Running title: OPA1 in LADC
Optic atrophy 1 increases cisplatin resistance via inhibition of
caspase-dependent cell death
Hsin-Yuan Fang1,2, Yu-Ting Wang3, Kuan-Chih Chow3,*, Shiow-Her Chiou4, Hsiu-Ching Yang3,5, Chih-Yi Chen5, Tze-Yi Lin6, I-Ping Chiang6, Yung-Yen Chiang7, and Wen-Je Ko1
1
Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan, 2
Departments of Surgery, China Medical University Hospital, China Medical University, Taichung, Taiwan, 3Graduate Institute of Biomedical Sciences and 4Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan,
5
Comprehensive Cancer Center, China Medical University Hospital, China Medical
University, Taichung, Taiwan, 6Departments of Pathology, China Medical University Hospital, China Medical University, Taichung, Taiwan, 7Department of Dental Laboratory Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan.
Abstract: word counts, 171; Text: pages 16, word counts: 4226 Table: 1; Figures: 5
*Corresponding author. Graduate Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan. Tel.: +886-4-22840896 x 118; Fax: +886-4-22859270; E-mail: [email protected]
Abstract
Optic atrophy 1 (OPA1) protein, a 112-kDa GTPase, is involved in the mitochondrial inner membrane fusion and anticancer drug-mediated cytotoxicity, which implicate an association with disease progression of cancer. In this study we investigated the prognostic value of OPA1 expression in patients with lung adenocarcinomas (LADC). Using
immunohistochemical staining, expression of OPA1 was determined in 286 LADC patients. Expression of OPA1 was confirmed by immunoblotting. Relationship between OPA1 expression and clinicopathological parameters was analyzed by a statistical analysis. Difference of survivals between different groups was compared by the log-rank test. The results showed that OPA1 overexpression was detected in 219 (76.6%) of LADC patients. A significant difference was found in cumulative survival between patients with high OPA1 levels and those with low OPA1 levels (p = 0.0016). In the in vitro experiments with cell lines, silencing of OPA1 expression decreased cisplatin resistance, which was further shown
through increased release of cytochrome c and activation of caspase-dependent pathway. In conclusion, OPA1 is highly expressed in lung adenocarcinomas, and indicates with poor prognosis.
Keywords: mitochondrial inner membrane, fusion, cytochrome c, prognosis, mitochondrial outer membrane, apoptosis-inducing factor
Introduction
Mitochondrion is one of the major organelles responsible for intracellular energy supply and regulation of programmed cell death (Aon et al, 2006). Because of the lack of DNA repair mechanism and the necessity to maintain integrity of the genome which encodes only 13 vital proteins for ATP synthesis (Wallace, 1999), mitochondria constantly exchange DNA with one another by fusion and fission (Twig et al, 2008). Two proteins, dynamin-related protein 1 (DRP1) and human homolog of yeast mitochondria fission protein 1 (hFis1), are essential for mitochondrial fission (Yoon et al, 2003). Three proteins, mitofusin (Mfn)-1, Mfn-2 and optic atrophy 1 (OPA1), are critical for mitochondrial fusion (Cipolat et al, 2004). Among them, Mfn-1 and Mfn-2 are imperative for the fusion of the mitochondrial outer membrane (MOM), while OPA1, behaving like its yeast homolog Mgm1, is crucial for the fusion of the mitochondrial inner membrane (Griparic et al, 2004).
OPA1 is a dynamin-related guanosine triphosphatase (GTPase). The gene is located on chromosome 3q28-q29, comprising of 31 exons (Davies and Votruba, 2006). The alternative splicing of exons 4, 4b and 5b produces eight transcript variants
(http://www.ncbi.nlm.nih.gov/nucleotide/GeneID: 4976). Therefore, depending upon the type of transcript variants the molecular mass of OPA1 has been shown to range from 107- to 118-kDa (924~1015 amino acid residues). Unlike other members of the dynamin family though, OPA1 does not contain the pleckstrin homology region, the GTPase effector domain or the proline-rich stretch at the carboxyl terminus (Chen and Chan, 2005; Davies and Votruba, 2006; Tondera et al, 2009). In addition, OPA1 mutations, which are frequently detected in patients with autosomal dominant optic atrophy (ADOA), are correlated with progressive loss of visual acuity, color vision and central vision field, as well as temporal discoloration of optic disc, ascending atrophy and dysmyelination of optic nerve (Chen and Chan, 2005; Davies and Votruba, 2006). In the in vitro experiment, silencing of OPA1
expression by siRNA provokes a series of mitochondrial events, e.g., change of mitochondrial morphology, increase of mitochondrial fragmentation, disruption of mitochondrial cristae as well as release of cytochrome c and induction of apoptosis, suggesting that OPA1 is an anti-apoptotic factor (Olichon et al, 2004; Frezza et al, 2006; Ishihara et al, 2006).
Recently, Todera et al. showed that pro-fusion proteins, OPA1 and Mfn-1, were required
for cell protection against apoptotic stress induced by actinomycin D or UV irradiation (Olichon et al, 2004). Moreover, remodeling of mitochondrial nucleoids, replication complex in mitochondrial matrix of which the structural integrity was maintained by OPA1 and Mfn-1, increased cell resistance to anticancer drug doxorubicin (Garrido et al, 2003; Ashley and Poulton, 2009). Interestingly, remodeling of mitochondrial nucleoids is regulated by p53 and ataxia telangiectasia mutated (ATM) protein, indicating that mitochondrial nucleoid and genomic DNA respond to DNA damage in a synchronous fashion. However, OPA1 has not been studied in non-small cell lung cancer, in particular, the lung adenocarcinoma, of which the incidence and mortality have increased dramatically in the last two decades in Taiwan (Annual reports of the Department of Health, the Executive Yuan, Republic of China, 2006). Although cigarette smoking has been shown to be a key factor of disease progression and treatment failure (Chen et al, 2006), a portion of patients who do not respond well to the radiation- and chemo-therapy in Taiwan are women and nonsmokers (Toh et al, 2006). Treatment failures of LADC, which are associated with DNA repair- and hypoxia-mediated drug- and radiation-resistance as well as with the rapid disease progression (Shannon et al, 2003; Chen et al, 2006; Toh et al, 2006; Maruyama et al, 2009), are major causes of the high disease-related mortality.
In this study, we used immunohistochemistry and immunoblotting to determine the expression level of OPA1 in LADC cells and pathological specimens. We also evaluated the statistical correlation between the expression of OPA1 and the clinicopathological parameters
as well as the prognostic significance of OPA1 expression in LADC patients. The effect of OPA1 expression on cisplatin sensitivity was characterized in vitro. Our results showed that expression of optic atrophy 1 protein could increase cisplatin resistance, and this effect could be through the inhibition of caspase-dependent cell death in lung adenocarcinoma cells.
Results
Expression of OPA1 in LADC cells determined by RT-PCR
Expression of OPA1 was examined by RT-PCR in one HeLa and nine lung cancer cell lines. OPA1 was detected in all cell lines (Figure 1). Following sequence analysis, which was performed using fluorescence-labeled dideoxy nucleotides (by Mission Biotech, www.missionbio.com.tw, Taipei, Taiwan), and DNA sequencing ladder read by an ABI PRISM 3700 DNA Analyzer (CD Genomics, Shirley, NY, USA), nucleotide sequence homology of cDNA fragments from the nine cell lines matched that of OPA1: BC075805,
Homo sapiens optic atrophy 1, autosomal dominant (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Expression of OPA1 in LADC cell lines
Following determination of specificity and sensitivity, monoclonal antibodies were used to detect OPA1 expression in the HeLa and nine lung cancer cell lines. Two protein bands (82- and 110-kDa), corresponding to the anticipated molecular mass of OPA1, were recognized in all the cell lines (Figure 2A). An 82-kDa protein was highly expressed in A549, H520 and H2009 cells. To determine the identity of the two proteins, cell lysate from H520 was precipitated with antibodies to OPA1 and the respective protein bands were subjected to analysis of matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF). The peptide mass fingerprints of both 82-kDa and 110-kDa proteins matched those of OPA1: GenBank| EAW78064.1, optic atrophy 1 (autosomal dominant), isoform CRA a (Homo sapiens). The matched peptides covered 33.8% (325/960 amino acids) of the protein. These data indicated that both 82-kDa and 110-kDa proteins were OPA1 (EAW78064.1) in the LADC cells.
Immunocytochemical staining showed that OPA1 was abundantly present in cytoplasm. The granular appearance of subcellular structures suggested that OPA1 could be present in
mitochondria (Figure 2B). An immunoblotting of sucrose gradient separated organelle fractions confirmed that OPA1 was localized in the mitochondrial fraction (Figure 2C). A MitoTracker® Green (Molecular Probes, Eugene, OR) uptake and confocal fluorescence immunocytochemistry (Figure 2D) further validated that OPA1 was mainly localized in the mitochondria. However, some of OPA1 signals did not co-localize with mitochondria, the grainy appearance suggested that they might be present in other organelles. Using sucrose gradient ultracentrifugation to separate organelle fractions showed that some of OPA1, as determined by immunoblotting, was detected in the mitochondria-associated membrane and light membrane fractions (Figure 2E), in particular when expression of DRP1 or Mfn-2 was inhibited by siRNA, indicating that OPA1 could also be located in the endoplasmic reticulum and mitochondria-associated membrane.
Pathological expression of OPA1 in lung adenocarcinomas
Using immunoblotting, we detected that the major OPA1 expressed in LADC specimens was the 82-kDa OPA1 (Figure 3A). Immunohistochemistry identified OPA1 in 219 (76.6%) of LADC pathological specimens. The signal was predominantly localized in cancer cells (Figure 3B1), but not in the nearby lymphocytes or in the non-tumor lung tissue (NTLT) (Figure 3B2). OPA1 expression was also detected in 54.7% (41/75) of metastatic lymph nodes (data not shown). Statistical analysis showed that overexpression of OPA1 correlated with patient’s gender, cigarette smoking habit, tumor stage, lymphovascular invasion, as well as expression of DDH I/II and mitochondria-associated proteins (Table 1), suggesting that OPA1 expression was associated with cell growth and metastatic potential of LADC (21,22). Interestingly, among the 219 patients who had high levels of OPA1, 97 (44.3%) patients had tumor recurrence during follow-up examination. Among the 67 patients who had low levels of OPA1, 8 (11.9%) patients had recurrence. All 105 patients who had recurrence developed
tumors within 24 months after operation. The risk of recurrence for patients with high levels of OPA1 was 3.709-fold higher than that for patients with low levels of OPA1 (p < 0.001). Survival of patients with low OPA1 levels was significantly better than that with high OPA1 levels (Figure 3C). The hazard ratio between the 2 groups was 1.69, and the difference in cumulative survival was significant (p = 0.0016) by the log-rank test. Multivariate analysis revealed that the difference in OPA1 expression between the 2 groups was highly significant as well (p = 0.004).
OPA1 in LADC cells is phosphorylated by PKC, and phosphorylation is essential for maintaining OPA1 stability
Previously, we have shown that phosphorylation of mitochondria-associated proteins, e.g., protein dynamin-related protein 1 (DRP1) and the ATPase family, AAA domain containing 3A (ATAD3A), was critical for maintaining protein stability (Chiang et al, 2009; Fang et al, 2010). Phosphorylation of DRP1was regulated by hypoxia-related AMP-activating protein kinase (AMPK) and that of ATAD3A was mediated by protein kinase c (PKC). We expected that OPA1 could be phosphorylated as well. We then ran OPA1 sequence through a NetPhos program to predict for phosphorylation sites (http://www.cbs.dtu.dk/services/NetPhos/), and a NetPhosK program to predict for specific kinase (http://www.cbs.dtu.dk/services/NetPhosK/) in eukaryotic proteins, and the results showed that for OPA1 (variant 1) the most probable kinase was PKC at Thr503, Ser708, Thr816, Thr893 and Thr929 (Supplementary Figure S1).
LADC cell lysates were treated with calf intestinal alkaline phosphatase (CIP) before immunoblotting, the amount of the 110-kDa protein was reduced, but that of the 82-kDa protein was increased (Figure 4A). These results suggest that the110-kDa OPA1 could be a phosphorylated form. LADC cells were therefore treated with a panel of kinase inhibitors. As shown in Figure 4B, addition of calphostin C, a PKC inhibitor, reduced the intensity of
82-kDa and 110-kDa protein bands, indicating that PKC was the kinase for OPA1
phosphorylation, and that phosphorylation was essential for maintaining OPA1 stability. For identifying the PKC isozyme that was responsible for OPA1 phosphorylation, H2009 cells, which expressed intermediate levels of OPA1, were transfected with plasmids carrying various genes of PKC isozyme. Interestingly, levels of the 110-kDa protein increased in cells expressed ectopical PKC isozymes (Figure 4C). These results confirmed that PKC was accountable for OPA1 phosphorylation. Addition of 10 μM nicotine at 37°C for 4 h increased levels of 110-kDa OPA1 (Figure 4D) and cisplatin resistance (Figure 4E).
Biosynthesis of OPA1 increases during S phase of cell cycle progression and silencing of OPA1 expression increases drug sensitivity
PKC activity is frequently associated with growth factor receptor-related cellular events and cell cycle progression (Hirai et al, 1989; Black, 2000). Therefore, findings of PKC
involvement in OPA1 phosphorylation suggested that OPA1 expression could be regulated by a cell cycle progression- or a growth factor receptor-related pathway. We therefore studied OPA1 expression pattern during cell cycle progression by the serum starvation-reactivation and the double-thymidine block methods. Results of double-thymidine block and release showed that levels of OPA1 protein, like other mitochondria-related proteins, e.g., DRP1 and ATAD3A (21, 22), increased during the S phase (four hours after release from DTB) and decreased in the G1 phase (16 hours after release from DTB) (Figure 5A), indicating that biosynthesis of OPA1 was during the S phase of cell cycle progression. Serum starvation markedly reduced 110-kDa OPA1 (Figure 5B). Replenish of serum increased both the 82- and 110-kDa proteins, suggesting that in addition to cell cycle progression OPA1 expression was regulated by growth factor receptor-related proliferation stimulation as well, by which OPA1 appeared rapidly following serum re-activation.
Previous studies have suggested that OPA1 plays an anti-apoptotic role in response to stress challenge (Garrido et al, 2006; Ashley and Poulton, 2009). As anticipated, knockdown of OPA1 (OPA1kd) expression by siRNA (Figure 5C) increased cisplatin sensitivity (Figure 5D). The effect of OPA1 silencing on cisplatin sensitivity persisted through cell cycle (Figure 5E). Very little cytotoxicity was observed in H23 cells until 12 hrs post serum stimulation. Maximal cell killing occurred between 15-21 hrs after serum re-activation, but around 24 hrs there was again a reduction in cisplatin cytotoxicity. Reduction of OPA1 expression increased 5-30% of cisplatin sensitivity, suggesting that besides nuclear toxicity, cisplatin might have toxicity to mitochondria as well. Results of confocal fluorescence immunocytochemistry (Figure 5F) and immunoblotting of sucrose gradient-separated organelle fractions (Figure 5G) showed that OPA1 silencing increased cytoplasmic cytochrome c, but not cytoplasmic nor nuclear apoptosis-inducing factor (AIF) following staurosporine (STS) treatment, suggesting that OPA1 deficiency might increase caspase-dependent cell death. We added sub-lethal doses of STS to examine this postulation. Interestingly, although treatment with sub-lethal doses of STS provoked very diminutive effect on caspase 3 activation (Figure 5H1) or degradation of poly (ADP-ribose) polymerase (PARP) (Figure 5H2) in wild-type A540 cells, it did induce greater effect on enzyme activation and substrate disintegration of
Discussion
Our results show that OPA1expression is frequently elevated in LADC. Increased OPA1 expression in LADC patients significantly correlates with patient’s gender, cigarette smoking habit, tumor stage and higher incidence of tumor recurrence as well as reduced drug
sensitivity, all of which indicate poor prognosis.
As shown in the immunoblotting experiment, besides an 82-kDA protein, OPA1 signals of size between 82- and 110-kDa were detected in both the pathological specimens and LADC cell lines. After CIP treatment, the intensity of the protein bands above 82-kDa reduced and that of the 82-kDa protein increased, indicating that the protein bands above 82-kDa OPA1 was phosphorylated. However, the 82-kDa protein was also sensitive to CIP, suggesting that the 82-kDa OPA1 was phosphorylated as well. Treatment with calphostin C, a pan-PKC inhibitor, reduced phosphorylation and protein level of 82- and 110-kDa OPA1, further implying that PKC could be the kinase that catalyzes OPA1 phosphorylation
(Kobayashi et al, 1989), and that post-translational modification is essential for maintaining OPA1 stability. Ectopic PKC expression showed that all the PKC isozymes were capable for OPA1 phosphorylation. These data corresponded well with the current results of
clinicopathological analyses that OPA1 expression correlated with patient’s habit of cigarette smoking, which could directly or indirectly influence PKC activity (Gopalakrishna et al, 1994; Lee et al, 2001).
Moreover, PKC activity is frequently associated with growth factor receptor-related cellular events and cell cycle (Hirai et al, 1989; Black, 2000; Fima et al, 2001). By showing that OPA1 expression is up-regulated during S phase of cell cycle progression and that OPA1 is phosphorylated by PKC, our work suggests that phosphorylation may be a general
mechanism for maintaining protein stability. These data also support our recent findings that mitochondria-related proteins, e.g., DRP1 and ATAD3A, of which the expression is elevated
during S phase, are phosphorylated by PKC, and associated with increased drug resistance (Chiang et al, 2009; Fang et al, 2010). It is therefore worth noting that knockdown of OPA1 expression by siRNAs (OPA1kd) decreased cisplatin resistance. These results clearly indicate that the OPA1 plays an important role in the drug sensitivity of LADC, which then reflects in patient’s survival. Patients with low OPA1 level are more sensitive to cisplatin-based
chemotherapy and have a better prognosis. In contrast, those with high OPA1 level are more resistant to chemotherapy and, thus, have a poorer prognosis. The exact mechanism by which OPA1 contributes to cisplatin resistance, however, is not yet clear.
By showing that antioxidants delayed the onset of mitochondrial dysfunction in OPA1 +/-drosophila, Tang et al. suggested that OPA1 mutations increased the production of oxygen reactive species (ROS) (Tang et al, 2009). Using electron microscopy, Olichon et al. showed that OPA1 deficiency led to perturbation of both mitochondrial inner and outer membranes (Olichon et al, 2003), implying that OPA1 could be essential for maintaining the
mitochondrial membrane integrity and for preventing ROS leakage (Olichon et al, 2003; Griparic et al, 2004). High intracellular level of ROS might initiate the aberrant autophagic cell death (White et al, 2009). Interestingly, ROS induced AKR1C2 expression via activation of antioxidant response element (Burczynski et al, 200335, Lou et al, 2006), and AKR1C1/2 overexpression was frequently detected in LADC (Hsu et al, 2001). It is possible that in the absence of polycyclic aromatic hydrocarbons (PAH), AKR1C1/2 may catabolize the
excessive endogenous or cisplatin-induced ROS (Deng et al, 2002). It is worth noting that aldoketo reductase contains PGF synthase activity and PGF2α level is proportional to AKR1C expression (Nakashima et al, 2003; Wang et al, 2007). AKR1C is a down-stream enzyme of cytochrome p450 1A1 and epoxide hydrolase, which convert hydroxylated PAH into catechols (Vogel et al, 1980). When cells were under hypoxic condition, the reduced
AKR1C to catalyze the intrinsic substrates toward prostaglandin F (PGF) synthesis. In addition to the elevated synthesis of PGF2α, hypoxia increased expression of hepatocyte growth factor (HGF) and interleukin (IL)-8 as well (Chiang, 2009). HGF, IL-8 and PGF2α consequently induced cancer cell’s growth and metastasis during disease progression (Chen
et al, 2006; Chiang, 2009). Our results not only supported the recent findings by Seo et al.
that AKR1C1/2 was highly expressed in the cancer stem cell population of LADC cell line A549 (Seo et al, 2007), but also provided a reasonable explanation for the increased drug resistance and elevated proliferation in cancer stem cells (Hsu et al, 2001; Shannon et al, 2006; Chen et al, 2006), suggesting that overexpression of AKR1C1/2 and OPA1 could be key features of cancer stem cells.
In fact, mitochondria are one of the major intracellular targets for clinically important anticancer drugs, such as cisplatin (Yang et al, 1997) and etoposide (Park and Kim, 2005). Current evidence suggests that in addition to the nuclear targets, these drugs may directly influence mitochondrial membrane functions, which are imperative for preventing the release of cytotoxic cytochrome c to initiate caspase-dependent cell death. The present study sheds further light on the status of mitochondrial OPA1protein in the cell cycle and how this affects cisplatin sensitivity. The increased cisplatin sensitivity in OPA1kd cells when compared to those which are logarithmically growing is consistent with the observations of others that OPA1, like ATAD3A and DRP1, could be an anti-apoptotic factor (Olichon et al, 2006; Chiang et al, 2009; Fang et al, 2010). Moreover, our results suggest that mitochondria and nucleus may have different effects on drug sensitivity of the cells. Elegant studies by Olichon
et al and Griparic et al showed that OPA1 deficiency increased deformation of mitochondria ,
inflation of mitochondrial cristae and abrasion of MOM (Olichon et al, 2003; Griparic et al, 2004), indicating that in addition to mitochondrial inner membrane fusion OPA1 might have a role for maintaining the integrity of MOM, i.e., by reducing ROS-associated membrane
damage or facilitating the phospholipid supply for MOM (Fang et al, 2010). The MOM abrasion increased the release of cytochrome c (caspase-dependent cell death), but not AIF (caspase-independent cell death), when cells were challenged with genotoxic stress, supporting the report by Frezza et al that OPA1 guarded the corralled cytochrome c in the enclosed cristae (Frezza et al, 2006). The mechanisms of differential release of cytochrome c and AIF, however, remain to be resolved.
In conclusion, immunoblotting and immunohistochemical staining reveal abundant expression of OPA1 in the LADC cells. Pathological results indicate that OPA1 expression correlates with patient’s gender, cigarette smoking habit, tumor stage and expression of AKR1C1/2, the features which are closely associated with the increased drug resistance and metastatic potential as well as the poor prognosis. In the in vitro experiments using LADC cells, our results show that OPA1 expression is tightly regulated during cell cycle, and OPA1 is phosphorylated by PKC. Silencing of OPA1 expression increases the level of cisplatin sensitivity, possibly via caspase-dependent cell death pathway, in LADC cell lines.
Materials and Methods
Tissue specimens and lung adenocarcinoma cell (LADC) lines
From January 2001 to December 2004, tissue specimens were collected from 286 patients with newly diagnosed LADC. Samples from all patients, for whom at least one follow-up examination or death was documented, were pathologically confirmed LADC. The stage of the disease was classified according to the new international staging system for lung cancer (Detterbeck et al, 2009). The protocols of these studies had been approved by the Medical Ethics Committee at China Medical University Hospital (DMR99-IRB-062).
Nine NSCLC cell lines (H23, H125, H226, H838, H1437, H2009, H2087, A549 and H520) were used for the in vitro evaluation of gene expression. H23, H838, H1437, H2009, H2087 and A549 are LADC cells, and H125, H226 and H520 are epithelial type cells. Cells were grown at 37°C in a monolayer in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 100 I.U./ml penicillin and 100 μg/ml streptomycin.
Reverse transcription-polymerase chain reaction (RT-PCR)
Following total RNA extraction and synthesis of the first-strand cDNA, an aliquot of cDNA was subjected to 35 cycles of PCR to determine the integrity of β-actin mRNA (Chen et al, 2006). The cDNA used in the following RT-PCR was adjusted according to the quality and quantity of β-actin mRNA. The primer sequences were selected by the web software Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3). For all the OPA1 variants, the primers are: OPA1s: 5'-GGAACAGCTCTGAAAGCATT-3' [sense primer, nucleotides (nts) 1737-1756,
NM_130837.1] and OPA1a: 5'-AACTTTCTTGAGGTCTTCCG-3' (antisense primer, nts 3046-3027). The anticipated cDNA fragments are 1310 bp. For differentiating OPA1 variants, the primers are: OPA1vs: 5'-GGATTGTGCCTGACATTGTG-3' (sense primer, nts 456-475, NM_130837.1) and OPA1va: 5'-TGCCTTTGTCATCTTTCTGC-3' (antisense primer, nts
983-964). The anticipated cDNA fragments are 528 bp (variant 8), 474 bp (variant 7), 420 bp (variant 6), 417 bp (variant 5), 366 bp (variant 4), 363 bp (variant 1), 309 bp (variant 3) and 255 bp (variant 2).
Immunoprecipitation, gel electrophoresis and protein analysis by MALDI-TOF
Total cell lysate was prepared by mixing 5 × 107 cells/100 μl phosphate-buffered saline with equal volume of 2 × NP-40 lysis buffer [40 mM Tris-HCl, pH 7.6, 2 mM EDTA, 300 mM NaCl, 2% NP-40, and 2 mM phenylmethylsulfonylfluoride (PMSF)]. Protein G sepharoseTM (Amersham Biosciences AB, Uppsala, Sweden) was pre-washed before mixing with 500 μg of total cell lysate. The reaction mixture was incubated at 4°C for 60 minutes, and then centrifuged at 800 × g for 1 minute. The supernatant was reacted with 5 μg of purified monoclonal antibodies and 20 μl of fresh protein G sepharose at 4°C for 18 hours. The reaction mixture was centrifuged at 800 × g for 1 minute. Following removal of the supernatant, the precipitate was washed with 1 × PBS, and dissolved in loading buffer (50 mM Tris, pH 6.8, 150 mM NaCl, 1 mM disodium EDTA, 1 mM PMSF, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue and 1% SDS). Eletrophoresis was carried out in two 10% polyacrylamide gels with 4.5% stacking. One gel was processed for immunoblotting (Chen et al, 2006), and the other gel was stained with Coomassie blue. Protein bands on the Coomassie-stained gel, which corresponded to the immunoblotting-positive bands, were extracted from the gel for identification by MALDI-TOF on a Voyager-DETM pro biospectrometry workstation (Applied Biosystems, Milpitas, CA, USA). Fragments of peptide fingerprints were matched with those on the SwissProt database by MS-fit
(ProteinProspector 4.0.5., The Regents of the University of California). After electrophoresis, proteins on the first gel were transferred to a nitrocellulose membrane for immunoblotting. The membrane was probed with specific antibodies. The signal was amplified by
biotin-labeled goat anti-mouse IgG, and peroxidase-conjugated streptavidin. The protein was visualized by exposing the membrane to an X-Omat film (Eastman Kodak, Rochester, NY) with enhanced chemiluminescent reagent (NEN, Boston, MA).
Immunoblotting analysis and immunostaining
Immunoblotting and immunohistochemistry were performed as described previously (Chen et
al, 2006). Antibodies for β-actin were obtained from Chemicon International (Temecula, CA).
Antibodies to OPA1 were raised in the laboratory. For immunocytochemical staining, the cells were grown overnight on slide, and then fixed with cold methanol/acetone at 4°C for 10 minutes before staining. For and immunohistochemical staing, the pathological slides were processed by a routine procedure. Following inactivation of endogenous peroxidase, the immunological staining was performed by an immunoperoxidase method (Chen et al, 2006). Antibodies for aldoketo reductase (AKR) 1C1/2 [also called dihydrodiol dehydrogenase (DDH) I/II], DRP1, ATAD3A, Mfn-1 and Mfn-2 were raised and characterized in our laboratory (Lin, 2005; Chiang et al, 2009; Fang et al, 2010). Antibodies to OPA1 were not added for the negative control group.
Slide evaluation of OPA1 expression by immunohistochemical staining
In each pathological section, non-tumor lung tissue served as internal negative control. Slides were evaluated by two independent pathologists blinded to the clinicopathological knowledge. The ImmunoReactive Scoring system was adapted for this study (Remmele and Schicketanz, 1993). Briefly, a specimen was considered having strong signals when more than 50% of cancer cells were positively stained; intermediate, if 25-50% of the cells stained positive; weak, if less than 25% or more than 10% of the cells were positively stained; and negative, if less than 10% of the cancer cells were stained. Cases with strong and intermediate OPA1
signals were classified as OPA1+, and those with weak or negative OPA1 signals were classified as OPA1- (Chen et al, 2006; Chiang et al, 2009).
Colony assay
Cells were treated at different time point during serum stimulation with various concentration of cisplatin at 37°C for 4 h, wash twice with warm medium for 10 min, and then trypsinized. Viable cells, as determined by trypan blue dye exclusion, were seeded quantitatively into 6-well plates with 5 ml of medium and incubated at 37°C and in 5% CO2 over 7-10 days. Colonies were stained with 2% crystal violet in methanol (with 3% glacial acetic acid). Percent survival was determined by comparison to PBS-treated controls. For gene silencing experiments, cells were harvested 48 hours following siRNA treatment.
Statistical analysis
Correlation of OPA1 level with clinicopathological factors was analyzed by either the Chi-Square test or the Fisher’s exact test. Survival curves were plotted using the
Kaplan-Meier estimator (Kaplan and Meier, 1958). Statistical difference in survival between different groups was compared by the log rank (Mantel, 1966) test. Statistical analysis was performed using GraphPad Prism5 statistics software (San Diego, CA). Statistical
Acknowledgements
This study was supported, in part, by the Comprehensive Academic Promotion Projects, Department of Education (NCHU 995002 to K.C. Chow), and, in part, by the Taiwan Department of Health, China Medical University Hospital, Cancer Research of Excellence (DOH99-TD-C-111-005). RNAi for silencing OPA1 gene expression was obtained from the National RNAi Core Facility in the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taipei, Taiwan, supported by the National Research Program for Genomic Medicine Grants of NSC (NSC 97-3112-B-001-016).
Conflict of interest
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Figure legends
Figure 1 Expression of OPA1 detected by RT-PCR in cancer cells. Expression of OPA1 mRNA was detected by RT-PCR in one HeLa and eight LADC cell lines. Expression of β-actin was used as a monitoring standard for the relative expression ratio of OPA1 mRNA.
Figure 2 Characterization of monoclonal antibodies to OPA1. (A) Immunoblotting revealed that monoclonal antibodies raised against recombinant OPA1 recognized 2 protein bands of approximately 95-kDa. Expression of the 82-kDa OPA1 was detected in all nine human lung cancer cell lines, high in H520, intermediate in H23, H125, H226, H1437, H2009, H2087 and A549 (relative to H838), and low in H838 cells. Expression of the 110-kDa protein was detected in eight human lung cancer cell lines, but not in H2009 cells. Cell lysate from H23 were precipitated by OPA1-specific monoclonal antibodies and protein G sepharoseTM. Both the 82- and 110-kDa proteins were characterized by MALDI-TOF. The peptide mass
fingerprints of 82-kDa and 110-kDa proteins matched that of OPA1: MS-Fit search
(http://prospector.ucsf.edu/): EAW78064.1, optic atrophy 1 (autosomal dominant), isoform CRA a (Homo sapiens). (B) Immunocytochemical staining showed that OPA1 was
abundantly present as distinct granules in the cytoplasm of A549 cells, which suggests that OPA1 is located in mitochondria. (C) Immunoblotting of H23 subcellular fractions, which were separated by sucrose gradient ultracentrifugation. Cytosol, cytosolic fraction; COX IV, cytochrome c Oxidase IV of mitochondria; (D) Confocal fluorescence immunocytochemical staining of A549 cells. OPA1 was detected by specific monoclonal antibodies labeled with FITC. Mitochondria were labeled with MitoTracker® Red CMXRos dye. Nuclei were stained with fluorescent dye 4', 6-diamidino-2-phenylindole (DAPI). A merged image of the first, second and third columns, and the magnification of a specific cell confirm that OPA1 is located in mitochondria. The white bar represents 10 μm. (E) OPA1, as determined by
immunoblotting, was detected in the fractions of light membrane (consisted mainly of endoplasmic reticulum) and mitochondria-associated membrane, which were separated using sucrose gradient ultracentrifugation. This phenomenon was particularly evident when
expression of DRP1 or Mfn-2 was inhibited by siRNA.
Figure 3 Correlation between OPA1 expression and survival of LADC patients. (A)
Expression of OPA1 was determined by immunoblotting. Expression of β-actin was used as a monitoring standard for the relative expression of OPA1. N: non-tumor lung tissue (NTLT), T: tumor fraction of surgical resections. (B) Representative examples of OPA1 expression in pathological specimens as detected by immunohistochemical staining (crimson precipitates). Expression of OPA1 was detected in (B1) the LADC tumor nest, but not in (B2) the NTLT. (C) Comparison of Kaplan-Meier product limit estimates of survival analysis in LADC patients. Patients were divided into 2 groups depending on the expression of OPA1. The difference in survival between the 2 groups was compared by the log rank test, and the p value was 0.0016.
Figure 4 Expression and post-translational modification of OPA1 in LADC cells. (A) Two bands of OPA1 protein were detected in LADC cells and pathological specimens by immunoblotting. However, RT-PCR showed that only one mRNA was detected in these samples, suggesting that the 110-kDa protein could be a phosphorylated OPA1. H23 cell lysate were treated with calf intestinal alkaline phosphatase (CIP) before immunoblotting; the 110-kDa protein band became less intense, while the 82-kDa protein band became more intense. (B) The effect of kinase inhibitor on OPA1phosphorylation. The H23 cells were treated with a panel of serine/threonine kinase inhibitors before immunoblotting. Only treatment with 5 μM calphostin C, a protein kinase C (PKC) inhibitor, reduced the intensity
of the 110-kDa and 82-kDa protein bands. The results suggested that the PKC-mediated phosphorylation was essential for maintaining OPA1 stability. (C) Different isozymes of PKC were ectopically expressed in H2009 cells before immunoblotting. Intensity of the 110-kDa protein increased in cells that ectopically expressed PKC. (D) Addition of 10 μM nicotine at 37°C for 4 h induced OPA1 phosphorylation. (E) Increase in OPA1 phosphorylation
increased cisplatin resistance. {, control H23 cells; z, H23 cells treated with 10 μM of nicotine prior to cisplatin challenge.
Figure 5 OPA1 expression during cell cycle progression and serum starvation, and the association of its expression with cisplatin cytotoxicity. (A) OPA1 expression in different phases of cell cycle progression. HeLa cells were treated with double thymidine block (DTB) to synchronize cells at the late G1 phase. OPA1 levels increased about four hours (S phase) and decreased about 16 hours (G1 phase) after release from DTB. (B) H23 cells were cultured in medium containing 0.25% fetal calf serum (FCS) for 24-48 h, and OPA1
expression decreased at 48 h of serum starvation (left panel). OPA1 levels increased about 12 h (S phase) and decreased about 20 h (G1 phase) after addition of fresh FCS. (C) Knockdown of OPA1 expression by siRNAs (OPA1kd) for 72 hours reduced the protein level of OPA1 in H23 cells as determined by immunoblotting analysis. (D) Silencing of OPA1 expression increased cisplatin sensitivity in H23 cells. z, H23, wild-type; {, H23, OPA1kd. F-test, p < 0.01 (E) Serum-stimulated H23 cells were treated at different time point with cisplatin at 37°C for 4 h, wash twice with warm medium for 10 min, and then trypsinized. Viable cells were seeded quantitatively into 6-well plates with 5 ml of medium and incubated at 37°C and in 5% CO2 over 7-10 days. Colonies were stained with 2% crystal violet in methanol. Percent survival was determined by comparison to PBS-treated controls. Results shown are from cells treated with 5 μM cisplatin. {, H23 cells were cultured in 10% serum medium, wild-type; ,
H23, OPA1kd; (F) Confocal fluorescence immunocytochemistry and (G) immunoblotting of sucrose gradient-separated organelle fractions showed that silencing of OPA1 increased cytoplasmic cytochrome c, but not cytoplasmic nor nuclear apoptosis-inducing factor (AIF) following treatment of cells with low dose (0.1 μM) of staurosporine (STS) for 4 hr,
suggesting that OPA1 deficiency might increase caspase-dependent cell death. For retarding nuclear translocation of AIF, we also suppressed expression of human homolog of yeast Rad23A (hHR23A) by siRNA. (H1) Addition of low doses (0.1 or 0.25 μM) of STS for 4 hr provoked very diminutive effect on caspase 3 activation or degradation of poly (ADP-ribose) polymerase (PARP) in wild-type A549 cells. (H2) However, addition of sub-lethal doses (0.1 or 0.25 μM) of STS induced caspase 3 activation and PARP disintegration in OPA1kd cells. It is worth noting that silencing of OPA1 increased non-specific degradation of PARP.
patients Parameter Expression of OPA1 p value/correlation§ High (n = 219) Low (n = 67) Gender Male (n = 224) 178 46 0.041†/0.13 Female (n = 62) 41 21 Cigarette Smoking Smoker (n = 154) 139 15 < 0.001†/0.35 Non-smoker (n = 132) 80 52 Stage I (n = 89) 81 8 <0.001‡/0.44 II (n = 122) 101 21 III (n = 75) 37 38 Cell differentiation Well (n = 39) 27 12 0.504‡/-0.052 Moderate (n = 169) 131 38 Poor (n = 78) 61 17 Lymphovascular invasion Positive (n = 216) 178 38 < 0.001†/0.242 Negative (n = 70) 41 29 Expression of AKR1C1/2 AKR1C1/2+ (n = 237) 208 29 < 0.001†/0.581 AKR1C1/2- (n = 49) 11 38
proteins DRP1nuc+ (n = 226)* 191 35 < 0.001†/0.364 DRP1nuc- (n = 60)* 28 32 ATAD3A+ (n = 242) 212 30 < 0.001†/0.611 ATAD3A - (n = 44) 7 37 Mfn-1+ (n = 203) 191 12 < 0.001†/0.647 Mfn-1 - (n = 83) 28 55 Mfn-2+ (n = 177) 158 19 < 0.001†/0.382 Mfn-2- (n = 109) 61 48
†Two-sided p value determined by F2 test
‡Two-sided p value determined by Fisher’s exact test
*Nuclear expression of DRP1 labeled as DRP1nuc+; Cells which did not express DRP1 or the DRP1 was in the cytoplasm was labeled as DRP1nuc-.
§
The value was determined by the Spearman Correlation, which was based on normal approximation, and not assuming the null hypothesis.
