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Introduction
Hypoxia is common in solid tumors and is associated
with an aggressive phenotype, and is resistant to
therapy[1]. Hypoxic regions are also common features within rapidly
growing malignant tumors, such as non-small cell lung
cancer (NSCLC)[2]. Cancer cells undergo genetic and adaptive
changes that allow them to survive and even proliferate in
the hypoxic environment[3]. The transcriptional complex
hypoxia-inducible factor (HIF) has emerged as a key regulator
mediating many cellular responses necessary to adapt to
changes in the hypoxic environment[4_6]. In normoxia, the
HIF-1α units are unstable since 2 prolyl residues within the
oxygen-dependent degradation domains of HIF-1α subunits
are hydroxylated by prolyl hydroxylases and dioxygen as a
co-substrate[6]. However, in hypoxia, as frequently occurs
within tumors, there is insufficient oxygen to allow this
process resulting in HIF-1α stabilization and translocation to
the nucleus, where it is able to bind HIF-1α. The complex
then recruits co-activators that bind to specific DNA
hypoxia response elements (HRE), resulting in increased mRNA
transcription[7]. Overexpression of
HIF-1α, a "master" gene in hypoxia, is a frequent occurrence in many tumors,
including NSCLC[8]. However, the pathways that regulate the
HIF-1α expression are unclear today.
Reports have shown several pathways are regulated by
hypoxia and many of the known oncogenic signaling
pathways[9] overlap with hypoxia-induced pathways. One of the
most consistently hypoxia-inducible genes has been
identified as embryo-chondrocyte expressed gene 1
(DEC1)[6].
DEC1[10], also called the enhancer of split and hairy
related protein-2[11] or stimulated with retinoic
acid-13[12], has been identified independently by 3 research laboratories,
each thoroughly studied on a different system of mammalian
differentiation, which is located at chromosome
3p25.3_26[13]. Although the role of DEC1 in human physiology is not
thoroughly clear, it has been reported to have roles in
proliferation[12],
apoptosis[14] and cell
differentiation[15]. Recent
studies[16,17] have also supposed there might be a possible
relationship between DEC1 and HIF-1α expression by
immuno-histochemistal detection of the 2 proteins in many solid
tumors, such as breast cancer and non-small lung cancer.
Thus, DEC1, which might associate with HIF-1α and its
expression in human tumors, might be a direct marker of tumor
hypoxia.
DEC1 expression in normal and tumor lung tissues and
cancer cells has rarely been reported. The potential role of
DEC1 in A549 cells and whether DEC1 is linked to
HIF-1α are unclear. Therefore, in order to further characterize the
significance of DEC1 in normal and neoplastic lung tissues and
A549 cells, we investigated the expression of DEC1 and its
mechanism in lung adenocarcinoma.
Materials and methods
Patients and resources Ethical approval was obtained
for this study from the Local Trials Committee. Paraffin
wax-embedded material lung adenocarcinoma samples
(n=82) and normal lung specimens (n=15) were obtained from Renmin
Hospital of Wuhan University (Wuhan, China) between year
2002 and 2004 and were studied by using serial sections of 4
µm thickness. The patients' ages ranged from 35 to 72 years.
Immunohistochemistal detection of DEC1 and
HIF-1α DEC1 protein was detected using the rabbit polyclonal
antiserum CW27[16]. CW27 antibody was a gift from Dr SB
FOX[7] (Nuffield Department of Clinical Laboratory Sciences, John
Radcliffe Hospital, Headley Way, UK). The CW27 and
HIF-1α antibodies (Boster, Wuhan, China) were applied to the
sections at dilution of 1:1000 and 1:200, respectively. The
primary antibody was applied for 1 h at 37 °C. The sections
were incubated with a secondary mouse anti-rabbit antibody
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 15 min
at 37 °C. Then the sections were performed by the
3,3'-diaminobenzidine (DAB) method. All positive controls
displayed an extensive and intensive positive combined nuclear
and cytoplasmic staining in more than 80% of cells. All
negative controls lacked cells displaying an immune reaction. The
immunohistochemical results for the protein were
classified as follows: _, no staining; +/++, nuclear staining in 1%_50%
of cells and/or with weak cytoplasmic staining; +++,
nuclear staining in more than 50% of cells and/or with strong
cytoplasmic staining.
Cell culture The human lung adenocarcinoma A549 cell
line, obtained from Wuhan University Cell Collection
(Wuhan, China), was cultured in RPMI-1640 medium
supplemented with 10% fetal calf serum (FCS) (Gibco, Grand Island,
USA) and was passaged at 80%_90% confluence with PBS
containing 0.25% trypsin and 1% EDTA. The cell was
maintained at 37 °C in a humidified atmosphere with 21%
O2, 74% N2, and 5%
CO2. For the hypoxic condition, cell culture dishes
were transferred to a Galaxy R CO2 incubator (RS Biotech Co,
Ayrshire, Scotland, England), which was cultured with 1%
O2, 5% CO2, and 94%
N2 for 12 h, 24 h, and 48 h.
Plasmid construction A cDNA fragment coding for the
full open reading frame of human DEC1 was cloned by
RT-PCR from A549 cells at hypoxia for 48 h. Two specific
primers for DEC1 were extended to include appropriate
endonuclease sites to facilitate cloning (Table 1). The sense and
antisense DEC1 cDNA were respectively ligated into the
plasmid pcDNA3.1 (Invitrogen, Carlsbad, California, USA) to
construct the pcDNA-DEC1+ and pcDNA-DEC1_ plasmids
through the T4 DNA ligase (TaKaRa, Dalian, China). The
resulting constructs were subjected to sequencing analyses.
Stable translation A549 cells
(1×108 cells/L) were seeded into 24-well cell culture plates. The cells were transfected
with 1 µg DNA per well using Lipofectamine 2000 (Invitrogen,
Carlsbad, California, USA) in serum-free Opti-MEMI medium
(Invitrogen, Carlsbad, California, USA), according to the
manufacturer's protocol. Selection for stably transfected
cells with 0.8 g/L G418 started 2 d after transfection. After 4
weeks of screening, these polyclonal, stably transfected cells
were trypsinized. Monoclones were picked into a 6-well plate
and cultured in RPMI-1640 medium with 10% FCS.
HIF-1α antisense oligonucleotide (AS-ODN) treatment
The effect of HIF-1α in epithelial cells was accomplished by
antisense oligonucleotide loading as described
previously[18], using phosphorothioate derivatives of antisense (5'-GCC GGC
GCC CTC CAT-3') or control sense (5'-ATG GAG GGC GCC
GGC-3') oligonucleotides. The stably transfected
pcDNA-DEC+/_ A549 cells were relatively washed in serum-free
medium. Then phosphorothioate control or antisense
oligonucleotides (10 µmol/L) were transfected into the cells.
After 4 h incubation at 37 °C, the transfected cells were
exposed to hypoxia for 24 h.
MTT assays A549 cells and the stably transfected
pcDNA-DEC+/_ A549 cells in the exponential phase of growth
were relatively plated into 96-well plates at
1×104 cells per well. After normoxia and hypoxia for 12 h, 24 h, and 48 h, The
cell viability was determined by MTT
assay[19].
Flow cytometric detection of apoptotic cells
After the A549 cells and the stably transfected pcDNA-DEC+/_ A549
cells were incubated with hypoxia for 0 h, 12 h, 24 h, and 48 h,
the cells were analyzed by flow cytometry using a FACScan
(Beckman Coulter, Chaska, MN, USA).
RT-PCR detection of DEC1 and HIF-1α Reverse
transcription of total RNA was performed using M-MLV reverse
transcriptase (Promega, San Luis Obispo, CA), and each PCR
reaction was performed with a TaKaRa
TaqTM hot start version (TaKaRa). The specific primers of
HIF-1α, anti-sense DEC1, and β-actin synthesized by Sangon Co (Shanghai,
China) are shown in Table 1. RT-PCR detection of the
HIF-1α transcript was performed using 2 µg cDNA at 94 °C for
the 5 min initial denaturation step, followed by 32 cycles at
94 °C for 50 s, 57.6 °C for 50 s, and 72 °C for 50 s. The
antisense DEC1 reaction condition of PCR was as follows:
94 °C for the 5 min initial denaturation step, followed by 30
cycles at 94 °C for 50 s, 57.2 °C for 50 s, and 72 °C for 50 s. As
a control for the equal amount of template, the β-actin gene
was amplified using 2 µg cDNA as a template in 32 PCR
cycles at 59.5 °C annealing temperature. Each band was
analyzed on GDS8000 image analysis system (American UVP
Co, Upland, USA). β-actin staining served as the internal
standard.
Western blotting detection of HIF-1α and DEC1
Western blotting was performed as previously described with
some modifications[20]. The cell pellets were homogenized in
extraction buffer (50 mmol/L Tris-HCl, pH6.8, 0.1% SDS, 150
μmol/L NaCl, 100 mg/L phenylmethylsulfonyl fluoride, 1
mg/L aprotinin, 1% NP-40 and 0.5% sodium orthovanadate), and
incubated at 4 °C for 30 min, and centrifuged 20 min at
12 000 g/min. The total protein in the cell lysate was measured with
the Bio-Rad colorimetric kit (Bio-Rad, Hercules, CA, USA).
For Western blotting, 50 µg protein lysate was separated in
10% SDS-PAGE and transferred onto nitrocellulose
membranes (0.45 µm, Millipore, Billerica, MA, USA) The
membranes were incubated for 24 h at 4 °C with the antibody
CW27 and anti-HIF-1α (Santa Cruz Biotechnology,
USA), respectively. The CW27 and HIF-1α antibodies were used
at the concentration of 1: 500 and 1:450. Detection of
monoclonal and polyclonal antibodies was performed using
horseradish peroxidase-conjugated goat anti-rabbit
immunoglobulins for 1 h at room temperature, respectively. Signals
were detected with an enhanced chemiluminescence kit
(Amersham Pharmacia, Buckinghamshire, UK). β-actin
staining served as the internal standard for all
membranes.
Immunocytochemistal detection of DEC1 and
HIF-1α A549 cells (1×108 cells/L) were seeded into 6-well cell culture
plates in 2 mL RPMI-1640 medium with 10% FCS to attain
70%_80% confluence. These cells were divided into 4 groups
treated for: 0 h, 12 h, 24 h, and 48 h group at hypoxia,
respec-tively. These cells were respectively fixed in 4%
paraformaldehyde for 10 min, and stained for immunocytochemistry by
following the procedures used for immunohistochemistry.
The primary CW27 and HIF-1α antibodies were applied to
sections at dilutions of 1:1000 and 1:150, respectively.
Statistical analysis Results were shown as mean±SD of
the number of experiments indicated in the figure legends.
Statistical analysis was done by Student's
t-tests, ANOVA, and Tukey's
post-hoc tests. Statistical significance was set
at a level of P<0.05.
Results
Expression of DEC1 and HIF-1α in normal and tumor
lung tissue DEC1 was weakly expressed in the normal lung
tissues and specifically located in the nuclei and cytoplasm
in 48.8% lung adenocarcinoma. HIF-1α was expressed in 3
(20%) normal human lung tissues, and was evaluated in 43
(52.4%) lung adenocarcinomas (Figure 1). There was a
statistically significant correlation between the expression of
HIF-1α and DEC1 (Table 2, P<0.05).
Inhibitory effect of hypoxia on the growth of A549 cells
and the stably transfected pcDNA-DEC+/_ A549 cells
No changes of the growth of A549 cells were observed in the
normoxic groups. However, the prolioferation of A549 cells
at hypoxia was strongly inhibited in a time-dependent
manner (Figure 2, Table 3, P<0.05). Moreover, the growth of the
stably transfected pcDNA-DEC+ A549 cells was higher than
those of A549 cells at the same time point at hypoxia. The
growth of the stably transfected pcDNA-DEC_ A549 cells
was lower than those of A549 cells at the same time point at
hypoxia (P<0.05).
Hypoxia induced apoptosis in A549 cells and the stably
transfected pcDNA-DEC+/- A549 cells When exposed to
hypoxemic environment for 0 h, 12 h, 24 h, and 48 h, apoptotic
A549 cells were detected by flow cytometry. Compared to
the normoxic groups, these apoptosis ratios in A549 cells
obviously increased in a time-dependent manner (Figures
3_5, Table 4, P<0.05). Moreover, the lower apoptosis ratio of
the stably transfected pcDNA-DEC+ A549 cells and the higher
apoptosis ratio of the stably transfected pcDNA-DEC_ A549
cells were detected at the same time point at hypoxia
(P<
0.05).
Hypoxia increased DEC1 and HIF-1α gene
transcription in A549 cells The gene transcription levels of both
DEC1and HIF-1α at hypoxia were higher than those under
normoxia conditions (Figure 6, P<0.01). Through the linear
correlation analysis about the aboved-mentioned results,
DEC1 mRNA probably strongly related with HIF-1α mRNA
for 24 h at hypoxia (r=0.822, P<0.01).
Hypoxia increased DEC1 and HIF-1α expression in A549
cell line Under a normoxic environment, DEC1 expression
was detected at a low level, while HIF-1α was expressed in
the nuclei and cytoplasm of A549 cells. And both DEC1 and
HIF-1α expression were induced by hypoxia in A549 cells in
a time-dependent manner (Figure 7, 8). The staining
intensity was stronger in the 48 h group than that of the 12 h and
24 h group (P <0.05).
HIF-1α mRNA and protein expression in the stably
transfected pcDNA-DEC+/_ A549 cells There was no obvious
difference of HIF-1α expression between the non-transfected
and empty plasmid transfected groups. However,
HIF-1α showed low gene transcription level in the pcDNA-DEC+
group (Figure 9, P<0.05), and high gene transcription level
of HIF-1α was detected in the pcDNA-DEC_ group compared to the non-transfected group
(P<0.05). After normalization with β-actin, the
HIF-1α protein significantly decreased in the cells transfected with plasmid pcDNA-DEC+
(Figure 10, P<0.05). On the contrary,
HIF-1α protein increased in the cells transfected with plasmid pcDNA-DEC_
(P<0.05). These results were in accordance with the mRNA
expression.
DEC1 mRNA and protein expression in the
stably transfected pcDNA-DEC+/_ A549 cells with or without
HIF-1α AS-ODN treatment This densitometric analysis revealed
there was no obvious different in the intensity of DEC1 mRNA
and protein levels with or without HIF-1α AS-ODN in the
stably transfected pcDNA-DEC+ A549 cells (Figure 11, 12,
P>0.05). The similar results were also found in the stably
transfected pcDNA-DEC_ A549 cells.
Discussion
In this study, the HIF-1α protein was strongly expressed
in the lung adenocarcinoma and A549 cells. The growth of
A549 cells was inhibited at hypoxia in a time-dependent
manner through the MTT test. Moreover, hypoxia could
obviously induce A549 cells apoptosis. We also found the mRNA
and protein levels of the HIF-1α increased at hypoxia. These
results reveal that HIF-1α plays an important role in the
growth of A549 cells at hypoxia.
We also studied a new factor, DEC1. DEC1 is a new and
structurally different class of the helix-loop-helix (HLH)
protein. We examined the expression of the DEC1 and
HIF-1α protein, which were both induced by hypoxia. These
results were accorded with the previous
study[21]. The DEC1 protein was variably expressed in lung adenocarcinomas.
DEC1 was weakly expressed in the normal lung tissues.
Previous study using immunohistochemical methods have
reported a variable positivity of DEC1, ranging from 0 to
70% in the non-small cell lung
cancer[16]. The data indicate that DEC1 play an important role in the process of lung
adenocarcinoma progression. The data also reveals DEC1 in the
lung adenocarcinoma directly links to HIF-1α, providing
strong evidence that DEC1 is indeed another marker of
tumor hypoxia.
Our results show that hypoxia significantly increases the
mRNA and protein levels of DEC1 and HIF-1α in the lung
adenocarcinoma A549 cells in a time-dependent manner. We
observed the strong protein expression of DEC1 until 48 h at
hypoxia, and the HIF-1α protein was strongly expressed from
12 h at hypoxia. The upregulation of DEC1 and
HIF-1α reveals that DEC1 and HIF-1α probably act as the key
mediators of the hypoxia-regulated process. Moreover, we found
DEC1 mRNA was significantly associated with HIF-1α mRNA
at hypoxia for 24 h. This data further indicate that DEC1
regulates HIF-1α or that HIF-1α has an effect on DEC1 at
hypoxia.
We constructed the pcDNA-DEC+ and pcDNA-DEC_ plasmids and selected the stably transfected pcDNA-DEC+
and pcDNA-DEC_ A549 cells lines. The MTT test showed
hypoxia could inhibit the growth of the stably transfected
pcDNA-DEC+ and pcDNA-DEC_ A549 cells in the
time-dependent manner. Moreover, the growth of the stably
transfected pcDNA-DEC+ A549 cells was faster than those of
A549 cells. On the contrary, the growth of the stably
transfected pcDNA-DEC_ A549 cells was lower than those of
A549 cells. We also found hypoxia could induce the
apoptosis of the stably transfected pcDNA-DEC+ and
pcDNA-DEC_ A549 cells in a time-dependent manner.
Compared to the A549 cells, the stably transfected pcDNA-DEC+
A549 cells showed a lower percentage of apoptosis while
the stably transfected pcDNA-DEC_ A549 cells showed a
higher percentage of apoptosis.
To investigate the interaction between DEC1 and
HIF-1α, we carried out RT-PCR and Western blotting to detect
the mRNA and protein levels of HIF-1α. HIF-1α showed low
expression level in the pcDNA-DEC+ group, but a high level
of HIF-1α was detected in the pcDNA-DEC_ group. Thus, it
suggests that DEC1 might be the target gene of
HIF-1α, and negative feedback exists between DEC1 and
HIF-1α.
To confirm this conclusion, we designed the antisense
and control sense oligonucleotides of HIF-1α. At mRNA
and protein levels, there was no obvious different intensity
of DEC1 with or without HIF-1α AS-ODN in the
pcDNA-DEC+ or pcDNA-DEC_ groups. Through the treatment with
the HIF-1α AS-ODN, we found that HIF-1α AS-ODN had no
effect on DEC1expression in the stably transfected
pcDNA-DEC+/_ A549 cells. So we identified that DEC1 may
down-regulate HIF-1α at both mRNA and protein levels at hypoxia
in A549 cells. However, more research on the relationship
between DEC1 and HIF-1α is still required in the future.
In conclusion, our results reveal that DEC1 has
significant roles in the process of lung adenocarcinoma
progression and is another marker of tumor hypoxia associated with
HIF-1α. DEC1 downregulates HIF-1α at both mRNA and
protein levels at hypoxia in the lung adenocarcinoma A549
cells.
Acknowledgments
We are grateful for excellent technical assistance from
State Key Laboratory of Virology, Medical School, Wuhan
University, Wuhan, China.
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