Introduction
Apoptosis, programmed cell death, plays a central role in the development and homeostasis of all multicellular organisms [
1,
2]. In humans, both excessive and insufficient apoptosis can lead to severe pathological consequences. Suppression of the apoptotic mechanism causes autoimmune diseases and is a hallmark of cancer [
3,
4]. The inhibitor of apoptosis (IAP) gene family encodes proteins that have emerged as key, intrinsic inhibitors of the caspase cascade and thus represent critical regulatory factors in apoptosis signaling [
5]. Of the known IAP, X-linked inhibitor of apoptosis (XIAP) protein is the best characterized and the most potent inhibitor of apoptosis [
6]. XIAP-associated factor 1 (XAF1) was identified as a novel negative regulator of XIAP based on its ability to bind with XIAP [
7]. XAF1 is shown to dramatically sensitize cancer cells to a wide spectrum of apoptotic triggers such as TNF-related apoptosis-inducing ligand (TRAIL) and etoposide treatments [
8]. XAF1 was implicated as a tumor suppressor based on the observation of lower expressions in various cancer cell lines. The loss of XAF1 is associated with malignant tumor progression in a variety of cancers [
9,
10]. However, so far, the mechanisms by which XAF1 expression is regulated in digestive system cancer are not well known. In this study, we attempted to study the transcriptional regulation of XAF1 expression in digestive system cancer cell lines.
Methods
Cell culture
Human normal fetal liver cell line L02, human embryonic kidney (HEK293) cells, human hepatoma cell line HepG2, human colon cancer cell line LoVo, and human gastric cancer cell line AGS were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal calf serum at 37°C in a humidified atmosphere containing 5% CO
2. For heat stress (HS) treatment, the human cancer cells were incubated in complete medium at 42°C HS medium for 30 min, followed by culture in normal medium at 37°C for 24 h [
11,
12].
Reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted from all cell lines using Trizol reagent. Two micrograms of RNA was converted to cDNA by reverse transcription using Oligo(dT)15 primer and Moloney murine leukemia virus (MMLV) reverse transcriptase. Of the 20-mL resulting complementary DNA, 1 mL was used in each PCR. The primers used to amplify XAF1 mRNA were as follows: forward, 5'-TCAGCAGTTGGAAGACACAG-3', and reverse, 5'-TCAGCAGTTGGAAGACAGC-3'. The primers used to amplify b-actin were as follows: forward, 5'-TGGCACCACACCTTCTACAA-3', and reverse, 5'-AGCCTGGATAGCAACGTACA-3'. The PCR products were subjected to electrophoresis on 1.5% agarose gel and visualized by GoldView staining.
Amplification and subcloning of 1395–bp 5′ flanking region of XAF1 gene
Human genomic DNA was extracted from cancer cells. The primers used to amplify 5′ flanking region of XAF1 gene were: forward, 5'-TTTTTAGTAGAGACGGGGTTTCAC-3', and reverse, 5'-TTCGGTTGAGTTTCGTTTCTTGC-3'. PCR amplified fragment was about 1395 bp that was subcloned into a T/A clone vector of pBS-T (Tianjin, China) to form pBS-T-XAF1p. The resultant construction was confirmed by enzyme digestion and sequence analysis.
Construction of luciferase reporter plasmid derived from XAF1 promoter
The 1395-bp fragment was excised from pBS-T-XAF1p with KpnⅠ and SacⅠ and ligated into the equivalent site of pGL3-basic vector to form pGL3-XAF1p. The resultant construction was confirmed by enzyme digestion.
Construction of green fluorescence protein reporter plasmid derived from XAF1 promoter
The 1395-bp fragment was excised from pBS-T-XAF1p with XhoⅠ and BamHⅠ and ligated into the equivalent site of pEGFP-1 vector to form pEGFP-XAF1p. The resultant construction was confirmed by enzyme digestion.
Transient transfection
Transient transfection was performed using the Lipofectamine2000 reagent (Gibco) according to the manufacturer’s instructions. Cells of 1 × 105 were seeded in each well of 24-well plates. The cells were incubated until 70%-80% confluence. Cells in each well were transfected with 0.8 μg plasmids DNA by 1 μL Lipofectamine2000 reagent. The Renilla luciferase reporter pRL-TK plasmid was cotransfected as an internal control in pGL3 construction group. pEGFP-N1 was used as a positive control in pEGFP construction group. EGFP positive cells were detected using an inverted fluorescence microscope (Olympus IX71, Japan) at an excitation wavelength of 488 nm.
Dual luciferase reporter assay
Luciferase activity of the reporter gene was evaluated in a variety of cell lines using the Dual-Luciferase Reporter Assay kit (Promega Corp., USA). After a 24-h recovery period, transfected cells were harvested and lysed in the presence of 100 mL of passive lysis buffer. The firefly and Renilla luciferase activities were measured using a luminometer Lumat LB 9507 (EG & G, Perth, Australia). Cell lysate of 20 mL was transferred into the luminometer tube containing 100 mL LAR II, and firefly luciferase activity was firstly measured; then, Renilla luciferase activity was measured after 100 mL of stop reagent was added. Promoter activity was presented as the fold of relative luciferase unit (RLU) compared with the pGL3-basic vector as control. RLU indicates values of firefly luciferase unit/values of Renilla luciferase unit. All the values were expressed as a mean of three independent experiments.
Statistical analysis
Quantitative data were expressed as or median (range). Differences between groups were analyzed using the t test. All statistical analyses were accomplished by SPSS 13.0 software (SPSS, Chicago, USA). P value of less than 0.05 was considered statistically significant.
Results
XAF1 mRNA expression in five cell lines
The levels of XAF1 mRNA in HEK293, L02, HepG2, LoVo, and AGS cells were as follows: 1.041 ± 0.153, 1.179 ± 0.192, 0.364 ± 0.046, 0.457 ± 0.037, and 0.409 ± 0.046, respectively. XAF1 mRNA was strongly expressed in HEK293 and L02 cells but weakly expressed in digestive system tumor cell lines.
Amplification and subcloning of 1395–bp 5′ flanking region of XAF1 gene
As shown in Fig. 1, the 1395–bp fragment was amplified by PCR in electrophoresis. Sequencing of the full insert confirmed the identity to the published sequence.
Construction and identification of pGL3-XAF1p and pEGFP-XAF1p
Recombinant plasmids pGL3-XAF1p and pEGFP-XAF1p were confirmed by restriction enzyme digestions (Fig. 2).
Green fluorescence protein expression driven by XAF1 promoter in five cell lines
A large number of EGFP-positive cells could be observed by fluorescence microscopy in 5 cell lines transfected with pEGFP-N1. However, extremely weak green fluorescence was observed in digestive system tumor cell lines transfected with pEGFP-XAF1p as compared with that in HEK293 and L02 transfected with pEGFP-XAF1p.
Luciferase expression driven by XAF1 promoter in variety of cell lines
The activities of pGL3-XAF1p were 6.97 ± 0.74, 6.29 ± 0.55, and 7.13 ± 0.89 in HepG2, LoVo, and AGS cells, and 15.64 ± 0.45 and 13.74 ± 0.61 in HEK293 and L02 cells, respectively (Fig. 3). Luciferase reporter assay showed that XAF1 promoter activity was much lower in digestive system tumor cell lines compared with that of HEK293 and L02 cells, which was similar to the results of GFP reporter gene analysis. After treatment with HS, the activities of pGL3-XAF1p in HepG2, LoVo, and AGS cells were 2.08 ± 0.19, 2.80 ± 0.26, and 3.14 ± 0.28, respectively. The stressed cancer cells had significantly lower activity of XAF1 promoter than the unstressed cancer cells (HepG2: P = 0.008, LoVo: P = 0.041, AGS: P = 0.01, respectively. t-test, 2-tailed) (Fig. 4).
Discussion
It is well known that the regulation of gene expression is a multistep process in eukaryotes, and the transcriptional regulation plays an important role in it. The interaction between transcription factors and their DNA recognition sites is important for the regulation of the network that controls gene expression [
13]. Reporter gene technology developed in recent decades, especially in the past 10 years, has been identified as the most basic tool for the study of gene promoter activity.
The term reporter gene is used to define a gene with a readily measurable phenotype that can be distinguished easily from background of endogenous proteins [
14]. Dual reporters are used to make relational or ratiometric measurements within an experimental system. Typically one reporter served as an internal control to which measurement of the other reporter is normalized. By this method, it is possible to minimize inherent variability that can undermine experimental accuracy. On the one hand, the superior dual-reporter technology integrating the assay of firefly luciferase with the Renilla luciferase assay offers the exceptional speed, sensitivity, and linearity achievable of two luciferase reporter assays in a single-tube format. On the other hand, GFP reporter gene offers a feature that is easy to assay. So these two different reporter gene technologies were applied in this study to monitor XAF1 promoter activity in a variety of cell lines.
In humans, XAF1 exists as a single gene with eight exons, which are localized on chromosome 17p13.2. XAF1 is ubiquitously expressed in normal tissues but is found at extremely low levels in the majority of the NCI 60 cell line panel of cancer cells, suggesting a potential tumor suppressor role [
7]. Recent studies demonstrated that XAF1 levels are drastically decreased in digestive system cancer such as gastric cancers [
9] and colon cancers [
15]. In this study, the results of RT-PCR analysis also showed that XAF1 mRNA was underexpressed in digestive system cancer cell lines. The reduction of XAF1 mRNA expression in digestive system cancer cell lines implicates that XAF1 plays a role in the malignant transformation process. Therefore, it is very important to study the regulation of tumor suppressor XAF1 expression in human digestive system cancers.
It was reported that the transcription initiation site of XAF1 gene was located in the -26 nt adenosine upstream of the ATG initiator by 5′-RACE, indicating the presence of a potential repressor element between -592 nt and -1414 nt [
12].
Given the importance of promoter activity of gene transcription, 1395-bp fragment of XAF1 gene promoter was amplified by PCR in the present study. To evaluate its promoter activity, the 1395-bp fragment was cloned into pGL3-basic vector and pEGFP-1 vector, respectively. Our results have shown that pGL3-XAF1p exhibited a much lower level of luciferase transcription in digestive system tumor cell lines compared with HEK293 and L02 cells, and the number of EGFP-positive cells in digestive system tumor cell lines transfected with pEGFP-XAF1p was less than that of HEK293 and L02 cells. That is to say, the profile of XAF1 promoter activity was almost parallel with the profiles of XAF1 mRNA in 5 cell lines.
Stress agents and conditions include physiological, environmental, or pathological stimuli [
16-
18]. It was well known that cancer cells have encountered higher level of stress pressure, both exogenous and endogenous [
19,
20]. Some stress agents and conditions, such as oxidative stress, have been considered to be a tumorigenic agents at low concentrations [
21,
22]. Heat-shock transcription factors were originally characterized as regulators of the expression of heat-shock protein or non–heat-shock protein, through binding to specific sequences (“heat-shock element” [HSE]), typically a pentanucleotide nGAAn structure oriented in inverted dyad repeats [
23-
25]. nGAAn/nTTCn contigs were also found between -592 nt and -1414 nt of XAF1 promoter region. As XAF1 was a proapoptotic protein, treatment with HS reduced the activity of XAF1 promoter and induced low expression of XAF1 in digestive tumor cell lines. Meanwhile, low expression of XAF1 promoted cytoprotection under stress in digestive cancer cells.
These findings provide a basis for future studies addressing the precise molecular mechanism for the transcriptional repression of XAF1 in digestive system tumors.
Acknowledgements
This study was supported by grants from the National Natural Sciences Foundation of China (Grant No. 30872237), Research Fund for the Doctoral Program of Higher Education of China (No. 20070487007), and the National Program for Basic Research (973 project) of China (No. 2007CB512900). The authors thank Prof. Xianxi LIU from the Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University at Jinan, China, for providing the plasmids, pGL3-basic, and pRL-TK.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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