblasts derived from wild-type and HO1−/− mice (Fig. 4). We measured 55Fe uptake and release as described for Fig. 3. Deletion of HO1 resulted in increased 55Fe accumulation and decreased 55Fe efflux. Together, these results show that HO expression and activity correlate with cellular iron efflux. HO1 prevents cell death by regulating cellular iron. Cellular iron is toxic because it contributes to the formation of free radicals, with consequent damage to DNA, proteins and lipids15,23,24. Thus, our finding that HO1 activity reduces cellular iron levels suggests a mechanism by which HO1 induction may protect cells against toxicity. Iron, particularly non-ferritin iron, is known to contribute to the free-radical formation that is involved in apoptosis 14,15. To determine whether the mechanism of HO1-mediated cytoprotection is related to HO1’s ability to deplete intracellular iron, we treated serum-deprived HO1−/− fibroblasts with iron chelators. Addition of 10 µM deferoxamine or 100 µg ml–1 apotransferrin 154 © 1999 Macmillan Magazines Ltd NATURE CELL BIOLOGY | VOL 1 | JULY 1999 | cellbio.nature.com articles a 30 20 ∗ ∗∗∗ ∗∗∗ HO1/CPR-293 10 0 20 40 60 Time (min) 80 55 0 75 3 Fe uptake (c.p.m. per 10 cells) CPR-293 55 Fe uptake (c.p.m. per 103 cells) a ∗∗∗ ∗∗∗ –/– HO1 ∗∗ 50 WT 25 0 20 0 40 60 80 Time (min) ∗∗∗ b 20 HO1/CPR-293 ∗ 15 10 CPR-293 55 5 0 30 60 90 WT 30 Fe released (%) Fe released (%) 55 b ∗∗∗ 25 ∗∗∗ ∗∗∗ 20 –/– HO1 ∗∗∗ 10 120 Time (min) 20 0 Figure 3 HO1 transfection regulates 55Fe uptake and release in HEK-293 cells. Cell lines stably expressing either CPR or CPR plus HO1 were established as described in Methods. a, Uptake of 55Fe is reduced by HO1 transfection. 55Fe uptake was measured by incubation of the indicated cell line (1 × 106 cells per well) in 2 ml DMEM/10% FCS with 10 µCi 55Fe. At the indicated times, 55Fe uptake was determined by collecting washed cells in PBS/1% Triton-X100 and analysing them by liquid scintillation spectrometry. The data shown are the means of triplicate determinations that varied by <10%. This experiment was repeated three times with the same results. b, 55Fe release is increased by HO1 transfection. Following incubation with 55 Fe-uptake media to equilibrium for 60 min as in a, 55Fe release was initiated, after washing the cells, by the addition of 2 ml fresh DMEM/10% FCS. The fraction of 55Fe released was determined by duplicate sampling (10-µl aliquots) of the media at the indicated times followed by liquid scintillation spectrometry. At the end of the experiment, the total 55Fe accumulated was determined and the percentage released was calculated. The data shown are the means of triplicate determinations that varied by <10%. * P<0.05; *** P<0.001. This experiment was repeated three times with the same results. 100 75 50 +b (10 ilirub nM in ) 25 Figure 5 Iron chelation, but not incubation with cGMP or bilirubin, blocks serum-deprivation-induced apoptosis in HO1−/− fibroblasts. HO1−/− fibroblasts were deprived of serum as in Fig. 1 except that some cells were simultaneously incubated with 10 nM bilirubin, 10 µM 8-Br-cGMP, 10 µM deferoxamine, or 100 µg ml–1 apotransferrin. a, Nuclear morphology was visualized with Hoechst 33258 staining. b, The percentage of apoptotic cells under each treatment condition was determined by NATURE CELL BIOLOGY | VOL 1 | JULY 1999 | cellbio.nature.com +tr (10 ansf 0 µ erri gm n l –1 ) +transferrin (100 µg ml–1) +d (10 efero µm xam ) ine +deferoxamine (10 µm) +8 (10 Br cG µM MP ) +8Br cGMP (10 µM) 80 b Apoptotic cells (%) +bilirubin (10 nM) 60 Figure 4 Genetic deletion of HO1 regulates 55Fe uptake and release in primary fibroblasts. Primary cultures of fibroblasts were established as described in Methods. a, 55Fe uptake is increased in HO1−/− fibroblasts. 55Fe uptake was measured as described for Fig. 3. The data shown are the means of duplicate or triplicate determinations that varied by <10%. This experiment was repeated with the same results. b, 55Fe release is decreased in HO1−/− fibroblasts. 55Fe release was measured as described for Fig. 3. The data shown are the means of duplicate or triplicate determinations that varied by <10%. ** P<0.01; *** P<0.001. This experiment was repeated with the same results. HO1–/–, –serum a 40 Time (min) cell counting, with the experimenter blind to the treatment condition. Bilirubin and cGMP had no effect on the number of condensed, apoptotic, nuclei after serum deprivation. Deferoxamine and apotransferrin afforded substantial protection, reducing the percentage of apoptotic nuclei to basal levels observed in the presence of serum. The data shown in a are examples typical of many microscopic fields studied in two separate experiments. © 1999 Macmillan Magazines Ltd 155 articles reduces serum-deprivation-induced cell death to wild-type levels (Fig. 5a,b). Conceivably, products of HO1 enzymatic activity might account for the protective affect of HO1. However, incubation of HO1−/− fibroblasts with a cyclic GMP analogue, 8-bromo cGMP, to mimic production of carbon monoxide, or with bilirubin failed to protect against serum-deprivation-induced cell death (Fig. 5a,b), indicating that none of the products of HO1 activity account for HO1’s cytoprotective effect. Thus, HO1 is necessary to protect cells from serum deprivation-induced cell death, and this protective activity is linked to its effect on intracellular iron. Discussion Following cellular stress in various tissues, HO1 is rapidly induced and degrades haem, releasing substantial quantities of free iron. Through the Fenton reaction, iron gives rise to hydroxyl radicals. Thus, the release of free iron associated with HO1 induction would be expected to be toxic23, yet we have found HO1 activity to be protective. Our discovery that HO1 activity is linked to the extrusion of iron from cells explains this apparent paradox and provides a mechanism for cellular protection following HO1 induction. Inhibition of iron release by genetic deletion of HO1 and by the HO inhibitor SnPPIX indicates that haem itself may be the source iron for iron release or mobilization from cells. Thus, our data indicate that HO1-mediated mobilization of cellular iron is an important mechanism controlling cell survival following stress. The detailed molecular mechanisms underlying HO1’s participation in cellular iron extrusion are unclear. Cellular iron uptake occurs through both transferrin-dependent and transferrin-independent pathways, and many of the molecules involved in these pathways are known20,25–27. In addition, iron re-utilization is a welldescribed physiological phenomenon28. Thus, humans normally require only about 1 mg iron per day from dietary sources, although more than 100 times this quantity is delivered to the blood from tissues daily29. Indeed, for more than 30 years, HO activity has been known to be a rate-limiting step in the conversion of haemoglobinderived haem to bilirubin and in the re-utilization of iron from haemoglobin for the synthesis of new red blood cells30,31. Nonetheless, specific molecular mechanisms for transmembrane transport of iron from the cytosol into the endoplasmic reticulum (part of the secretory pathway) or extracellular fluid have not been described. We have recently identified an iron-transporting ATPase in liver microsomal membranes that is induced by iron, co-localizes with HO1 and may act in association with HO1 to mediate the release/ mobilization of cellular iron32. Together with HO1, this Fe–ATPase may mediate the mobilization of cellular iron for iron re-utilization, acting to reduce iron amounts within cells and thereby protecting cells under stress. A role for HO1 induction in cardiac xenograft survival has also been described33. It has yet to be determined whether HO1-dependent iron mobilization determines the xenograft survival observed in this experimental model. HO2 is a constitutively expressed HO isoform that is enriched in neurons22,34–36. In lung tissue, HO2 is associated with the accumulation of iron that follows exposure to high oxygen levels37. The inhibition of HO2 by SnPPIX in CPR-293 cells indicates that HO2 may also affect iron deposition in some circumstances. HO3 is a newly described HO isoform with minimal catalytic activity and unclear physiological function38. Whether the iron-mobilizing and antiapoptotic functions described here are specific to the inducible HO1 or are a common feature of all HOs remains to be established. h Methods Unless otherwise indicated, all chemicals were from Sigma. Assay of HO enzymatic activity. HO activity was measured using [55Fe]haemin (NEN Life Science Products). Briefly, membrane fractions from cells (10–200 µg protein) were incubated with NADPH (1 mM) and [55Fe]haemin (20,000 c.p.m.) in 156 100 µl 50 mM HEPES, pH 7.4, and 1 mM EDTA. Routinely, reactions were incubated for 8 min at 25 °C and stopped with the addition of 1 ml ice-cold 50 mM HEPES, pH 7.4, containing 10 µM SnPPIX (LC laboratories, Woburn, MA). 55Fe released by HO was quantified by applying the entire reaction mixture to an anion-exchange column (Dowex AG1X-8, Bio-Rad) (0.5 ml bed volume) to remove intact [55Fe]haemin. Free 55Fe was eluted by addition of 1 ml 50 mM HEPES, pH 7.4, plus 1 M NaCl and quantified by liquid scintillation spectrometry. Establishment of stable cell lines and primary culture of murine fibroblasts. HEK-293 cells were cultured according to standard techniques in DMEM medium supplemented with 10% FCS, penicillin and streptomycin, and glutamine. Stable transfection of cell lines was done essentially as described39. Briefly, human CPR and human HO1 complementary DNAs were subcloned into the cytomegalovirus (CMV)-based expression vector pRK5. Using pRSVneo as a selectable marker, CPR–pRK5 was transfected into HEK293 cells. Stable transformants were selected with G418, resulting in the cell line HEK293-CPR, designated CPR-293 here. The CPR-293 cell line was then transfected with pZeoSV2 and HO1–pRK5, followed by selection in both G418 and zeocin (Invitrogen). The resulting cell line, HEK-293-CPR/HO1, is designated HO1/CPR-293 here. General cell viability was assessed by determining cell growth rates (doubling times), studying trypan-blue exclusion, and monitoring basal lactate dehydrogenase (LDH) release (Sigma Diagnostics). CPR-293 and HO1/CPR-293 cells divide somewhat more slowly than do wild-type HEK-293 cells. Doubling times of 41, 39 and 34 h were observed for CPR-293, HO1/CPR-293, and wild-type HEK-293 cells, respectively. More than 95% of cells from all cell lines excluded trypan blue under normal culture conditions. No significant LDH release could be detected in either CPR-293 or HO1/CPR-293 cells under normal culture conditions. Thus, the transfection of CPR alone or together with HO1 does not appear to affect general cell viability. Primary cultures of fibroblasts were established from abdominal skin samples of HO1 −/− mice and their wild-type littermates by the Genetics Resource Core Facility, Cell Culture Laboratory, at The Johns Hopkins University School of Medicine, according to established protocols. Cells were maintained in standard cell-culture media (DMEM, 10% FCS, penicillin, streptomycin and glutamine) before experiments. Although we did observe variable rates of growth of various fibroblast cell lines (both HO1+/+ and HO1 −/−), we established several cell lines and chose cell lines with similar growth rates for experiments. Once cultured to confluence, >90% of cells excluded trypan blue, and no significant spontaneous LDH release could be detected. Induction of apoptosis. Apoptosis was induced by simple serum deprivation for most experiments as indicated. In other experiments, primary fibroblasts or HEK293-derived cell lines were treated for 4–96 h with 200 nM staurosporine (Calbiochem) or 100 µM etoposide before determination of nuclear morphology and/or DNA laddering. Nuclear morphology. Nuclear morphology of cells was determined by standard fluorescence light microscopy using the cellpermeable fluorescent dye Hoechst 33258 (Molecular Probes). After incubation under the indicated conditions for the indicated times, cells were collected by gentle scraping with a rubber policeman. The cells were washed two times with PBS and the resulting suspension was stained with a 1:500 dilution of the Hoechst dye for 5 min at 25 °C. The cells were again washed with PBS before mounting on slides for light microscopy. The percentages of normal and apoptotic cells were determined by counting multiple high-power fields using fluorescence microscopy with the experimenter blind to cell type or treatment condition. Statistical significance was determined using Student’s paired t-test. DNA-ladder formation. DNA ladders were visualized essentially as described40. Briefly, cells were resuspended in buffer (5 mM Tris-Cl, pH 7.4, 20 mM EDTA, 0.5% Triton-X100), and incubated on ice for 20 min. Samples were then centrifuged at 27,000g for 20 min to remove intact genomic DNA. Thus, samples without significant apoptosis will not have any DNA present. The supernatant was extracted with phenol/chloroform, and nucleic acids were precipitated with ethanol. The pellet was resuspended and incubated at 37 °C in 1% RNase without DNase (Boehringer Mannheim) for 1 h. The samples were then electrophoresed on a 2% agarose (LMP agarose, Gibco) gel made with ethidium bromide to visualize DNA. Atomic emission spectrometry. Liver samples were analysed for iron content using a spectroanalytical spectroflame end-on-plasma (EOP) instrument operating at 1,300 W. The analysis was carried out using the 259.921-nm line, which afforded high sensitivity and selectivity. Liver samples were prepared for AES by homogenization with a polytron homogenizer (Brinkmann) followed by solubization in 50 mM HEPES, pH 7.4, and 2% TritonX100 and then centrifugation at 100,000g. The resulting supernatants were filtered, diluted 100-fold with deionized water, and introduced into the plasma using a Meinhard nebulizer. Four replicate samples of both standards and unknowns were averaged. Negligible matrix effects were observed at the concentrations used in this analysis. 55 Fe uptake and release. FeCl3 was obtained from NEN Life Science Products (6.7 Ci mmol–1). 55Fe uptake and release were measured essentially as described19, with some modifications. Briefly, cells were cultured in standard 6well plates and incubated (1 × 106 cells per well) with 10 µCi 55FeCl3 in 2 ml per well DMEM/10% FCS for the indicated times. Complete media, including FCS, were used to ensure adequate levels of physiological chelators for 55Fe, as ferric hydroxide complexes form readily under physiological conditions and have extremely poor solubility. Transferrin concentration in serum is 4 g l–1 (50 µM) transferrin, and transferrin saturation is 30%. As we used 10% serum in our experiments and as transferrin binds two moles iron per mole transferrin, transferrin is available to bind up to 7 µM iron. In our experiments, by adding 10 µCi 55Fe with a specific activity of 6.7 Ci mmol–1, the iron concentration increases to 740 nM above physiological levels. Thus ample transferrin is available for binding iron. In addition, to ensure physiological labelling of cells, we labelled cells with 55Fe–transferrin in PBS and obtained results similar to those obtained with complete media containing serum. Before collecting the cells, we washed attached cells with 2 × 2 ml ice-cold PBS/100 µM deferoxamine. In preliminary experiments, we found that 55 © 1999 Macmillan Magazines Ltd NATURE CELL BIOLOGY | VOL 1 | JULY 1999 | cellbio.nature.com articles washing cells with PBS alone produced the same results, although background levels of cell-associated 55 Fe were ~15% higher. We also evaulated the possibility of washing cells with PBS/100 µM EDTA. Although EDTA, like deferoxamine, reduced background levels of cell-associated 55Fe, EDTA tended to cause some cells to detach from the plate, leading to less accurate replicates. We then added 2 ml ice-cold PBS/1% Triton-X100 and incubated the cells on ice for 20 min to allow complete lysis. Lysed cells from each well were collected and the amount of 55Fe was determined by liquid scintillation spectrometry. For routine experiments, determinations were made in either duplicate or triplicate with less than 10% variability. For 55Fe release, cells were incubated to equilibrium (60–90 min) under 55Fe-uptake conditions, and then washed with 2 × 2 ml PBS/100 µM deferoxamine (37 °C) before addition of 2 ml DMEM/10%FCS. 55 Fe release from each well was quantified by removing 10-µl aliquots in duplicate from the media at the indicated times. The percentage of accumulated 55Fe released was calculated after determination of the total 55Fe accumulated by collecting the cells at the end of the experiment. These experiments were also routinely done in duplicate or triplicate, with less than 10% variability. Data were analysed using GraphPad Prism software, version 2.01 (GraphPad Software Inc., San Diego, CA), and significance was analysed using a paired, two-tailed, Student’s t-test. RECEIVED 26 JANUARY 1999; REVISED 24 MAY 1999; ACCEPTED 26 MAY 1999; PUBLISHED 10 JUNE 1999. 1. 2. 3. 4. 5. Lindquist, S. & Craig, E. A. The heat-shock proteins. Annu. Rev. Genet. 22, 631–677 (1988). Lindquist, S. 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