Down-Regulation of FXYD3 Expression in Human Lung Cancers
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080571
ISSN1525-2191
AutoresKoji Okudela, Takuya Yazawa, Jun Ishii, Tetsukan Woo, Hideaki Mitsui, Tomoyasu Bunai, Masashi Sakaeda, Hiroaki Shimoyamada, Hanako Sato, Michihiko Tajiri, Nobuo Ogawa, Munetaka Masuda, Haruhiko Sugimura, Hitoshi Kitamura,
Tópico(s)Renal and related cancers
ResumoFXYD3 is a FXYD-containing Na,K-ATPase ion channel regulator first identified as a protein overexpressed in murine breast tumors initiated by oncogenic ras or neu. However, our preliminary study revealed that FXYD3 expression was down-regulated in oncogenic KRAS-transduced airway epithelial cells. This contradiction led us to investigate the role of FXYD3 in carcinogenesis of the lung. FXYD3 mRNA and protein levels were lower in most of the lung cancer cell lines than in either the noncancerous lung tissue or airway epithelial cells. Protein levels were also lower in a considerable proportion of primary lung cancers than in nontumoral airway epithelia; FXYD3 expression levels decreased in parallel with the dedifferentiation process. Also, a somatic point mutation, g55c (D19H), was found in one cell line. Forced expression of the wild-type FXYD3, but not the mutant, restored the well-demarcated distribution of cortical actin in cancer cells that had lost FXYD3 expression, suggesting FXYD3 plays a role in the maintenance of cytoskeletal integrity. However, no association between FXYD3 expression and its promoter's methylation status was observed. Therefore, inactivation of FXYD3 through a gene mutation or unknown mechanism could be one cause of the atypical shapes of cancer cells and play a potential role in the progression of lung cancer. FXYD3 is a FXYD-containing Na,K-ATPase ion channel regulator first identified as a protein overexpressed in murine breast tumors initiated by oncogenic ras or neu. However, our preliminary study revealed that FXYD3 expression was down-regulated in oncogenic KRAS-transduced airway epithelial cells. This contradiction led us to investigate the role of FXYD3 in carcinogenesis of the lung. FXYD3 mRNA and protein levels were lower in most of the lung cancer cell lines than in either the noncancerous lung tissue or airway epithelial cells. Protein levels were also lower in a considerable proportion of primary lung cancers than in nontumoral airway epithelia; FXYD3 expression levels decreased in parallel with the dedifferentiation process. Also, a somatic point mutation, g55c (D19H), was found in one cell line. Forced expression of the wild-type FXYD3, but not the mutant, restored the well-demarcated distribution of cortical actin in cancer cells that had lost FXYD3 expression, suggesting FXYD3 plays a role in the maintenance of cytoskeletal integrity. However, no association between FXYD3 expression and its promoter's methylation status was observed. Therefore, inactivation of FXYD3 through a gene mutation or unknown mechanism could be one cause of the atypical shapes of cancer cells and play a potential role in the progression of lung cancer. We recently proposed that potential tumor suppressors lie hidden downstream of oncogenic KRAS, based on the finding that a mutated KRAS (V12) induced severe growth suppression in primary and immortalized bronchial epithelial cells.1Okudela K Yazawa T Woo T Sakaeda M Ishii J Mitsui H Shimoyamada H Sato H Tajiri M Ogawa N Masuda M Takahashi T Sugimura H Kitamura H Down-regulation of DUSP6 expression in lung cancers: its mechanism and potential role in carcinogenesis.Am J Pathol. 2009; 175: 867-881Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar We have identified several candidate tumor suppressors, including dual specificity phosphatase 6 (DUSP6)1Okudela K Yazawa T Woo T Sakaeda M Ishii J Mitsui H Shimoyamada H Sato H Tajiri M Ogawa N Masuda M Takahashi T Sugimura H Kitamura H Down-regulation of DUSP6 expression in lung cancers: its mechanism and potential role in carcinogenesis.Am J Pathol. 2009; 175: 867-881Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar and insulin-like growth factor binding protein (IGFBP)-2/4,2Sato H Yazawa T Suzuki T Shimoyamada H Okudela K Ikeda M Hamada K Yamada-Okabe H Yao M Kubota Y Takahashi T Kamma H Kitamura H Growth regulation via insulin-like growth factor binding protein-4 and -2 in association with mutant K-ras in lung epithelia.Am J Pathol. 2006; 169: 1550-1566Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar through comprehensive gene expression analyses. The results prompted us to further identify novel tumor suppressors from among the downstream targets of oncogenic KRAS. Mammary tumor 8 kDa (MAT8)/FXDY domain-containing ion transport regulator 3 (FXYD3), a FXYD-containing Na,K-ATPase ion channel regulator, was first identified as a protein highly expressed in murine breast tumor initiated by oncogenic ras (v-Ha-ras) or neu (her2, egfr2)1Okudela K Yazawa T Woo T Sakaeda M Ishii J Mitsui H Shimoyamada H Sato H Tajiri M Ogawa N Masuda M Takahashi T Sugimura H Kitamura H Down-regulation of DUSP6 expression in lung cancers: its mechanism and potential role in carcinogenesis.Am J Pathol. 2009; 175: 867-881Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 2Sato H Yazawa T Suzuki T Shimoyamada H Okudela K Ikeda M Hamada K Yamada-Okabe H Yao M Kubota Y Takahashi T Kamma H Kitamura H Growth regulation via insulin-like growth factor binding protein-4 and -2 in association with mutant K-ras in lung epithelia.Am J Pathol. 2006; 169: 1550-1566Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 3Travis WD Brambilla E Muller-Hermelink HK Harris CC World Health Organization classification of tumors. 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27: 1017-1024Crossref PubMed Scopus (9) Google Scholar, 12Arimochi J Ohashi-Kobayashi A Maeda M Interaction of Mat-8 (FXYD-3) with Na+/K+-ATPase in colorectal cancer cells.Biol Pharm Bull. 2007; 30: 648-654Crossref PubMed Scopus (21) Google Scholar, 13Morrison BW Moorman JR Kowdley GC Kobayashi YM Jones LR Leder P Mat-8, a novel phospholemman-like protein expressed in human breast tumors, induces a chloride conductance in Xenopus oocytes.J Biol Chem. 1995; 270: 2176-2182Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar Recent studies found FXYD3 to be overexpressed breast, prostate, and pancreatic cancers,5Morrison BW Leder P Neu and ras initiate murine mammary tumors that share genetic markers generally absent in c-myc and int-2-initiated tumors.Oncogene. 1994; 9: 3417-3426PubMed Google Scholar, 14Kayed H Kleeff J Kolb A Ketterer K Keleg S Felix K Giese T Penzel R Zentgraf H Büchler MW Korc M Friess H FXYD3 is overexpressed in pancreatic ductal adenocarcinoma and influences pancreatic cancer cell growth.Int J Cancer. 2006; 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20: 3-15Crossref PubMed Scopus (47) Google Scholar and by treatment with an anticancer drug, 5-fluofouracil, in colon and/or breast cancer cells.17Maxwell PJ Longley DB Latif T Boyer J Allen W Lynch M McDermott U Harkin DP Allegra CJ Johnston PG Identification of 5-fluorouracil-inducible target genes using cDNA microarray profiling.Cancer Res. 2003; 63: 4602-4606PubMed Google Scholar, 18Bibert S Roy S Schaer D Felley-Bosco E Geering K Structural and functional properties of two human FXYD3 (Mat-8) isoforms.J Biol Chem. 2006; 281: 39142-39151Crossref PubMed Scopus (22) Google Scholar Furthermore, contrary to initial observations, our reduced preliminary study revealed that its expression was rather in oncogenic KRAS-transduced immortalized human airway epithelial cells. Thus, FXYD3 could play diverse roles in different instances and its significance in carcinogenesis could vary in type of cancer. To elucidate the potential role of FXYD3 in carcinogenesis of the lung, we here examined its expression, gene mutation, and biological function in lung cancer cell lines and/or primary human lung cancers, as well as analyzed the correlation between its expression levels and a variety of clinicopathologic parameters. The immortalized human airway epithelial cell line (16HBE14o, Simian virus 40 [SV40]-transformed human bronchial epithelial cells) described by Cozens et al19Cozens AL Yezzi MJ Kunzelmann K Ohrui T Chin L Eng K Finkbeiner WE Widdicombe JH Gruenert DC CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.Am J Respir Cell Mol Biol. 1994; 10: 38-47Crossref PubMed Scopus (782) Google Scholar was kindly provided by D.C. Gruenert (California Pacific Medical Center Research Institute). A subclone of 16HBE14o cells, described as NHBE-T in this paper, was used for experiments. The immortalized airway epithelial cell lines (HPL1D and HPL1A, SV40-transformed human small airway epithelial cells) established by Masuda et al,20Masuda A Kondo M Saito T Yatabe Y Kobayashi T Okamoto M Suyama M Takahashi T Takahashi T Establishment of human peripheral lung epithelial cell lines (HPL1) retaining differentiated characteristics and responsiveness to epidermal growth factor, hepatocyte growth factor, and transforming growth factor beta1.Cancer Res. 1997; 57: 4898-4904PubMed Google Scholar was kindly provided by Takahashi T (Nagoya University School of Medicine). Human lung cancer cell lines (A549, H358, H1299, H2087, H526, H82, H1688, and H460), immortalized human lymphoblasts generated from the same person as the H2087 lung cancer cells, human lung fibroblasts (WI-38), and a human embryonic kidney cell line (HEK293T) were purchased from American type culture collection (ATCC, Manassas, VA). Human lung cancer cell lines, LC/MS, LCD, LC2/ad, Lu65, Lu134A, Lu135, Lu139, Lu140, ABC1, HLC1, and LCKJ were purchased form Riken cell bank (Tsukuba, Japan). Human lung cancer cell lines, PC1, PC3, PC9, PC13, and HARA were obtained form Riken cell bank (Tsukuba, Japan), and Immuno-Biological laboratories Co. (Gunma, Japan), respectively. Human lung cancer cell lines, TKB1, TKB2, TKB4, TKB5, TKB6, TKB7, TKB9, TKB12, TKB15, TKB16, TKB17, and TKB20, were kindly provided by Kamma (Kyorin University School of Medicine). These cells were cultured and grown in Dulbecco's Modified Eagle Medium (Sigma Aldrich, St. Louis, MO) or RPMI1640 medium (Sigma) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Sigma), 100 units/ml of penicillin (Sigma), and 100 μg/ml of streptomycin (Sigma). Primary small airway epithelial cells (SAEC) and bronchial epithelial cells (NHBE) were purchased from Sanko Kagaku (Tokyo, Japan), and grown in defined keratinocyte serum free medium (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) coding for the wild-type (KRAS/G12) and mutated KRAS (KRAS/V12) were PCR-amplified (PrimeSATR HS DNA polymerase, Takara Bio Inc., Kyoto, Japan) with primers having BamHI restriction sites, forward (F) 5′-CTCCGCGGATCCAAGCTTGCTGAAA-3′ and reverse (R) 5′-AGGGGCGGATCctcattacataa-3′ (BamHI restriction sites italicized), using plasmid vectors, pcDNA3.1-KRAS/G12 and -KRAS/V12 described elsewhere1Okudela K Yazawa T Woo T Sakaeda M Ishii J Mitsui H Shimoyamada H Sato H Tajiri M Ogawa N Masuda M Takahashi T Sugimura H Kitamura H Down-regulation of DUSP6 expression in lung cancers: its mechanism and potential role in carcinogenesis.Am J Pathol. 2009; 175: 867-881Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar as templates. The amplified fragments were inserted into the BamHI site of pQCXIH retroviral vectors (BD Clontech, Palo Alto, CA). Enhanced green fluorescent protein (EGFP) cDNA was PCR-amplified (PrimeSATR HS DNA polymerase, Takara) using the pIRES2-EGFP vector (BD Clontech) as a template with mismatch primers having EcoRI restriction sites, F, 5′-ATGGGAATTCCCgggGTGAGCAAGGGCGAGGA-3′ (fist methionine [ATG] was converted to glycine [GGG], M1G) or 5′-ATGGaattcACCATGGTGAGCAAGGGCGAGGAG-3′; R, 5′-ATCTAGGAATTCGGCCGCTTTACTTGTA-3′ (EcoRI restriction sites italicized). It was inserted into the EcoRI site of pQCXIN (BD Clontech). FXYD3 variant 1 cDNA (GENE BANK ACCESSION# NM_005971) and a mutant (FXYD3 g55c [D19H]) were PCR-amplified (LA TaqDNA polymerase, Takara) with primers, F 5′-GGCCAGCGCTCTGACATGCAGAA-3′ and R 5′-CTGGGGACAGAGAACGGTCCTCC-3′, using cDNA reverse-transcribed (SuperScript III, Invitrogen, Carlsbad, CA) from total RNA of H2087 cells. After the adenylation of 3′ terminals with TaqDNA polymerase (Takara), the amplified fragment was subcloned into a pT7Blue vector (Novagen, Darmstadt, Germany). FXYD3 cDNA was PCR-amplified again (PrimeSATR HS DNA polymerase, Takara) using pT7Blue/FXYD3 as a template with mismatch primers having BamHI restriction sites, F 5′-ACCCGGGGATCCGATTGGCCAG-3′ and R 5′-CAGCTGGTGGATCCTCCTCCGCTTTG-3′ (stop codon [TGA] was converted into glycine [GGG]) (BamHI restriction sites italicized, mutant in bold). It was inserted into the BamHI site of pQCXIN/EGFP (M1G). A candidate promoter region (−2539 to −1615, −1615 to −5, −1159 to −5, −760 to −5, or −461 to −5 of the FXYD3 gene locus [NC_000019] taking the first base of the transcription start site as + 1), was PCR-amplified (PrimeSATR HS DNA polymerase, Takara) with primer sets: F-2539 5′-GGTGCTGAGACCACAGCGCTGTTC-3′ and R-1615, 5′-TGGGGGACCAGCAATTAGAGACTTC-3′; F-1615, 5′-TCTAATTGCTGGTCCCCCAGGTTGAAAC-3′ and R-4, 5′-GTCCCCTTCCAGGCCACATCTTGCCG-3′; F-1159, 5′-TGTACACGCCCCTACTCGCTCTCGG-3′ and R-5, 5′-GTCCCCTTCCAGGCCACATCTTGCCG-3′; F-760 5′-AGCGATGACAAGTGGGGGGTCCTTC-3′ and R-5 5′-GTCCCCTTCCAGGCCACATCTTGCCG-3′; F-461 5′-TGTCTAAGAAGGCGGAATTGAGGGG-3′ and R-5 5′-GTCCCCTTCCAGGCCACATCTTGCCG-3′, using genomic DNA extracted from nontumorous parts of surgically resected lung as a template. The products were subcloned into the pT7Blue vector (Novagen). They were cut out with HindIII (Takara) and KpnI (Takara), and inserted into pGL4.1 (Promega, Madison, WI). The accuracy of all of the constructs was verified by DNA sequencing (Dye-deoxy DNA sequencing kit, Amersham life science, Piscataway, NJ). The pQCXIH or pQCXIH-based vectors and the pCL10A1 retroviral packaging vector (Imgenex, San Diego, CA) were cotransfected into HEK293T cells with Lipofectoamine 2000 reagent (Invitrogen). After 24 hours, the conditioned medium was recovered as a viral solution. Desired genes were introduced by incubating cells with the viral solution containing 10 μg/ml of polybren (Sigma) for 24 hours. Cells stably expressing desired genes were selected with 1000 μg/ml of neomycin (G418, Invitrogen) or 500 μg/ml of Hygromycin B (Invitrogen) for 7 days. Selected pooled clones were used for biological analyses. First-strand cDNA was synthesized from total RNA using the SuperScript First-Strand Synthesis System according to the protocol of the manufacturer (Invitrogen). Fragments of FXYD3 and ACTB (β-actin) were PCR-amplified with a LA TaqPCR kit using the following primers: for FXYD3 forward (F) 5′-GGCCAGCGCTCTGACATGCAGAA-3′ and reverse (R) 5′-CTGGGGACAGAGAACGGTCCTCC-3′, and for ACTB, F 5′-TGGCACCCAGCACAATGAA-3′ and R 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. First-strand cDNA was synthesized by the method described above. The cDNA generated was used as a template in real-time PCR with SYBR Premix EXTaq (Takara) and run on a Thermal Cycler DICE real-time PCR system (Takara). The primer set used for the detection of FXYD3 was forward (F) 5′-GGCCAGCGCTCTGACATGCAGAA-3′ and reverse (R) 5′-CTGGGGACAGAGAACGGTCCTCC-3′. That used for ACTB was F 5′-CTGGGGACAGAGAACGGTCCTCC-3′ and R 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. The means and standard deviations of copy numbers of FXYD3 relative to ACTB mRNA were statistically obtained from triplicate reactions. Genomic DNA was extracted from lung cancer cell lines and tumor cells microscopically dissected (PALM-MCB laser microdissection system, Carl Zeiss, Jena, Germany) from primary lung cancer tissues (90% ethanol-fixed materials) by a method described elsewhere.3Travis WD Brambilla E Muller-Hermelink HK Harris CC World Health Organization classification of tumors. Pathology and genetics of the lung, pleura, thymus and heart. IARC Press, Lyon2004Google Scholar Coding regions of the FXYD3 gene were PCR-amplified (LA TaqDNA polymerase, Takara) using the genomic DNA as a template with the following sets of primers: for exon 3, forward (F) 5′-CAGCTGCGGCTTATCTCTCAGCCCA-3′ and reverse (R) 5′-AGATGCTGTTCTAACATTTACCACc-3′; for exons 4 and 5, F 5′-CCTGTCCCGCAGGAGACCCTTT-3′ and R 5′-TCTTTGTCCTGTGATGCTCACg-3′; for exons 6 and 7, F 5′-GTCTGTTTTCTTATGGCGGTGTC-3′ and R 5′-AGCCCTTTCACCCTGAAAAGCG-3′; and for exons 8 and 9, F 5′-ATGTCTGGGCAGGCTAAGAACCC-3′ and R 5′-TTCCATCCTGGAGTTCAAGTTTCT-3′. Surplus primers and nucleotides were removed enzymatically with EXO-SAP-IT (Amersham). The purified PCR products were subjected to a dye-terminator reaction with Big Dye version 3.1 (ABI, Foster, CA). For the dye-terminator reaction, the same primers as for the PCR-amplification were used. The DNA sequence was analyzed with an ABI 3100 DNA sequencer (ABI). Whole cell lysate was subjected to SDS-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes (Amersham). The membranes were incubated with nonfat dry milk in 0.01 M/L Tris-buffered saline containing 0.1% Tween-20 to block non-immunospecific protein binding, and then with 0.1 μg/ml of primary antibodies (EGFP, Santa Cruz, Santa Cruz, CA; FXYD3, ALTAS Antibodies, Stockholm, Sweden; β-actin, Sigma). After being washed with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with animal-matched horseradish peroxidase-conjugated secondary antibodies (Amersham). Immunoreactivity was visualized with the enhanced chemiluminescence system (ECL, Amersham). Cells were treated either with 10 μmol/L of 5-azacytidine (AZA, Sigma) for 72 hours by exchanging the medium everyday or with 300 ng/ml of trichostatin A (Wako, Osaka, Japan) for 24 hours. In addition, cells were treated also with AZA for 48 hours and then with a combination of AZA and trichostatin A for an additional 24 hours. Genomic DNA was extracted from cultured cells by a method described previously,3Travis WD Brambilla E Muller-Hermelink HK Harris CC World Health Organization classification of tumors. Pathology and genetics of the lung, pleura, thymus and heart. IARC Press, Lyon2004Google Scholar and was subjected to bisulfate conversion treatment using a MethylEasy DNA bisulfate modification kit (Human Genetic Signatures, Macquarie Park, Australia) according to the manufacturer's instructions. The regions from −1615 to −1159 and from −465 (or −175) to −5 of the FXYD3 gene were PCR-amplified (TaqHS DNA polymerase, Takara) using the bisulfate converted DNA as a template with the following sets of primers: forward (F)−1663 5′-TATTTTTAAGATGTAAAATTTAAAAA-3′ and reverse (R)−1140 5′-TGTTAATTTTTAAAAATATTTTATTTA-3′; F-465 5′-TGTTTAGGGTAGAGTTATTTTTTTT-3′ and R-5 5′-AAAACCTCCAAAACCATACAATAAA-3′; F-175 5′-AGGGTAGGGAGTAGGTTATGATTAg-3′ and R-5 5′-AAAACCTCCAAAACCATACAATAAA-3′, and were subcloned. Ten clones were randomly chosen. The conversion of nucleotide at CpG sites was examined for by DNA sequencing (Dye-deoxy DNA sequencing kit, Amersham). MOCK (EGFP) or FXYD3-EGFP was retrovirally transduced into H1299 cells. After the selection treatment, the surviving cells were harvested and counted, and 1.0 × 104 cells were re-seeded onto a 10 cm dish. After 12 days, the cells were fixed with methanol and stained with Giemsa. The selected cells (1.25 × 104) were cultured and grown in 1 ml of 0.3% soft agar in 3.5 cm culture dishes for 3 weeks, then fixed with a buffered 4% paraformaldehyde solution. The selected cells (2.5 × 105) were seeded onto 3.5 cm culture dishes and grown for 10 days. The cells were counted every 2 days. Genomic DNA was extracted from fresh frozen lung cancer tissues or paraffin sections by methods described previously.21Ward R Hawkins N O'Grady R Sheehan C O'Connor T Impey H Roberts N Fuery C Todd A Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.Am J Pathol. 1998; 153: 373-379Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 22Okudela K Hayashi H Ito T Yazawa T Suzuki T Nakane Y Sato H Ishi H KeQin X Masuda A Takahashi T Kitamura H K-ras gene mutation enhances motility of immortalized airway cells and lung adenocarcinoma cells via Akt activation: possible contribution to non-invasive expansion of lung adenocarcinoma.Am J Pathol. 2004; 164: 91-100Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar For the detection of KRAS codon 12 mutations, the REMS PCR described by Ward et al21Ward R Hawkins N O'Grady R Sheehan C O'Connor T Impey H Roberts N Fuery C Todd A Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.Am J Pathol. 1998; 153: 373-379Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar was partially modified. Briefly, the reaction mixture, a final volume of 5 μl, containing 0.5 μl of 10 ng/μl DNA solution (1 ng/μl), 0.5 μl of 10×PCR buffer (1×), 0.4 μl of each 2.5 mmol/L dNTP (0.2 mmol/L), 0.04 μl of 50 μmol/L primers (three primer sets described by Ward R et al,21Ward R Hawkins N O'Grady R Sheehan C O'Connor T Impey H Roberts N Fuery C Todd A Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.Am J Pathol. 1998; 153: 373-379Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar each 0.4 μmol/L), 0.025 μl of TaqHS DNA polymerase (Takara), 0.4 μl of BstN1 (New England BioLabs, Northbrook, IL) (0.8 units/μl), and 2.935 μl of distilled water (final volume, 5 μl), was PCR-cycled on a Thermal Cycler Dice (Takara) with the following program; 94°C for 2 minutes (one cycle), 92°C for 20 seconds −60°C for 3 minutes (35 cycles), held at 4°C. The products were resolved by electrophoresis in 5% agar. Genomic DNA from A549 (mutated KRAS) and H1299 (wild-type KRAS) mixed indicated ratio (A549/H1299 = 1/0, 1/1, 1/10, 0/1), and from H358 (mutated KRAS), and pQCXIH vectors bearing wild-type (G12) and mutated KRAS (V12), served as experimental controls. The pGL4.1-based vectors constructed above and a pGL4.7-TK Renilla (Promega, Madison) were co-transfected into NHBE-T cells with Lipofectamine 2000 (Invitrogen). After 24 hours, the cells were solved and luminosity was measured using a Dual Luciferase Reporter Assay system (Promega) on a luminometer (TD-20/20, Turner BioSystems, Sunnyvale, CA). The luminosity derived from the pGL4.1-based vectors was corrected with that from the pGL4.7-TK Renilla, to normalize the transfection efficiency. Luminosity relative to that of the empty pGL4.1 vector was determined as promoter activity. All cases examined were of lung cancer patients who underwent surgical resection at Kanagawa Cardiovascular and Respiratory Disease Center Hospital during the period from 2001 to 2008. Informed consent for research use was obtained from all of the subjects providing materials. Sixty-five cases (adenocarcinomas [ADC, 36], squamous cell carcinomas [SQC, 21], adenosquamous carcinomas [ASC, 2], large cell carcinomas [LCC, 3], and small cell carcinomas [SCC, 3]), were analyzed for FXYD3 and Ki-67 expression by immunohistochemistry, and for KRAS codon 12 mutations by restriction endonuclease-mediated selective PCR (a representative result is shown: see Supplemental Figure S1 at http://ajp.amjpathol.org). ADCs were classified into histological subtypes according to the criteria of the World Health Organization.3Travis WD Brambilla E Muller-Hermelink HK Harris CC World Health Organization classification of tumors. Pathology and genetics of the lung, pleura, thymus and heart. IARC Press, Lyon2004Google Scholar If a case was of mixed type, the major component was described as the subtype in the present study. Maximal tumor sections were subjected to immunohistochemistry. Four-micron–thick, formalin-fixed, paraffin-embedded tissue sections were deparaffinized and rehydrated, and boiled in citrated buffer (0.01 M/L, pH 6.0) to retrieve the masked epitope. Then, the sections were incubated with 3% hydrogen peroxide, followed by 5% goat serum to block endogenous peroxidase activities and nonimmunospecific protein binding. The sections were incubated with the primary antibody against either FXYD3 (ATLAS Antibodies) or Ki-67 (MIB1, DAKOcytomation, Dako, Ely, UK). Immunoreactivity was visualized with the Envision detection system (Dako), and the nuclei were counterstained with hematoxylin. The specificity of the anti-FXYD3 antibody was confirmed by an experiment of immunocytochemistry with sections cut out from paraffin-embedded H1299 cells transduced with the FXYD3-EGFP construct (see Supplemental Figure S2 at http://ajp.amjpathol.org.). Also, the specificity of an immunoreactive signal of FXYD3 in tissue sections was further confirmed by immunohistochemistry using nonimmunized rabbit serum instead of the primary specific antibody against FXYD3 (see Supplemental Figure S2 at http://ajp.amjpathol.org.). The expression of FXYD3 in lung cancer cells was categorized as, negative (0), weak (1), or strong (2). Weak expression was defined as a level similar to that in bronchial or alveolar epithelial cells. Strong expression was defined as a signal of unequivocally greater signal intensity than that in bronchial epithelial cells. The expression score was determined by averaging the degree of staining corresponding to the areal proportion. A labeling index of MIB1 was calculated as the proportion of positive cells by counting 500 to 1000 cancer cells. Those cases with a labeling index of less than 0.15 and of 0.15 or more were classified as lower and higher expressers, respectively. Differences in FXYD3 expression scores among clinicopathologic subgroups were analyzed with Student's t-test. Correlations of FXYD3 expression levels with clinicopathologic parameters, KRAS gene mutations, and MIB index levels were analyzed with a χ2 test. P values of less than 0.05 were considered significant. Cells (2.5 × 105) were seeded onto 3.5-cm culture dishes (Iwaki, Tokyo, Japan) and grown for 10 days. The cells were counted every 2 days without a change of medium until the finish of the experiments. Cells (1.0 × 104) were seeded onto 10 cm culture dishes (Iwaki), and grown for 12 days. The cells were fixed with methanol and stained with Giemsa, and colonies more than 0.5 mm in diameter were counted. Cells (1.25 × 104) were cultured and grown in 1 ml of Dalbecco's Modified Eagle Medium-based 0.3% agar (agar noble; Becton Dickinson, Sparks, MD) conta
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