|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 1 March 2004, Vol. 103, No. 5, pp. 1876-1882. Prepublished online as a Blood First Edition Paper on November 20, 2003; DOI 10.1182/blood-2003-06-1859.
NEOPLASIA Anoxic induction of ATF-4 through HIF-1–independent pathways of protein stabilization in human cancer cellsFrom Cancer Research UK, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; the Division of Genomic Medicine, Section of Oncology, Sheffield Medical School, Sheffield, United Kingdom; and the Department of Molecular and Cellular Biochemistry, Neurobiotechnology Center, Ohio State University, Columbus.
Hypoxia is a key factor in tumor development, contributing to angiogenesis and radiotherapy resistance. Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor regulating the response of cancer cells to hypoxia. However, tumors also contain areas of more severe oxygen depletion, or anoxia. Mechanisms for survival under anoxia are HIF-1  independent in Caenorhabditis elegans and, thus, differ from the hypoxic response. Here we report a differential response of cancer cells to hypoxia and anoxia by demonstrating the induction of activating transcription factor-4 (ATF-4) and growth arrest DNA damage 153 (GADD153) protein specifically in anoxia and the lack of induction in hypoxia. By applying RNAi, ATF-4 induction in anoxia was shown to be independent of HIF-1, and desferrioxamine mesylate (DFO) and cobalt chloride induced HIF-1 but not ATF-4 or GADD153. Furthermore, the inductive response of ATF-4 and GADD153 was not related to alterations in or arrest of mitochondrial respiration and was independent of von Hippel-Lindau (VHL) disease mutations. In reoxygenated anoxic cells, ATF-4 had a half-life of less than 5 minutes; adding the proteasome inhibitor to normoxic cells up-regulated ATF-4 protein. Extracts from primary human tumors demonstrated more ATF-4 expression in tumors near necrotic areas. Thus, this study demonstrates a novel HIF-1–independent anoxic mechanism that regulates ATF-4 induction at the protein stability level in tumor cells.
Cells from humans to Caenorhabditis elegans have a sensing pathway for hypoxia involving the hypoxia-inducible factor-1 (HIF-1) complex.1,2 HIF-1 is a heterodimer that includes HIF-1 and the aryl hydrocarbon receptor nuclear translocator (ARNT).3 In normoxia, prolyl hydroxylase modifies HIF-1 by hydroxylation of 2 prolyl residues,4 leading to its interaction with the von Hippel-Lindau protein (pVHL) and resulting in ubiquitination and degradation in the proteasome. In hypoxia, hydroxylation is blocked, and HIF-1 is stable.5,6 Mild levels of hypoxia (5%-0.5% O2) result in the up-regulation of HIF-1,7 which activates a gene transcription program, including the switch-on of glycolysis,8 and angiogenic factors, such as vascular endothelial growth factor (VEGF),9 that serve to protect the cell from the damaging effects of hypoxia.
HIF-1 The basic region-leucine zipper (bZip) factor ATF-4 has been shown to be up-regulated in primary rat fibroblasts under anoxia,23 but whether this is HIF-1 regulated remains unknown. ATF-4, which generally binds the cyclic adenosine monophosphate (cAMP)–responsive element (CRE),26 showed increased DNA binding activity to a variant site under anoxia.23 Binding of ATF-4 to variant sites during conditions of stress can activate the expression of cognate target genes such as growth arrest DNA damage/C-EBP homologous protein (GADD153/CHOP).27,28 Posttranslational regulation by protein degradation is one crucial mechanism of the hypoxic signaling pathway leading to HIF-1 induction. Considering posttranslational regulation by protein degradation, C/ATF, the mouse homologue of human ATF-4, has been shown to be an unstable protein with a half-life of less than 30 minutes.29 Recently, it has been shown that human ATF-4 is an unstable protein targeted to proteasomal degradation through phosphorylation-dependent interaction with the SCFtrcp ubiquitin ligase.30 To investigate the differential response of human cancer cells to hypoxia and anoxia and to determine different regulatory pathways to the hypoxia/HIF-1 system, the expression of ATF-4 and its target gene GADD153 in normoxia (21% O2), severe hypoxia (0.1% O2), and anoxia (less than 0.1% O2) was studied. This report describes the findings that ATF-4 is induced by anoxia rather than hypoxia and that this induction does not involve HIF-1 or electron transport but occurs mainly by stabilization of the protein, which has a half-life after anoxia reoxygenation of less than 5 minutes.
Cells The human breast cancer cell lines MCF-7, T47D, MDA-MB 231, 435, 468, the human melanoma cell line LB-4, and the VHL mutant renal cancer cell line 786-0 were provided by Cancer Research UK (London, United Kingdom). Murine neuroblastoma 2a cell line was a gift from Professor Ernst Wagner (Ludwig-Maximilian University, Munich, Germany). Cells were maintained in Dulbecco modified Eagle medium (DMEM, with 4.5 mg/mL glucose) supplemented with 10% (vol/vol) fetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL), and 4 mM L-glutamine (Gibco, Paisley, United Kingdom). Previously, 786-0 renal cells had been described,31 and they were supplemented with 500 µg/mL G418 for selection. Reagents and plasmids
Rabbit polyclonal antibody to ATF-4 (sc-200) and mouse monoclonal antibody to GADD153 (sc-7351) were from Santa Cruz Biotechnology (Calne, United Kingdom). The monoclonal antibody to HIF-1 Hypoxic incubation For hypoxic incubation, cells were placed in a Heto-Holten cell house 170 (Heto-Holten, Surrey, United Kingdom) incubator set at 0.1% O2. Hypoxic conditions were achieved with 95% N2/5% CO2. The oxygen level was monitored with an in-built O2 sensor. Cells were trypsinized from a 70% to an 80% dense culture, and 1.5 to 2 x 106 cells in 100 mm tissue culture Petri dishes with 6 to 7 mL culture media were incubated immediately in hypoxia or were incubated first in a normal (normoxia) 5% CO2 incubator for 20 to 24 hours and then placed for the corresponding times in hypoxia. In all cases cells were approximately 60% to 70% confluent. The same treatment was used for cells to be incubated in normoxia (21% O2). Anoxic incubation A humidified gas-sorted anoxic incubator-gloved box (InVivo2 400; Ruskin, Leeds, United Kingdom) was used for anoxic experiments. The gas was sorted using a Ruskin Microaerophilic gas sorter, resulting in 5% H2, 5% CO2, and 90% N2. The humidity level was controlled by placing 4 200 x 25-mm tissue culture Petri dishes (Becton Dickinson Labware, Oxford, United Kingdom) filled with dH2O in the gloved box and using an in-built humidity sensor to ensure the humidity level was greater than 90%. Two previously unused (ie, not reheated) palladium catalysts were used to scavenge traces of oxygen. Anoxic conditions were controlled using an anaerobic indicator (Oxoid, Basingstone, United Kingdom) that demonstrated anoxic conditions within 10 to 15 minutes of gas purging and an O2 sensor installed in the gloved-box incubator that demonstrated 0% oxygen within 15 minutes of gas purging. After the indicators showed satisfactory anoxic conditions, gas was purged in the gloved box for another 45 minutes. Cells (1.5 x 106) with 6 to 7 mL media in 100-mm Petri dishes were placed in the chamber immediately or after 24 hours of normoxic incubation, with approximately 70% confluence. RNAi treatment of cells and transfection procedures
Cells were plated onto 100-mm Petri dishes and were grown to approximately 50% confluence before transfection. HIF-1 To test the anti–ATF-4 antibody used in this study, MCF-7 cells were transfected with 1 µg pCG-ATF4 by using Fugene-6 according to the manufacturer's protocol (Roche Diagnostics, Sussex, United Kingdom). After 24 hours, cells were lysed by using a urea-denaturing buffer (described below) and analyzed for ATF-4 expression. Immunoblot analysis All cell extracts were made in a cold room (4°C). The cell lysis buffer used for preparing total cell extracts was a urea-denaturing buffer (6.7 M urea, 10 mM Tris-HCl, pH 6.8, 5 mM dithiothreitol [DTT], 1% sodium dodecyl sulfate [SDS], and 10% glycerol) supplemented with Complete mini-protease inhibitor cocktail tablets (Roche Diagnostics). Cultured cells were washed rapidly once with ice-cold phosphate-buffered saline (PBS), scraped off, and centrifuged at 13 000 rpm for 20 seconds, and 250 µL urea buffer was added directly to the cell pellet. This was then homogenized rapidly on ice for 15 seconds by using the Ultra-Turrax homogenizer at full speed (IKA, Düsseldorf, Germany). Nuclear and cytoplasmic extracts were based on the method described by Schreiber et al32 but slightly modified. Cells were washed rapidly once with ice-cold phosphate-buffered saline (PBS), scraped off, and centrifuged for 20 seconds at 13 000 rpm. Cells were resuspended in 4 packed cell volumes of ice-cold buffer A (10 mM HEPES, pH 7.5, 0.1 mM EDTA [ethylenediaminetetraacetic acid], pH 8, 0.1 mM EGTA [ethyleneglycotetraacetic acid], pH 8, 10 mM KCl) (Sigma) supplemented with Complete mini-protease inhibitor cocktail tablets (Roche Diagnostics) for cytoplasmic extracts. For nuclear extracts, the cell pellet was resuspended in buffer C (20 mM HEPES, pH 7.5, 1 mM EDTA, pH 8), 1 mM EGTA, pH 8, 0.4 M NaCl) supplemented in Complete mini-protease inhibitor cocktail tablets (Roche Diagnostics). The volume of buffer C used was the same as buffer A. To make tumor and normal tissue extracts, tumor tissue (or paired tumor/normal tissue) from patients with invasive breast cancer was frozen in liquid nitrogen immediately after surgery and stored at –80°C until extraction. Then 100 to 1000 mg frozen tissue was pulverized on dry ice to a fine powder, and ice-cold extraction buffer (20 mM HEPES, pH 7.4, 1.5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 0.5 mM benzamidine, and 10 µg/mL ovomucoid trypsin inhibitor) was added at a 0.2 g tissue/2 mL buffer ratio. The resultant suspension was homogenized on ice with an Ultra-Turrax. The mixture was then centrifuged at 13 000 rpm at 4°C for 30 seconds, and the supernatant (cytosol) was collected and centrifuged at 13 000 rpm at 4°C for 30 minutes. The pellet was then resuspended in a urea-denaturing buffer supplemented with Complete mini-protease inhibitor cocktail tablets (Roche Diagnostics) and was homogenized on ice with an Ultra-Turrax. This was then centrifuged at 4°C for 5 minutes at 13 000 rpm, and the supernatant was analyzed for ATF-4. The detergent-compatible (DC) protein assay (Bio-Rad, Hertfordshire, United Kingdom) was used to estimate the protein concentration of extracts according to the manufacturer's protocol. Nuclear/cytoplasmic (25 µg/lane) or total cell extracts (50-60 µg/lane) were subjected to reducing (10%-12%) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS). Resolved proteins were then electroblotted (semidry) onto a Millipore Immobilon-P transfer membrane (PVDF microporous membrane). Bound antibodies were detected using the chemiluminescent substrate ECL+Plus (Amersham Biosciences, Buckinghamshire, United Kingdom). RNase protection assay RNA was extracted from the cells using the tri-reagent method according to the manufacturer's protocol (Sigma). The human ATF-4 gene (316 bp) was subcloned from the pCG-ATF-4 plasmid into the KpnI and EcoRV restriction sites of the bluescript II KS (Stratagene). This ATF-4 fragment was a portion of the human ATF-4 coding region from positions 565 to 881, which served as the DNA template for generating 32P-labeled RNA probes. To generate 32P-labeled RNA probes, the bluescript II KS containing the ATF-4 fragment was linearized by using the EcoRV restriction enzyme (New England Biolabs, Hertfordshire, United Kingdom). Constitutively expressed U6 small nuclear RNA served as an internal control. RPA was carried out as described previously.33 ATF-4 immunostaining of human tumors Five-micrometer sections were cut from 10 ductal invasive carcinomas of human breast, obtained from the Cancer Research UK, Oxford Cancer Center. After dewaxing and rehydration, endogenous peroxidase was blocked using 3% H2O2 in methanol for 20 minutes, followed by antigen retrieval (immersion in 0.01 M trisodium citrate and microwaving on high power for 10 minutes [800-W microwave]). After washing in PBS and incubation with 5% normal horse serum (Vector Laboratories, Peterborough, United Kingdom) for 20 minutes, sections were incubated for 2 hours at room temperature in a negative control antibody (purified rabbit immunoglobulin fraction) at a concentration of 2 µg/mL (DAKO) or in a polyclonal antihuman ATF-4 antibody (Santa Cruz) at the same concentration (2 µg/mL). The reaction was then detected using a standard 3-stage immunoperoxidase technique (Vector Laboratories Elite ABC kit) with the brown chromogen, diamino benzidine (DAB). Sections were counter-stained in hematoxylin (British Drug Houses [BDH]), dehydrated, cleared in xylene, and mounted in DPX (BDH).
Induction of ATF-4 by anoxia Transfection of MCF-7 cells with the pCG-ATF-4 expression plasmid resulted in a markedly increased expression of a 50-kDa protein (Figure 1A). The same 50-kDa ATF-4 protein was up-regulated in other cancer cells incubated for 16 hours in anoxia (Figure 1A-B). Anoxically induced ATF-4 was nuclear, and no differences in ATF-4 levels between normoxic and anoxic cytoplasmic extracts were detected (Figure 1C). The high expression detected in normoxia in the transfectant likely represents effective transcription and translation from the promoter and the saturation of proteasomal degradation.
ATF-4 mRNA level in normoxia and anoxia RPA demonstrated that ATF-4 mRNA was present at similar levels in normoxia and anoxia in MDA-MB 435 cells (Figure 2A). In a synchronous control, immunoblot analysis demonstrated that ATF-4 protein was induced in anoxia (Figure 2B), suggesting that ATF-4 induction in anoxia is not at the level of transcription or RNA stability.
Effect of reoxygenation and proteasome inhibition on ATF-4 protein level
After 16 hours of exposure to anoxia, MDA-MB435 and LB-4 cells were placed for 40 minutes in an incubator with normal 5% CO2 (normoxia, 21% O2). This reoxygenation resulted in a decline in ATF-4 protein levels to those similar for normoxic cells (Figure 3A), suggesting that anoxic ATF-4 protein is unstable on reoxygenation after anoxia. This was further confirmed by reoxygenating the cells through replacing the anoxic media with normoxic media and placing the cells in a normoxic incubator for 5 minutes. Within 5 minutes, ATF-4 and HIF-1
Induction of ATF-4 by anoxia and relation to GADD153/CHOP10
ATF-4 expression in hypoxia and anoxia and correlation with GADD153 and HIF-1
Time course of anoxic induction of ATF-4. Figure 4B shows that HIF-1 ATF-4 induction in primary tumor extracts, paired primary tumor and normal tissue extracts, and expression near necrotic areas. Extracts made from patient primary tumors demonstrated ATF-4 expression similarly to the observations made with the in vitro cell culture extracts (Figure 5A). Variations were observed among patient extracts. Some demonstrated greater levels of the 50-kDa ATF-4 protein than others, suggesting that anoxic stress may be more pronounced in some tumors than in others. Interestingly, GADD153 expression was also observed in the tumors, with the greatest levels evident in patients 1 and 4, who also showed the greatest level of ATF-4 expression (Figure 5A). In addition, paired tumor (T)/normal (n) tissue extracts demonstrated ATF-4 expression to be greater in tumors than in normal tissues (Figure 5B). ATF-4 expression was analyzed further through immunostaining of 10 ductal invasive carcinomas of the breast. Enhanced ATF-4 expression (brown) was shown by tumor cells around areas of necrosis (Nec) known to be anoxic (Figure 5C), though it should be noted that ATF-4 staining was not restricted to these areas alone because it was also seen in other tumor areas in some tumors. No such staining was seen when the primary (ATF-4) antibody was replaced with the same concentration of a purified immunoglobulin fraction from a nonspecific rabbit serum (not shown). Nuclei were highlighted in sections using hematoxylin (blue).
Induction of ATF-4 does not involve HIF-1
HIFs are another group of oxygen-sensitive transcription factors, regulated by proteolysis through the VHL protein. To investigate the relation of the ATF-4 anoxic response to the HIF proteolytic pathway, renal cell lines with mutant VHL were used. The 50-kDa ATF-4 protein was not induced constitutively in parental 786-0 cells (ie, VA, lacking pVHL) or 786-0 cells with pVHL expression vector, but it was up-regulated in both cells under anoxia only (Figure 6A). These cell lines express HIF-2
Treating the cells with RNAi blocked HIF-1
Desferrioxamine or cobalt chloride (200 µM) did not up-regulate ATF-4 or GADD153 but did induce HIF-1
Tunicamycin (1 mg/mL) induced GADD153 and the 50-kDa ATF-4 protein but failed to induce HIF-1
Finally, glucose deprivation using glucose-free DMEM for 16 hours, which deprives cells of glycolytic and oxidative phosphorylation substrates, did not induce HIF1-
The data presented in this paper suggest that cells can sense and discriminate between severe hypoxia (0.1% O2) and anoxia in an HIF-1–independent manner. The hypoxia mimetics DFO and CoCl2 failed to induce ATF-4. The observations that hypoxia did not induce ATF-4 but did induce HIF-1 and that blocking HIF-1 induction in anoxia did not prevent the anoxic ATF-4 response suggest that cells can respond to anoxia independently of the hypoxic HIF-1 signaling pathway. Our data indicate the anoxic ATF-4 protein to be unstable in normoxia and the protein degradation mechanism to be a major pathway controllingATF-4 induction in cancer cell lines under anoxia.ATF-4 has been shown to contain a nuclear targeting signal in the C-terminus,34 which may account for the nuclear translocation of increased levels of ATF-4 protein during anoxic stress. ATF-4 mRNA was expressed at similar levels under normoxia and anoxia in MDA-MB 435 cells. This finding is concordant with the observation that ATF-4 mRNA is abundant in human tumor cell lines in normoxia.35 It also suggests primarily that ATF-4 up-regulation is not caused by an increase in mRNA transcription but rather by posttranscriptional events. However, this may be cell type dependent because previously it had been shown that anoxia results in increased levels of ATF-4 mRNA in primary rat fibroblasts.23
One pathway that may contribute to ATF4 regulation is that induced by endoplasmic reticulum (ER) stress, which can be initiated by several mechanisms, including unfolded proteins, tunicamycin, and calcium ionophores. Whether ER stress through tunicamycin mimics anoxic signaling pathways that up-regulate ATF-4 is uncertain, but several proteins induced by ER stress, such as ORP 150 or ATF-6, are also induced in anoxic conditions.36,37 Protein disulfide isomerase (PDI), which is necessary for disulfide bond formation and, hence, protein folding, is also induced maximally in anoxia.38 In addition, the endoplasmic reticulum resident kinase PERK has been shown recently to become hyper-phosphorylated under hypoxia and anoxia, independent of HIF-1
Our observations are similar in some respects to those of Dong et al,22 who showed recently the up-regulation of another factor, the antiapoptotic protein IAP-2, in anoxia through an HIF-1–independent pathway. Milder hypoxic conditions (2% O2) than those used in our experiments (0.1% O2) were shown to induce HIF-1 We demonstrated ATF-4 expression to be greater in tumors than in normal tissue, but the function of ATF-4 in human tumors remains unknown. It has been shown in ATF-4 knock-out transgenic mice that ATF-4 is critical for processes that require high-level proliferation, such as during fetal-liver hematopoiesis.42 Other knock-out transgenics have suggested ATF-4 to be critical for preventing p53-dependent apoptosis in anterior lens epithelial cells.43,44 Recently, transgenic mice that specifically overexpressed ATF-4 in mammary epithelial cells demonstrated ATF-4 to function as an antiproliferation and a proapoptotic factor during mammary gland development,45 roles that may be relevant to cancer when expressed in anoxic areas. ATF-4 was also shown to block differentiation, another pathway important in cancer development. Thus, the role of ATF-4 in cancer development and progression must be investigated, and the use of ATF-4 as a potential marker to predict the severity of hypoxia in perinecrotic tumor areas must be considered.
ATF-4 is a substrate for
Submitted June 16, 2003; accepted October 24, 2003.
Prepublished online as Blood First Edition Paper, November 20, 2003; DOI 10.1182/blood-2003-06-1859.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Adrian L. Harris, Cancer Research UK, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, United Kingdom; e-mail: aharris.lab{at}cancer.org.uk.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
-tubulin (as an internal control). (B) MDA-MB435 cells were incubated under anoxia for various times, as indicated, and were analyzed by immunoblot for the indicated proteins. Cells from normoxic conditions were included as a control. N indicates normoxia; H, hypoxia; A, anoxia.
B