Gene expression profiles of proliferating vs. G1/G0 arrested human leukemia cells suggest a mechanism for glucocorticoid-induced apoptosis MARTIN TONKO,*,† MICHAEL J. AUSSERLECHNER,* DAVID BERNHARD,* ARNO HELMBERG,* AND REINHARD KOFLER*,†,1 *Institute of General and Experimental Pathology, Division of Molecular Pathophysiology, University of Innsbruck, A-6020 Innsbruck, Austria; and †Tyrolean Cancer Research Institute, Innsbruck, Innrain 66, A-6020 Innsbruck, Austria Glucocorticoids (GC) have pronounced effects on metabolism, differentiation, proliferation, and cell survival (1). In certain lymphocytes and lymphocyterelated malignancies, GC inhibit proliferation and induce apoptotic cell death, which has led to their extensive use in the therapy of malignant lymphoproliferative disorders (2). Most of these effects result from regulation of gene expression via the GC receptor (GR), a ligand-activated transcription factor (3). Although hundreds of genes are regulated by GC (1), how certain biological GC effects relate to individual gene regulation remains enigmatic. To address this question with respect to GC-induced cell cycle arrest and apoptosis, we applied DNA chip technology (4, 5) to determine gene expression profiles in proliferating and G1/G0-arrested (by conditional expression of the CDK inhibitor p16/INK4a) acute lymphoblastic T cells undergoing GC-induced apoptosis. Of 7074 genes tested, 163 were found to be regulated by dexamethasone in the first 8 h in proliferating cells and 66 genes in G1/G0-arrested cells. An almost nonoverlapping set of genes (i.e., only eight genes) was coordinately regulated in proliferating and arrested cells. Analysis of the regulated genes supports the concept that GC-induced apoptosis results from positive GR autoregulation entailing persistent down-regulation of metabolic pathways critical for survival.—Tonko, M., Ausserlechner, M. J., Bernhard, D., Helmberg, A., Kofler, R. Gene expression profiles of proliferating vs. G1/G0 arrested human leukemia cells suggest a mechanism for glucocorticoid-induced apoptosis. FASEB J. 15, 693– 699 (2001)
ABSTRACT
Key Words: glucocorticoid-induced gene regulation 䡠 DNA chip expression profiling 䡠 acute lymphoid leukemia 䡠 pathophysiology
To identify glucocorticoid (GC) -regulated genes involved in GC-induced inhibition of cell cycle progression and apoptosis, we used the CCRF-CEM acute lymphoblastic T cell leukemia model (6). GCtreated CCRF-CEM cells undergo cell cycle arrest after ⬃24 –36 h, followed by apoptosis starting at ⬃36 h and being complete at ⬃72 h (7, 8). When such cells are arrested in G1/G0 by conditional expression of the 0892-6638/01/0015-0693 © FASEB
cyclin-dependent kinase (CDK) inhibitor p16/INK4a, they are still sensitive (in fact, considerably more sensitive [9]) to GC. We reasoned that the expression profile alteration induced by GC in arrested cells might differ from that in proliferating cells. Since both populations undergo apoptosis, we assumed that commonly regulated genes more likely relate to cell death, thereby narrowing down the number of candidate genes for functional analysis. Following this rationale, we prepared mRNA from proliferating and G1/G0arrested CCRF-CEM derivatives that were either untreated or exposed to 10⫺7 M dexamethasone for 2 or 8 h. The effect of GC on cell cycle progression and survival followed the above-mentioned and previously published (7, 8) kinetics (data not shown). The mRNAs were reverse-transcribed into red and green fluorescent cDNA probes and hybridized pairwise to DNA chips containing 7074 genes (Incyte Genomics, Inc., St. Louis, Mo.). Untreated G1/G0-arrested cells (CEMC7H2– 6E2-p16) [9] were compared with either 2 or 8 h treated G1/G0-arrested cells, and untreated proliferating cells (CEM-C7H2) with their counterparts exposed to dexamethasone for 2 or 8 h.
MATERIALS AND METHODS Cell culture and reagents CCRF-CEM-C7H2 (8) and CCRF-CEM-C7H2– 6E2-p16 [9] cells were maintained in RPMI 1640 containing 10% fetal calf serum (Gibco BRL, Paisley, U.K.), 100 U/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine (Gibco BRL) at 5% CO2 at 37°C in saturated humidity. All reagents (dexamethasone, doxycycline) were from Sigma (Vienna, Austria). Proliferating CEM-C7H2 cells were treated with 10⫺7 M dexamethasone and probes were removed for mRNA preparation after 0, 2, and 8 h treatments. From the same cells, probes were taken at 0, 24, 48, and 72 h for FACS analyses to check cell cycle and extent of apoptosis in the respective cells. The same procedure was used for CEM-C7H2– 6E2-p16 cells, 1 Correspondence: Tyrolean Cancer Research Institute, Innrain 66, 6020 Innsbruck, Austria. E-mail: Reinhard.Kofler@ uibk.ac.at
693
TABLE 1. Alphabetical list of GC-regulated genes found by DNA chip analysisa CEM-C7H2 Gene Name
2-amino-3-ketobutyrate-CoA ligase 3-phosphoglycerate dehydrogenase A kinase anchor protein, 149 kD A kinase anchor protein (AKAP100) acid phosphatase 2, lysosomal acyl-CoA thioester hydrolase ADE2 AIM1/absent in melanoma 1 aldolase A Apolipoprotein E Arylsulfatase C, isozyme S ATP citrate lyase ATP synthase Bactericidal/permeability-increasing protein bcl-2 binding component 3 (bbc3) bradykinin receptor B2 brain secretory protein HSEC10 brain-expressed HHCPA78 homolog bystin-like c-myc c-myc binding protein (MBP-1) C8FW phosphoprotein cadherin 15 casein kinase 2, alpha 1 polypeptide caspase 4 CD1B, b polypeptide CD53 CD58 CD74 Chromosome 18 open reading frame 1 collagen, type XVI, alpha 1 cullin 1 cyclin D3 cyclin G2 cyclin-dependent kinase 9 deafness, autosomal dominant 5 deleted in malignant brain tumors 1 Dipeptidylpeptidase IV (CD26) DNA (cytosine-5-)-methyltransferase 2 down syndrome candidate region 1 Dp-2 (E2F dimerization partner 2) Dyskeratosis congenita 1, dyskerin eph, receptor tyrosine kinase ephB6 epithelial membrane protein 3 eukaryotic translation initiation factor 4A EVI2A Extracellular matrix protein (MFAP3) Faciogenital dysplasia (FGD1) FAK/focal adhesion kinase fatty acid binding protein 5 flotillin-1 fructose-bisphosphatase 1 Fucosyltransferase 3 Galactosidase, alpha galectin 9 glucan (1,4-alpha-), branching enzyme 1 Glucocorticoid receptor (GR) Glutaredoxin (thioltransferase) Glycophorin A Glycoprotein receptor gp330 precursor gpp130 Grancalcin
CEM-6E2 (⫹p16/G1)
GenBank
0/2
0/8
0/2
0/8
AI028459 AF006043 X97335 AB002309 X15525 U91316 X53793 U83115 M11560 M12529 M16505 X64330 D14710 J04739 U82987 AA195072 U85946 S73591 L36720 K02276 M55914 AJ000480 D83542 M55265 U28014 M28826 M37033 Y00636 M13560 AF009424 M92642 AF062536 M92287 U47414 AI004271 AF007790 AJ000342 X60708 AJ223333 U85267 U18422 U59151 M18391 D83492 U87947 D30655 M55267 L35251 U11690 L13616 M94856 AI041629 U21931 U27333 U78027 AB006782 L07956 M10901 X76648 L31860 U33837 U55853 M81637
b.t. ⫺1.3 2 2.1 1.1 ⫺1.5 ⫺1.1 1.2 ⫺1.5 1.4 ⫺1.2 ⫺1 ⫺1.1 b.t. ⴚ2 b.t. ⫺1.1 ⫺1 ⫺1.1 1.3 ⫺1.4 1.4 ⫺1 ⫺1.5 1.5 b.t. b.t. b.t. 1.3 b.t. 1.6 b.t. ⫺1.3 1 b.t. b.t. 1.2 ⫺1 1.3 1.2 1.1 1.3 b.t. ⫺1.5 1.1 1.1 1.4 b.t. 1.1 b.t. 1.4 1.2 1.5 b.t. 1.2 ⫺1.1 ⫺2.3 1.8 1.5 ⫺1 2 b.t. ⫺1.8
2.2 ⴚ2 1.3 b.t. 2 ⫺1.4 ⫺1.9 1.9 ⴚ3.1 2.1 ⴚ2.2 ⴚ2 ⴚ2 2.1 ⫺1.4 b.t. ⫺1.1 ⫺1.1 ⫺1 ⴚ2 ⫺1.9 2.1 2.2 ⴚ2.1 2.4 b.t. b.t. 2.3 2.1 1.4 2.3 2.1 ⫺1.3 2.1 b.t. b.t. 2 2 2.1 1.3 ⴚ2.2 ⫺1.3 2.2 ⴚ2.6 2.1 ⫺1 2 2 2 2.1 ⫺1 ⫺1.2 2 2.1 2.2 ⴚ2.1 ⴚ3.9 2.4 2.9 2 b.t. 2.1 ⫺1.6
1.1 1.2 1.2 1.2 1.3 2.1 ⫺1.6 2.3 1.3 1 ⫺1.1 1.2 ⫺1 ⫺1.1 1.1 1.2 ⴚ2 1.8 ⫺1.4 ⫺1 ⫺1.2 1.6 ⫺1.3 ⫺1.1 1.1 ⫺1.4 1.2 1.1 1.3 1.3 1.3 1.1 ⫺1.4 ⫺1 ⴚ2.3 1.6 1.8 1.6 1.2 2.4 1 1.1 1 ⫺1.4 1.5 ⫺1.4 1.1 1.4 1.1 1.5 ⫺1.1 ⴚ2 1.2 1.1 1.1 ⫺1.1 ⫺1.2 3 1.1 1.5 1.4 1.3 2
1.1 ⫺1.1 ⫺1.2 ⫺1.1 1.2 1.5 ⴚ2.7 10.4 ⫺1.4 1.1 ⴚ2.8 ⫺1.1 ⫺1.5 ⫺1.1 ⫺1.2 2.2 ⫺1.1 2.6 ⴚ2.5 ⫺1.9 ⴚ2.3 1.6 ⫺1.5 ⫺1.9 1.1 ⴚ2 2 ⫺1.1 1 2 1.3 ⫺1 ⴚ2.2 1.1 ⫺1.1 2.2 1.5 1.6 1 3.5 ⫺1.6 ⴚ2.3 1.2 ⫺1.8 1.1 ⴚ2.2 1.3 1.2 1.2 1.3 ⴚ2 ⫺1.6 1.1 ⫺1 ⫺1 ⫺1.3 ⫺1.9 3.1 1.5 1.3 1.2 1.1 1.3
Continued on next page 694
Vol. 15
March 2001
The FASEB Journal
TONKO ET AL.
TABLE 1.(continued) CEM-C7H2 Gene Name
heat shock 60 kDa protein 1 heat shock 90 kDa protein 1, alpha heparan sulfate 3-O-sulfotransferase-1 Hexokinase 1 HRY/homolog of Drosophila hairy gene IB alpha inducible protein integrin alpha6 integrin, alpha 4 (CD49D) interferon stim. T cell alpha chemoattractant interferon gamma-inducible protein 16 Interferon-inducible protein 1-8U interleukin 7 receptor interleukin 8 receptor, beta isoleucine-tRNA synthetase kinesin 1 (110–120 kDa) lactate dehydrogenase A-LDH lamin B receptor leucine zipper protein/LZ leukemia associated gene 2 leukemia inhibitory factor receptor malic enzyme 2 Tau mox1 msh (Drosophila) homeo box homolog 1 myocilin (TIGR) natural killer cell transcript 4 NFAT4c ninjurin 1 nitric oxide synthase 3 (endothelial cell) NK-tumor recognition molecule-related prot. nonmetastatic cells 1 (NM23A) Not Present in UniGene orphan nuclear hormone receptor Parathymosin Parathyroid hormone-like hormone PEPT 2 periplakin Peroxisomal farnesylated protein Phosphoglycerate kinase 1 Phosphorylase kinase, beta Plasminogen activator inhibitor, type II PP2A Proteasome subunit, beta type, 9 PTEN putative oncogene protein rag1 RaIB ran binding protein 2-like 1 regulatory factor X, 5 renal cell carcinoma antigen RAGE-1 ribosomal protein L37 ribosomal protein L7a RNA binding protein Etr-3 RNA polymerase I 40 kDa subunit RNA polymerase III subunit (RPC39) S-adenosylmethionine synthetase sal (Drosophila)-like 2 sap-1 (SRF accessory protein 1) Semaphorin F SGN3/COP9
CEM-6E2 (⫹p16/G1)
GenBank
0/2
0/8
0/2
0/8
M22382 M16660 AF019386 AF016365 L19314 M69043 L47738 X53586 X16983
⫺1.2 ⫺1.3 b.t. ⫺1.5 1.4 1.3 ⫺1.3 1 ⫺1
⫺1.6 ⫺1.8 2 ⴚ2 b.t. 1.9 ⴚ2.1 2.9 1
⫺1.1 ⫺1 1.2 1 ⫺1.1 2.3 1.3 4.3 ⫺1.4
ⴚ2.3 ⴚ2.3 ⫺1.1 ⫺1.1 ⴚ2.1 4 ⫺1.3 7 ⴚ2.5
AF002985 M63838 J04164 M29696 L19593 U04953 AA812064 X02152 AA725870 Z50781 Y15228 X61615 M55905 AF047863 U10492 M97676 Z97171 M59807 L41067 U91512 M93718
1.4 ⫺1.3 ⫺1.2 b.t. b.t. ⫺1.1 b.t. ⫺1.4 b.t. 3.9 1.3 1.5 1.2 1.4 1.4 1.1 1.1 ⫺1.1 ⫺1.1 1.2 1.1
2 ⫺1.3 ⫺1.4 b.t. b.t. ⴚ2 2.1 ⴚ2.5 2.5 5.6 2 2 2 2.1 2 2 2.2 ⫺1.6 ⫺1.7 2.1 2
1.4 ⫺1.5 ⫺1.4 2.2 ⴚ2 1.1 1.4 ⫺1.8 1.1 5.3 1.1 1.4 1.2 1 ⫺1.2 1.2 1.5 ⫺1.6 ⫺1.6 1.3 1.3
1.6 ⴚ2.3 ⴚ2.2 7.1 1.1 ⫺1.4 1.2 ⴚ2.5 1 8 ⫺1 1.4 ⫺1.2 1.1 1.3 1 1.3 ⴚ2.9 ⴚ2.7 1.2 ⫺1.2
U82939 X17620 AI027597 X56199 M24398 J03580 S78203 AF001691 AA039519 V00572 X84908 Y00630 U37352 Z14977 U92436 AF026816 M29474 M35416 AF012086 X85786 U46194 D23661 M36072 U69546 AF008442 U93869 X68836 X98834 M85164 U52840 AF031647
1.1 ⫺1 b.t. 1 ⫺1.1 1.2 1.2 b.t. b.t. ⫺1.3 1.1 b.t. 2 ⫺1 ⫺1.2 ⫺1.1 1.9 b.t. 1.1 ⫺1.2 ⫺1.2 1 ⫺1.4 1.4 1.4 b.t. 1.4 b.t. 1.1 ⫺1.1 1.4
2.2 ⫺1.1 2.1 ⫺1.1 ⫺1.8 2.2 2.5 2.1 2 ⴚ2 2.1 b.t. 1.2 1.1 1.4 b.t. 1.8 2 2 1.3 1.4 ⫺1.2 ⴚ2 1.4 2 2 b.t. b.t. 2 2 ⫺1.2
1.2 ⫺1.3 ⫺1.9 ⴚ2.1 ⫺1.2 ⫺1.1 1.5 ⫺1 ⫺1.1 1.1 ⫺1 ⴚ2 1.2 ⫺1.1 1 ⫺1.8 ⫺1.2 1.3 ⫺1 ⫺1.9 ⫺1.4 ⫺1.1 1.2 1.5 1.5 ⫺1.2 ⫺1.7 2 1.1 1.1 ⫺1.2
⫺1.1 ⴚ2.3 ⫺1.4 ⫺1.3 ⴚ2 ⫺1.4 1.2 1.1 ⫺1.2 ⫺1.6 1.1 ⴚ2.5 1.1 ⴚ2 2.1 ⴚ2.3 ⴚ2.4 1.1 ⫺1.1 ⴚ2.2 ⴚ2 ⴚ2 ⫺1.4 2 1.1 ⫺1 ⴚ2 1.7 1.1 1.5 ⴚ2.1
Continued on next page DNA-CHIP ANALYSIS DURING GC-INDUCED APOPTOSIS
695
TABLE 1.(continued) CEM-C7H2 Gene Name
SOCS1/JAK binding protein Squalene synthase T cell receptor, alpha (V, D, J, C) T cell receptor, delta (V, D, J, C) T complex protein 1, ␣ subunit TBP-associated factor 172 TIM17 preprotein translocase Triosephosphate isomerase 1 tuberous sclerosis 1 tumor necrosis factor 8 U5 snRNP-specific protein, 116 kD ubiquinol-cytochr. c reductase core protein II ubiquitin carrier protein (E2-EPF) Utrophin vav 2 oncogene vesicle docking protein p115 YAF2/YY1-associated factor 2 zinc finger protein 84 (HPF2) zinc finger protein PLZF zinc finger protein, X-linked
CEM-6E2 (⫹p16/G1)
GenBank
0/2
0/8
0/2
0/8
AB000734 X69141 X64643 M21624 X52882 AF038362 X97544 U47924 AF013168 L09753 D21163 J04973 M91670 X69086 S76992 D86326 U72209 M27878 Z19002 X59739
b.t. ⫺1.6 1.4 1.2 ⫺1.1 1.2 1.5 ⫺1.2 ⫺1 b.t. ⫺1.1 ⫺1.1 b.t. ⫺1.1 1.6 b.t. b.t. 1.2 b.t. b.t.
2.3 ⴚ3.7 2.2 2.1 ⴚ2.2 ⫺1.2 1 ⫺1.6 2.1 b.t. ⴚ2 ⫺1.7 b.t. 2.2 2.3 2.3 2.1 2 2.2 2
2 1.1 1.3 1.2 1.1 ⴚ2.2 ⫺1.4 ⫺1.4 1.1 1.3 1.3 ⫺1.4 ⫺1.5 1.5 1.4 1 2.1 ⫺1 1.1 1
2.7 ⫺1.5 1.4 1 ⫺1.3 ⫺1.4 ⴚ2 ⴚ2.3 1.2 2.2 1.1 ⴚ2.2 ⴚ2 1.5 1.2 ⫺1 2.4 ⫺1.1 1 ⫺1.1
a Alphabetic list of genes (except ESTs) regulated after 2 and 8 h GC treatment along with their GenBank accession number (www.ncbi.nlm.gov). CEM-C7H2 reflects proliferating cells, CEM-6E2(⫹p16/G1) reflects G1-arrested cells. Regulation is expressed as ratio between the values for 0 and 2 h (0/2 h) or 0 and 8 h (0/8 h). Negative ratios indicate downregulation. Values above 2.0 were considered significant (boldface). b.t., below threshold. A complete table of all regulated genes including ESTs, is available on www.tilak.or.at/tkfi.
except that these cells were arrested in G1/G0 by 24 h doxycycline treatment (200 ng/ml) before adding dexamethasone. In the concentration used, doxycycline has no detectable effect on cell cycle progression or apoptosis (9, 10, 11). G1/G0 arrest was checked by FACS analysis. Apoptosis and cell cycle analyses Nuclear staining with propidium iodide in concert with forward/sideward scatter analysis was used for detection and analysis of cell cycle phase and apoptosis (12, 13). Briefly, cells were centrifuged and pellets were resuspended in 0.75 ml hypotonic propidium iodide solution. The tubes were kept at 4°C in the dark overnight. Nuclear fluorescence and forward/sideward scatter were analyzed with a Becton Dickinson FACScan.
Chip analysis Total RNA was extracted with TriReagent™ (LPS, Moonachie, N.J.). From this, mRNA was extracted using Quiagen Oligotex™ columns (Valencia, Calif.). 600 ng mRNA (50 ng/l) was sent on dry ice to Incyte Genomics, who performed mRNA labeling, hybridizations and primary data preparation. Analyses of the resulting image and data files were performed in our lab using conventional data analysis programs. Northern blotting Total RNA was extracted with TriReagent™ (LPS) from 5 ⫻ 106 cells. Eight g of RNA were separated by electrophoresis on a denaturing 1% agarose gel containing formaldehyde in 4-morpholinopropanesulfonic acid buffer, and blotted over-
TABLE 2. Genes coregulated by GC in proliferating and in arrested cellsa CEM-C7H2 GeneName
Leucine zipper (20) integrin alpha6 (24) GR ESTs SOCS1/JAKbp (25) YAF2 (26) LDH A Arylsulfatase C (27) a
696
CEM-6E2 (⫹p16/G1)
GenBank
0h
8h
Factor regul. 0/8
Z50781 X53586 M10901 AA682502 AB000734 U72209 X02152 M16505
198 360 403 141 155 135 10261 778
1103 1059 951 326 354 286 4062 356
5.6 2.9 2.4 2.3 2.3 2.1 ⴚ2.5 ⴚ2.2
0h
8h
Factor regul. 0/8
1452 1383 2452 732 535 667 11206 3414
11652 9722 7592 1729 1467 1593 4400 1211
8.0 7.0 3.1 2.4 2.7 2.4 ⴚ2.5 ⴚ2.8
In the column headed 0 h and 8 h, the absolute expression values are depicted. For further explanation, see legend to Table 1.
Vol. 15
March 2001
The FASEB Journal
TONKO ET AL.
night onto Zetabind™ nylon membranes (Cuno, Meridien, Colo.) according to standard protocols. RNA was cross-linked to membranes by UV. Filters were prehybridized in phosphateblocking buffer containing sodium dodecyl sulfate and bovine serum albumin at 65°C for 3 h, and hybridized for another 12 h to the respective heat-denatured probes. The probes were labeled with ␣-32P dATP using a Promega (Madison, Wis.) random priming DNA labeling kit. Northern-probes were derived from clones purchased by Incyte Genomics.
RESULTS The complete expression profiles can be accessed through the internet (www. tilak.or.at/tkfi). In Table 1, we provide a summary of all genes whose regulation was considered significant (ⱖ twofold, as recommended by Incyte Genomics). Using this criterion, 163 genes were regulated by dexamethasone in proliferating cells and 66 genes in G1/G0-arrested cells. However, only eight genes were coordinately regulated both in proliferating and arrested cells (Table 2). To test the reliability of the above data, we confirmed the regulation of some individual genes by Northern blot experiments. As shown in Fig. 1, all such tests (i.e., LDH, leucine zipper, EphB6, glucan-1,4␣-branching enzyme, squalene synthase) confirmed the results of the DNA chip analysis-derived data. To test the data validity as a whole, we compared our data with reports on GC-regulated genes in the literature. Whereas our previous analysis recorded 225 GC-regulated genes in 1996 (1), our recent PubMed search resulted in 363 GC-regulated genes, 220 of which could be unambiguously assigned to genes present on the chip (see www.tilak.or.at/tkfi). 12% (26 genes) were clearly regulated by chip criteria (ⱖ twofold); an additional 29% (64 genes) showed evidence for regulation (1.6- to 1.9-fold regulation). Since the data on GC-regulated genes in the literature derived from multiple tissues, species, and conditions, the observed congruence is remarkable and, although not allowing conclusions for any individual regulation, strongly supports the validity of the data set as a whole.
DISCUSSION
Figure 1. Chip images and corresponding Northern blots of GC-regulated genes. Shown are the leucine zipper (LZ, top panel) (20), LDH-A (second panel) (17), EphB6 (third panel) (21), glucan-1,4␣-branching enzyme (Gluc 1,4␣ BE, fourth panel), and squalene synthase (SS, bottom panel) as chip spots and respective Northern bands (untreated: 0 and 8 h GC treated; see also Table 1). C7H2 reflects proliferating cells; 6E2(⫹p16/G1) reflects cells arrested in G1 by conditional expression of p16/INK4a. As loading control, ethidium bromide (EtBr) stained 28S rRNA bands are shown.
When relating the above expression profile changes to GC-induced cell cycle arrest and apoptosis, we were struck by the observation that the GC receptor (GR) was among the very few genes regulated in both proliferating and G1/G0-arrested cells. Its marked up-regulation was particularly stunning, because GC downregulate their receptors in most tissues investigated (14). Supporting our data, GC up-regulation was observed and implicated in GC-induced cell death many years ago (15), and its significance for GC-induced leukemia apoptosis was suggested recently in elegant experiments (16). Assuming that GR up-regulation (or lack of down-regulation) is a critical prerequisite for cell death, we propose that the subsequent continuous
repression of various metabolic pathways (Fig. 2) might have a role in cell cycle arrest and ultimately lead to cell death. Particularly critical in this regard might be the down-regulation of the lactate dehydrogenase (LDH) gene seen in proliferating and G1/G0-arrested cells. This enzyme controls glycolysis in cancer cells (17, 18), a pathway preferentially used by (and perhaps critical for) malignant cells to generate ATP, as originally reported in 1926 by Otto Warburg (‘Warburg effect’) (19). Given that our analysis covered only ⬃10% of the genes in the human genome, other pathways controlled by genes not on the chip may also be affected. Obviously, this concept needs to be tested in additional cell lines and, most important, in cells from afflicted
DNA-CHIP ANALYSIS DURING GC-INDUCED APOPTOSIS
697
Figure 2. Overview of metabolic pathways affected by GC treatment. GC regulate mRNAs for enzymes important in glycolysis: hexokinase 1 [1] (AF016365); glucan-1,4␣-branching enzyme [2] (L07956); fructose-1,6-bisphosphatase [3] (U21931); aldolase A [4] (M11560); triosephosphate isomerase [5] (U47924); phospho-glycerate kinase 1 [6] (V00572); lactate dehydrogenase A [8] (LDH, X02152); amino acid biosynthesis: 3-P-glycerate-dehydrogenase [7] (AF006043); malic enzyme 2 [9] (M55905); cholesterol: ATP citrate lyase [10] (X64330); Hydroxymethylglutaryl (HMG) CoA-reductase (11) (M11058); squalene synthase [12] (X69141); steroid biosynthesis: arylsulfatase [13] (M16505); and Inositol monophosphate (IMP) production: ADE2 [14] (X53793). mRNAs for all mentioned enzymes are down-regulated by GC except for fructose-1,6-bisphosphatase [3] and malic enzyme 2 [9] which are up-regulated. As can be seen in Tables 1 and 2, only lactate dehydrogenase A [8] and arylsulfatase [13] are regulated in cycling and in arrested cells. HMG CoA reductase [11] was previously described by us and others to be regulated by GC (22, 23), and shows a tendency to be regulated on the chip (see www.tilak.or.at/tkfi). Dotted arrows indicate additional regulatory steps in between, normal arrows indicate direct reactions; double arrows symbolize reactions possible in both directions.
patients treated with GC in vivo, a subject currently under investigation in our laboratory. Moreover, details of the proposed ‘metabolic disaster’ need to be delineated as well as the question of whether GC-induced apoptosis follows the same mechanistic principle in malignant and normal, and proliferating and arrested lymphocytes. In conclusion, our data provide for the first time a comprehensive insight into the complex network of GC-regulated genes in human leukemia cells and suggest a testable hypothesis for the mechanism of GCinduced apoptosis. The authors thank S. Geymayer, W. Doppler, and A. Amberger for valuable discussions and critical reading of the manuscript and I. Jaklitsch for technical assistance. This study was supported by grants from the Austrian Science Fund (SFB-F002, P11964-Med, and P11306). 698
Vol. 15
March 2001
REFERENCES 1. 2. 3. 4.
5.
Geley, S., Fiegl, M., Hartmann, B. L., and Kofler, R. (1996) Genes mediating glucocorticoid effects and mechanisms of their regulation. Rev. Physiol. Biochem. Pharmacol. 128, 1–97 Bansal, N., Houle, A., and Melynkovych, G. (1991) Apoptosis: mode of cell death induced in T cell leukemia cell lines by dexamethasone and other agents. FASEB J. 5, 211–216 Hollenberg, S. M., and Evans, R. M. (1988) Multiple and cooperative trans-activating domains of the human glucocorticoid receptor. Cell 55, 899 –906 Iyer, V., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T., Lee, J. F. C., Trent, F. M., Staudt, L. M., Hudson, J., Jr., Boguski, M. S., Lashkari, D., Shalon, D., Botstein, D., and Brown, P. O. (1999) The transcriptional program in the response of human fibroblasts to serum. Science 283, 83– 87 Alizadeh, A. A., Eisen, M. B., Davis, R. E., Ma, C., Lossos, S., Rosenwald, A., Boldrick, J. C., Yang, L., Marti, G. R., Moore, T., Hudson, J.Jr, Lu, L., Lewis, D. B., Tibshirani, R., Shelock, G., Chen, W. C., Greiner, T. C., Weisenburger, D. D., Armitage, J. O., Warnke, R., Levy, R., Wilson, W., Grever, M. R., Byrd, J. C., Botstein, D., Brown, P. O., and Staudt, L. M. (2000) Distinct
The FASEB Journal
TONKO ET AL.
6.
7.
8.
9.
10.
11.
12.
13.
14. 15.
16.
types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature (London) 405, 503–511 Foley, G. E., Lazarus, H., Farber, S., Uzman, B. G., Boone, B. A., and McCarthy, R. E. (1965) Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer 18, 522–529 Geley, S., Hartmann, B. L., Hala, M., Strasser-Wozak, E. M. C., Kapelari, K., and Kofler, R. (1996) Resistance to glucocorticoidinduced apoptosis in human T cell acute lymphoblastic leukemia CEM-C1 cells is due to insufficient glucocorticoid receptor expression. Cancer Res. 56, 5033–5038 Strasser-Wozak, E. M., Hattmannstorfer, R., Hala, M., Hartmann, B. L., Fiegl, M., Geley, S., and Kofler, R. (1995) Splice site mutation in the glucocorticoid receptor gene causes resistance to glucocorticoid-induced apoptosis in a human acute leukemic cell line. Cancer Res. 55, 348 –353 Ausserlechner, M. J., Obexer, P., Wiegers, G. J., Hartmann, B. L., Geley, S., Kofler, R. (2001) The cell cycle inhibitor plb/INK4A sensitizes lymphoblastic leukemia cells to apoptosis by physiologic glucocorticoid levels. J. Biol. Chem. In press. Hartmann, B. L., Geley, S., Loffler, M., Hattmannstorfer, R., Strasser-Wozak, E. M., Auer, B., and Kofler, R. (1999) Bcl-2 interferes with the execution phase, but not upstream events, in glucocorticoid-induced leukemia apoptosis. Oncogene 18, 713–719 Bernhard, D., Ausserlechner, M. J., Tonko, M., Loffler, M., Hartmann, B. L., Csordas, A., and Kofler, R. (1999) Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts. FASEB J. 13, 1991–2001 Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., and Riccardi, C. A. (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271–279 Strasser-Wozak, E. M., Hartmann, B. L., Geley, S., Sgonc, R., Bock, G., Santos, A. J., Hattmannstorfer, R., Wolf, H., Pavelka, M., and Kofler, R. (1998) Irradiation induces G2/M cell cycle arrest and apoptosis in p53-deficient lymphoblastic leukemia cells without affecting Bcl-2 and Bax expression. Cell Death Diff. 5, 687– 693 Schmidt, T. J., and Meyer, A. S. (1994) Autoregulation of corticosteroid receptors. How, when, where, and why? Receptor 2, 229 –257 Eisen, L. P., Elsasser, M. S., and Harmon, J. M. (2048) (1988) Positive regulation of the glucocorticoid receptor in human T cells sensitive to the cytolytic effects of glucocorticoids. J. Biol. Chem. 263, 12044 –1 Ramdas, J., Liu, W., and Harmon, J. M. (1999) Glucocorticoidinduced cell death requires autoinduction of glucocorticoid
DNA-CHIP ANALYSIS DURING GC-INDUCED APOPTOSIS
17.
18. 19. 20.
21. 22.
23.
24.
25.
26.
27.
receptor expression in human leukemic T cells. Cancer Res. 59, 1378 –1385 Shim, H., Dolde, C., Lewis, B. C., Wu, C. S., Dang, G. Jungmann, R. A., Dalla-Favera, R., and Dang, C. V. (1997) c-Myc transactivation of LDH-A: implications for tumor metabolism and growth. Proc. Natl. Acad. Sci. USA 94, 6658 – 6663 Dang, C. V., and Semanza, G. L. (1999) Oncogenic alterations of metabolism. Trends Biochem. Sci. 24, 68 –72 Warburg, O. (1926) The Metabolism of Tumors, Springer, New York Vogel. P., Ma¨gert, H.-J., Cielak, A., Adermann, K., and Forssman, W.-G. ( 1996) hDIP—a potential transcriptional regulator related to murine TSC-22 and Drosophila shortsighted (shs)—is expressed in a large number of human tissues: BBA 1309, 200 –204 Mellitzer, G., Xu, Q., and Wilkinson, D. G. (1999) Eph receptors and ephrins restrict cell intermingling and communication. Nature (London) 400, 77 Helmberg, A., Fassler, R., Geley, S., Johrer, K., Kroemer, G., Bock, G., and Kofler, R. (1990) Glucocorticoid-regulated gene expression in the immune system. Analysis of glucocorticoidregulated transcripts from the mouse macrophage-like cell line P388D1. J. Immunol. 145, 4332– 4337 Simonet, W.S., Ness, G.C. (1989) Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA in rat liver. Glucocorticoids block the stabilization caused by thyroid hormones. J. Biol. Chem. 264, 569 –573 Stoolman, L. M., Wang, T.-L., Situ, R., and Varani, J. (1993) Regulation of fibronectin and laminin binding activity in cultured human lymphoblastic cell lines. J. Cell. Physiol. 154, 593– 600 Marine, J.-C., Topham, D. J., MacKay, C., Wang, D., Parganas, E., Stravopodis, D., Yoshimura, A., and Ihle, J. N. (1999) SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality. Cell 98, 609 – 616 Kalenik, J. L., Chen, D., Bradley, M. E., Chen, S. J., and Lee, T.-C. (1995) Yeast two-hybrid cloning of a novel zinc finger protein that interacts with the multifunctional transcription factor YY1. NAR 25, 843– 850 Stephens, J. L., and Pieringer, R. A. (1984) Regulation of arylsulfatase A and sulphogalactolipid turnover by cortisol in myelinogenic cultures of cells dissociated from embryonic mouse brain. Biochem. J. 219, 689 – 697 Received for publication May 24, 2000. Revised for publication September 5, 2000.
699
