1171Research Article

IntroductionMajor features of lung adenocarcinoma (LADC) are rapid growthand high metastatic potential as well as resistance to irradiation andchemotherapy (Rosell et al., 2006). Cell proliferation and metastasisare regulated by the balance between growth factors and inhibitorymolecules (Schaefer et al., 2007). Using suppression subtractivehybridization (SSH), microarray and hierarchical clustering toinvestigate gene expression patterns in patients with lung cancer,we found that hepatocyte growth factor (HGF) and HGF receptor(HGFR, or product of proto-oncogene met, MET) were highlyexpressed in advanced LADC patients who smoked. Cigarettesmoking was further shown to be a key factor of disease progressionand treatment failure (Chen et al., 2006). However, a portion ofpatients who did not respond well to therapies in Taiwan werewomen and nonsmokers (Sun et al., 2007).

We used the same strategy to identify genes that were highlyexpressed in LADC. We then subtracted this LADC-specific genepool from smoking-related genes. The resulting genes weresubcategorized for ATPase and GTPase. The genes, of which theenzyme activity was activated by receptors, such as hepatocyte growthfactor receptor (HGFR), epidermal growth factor receptor (EGFR)and HER2/neu, were excluded. Using this procedure, we identifiedthree genes, encoding for dynamin-related protein 1 (DRP1) (Chianget al., 2009), mitofusin 2 (Mfn-2) (de Brito and Scorrano, 2008a) andthe ATPase family, AAA domain-containing protein 3 (ATAD3)(Hubstenberger et al., 2008), which were upregulated in LADC. DRP1and Mfn-2 are GTPases, and ATAD3 is an ATPase.

Three types of ATAD3 have been documented in the NCBIdatabase (http://www. ncbi.nlm.gov): a 66-kDa ATAD3A

(BC033109), a 72.6-kDa ATAD3B (NM_031921) and a 46-kDaATAD3C (NM_001039211; the differences in protein sequencesamong ATAD3A, 3B and 3C are summarized in supplementarymaterial Fig. S1A). Although protein sequence alignment indicatesthat ATAD3A and 3C are truncated isoforms of ATAD3B, they areencoded by different genes located in non-overlapping regions onchromosome 1 (supplementary material Fig. S1B,C). Moreover,ATAD3A contains 16 exons, 3B 15 exons and 3C 12 exons,indicating that ATAD3A and 3C are not alternatively splicedvariants of ATAD3B.

Using autoantibody-mediated identification of antigens(AMIDA), Gires et al. detected overexpression of KIAA1273/TOB3(ATAD3B) in patients with head and neck cancer (Gires et al., 2004).Applying phage display to probe tumor-associated antigens, Geuijenet al. identified ATAD3A in acute myeloid leukemic (AML) blasts(Geuijen et al., 2005). Inhibition of ATAD3B expression by siRNAincreased apoptosis (Schaffrik et al., 2006), but whether ATAD3Bor ATAD3A were directly involved in programmed cell death, wasnot clear. In this study, we determined the expression level ofATAD3A in LADC cells and pathological specimens. Thecorrelation between ATAD3A expression and patient survival wasevaluated statistically. The effect of ATAD3A on cell growth andapoptosis was characterized in vitro.

ResultsExpression of ATAD3A in LADC cells determined byRT-PCRExpression of ATAD3A was examined by RT-PCR in one HeLaand eight lung cancer cell lines. ATAD3A was detected in all cell

ATPase family AAA domain-containing 3A is a novelanti-apoptotic factor in lung adenocarcinoma cellsHsin-Yuan Fang1,2, Chia-Ling Chang3, Shu-Han Hsu3, Chih-Yang Huang4, Shu-Fen Chiang4,Shiow-Her Chiou4, Chun-Hua Huang3, Yi-Ting Hsiao3, Tze-Yi Lin5, I-Ping Chiang5, Wen-Hu Hsu6,Sumio Sugano7, Chih-Yi Chen2, Ching-Yuang Lin2, Wen-Je Ko1,* and Kuan-Chih Chow3,*1Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan2Departments of Surgery, China Medical University Hospital, China Medical University, Taichung, Taiwan3Graduate Institute of Biomedical Sciences, and 4Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung,Taiwan5Department of Pathology, China Medical University Hospital, Taichung, Taiwan6Division of Thoracic Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan7Laboratory of Functional Genomics, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, the University of Tokyo,Tokyo, Japan*Authors for correspondence (wenje@ha.mc.ntu.edu.tw; kcchow@dragon.nchu.edu.tw)

Accepted 3 January 2010Journal of Cell Science 123, 1171-1180 © 2010. Published by The Company of Biologists Ltddoi:10.1242/jcs.062034

SummaryAAA domain-containing 3A (ATAD3A) is a member of the AAA-ATPase family. Three forms of ATAD3 have been identified: ATAD3A,ATAD3B and ATAD3C. In this study, we examined the type and expression of ATAD3 in lung adenocarcinoma (LADC). Expressionof ATAD3A was detected by reverse transcription-polymerase chain reaction, immunoblotting, immunohistochemistry and confocalimmunofluorescent microscopy. Our results show that ATAD3A is the major form expressed in LADC. Silencing of ATAD3A expressionincreased mitochondrial fragmentation and cisplatin sensitivity. Serum deprivation increased ATAD3A expression and drug resistance.These results suggest that ATAD3A could be an anti-apoptotic marker in LADC.

Key words: ATAD3A, Lung adenocarcinoma, Mitochondrial fragmentation, Cisplatin sensitivity

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lines (Fig. 1A). In eight pairs of lung cancer biopsy samples,overexpression of ATAD3A was detected in six LADC samples (Fig.1B). Following sequence analysis, which was performed usingfluorescently labeled dideoxy nucleotides (Mission Biotech,www.missionbio.com.tw, Taipei, Taiwan), and a DNA sequencingladder read using an ABI PRISM 3700 DNA Analyzer (CDGenomics, Shirley, NY), nucleotide sequence homology of cDNAfragments from the nine cell lines and four LADC specimens was

searched using a web program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). They matched that of ATAD3A: NM_033109, Homosapiens ATPase family, AAA domain containing 3A (ATAD3A).No mutation was detected (GenBank, BankIt1285471, GU189416).ATAD3B and ATAD3C were not detected by RT-PCR or DNAsequencing in LADC cells (data not shown).

Expression and subcellular distribution of ATAD3A inLADC cellsFollowing determination of specificity and sensitivity, monoclonalantibodies were used to detect ATAD3A expression in LADC cells.A 66-kDa protein band corresponding to the anticipated molecularmass of ATAD3A was recognized in all the cell lines (Fig. 2A). A70-kDa protein was only highly expressed in H23 and H2087 cells,but it was not detected in A549, HeLa or mouse embryonicfibroblasts. To determine the identity of the two proteins, cell lysatefrom H23 was immunoprecipitated and the respective proteinband was subjected to analysis using matrix-assisted laserdesorption/ionization-time-of-flight mass spectrometry (MALDI-TOF). The peptide mass fingerprints of both the 66-kDa and 70-kDa proteins matched (MS-Fit search; http://prospector.ucsf.edu/):those of the full-length ATAD3A: GenBank CAI22955.1, Homosapiens, ATPase family AAA domain-containing 3A. The matchedpeptides covered 36% (211/586 amino acids) of the protein(supplementary material Fig. S2B).

Both the 66- and 70-kDa proteins were characterized by MALDI-TOF. The peptide mass fingerprints of both the 66-kDa and70-kDa proteins matched that of AATD3A: MS-Fit search(http://prospector.ucsf.edu/): CAI22955, ATPase family, AAA domain

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Fig. 1. Expression of ATAD3A in cancer cells as detected by RT-PCR.(A)Expression of ATAD3A mRNA was detected by RT-PCR in one HeLa andeight lung cancer cell lines. (B)In eight pairs of lung cancer biopsy samples,overexpression of ATAD3A mRNA was detected in six of the LADC samples.Expression of -actin was used as a monitoring standard for the relativeexpression ratio of ATAD3A mRNA. N, non-tumor lung tissue; T, tumorfraction of surgical resections.

Fig. 2. Characterization of monoclonal antibodies toATAD3A. (A)Immunoblotting revealed that monoclonalantibodies raised against recombinant ATAD3Arecognized two protein bands of approximately 66 kDa.Expression of the 66-kDa ATAD3A was detected in alleight human lung cancer cell lines: high in H23, H226,H838, H2009, H2087, and SK-MES-1 (relative to A549),and low in A549 and H1437 cells. Expression of the 70-kDa protein was high in H23 and H2087 cells. Cell lysatefrom H23 were precipitated by ATAD3A-specificmonoclonal antibodies and protein-G-SepharoseTM (seealso supplementary material Fig. S2B).(B)Immunocytochemical staining showed that ATAD3Awas abundantly present as distinct granules in thecytoplasm, which suggests that ATAD3A is located inmitochondria. (C)Confocal fluorescenceimmunocytochemistry of H2087 and A549 cells.ATAD3A was detected by specific monoclonal antibodieslabeled with FITC. Mitochondria were labeled withMitoTracker® Red CMXRos dye. Nuclei were stainedwith the fluorescent dye DAPI (4�,6-diamidino-2-phenylindole). A merged image of the first, second andthird columns, and the magnification of specific cellsconfirm that ATAD3A is located in mitochondria.(D)Immunoblotting of SK-MES1 subcellular fractions,which were separated by sucrose gradientultracentrifugation. LM, light membrane fraction of theER; MAM, mitochondria-associated membrane of theER; AIF, apoptosis-inducing factor; GRP78, 78-kDaglucose-regulated protein; COX IV, cytochrome coxidase IV of mitochondria.

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containing 3A [Homo sapiens] (supplementary material Fig. S2B);however, they covered only 25.0% (167/648 AAs) of the ATAD3B.Moreover, three MALDI-TOF resultant fragments did not match withATAD3B (the mismatched sequences are shown in supplementarymaterial Fig. S2C,D). These data indicated that both 66-kDa and 70-kDa proteins were ATAD3A (CAI22955), and that the 70-kDa proteincould be a post-translationally modified ATAD3A in LADC cells.The 85-kDa protein, which was determined by MALDI-TOF, wasidentified as DRP1, suggesting that ATAD3A interacted with DRP1(supplementary material Fig. S2B1 and Fig. S2B3).

Immunocytochemical staining showed that ATAD3A wasabundantly present in cytoplasm. The granular appearance ofsubcellular structures suggested that ATAD3A could be present inmitochondria (Fig. 2B). A MitoTracker Red CMXRos (MolecularProbes, Eugene, OR) uptake assay and confocal fluorescenceimmunocytochemistry (Fig. 2C) as well as immunoblotting ofsucrose gradient-separated organelle fractions (Fig. 2D) confirmedthat ATAD3A was localized in light membrane and mitochondria-associated membrane fractions of endoplasmic reticulum (ER) aswell as in mitochondrial fraction. In addition to ATAD3A, apoptosis-inducing factor (AIF) and glucose response protein (GRP) 78 weredetected in the same fractions. The results showed that as suggestedby the web prediction program (http://www.ch.embnet.org/software/TMPRED_form.html), ATAD3A (BC033109) carried atransmembrane domain with a coiled-coil domain exposed to thecytoplasm which could interact with DRP1 and/or Mfn-2(supplementary material Fig. S2E and Fig. S3A-C).

Pathological expression of ATAD3A in lungadenocarcinomasUsing immunoblotting, we identified that the major type of ATAD3expressed in LADC specimens was the 66-kDa ATAD3A (Fig. 3A).Immunohistochemistry detected ATAD3A in 93 (86.9%) of thepathological samples from patients with LADC. The signal waspredominantly localized in cancer cells (Fig. 3B1), but not in non-tumor lung tissue (NTLT; Fig. 3B2). ATAD3A expression was alsodetected in 89.3% (50/56) of metastatic lymph nodes (data notshown). Statistical analysis showed that overexpression of ATAD3Ain tumors correlated with tumor stage and lymphovascularinvolvement (Table 1), suggesting that ATAD3A expression couldbe associated with the metastatic potential of LADC. Interestingly,among the 93 patients who had high levels of ATAD3A, 39 (41.9%)patients had tumor recurrence during follow-up examination.Among the 14 patients who had low levels of ATAD3A, three hadtumor recurrence (21.4%). All 42 patients who had recurrencedeveloped tumors within 24 months of the operation. The risk ofrecurrence for patients with high levels of ATAD3A was 3.01-foldhigher than that for patients with low levels of ATAD3A (P0.045).Survival of patients with low ATAD3A levels was significantlybetter than that of patients with high ATAD3A levels (Fig. 3C). Thehazard ratio between these two groups was 2.415, and the differencein cumulative survival was significant (P0.0027). Multivariateanalysis, however, revealed that the difference in ATAD3Aexpression between the two groups was marginal (P0.052).

ATAD3A in LADC cells is phosphorylated by PKC, andphosphorylation is essential for ATAD3A stabilityAs shown previously, ATAD3A appeared as a 66-kDa form in A549and HeLa cells as well as in mouse embryonic fibroblasts. The 70-kDa form, however, was only highly expressed in H23 and H2087cells. In H1437, H226, H838 and H2009 cells, expression level of

ATAD3A varied. Moreover, the amino acid sequence of the 70-kDaprotein, which was frequently detected in lung cancer cell lines, butnot in pathological specimens (Fig. 4A, also refer to Fig. 2A and Fig.

Fig. 3. Correlation between ATAD3A expression and survival in patientswith LADC. (A)Expression of ATAD3A was determined by immunoblotting.Expression of -actin was used as a monitoring standard for the relativeexpression of ATAD3A. N, non-tumor lung tissue; T, tumor fraction ofsurgical resections; numbers above the lanes are the patients’ sample numbers.(B) Representative examples of ATAD3A expression in pathologicalspecimens as detected by immunohistochemical staining (crimson precipitatesin cytoplasm). Expression of ATAD3A was detected in LADC tumor nests(B1), but not in non-tumor lung tissue (B2). B3 is a negative control of B1,and B4 a negative control of B2. Antibodies to ATAD3A were not added in thenegative control groups. (C)Comparison of Kaplan-Meier product limitestimates of survival analysis in patients with LADC. Patients were dividedinto two groups based on ATAD3A expression. Survival difference betweenthe two groups was compared by a log rank test. P0.0027.

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3A), matched that of ATAD3A (supplementary material Fig. S2B-D), suggesting that the 70-kDa protein might not be an ATAD3B,but a phosphorylated ATAD3A. To resolve the issue, we ran theprotein sequence of ATAD3A through a web NetPhos program topredict for phosphorylation sites (http://www.cbs.dtu.dk/services/NetPhos/), and a NetPhosK program to predict specific kinase(http://www.cbs.dtu.dk/services/NetPhosK/) in eukaryotic proteins.The results showed that for ATAD3A (BC033109), the most probablekinase was protein kinase C (PKC) at the possible phosphorylationsites Thr335, Thr338, Thr359, or PKA at Thr118 (supplementarymaterial Fig. S4A). For ATAD3AL (NM-018188) and ATAD3B, themost probable kinase was PKA (supplementary material Fig. S4B,C).The most probable kinase for ATAD3C was PKC at position Thr184(supplementary material Fig. S4D). However, since this segment islocated inside the membrane of the prospective vesicle, the predictionmight not be applicable. When the cell lysate of H23 cells was treatedwith calf intestinal alkaline phosphatase (CIP) before immunoblotting,signals of the 70-kDa protein band reduced markedly (Fig. 4B1).Treatment with CIP also reduced the 66-kDa protein to 63 kDa,suggesting that both the 66-kDa and 70-kDa proteins werephosphorylated. When ATAD3A antibody-precipitated proteins wereprobed with antibodies specific to phosphoserine/threonine (S-P/T-P)or phosphotyrosine (Upstate, Millipore Corporate, Billerica, MA),both 66-kDa and 70-kDa protein bands were positive for S-P/T-P(Fig. 4B2), but not phosphotyrosine (data not shown), indicating thatthe phosphorylated residues in both the 66-kDa and 70-kDa proteinswere at serine or threonine.

To search for the kinase that is responsible for ATAD3Aphosphorylation, we treated H23 cells with a panel of kinaseinhibitors. As shown in Fig. 4C1, only addition of calphostin C, aprotein kinase C (PKC) inhibitor, reduced the intensities of the 70-kDa and 66-kDa protein bands, indicating that PKC is the major kinasefor ATAD3A phosphorylation. The results excluded a possibility ofPKA in ATAD3A phosphorylation. Since calphostin C treatmentreduced 70-kDa and 66-kDa proteins in a dose-dependent fashion(Fig. 4C2), the data also suggested that phosphorylation was essentialfor maintaining ATAD3A stability. To identify the PKC isozyme thatphosphorylated ATAD3A, A549 cells, which only expressed 66-kDaATAD3A, and H23 cells were transfected with plasmids carrying

various isozyme of PKC genes. Interestingly, level of the 70-kDaproteins increased in cells that ectopically expressed PKC, PKCand PKC (Fig. 4D1,2). These results confirmed that PKC wasresponsible for ATAD3A phosphorylation.

Biosynthesis of ATAD3A increases during S phase of cellcycle progression or under serum starvationBecause PKC activity is associated with growth factor receptor-relatedcellular events and cell cycle progression (Hirai et al., 1989; Black,2000), the fact that PKC is involved in ATAD3A phosphorylationsuggests that ATAD3A expression may be regulated by growth factorreceptor- or cell cycle progression-related pathways. We thereforeanalyzed the expression pattern of ATAD3A during cell cycleprogression by double-thymidine block (DTB) and serum starvation-reactivation methods. Results of DTB and release showed that levelsof ATAD3A increased in S phase (4 hours after release from DTB)and decreased in the G1 phase (16 hours after release from DTB;Fig. 5A), indicating that ATAD3A biosynthesis was at S phase ofcell cycle progression. Surprisingly, serum starvation increased levelsof ATAD3A as well. The increase was dose (Fig. 5B1) and timedependent (Fig. 5B2). Moreover, increase of ATAD3A during serumstarvation was associated with increase of cisplatin resistance (Fig.5C). However, serum starvation did not increase levels of ATAD3AmRNA, but it did increase expression of a panel of metastasis- andangiogenesis-related genes, including those for HGF, vascularendothelial growth factor-B and matrix metalloproteinases (Table 2).Increase of AATD3A protein level could be a translational activation.The results showed that serum starvation induced growth arrest ofcells at G1 phase and down-regulated expression of replication-relatedgenes, which reflected reduced DNA damage and less drug toxicity(Chow and Ross, 1987; Chen et al., 2008). The loss of function effectof ATAD3A on drug sensitivity is yet to be resolved.

Silencing of ATAD3A expression increases drugsensitivity, mitochondrial fragmentation, and reducescommunication between endoplasmic reticulum andmitochondriaAs anticipated, silencing of ATAD3A expression by siRNA (Fig.6A1) increased cisplatin sensitivity (Fig. 6A2). Moreover,

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Table 1. Correlation of ATAD3A expression with clinicopathological parameters in patients with LADC

Expression of ATAD3A P value

Parameter High (n93) Low (n14) Univariate Multivariate

GenderMale (n84) 75 9 0.165† 0.498Female (n23) 18 5

Cigarette smokingSmoker (n78) 70 8 0.155† 0.463Non-smoker (n29) 23 6

StageI (n26) 20 6 0.030‡ 0.057II (n31) 25 6III (n50) 48 2

Cell differentiationWell (n17) 12 5 0.075‡ 0.141Moderate (n60) 53 7Poor (n30) 28 2

Lymphovascular invasionPositive (n81) 75 6 0.002† 0.017Negative (n26) 18 8

†Two-sided P value determined by c2 test. ‡Two-sided P value determined by Fisher’s exact test.

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knockdown of ATAD3A (ATAD3Akd) expression increasedmitochondrial fragmentation (Fig. 6B1), and decreasedcolocalization of the ER [ER was visualized by ER retention signalKDEL-conjugated green fluorescence protein (GFP); Fig. 6B2,upper row] and mitochondria (Fig. 6B2, upper row, redfluorescence). Silencing of ATAD3A expression reduced the totalamount of mitochondria as well, but it increased the amount ofenlarged ER (Fig. 6B2, center row). These results suggested thatATAD3A could be detected on both ER and mitochondria, and likemitofusin-2 (Mfn-2) and DRP1, ATAD3A could be involved inmitochondrial shaping, i.e. fission and fusion, as well ascommunication between ER and mitochondria (Fig. 6B2, upperrow). Interestingly, knockdown of DRP1 (DRP1kd) also increasedthe amount of prominently enlarged ER (Fig. 6B2, bottom row).The presence of prominently enlarged ER in DRP1kd cells was

confirmed by electron microscopy (Fig. 6B3A,B). In ATAD3Akd

cells, we identified two notable features, small vesicles around thedilated ER that appeared to be budding off from the ER (Fig. 6B3C)and mitochondria encased in vacuoles (Fig. 6B3D). Although thecorresponding features of engulfed mitochondria in double-labeledconfocal fluorescence micrographs were not detected, it is possiblethat the enzyme activity in vacuole-encased mitochondria wasobscured. As noted above, ATAD3A, Mfn-2 and DRP1 were allinvolved in mitochondrial shaping. Results of a PSORT II predictionprogram (http://psort.ims.u-tokyo.ac.jp/) further showed that allthree molecules contained notable stretch of coiled-coil(supplementary material Fig. S4A-C), suggesting that these proteinsmight interact with each other, and act together in communicationbetween the ER and mitochondria. Using immunoprecipitation andimmunoblotting, Mfn-2 and ATAD3A were co-precipitated by the

Fig. 4. Expression and post-translational modification of ATAD3A in LADC cells. (A)Comparison of ATAD3A expression between LADC cells andpathological specimens as measured by immunoblotting. A 70-kDa protein was detected in lung cancer cell lines H23 and H2087, but not in pathologicalspecimens. (B)The 70-kDa protein is a phosphorylated ATAD3A. (B1)Treatment of H23 cell lysate with calf intestinal alkaline phosphatase (CIP) beforeimmunoblotting decreased signals of 70-kDa protein, and the molecular mass of the 66-kDa protein to 63-kDa. (B2)Cell lysate of H23 was immunoprecipitatedwith antibodies specific to ATAD3A before immunoblotting, which was probed with antibodies specific to phosphoserine/threonine or phosphotyrosine. Both the66- and 70-kDa protein bands were positive for phosphoserine/threonine (pT/S). These results suggested that both proteins were phosphorylated ATAD3A. (C)Theeffect of the kinase inhibitor on phosphorylation of ATAD3A. (C1)The H23 cells were each treated with a panel of serine/threonine kinase inhibitors beforeimmunoblotting. Only treatment with 5M calphostin C, a protein kinase C (PKC) inhibitor, at 37°C for 2 hours reduced the intensity of the 70-kDa and 66-kDaprotein bands. (C2)H23 cells were treated with various concentrations of calphostin C at 37°C for two hours before immunoblotting. Intensity of the 70-kDaprotein decreased at concentrations of calphostin C greater than 0.32M; whereas that of the 66-kDa protein were reduced at concentrations higher than 0.64M.The results suggested that PKC-mediated phosphorylation was essential to maintaining ATAD3A stability. (D)Different isozymes of PKC were ectopicallyexpressed in A549 (D1) and H23 (D2) cells before immunoblotting. Intensity of the 70-kDa protein increased in cells that expressed PKC, PKC and PKC.

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respective antibodies (Fig. 6C1). Moreover, using the same methodto react with mitochondria-associated membrane and lightmembrane fractions of sucrose gradient ultracentrifugation beforeimmunoblotting, mitochondrial proteins, such as optic atrophyprotein 1 (OPA1), AIF and Mfn-2, were co-precipitated byantibodies specific to ATAD3A (Fig. 6C2), suggesting that DRP1,Mfn-2 and ATAD3A were involved in a, yet to be determined,protein transport between the ER and mitochondria. In this case,silencing expression of Mfn-2, an important molecule for membranefusion, would increase darkening of the ER, mitochondrialfragmentation, the number of small vesicles (Fig. 6D1,2), andcisplatin sensitivity (data not shown). Increased darkening of theER would suggest protein accumulation in the organelle. Takentogether, the data suggested that these proteins played a pivotal rolein maintaining normal morphology of the ER and mitochondria. Adefect in these proteins would increase drug sensitivity and cellapoptosis.

DiscussionOur results indicate that the ATAD3A is the major type of ATAD3family detected in LADC. Overexpression of ATAD3A in patientswith LADC correlated with significantly higher incidence of earlytumor recurrence and increased drug resistance, which ultimatelyreflected poor survival.

By demonstrating that the 70-kDa protein, which was frequentlydetected in LADC cell lines, was sensitive to CIP, our data suggestedthat the 70-kDa protein was a phosphorylated ATAD3A. Interestingly,the 66-kDa protein was sensitive to CIP as well, indicating that 66-kDa ATAD3A was also phosphorylated, and that the phosphorylationsites were at serine/threonine residues. Treatment with calphostin C,a pan-PKC inhibitor (Kobayashi et al., 1989), for 2 hours reducesphosphorylation and protein level of both 66- and 70-kDa ATAD3A,confirming that PKC is the kinase that catalyzes ATAD3Aphosphorylation, and that phosphorylation is essential forATAD3A stability. These findings corresponded well withimmunoblotting results of patients’ biopsy specimens, and suggestedthat ATAD3A detected in non-tumor lung tissue was a 63-kDa protein,which might not be readily phosphorylated and was labile. Inaddition, ectopic expression of PKC showed that the PKC isozymesresponsible for ATAD3A phosphorylation were PKC, PKC andPKC (Hirai and Chida, 2003). It is therefore worth noting thatATAD3A expression was upregulated in S phase of cell cycle.However, serum starvation also increased ATAD3A. It is unclear howserum starvation induces ATAD3A expression, but the findingscorrespond well with pathological observations that expression ofATAD3A increases with disease progression of LADC. Our previousresults showed that hypoxia increased expression of hepatocytegrowth factor (HGF) and interleukin-8 as well as synthesis ofprostaglandin F2 (Chiang, 2009). Since rapid growth of cancer cellsduring disease progression often results in inadequate supply ofoxygen and nutrients in the tumor nest, our findings provide furtherexplanation of how short-term serum deprivation-induced genes workin concert on survival and possibly metastasis of cancer cells (Tavalucet al., 2007).

As noted previously, using AMIDA, Gires et al. detectedoverexpression of ATAD3B in patients with head and neck cancer(HNC) (Gires et al., 2004). In addition, Schaffrik et al. showed thattwo forms of ATAD3B, ATAD3Bl (large) and ATAD3Bs (small), weredetected in HNC (Schaffrik et al., 2006). Unlike ATAD3Bl andATAD3Bs, which differ at their N-termini, the discrepancy betweenATAD3A and 3B is at their C-termini (supplementary material Fig.S1A-D). Using MALDI-TOF to determine specific antibody-precipitated proteins, we found that peptide mass fingerprints of boththe 66-kDa and 70-kDa proteins match those of ATAD3A, includingthe N-terminus (supplementary material Fig. S2A-D). However, threeMALDI-TOF fragments did not match ATAD3B or ATAD3AL

(NM_018188), indicating that the ATAD3A overexpressed in LADCcells is ATAD3A (BC033109). Applying phage display, Geuijen etal. identified ATAD3A as a significant tumor-associated antigen inAML blasts (Geuijen et al., 2005). Our observations support theirdata, indicating that AML and LADC might overexpress ATAD3Ato facilitate cell growth and possibly metastasis. Inhibition ofATAD3A expression, by contrast, increases apoptosis and drugsensitivity, suggesting that ATAD3A is an anti-apoptotic factor.

It is interesting to note that silencing of ATAD3A expressionincreased mitochondrial fragmentation and mitochondria-containingautophagic vacuoles, phenomena that are frequently detected inMfn-2 knockout-associated and DRP1-related apoptotic cells(Honda et al., 2005; de Brito and Scorrano, 2008a; Gomez-Lazaro

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Fig. 5. ATAD3A expression during cell cycle progression and serumstarvation. (A)ATAD3A expression in different phases of cell cycleprogression. HeLa cells were treated with double thymidine block (DTB) tosynchronize cells at the late G1 phase. ATAD3A levels increased by about 4hours (S phase) and had decreased by 16 hours (G1 phase) after release fromDTB. (B)Serum starvation increases ATAD3A expression. (B1)ATAD3Aexpression increased in H23 cells when the concentration of fetal calf serum(FCS) was decreased from 2% to 1%. (B2)H23 and H2087 cells were culturedin medium containing 0.25% FCS for 24-48 hours, and ATAD3A expressionincreased starting from 24-hour of serum starvation. (C)Serum starvationreduced cisplatin cytotoxicity. H23 cells were grown in medium with 10% or0.25% FCS for 24 hours before addition of cisplatin. Cisplatin resistanceincreased when cells were cultured in low serum medium (0.25%, blackcircles). White circles, H23 cells were cultured in 10% serum medium.

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et al., 2008; Knott et al., 2008; Chiang et al., 2009), implying thatATAD3A could be involved in mitochondrial fusion. Recently, deBrito and Scorrano showed that Mfn-2, an essential mitochondrialfusion protein, was detected on the ER, in particular in themitochondria-associated membrane and the mitochondrial outermembrane. They suggested that the coiled-coil structure on thecytoplasmic side of Mfn-2 on both organelles tethered the twoorganelles together to coordinate Ca2+ flow (de Brito and Scorrano,2008b; Merkwirth and Langer, 2008). However, results of a PSORTII prediction program (http://psort.ims.u-tokyo.ac.jp/) showed thatalthough a cleavage site for mitochondrial presequence was detectedbetween amino acid residues 20 and 21 (KRH|MA), no evidentmitochondrial targeting sequence or ER membrane retention signalwas identified in Mfn-2. On the contrary, two peroxisomal targetingsignals, KINGIFEQL starting at amino acid residue 38 andRLKFIDKQL at residue 400 (supplementary material Fig. S4A),were found in the protein. Since peroxisomes are derived from ER,their findings suggested that Mfn-2 could be targeted to bothorganelles by a, yet to be determined, factor or the protein may takean alternative route (Pfanner and Geissler, 2001) via the ER beforetransport to mitochondria.

Like Mfn-2, ATAD3A is a transmembrane protein containing anextensive coiled-coil in the cytoplasmic domain of the N-terminus(supplementary material Fig. S4B). Moreover, like Mfn-2 andATAD3A, DRP1, which is essential for mitochondrial fission, alsocontains a stretch of coiled-coil (supplementary material Fig. S4C).Interestingly, knockdown of DRP1 increased bulging of the ER,which was detected by ectopic expression of a KDEL-conjugatedGFP. Electron micrographs confirmed that the ballooning structureobserved by fluorescence microscopy was the ER, suggesting thatDRP1 was involved in configuration changes of the ER. WhenDRP1 activity was kept undisturbed, knockdown of ATAD3Aincreased the number of transport vesicles, which appeared to be

budding off of a dilated region of ER. Knockdown of any geneconcomitantly changed the morphology of the ER and mitochondria,suggesting a functional connection between the two organellesinvolving Mfn-2, DRP1 and ATAD3A.

It is worth noting that ATAD3A and DRP1 are concurrentlyupregulated in the early S phase of cell cycle progression in LADCcells (Chiang et al., 2009). Elegant studies by Shiao et al. (Shiaoet al., 1995) and Jackowski (Jackowski, 1996) showed that the levelof phospholipids that are synthesized in the ER and transported tomitochondria via mitochondria-associated membranes also increasesin the early S-phase of cell cycle progression. Moreover, usingsucrose gradient ultracentrifugation, ATAD3A, glucose responseprotein 78 (GRP78) (Sun et al., 2006) and apoptosis AIF (Chen etal., 2008) were detected in fractions of light membrane,mitochondria-associated membrane and mitochondria. Thesedata, considered together with studies using confocalimmunofluorescence microscopy (CIM) and electron microscopy(EM), suggest that ATAD3A, an ATPase, could be required for analternative transport of proteins, such as GRP78, AIF andmembrane-anchored Mfn-2, from the ER to the mitochondria. Weare examining such a possibility in an ongoing study.

In conclusion, immunoblotting and immunohistochemistryrevealed abundant expression of ATAD3A in lung adenocarcinomacells. Pathological results suggest that ATAD3A expression isassociated with lymphovascular invasion, which reflects the increasedmetastatic potential of LADC and poor prognosis of patients. In vitro,serum starvation increased expression of ATAD3A and the level ofcisplatin resistance in lung adenocarcinoma cells. Our finding thatATAD3A was present in the mitochondria-associated membrane ofthe ER and mitochondria, and that silencing of ATAD3A increasedtransport of vesicle-like figures and mitochondrial fragmentation aswell as cisplatin sensitivity suggest that in addition to materialtransport between the ER and the mitochondria ATAD3A might play

Table 2. Metastasis- and replication-related cytokine, growth factor and transcription factor genes in H23 cells following serumstarvation for 24-48 hours

Serum starvation

Description of the gene 24 hours 48 hours

Metastasis-related genesMMP-2 (gelatinase A, type IV collagenase) 2.895 6.937MMP-10 (stromelysin 2) – 2.108MMP-11 (stromelysin 3) – 3.751MMP-14 – 4.817MMP15 – 2.275Homo sapiens metastasis related protein (MB2) [AF100640] 12.53 3.093Homo sapiens metastasis associated lung adenocarcinoma transcript 1 (MALAT1) – 2.273Metastasis-associated protein MTA3 – 2.601

Replication-related proteinsTopoisomerase II – 0.155Karyopherin 5 – 0.155Centrosomal protein Q – 0.16Topoisomerase II binding protein – 0.183CHK1 – 0.197

Transcription factor/modulatorHistone deacetylase 5 (HDAC5) – 4.839ATPase family, AAA domain containing 2 (ATAD2) 0.385 0.092

Cytokines Tumor necrosis factor superfamily member 10 (TNFSF10) 2.036 –Homo sapiens prostaglandin I2 (prostacyclin) synthase – 5.771Homo sapiens C1q and tumor necrosis factor related protein 6 (C1QTNF6) – 2.336Homo sapiens tumor necrosis factor (ligand) superfamily, member 12 (TNFSF12) – 2.795Transforming growth factor, beta (TGF1) – 2.10Vascular endothelial growth factor-B (VEGF-B) – 2.007Hepatocyte growth factor (HGF) – 2.392

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Fig. 6. See next page for legend.

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a role in drug resistance of lung cancer cells, in particular duringdeprivation of nutrients. At that stage, the cancer cells might stopproliferating and prepare for moving out of the gradually deterioratingmicroenvironment.

Materials and MethodsTissue specimens and non-small cell lung cancer cell (NSCLC) linesThe patients in this study were from the same cohort used in the previous study. Theprotocols of both studies were approved by the Medical Ethics Committee. All clinicaldata were identical to those in the previous study (Chen et al., 2006).

Eight NSCLC cell lines (H23, H226, H838, H1437, H2009, H2087, A549 andSK-MES-1) were used for the in vitro evaluation of gene expression. H23, H838,H1437, H2009, H2087 and A549 are LADC cells, and H226 and SK-MES-1 areepithelial type cells. HeLa is a uterine cervical epithelial cell line. Cells were grownat 37°C in a monolayer in RPMI 1640 supplemented with 10% fetal calf serum (FCS),100 IU/ml penicillin and 100 g/ml streptomycin.

Reverse transcription-polymerase chain reaction (RT-PCR)Following total RNA extraction and synthesis of the first-strand cDNA, an aliquotof cDNA was subjected to 35 cycles of PCR to determine the integrity of -actinmRNA (Chen et al., 2006). The cDNA used in the following RT-PCR was adjustedaccording to the quality and quantity of -actin mRNA. The primer sequences wereselected using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3). For ATAD3A, theprimers were: ATAD3As: 5�-GGTCTACTCAGCCAAGAAT-3� (sense primer, nts889-907, BC033109) and ATAD3Aa: 5�-CACTTCCTCCCGTAGTCAAA-3�(antisense primer, nts 1633-1653). The primers for ATAD3B were: ATAD3Bs: 5�-

GGTCTACTCAGCCAAGAAT-3� (sense primer, nts 881-899, NM_031921) andATAD3Ba: 5�-GCGCATCTTCTGTCGGTACT-3� (nts 1792-1811, NM_031921) andthose for ATAD3C were: ATAD3Cs: 5�-GTGACAGACCGGGACAAAGT-3� (senseprimer, nts 1188-1207, NM_001039211) and ATAD3Ca: 5�-CACTTCCTC CCGT -AGTCAAA-3� (antisense primer, nts 2014-1995). ATAD3A and 3B shared the samesense primer, but had the different antisense primers. ATAD3A and 3C had the differentsense primers, but shared the same antisense primer. The anticipated cDNA fragmentswere 765 base-pairs (bp) for ATAD3A, 931 bp for ATAD3B and 827 bp for ATAD3C.For N-terminal ATAD3A, the primers were: ATAD3A Ns: 5�-TGCGAGC AT -GTCGTGGCTCTTCGG-3� (sense primer, nts 84-107, BC033109) and ATAD3A Na:5�-GGGACGTCTCCCTCACTAGG-3� (antisense primer, nts 958-939).

Immunoprecipitation, gel electrophoresis and protein analysis by MALDI-TOFTotal cell lysate was prepared by mixing 5�107 cells/100 l phosphate-buffered salinewith 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-washedbefore mixing with 500 g of total cell lysate. The reaction mixture was incubatedat 4°C for 60 minutes, and then centrifuged at 800 g for 1 minute. The supernatantwas reacted with 5 g of purified monoclonal antibodies and 20 l of fresh protein-G-Sepharose at 4°C for 18 hours. The reaction mixture was centrifuged at 800 g for1 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 mMdisodium EDTA, 1 mM PMSF, 10% glycerol, 5% -mercaptoethanol, 0.01%Bromophenol Blue and 1% SDS). Electrophoresis 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 bandson the Coomassie-stained gel, which corresponded to the immunoblotting-positivebands, 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 databaseby MS-fit (ProteinProspector 4.0.5., The Regents of the University of California).After electrophoresis, proteins on the first gel were transferred to a nitrocellulosemembrane 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 anX-Omat film (Eastman Kodak, Rochester, NY) with enhanced chemiluminescentreagent (NEN, Boston, MA).

Immunoblotting analysis and immunocytochemistryImmunoblotting and immunohistochemistry were performed as described previously(Chen et al., 2006). Antibodies for -actin were obtained from Chemicon International(Temecula, CA). Antibodies to ATAD3A were raised in the laboratory (supplementarymaterial Fig. S1). For immunocytochemistry, the cells were grown overnight on slides,and then fixed with cold methanol-acetone at 4°C for 10 minutes before staining.Immunological staining was performed by an immunoperoxidase method. Antibodiesto ATAD3A were not added for the negative control group.

Preparation and characterization of mouse polyclonal and monoclonalantibodiesDNA sequence corresponding to C-terminal amino acids 281-534 of ATAD3A wasamplified by primer sequences containing BamHI (sense) and HindIII (antisense)restriction sites. The primer sequences were ATAD3A-forward: 5�-ATCGGATCC -ATGGTCTACTCAGCCAAGAAT-3� (BamHI site is underlined) and ATAD3A-reverse: 5�-ATCAAGCAACTATCACTTCCTCCCGTAGTCAAA-3� (HindIII site isunderlined).

The restriction fragment of ATAD3A was cloned into an expression vector pET-32b+ (pET32+-AIF; Promega KK, Tokyo, Japan). Bacterial colonies containingpET32+-ATAD3A was selected, and induced by isopropyl--D-thiogalactopyranoside(IPTG) to mass-produce recombinant protein fragments of ATAD3A. The recombinantprotein was purified using a nickel-affinity column. Affinity-purified ATAD3A wasused to immunize BALB/c mice, and sensitivity of antiserum (OD405>0.3 at 1:6000dilutions) was measured by enzyme-linked immunosorbent assay (ELISA). Specificityof antibodies was determined by the appearance of a 66-kDa band in immunoblotsof lung cancer cell extract (Geuijen et al., 2005). Monoclonal antibodies were producedby a hybridoma technique using mouse myeloma cells NS1, and ATAD3A-specificantibodies were screened by the above-mentioned methods. In some cells, two proteinbands, a 66-kDa and a 70-kDa one, were detected by the antibodies in theimmunoblotting. Sensitivity of the antibodies, which was measured by a seriallydiluted mouse ascites, reached 1:51,200 dilutions (supplementary material Fig. S2A1).In mouse tissues, the antibodies recognized a 66-kDa protein in tissue extracts fromheart, lung, muscle and spleen. The 70-kDa protein was only detected in kidney andliver (supplementary material Fig. S2A2). In order to determine the identity of the66-kDa and 70-kDa human proteins, the respective bands were excised from aCoomassie-stained gel and subjected to an analysis by matrix-assisted laserdesorption/ionization and time-of-flight mass spectrometry (MALDI-TOF). Theresults showed that both immunoprecipitated 66-kDa and 70-kDa proteins matchedATAD3A (CAI22955; MS-Fit data shown in supplementary material Fig. S2B). Thepeptides matched 36.0% (211/586AAs) of ATAD3A. However, they matched only

Fig. 6. Influence of ATAD3A expression on cell features. (A)Decrease ofATAD3A expression increased cisplatin cytotoxicity. (A1)Knockdown (kd) ofATAD3A expression by siRNAs (ATAD3Akd) for 96 hours reduced the proteinlevel of ATAD3A in A549 and H838 cells as determined by immunoblottinganalysis. (A2)Silencing of ATAD3A expression increased cisplatin sensitivityin A549 and H838 cells. White squares, H838 wild-type; black squares, H838,ATAD3Akd; white triangles, A549, wild-type; black triangles, A549,ATAD3Akd. F-test, P<0.01. (B)Decrease of ATAD3A expression changed theultrastructure of the cells. (B1)Knockdown of ATAD3A expression by siRNAs(ATAD3Akd) increased mitochondrial fragmentation in H838 cells.(B2)Knockdown of ATAD3A expression (center row) decreasedcolocalization (yellow fluorescence, columns 3 and 4) of ER (KDEL-conjugated GFP, upper row) and mitochondria (red fluorescence, upper row).Silencing of ATAD3A expression reduced the number of mitochondria, but itincreased the enlarged ER (column 4, center row, white arrow). These resultssuggested that ATAD3A could be involved in communication betweenmitochondria and the ER. Knockdown of DRP1 (DRP1kd) increased theprominently enlarged ER (column 4, bottom row). N, nucleus; (B3) Functionof ATAD3A and DRP1 in organelle morphology as observed by electronmicroscopy. (B3A) Electron micrographs of H838 cells revealed manymitochondria associated with the ER, which could be mitochondria-associatedmembrane (MAM; black arrows). (B3B) In DRP1kd H838 cells, mitochondriawere elongated (white arrows), and many ER structures were prominentlydilated (black arrow). In ATAD3Akd H838 cells, two notable features wereidentified: (B3C) small vesicles (white arrows) that appeared to be budding offfrom the dilated ER (black arrows); and (B3D) mitochondria encased invacuoles (white arrows). However, the features of engulfed mitochondria werenot detected in double-labeled confocal fluorescence micrographs. N, nucleus;*, dilated peroxisomes. (C)Interaction between ATAD3A and Mfn-2.(C1)Immunoprecipitation and immunoblotting showed that ATAD3A-specificmonoclonal antibodies and protein-G-Sepharose co-precipitated Mfn-2 in thecell lysate. IP, immunoprecipitation; IB, immunoblotting; M, protein marker;S, supernatant; P, IP pellet. (C2)Immunoprecipitation and two-dimensionalimmunoblotting showed that ATAD3A-specific monoclonal antibodies andprotein-G Sepharose co-precipitated Mfn-2, OPA1 and AIF in light membraneand MAM fractions of sucrose gradient ultracentrifugation. (D)Effect of Mfn-2 silencing on organelle morphology. (D1)Electron micrograph of controlH838 cells. (D2)In Mfn-2kd H838 cells, vacuoles (arrowheads) and silhouetteof transport-vesicle-like figures (arrows) increased. Mitochondria were smallerin Mfn-2kd H838 cells than those in the control group, which it has beensuggested is due to mitochondrial fragmentation. Gene silencing was carriedout by siRNA treatment for 48 hours before cell harvest.

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25.0% (167/648AAs) of ATAD3B, and three MALDI-TOF fragments did not matchATAD3B (the mismatched sequences are noted in supplementary material Fig. S2Cand D). The results excluded the possibility that LADC cells expressed ATAD3B.

It is worth noting that two different ATAD3As, ATAD3A (BC033109) and ATAD3A(NM_018188), are listed in GenBank (http://www.ncbi.nlm.nih.gov/entrez). Thedifference between ATAD3A (BC033109) and ATAD3A (NM_018188) is an insert ofa peptide fragment containing 48 extra amino acid residues between Lys94 and Glu143in ATAD3A (NM_018188), which has not been identified in ATAD3A (BC033109),3B (NM_031921) or 3C (NM_001039211) (as shown in supplementary material Fig.S1D). Our results of MALDI-TOF analysis of immunoprecipitated proteins show thatthe ATAD3A, present in LADC, is ATAD3A (BC033109). To avoid confusion, werenamed ATAD3A (NM_018188) as ATAD3AL in this manuscript.

Slide evaluation of ATAD3A expression by immunohistochemical stainingIn each pathological section, non-tumor lung tissue served as an internal negativecontrol. Slides were evaluated by two independent pathologists with no knowledgeof the clinicopathology of the specimens. The ImmunoReactive Scoring system wasadapted for this study (Remmele and Schicketanz, 1993). Briefly, a specimen wasconsidered to have strong signals when more than 50% of cancer cells were positivelystained; 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 ATAD3A signalswere classified as ATAD3A+, and those with weak or negative ATAD3A signals wereclassified as ATAD3A– (Chen et al., 2006; Chiang et al., 2009).

Statistical analysisCorrelation of ATAD3A level with clinicopathological factors was analyzed by eitherthe c2-test or the Fisher’s exact test. Survival curves were plotted using the Kaplan-Meier estimator (Kaplan and Meier, 1958). Statistical difference in survival betweendifferent groups was compared by the log rank test (Mantel, 1966). Statistical analysiswas performed using GraphPad Prism5 statistics software (San Diego, CA). Statisticalsignificance was set at P<0.05.

Electron microscopyElectron microscopy was carried out using a routine protocol. Briefly, cells werefixed with 2.5% glutaraldehyde (EM grade, Sigma) in 100 mM phosphate buffer(PB; pH 7.2), incubated at 4°C overnight. The cells were washed with PB three timesbefore post-fixation with 1% osmium tetroxide in PB for 2 hours. After removal ofthe fixative with distilled water, the cells were suspended in 2% molten agar. Theagar blocks were trimmed and dehydrated in a serial dilution of ethanol for 15 minuteseach. The blocks were further dehydrated with 100% ethanol three times, for 15minutes each, and infiltrated with 100% ethanol-LR white (1:1) mixture overnight.The blocks were changed to the pure LR white (Agar Scientific Ltd., Essex, England)and infiltration was continued at 4°C for 24 hours, before transferring to capsulesfilled with LR white, which were polymerized and solidified at 60°C for 48 hours.The resin blocks were trimmed and cut with an ultramicrotome (Leica Ultracut R,Leica Mikrosysteme GmbH, Vienna, Austria). The thin sections were transferred to200 mesh copper grids, and stained with 2% uranyl acetate for 30 minutes, and 2.66%lead citrate (pH 12) for 10 minutes, before observation with an electron microscope(JEM1400, JEOL USA, Inc., Peabody, MA) at 100-120 kV. For gene silencingexperiments, cells were harvested 48 hours following siRNA treatment.

We thank Dr Jae-Won Soh (Department of Chemistry, Inha University,Incheon, Korea) for generously providing pHACE-PKC-CAT, pHACE-PKC-CAT, pHACE-PKC-CAT, pHACE-PKC-CAT, pHACE-PKC-CAT and pHACE-PKC-CAT, and Dr Jiuping Ding (Key Laboratory ofMolecular Biophysics, Huazhong University of Science and Technology,Wuhan, Hubei, China) for KDEL-conjugated GFP. RNAi for silencingDRP1 or ATAD3A gene expression was obtained from the National RNAiCore Facility in the Institute of Molecular Biology/Genomic ResearchCenter, Academia Sinica, Taipei, Taiwan, supported by the NationalResearch Program for Genomic Medicine Grants of NSC (NSC 97-3112-B-001-016). This study was supported, in part, by the ComprehensiveAcademic Promotion Projects (NCHU 975014 to K.C.C.).

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/123/7/1171/DC1

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