Abstract
Background
Six human lung cancer cell lines (SNU-371, SNU-963, SNU-1327, SNU-1330, SNU-2292 and SNU-2315) were newly established through primary cell cultures. These cell lines were derived from a pulmonary blastoma, a small cell lung cancer, three adenocarcinomas and a squamous cell carcinoma of the lung of six Korean lung cancer patients.
Methods
The histopathology of the primary tumors and their in vitro growth characteristics were described. DNA fingerprinting analysis and genetic alterations in the p53, β-catenin, TGFβRII, K-ras and EGFR genes were conducted. mRNA expressions levels of E-cadherin, COX-2, MDR1, MXR, CGA, synatophysin and TTF1 genes were investigated and sensitivity to anticancer drugs was screened.
Results
Five cell lines grew as adherent cells and one cell line grew as floating aggregates. All lines were free of mycoplasma or bacteria and were proven unique by DNA fingerprinting analysis. A significant polymorphism at codon 72 (Arg to Pro) of the p53 gene was found in one line (SNU-1327) and a mutation at codon 176 was found in SNU-2292. No mutations in the K-ras, β-catenin and TGF-βRII genes were observed. E-cadherin was not expressed in SNU-371 and COX-2 was overexpressed in SNU-1330, SNU-2292 and SNU-2315 cell lines. MDR1 was overexpressed in SNU-371 and SNU-2292 cell lines and MXR was overexpressed in SNU-1327 cell line. Interestingly, the SNU-371 cell line derived from a pulmonary blastoma and which overexpressed MDR1 displayed cross resistance for several anticancer drugs. Neuroendocrine markers, chromogranin A and synaptophysin, were overexpressed in the small cell lung cancer cell line, SNU-963 and thyroid transcription factor-1 was also over expressed in this cell line. Two mutations (p.Glu746_Ser752delinsVal and p.Glu746_Ala750del) in exon 19 of EGFR were found in SNU-1330 and SNU-2315 cell lines, respectively.
Conclusion
These well-characterized lung cancer cell lines may be useful tools for investigations of the biological characteristics of lung cancers, particularly for investigations related to mutations of EGFR.
Keywords: Establishment, Lung cancer, Cell line, EGFR gene
Introduction
Lung cancer is the most frequent cause of cancer-related death in the United States [1]. In Korea, Lung cancer is the second and fifth most common cancer in males and females, respectively [2]. Environmental air pollutants such as tobacco smoke and asbestos are accepted as the major causes of the disease. Most lung cancer is of epithelial origin, with a few exceptions such as mesothelioma, which is derived from a non-epithelial cell type attributable to asbestos exposure. Epithelial lung cancer is generally classified into two major types: small cell lung cancer (SCLC) and non-small cell lung cancer. An understanding of the biologic nature of these neoplasms is needed to develop therapeutic methods and to improve the prognosis of those with lung cancer. For these purposes, permanently growing cell lines can be an invaluable tool because of their usefulness in a variety of experimental situations. Examples include the understanding of tumor biology and investigation of genetic, epigenetic changes and malignant transformations associated with development of various cancers. Genetic alterations of Ras, p14, p16, p53, LKB1/STK11 and PTEN genes, tumor suppressor genes at chromosome 3p, and amplifications of myc proto-oncogene have been linked to lung cancer (reviewed in ref. [3]). Recent reports have demonstrated that clinical responsiveness to gefitinib, an epidermal growth factor receptor (EGFR)-targeting agent, is closely related with EGFR mutations in the kinase domain (reviewed in ref. [4]). Furthermore, EGFR mutation is strongly predictive of prolonged survival in NSCLC patients treated with gefitinib [5].
In the present study, we report the establishment and characterization of six new human lung cancer cell lines derived from patients with pulmonary blastoma, SCLC, adenocarcinoma and squamous cell carcinoma. We describe the cell phenotypes, including the histopathology of the primary tumors and their in vitro growth characteristics.
We also conducted a DNA fingerprinting analysis to verify the authenticity of each line, and screened each for genetic alterations in the p53, β-catenin, transforming growth factor beta II (TGFβRII), K-ras and EGFR genes. In addition, we investigated the mRNA expressions levels of E-cadherin, cyclooxygenase-2 (COX-2), multidrug resistance 1 (MDR1), mitoxantrone resistance (MXR), chorionic gonadotrophin alpha (CGA), synatophysin and thyroid transcription factor 1 (TTF1) genes, and screened for sensitivity to anticancer drugs in the newly established lung cancer cell lines.
Materials and methods
Cell culture
Cell lines were established from a pulmonary blastoma, a small cell lung cancer, three adenocarcinomas and a squamous cell carcinoma of the lung. Solid tumors were finely minced with a scissors and dissociated into small aggregates by pipetting. Appropriate amounts of finely minced neoclassic tissue fragments were seeded into 25 cm2 flasks. Pleural effusions were collected, pelleted, washed, and resuspended in growth medium. Tumor cells were initially cultured in ACL-4 medium supplemented with 5% heat-inactivated fetal bovine serum (AR5). ACL-4 is a fully defined medium formulated for the selective growth of human lung adenocarcinoma [6, 7]. This medium has also proven useful in the establishment of biliary tract cancer cell lines [8] and thyroid cancer cell lines [9]. After establishment, the cell lines were maintained in RPMI-1640 containing 10% heat-inactivated fetal bovine serum (R10). Initial cell passages were performed when heavy tumor cell growth was observed, and subsequent passages were performed every 1 or 2 weeks. Adherent cultures were passaged at subconfluence after trypsinization. If stromal-cell growth was noted in the initial cultures, differential trypsinization was used to obtain a pure tumor cell population. Cultures were maintained in humidified incubators at 37°C in an atmosphere of 5% CO2 and 95% air. A-549, NCI-H1793, SK-MES1 and HeLa cell lines obtained from the Korean Cell Line Bank (KCLB) were used as polymerase chain reaction (PCR) controls.
Growth properties and morphology in vitro
Population doubling times were determined by seeding 5 × 104–3 × 105 viable cells into 25 cm2 flasks and counting daily for 14 or more days. Cultures were fed every 3 or 4 days and 24 h prior to counting. Cell viability was determined by a standard dye exclusion method using 0.4% Trypan Blue. PCR and microscopic examination were used to test for mycoplasma (e-Myco mycoplasma detection kit; Intron Biotechnology, Gyonggi, Korea) and bacterial contamination. For morphological studies, the cells were grown on 75 cm2 culture flasks and observed daily by phase-contrast microscopy.
Nucleic acid isolation and cDNA synthesis
Genomic DNA and total RNA were isolated from washed cell pellets. Total genomic DNA was extracted according to the standard sodium dodecyl sulfate-proteinase K procedure, and total cellular RNA was extracted according to the manufacturer’s instructions (Intron Biotechnology). For cDNA synthesis, 2 μg of total RNA was reverse transcribed using random oligo (dT) primer, dNTPs, and 1 μl (200 units) of Superscript™ II reverse transcriptase (Life Technologies, Gaithersburg, MD) in a final volume of 20 μl for 75 min at 42°C after a 10 min denaturation at 70°C. Eighty microliters of distilled water was then added to the reverse transcription reaction.
DNA profiles
DNA was amplified by PCR at loci containing the highly polymorphic microsatellite markers, D1S1586 and D3S1765. PCR was performed in a final volume of 25 μl containing 50 mM KCl, 10 mM Tris–HCl (pH 7.5), 1.5 mM MgCl2, 0.01% (w/v) gelatin, 1 mM of each deoxynucleotide triphosphate, 20 pmol of each primer, 0.5 units of AmpliTaq polymerase (Perkin-Elmer Cetus, Norwalk, CT), 1 μCi[α-32P]-dCTP (Amersham, Aylesbury, UK), and 100 ng of DNA. PCR conditions used were an initial denaturation at 95°C (5 min), and 30 cycles of 94°C (1 min), 53°C–57°C (1 min) and 72°C (1 min), followed by a final elongation at 72°C (7 min). PCR products were denatured using 95% formamide and electrophoresed on a sequencing gel for 2 h at a constant 60 W. Gels were dried and visualized autoradiographically. DNA was also amplified using AmpFlSTR identifier PCR amplification kit (Applied Biosystems, Foster City, CA). A single PCR amplified 15 tetranucleotide repeat loci (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX and vWA) and Amelogen gender determining marker at loci containing highly polymorphic microsatellite markers. Amplified products were analyzed using ABI 3730 Genetic analyzer (Applied Biosystems).
Detection of p53, β-catenin, TGF-βRII, K-ras and EGFR mutations
Mutational screening of exons 4–8 of p53 was performed by PCR-based single-strand conformation polymorphism (PCR-SSCP) analysis. Oligonucleotide primers for the genomic PCR and PCR procedures were as described previously [10]. PCR-SSCP analysis to screen mutations of the β-catenin gene was carried out by designed primers for amplification of exons 3, 5 and 6, since previously-reported mutations in the β-catenin gene are concentrated at these exons [11, 12]. Mutation screening of exon 3 in the TGF-βRII gene was also performed by direct sequencing analysis. The primers and conditions used for PCR were as described previously [13]. Mutations of codons 12 and 13 in the K-ras gene were screened by direct sequencing analysis [8]. Mutations of the epidermal growth factor receptor gene (EGFR) were also screened through exons 18 and 21 by direct sequencing analysis [14].
Non-radioisotopic PCR reactions were carried out in 25 μl containing 100 ng genomic DNA, 2.5 pmoles of each primer, four dNTPs at 250 μM each, 0.5 units of Taq polymerase and PCR reaction buffer (Boehringer Mannheim, Mannheim, Germany). Reactions were initiated by denaturation for 5 min at 94°C, and amplification was conducted over 35 cycles in a Perkin Elmer Cetus 9600 programmable thermal cycler (Roche Molecular Systems, Nutley, NJ). For SSCP, genomic DNA was amplified in a final volume of 10 μl using the same PCR procedure as described above, except for the addition of [α-32P]-dCTP. PCR products were electrophoresed in a non-denaturing SSCP gel, and allowed to separate over 12–16 h in a cool room (16°C) at a constant 300 V. After electrophoresis, the gel was transferred to 3 M Whatman paper, dried, and autoradiographed. Samples showing abnormal bands by SSCP were subjected to DNA sequencing analysis. Fresh PCR products were sequenced using a Taq dideoxy terminator cycle sequencing kit on an ABI 3730 DNA sequencer (Applied Biosystems).
RT-PCR analysis and MTT assay
For the mRNA expression analysis of E-cadherin [15], COX-2 [16], MDR1[17], MXR [18], CGA [19], synaptophysin [20] and TTF1 [21] genes in the six cell lines, cDNA was amplified in 25 μl of a PCR reaction mix using 1 μl of reverse-transcription (RT) reaction, the primers and 0.5 units of Taq DNA polymerase. PCR amplification was carried out in a Perkin Elmer Cetus 9600 programmable thermal cycler (Roche Molecular Systems). Primers for β-actin were used to confirm RNA integrity. Both gene and β-actin RT-PCR reactions used the same cDNA synthesis. Amplified DNA fragments were fractionated in a 2% agarose gel and stained with ethidium bromide. A standard colorimetric assay using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to assess the cytotoxicity of doxorubicin, 5-FU, mitomycin-C, cisplatin and VP16 in SNU-371, SNU-1327 and SNU-1330 cell lines.
Results
Culture characteristics
We tried to establish lung cancer cell lines in 26 lung cancer samples from 1989 to 2007. After establishing cancer cell lines, cancer cell lines were frozen in liquid nitrogen until further characterization. The culture success rate was 23% (6 of 26 trials). The approximate average time for establishing a cancer cell line was 6 months. Cell lines of pulmonary blastoma (n = 1), SCLC (n = 1), adenocarcinoma (n = 3) and squamous cell carcinoma (n = 1) derived from Korean patients were established in AR5 medium. The cell lines were designated Seoul National University (SNU)-371, SNU-963, SNU-1327, SNU-1330, SNU-2292 and SNU-2315. After establishment, the six cell lines were maintained and passaged in RPMI1640 medium supplemented with 10% fetal bovine serum. Population doubling times ranged from 39 to 149 h and about 85% of the populations remained viable. All cell lines were free of contamination from either bacteria or mycoplasma (Tables 1 and 2).
Table 1.
Origin, in vitro, and in vivo characteristics of SNU human lung cancer cell lines
| Cell line | Culture date | Sex/Age | Tumor source | Tumor type |
|---|---|---|---|---|
| SNU-371 | ’90.3.22. | M/44 | primary lung cancer | pulmonary blastoma |
| SNU-963 | ’92.10.19. | M/78 | primary lung cancer | small cell lung cancer |
| neuron specific enloase (+), chromongranin (+) | ||||
| previous chemo-radiation therapy | ||||
| SNU-1327 | ’94.11.15. | M/84 | pleural effusions | adenocarcinoma |
| SNU-1330 | ’94.12.2. | M/49 | pleural effusions | squamous cell carcinoma |
| SNU-2292 | ’06.6.13 | F/39 | pleural effusions | adenocarcinoma |
| SNU-2315 | ’07.1.18 | M/41 | lymph node | adenocarcinoma |
Table 2.
In vitro characteristics of 6 SNU lung cancer cell lines
| Cell line | Growth pattern | Viability | Doubling time | Cell morphology |
|---|---|---|---|---|
| SNU-371 | Adherent | 87% | 98 h | Pleomorphic |
| SNU-963 | Floating aggregates | 78% | 149 h | Round to oval |
| SNU-1327 | Adherent | 92% | 42 h | Pleomorphic |
| SNU-1330 | Adherent | 89% | 39 h | Polygonal |
| SNU-2292 | Adherent | 82% | 72 h | Pleomorphic |
| SNU-2315 | Adherent | 85% | 141 h | Pleomorphic |
In vivo characteristics
The original tumor of SNU-371 showed a biphasic pattern, being composed of tubular glands and cellular stroma. The glandular epithelium had eosinophilic cytoplasm and subnuclear vacuoles. The stromal cells were oval-to-spindle shaped, floating in a variable amount of myxoid extracellular material. Mitotic figures were abundant in the glandular as well as in stromal cells. The cell line was derived from pulmonary blastoma (Fig. 1b). The original tumor cells of SNU-963 were small with scant cytoplasm, ill-defined cell borders, finely granular chromatin and no nucleoli. The cells were round-to-oval and mitotic figures were frequently evident. Immunohistochemistry on chromogranin showed positivity in the cytoplasm (Fig. 1d). The original tumor of the SNU-1330 cell line was squamous cell carcinoma, being composed of solid sheet of polygonal cells with inconspicuous evidence of keratinization.
Fig. 1.
a Phase-contrast photography of a tissue-culture specimen of the pulmonary blastoma (SNU-371). Note the spindle or pleomorphic cells. b Original tumor of SNU-371 showed biphasic pattern composed of tubular glands and cellular stroma (H&E stain). c Phase-contrast photography of the tissue culture of the small cell lung cancer (SNU-963). Note the large floating aggregates consisted of round or oval cells. d The original tumor cells of SNU-963 composed of round to oval cancer cells. e Phase-contrast photography of the tissue culture of the adenocarcinoma of the lung (SNU-1327). Note the polymorphic epithelial cells with spindle or polygonal shapes growing as monolayers. f Culture of the tissue specimen of the squamous cell carcinoma of the lung (SNU-1330). Note the polygonal cells with the prominent nucleoli. g Cell culture photography of SNU-2292. h Phase-contrast photography of SNU-2315 derived from adenocarcinoma of lung
In vitro morphology
In vitro, three cell lines grew as adherent monolayers and the SNU-963 cell line grew as floating aggregates and adherent cells (Fig. 1a, c, e–h). SNU-371 consisted of spindle or pleomorphic cells with large, round nuclei containing several nucleoli and grew as monolayers (Fig. 1a). The SNU-963 cell line consisted of round or oval cells and grew as floating aggregates and adherent cells (Fig. 1c). The SNU-1327 cell line consisted of pleomorphic, spindle- or polygonal-shaped epithelial cells that grew as monolayers (Fig. 1e). The SNU-1330 cell line consisted of polygonal cells with prominent nucleoli (Fig. 1f). SNU-2292 and SNU-2315 cell lines grew as monolayers consisting of polygonal- or pleomorphic-shaped cells (Fig. 1g and h) (Table 2).
DNA profiles
DNA profiles using the two highly polymorphic microsatellite markers and AmpFlSTR identifier PCR amplification kit showed that the six lung cancer cell lines were unique and unrelated (Fig. 2, Table 3). These results helped exclude the possibility of cross-contamination between the cell lines.
Fig. 2.
DNA-fingerprinting analysis of lung cancer cell lines using two highly-polymorphic microsatellite markers. D1S1586 and D3S1765 STR markers can amplicate GTA and GATA repeats in each locus. The expected amplicon sizes of D1S1586 and D3S1765 are 91-118 bp and 192-212 bp. a D1S1586, b D3S1765. The four SNU lung cancer cell lines are unique and unrelated
Table 3.
DNA fingerprinting analysis using 16 STR loci for newly established 6 lung cancer cell lines
| Loci | SNU-371 | SNU-963 | SNU-1327 | SNU-1330 | SNU-2292 | SNU-2315 |
|---|---|---|---|---|---|---|
| D8S1179 | 14 | 15,16 | 13,14 | 12 | NT | NT |
| D21S11 | 29,32 | 30 | 29 | 30 | NT | NT |
| D7S820 | 5,12 | 11,12 | 8,13 | 8 | 11 | 10,11 |
| CSF1P0 | 10,12 | 13 | 12 | 12 | 11,13 | 10 |
| D3S1358 | 15 | 15 | 16 | 16 | 16 | 17 |
| TH01 | 7 | 7 | 9 | 9 | NT | NT |
| D13S317 | 9 | 11 | 9 | 8 | 11 | 9 |
| D16S539 | 12 | 10 | 9,11 | 10 | NT | NT |
| D2S1338 | 18,24 | 19 | 19,20 | 24 | NT | NT |
| D19S433 | 14 | 14.2 | 13 | 13 | NT | NT |
| vWA | 14,19 | 17,18 | 18,19 | 14 | 17,18 | 17 |
| TPOX | 8,9 | 9,11 | 10,11 | 8 | 8 | 11 |
| D18S51 | 13,17 | 13,15 | 20 | 13 | NT | NT |
| Amelogenin | X,Y | X,Y | X,Y | X,Y | X,X | X,Y |
| D5S818 | 12 | 11 | 11 | 12 | 11 | 10,13 |
| FGA | 21 | 23 | 18 | 22 | 23,24 | 23,15 |
numbers indicate the number of short tandem repeats at STR loci on each chromosome; NT not tested
Alterations in p53, β-catenin, TGF-βRII and K-ras genes
SNU-1327 and SNU-2292 cell lines had mutations in the p53 gene. The SNU-1327 cell line had a homozygous missense mutation from arginine (CGC) to proline (CCC) at codon 72 of exon 4 and the SNU-2292 cell line had a homozygous missense mutation from cysteine (TTC) to phenylalanine (TGC) at codon 176 in exon5 (Fig. 3). There were no mutations in TGF-βRII, β-catenin and K-ras genes.
Fig. 3.
Mutational analysis of the p53 gene in the lung cancer cell lines. a DNA sequencing analysis of exon 4 shows a polymorphism in SNU-371 and SNU-1327 cell lines. b SNU-2292 cell line has a mutation in exon 5
Mutations in EGFR gene
Mutations in EGFR gene were found in the SNU-1330 and SNU-2315 cell lines. A small deletion from nucleotides 2237–2255, which resulted in elimination of codons 746–752, and a thymidine insertion that resulted in addition of valine after codon 745 were found in exon 19 of EGFR in SNU-1330 (p.Glu746_Ser752delinsVal). This cell line was homozygous for this mutation (Fig. 4). The other small deletion from nucleotides 2235–2249, which resulted in elimination of codons 746–750, was found in exon 19 of EGFR in SNU-2315 (p.Glu746_Ala750del). This cell line was heterozygous for this mutation (Fig. 4).
Fig. 4.
Mutations in EGFR gene in lung cancer cell lines. Two mutations show small deletions in exon 19 of EGFR gene in lung cancer cell lines, SNU-1330 and SNU-2315
Expression of E-cadherin, COX-2, MDR1, MXR, CGA, synaptophysin and TTF1 genes
E-cadherin was not expressed in the SNU-371 cell line and COX-2 was expressed in SNU-1330, SNU-2292 and SNU-2315 cell lines. MDR1 was expressed in SNU-371 and SNU-2292 cell lines and MXR was overexpressed in SNU-1327 cell line and weakly expressed in the SNU-1330, SNU-2292 and SNU-2315 cell lines (Fig. 5). CGA, synaptophysin and TTF1 were expressed in SNU-963 cell line (Fig. 6). CGA was also expressed in the SNU-1327 cell line and TTF1 was expressed in SNU-2292 and SNU-2315 cell lines. The results are summarized in Table 4. The SNU-371 cell line derived from a pulmonary blastoma showed more cross resistance for several anticancer drugs than other cell lines (Table 5).
Fig. 5.
RT-PCR analysis of E-cadherin, COX-2, MDR and MXR genes in SNU lung cancer cell lines. Note the no expression of E-cadherin in SNU-371 cell line, expressions of COX-2 in SNU-1130 cell line, expressions of MDR1 in SNU-371 and SNU-2292 and expressions of MXR in SNU-1327, SNU-1330, SNU-2292 and SNU-2315 cell lines (35 cycles of PCR), A549, NCI-H1793 and SK-MES1 cell lines were used for controls. β-actin was amplified as an internal control (30 cycles of PCR)
Fig. 6.
RT-PCR analysis of CGA, synaptophysin and TTF1 genes in SNU lung cancer cell lines. Note the expressions of CGA, synaptophysin and TTF1 in small cell lung cancer cell line, SNU-963 (35 cycles of PCR). β-actin was amplified as an internal control (30 cycles of PCR)
Table 4.
Expressions of genes in newly established lung cancer cell lines. -∼+++ means a relative expression density of each gene in cell lines
| Cell line | E-cadherin | COX-2 | MDR1 | MXR | CGA | Synaptophysin | TTF1 |
|---|---|---|---|---|---|---|---|
| SNU-371 | − | − | + | − | − | − | − |
| SNU-963 | + | − | − | − | +++ | +++ | +++ |
| SNU-1327 | + | − | − | +++ | ++ | + | − |
| SNU-1330 | + | + ++ | − | + | − | − | − |
| SNU-2292 | + | + + | + | + | − | − | + |
| SNU-2315 | + | + | + | + | − | − | ++ |
Table 5.
Comparison of the in vitro antitumor activity of doxorubicin, 5-FU, mitomycin-C, cisplatin and VP-16 in SNU-371, SNU-1327 and SNU-1330 lung cancer cell lines
| Cell line | Doxorubicin | 5-FU | Mitomycin-C | Cisplatin | VP-16 |
|---|---|---|---|---|---|
| SNU-371 | 1.92 ± 0.74 | >500 | 11.72 ± 2.91 | 22.2 ± 8.69 | >200 |
| SNU-1327 | 0.05 ± 0.01 | 0.20 ± 0.08 | 0.09 ± 0.02 | 0.38 ± 0.09 | <0.02 |
| SNU-1330 | 0.42 ± 0.16 | 30.1 ± 17.8 | 0.35 ± 0.16 | 8.51 ± 4.87 | >200 |
Discussion
Advances in cell culture methods have made it possible to establish a variety of human carcinoma cell lines from surgical and autopsy tissues, peritoneum effusion and biopsy specimens. Moreover, because pure cells in culture can be used for a variety of studies, which cannot be carried out using tissue specimens, the study of permanent cell lines established from human cancers has played a major role in our understanding of the biology of cancer (reviewed in ref. [8]). Similarly, cell lines originated from lung cancers have been used worldwide in many studies regarding lung carcinogenesis.
In this paper, we describe six newly-established lung cancer cell lines derived from a pulmonary blastoma (SNU-371), a small cell lung cancer (SNU-963), three adenocarcinomas (SNU-1327, SNU-2292 and SNU-2315) and a lung squamous cell carcinoma (SNU-1330).
When new cell lines are being established, DNA fingerprinting analysis for authentication of cell lines and presence of mycoplasma and other bacteria should be tested. During cell culture, cross-contamination between cell lines is widely prevalent and continues to be an ongoing problem. The cross-contamination of cultures has plagued many researchers, often leading to mistaken results, retractions of results, and some out-and-out falsification of data and results following inadvertent use of the wrong cells. DNA fingerprinting has proven a valuable approach to authenticating and characterizing cell lines [22]. In our study, DNA fingerprinting profiles established using two microsatellite markers and AmpFlSTR identifiler showed that each profile of the six cell lines is indeed unique. None of the cells lines showed mycoplasma or bacterial contamination.
The primary tumors from which the present cell lines were derived possessed distinctive morphological features. Moreover, the resulting carcinoma cells showed characteristic morphological features in culture that were maintained through numerous passages as the lines were serially propagated. Five cell lines each grew as a monolayer and a SCLC cell line, SNU-963, grew as floating aggregates and adherent cells.
Pulmonary blastoma is one of the rarest lung cancers, which grows as a tumor consisting of malignant fetal-type glands and stroma. The concept of pulmonary blastoma was expanded by a report of ‘pulmonary endodermal tumor resembling fetal lung’ (i.e., a tumor consisting exclusively of fetal-type epithelial components). Genetic alterations in pulmonary blastomas are largely unknown. Previously, p53 mutations in a subset of tumors and the absence of K-ras mutation have been reported (reviewed in ref. [23]). There are no reports of pulmonary blastoma cell lines in the literature. The presently-reported pulmonary blastoma cell line, SNU-371, is thus a unique and noteworthy accomplishment.
The original tumor of SNU-371 showed characteristics of pulmonary blastoma, (i.e., a biphasic pattern, being composed of tubular glands and cellular stroma). This cell line was composed of spindle-shaped or pleomorphic cells and grew as a monolayer. We think that this cell line will be very useful for studies on the biological behavior of pulmonary blastoma.
β-catenin is a submembranous component of the adherens junction, and it also acts as a transcriptional activator of the Wnt signaling pathway. Mutations of β-catenin have been reported in various tumors but are rare in lung cancers. β-catenin mutations in pulmonary blastomas were recently described [23]. However, no β-catenin mutations were presently observed.
p53 tumor suppressor protein is the most commonly mutated protein in diverse cancers and has been implicated in the late stage of malignant transformation [24]. Mutations in the p53 gene are extremely frequent in SCLCs (75%–100%), whereas in NSCLCs the frequency varies among the histological types (reviewed in ref. [3]). Of the six newly established cell lines, we detected two mutations in the p53 gene. Two cell lines (SNU-1327 and SNU-2292) were derived from adenocarcinomas. The mutation in SNU-1327 was a homozygous missense mutation from arginine (CGC) to proline (CCC) at codon 72 of p53. The Arg72 form has a high risk of developing into other forms of cancer compared to the Pro72 form [25]. The Arg72 variant induces apoptosis markedly more than does the Pro72 variant and the two polymorphic variants of p53 are functionally distinct. These differences may influence cancer risk or treatment [26]. A missense mutation from cysteine (TGC) to phenylalanine (TTC) at codon 176 in exon 5 of p53 in SNU-2292 cell line was a G → T transversion located in the sequence-specific DNA binding region (codons 100–293). This mutation has been reported in lung cancers and oral cancers [27–29].
Transforming growth factor β (TGF-β) exerts a growth inhibitory effect on many cell types through binding to types I and II receptors. Resistance to TGF-β due to lack of type II receptor has been described in some cancer types including SCLC. Of 35 SCLC and NSCLC tumor specimens with chromosome 3p loss examined for TGF-βRII mutations, only one SCLC tumor harbored a mutation in the poly-A tract (reviewed in ref. [30]). In our lung cancer cell lines, the mutations in exon 3 of TGF-βRII gene were not found by direct sequencing analysis.
E-cadherin has been termed a “metastasis suppressor” gene, because the E-cadherin protein can suppress tumor cell invasion and metastasis [15]. E-cadherin expression is reduced or silenced in carcinomas of the breast, liver and many cell lines including those from colon, stomach and prostate (reviewed in ref. [15]). Of our six lung cancer cell lines, E-cadherin was not expressed in SNU-371, a pulmonary blastoma cell line. The association between no expression of E-cadherin and hypermethylation of the promoter remains to be studied in this cell line.
COX-2 expression is induced during inflammation by pro-inflammatory cytokines and growth factors and is detectable in most tissues [16]. COX-2 expression takes part in tumor angiogenesis and is a significant poor prognostic factor in surgically resected NSCLC [31]. These authors concluded that COX-2 inhibitor, either in combination therapy with other agents or for chemoprevention, may be effective via suppression of angiogenesis in this fatal disease. In our experiments, COX-2 was overexpressed in SNU-1330, SNU-2292 and SNU-2315 cell lines.
The most frequently reported alteration associated with multidrug resistance is the increased expression of a 170-kDa membrane protein termed the P-glycoprotein. P-glycoprotein is encoded by MDR1 and functions as an energy-dependent drug efflux pump that reduces intracellular drug accumulation, thereby causing resistance to many structurally different drugs (reviewed in ref. [32]). Of our cell lines, MDR1 was overexpressed in the pulmonary blastoma-derived SNU-371 and SNU-2292 cell lines, suggesting that the cells might be resistant to unrelated anticancer drugs. Use of several anticancer drugs demonstrated that the SNU-371 cell line was more resistant to several anticancer drugs than the other tested cell lines.
BCRP, also called ABCG2, ABCP or MXR, is an ABC transporter that has an N-terminal ATP biding domain and a C-terminal transmembrane domain. BCRP/MXR overexpression has been reported in various drug-resistant cells selected with mitoxantrone, doxorubicin, topotecan and NB-506 [18]. BCRP/MXR presumably acts as an efflux pump, resulting in decreased intracellular concentrations of these anticancer agents (reviewed in ref. [33]). In our experiments, BCRP/MXR was overexpressed in the SNU-1327 cell line and weakly expressed in the SNU-1330, SNU-2292 and SNU-2315 cell lines. MDR1 was overexpressed only in the SNU-371 cell line. Mutations in the kinase domain of EGFR are closely associated with enhanced responsiveness to the EGFR tyrosine kinase inhibitor, gefitinib, in patients with NSCLC. Activations of EGFR transfers signals through the ERBB2, RAS and RAF proteins in the downstream pathway (reviewed in ref. [4, 34]). In our study, mutations in EGFR were evident in a squamous carcinoma cell line (SNU-1330) and an adenocarcinoma cancer cell line (SNU-2315). Two mutations (p.Glu746_Ser752delinsVal in SNU-1330 and p.Glu746_Ala750del in SNU-2315) were detected in the kinase domain of EGFR, consistent with previous studies [5, 14, 35].
The presently described lung cancer cell lines should prove useful in investigation of the biological characteristics of lung cancer especially those related to anticancer drug resistance mechanism associated with overexpression of COX2, MDR1 and MXR, and EGFR mutations gene in lung cancer.
Conclusions
We report the characteristics of six lung cancer cell lines (SNU-371, SNU-963, SNU1327, SNU-1330, SNU-2292 and SNU-2315) established from a pulmonary blastoma, a small cell lung cancer, three adenocarcinomas and a squamous cell carcinoma of the lung of six Korean lung cancer patients. These lung cancer cell lines were well characterized and these may be useful tools for investigations of the biological characteristics of lung cancers, particularly for investigations related to mutations of EGFR, because lung cancer cell lines with mutations in the kinase domain of EGFR can be widely used for study of responsiveness to the new EGFR tyrosine kinase inhibitors and resistant mechanisms to the these inhibitors. These cell lines will be deposited to Korean Cell Line Bank (KCLB, http://cellbank.snu.ac.kr) and widely available to the scientific community through the KCLB.
Acknowledgments
This work was supported by the S.N.U. foundation & overhead research fund, Korean Cell Line Research Foundation research fund and Priority Research Centers Program National Research Foundation of Korea grant, Ministry of Education, Science and Technology (2009-0093820).
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