BNIP3 Plays a Role in Hypoxic Cell Death in Human Epithelial Cells That Is Inhibited by Growth Factors EGF and IGF

BNIP3 plays a role in hypoxic cell death in human epithelial cells that is inhibited by growth factors EGF and IGF

Shilpa Kothari1,4, Jeannick Cizeau1, Eileen McMillan-Ward1, Sara J Israels1, Michelle Bailes1, Karen Ens2,3, Lorrie A Kirshenbaum2,3 and Spencer B Gibson*,1,4,

1Manitoba Institute of Cell Biology, 675 McDermot Ave., Canada; 2Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, Canada; 3Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; 4Department of Biochemistry and Medical Genetics, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

Hypoxic regions within solid tumors are often resistant to Introduction chemotherapy and radiation. BNIP3 (Bcl-2/E1B 19 kDa interacting protein) is a proapoptotic member of the Bcl-2 Hypoxic microenvironments often emerge in solid family that is expressed in hypoxic regions of tumors. tumors derived from epithelial cells. These hypoxic During hypoxia, BNIP3 expression is increased in many regions often correlate with poor prognosis due to the cell types and upon forced overexpression BNIP3 induces ability of cells within these regions to become resistant cell death. Herein, we have demonstrated that blockage of to chemotherapeutic agents and radiation therapy hypoxia-induced BNIP3 expression using antisense oligo- (Caro, 2001; Knowles and Harris, 2001; Harris, 2002; nucleotides against BNIP3 or blockage of BNIP3 Hochachka et al., 2002). By understanding the regula- function through expression of a mutant form of BNIP3 tion of hypoxia-induced cell death, effective treatments inhibits hypoxia-induced cell death in human embryonic against cancerous tumors can be developed. kidney 293 cells. We have also determined that hypoxia- Bcl-2/E1B 19 kDa interacting protein (BNIP3) is a mediated BNIP3 expression is regulated by the transcrip- unique member of the Bcl2 family members that is tion factor, hypoxia-inducible factor-1a (HIF-1a)in upregulated under hypoxic conditions (Chen et al., 1997; human epithelial cell lines. Furthermore, HIF-1a directly Cizeau et al., 2000). Forced overexpression of BNIP3 binds to a consensus HIF-1a-responsive element (HRE) in induces cell death characterized by localization at the the human BNIP3 promoter that upon mutation of this mitochondria, opening of the permeability transition HRE site eliminates the hypoxic responsiveness of the (PT) pore, loss of membrane potential (Dcm) and promoter. Since BNIP3 is expressed in hypoxic regions of reactive oxygen species (ROS) production (Vande Velde tumors but fails to induce cell death, we determined et al., 2000). Electron microscopy (EM) images show whether growth factors block BNIP3-induced cell death. extensive cytoplasmic vacuolization and the formation Treatment of the breast cancer cell line MCF-7 cells with of electron-dense bodies following BNIP3 expression. In epidermal growth factor (EGF) or insulin-like growth addition, BNIP3-induced cell death is independent of factor effectively protected these cells from BNIP3- cytochrome c release from the mitochondria and caspase induced cell death. Furthermore, inhibiting EGF receptor activation in fibroblast and epithelial cells (Vande Velde signaling using antibodies against ErbB2 (Herceptin) et al., 2000). BNIP3 is also implicated in ischemia- resulted in increased hypoxia-induced cell death in induced apoptosis in rat cardiomyocytes (Kubasiak MCF-7 cells. Taken together, BNIP3 plays a role in et al., 2002; Regula et al., 2002). It remains to be hypoxia-induced cell death in human epithelial cells that determined, however, whether hypoxia-mediated BNIP3 could be circumvented by growth factor signaling. expression in human epithelial cells is involved in Oncogene (2003) 22, 4734–4744. doi:10.1038/sj.onc.1206666 hypoxia-induced cell death. BNIP3 structure is similar to other Bcl2 family Keywords: hypoxia; cell death; tumor; BNIP3; EGF; members since it contains a C-terminal transmembrane IGF (TM) domain that targets the protein to the mitochondria and is required to induce cell death (Vande Velde et al., 2000). The BNIP3 protein also contains a Bcl-2 homology 3 (BH3) domain as determined by sequence consensus with other BH3 domains in the Bcl-2 family, but in contrast to other Bcl-2 family members, deletion of the domain does not affect its killing activity (Ray et al., 2000). BNIP3-induced cell death is blocked by *Correspondence: SB Gibson; Manitoba Institute of Cell Biology, 675 McDermot Ave., University of Manitoba, Manitoba, Winnipeg, overexpression of Bcl2, but unlike other pro-cell death Canada; E-mail: [email protected] Bcl2 family members, Bcl2 fails to associate with the Received 14 January 2003; revised 1 April 2003; accepted 2 April 2003 BH3 domain of BNIP3 instead Bcl2 associates BNIP3 regulates hypoxia-induced cell death S Kothari et al 4735 with the TM domain of BNIP3 (Vande Velde et al., BRL) supplemented with 10% FBS, 1% MEM sodium 2000). Thus, the function of the BH3 domain in BNIP3 pyruvate (GIBCO, BRL), 1% HEPES (GIBCO, BRL) and is still unclear. 1% l-glutamine (GIBCO, BRL) and antibiotics unless Besides BNIP3, the transcription factor, hypoxia- otherwise stated. Chinese Hamster Ovary (CHO) parental cell inducible factor-1a (HIF-1a) is increased and activated line and CHO HIF-1a-defective cell line (CHO KA13) (gift from A Harris, Institute of Molecular Medicine, UK) were during hypoxia (Piret et al., 2002; Semenza, 2002a). cultured in F:12 medium supplemented with 10% FBS, 1% l- Under oxygenated (normoxic) condition, HIF-1a is glutamine (GIBCO, BRL) and antibiotics. All cell lines were targeted for degradation, but in the absence of oxygen, grown in a humidified incubator in the presence of 5% CO2 at this degradation is blocked resulting in the translocation 371C. Cells were maintained under hypoxic conditions at 371C of the transcription factor to the nucleus and the within a hypoxic chamber (Forma Scientific, Marietta, OH, activation of target genes (Giatromanolaki and Harris, USA) filled with 5% CO2 balanced with N2. 2001; Hon et al., 2002; Piret et al., 2002; Sang et al., 2002; Semenza, 2002b). Overexpression of von Hippel– Western blot analysis Lindau protein that suppresses HIF-1 levels inhibits hypoxia-induced BNIP3 expression (Sowter et al., 2001). MCF-7 and HEK293T cells were homogenized in a lysis buffer In rodent fibroblasts, HIF-1a activation was demon- containing 50 mm Tris HCl (pH 7.4), 250 mm NaCl, 0.5% NP40, 50 mm NaF, 15 mm sodium pyrophosphate, 1 mm strated to activate the rodent BNIP3 promoter (Bruick, m 2000). This activation is regulated by a putative HIF-1a- glycerophosphate, 1 m Na3VO4, 500 nM okadeic acid, 20 mg aprotinin/ml, 0.7 mg pepstatin/ml, 5 mg leupeptin/ml, and 1 mm responsive element (HRE) in this promoter. The ability PMSF. Protein extracts were resolved on a 10% SDS– of HIF-1a to directly bind to this HRE site or the polyacrylamide gel electrophoresis (SDS–PAGE) gel and hypoxia regulation of the human BNIP3 promoter transferred electrophoretically onto a nitrocellulose membrane remains to be determined. (Bio-Rad laboratories Canada Ltd., Mississauga, ON, Cana- Many tumors express growth factors and their da). BNIP3 protein and HIF-1a protein were detected using a corresponding receptors that contribute to cancer mouse anti-human BNIP3 monoclonal antibody (Ray et al., progression (Kumar, 1998; Hung and Lau, 1999; 2000) and a mouse anti-human HIF-1a monoclonal antibody Furstenberger and Senn, 2002). Two such growth (BD Transduction Laboratories, Mississauga, ON, Canada) factors that contribute to tumor progression are respectively. Goat anti-mouse horseradish peroxidase (Bio- epidermal growth factor (EGF) and insulin-like growth Rad laboratories Canada Ltd., Mississauga, ON, Canada) and enhanced chemiluminescence developing reagents (Perkin- factor (IGF) (Hung and Lau, 1999; Furstenberger and Elmer Life Science) were used to detect the immunoreactive Senn, 2002). Treatment with these growth factors bands. protects cells from various apoptotic stimuli (Kulik et al., 1997; Gibson et al., 1999; Gibson et al., 2002). BNIP3 is expressed in hypoxic regions of epithelial- b-galactosidase viability assay derived tumors, but fails to induce cell death in these 5 hypoxic cells (Sowter et al., 2001). The ability of many HEK293T (1 Â 10 cells) were seeded in six-well plates. The next day, using lipofectamine reagent (Invitrogen) cells were growth factors to protect epithelial-derived cells against transfected with 0.2 mg of pcDNA3-b-galactosidase (pcDNA3- BNIP3 and hypoxia-mediated cell death is, however, b-gal) plasmid plus 1 mg of pcDNA3-BNIP3DTM. After 4 h unknown. transfection, cells were incubated either in DMEM under In this study, we have determined that BNIP3 normoxic or hypoxic conditions for 48 h. Following incuba- expression plays a role in hypoxia-induced cell death tion, cells were fixed in 0.2% glutaraldehyde and washed three in human epithelial-derived cells. In addition, hypoxia- times with 0.1 m phosphate buffer saline (PBS) and stained in induced BNIP3 expression is mediated by HIF-1a X-gal buffer (0.5 mg/ml 5-bromo-4-chloro-3-indolyl-b-d-ga- m m binding to a HRE site in the human BNIP3 promoter. lactopyranoside, 3 m K3Fe(CN)6,3m K4Fe(CN)63H2O, m m Finally, we have determined that BNIP3- and hypoxia- 1m MgCl2 in 0.1 PBS) to detect for b-galactosidase induced cell death is blocked by growth factor signaling expression as described previously (Miura and Yuan J, 2000). The percentage of dead cells was calculated by assessing the in human epithelial-derived cells. This suggests that number of round, condensed, blue cells in the total population BNIP3 plays a role in hypoxia-induced cell death, of flat blue cells. A total number of at least 200 cells were but is blocked by growth factor signaling in cancer counted for each experiment. To assess for the effect of EGF cells. on BNIP3 overexpressing cells, MCF-7 (8 Â 104 cells) were seeded in six-well plates for 24 h. The next day cells were incubated with EGF (1 mg/ml) (Sigma Aldrich, Oakville, ON, Canada) for 1 h. Using lipofectamine reagent, cells were Materials and methods transfected with 0.2 mg of pcDNA3-b-galactosidase and 1 mg of pcDNA3-BNIP3-30T7 in the presence of EGF (1 mg/ml). Cell culture After 4 h transfection, DMEM containing serum and EGF Human embryonic kidney (HEK) 293 and 293T (middle T (1 mg/ml) were added to the cells and incubated at 371C. antigen transformed) cells were cultured in Dulbecco’s Following incubation, cells were stained as described pre- modified essential medium (DMEM) (GIBCO, BRL) supple- viously to assess for b-galactosidase expression. The percen- mented with 10% fetal bovine serum (FBS) (GIBCO, BRL) tage of dead cells was calculated by assessing the number of and 100 U/ml penicillin/streptomycin (GIBCO, BRL) unless round, condensed, blue cells in the total population of flat blue otherwise stated. Breast carcinoma MCF-7 cells were main- cells. A total number of at least 200 cells were counted for each tained in a-minimal essential medium (a-MEM) (GIBCO, experiment.

Oncogene BNIP3 regulates hypoxia-induced cell death S Kothari et al 4736 Acridine orange cell death assay EDTA) at 41C. Gels were vacuum dried and autoradiographed at À801C for 18–24 h. MCF-7 cells were treated with antibody against ErbB1 (Herceptin) (Roche, Mississauga, ON, Canada) ranging from 0.1 to 100 mg/ml for 0–96 h. Cells were then resuspended in Antisense experiments 100 ml of media by gently vortexing, and 4 ml of acridine orange Antisense oligonucleotide (50-GCGCTCCGTTCTGCGA- (100 mg/ml) and ethidium bromide (100 mg/ml) were added. A CATG-30) (Sigma Genesys, Oakville, ON, Canada) and sense 10 ml aliquot was removed and placed on a microscope slide, oligonucleotide (50-GTACAGCGTCTTGCCTCGCG-30) were and a coverslip was applied over the cells. The slide was viewed complimentary to bases À1to þ 19 of the human Bnip3 gene. on a fluorescence microscope using a fluorescein filter. The Random sequence oligonucleotide (50-GCAGTCAGC- percentage of dead cells was calculated by counting the GACGTCGAAGC-30) contained the same bases in a number of orange cells and cells containing bright green local scrambled sequence. Oligonucleotides were phosphorothioate DNA condensation in a population of diffused green cells. A modified to prevent degradation. Oligonucleotides were total number of at least 250 cells were counted for each assay. diluted in Opti-MEM (Gibco BRL) mixed with oligofectamine (Invitrogen) and incubated with HEK293 cells for 4 h then BNIP3 promoter isolation hypoxic medium was added and cells were further incubated under hypoxia for 24–48 h. At indicated times, cell death was Recombinant luciferase plasmids were constructed as de- determined via Trypan blue exclusion assay or cells were lysed scribed in Chen et al. (1997). Briefly, BAC clone and protein extracts were analysed for BNIP3 expression as H_NH0045A17 containing the human BNIP3 genomic se- described above. quence was used as template with appropriate primers to amplify 1193 bp of the BNIP3 promoter (À12 to À1205 bp). Trypan blue cell death assay Primers were designed to incorporate appropriate restriction enzyme sites at the 50 and the 30 ends by polymerase chain Trypan blue exclusion assays were performed to identify reaction (PCR). Following restriction digestion, the inserts compromised plasma membranes. Culture media were re- were ligated upstream of the luciferase gene into the pGL3- moved and replaced with 0.4% Trypan blue in PBS for 5 min basic (Promega, Madison, WI, USA) reporter vector. BNIP3 at 371C. The percentage of dead cells was calculated by promoter HRE mutants were generated by PCR, using site- counting the number of blue cells in a population of clear cells. directed mutagenesis that mutated each of the two potential A total number of at least 200 cells were counted for each HRE. The BNIP3 promoters containing mutated HRE were assay. ligated into the pGL3-basic reporter vector. Electron microscopy Transfection and reporter assays HEK293T cells with or without EGF stimulation (4 h) or HEK293, MCF-7 and CHO parental and CHO KA13 cells pepstatin A (1 h) treatment were transfected with BNIP3. After were plated onto 6 mm plates (1 Â 105 cells) in 5 ml of DMEM, 12 h of transfection, cells were collected and fixed in 2% a-MEM or F12 media supplemented with 10% FBS for 48 h paraformaldehyde, 0.1% glutaraldehyde in 0.1 m sodium before transfection. Cells were transfected using the lipofecta- cacodylate for 2 h, postfixed with 1% OsO4 for 1.5 h, washed mine reagent. For each transfection, 0.9 mg of the pGL3- and finally stained for 1 h in 3% aqueous uranyl acetate. The BNIP3 plasmid and 0.1 mg of pCDNA3-b-gal reporter vector samples were then washed again, dehydrated with graded or 0.5 mg of pGL3-BNIP3 and 2.5 mg of pCEP4-HIF-1a and alcohol, and embedded in Epon-Araldite resin (Canemco Inc.). 0.1 mg pCDNA3-b-gal were added. Cells were incubated for Ultrathin sections were cut on a Reichert ultramicrotome, 24 h under normoxic conditions or 4 h under normoxic counterstained with 0.3% lead citrate and examined on a conditions followed by 20 h under hypoxic conditions. Cells Philips EM420 electron microscope. were lysed in 400 ml of cell culture lysis buffer (Promega Madison, WI, USA) and the lysate was analysed for luciferase and b-galactosidase activities. The luciferase assay and b- Results galactosidase activity assay were performed according to the protocols from the manufacturer (Promega Madison, WI, USA). Transfection efficiency was normalized on the basis of In human HEK293 and MCF-7 cells, BNIP3 protein b-galactosidase activity. increases during hypoxia Under hypoxic conditions, BNIP3 protein levels in- Electrophoretic mobility shift assay crease in rodent fibroblasts and mouse cardiomyocytes Oligonucleotide probes were generated by 50 end labeling with cells (Guo et al., 2001; Kubasiak et al., 2002; Regula [g-32P]ATP (Perkin-Elmer) and T4 polynucleotide kinase et al., 2002). BNIP3 protein was also shown increased in (BioLabs). Binding reactions were carried in a total volume human solid tumors (Sowter et al., 2001). To determine of 20 ml containing 5 mg of protein extract and 0.1 mgof whether BNIP3 protein levels increase in human denatured herring sperm DNA (Sigma) in 10 mm Tris-HCl (pH epithelial-derived cells under hypoxia, we exposed m m m m 7.5), 50 m KCl, 50 m NaCl, 1 m MgCl2,1m EDTA, MCF-7 and HEK293 cells to hypoxic conditions over m 5m DTT and 5% glycerol. Anti-HIF-1a (Santa Cruz Inc.) a 60 h time course in a hypoxic chamber and analysed was mixed to extracts prior to probe where indicated. After BNIP3 expression in these cells by Western blotting. preincubation for 5 min at room temperature, probes were added to the binding reactions and the incubation was Hypoxic exposure resulted in an increased BNIP3 continued for an additional 30 min. The reaction mixtures expression in MCF-7 cells at 12 h. BNIP3 expression were then loaded onto a 5% nondenaturing polyacrylamide remained increased for 48 h, and declined after 60 h gel. Electrophoresis was performed at 180 V in a 0.3 Â TBE (Figure 1). In HEK293 cells, BNIP3 expression in- (1 Â TBE is 89 mm Tris-HCl, 89 mm boric acid and 5 mm creased at 24 h and was maximally increased after 60 h

Oncogene BNIP3 regulates hypoxia-induced cell death S Kothari et al 4737 MCF-7 HEK293 a 0 12 24 36 48 60 hours 0 12 24 36 48 60 hours

BNIP3 Non treated Random Sense Antisense

BNIP3

β-actin

β-actin Figure 1 BNIP3 protein levels accumulate during hypoxia. MCF- 7 and HEK293T cells were incubated under hypoxia for a 60 h time course. Protein lysates were isolated and Western blotted with b 40 monoclonal anti-BNIP3 antibody. The blots were stripped and 35 reprobed for b-actin to determine equal loading. Results are representative of three independent experiments 30 25 20 15 ∗ (Figure 1). No change was detected in b-actin levels in 10 % OF CELL DEATH either of these cell lines over the time course (Figure 1). 5 These results indicate that BNIP3 expression increases 0 during hypoxia in human epithelial-derived cells. non- Random BNIP3 BNIP3 transfected oligonucleotide Sense Anti-sense oligonucleotide oligonucleotide Hypoxia-induced cell death is blocked by either antisense oligonucleotide against BNIP3 or expression of human c 35 BNIP3 protein lacking the transmembrane domain vector alone 30 BNIP3∆TM BNIP3 is induced under hypoxic condition and upon forced overexpression of BNIP3, cells undergo cell death 25 (Cizeau et al., 2000; Vande Velde et al., 2000; Regula et al., 2002). Furthermore, by blockage of BNIP3 ∗ 20 expression in mouse cardiomyocytes, ischemia-induced cell death is inhibited (Kubasiak et al., 2002). It is, 15 however, unknown if BNIP3 is involved in hypoxia- induced cell death in human epithelial-derived cells. % OF CELL DEATH 10 Using antisense oligonucleotides against BNIP3 to reduce expression, we determined if hypoxia-induced cell death was affected in HEK293 cells. For 48 h, 5 HEK293 cells were treated with random, sense and 0 antisense oligonucleotide against human BNIP3, and Normoxia Hypoxia exposed to hypoxic conditions. To confirm that antisense oligonucleotides against BNIP3 are blocking Figure 2 Treatment with antisense oligonucleotides against BNIP3 or expression of dominant-negative BNIP3 reduce hypox- its hypoxia induction, cells were lysed and Western ia-induced cell death. (a) HEK293 cells were incubated with blotted for BNIP3 expression. Only treatment with random, sense and antisense oligonucleotides against BNIP3. Cells antisense oligonucleotides resulted in a significant were subjected to hypoxia for 48 h. Proteins were isolated and decreased expression of BNIP3 protein as compared Western blotted with monoclonal anti-BNIP3 antibody. The blots cells treated with random or sense oligonucleotides were stripped and reprobed for b-actin to determine equal loading. (b) Cells were treated with oligonucleotides as in (a). After a 48 h under hypoxic conditions (Figure 2a). Oligonucleotide hypoxic incubation, cell death was measured by trypan blue treatments had no significant effect on b-actin levels exclusion assay. Differences between cells treated with random (Figure 2a). The amount of hypoxia-induced cell death oligonucleotide and antisense oligonucleotide were statistically was determined by trypan blue exclusion assay as significant with a P-value of o0.005. The error bars represent standard deviation between three independent experiments. (c) described in Materials and methods section. At 48 h, HEK293 cells were transfected with the dominant-negative hypoxic treatment resulted in 29, 28 and 27% cell death BNIP3DTM form of BNIP3 in a pcDNA3 expression vector or in nontransfected, random oligonucleotide-transfected vector alone in combination with b-galactosidase cDNA in and sense oligonucleotide-transfected cells, respectively pcDNA3. Cells were incubated under hypoxia for 48 h, and cell (Figure 2b). In contrast, cells transfected with the death was measured as changes in the morphology in b- galactosidase-positive cells. Differences between cells transfected antisense oligonucleotide against BNIP3 showed 12% with BNIP3DTM and vector alone were statistically significant cell death (Figure 2b). This is comparable to the level of with a P-value of o0.01. The error bars represent standard cell death under normoxia conditions (data not shown). deviation between three independent experiments Thus, the lower expression of BNIP3 protein in cells treated with antisense oligonucleotides correlated with a reduction in hypoxia-induced cell death.

Oncogene BNIP3 regulates hypoxia-induced cell death S Kothari et al 4738 Deletion of the transmembrane domain of BNIP3 a (BNIP3DTM) fails to induce cell death upon over- 24 h 48 h 72 h expression (Ray et al., 2000). In addition, BNIP3DTM NHNHNH acts as a dominant-negative protein blocking endogen- ous BNIP3 from integrating into the mitochondria (Ray HIF-1α et al., 2000). BNIP3DTM was transiently expressed in HEK293 cells and cells were exposed to hypoxic BNIP3 conditions for 48 h. Hypoxia-induced cell death was reduced from 30% for cells transfected with vector alone as a negative control to 18% for cells expressing β-actin BNIP3DTM (Figure 2c). Transfection of vector alone into HEK293 cells resulted in similar levels of cell death b HIF-1α compared to untransfected cells (data not shown). These results suggest that blockage of BNIP3 activity results in reduced hypoxia-induced cell death. Normoxia Normoxia Hypoxia Hypoxic induction of human BNIP3 is mediated by Hypoxia transcription factor HIF-1a BNIP3 One of the major mediators of hypoxia gene regulation is the transcription factor HIF-1a (Harris, 2002; Leek β-actin et al., 2002). In rodent fibroblasts, BNIP3 upregulation is mediated by HIF-1a (Bruick, 2000). Hypoxic regula- Figure 3 Hypoxic induction of human BNIP3 is mediated by tion of the human BNIP3 protein is, however, unknown. transcription factor HIF-1a.(a) MCF-7 cells were incubated under MCF-7 cells were incubated under hypoxia or normoxia hypoxic condition over a 72 h time course. At indicated times, cells for a 72 h time course. Hypoxic exposure resulted in an were lysed and protein lysates were Western blotted with increased expression of both BNIP3 and HIF-1a monoclonal anti-HIF-1a antibody. The blots were than stripped and reprobed for BNIP3 with monoclonal anti-BNIP3 antibody. proteins in MCF-7 cells. Furthermore, increased HIF- The blots were also analysed for b-actin expression to ensure equal 1a expression was detected at 24 h, while BNIP3 protein loading. Results are representative of three independent experi- was only detected after 48 h. At 72 h, BNIP3 expression ments. (b) HEK293T cells were either untransfected or transiently was still increased, whereas HIF-1a expression was transfected with HIF-1a cDNA in pCEP expression vector as significantly decreased (Figure 3a). Neither BNIP3 nor indicated. Cells were incubated under hypoxic or normoxic conditions for 24 h. Protein lysates were isolated and Western HIF-1a was detectable in cells cultured under normoxic blotted with anti-BNIP3 antibody. The blots were than stripped conditions (Figure 3a). This indicates that HIF-1a might and reprobed for b-actin. Results are representative of three regulate BNIP3 expression. independent experiments To determine whether BNIP3 upregulation was HIF- 1a dependant, MCF-7 cells were transiently tranfected with HIF-1a and incubated under normoxic conditions. luciferase activity of the BNIP3 promoter compared This resulted in increased expression of BNIP3 under to normoxic conditions (Figure 4c). In the CHO normoxic conditions after 24 h (Figure 3b). BNIP3 HIF-1a-defective cells, absence of functional HIF-1a expression increased further under hypoxic conditions lead to a complete loss of hypoxia-mediated responsive- (Figure 3b). This difference in the amount of BNIP3 ness of the BNIP3 promoter (Figure 4c). To confirm expression could be due to only 30% of the cells being that loss of hypoxia responsiveness was due to the transfected with HIF-1a whereas all cells were exposed absence of a functional HIF-1a, HIF-1a was transfected to hypoxia (data not shown). These results indicate that in the CHO HIF-1a-defective cells. The restoration HIF-1a overexpression increases BNIP3 protein levels. of functional HIF-1a resulted in a fourfold induction of Previously, it was demonstrated that the rodent 0.5 kb luciferase activity from the BNIP3 promoter at BNIP3 promoter was activated by HIF-1a (Bruick, 24 h post-transfection under both normoxic and hypoxic 2000). We have isolated a human 1.2 kb BNIP3 conditions. This induction was similar to the increase promoter and placed it upstream of a luciferase reporter in luciferase activity in CHO parental cells exposed gene (Figure 4a). The promoter activity was monitored to hypoxic conditions (Figure 4c). These results in MCF-7, CHO parental and CHO HIF-1a-defective indicate that the human BNIP3 promoter is activated cells. Luciferase expression was induced 15-fold when in response to hypoxia mediated by the transcription MCF-7 cells were incubated for 19 h under hypoxic factor HIF-1a. conditions or when cells were cotransfected with HIF-1a (Figure 4b). Furthermore, expression with a dominant- Human BNIP3 promoter has a HRE that binds to negative form of HIF-1a blocked hypoxia-induced transcription factor HIF-1a and controls its hypoxic activation of the BNIP3 promoter. In addition, CHO induction parental and CHO HIF-1a-defective cells were transfected with the BNIP3 promoter construct. In CHO HIF-1a binds to a consensus 50-RCGTG-30 site on parental cells, hypoxia-induced a fourfold increase in target gene promoters to activate their transcriptional

Oncogene BNIP3 regulates hypoxia-induced cell death S Kothari et al 4739 HRE 1 HRE 2 ab-610 -246 ATG 14000 Normoxia Human 12000 Hypoxia -1200 +1 10000 HRE 1 HRE 2 -234 -156 ATG 8000 6000 Rat -500 +1 4000

Relative Luciferase Units 2000

0 Conserved region between the human and rodent BNIP3 BNIP3-promoter + + + + + + HIF-1α - - + + - - HRE: HIF-1 response element HIF-1αDN - - - - + +

c d 10000 Normoxia

5000 Hypoxia Normoxia 8000

4000 Hypoxia 6000 3000 4000 2000

Relative Luciferase Units 2000 1000 Relative Luciferase Units

0 0 parental cells HIF-1 defective HIF-1 defective Wild type BNIP3 HRE1 mutated HRE2 mutated cells cells transfected promoter promoter promoter with HIF-1

e Hypoxia Normoxia Hypoxia HIF Antibody Hypoxia Normoxia -+ control control HRE2 HRE2 HRE1 HRE1 HRE2 HRE2 Mutated HRE2 Mutated HRE2

HIF-1α

Non-specific binding

Probe

Figure 4 HIF-1a regulates human BNIP3 promoter activation. (a) Schematic diagram of human and rodent BNIP3 promoter. Promoters contain a 18 bp homologous sequence. Both promoters contain two HREs. The functional HRE in rodent is located within the conserved sequence. (b) MCF-7 cells were transfected with a luciferase reporter vector (pGL3-basic) regulated by the BNIP3 promoter in combination with a b-galactosidase vector in pcDNA3. When indicated, cells were also cotransfected with HIF-1a cDNA, HIF-1a dominant-negative (DN) or pCEP expression vector alone. Cells were incubated under hypoxia or normoxia conditons for 20 h. Cells were then lysed and luciferase and b-galactosidase activities measured as described in Materials and methods section. Results represent average relative luciferase activity of three independent experiments and error bars represent standard deviation. (c) CHO parental and CHO HIF-1a-defective cells were transfected with a luciferase reporter construct containing the BNIP3 promoter in combination with b-galactosidase cDNA in a pcDNA3 expression vector. When indicated, cells were also transfected with HIF-1a cDNA in a pCEP expression vector. After incubation under hypoxia or normoxia for 20 h, luciferase and b-galactosidase activities were determined. Results represent average relative luciferase activity of three independent experiments and error bars represent standard deviation. (d) Luciferase reporter constructs containing the BNIP3 wild-type promoter or the BNIP3 promoter containing a mutation at HRE1 or HRE2 were transfected in HEK293T cells in combination with b-galactosidase in a pcDNA3 expression vector. Cells were incubated under hypoxia or normoxia for 20 h. Cells were lysed and luciferase and b-galactosidase activities determined. Results represent average relative luciferase activity of three independent experiments and error bars indicate standard deviation. (e) MCF-7 cells were incubated under either hypoxic or normoxic conditions for 24 h prior to protein extract preparation. Extracts were mixed with probes containing the human HRE2 site, the mutated HRE2 site, the HRE1 site and a probe containing a consensus HIF- 1a binding site (W18). Where indicated extracts were mixed with anti- HIF-1a antibodies prior to incubation with HRE 2 probes. Binding reactions were analysed by EMSA as described in Materials and methods section

Oncogene BNIP3 regulates hypoxia-induced cell death S Kothari et al 4740 activity (Giatromanolaki and Harris, 2001; Piret et al., Senn, 2002; Gibson et al., 2002). We determined whether 2002). In the rodent BNIP3 promoter, a HRE site growth factors provide a survival response against located at À234 bp was found to be essential for BNIP3-induced cell death. MCF-7 cells were treated transcriptional activation (Bruick, 2000). Analysis with 1 mg/ml of EGF or IGF in 10% FBS media for 1 h of the BNIP3 promoter showed two possible HRE and transiently cotranfected with BNIP3 and b-galacto- sites (HRE1: 50-CACGTC-30 located at À206 bp and sidase plasmids in the presence of EGF or IGF. HRE2: 50CACGTG-30 located at À609 bp) that Overexpression of BNIP3 increased cell death from are almost identical to the consensus sequence. Com- 13% of cells after 12 h to 35% after 36 h. In the presence parison between the rodent and human BNIP3 promo- of EGF or IGF, the percentage of BNIP3-induced cell ter revealed that an 18 bp sequence was identical death was reduced to 7% at 12 h and 23% at 36 h between the two species. This sequence contains a (Figure 5a). This reflects a 40% protection against consensus HRE site found essential for hypoxic activa- BNIP3-induced cell death at 12 and 18 h, and 30% tion of the rodent promoter (Figure 4a). HEK293 cells protection at 24 and 36 h post-transfection. Treatment were transfected with the human wild-type BNIP3 with EGF or IGF failed to change the expression levels promoter and exposed to normoxic conditions. This of BNIP3 in transfected cells (data not shown). In established a background luciferase activity of addition, HEK293T cells were also determined for EGF 1000 RLU. Under hypoxic conditions, the promoter protection against BNIP3-induced cell death and activity increased. Cells transfected with the BNIP3 showed similar results (data not shown). Thus, growth promoter mutated at HRE2 site and exposed for 19 h factors protect cells from BNIP3-induced cell death. under hypoxia failed to increase luciferase activity BNIP3-transfected cells were characterized by (700 RLU, Figure 4d). In contrast, luciferase activity rounded mitochondria with destroyed cisternae, exten- of cells transfected with the BNIP3 promoter mutated at sive cytoplasmic vacuolization and electron-dense re- the HRE 1 site was increased under hypoxia to similar gions (Vande Velde et al., 2000). EGF stimulation may levels as the wild-type promoter for BNIP3 (Figure 4d). affect these events. To determine this, HEK293T cells This suggests that the HRE2 site in the BNIP3 promoter were tranfected with BNIP3. After 12 h post-transfec- is required for hypoxia-induced activation of the tion, cells were examined under transmission EM as promoter. described in Materials and methods section. BNIP3- Since the BNIP3 promoter has a HRE site essential transfected cells appear to have collapsed mitochondria, for its hypoxia-mediated activation, it remains unknown cytoplasmic vacuolization and appearance of electron- in both rodent and human whether HIF-1a directly dense regions (Figure 5b). In contrast, BNIP3-trans- binds to the HRE2 site in the promoter. Using fected cells treated with EGF showed moderate swollen oligonucleotides containing the HRE2 site or a mutated mitochondria with intact internal structure, no cyto- HRE2 site, HIF-1a binding to these probes was plasmic vacuolization and no electron-dense regions determined by electro mobility shift assay as described (Figure 5b). Taken together, these data suggest that in Materials and methods section. A probe (W18) EGF protection involves blocking BNIP3-mediated containing a consensus HRE was used as a positive mitochondrial changes and cytoplasmic vacuolization. control. Both W18 and HRE2 probes showed binding to Indeed, EGF treatment reduces BNIP3-mediated loss of HIF-1a under hypoxic conditions. Mutation in the membrane potential and increase in ROS in the HRE2 site resulted in the loss of HIF-1a binding under mitochondria (data not shown). hypoxic conditions (Figure 4e). The binding was specific for HIF-1a since using anti-HIF-1a antibodies resulted in a supershift (Figure 4e). When the W18 and HRE2 BH3 domain of BNIP3 mediates growth factor protection probes were incubated with lysates from cells exposed to normoxic conditions, no binding could be detected. In BNIP3 contains several defined domains such as addition, using a probe for HRE1 site, there was no the PEST domain (amino acids 54–81), a BH3 domain detectable binding of HIF-1a under hypoxic conditions (amino acids 104–119) and a transmembrane domain (Figure 4e). This suggests that HRE2 site in the human (amino acids 164–184) (Yasuda et al., 1998; Cizeau et al., BNIP3 promoter binds to HIF-1a. 2000; Ray et al., 2000; Farooq et al., 2001; Kim et al., 2002). We determined whether the EGF protective Growth factors protect cells from BNIP3-mediated cell effects are mediated through these domains. Deletions death of the PEST domain, amino-terminus region or the BH3 domain in BNIP3 were transiently transfected in BNIP3 is a hypoxia-regulated protein that induces cell MCF-7 cells. Deletions of the amino-terminus region death upon overexpression in human epithelial-derived or the PEST domain of BNIP3 did not alter EGF- cells (Vande Velde et al., 2000). In epithelial-derived or IGF-mediated protection (Figure 6a). However, tumors, BNIP3 is expressed in hypoxic regions where deletion of the BH3 domain abrogated the effect of cells are resistant to cell death (Sowter et al., 2001). This EGF- or IGF-mediated protection. Similar results were suggests a possible survival mechanism blocking BNIP3- also found in HEK293T cells (Figure 6b). These results induced cell death in these cells. Growth factors provide suggest that EGF or IGF requires an intact BH3 survival responses to cells and contribute to cancer domain of BNIP3 to confer protection against BNIP3- progression (Chapman et al., 1999; Furstenberger and induced cell death.

Oncogene BNIP3 regulates hypoxia-induced cell death S Kothari et al 4741 a Control BNIP3 35 BNIP3+EGF BNIP3+IGF 30 * 25 *

20 * * 15

% OF CELL DEATH * * 10

0 12 hours 18 hours 24 hours 36 hours

b Control BNIP3 BNIP3+EGF

HEK293T cells

Mitochondria

Vacuoles

Figure 5 Growth factors block cell death mediated by BNIP3. (a) MCF-7 cells were untreated or pretreated with 1 mg/ml of EGF or IGF in 10% FBS media. The cells were then transfected with empty vector pcDNA3 (control) or a cDNA for BNIP3 in a pcDNA3 expression vector in combination with b-galactosidase for the times indicated. The level of cell death was determined by morphological changes to b-galactosidase-positive cells by light microscopy. Error bars represent standard deviation of three independent experiments. Difference between cells treated with EGF and IGF and untreated was statistically significant with a P-value of 40.005. (b) HEK293T cells were transfected with empty vector or cDNA for BNIP3 in a pcDNA3 expression vector. Cells were also untreated or treated with EGF (1 mg/ml) in 10% FBS media for 18 h. The cells were visualized using electron microscope. Black arrows indicate where mitochondria and vacuoles are located under the appropriate heading. White arrows indicate electron-dense bodies. The images of the whole cell were magnified 7500 times whereas images of mitochondria and vacuoles were magnified 350 000 times

Herceptin sensitizes cells to hypoxia-induced cell death MCF-7 cells that express ErbB2 were incubated with increasing concentrations of Herceptin and exposed to We have demonstrated that BNIP3 plays a role in hypoxia for 48 h. The amount of cell death increased hypoxia-induced cell death in human epithelial-derived from 10% for nontreated cells to 25% at 10 mg/ml cells. We have also determined that stimulation with Herceptin and 30% at 100 mg/ml Herceptin (Figure 7a). EGF confers protection against BNIP3-induced cell Under normoxic conditions, the amount of cell death death. This suggests that EGF signaling could prevent remained the same over the range of Herceptin hypoxia-induced cell death. Inactivation of EGF recep- concentrations used (Figure 7a) Furthermore, treatment tors has been previously shown to increase cell death in with 10 mg/ml Herceptin increased cell death from 10% epithelial-derived cells (Gibson et al., 1999, 2002). Using at 0 h, 20% at 48 h and 40% at 96 h in MCF-7 cells an inhibitory antibody against the EGF receptor ErbB2 incubated under hypoxic conditions (Figure 7b). In the (Herceptin) that is currently used in the treatment of absence of Herceptin treatment, the percentage cell breast cancer (Burris, 2000; Stebbing et al., 2000; Mrsic death of MCF-7 cells incubated under hypoxic condi- et al., 2001), we determined if hypoxia-induced cell tions was only slowly increasing over the time course death is altered following treatment with Herceptin. (Figure 7b). Using another antibody used in cancer

Oncogene BNIP3 regulates hypoxia-induced cell death S Kothari et al 4742

a a 45 16 Hypoxia Control EGF 40 14 IGF Normoxia 35 12 30 10 25

8 20

6 15 % OF CELL DEATH

% OF CELL DEATH 4 10 5 2 0 0 0 0.01 0.1 0.5 1 10 100 Vector BNIP3 ∆N- ∆N- ∆BH3 Alone term PEST Herceptin (µg/ml)

b 50 b 50 Control untreated EGF 40 40 herceptin 30 30 20

20 % OF CELL DEATH 10 % OF CELL DEATH 10 0 0 24487296 0 Time (hours) Vector BNIP3 ∆N- ∆N- ∆BH3 Alone term PEST Figure 7 Herceptin sensitizes cells to hypoxia-induced cell death. Figure 6 The BH3-like domain of BNIP3 mediates growth factor (a) MCF-7 cells were treated with increasing concentrations of protection. (a) MCF-7 cells or (b) HEK293T cells were untreated Herceptin as indicated. Cells were either incubated under hypoxic (control) or pretreated with EGF (1 mg/ml) in 10% FBS media for or normoxic conditions for 48 h. The amount cell death was 1 h. The cells were transfected with vector alone (pcDNA3) or the determined by acridine orange assays. Error bars represent various deletion mutants of the amino-terminus region, PEST standard deviation between three independent experiments. (b) domain, and BH3 domain in combination with b-galactosidase and MCF-7 cells were treated with 10 mg/ml Herceptin for a 72 h time incubated for 12 h. The amount of cell death was determined by course. At indicated times, cell death was determined by acridine morphological changes in b-galactosidase-positive cells. Error bars orange assays. Error bars represent standard deviation between represent standard deviation between three independent experi- three independent experiments. ments

treatment (Rituxmab) failed to sensitize cells to hypoxia- from hypoxic regions in tumors (Yu et al., 2002). Thus, induced cell death (data not shown). Herceptin treat- targeting hypoxic-mediated cell death machinery such as ment, however, failed to change hypoxia-induced BNIP3 is important in cancer treatment. BNIP3 expression levels (data not shown). These results BNIP3 is one of the most hypoxic-responsive genes indicate that inactivation of ErbB2 sensitizes cell to with its mRNA and protein levels increasing to high hypoxia-induced cell death. levels following hypoxia (Caro, 2001; Guo et al., 2001). Increased expression of BNIP3 induces cell death through mitochondrial dysfunction in many cell types including epithelial-derived cancer cells (Chen et al., Discussion 1999; Cizeau et al., 2000; Vande Velde et al., 2000). In the present study, we have determined that blockage The relevance of hypoxia in epithelial-derived tumors is of BNIP3 induction or function reduces the amount becoming increasingly important. Antiangiogenesis of hypoxia-induced cell death in human epithelial cells. therapy has shown that hypoxic regions can survive Furthermore, transcription factor HIF-1a is essential and repopulate the tumor after therapy (Yu et al., 2002). for BNIP3 transcriptional activation under hypoxia. Hypoxic tumor cells are also more resistant to che- It has been previously shown that rodent BNIP3 motherapy primarily due to the lack of an efﬁcient way promoter is activated by HIF-1a (Bruick, 2000). to deliver the drugs to these cells (Giatromanolaki and However, direct interaction between BNIP3 promoter Harris, 2001; Koukourakis, 2001; Goonewardene et al., and HIF-1a was not demonstrated. Herein, we 2002). Radiation that relies on the formation of ROS to have shown for the ﬁrst time that HIF-1a could directly induce apoptosis is also ineffective in hypoxic cells due binds to a HRE site on the human BNIP3 promoter to the low rate of oxidative phosphorylation (Koukour- and this binding is required for activation of the human akis, 2001). In addition, hypoxic cells are under constant BNIP3 promoter. Furthermore, 1.2 kb human and stress and are more likely to have genomic instability. 0.5 kb rodent BNIP3 promoter are not identical except Mutations in genes such as p53 have been shown to arise for a 24 bp sequence wherein a consensus sequence for a

Oncogene BNIP3 regulates hypoxia-induced cell death S Kothari et al 4743 HRE site is located. This indicates the importance able to protect cells against the accumulation of death of HIF-1a in BNIP3 activation under hypoxia. inducing proteins such as BNIP3. We have determined In addition, transcription factor PGLAL-2 induces that growth factors EGF and IGF protect cells against BNIP3 expression under hypoxic condition, but BNIP3-induced cell death by preventing mitochondrial HIF-1a is still required for full activation of the dysfunction, plasma membrane permeability and cyto- promoter (Mizutani et al., 2002). Taken together, plasmic vacuolization. Our results further suggest that BNIP3 is an important mediator of hypoxic-induced the BH3 domain of BNIP3 is required to confer growth cell death in human epithelial-derived cells mediated by factor protection against BNIP3-induced cell death. The HIF-1a. function of the BH3 domain of BNIP3, however, HIF-1a is one of the key regulators of genes that are remains controversial. Some studies have suggested that activated under low oxygen levels (Giatromanolaki and the BH3 domain is implicated in cell death and interacts Harris, 2001). Genes upregulated by HIF-1a encode with Bcl-2 (Yasuda et al., 1998). In contrast, other study proteins involved in glucose uptake, glucose metabo- showed that deletion of the BH3 domain does not lism, angiogenesis and erytropoesis (Giatromanolaki prevent cell death and is not required to interact with and Harris, 2001). Expression of these genes contributes Bcl-2 or Bcl-xL (Ray et al., 2000). The mechanism for to the adaptation against oxygen deficiency by optimiz- negative regulation of BNIP3-induced cell death is ing oxygen delivery and energy generation. This suggests unknown and will be further investigated. that initial hypoxic insult leads to the activation of genes Growth factors have been shown to protect cells to promote cell survival. HIF-1a, however, also activates from hypoxia-induced cell death (Clawson et al., 1999; the expression of pro cell death genes, such as BNIP3, Han and Holtzman, 2000; Jin et al., 2000; Seigel et al., and Nix that accumulate and induce cell death (Bruick, 2000). We have determined that BNIP3 is involved in 2000). We have shown that hypoxia-induced BNIP3 hypoxia-induced cell death and BNIP3-induced cell expression was detectable 24 h after initial upregulation death is blocked by growth factors. In tumors, the of HIF-1a and peaked after 72 h. This suggests that existence of hypoxic regions and high expression BNIP3 expression occurs later in the hypoxia response of growth factors and/or their receptors often correlate than other genes such as angiogenesis factor VEGF. with poor prognosis (Harris, 2002). We have further VEGF expression is increased only after 12 h of hypoxia determined that inhibition of ErbB2 signaling (data not shown). Indeed, after 48 h of hypoxia, 28% of using Herceptin, sensitizes MCF-7 cells to hypoxia- HEK293 cells undergo cell death whereas only 5% cell induced apoptosis without effecting hypoxia-induced death was detected at 24 h (data not shown) correspond- BNIP3 expression. This suggests that blocking growth ing with induction of BNIP3. Thus, HIF-1a activates factor signaling in hypoxic regions of tumor might both survival and death inducing genes in which the be sufficient for BNIP3 to induce cell death in these cells. balance between these genes would likely determine the This has implications in the use of Herceptin in treating cell’s fate under hypoxia. breast cancer where it might induce hypoxic cells to While cell death is the target of cancer therapies, cell undergo cell death. This could explain the existence of survival is the focus of treatment for ischemic injury of reduced angiogenesis in breast tumors treated with the heart. Deprivation of blood supply causes hypoxia Herceptin through modulation of hypoxic-regulated in the heart termed ischemia which is a major cause of angiogenic factors (Izumi et al., 2002). It is important heart failure (Hellermann et al., 2002). BNIP3 has also to note that Herceptin sensitization of cells to hypoxia- been shown to be upregulated and induces cell death in induced cell death could still be independent of BNIP3. mouse and rat cardiomyocytes under hypoxic conditions Therefore, further studies are required to determine (Kubasiak et al., 2002; Regula et al., 2002). The use of the role BNIP3 plays following hypoxia in epithelial- antisense oligonculeotides against BNIP3 reduces ische- derived cells. mia-induced cell death in these cardiomyocytes (Kuba- siak et al., 2002). Thus, similar to epithelial-derived cells, BNIP3 could play significant role in regulating cell death Acknowledgements in heart disease. We dedicate this manuscript in the memory of Dr Arnold Epithelial-derived tumors contain regions of hypoxia. Greenberg who was a mentor and friend. Dr A Greenberg Both HIF-1a and BNIP3 have been shown to be initiated the project in collaboration with Dr Gibson. This work is supported by Canadian Institutes of Health Research upregulated in these hypoxic regions where many cells operating grant. LKirshenbaum is a Canadian Research Chair die (Giatromanolaki and Harris, 2001; Sowter et al., in Molecular Cardiology. S Kothari is supported by a 2001; Harris, 2002). Some cells, however, survive and studentship from the Manitoba Health Research Council. J become resistant to cell death (Giatromanolaki and Cizeau is a recipient of the Shaw Communications/Lymphoma Harris, 2001). This suggests that a survival mechanism is Society of Canada Post-doctoral Fellowship.

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Oncogene