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Immunology logoLink to Immunology
. 2009 Sep;128(1 Pt 2):e905–e918. doi: 10.1111/j.1365-2567.2009.03104.x

Identification and characterization of the interferon-β-mediated p53 signal pathway in human peripheral blood mononuclear cells

Fanglin Zhang 1, Subramaniam Sriram 1
PMCID: PMC2753925  PMID: 19740351

Abstract

The relationship between the p53 signal pathway and the response of human peripheral blood mononuclear cells (PBMC) to interferon (IFN)-β has hitherto not been examined. Using an oligonucleotide microarray, we found differential expression of at least 70 genes involved in the p53 signal pathway, including p53, which regulate cell proliferation and cell death following stimulation with IFN-β. We verified our observations on a limited set of p53-regulated genes at the transcriptional and translational levels. We also examined the consequences of the activation of the p53 signal pathway by IFN-β in PBMC. When cultured in the presence of T-cell mitogens, IFN-β restricted the entry of lymphocytes from the G0/G1 phase to the S phase and reduced the number of cells in the G2 phase. The addition of IFN-β alone did not increase apoptosis. However, in the presence of actinomycin D, a DNA-damaging agent, addition of IFN-β enhanced the susceptibility of PBMC to apoptosis. These observations suggest that, in spite of the activation of a number of mutually overlapping pathways mediating cell death, cell cycle arrest was the most evident consequence of IFN-β signalling in PBMC.

Keywords: apoptosis, interferon-β, cell cycle arrest, lymphocytes, p53

Introduction

Interferon (IFN)-β belongs to a family of naturally occurring molecules that have pleiotropic effects on immune and non-immune cells.1,2 The receptor for IFN-β is widely expressed in tissues, and the interaction of IFN-β with its receptor leads to oligomerization of the receptor and phosphorylation of the receptor-associated tyrosine kinases Janus kinase 1 (Jak1) and tyrosine kinase 2 (Tyk2). This then leads to the phosphorylation of signal transducers and activators of transcription 1 (STAT1) and STAT2, which subsequently dimerize, translocate to the nucleus and activate the transcription of a number of IFN-stimulated genes.3,4 Most of the type 1 interferon-stimulated genes have IFN-stimulated response element (ISRE) sequences in the promoter region.5,6 Activation of the IFN-stimulated genes requires the binding of the activated STAT proteins with p48 to form a trimeric complex that is responsible for regulating IFN actions.

IFN-β is currently used as a therapeutic agent in the treatment of hepatitis induced by the hepatitis C virus, multiple myeloma and multiple sclerosis.79 In the three major clinical applications of IFN-β, therapeutic benefits have in large part been derived from strategies focused on the proliferation and expansion of the target cells. Not surprisingly, a number of studies that examined the activation of genes by IFN-β have focused on the expression and regulation of proteins that mediate cell proliferation and apoptosis. These studies have shown increased expression of tumour necrosis factor (TNF), Fas ligand, and TNF-related apoptosis-inducing ligand (TRAIL) by IFN-β.1014 IFN-β has also been shown to decrease the expression of Fas-associated death domain-like interleukin-1β-converting enzyme inhibitory protein (FLIP) and immunosuppressive acidic protein (IAP), two proteins that inhibit apoptosis.15,16 Although the induction of death receptors and their ligands has been surmised to be one of the principal mechanisms of action of IFN-β, direct evidence of the role of IFN-β in cell proliferation and apoptosis in human lymphocytes is lacking. More recently, IFN-β was shown to increase the induction of p53, a key protein involved in the activation of apoptosis in murine fibroblasts; however, the response of human peripheral blood mononuclear cells (PBMC) to the effects of IFN-β and in particular activation of the p53 pathway remains unexplored.14,1720

The tumour suppressor protein p53 is a key transcription factor that is involved in the regulation of cell proliferation and cell death.21,22 By preventing the proliferation of cells bearing damaged DNA, which if left unattended can lead to neoplasia, p53 facilitates repair of DNA and, if the damage cannot be repaired, it directs the cells towards apoptosis. Thus, p53 acts as a tumour suppressor, and this has been confirmed by the presence of mutations of the p53 gene in cancer.2325 It was believed that IFN-β-mediated activation of p53 following viral infection of tumour cells would lead to rapid apoptosis before viral expansion could occur, and thus restrict viral spread.26 This mechanism of action might explain the beneficial effects of IFN-β in the treatment of hepatitis caused by the hepatitis C virus.27 The mechanism of activation of p53 and its downstream signal pathway in PBMC and their role in regulating autoimmune diseases, including multiple sclerosis, remain unknown.

In our study, for the first time, we set out to examine, in a cohort of normal healthy individuals, the expression patterns and functions of the genes involved in the p53 signal pathway following culture with IFN-β and their effects on lymphocyte survival. Such studies, we believe, are important for the following reasons: (i) a detailed study of the activation of the p53 signal pathway in the PBMC of healthy donors by IFN-β is currently lacking; (ii) understanding of the outcome of p53 activation of PBMC in vitro will provide a basis for recognition of p53 activation pathways in the PBMC of patients on treatment with IFN-β for viral or autoimmune diseases, and (iii) evidence of defects in the p53 activation pathway may allow the identification of patients who show sub-therapeutic responses to IFN-β.

We show that, despite the activation of a number of proteins that have pro-apoptotic functions by IFN-β, the predominant effect on cell division was the induction of cell cycle arrest, and not apoptosis. These novel results have implications for the mechanism of action of IFN-β in the regulation of lymphocyte function in vivo.

Materials and methods

Subjects

The study group comprised 12 healthy volunteers who had no history of autoimmune disease and were not on any immunotherapy. The male to female ratio was 1 : 1; the ages of the subjects ranged from 30 to 60 years. Human subject studies were approved by the Committee for the Protection of Human Subjects of the Vanderbilt University Institutional Review Board.

Reagents

The RNA isolation kit and RNAse-free DNAse set were from Qiagen (Valencia, CA). cDNA was generated using Reverse Transcription Reagents (Applied Biosystems, Foster City, CA), and the iQ SYBR Green Supermix was from Bio-Rad Laboratories (Hercules, CA). The following primary antibodies were obtained and used in the indicated dilutions: mouse anti-human p53 antibody (DO-1) (1 : 2000), rabbit anti-human p21 antibody (1 : 2000), rabbit anti-human Bcl-2-associated X protein (Bax) antibody (1 : 2000), rabbit anti-human STAT1 antibody (1 : 5000), rabbit anti-human STAT2 antibody (1 : 5000), rabbit anti-human β-actin antibody (1 : 10 000), secondary horseradish peroxidase linked anti-mouse immunoglobulin (IgG) and anti-rabbit IgG (1 : 10 000); all these antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). The CD3-fluorescein isothiocyanate (FITC)-conjugated anti-human antibody and the Annexin V-FITC & 7-amino-actinomycin D (AAD) apoptosis detection kit were from BD Biosciences Pharmingen (San Jose, CA). DNAse-free RNAse and propidium iodide were from Roche Applied Science (Indianapolis, IN). IFN-β-1a was a gift from Serono Inc., (Rockland, MA). Actinomycin-D (Act D), phytohaemagglutinin (PHA) and Ficoll-Hypaque was purchased from Sigma-Aldrich (St Louis, MO).

Isolation and culture of PBMC

PBMC were isolated by density gradient centrifugation with Ficoll-Hypaque from freshly heparinized blood. The cells were washed in phosphate-buffered saline (PBS) and re-suspended at 1 × 106 cells/ml in complete RPMI-1640 medium containing 2 mmol glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (Invitrogen, Carlsbad, CA). The induction of p53 was examined at the following doses of IFN-β: 100, 1000 and 5000 IU/ml. The PBMC were cultured in the presence of 10 μg/ml PHA and Act D was used at a single dosage of 50 ng/ml.

Total RNA extraction and reverse transcription

Total RNA was extracted from PBMC using the RNeasy mini kit (Qiagen, Valencia, CA) and treated with the RNAse-free DNAse set, following the manufacturer’s recommendations. A Bioanalyzer microfluidic assay (Agilent Technologies, Palo Alto, CA) was applied to test RNA integrity. Spectrophotometric and fluorometric methods were combined to quantify RNA. cDNA was generated from RNA using Reverse Transcription Reagents (Applied Biosystems). One microgram of total RNA was reverse-transcribed in a total volume of 25 μl using 100 units of reverse transcriptase, 2·5 μl of 10 × reverse transcription buffer, 2·5 μl of 10 × random primer and 1·5 μl of 20 U/μl RNase inhibitor. The mixture was incubated for 10 min at 25°, 120 min at 37° and 5 seconds at 85° and then rapidly cooled on ice. The cDNA samples were stored at −20°.

Microarray analysis

To determine the differentially expressed genes in PBMC following culture with IFN-β, we used the GeneChip® Human Gene 1·0 ST (Affymetrix Inc., Santa Clara, CA). This chip contains 764 885 probes representing 28 869 genes, each of which is represented on the array by approximately 26 probes spread across the full length of the gene. Peripheral blood mononuclear cells were obtained from five healthy individuals. The isolated PMBC were cultured with IFN-β (1000 IU/ml) for 0, 24 and 48 hr. RNA samples were submitted to the Vanderbilt Microarray Shared Resource (Vanderbilt University, Nashville, TN, USA) for microarray analysis using the GeneChip Whole Transcript (WT) Sense Target Labeling Assay protocol (Affymetrix Inc., Santa Clara, CA). Briefly, a total of 100 ng of total RNA was reverse-transcribed to cDNA which was then used as a template in an in vitro transcription reaction followed by fragmentation of the single-stranded cDNA and labelling through a terminal deoxy-transferase reaction. The biotinylated cDNA (5 μg) was fragmented and hybridized to the Human Gene 1·0 ST Array, which was then scanned using genechip scanner 3000 7G Plus 2 and command console Software (AGCC) version 1·0 (Affymetrix Inc.). Generated CEL files (raw Affymetrix data) were imported into expression console (Affymetrix Inc.) and normalized by robust multi-array average (RMA)-sketch for quality control purposes.28 Normalized data were uploaded into partek genomics suites (Partek Inc., St Louis, MI) for statistical analysis. To identify significant differences in gene expression level among the groups, log2gene expression measurements for each gene on each chip were modelled using a multifactor mixed model in the partek genomics suites software. In order to increase sensitivity and allow identification of potentially important biological changes, we employed a lower level of stringency and set an adjusted P-value [false discovery rate (FDR)] cut-off of 0·2. The lists of differentially expressed genes were then classified according to their biological pathway and biological processes. This was achieved using the protein analysis through evolutionary relationships (PANTHER) Classification System to compare them with reference lists to look for enriched functional categories.29

Real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR)

Real-time quantitative PCR was carried out in an iCycler detection system (Bio-Rad laboratories, Hercules, CA) in a volume of 25 μl. The reaction mixture consisted of 12·5 μl of iQ SYBR Green Supermix, 200 nm of each primer, and 1 μl of cDNA template. Reactions were performed for 45 cycles (95° for 15 seconds, 60° for 30 seconds and 72° for 30 seconds) after an initial 3-min incubation at 95°. Primers for the different genes amplified are shown in Table 1. The primers for p53 comprised regions that overlapped the full length and the beta/gamma isoform of p53. All reactions were performed in duplicate. Values for each gene were normalized to the values of the internal control β-actin using the threshold cycle (Ct) method, and the fold change compared with the culture control was calculated.

Table 1.

Primers for quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR)

Forward primer Reverse primer
p53 CGTCAGAAGCACCCAGGACT CATCCTCCTCCCCACAACAA
p21 TCCTCTAGCTGTGGGGGTGA GAAGGTCGCTGGACGATTTG
BAX CAGCAAACTGGTGCTCAAGG CGGAGGAAGTCCAATGTCCA
MDM235 CAAGTTACTGTGTATCAGGCAGGG TCTGTTGCAATGTGATGGAAGG
NOXA ACCGCTGGCCTACTGTGAAG TGTGCTGAGTTGGCACTGAAA
PUMA GACCTCAACGCACAGTACGAG AGGAGTCCCATGATGAGATTGT
STAT1 TGCAAATGCTGTATTCTTCTTTGG TATGCAGTGCCACGGAAAGC
STAT2 CCTGCTGTGCTGGGAGGTAT GAAAGAAGCCACTGCCCTGA
β-actin GCCGAGGACTTTGATTGCAC TGGACTTGGGAGAGGACTGG

BAX, Bcl-2 associated X protein; MDM2, murine double minute 2; STAT, signal transducers and activators of transcription.

Western blot analysis

Cell lysates for western blotting were prepared by treating PBMC with 50 mm Tris (pH 8·0), 200 mm NaCl, 1% NP40 supplemented with 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mm NaF, 20 mmβ-glycerophosphate, 1 mm sodium vanadate, 1 mm dithiothreitol and 1 mm phenylmethysulphonyl fluoride. The cells were incubated on ice for 30 min and sonicated, before being centrifuged at 18 000 g for 15 min. The total protein concentration was measured according to the Bradford assay method (Bio-Rad Laboratories). Equal amounts of protein were loaded onto a 12% sodium dodecyl sulphate (SDS)–polyacrylamide gel in electrophoresis buffer (25 mm Tris-HCl, 250 mm glycine and 0·1% SDS) and separated at 100 V. Proteins were then transferred onto polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA) by electroblotting for 1 hr at 100 V. After blocking with blotto (1 × Tris buffer solution (TBS), 0·05% Tween-20 and 5% non-fat milk powder) for 2 hr, the membranes were probed with primary antibodies. After three washes, secondary horseradish peroxidase linked anti-mouse IgG or anti-rabbit IgG was added for 1 hr. Specific bands were visualized using enhanced chemiluminescence reagent and exposed to X-ray film. The intensity of the bands was quantified using wcif image j software (Wright Cell Imaging Facility, Toronto, Canada.). The ratio of the intensity of the band of the tested protein and that of β-actin was measured on the same membrane.

Detection of apoptosis

Apoptosis was analysed by labelling with the Annexin V-FITC & 7-AAD apoptosis detection kit. PBMC were cultured with IFN-β for 48 hr in either the presence or absence of DNA-damaging agent Act D for 24 hr before harvesting. At the end of the culture period, the cells were washed and stained with Annexin V-FITC and 7-AAD, and then were submitted to the BD LSRII flow cytometer (BD Biosciences, San Jose, CA). Data were analysed using bd facsdiva software (BD Biosciences) and cell apoptosis was determined by Annexin V+ and 7-AAD.

Cell cycle analysis

PBMC (2 × 106) were stained with CD3-FITC for 30 min, washed twice and fixed in 75% ethanol at 4° for 2 hr, and then washed in PBS and subjected to digestion with DNAse-free RNAse for 0·5 hr at 37°. Cells were re-suspended in 500 μl of PBS with propidium iodide, and then submitted to the BD LSRII flow cytometer. Flow cytometry data were analysed using flowjc software (FlowJo, Ashland, OR).

Statistic analysis

Results are expressed as mean ± standard deviation. Statistically significant differences among groups were identified using analysis of variance (anova). Specifically, we employed repeated measures anova for the data obtained in the western blotting, real-time RT-PCR and cell cycle experiments. The PROC MIXED procedure in sas (version 9·1; SAS Institute, Cary, NC) and the SIMULATE adjustment were used to compute adjusted P-values of all pairwise differences of three time point's measurements for each parameter such as p53, p21 and Bax. The data from apoptosis experiments were analysed using one-way anova in spss 11.0 software (SPSS, Chicago, IL) and P-values < 0·05 were considered significant.

Results

Activation of the p53 signal pathway by IFN-β

We examined the genes involved in the p53 signal pathway that were targeted by IFN-β using GeneChip® Human Gene 1·0 ST. A list of 8060 genes that showed a statistically significant change from baseline (FDR < 0·2) was generated. Among the 74 genes that were recognized as being involved in the p53 signal pathway, apoptosis and the cell cycle were two of the most highly represented biological processes (Tables 2 and 3, Fig. 1a). Of these genes, 16 were involved in the cell cycle process, 15 in apoptosis and 10 in both (Table 3). As shown in the heat map (Fig. 1a), there was an increase in p53 expression in cells cultured with IFN-β. The previously recognized downstream targets of p53, such as p21, PUMA, NOXA, Bax and growth arrest and DNA damage inducible gene 45 (Gadd45), were all shown to be induced by IFN-β. Genes that regulate the expression of death receptor-associated genes, such as those belonging to the TNF superfamily [Fas-associated protein with death domain (FADD), TNF receptor-associated factor 2 (TRAF2), TNF, apoptosis stimulating fragment (FAS), Fas ligand (FASLG)] and those involved in the common apoptotic pathway (apoptosis inducing factor 2 and Caspase 9), were also up-regulated. We also noted increased expression of murine double minute 2 (MDM2), a protein known to down-regulate apoptosis by inhibiting the actions of p53.

Table 2.

Genes in the p53 signalling pathway are involved in the response of PBMC to interferon (IFN)-β

Gene GenBank accession number P-value Definition
PML NM_033240 7·78E-11 Promyelocytic leukaemia
MCL1 NM_021960 8·60E-09 Myeloid cell leukaemia sequence 1 (BCL2-related)
BRCA2 NM_000059 1·44E-08 Breast cancer 2, early onset
STAT2 NM_005419 1·84E-07 Signal transducer and activator of transcription 2, 113 kDa
FAS NM_000043 6·30E-07 Fas (TNF receptor superfamily, member 6)
CDKN1A(p21) NM_078467 7·47E-07 Cyclin-dependent kinase inhibitor 1A (p21, Cip1)
PMAIP1(NOXA) NM_021127 2·21E-06 Phorbol-12-myriSTATe-13-acetate-induced protein 1
IGF1R NM_000875 3·39E-06 Insulin-like growth factor 1 receptor
TSC2 NM_000548 1·12E-05 Tuberous sclerosis 2
CCNA1 NM_003914 1·13E-05 Cyclin A1
FASLG NM_000639 1·40E-05 Fas ligand (TNF superfamily, member 6)
AIFM2 NM_032797 2·30E-05 Apoptosis-inducing factor, mitochondrion-associated, 2
PRKDC NM_006904 2·68E-05 Protein kinase, DNA-activated, catalytic polypeptide
SIRT7 NM_016538 2·83E-05 Homo sapiens sirtuin (silent mating type information regulation 2 homologue) 7
AKT3 NM_181690 4·22E-05 v-akt murine thymoma viral oncogene homologue 3 (protein kinase B, gamma)
RELA NM_021975 4·99E-05 v-rel reticuloendotheliosis viral oncogene homologue A (avian)
TSC1 NM_000368 6·25E-05 Tuberous sclerosis 1
C20orf74 NM_020343 8·63E-05 Chromosome 20 open reading frame 74
B2M NM_004048 0·000127075 Beta-2-microglobulin
MDM2 NM_002392 0·000162073 MDM2 p53 binding protein homologue (mouse)
MYST4 NM_012330 0·000243101 MYST histone acetyltransferase (monocytic leukaemia) 4
GADD45B NM_015675 0·000253463 Growth arrest and DNA-damage-inducible, beta
PRKAG2 NM_016203 0·000278452 Protein kinase, AMP-activated, gamma 2 non-catalytic subunit
RB1 NM_000321 0·000334025 Retinoblastoma 1 (including osteosarcoma)
PIK3CB NM_006219 0·000547966 Phosphoinositide-3-kinase, catalytic, beta polypeptide
MAPK13 NM_002754 0·000598988 Mitogen-activated protein kinase 13
IGF1R NM_000875 0·000800872 Insulin-like growth factor 1 receptor
HK2 NM_000189 0·000908611 Hexokinase 2
TRAF2 NM_021138 0·00102911 TNF receptor-associated factor 2
PCNA NM_002592 0·0011794 Proliferating cell nuclear antigen
BBC3(PUMA) AF354654 0·00126057 BCL2 binding component 3
GARNL1 NM_014990 0·00131323 GTPase activating Rap/RanGAP domain-like 1
NFATC2IP NM_032815 0·00131522 Nuclear factor of activated T-cells, cytoplasmic, calcin
PCAF NM_003884 0·0014721 p300/CBP-associated factor
PRKAG1 NM_212461 0·00155707 protein kinase, AMP-activated, gamma 1 non-catalytic subunit
CASP9 NM_001229 0·00194233 Caspase 9, apoptosis-related cysteine peptidase
CDK2 NM_001798 0·00232801 Cyclin-dependent kinase 2
DDB2 NM_000107 0·00252077 Damage-specific DNA-binding protein 2, 48 kDa
SIRT6 NM_016539 0·00268096 Sirtuin (silent mating type information regulation 2 homologue) 6 (S. cerevisiae)
BAX NM_004324 0·00335452 BCL2-associated X protein
STAT1 NM_007315 0·00344978 Signal transducer and activator of transcription 1, 91 kDa
SESN2 NM_031459 0·00395825 Sestrin 2
PPP2CA NM_002715 0·00404361 Protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform
UBTF NM_014233 0·00431895 Upstream binding transcription factor, RNA polymerase I
SIAH1 NM_001006610 0·00479628 Seven in absentia homologue 1 (Drosophila)
ATM NM_000051 0·00500669 Ataxia telangiectasia mutated
IGBP1 NM_001551 0·00532907 Immunoglobulin (CD79A) binding protein 1
RHEBL1 NM_144593 0·00552916 Ras homologue enriched in brain like 1
FADD NM_003824 0·00561322 Fas (TNFRSF6)-associated via death domain
FRAP1 NM_004958 0·00782508 FK506 binding protein 12-rapamycin associated protein 1
TP53 NM_000546 0·00814257 Tumour protein p53 (Li-Fraumeni syndrome)
PIK3CG NM_002649 0·00840817 Phosphoinositide-3-kinase, catalytic, gamma polypeptide
PPM1D BC042418 0·00952229 Protein phosphatase 1D magnesium-dependent, delta isoform
RHEB NM_005614 0·0104375 Ras homologue enriched in brain
DNMT1 NM_001379 0·0106164 DNA (cytosine-5-)-methyltransferase 1
RRAS NM_006270 0·0108314 Related RAS viral (r-ras) oncogene homologue
SIRT2 NM_012237 0·0115687 Sirtuin (silent mating type information regulation 2 homologue) 2
GADD45G NM_006705 0·013215 Growth arrest and DNA-damage-inducible, gamma
PPP2CB NM_001009552 0·0150046 Protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform
ZMAT3 NM_022470 0·015343 Zinc finger, matrin type 3
YWHAB NM_003404 0·0166223 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide
YWHAQ NM_006826 0·0168976 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide
TNF NM_000594 0·0177639 Tumour necrosis factor (TNF superfamily, member 2)
RPKAB1 NM_006253 0·020311 Protein kinase, AMP-activated, beta 1 non-catalytic subunit
NF1 NM_001042492 0·0224542 Neurofibromin 1
MLH1 NM_000249 0·0237558 mutL homologue 1, colon cancer, nonpolyposis type 2 (E. coli)
TOX4 NM_014828 0·0242946 TOX high-mobility group box family member 4
MAPK14 NM_001315 0·0277024 Mitogen-activated protein kinase 14
ZMAT2 NM_144723 0·0279075 Zinc finger, matrin type 2
KRAS NM_033360 0·0351255 v-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue
MTA2 NM_004739 0·0357651 Metastasis associated 1 family, member 2
BCL2A1 NM_004049 0·0384042 BCL2-related protein A1
RBL1 NM_002895 0·0424297 Retinoblastoma-like 1 (p107)
NFKB1 NM_003998 0·0430098 Nuclear factor of kappa light polypeptide gene enhancer

Table 3.

Identification of p53 response pathway genes that play a role in cell cycle arrest and apoptosis

Gene GenBank accession number Regulation by interferon-β P-value Definition
Apoptosis
PIK3CG NM_002649 0·008408 Phosphoinositide-3-kinase, catalytic, gamma polypeptide
PIK3CB NM_006219 0·000548 Phosphoinositide-3-kinase, catalytic, beta polypeptide
NFKB1 NM_003998 + 0·04301 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1
FADD NM_003824 + 0·005613 Fas (TNFRSF6)-associated via death domain
TRAF2 NM_021138 + 0·001029 TNF receptor-associated factor 2
TNF NM_000594 + 0·017764 Tumour necrosis factor (TNF superfamily, member 2)
BCL2A1 NM_004049 + 0·038404 BCL2-related protein A1
CASP9 NM_001229 + 0·001942 Caspase 9, apoptosis-related cysteine peptidase
AIFM2 NM_032797 + 2·3E-05 Apoptosis-inducing factor, mitochondrion-associated, 2
MCL1 NM_021960 + 8·6E-09 Myeloid cell leukaemia sequence 1 (BCL2-related)
FASLG NM_000639 + 1·4E-05 Fas ligand (TNF superfamily, member 6)
MDM2 NM_002392 + 0·000162 MDM2 p53 binding protein homologue (mouse)
FAS NM_000043 + 6·3E-07 Fas (TNF receptor superfamily, member 6)
BBC3(PUMA) AF354654 + 0·001261 BCL2 binding component 3
PMAIP1 (NOXA) NM_021127 + 2·21E-06 Phorbol-12-myriSTATe-13-acetate-induced protein 1
Cell cycle arrest
PRKDC NM_006904 2·68E-05 Protein kinase, DNA-activated, catalytic polypeptide
C20orf74 NM_020343 8·63E-05 Chromosome 20 open reading frame 74
TSC2 NM_000548 1·12E-05 Tuberous sclerosis 2
TSC1 NM_000368 6·25E-05 Tuberous sclerosis 1
MYST4 NM_012330 0·000243 MYST histone acetyltransferase (monocytic leukaemia) 4
GARNL1 NM_014990 0·001313 GTPase activating Rap/RanGAP domain-like 1
FRAP1 NM_004958 0·007825 FK506 binding protein 12-rapamycin associated protein 1
RBL1 NM_002895 0·04243 Retinoblastoma-like 1 (p107)
YWHAB NM_003404 0·016622 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide
YWHAQ NM_006826 + 0·016898 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide
RB1 NM_000321 + 0·000334 Retinoblastoma 1 (including osteosarcoma)
SESN2 NM_031459 + 0·003958 Sestrin 2
CDK2 NM_001798 + 0·002328 Cyclin-dependent kinase 2
PCNA NM_002592 + 0·001179 Proliferating cell nuclear antigen
CCNA1 NM_003914 + 1·13E-05 Cyclin A1
CDKN1A NM_078467 + 7·47E-07 Cyclin-dependent kinase inhibitor 1A (p21, Cip1)
Overlapping
IGF1R NM_000875 3·39E-06 Insulin-like growth factor 1 receptor
AKT3 NM_181690 4·22E-05 v-akt murine thymoma viral oncogene homologue 3 (protein kinase B, gamma)
IGF1R NM_000875 0·000801 Insulin-like growth factor 1 receptor
ATM NM_000051 0·005007 Ataxia telangiectasia mutated
GADD45G NM_006705 0·013215 Growth arrest and DNA-damage-inducible, gamma
TP53 NM_000546 + 0·008143 Tumour protein p53 (Li-Fraumeni syndrome)
NFATC2IP NM_032815 + 0·001315 Nuclear factor of activated T-cells, cytoplasmic, calcin
RELA NM_021975 + 4·99E-05 v-rel reticuloendotheliosis viral oncogene homologue A
BAX NM_004324 + 0·003355 BCL2-associated X protein
GADD45B NM_015675 + 0·000253 Growth arrest and DNA-damage-inducible, beta

+, up-regulation; −, down-regulation.

Figure 1.

Figure 1

Figure 1

Activation of the p53 signal pathway by interferon (IFN)-β in peripheral blood mononuclear cells (PBMC) at the transcription level. (a) Hierarchical cluster of 74 differentially regulated genes in the p53 signal pathway after culture of PBMC with IFN-β. Each row corresponds to a single gene and each column corresponds to the average relative expression level at each time-point, with 0, 24 and 48 hr from left to right. The values were transformed to a log2 scale and converted into colour intensity. Red indicates increased expression and blue indicates reduced expression. (b–g) Real-time reverse transcription–polymerase chain reaction (RT-PCR) values for p53 and its target genes following culture with IFN-β: (b) p53, (c) Bcl-2-associated X protein (Bax), (d) NOXA, (e) PUMA, (f) p21 and (g) MDM2; pooled data from seven individuals. The y-axis represents the fold increase in real-time values after normalization to β-actin. **P < 0·001; *P < 0·05 when compared with unstimulated cells at 0 hr.

To determine levels of p53 mRNA in cells cultured with IFN-β, we performed real-time RT-PCR on RNA isolated from the PBMC of seven individuals, which were cultured with 1000 IU/ml of IFN-β, using primers specific for p53, p21, PUMA, NOXA and MDM2. After 48 hr of culture with IFN-β, there were significant increases (P < 0·05) in the expression of p53 (2·3-fold), p21 (12-fold), PUMA (3·5-fold), NOXA (5·5-fold), MDM2 (5-fold) and Bax (2·8-fold), compared with PBMC cultured in medium alone. At 24 hr, only NOXA, p21 and MDM2 showed a statistically significant difference from 0 hr (Fig. 1b–g).

Induction of p53 and p53 targeted proteins was also examined using western blot assays. We examined the kinetics of induction of p53 following in vitro culture of PBMC with IFN-β from 12 healthy volunteers. The addition of 1000 U/ml IFN-β was sufficient for significant induction of p53 at 48 hr (Fig. 2a,b) and there was a time-dependant increase in the full-length and beta/gamma isoforms of p53. The increase in protein level was already significant at 24 hr and increased further at 48 hr (P < 0·05 compared with baseline). Densitometric studies for 12 individuals showed a 1·92-fold increase in the amount of full-length p53 and a 1·98-fold increase in the amount of the beta/gamma isoform, which was significant at 48 hr (Fig. 2b). Induction of p21 and Bax proteins using western blot assays was performed for 12 individuals. Densitometric analysis of western blots showed a 3·8-fold increase in p21 (P < 0·001) and a 1·39-fold increase in Bax (P < 0·05) following 48 hr of culture with IFN-β (Fig. 2d,e). These studies support the hypothesis that IFN-β may activate the p53 signal pathway in PBMC which is critical for cell proliferation and apoptosis.

Figure 2.

Figure 2

(a) Western blots showing the dose–response of p53 expression in response to interferon (IFN)-β at 48 hr, (b) western blots showing the induction of p53, p21 and Bcl-2-associated X protein (Bax) in peripheral blood mononuclear cells (PBMC) cultured with 1000 IU/ml IFN-β, (c–e) densitometric values of western blots of (c) p53, (d) p21 and (e) Bax, for 12 individuals, normalized to β-actin. **P < 0·001; *P < 0·05 when compared with unstimulated cells at 0 hr.

Differences in the induction of p53 and p53 isoforms following gamma irradiation (IR) and upon culture with IFN-β

As gamma IR is a potent inducer of p53, we compared the induction of p53 and its beta/gamma isoform following either treatment with gamma IR or the addition of 1000 IU/ml IFN-β for 48 hr. As shown in Fig. 3(a,c), gamma IR (10 Gy) of PBMC induced the expression principally of full-length p53. Treatment with IFN-β, in contrast, induced both the full-length and beta/gamma isoforms of p53. In three volunteers, the beta/gamma isoform was dominant over the full-length isoform (Fig. 3b,d). These studies suggest that the p53 activation patterns of IFN-βare different from those of genotoxic stress, the most well-known inducer of p53.

Figure 3.

Figure 3

Induction of p53 in peripheral blood mononuclear cells (PBMC) following gamma irradiation (IR) or after culture with interferon (IFN)-β. (a) Induction of p53 in PBMC from three individuals following culture with IFN-β (1000 IU/ml for 48 hr) and probing with anti-p53 antibody. (b) Densitometric analysis of p53 after normalization to β-actin. (c) Induction of p53 in PBMC from the same three individuals following gamma irradiation of PBMCs (10 Gy). (d) Densitometric analysis of p53 and its isomers, after normalization to β-actin. The y-axis in (b) and (d) represents the per cent increase in the signal of the full-length (FL) and beta/gamma (b/g) isoforms in cells subjected to either gamma irradiation or culture with IFN-β when compared with cells cultured in medium alone.

Induction of STAT1 and STAT2 by IFN-β

The IFN-β receptor uses the Jak-STAT pathway to transduce signals necessary for the transcription of IFN-responsive genes. Also, STAT1 and STAT2 form part of the heterotrimeric complex that binds to the promoter regions of p53. We examined whether STAT1 and STAT2 are targets for IFN-β, and thus act to amplify the IFN-β signalling pathway. We examined the expression of STAT1 and STAT2 following culture of PBMC with IFN-β from 12 healthy volunteers. As shown in Fig. 4(a–c), there was a significant increase in protein levels of both STAT1 and STAT2 as early as 24 hr after culture using western blotting techniques. Densitometric studies showed a twofold increase over baseline for the induction of STAT2, while STAT1 showed a 3·7-fold increase (P < 0·05 compared with untreated cells for both STAT1 and STAT2).

Figure 4.

Figure 4

Induction of signal transducers and activators of transcription 1 (STAT1) and STAT2 by interferon (IFN)-β. (a) Western blots of STAT1 and STAT2 proteins following culture of peripheral blood mononuclear cells (PBMC) with IFN-β from two individuals. (b) Densitometric values of protein levels for STAT1 and (c) densitometric values for STAT 2; pooled analysis for 12 individuals. (d, e) Results of real-time reverse transcription–polymerase chain reaction (RT-PCR) for (d) STAT1 and (e) STAT2 gene expression following culture of PBMC with IFN-β; pooled analysis for seven individuals. Results are expressed as fold increase in mRNA levels over that seen following culture of PBMC in medium alone. *P < 0·05; **P < 0·001 when compared with cells at 0 hr.

To determine whether the increased expression of STAT1 and STAT2 was attributable to an increase in the mRNA of the respective STAT1 and STAT2 genes, real-time RT-PCR using primers specific for STAT1 and STAT2 was performed on PBMC. In mRNA obtained from the PBMC of seven individuals cultured with IFN-β, real-time RT-PCR values showed a 5·6-fold increase in mRNA levels over baseline for STAT1 and a 5·4-fold increase for STAT2 at 48 hr (Fig. 4d,e). Kinetic studies showed that the increase in mRNA for both STAT1 and STAT2 was seen early, at 6 hr (P < 0·05 compared with untreated cells). These studies show that IFN-β induces rapid transcription of STAT1 and STAT2, thereby increasing the constitutive levels of the key signalling proteins necessary for the activation of the IFN-β receptor signalling pathway.

Induction of apoptosis in PBMC cultured with IFN-β

To examine the functional consequences of activation of p53, we investigated the apoptosis of PBMC following culture with IFN-β using flow cytometry. The addition of IFN-β to PBMC and culture for 48 hr did not increase apoptosis when compared with cells cultured in medium alone. As Act D is a known inducer of apoptosis in a number of cell lines,30 we examined the effects of addition of Act D to PBMC cultured with IFN-β. The addition of IFN-β did not increase the number of Annexin V-stained cells. The percentage of Annexin V+ 7-AAD cells increased from 10·63 to 25·50% in the presence of Act D. In the presence of both ActD and IFN-β, the percentage of Annexin V+ 7-AAD cells was 39·73% (P < 0·05; Fig. 5). These experiments showed that, although there was an increase in the expression of pro-apoptotic genes following culture with IFN-β (Fig. 1), a direct effect of IFN-β on apoptosis was not evident unless a DNA-damaging agent was added.

Figure 5.

Figure 5

Flow cytometric analysis of induction of apoptosis by interferon (IFN)-β: (a) cells treated with medium alone, (b) cells treated with 1000 IU/ml IFN-β for 48 hr, (c) cells treated with actinomycin D (50 ng/ml) for 24 hr, and (d) cells treated with IFN-β for 48 hr with actinomycin D added for the last 24 hr of culture. (e) Bar graph representing the apoptosis of peripheral blood mononuclear cells (PBMC) following culture with IFN-β in the presence or absence of actinomycin D. Data are representative of seven independent experiments. *P < 0·05; **P < 0·001 when compared with cells that were cultured with medium alone; ΔP < 0·05 compared with cultured with actinomycin D alone.

IFN-β prevents exit from the G0/G1 stage of the cell cycle

Our microarray analysis, along with the real-time RT-PCR and western blot experiments, showed that the expression of p21 was significantly elevated following culture with IFN-β. As p21 plays a critical role in inducing cell cycle arrest, we examined the effect of IFN-β on cell cycle progression. As the majority of fresh PBMC are non-proliferating (arrested in the G0/G1 stage), the cells were cultured with PHA to promote cell division in T cells, and the effect of the addition of IFN-β on cell cycle progression was examined using flow cytometry (Fig. 6). As expected, after the addition of PHA (10 μg/ml), the percentage of CD3+ lymphocytes in the G0/G1 stage in control cultures dropped from 92·2% at 0 hr to 52·2% at 24 hr, and was 36·7% at 48 hr (Fig. 6e). In CD3+ lymphocytes that were cultured with IFN-β (1000 IU/ml) and PHA (10 μg/ml), the percentage of cells in G0/G1 decreased from 91·7% at 0 hr to 63·9% at 24 hr and reduced further to 50·3% at 48 hr (Fig. 6e; P < 0·05 compared with cells that did not receive IFN-β, but were cultured with PHA). Also, the percentage of cells in G2 decreased from 24·9% when cultured with PHA alone to 19·65% when IFN-β was added with PHA (Fig. 6g,c,d; P < 0·05). The percentages of cells entering the S phase in cells that were treated with PHA and IFN-β were also lower compared with cells treated with PHA alone (Fig. 6c,d,f). These results show that, in the presence of PHA, IFN-β induces cell cycle arrest at G0/G1 and decreases the transition to the S phase, and thereby decreases the number of cells in the G2 phase.

Figure 6.

Figure 6

Flow cytometric analysis of cell cycle dynamics of CD3+ T lymphocytes stimulated with phytohaemagglutinin (PHA) in the presence or absence of interferon (IFN)-β: (a) control, cells cultured in medium alone; (b) cells cultured with IFN-β for 48 hr; (c) cells cultured with PHA for 48 hr and (d) cells cultured with IFN-β and PHA for 48 hr. The figure shows the profile for one representative from six individuals. Regulation of cell cycle progression by IFN-β: (e) G0/G1 phase, (f) S phase and (g) G2 phase. Error bars represent the mean and standard deviation of values for six individuals. *P < 0·05 for the comparison between cells cultured with PHA alone and cells cultured with PHA plus IFN-β.

Discussion

Using microarray techniques, complemented by real-time RT-PCR and western blot analyses, we show that IFN-β is capable of the activation of a number of genes involved in the p53 signalling pathway in human PBMC. The proteins that were activated downstream of p53 by IFN-β in our study are to some degree similar to those previously described as being activated by genotoxic stress.21,31,32 These include genes that control apoptosis, such as PUMA, NOXA and Bax, and those that induce cell cycle arrest, such as p21 and Sestrin 2. However, unlike the induction of apoptosis that follows activation of p53 after genotoxic stress, IFN-β induces cell cycle arrest in activated lymphocytes. The addition of IFN-β increased the sensitivity of lymphocytes to apoptosis in the presence of Act D. These observations suggest that DNA damage or other additional signals of cellular stress or damage may be necessary for IFN-β to mediate apoptosis in human lymphocytes.

The prevailing view regarding cell lines is that an increase in the constitutive levels of p53 allows time for DNA repair by inducing cell cycle arrest, or instructs the initiation of the cell death if the damage appears irreparable. In our study, the addition of IFN-β to PBMC cultured with PHA restricted the transition of cells from the G0/G1 phase to the S phase (induction of cell cycle arrest) and reduced the number of cells in the G2 phase. Inhibition of the transition from the G1 phase to the S phase involves the activation of a number of genes, of which p21 has been most extensively studied and is a key molecule involved in inhibiting cyclin-dependent kinase 1/2 (CDK1/2)33 The 12-fold increase in the expression of p21 suggests that this protein, along with other genes that regulate cell cycle arrest, as shown in Table 3, is critical for impeding the transition from G0/G1 to S in cells cultured with IFN-β. However, although a number of genes involved in the apoptotic process, such as BAX, PUMA and NOXA, were up-regulated, the cell death programme was not initiated.

The answer to the fundamental question of how activation of p53 leads to either cell cycle arrest or apoptosis is unclear, especially in light of the finding that induction and activation of the p53 signal pathway are not the result of double-stranded DNA breaks such as are seen with IFN-β. One possibility might relate to the activation of different isoforms of p53. Studies on tumour cell lines showed that transcription of p53 was regulated by a single promoter, producing the full-length transcript and two isoforms.34 More recently, an internal promoter of p53 was described, and at least six additional isoforms, some of which act to interfere with the transcription of the full-length protein, have been described.35 We have shown that IFN-β induces the expression of the full-length and beta/gamma isoforms of p53 in PBMC. We also observed that the pattern of induction of the full-length and beta/gamma isoforms seen following stimulation with IFN-β is distinct and different from that seen following genotoxic stress, which predominantly induces full-length p53 only. Thus, the expression of different isoforms of p53 induced by genotoxic injury or IFN-β may also alter the potency of the expression of target genes, which would skew the response towards either cell cycle arrest or apoptosis.

Another possibility might relate to the ability of p53 to bind additional transcription factors and recruit them to the promoter regions of p53 target genes.3639 A study examining the binding of p53 to different DNA-binding sites in yeast and mammalian systems showed a difference in the ability of p53 to bind sites derived from genes involved in cell cycle arrest and/or DNA repair when compared with genes regulating mitochondrial apoptotic pathways.40 These results suggest that, whereas only the binding site sequences are required for p53-dependent activation of the cell cycle arrest genes, additional transcription factors are needed for the induction and expression of many of the pro-apoptotic genes. Hence, recruitment of additional transcription factors to p53 targeted genes may differ between cells cultured with IFN-β and cells subjected to genotoxic stress. Activation of haematopoietic zinc finger protein (Hzf), a p53 target protein, results in the transactivation of pro-arrest genes over that of pro-apoptotic genes,41 and may be favoured in cells stimulated with IFN-β.

Cellular levels of p53 are regulated tightly at the levels of transcription, post-translational modification and degradation. The ISRE that is present in the promoter region of the p53 gene binds to the heterotrimeric complex consisting of STAT1 and STAT2, thereby regulating the transcription of p53.19,42 Our studies showed a rapid induction of STAT1 and STAT2 mRNA and proteins following culture with IFN-β. Considering that the induction of both p53 mRNA and protein was modest at 24 hr and significant at 48 hr, this suggests that the initial amplification of STAT1 and STAT2 is necessary for optimal transcription of the p53 gene. Post-translational modifications of p53 are a critical step in the regulation of cellular levels of p53. In normal resting cells, p53 is rapidly degraded following binding to MDM2, thus ensuring cell integrity.43 IFN-β appears to regulate the expression of p53 both at transcription, by increasing mRNA levels, and also at degradation, by increasing the levels of MDM2. Although the increase in the amount of p53 protein in IFN-β-activated cells may be modest, the cellular consequences of activation of downstream targets, especially p21, and the induction of cell cycle arrest were significant.

Our study indicates additional mechanisms by which IFN-β may provide therapeutic benefits in human disease. Although the potency and kinetics of p53 induction varied among donors, all donor cells showed an increase in p53 expression 48 hr after culture with IFN-β. In autoimmune diseases such as multiple sclerosis, by activating p53 targeted genes, IFN-β can induce cell cycle arrest and thereby restrict the expansion of putative autoreactive lymphocytes. Whether the p53 response governs the optimal clinical response is at present not known. In patients being treated for hepatitis caused by hepatitis C virus, activation of the p53 pathway may dictate the antiviral response in hepatocytes. Acting as an adjuvant, IFN-β, by inducing p53-related pro-apoptotic genes, can enhance the actions of chemotherapeutic drugs in inducing cell death and improve outcomes in the treatment of human neoplastic diseases.

Acknowledgments

This study was supported by a postdoctoral fellowship award from the National MS Society (FG 1737A1/1) to FZ and by an investigator originated grant from Serono Inc, and the William Weaver Fund.

Disclosures

Neither author has any conflict of interest to disclose.

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