Dp71 depleted HBE cells displayed increased DNA damage and apoptosis induced by H2O2
Human bronchial epithelium (HBE)-Dp71 anti-sense(AS)cells with stably transfected Dp71 siRNA plasmids were prepared for further exploration of Dp71 biological traits in cells other than PC12. HBE-Dp71AS cells displayed increased DNA damage induced by H2O2. Apoptosis of HBE-Dp71AS cells induced by H2O2 was increased via enhancing caspase 3, caspase 8 and caspase 9. HBE-Dp71AS cells also displayed decreased proliferation and clonogenic formation. RAD51 was proved to be a new binding partner of Dp71 by co-immunoprecipitation (Ip) and immunofluorescence. Reduced RAD51 mRNA and protein levels were observed in HBE-Dp71AS cells. Decreased lamin B1, focal adhesion kinase (FAK), phosphorylated focal adhesion kinase (p-FAK) and phosphorylated protein kinase B (p-AKT) were detected in the HBE-Dp71AS cells, which functioned together with RAD51 as the molecular explanations for the character alterations of HBE-Dp71AS cells.
KeywordsDp71 DNA damage Apoptosis RAD51 FAK
Protein kinase B
Duchenne muscular dystrophy
Focal adhesion kinase
Human bronchial epithelium
Quantitative real-time polymerase chain reaction
Single strand break
Dystrophin Dp71 is one of the most widely expressed isoforms of dystrophin, the pathogenic gene of Duchenne muscular dystrophy (DMD), an X-linked recessive disorder . Functioning as one of the most ubiquitously expressed isoforms of dystrophin, Dp71 is a 70- to 75-kDa protein located in all tissues except skeletal muscle [2, 3]. Previous research on Dp71 identified its crucial role for cell adhesion, neuronal differentiation and the cell cycle in PC12 cells. Dp71 was proved to be a putative tumor suppressive gene in gastric cancer [4, 5, 6]. Our preliminary clinical work also identified reduced Dp71 expression in lung cancer. Considering HBE as a usual cell model for pulmonary functional analysis, a shRNA strategy was used to knock down Dp71 in HBE to further clarify its biological significance. HBE-AS cells displayed increased DNA damage under oxidative stress, and decreased proliferation and clone formation capabilities. In a caspase-dependent way, HBE-AS cells displayed an increased apoptosis rate induced by H2O2. Our further characterization of HBE-AS cells identified RAD51, lamin B1, FAK and AKT to be the molecular explanations for the altered phenotypes of HBE-AS cells.
Material and methods
Construction of Dp71 short hairpin RNA plasmid
According to the open reading frame of the human Dp71 gene (NM_004015), one siRNA sequence (5′-gcactttaattatgacatc-3′) was selected. The scrambled sequence (5′-ttctccgaacgtgtcacgt-3′) which has no significant homology with human gene sequences was included as a negative control. Two complementary oligonucleotides for Dp71 (5′-gatcccgtctttagctgacctgaataactcgagttattcaggtcagctaaagactttttggat-3′ and 5′-agctatccaaaaagtctttagctgacctgaataactcgagttattcaggtcagctaaagacgg-3′), and for the negative control (5′-gatcccttctccgaacgtgtcacgtctcgagacgtgacacgttcggagaatttttggat-3′ and 5′-agctatccaaaaattctccgaacgtgtcacgtctcgagacgtgacacgttcggagaagg-3′), were synthesized by Invitrogen. Sense or antisense strands are in bold letters and stem loop sequences are in italics. They were annealed to generate double-stranded DNAs and ligated into the linearized shRNA (short hairpin RNA) eukaryotic expression vectors purchased from Genechem (Shanghai, China, containing hU6-MCS-CMV-GFP-SV40-Neomycin elements) to construct Dp71 shRNA or control empty shRNA vectors, which were termed Dp71AS and Dp71 empty shRNA vector (E) respectively. The nucleotide sequences of the plasmids were verified by automated DNA sequencing.
Cell culture and generation of stable transfectants
HBE was obtained from the Culture Center, Chinese Academy of Medical Sciences (Shanghai, China). HBE cells were cultured in the same condition as described previously . For stable transfectants, 5 μg of Dp71shRNA plasmid or 5 μg of control empty shRNA plasmid was mixed with 15 μl of Lipofectamine in serum- and antibiotics-free 1640, and the DNA/Lipofectamine mixture was added to the cell culture medium and incubated in the incubator for 4 h. The transfection mixture was removed and cells were maintained in 1640 supplemented with sera. Selection of stable transfectants was initiated with 600 μg/ml of G418 (Invitrogen) 48 h after transfection, a neomycin analog. The stable transfected HBE cells were named HBE-Dp71AS and HBE-Dp71E respectively.
Isolation of cell extracts and western blot analysis
Cultured cells were collected by centrifugation at 1200 rpm for 5 min, and washed twice with PBS. Protein extraction, concentration determination, 10% SDS-PAGE electrophoresis, and membrane incubation with the corresponding primary antibody (rabbit anti-dystrophin, rabbit anti-RAD51 polyclonal antibody purchased from Abcam; rabbit anti-FAK polyclonal antibody, p-FAK polyclonal antibody; rabbit anti-Akt polyclonal antibody, p-Akt polyclonal antibody; rabbit anti-phospho-histone H2AX (γH2AX; Ser 139) antibody (Bioworld Technology, Inc) was performed as described previously. After three washes with TBS-T, horseradish peroxidase-conjugated anti-rabbit IgG was used as the secondary antibody and developed using the ECL Western blotting analysis system (Amersham-Pharmacia).
Quantitative real-time polymerase chain reaction (QRT-PCR) and RT-PCR
The following primers were used and they produced a 157 bp PCR product for Dp71: 173 bp PCR product for FAK, 146 bp PCR product for lamin B1, 160 bp PCR products for RAD51 and 181 bp PCR product for 18 s. The primers are: LMNB1 (Human Accession NM_005573) F:5′tccaggagaaggaggagctg3′, R:5′ggtctcgtagagcgccttg3′; Dp71 (Human Accession NM_004017.2) F:5′ttggcagtcaaacttcggactc3′,R:5′gtgtcctctctcattggctttccag3′; FAK (Human Accession L13616.1) F: 5′ tccccagagctcctcaagaa 3′,R: 5′ tactcgctccattgcaccag3′; RAD51 (Human Accession D14134.1) F: 5′gggaagacccagatctgtca 3′, R: 5′catcactgccagagagacca 3′; Human 18S (NM_022551) F: 5′ aaatagcctttgccatcactgcc3′,R: 5′ gttcaagaaccagtctgggatc3′.
Cell viability assay
The cell viability was assessed by conducting the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assay and results interpretations were performed as described previously .
Plate colony formation assay
Clone formation assay was performed as described previously. Clone formation efficiency was calculated according to the formula: (clone number/plated cell number) × 100% .
Apoptosis of HBE, HBE-Dp71AS and HBE-Dp71E cells in the log growth phase were induced by 0.2 mM H2O2 (Sigma, St. Louis, USA) for 16 h. The cells then were harvested by trypsinization for flow cytometry. Apoptosis was quantified using the PE Annexin V apoptosis detection kit (BD Pharmingen, San Diego, USA) according to the manufacturer’s protocol. Cell analyses were made using a FACSCalibur flow cytometer (Becton-Dickinson, Mountain View, CA) and CellQuest software (BD Biosciences). Each assay was repeated 3 times .
Measurement of caspase 3, 8, 9 activities
The caspase fluorescent assay kits specific for caspase 3, caspase 8 and caspase 9 (BioVision, San Francisco, USA) were used to detect caspase activation by measuring the cleavage of a synthetic fluorescent substrate. Cell treatment and fold increases in caspase 3, caspase 8 and caspase 9 activities were determined as described previously .
Alkaline comet assay for DNA damage
To perform the comet assay, the cell suspension of each cell group was mixed with low melting-point agarose at 37 °C, to a final concentration of 0.7%. The mixture (15 μl) was pipetted onto slides pretreated with 0.5% normal-melting-point agarose, to retain the agarose cell suspension. The drop containing the cells was covered with a glass cover slip (24 mm × 24 mm) and left at 4 °C for 5 min. The cover slips were gently removed and the slides were then ready for processing. The alkaline comet assay was performed using the basic rationale of Singh et al. The slides were then incubated in the dark for 30 min in cold electrophoresis buffer (300 mM NaOH, 1 mM EDTA, 1% (v/v) DMSO, pH 13) to allow the DNA to unwind before electrophoresis at 25 V for 25 min. After neutralization with 0.5 M Tris–HCl (pH 8.0), the slides were stained with 50 μl ethidium bromide (30 μg/mL, Absin Bioscience Inc., China). Finally, the images were taken by fluorescence microscope and at least 120 randomly selected cells (30 cells from each of the three replicate slides) were analyzed per sample and analyzed using the Comet Assay Software Pect (CASP 1.2.3 beta 1) (http://casplab.com/download). Parameters of tail moment (% DNA in tail × tail length), tail length, and percent of DNA in tail, the most frequently used parameters in the comet assay, were used in this study.
Immunofluorescence and confocal microscopy analysis
The immunofluorescence and confocal microscopy analysis of Dp71, Rad51 and γ-H2AX in HBE was as follows: After the three HBE cells were cultured on glass coverslips for 24 h, cells were treated with 200 μM H2O2 for 30 min as described previously, treated cells and untreated cells were incubated overnight at 4 °C with the primary anti-dystrophin, anti-RAD51 and anti-γ-H2AX antibody. Cells were incubated for 10 min at 37 °C with 1 mg/ml 49,6-diamidino-2-phenylindole (DAPI) for counterstaining, After washing, coverslips were mounted on microscope slides with VectaShield (Vector Laboratories, Inc., Burlingame, CA, USA) and analyzed in a confocal and multiphoton microscope (TCS-SP5, Leica Microsystems, Heidelberg, Germany), using an oil immersion 636 objective. Co-localization of FITC, TRITC, and DAPI staining was analyzed in single optical sections obtained for two channels throughout the Z axis.
Total protein extracts in a final volume of 250 ml were incubated overnight at 4 °C with 5 μg of rabbit anti-lamin B1, 5 μg of rabbit anti-Dp71 antibody, 5 μg of rabbit anti-FAK, and 5 μg of rabbit anti-RAD51 antibody, previously bound to protein G magnetic beads (Millipore). An irrelevant rabbit polyclonal antibody bound to protein G magnetic beads was performed as a negative control. The immune complexes were precipitated by placing the tube into the magnetic stand (Millipore) and washing 3 times with 500 μl of PBS containing 0.1% Tween 20. Precipitated proteins were separated by SDS-PAGE and analyzed by Western blotting with mouse anti-lamin B1, mouse anti-Dp71 antibody, mouse anti-RAD51 and mouse anti-FAK antibody.
All assays were repeated 3 times to ensure reproducibility. Results were displayed as mean ± SE. One-way ANOVA and LSD were used to analyze all experimental data. All statistical analyses were performed with SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA). P < 0.05 was considered as indicating a statistically significant difference.
Establishment of Dp71 depleted HBE cell lines
HBE-Dp71 AS cells displayed increased DNA damage induced by H2O2
Dp71 knockdown HBE cells displayed increased H2O2-induced apoptosis via enhanced caspase 3, caspase 8 and caspase 9 activation
H2O2 can induce the apoptosis via activation of both the extrinsic death receptor apoptosis pathway and the intrinsic apoptotic pathway. Caspase 9 is an initiator of the intrinsic pathway of apoptosis; caspase 8 is an indispensable enzyme of the extrinsic pathway. The activation of both caspase 8 and 9 will ultimately result in the activation of the effector caspase 3. As shown in Fig. 3c, significant fold change of caspase 3 in HBE-Dp71AS was observed compared with HBE and HBE-Dp71E cells after they had been treated with H2O2. Significant fold changes of caspase 8 and 9 are shown in Fig. 3d and e. To sum up, the ablation of Dp71 increased the H2O2-induced apoptosis via enhancing the activations of caspase 3, 8 and 9.
Dp71 knockdown HBE cells displayed decreased proliferation rate
HBE-Dp71 AS cells displayed reduced clone formation capabilities
The anti-cell proliferation effect of Dp71 was assessed by a clonogenic formation assay. There was a significant reduction in the number and size of foci in HBE-Dp71 AS cells compared with HBE-Dp71E and HBE cells (Fig. 4b). The clone formation efficiency for HBE-Dp71E, HBE and HBE-Dp71AS cells was 85.33 ± 1.72, 88.73 ± 4.67 and 67.53% ± 3.45% respectively. The clone formation efficiency for HBE-Dp71AS cells was 20% less than that in HBE-Dp71E and HBE cells; the differences were statistically significant (Fig. 4c). Together with the results from the MTT assay, it is found that ablation of Dp71 protein can significantly inhibit the growth of HBE cells.
RAD51 interact with Dp71 and Lamin B1 in HBE
Immunoblot was carried out to quantify lamin B1 and RAD51 expression in HBE-Dp71AS cells. Compared with parental HBE cells and HBE-Dp71E cells, significant reduction of Dp71, lamin B1, RAD51 mRNA (Fig. 5e, f, and g) and protein (Fig. 5h) was observed. The endogenous RAD51 and lamin B1 expression levels were both reduced after the Dp71 expression was knocked down in HBE cells.
Dp71 depletion resulted in decreased FAK, p-FAK and p-AKT
QRT-PCR displayed reduced FAK mRNA in HBE-Dp71AS cells (Fig. 6c, d). Decreased FAK protein and p-FAK expression was also displayed by immuno-blotting in HBE-Dp71AS cells. Although AKT did not display any expression alteration, p-AKT displayed a significant reduction in HBE-Dp71AS cells (Fig. 6e). Knocking down Dp71 in HBE cells resulted in the reduction of cytoplasmic expression of FAK, p-FAK and p-AKT.
Being one of the most abundantly expressed dystrophin isoforms, Dp71 is distributed in all tissues except skeletal muscle. The deficiency of Dp71 is involved in mental retardation and retinal dysfunction of DMD patients [2, 12, 13]. For years, PC12 cells have been used as the traditional cell model to study the biological function of Dp71. In our further characterization of this ubiquitously expressed scaffolding protein, Dp71 was found to contain more functional diversity than expected. Being one of the newly identified tumor suppressive genes in gastric cancer, our preliminary work identified decreased Dp71 in lung cancer as well. As a usual cell model for pulmonary research, we used shRNA to knock down Dp71 and revealed the biological function of HBE-Dp71AS cells.
After the HBE-Dp71AS cells were stimulated by H2O2, the alkaline comet assay displayed that tail length and tail moment increased in HBE-Dp71AS cells compared with parental cells and HBE-Dp71E cells. More γ-H2AX foci were detected in the HBE-Dp71AS nucleus. That means more single strand break (SSB) and DSB occurred in HBE-Dp71AS cells under oxidative stress. Knocking Dp71 down increased the DNA damage induced by H2O2.
RAD51 was found to be a new binding partner of Dp71 in our further exploration of HBE-Dp71AS cells. Although cytoplasmic and nuclear interactions between Dp71 and RAD51 were detected in our research, the nuclear Dp71-RAD51 interaction plays a significant role in the increased DNA damage of HBE-Dp71AS cells induced by H2O2. Co-Ip proved the existence of Dp71-lamin B1-RAD51 protein complex in HBE. RAD51 is proved to be an essential protein for DNA repair by homologous recombination [14, 15]. Overexpression of Rad51 in different organisms and cell types increased homologous recombination (HR) and increased resistance to DNA damaging agents. Being an associating protein of Dp71, lamin B1 has been found to be a binding partner of RAD51, which promotes DSB repair and cell survival by maintaining the RAD51 protein stability [9, 10]. In HBE-Dp71AS cells, depletion of Dp71 results in Rad51, lamin B1 mRNA and protein reduction. RAD51 suffers both decreased expression and impaired stability, which leads to decreased HR and increased DNA damage stimulated by H2O2, and finally results in increased apoptosis.
Via interaction with lamin B1, the nuclear Dp71 is proved to affect the cell growth of PC12 and A549. Decreased lamin B1 expression also slows cell proliferation and induces premature senescence in WI-38 cells [6, 16]. In HBE-AS cells, significantly reduced lamin B1 explains the phenotypes of growth inhibition. Increased BRCA1 expression caused by decreased lamin B1 reduction leads to the overactivation of caspase 8 and 3, combined with impaired expression and function of RAD51. The apoptosis rate of HBE-Dp71AS cells increased under H2O2 stimulation.
For the first time, it is reported that increased DNA damage occurs in Dp71 depleted cells. With the accumulation of studies showing that many of the human hereditary and non- hereditary cancer predisposition diseases are caused by germ-line mutations in DNA repair genes, the pivotal role that DNA repair plays in the in vivo tumorigenesis process becomes evident. We have proved the in vitro reduction of RAD51 and FAK from the transcription level in HBE-Dp71AS cells. RAD51 and FAK are two important proteins participating in DNA damage repair. Such carcinogens as ROS produced by in vivo metabolism attack DNA and cause a variety of DNA lesions. Unrepaired lesions cause gene mutations and chromosomal damage, which may lead to tumor initiation and progression. However, more experimental evidence is needed to prove the role Dp71 plays in the in vivo tumorigenesis.
Being one of the most ubiquitously expressed isoforms of the dystrophin family, our newly revealed biological traits of HBE-Dp71AS cells suggest a much broader role played by Dp71 in many pathophysiological process, which adds some fresh color to this “old” protein.
Depletion of Dp71, a widely expressed isoform of dystrophin, displays increased DNA damage, decreased proliferation, and increased apoptosis during oxidative stress via decreasing RAD51, lamin B1, pAKT, FAK and pFAK expression.
SPT and JLX conceived and designed the experiments and wrote the manuscript. SCT, XFX and LX performed the experiments. SZH performed the statistical analysis. All authors read and approved the final manuscript.
This study is supported by the National Natural Science Fund of China (Grant No. 81870071), Hunan Natural Science Fund of China (Grant No.2018JJ2628).
Ethics approval and consent to participate
There were no human tissue or animal experiments in this study.
Consent for publication
The authors declare that they have no competing interests.
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