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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Malignant pleural mesothelioma (MPM) is an asbestos-related malignancy that is highly resistant to current therapeutic modalities. We established four MPM cell lines (ACC-MESO-1, ACC-MESO-4, Y-MESO-8A and Y-MESO-8D) from Japanese patients, with the latter two from the same patient with biphasic-like characteristics of MPM, showing epithelial and sarcomatous phenotypes, respectively, in cell culture. These cells grew well in RPMI-1640 medium supplemented with 10% fetal bovine serum under 5% CO2. Mutation and expression analyses demonstrated that the tumor suppressor gene NF2, which is known to be one of the most frequently mutated in MPM, is mutated in ACC-MESO-1. We detected homozygous deletion of p16INK4A/p14ARF in all four MPM cell lines. However, mutations of other tumor suppressor genes, including TP53, and protooncogenes, including KRAS, NRAS, BRAF, EGFR and HER2, were not found in these cell lines. Polymerase chain reaction amplification of the simian virus 40 sequence did not detect any products. We also analyzed genetic alterations of six other MPM cell lines and confirmed frequent mutations of NF2 and p16INK4A/p14ARF. To characterize the biological differences between Y-MESO-8A and Y-MESO-8D, we carried out cDNA microarray analysis and detected genes that were differentially expressed in these two cell lines. Thus, our new MPM cell lines seem to be useful as new models for studying various aspects of the biology of human MPM as well as materials for the development of future therapies. (Cancer Sci 2006; 97)

Abbreviations:
ATCC

American Type Tissue Culture Collection

IL8

interleukin 8

MM

malignant mesothelioma

MPM

malignant pleural mesothelioma

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

SDS

sodium dodecylsulfate

SSC

saline-sodium citrate

SSCP

single-strand conformation polymorphism

STR

short tandem repeat

SV40

simian virus 40

TSG

tumor suppressor gene

Malignant mesothelioma is an aggressive neoplasm arising from mesothelial cells that most often occurs in the pleural cavity as MPM. MPM is considered to be associated with previous exposure to asbestos fibers. Owing to the long latency period after exposure and the widespread use of asbestos fibers for many years, the incidence of MPM is projected to rise sharply worldwide in the next two decades.(1) In Japan, 500 patients with MM died in 1995, and that number increased to approximately 900 patients in 2003.(2)

Several clinical problems regarding the diagnosis, pathophysiology and treatment of MM remain unsolved. In particular, MM has been demonstrated to be resistant to all conventional therapy regimens, including chemotherapy, radiotherapy and surgery, and the prognosis of patients remains very poor.(3) The discrepancy between the rising incidence of MM and the lack of success of new more effective therapeutic strategies may be related at least in part to inadequate knowledge of the biological properties of this tumor. It is hoped that a better understanding of MM biology may provide the rationale for new therapeutic strategies. In this regard, the development of tumor cell lines has been an important tool in setting up suitable in vitro models for studying the biological properties of many tumors and to assess tumor sensitivity to various drugs or biological response modifiers. However, as opposed to lung cancer, for example, where several hundred cell lines have been established, a relatively small number of MPM cell lines have been established,(4–8) and only a few cell lines are available in tissue culture banks such as the ATCC. Furthermore, according to previous reports, only a few cell lines were established from Japanese patients with MPM.(9)

In the present study, four MPM cell lines, designated ACC-MESO-1, ACC-MESO-4, Y-MESO-8A and Y-MESO-8D (the latter two being from the same patient with biphasic-like characteristics of MPM), were established from Japanese patients, and their genetic alterations were analyzed. The TSG and protooncogenes analyzed were NF2, p16INK4A/p14ARF, TP53, KRAS, NRAS, BRAF, EGFR and HER2, the first three of which were reported to be inactivated in MPM.(10) We found a point mutation of NF2 in ACC-MESO-1 and homozygous deletion of p16INK4A in all four cell lines. As there has been no prior report of two distinct morphologically different MPM cell lines being established from the same patient, we characterized the biological and genetic properties of Y-MESO-8A and Y-MESO-8D in detail, including tumorigenicity in nude mice, and found different gene expression profiles between these cell lines, with some genes encoding molecules involved in cell structural activity or cell adhesion being preferentially expressed in one cell line rather than the other.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patient and establishment of cell lines

Three Japanese patients with pleural thickening or pleuritis were diagnosed with malignant mesothelioma through routine histopathological examination of haematoxylin–eosin staining and/or immunohistochemical studies (including carcinoembryonic antigen [CEA], vimentin and carletinin). ACC-MESO-1 was established from a 61-year-old Japanese woman, ACC-MESO-4 from a 59-year-old Japanese man, and Y-MESO-8 A and Y-MESO-8D from a 60-year-old Japanese man. The patient with ACC-MESO-4 had a history of asbestos exposure, but the remaining two patients did not have any obvious history. Cell cultures were established using a method similar to that described previously,(11) with approval by local ethical committees. Briefly, after collection of the materials, the pleural effusion of 20 mL or dissected tumor samples were transferred into a 75-cm2 culture flask. They were incubated at 37°C in a humidified incubator containing 5% CO2 with replacement of fresh RPMI-1640 medium (Sigma Aldrich, Irvine, UK) supplemented with 10% fetal bovine serum (Equitech-Bio, Ingram, TX, USA) and 1 × antibiotic-antimycotic (Gibco BRL Life Technologies, Rockville, MD, USA). Thereafter, the medium was replaced twice a week. Significant contaminant cells, such as fibroblast cells, were removed by scraping.

During the subsequent period of continuous propagation by culture, the cells were sampled at intervals, resuspended in the Cell Banker freezing medium (Juji Field, Tokyo, Japan), and stored in liquid nitrogen. After thawing, the stored cells could be propagated in culture without noticeable change in growth and morphology. Tumor cells grown in the flasks were examined directly with an inverted microscope, and phase-contrast photographs were taken periodically.

Source of other cell lines

Three MPM cell lines (NCI-H28 [CRL5820], NCI-H2373 [CRL5943] and MSTO-211H) were purchased from ATCC (Rockville, MD, USA). The other three MPM cell lines (NCI-H290, NCI-H513 and NCI-H2052) and a lung cancer cell line of an adenocarcinoma (NCI-H358) were gifts from Dr Adi F. Gazdar. All cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum and 1 × antibiotic-antimycotic at 37°C in a humidified incubator with 5% CO2.

Preparation of DNA and RNA

DNA and RNA were prepared from cell lines by standard techniques.(12) Random-primed, first-strand cDNAs were synthesized from 2 µg of total RNA using Superscript II according to the manufacturer's instructions (Invitrogen, New York, NY, USA).

Mutation analysis

Mutation analyses were carried out either by direct sequencing after genomic PCR amplification and/or SSCP analysis followed by sequencing using aberrant bands. Sequencing analysis was carried out using an Applied Biosystems Model 3100 DNA sequencer (Perkin-Elmer Cetus, Norwalk, CT, USA) with a PCR primer and a BigDye terminator Cycle sequencing FS Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). SSCP analysis was carried out on mutation detection enhancement (MDE) gels (FMC Bioproducts, Rockland, ME, USA) containing 10% glycerol, as described previously.(13)

For TP53 mutation, PCR-SSCP analysis was carried out for exons 2–11 using genomic DNA to cover the entire coding frame of TP53. Primers used were as described previously.(14)

For NF2 mutation, direct sequencing was carried out, and primers used were as follows: NF2exon1S, 5′-AGGCCTGTGCAGCAACTC-3′; NF2exon1AS, 5′-GAGAACCTCTCGAGCTTCCAC-3′; NF2exon2S, 5′-GAGAGTTGAGAGTGCAGAG-3′; NF2exon2AS, 5′-TCAGCCCCACCAGTTTCATC-3′; NF2exon3S, 5′-GCTTCTTTGAAGGTAGCACA-3′; NF2exon3AS, 5′-GGTCAACTCTGAGGCCAACT-3′; NF2exon4S, 5′-CCTCACTTCCCCTCACAGAG-3′; NF2exon4AS, 5′-CCCATGACCCAAATTAACGC-3′; NF2exon5S, 5′-ATCTTTAGAATCTCAATCGC-3′; NF2exon5AS, 5′-AGCTTTCTTTTAGACCACAT-3′; NF2exon6S, 5′-CATGTGTAGGTTTTTTATTTTGC-3′; NF2exon6AS, 5′-GCCCATAAAGGAATGTAAACC-3′; NF2exon7S, 5′-CAGTGTCTTCCGTTCTCC-3′; NF2exon7AS, 5′-AGCTCAGAGAGGTTTCAA-3′; NF2exon8S, 5′-CCACAGAATAAAAAGGGCAC-3′; NF2exon8AS, 5′-GATCTGCTGGACCCATCTGC-3′; NF2exon9S, 5′-GTTCTGCTTCATTCTTCC-3′; NF2exon9AS, 5′-GTAATGAAAACCAGGATC-3′; NF2exon10S, 5′-CCTTTTAGTCTGCTTCTG-3′; NF2exon10AS, 5′-TCAGTTAAAACAAGGTTG-3′; NF2exon11S, 5′-TCGAGCCCTGTGATTCAATG-3′; NF2exon11AS, 5′-AAGTCCCCAAGTAGCCTCCT-3′; NF2exon12S, 5′-CCCACTTCAGCTAAGAGCAC-3′; NF2exon12AS, 5′-CTCCTCGCCAGTCTGGTG-3′; NF2exon13S, 5′-GGTGTCTTTTCCTGCTACCT-3′; NF2exon13AS, 5′-GGGAGGAAAGAGAACATCAC-3′; NF2exon14S, 5′-TGTGCCATTGCCTCTGTG-3′; NF2exon14AS, 5′-AGGGCACAGGGGGCTACA-3′; NF2exon15S, 5′-TCTCACTGTCTGCCCAAG-3′; NF2exon15AS, 5′-GATCAGCAAAATACAAGAAA-3′; NF2exon16S, 5′-CTCTCAGCTTCTTCTCTGCT-3′; NF2exon16AS, 5′-CCAGCCAGCTCCTATGGATG-3′; NF2exon17S, 5′-GGCATTGTTGATATCACAGGG-3′; and NF2exon17AS, 5′-GGCAGCACCATCACCACATA-3′. Numbers after exon indicate exon number, and S and AS indicate sense and antisense, respectively.

Polymerase chain reaction of p16INK4A was carried out using the primer sets: p16ex1S, 5′-TGCCACATTCGCTAAGTGCT-3′; p16ex1AS, 5′-GCTGGCGGAAGAGCCC-3′; p16ex2S, 5′-GTGGACCTGGCTGAGGAGC-3′; p16ex2AS, 5′-TCTCAGGGTACAAATTCTCAGATCAT-3′; p16ex3S, 5′-AAGAAAAACACCGCTTCTGCC-3′; and p16ex3AS, 5′-TCCCTAGTTCACAAAATGCTTGTC-3′.

For KRAS, NRAS and BRAF mutations, direct sequencing was carried out, and the primers for KRAS and NRAS were as described previously.(15) PCR of BRAF was carried out using the following primer sets: BRAF11S, 5′-TTCTGTTTGGCTTGACTTGAC-3′ and BRAF11AS, 5′-CTATTATGACTTGTCACAATGTCACC-3′ for exon 11; and BRAF15S, 5′-TCATAATGCTTGCTCTGATAGGA-3′ and BRAF15AS, 5′-GGCCAAAAATTTAATCAGTGGA-3′ for exon 15.

Tumorigenecity in nude mice

The cultured cells (4 × 106) were washed, resuspended in 0.2 mL of PBS, and injected subcutaneously into the left flank of 10 6-week-old BALB/c (nu/nu) female nude mice. As a control, 0.2 mL of PBS alone was similarly injected into the right flank of the nude mice. The animals were examined every week for the development of tumors. Tumor volume was calculated as length × height × width × 0.5. All animal care was in accordance with institutional guidelines. After the single tumor-bearing mouse was killed, the tumor tissue was excised, fixed in 10% formalin and processed for routine histopathological examination.

Genetic analysis

To confirm that there was no cross-contamination of cell lines, the uniqueness of the established cell line was evaluated by analysis of STR polymorphisms using the AmpFISTR Identifiler Kit (Applied Biosystems). This kit includes 16 STR loci, which are D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, Amelogenin, D5S818 and FGA.

Western blot analyses

Preparation of total cell lysates and western blotting were carried out as described previously.(16) In brief, cells growing subconfluently were rinsed twice with PBS, lysed in SDS sample buffer (62.5 mM Tris pH 6.8, 2% SDS, 2% 2-mercaptoethanol, 10% glycerol) and homogenized. Total cell lysate protein (15 µg) was subjected to SDS-polyacrylamide gel electrophoresis and transferred to poly(vinylidene fluoride) (PVDF) membranes (Millipore, Bedford, MA, USA). Following blocking with 5% non-fat dry milk, the filters were incubated with the primary antibody, washed with PBS, reacted with the secondary antibody, and then detected with ECL (Amersham Biosciences, Buckinghamshire, UK). The primary antibodies used were anti-E-cadherin antibody (C20820; Pharmingen/Transduction Laboratories, San Diego, CA, USA), anti-N-cadherin antibody (C70320; Pharmingen/Transduction Laboratories), and anti-ERC/mesothelin antibody (IBL, Gunma, Japan).

Microarray analysis

The fluorescently labeled cRNA targets were prepared by incorporation of CyDye-NTP through in vitro transcription reaction. Aliquots of total RNA (4 µg) from Y-MESO-8A and Y-MESO-8D were labeled using RNA Transcript SureLABEL Core Kit (TAKARA BIO, Otsu, Japan) with Cy5-UTP and Cy3-UTP (Amersham Biosciences), respectively, in each paired case. We used the commercially available IntelliGene HS Human Expression chip (TAKARA BIO) to carry out microarray analysis.

Labeled probes were mixed with hybridization solution (6 × SSC, 0.2% SDS, 5 × Denhardt's solution, 0.1 mg/mL denatured salmon sperm DNA, 50% formamide). After hybridization for 16 h at 65°C, the slides were washed twice in 2 × SSC and 0.1% SDS for 5 min at 65°C, once in 2 × SSC and 0.1% SDS for 5 min at 65°C, and once in 0.05 × SSC for 5 min at room temperature. The slides were scanned using the Affymetrix 428 scanner (Affymetrix, Santa Clara, CA, USA). The signal intensity of hybridization was evaluated photometrically by the ImaGene computer program (BioDiscovery, El Segundo, CA, USA) and normalized to the averaged signals of housekeeping genes (or global normalization). A cut-off value for each expression level was calculated according to the background fluctuation.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Establishment of MPM cell lines

The new MPM cell lines ACC-MESO-1, ACC-MESO-4, Y-MESO-8A and Y-MESO-8D were established successfully from a 61-year-old Japanese woman, a 59-year-old Japanese man, and a 60-year-old Japanese male (Fig. 1). Microscopically, the original tumors of ACC-MESO-1 and ACC-MESO-4 mainly consisted of epithelioid cells, and that of Y-MESO-8A and Y-MESO-8D mainly consisted of spindle cells. Because the primary cultures of Y-MESO-8 showed several colonies with different morphological types, they were subcloned. Y-MESO-8A showed polygonal and epithelial-like morphology, whereas Y-MESO-8D showed spindle-like morphology (Fig. 1c,d). These cell lines grew as adherent monolayers and maintained a consistent morphology from the primary culture to the following passages. After thawing, the cryopreserved cells were able to propagate in culture without noticeable change in growth and morphology.

image

Figure 1. Morphology of the four malignant pleural mesothelioma (MPM) cell lines. Micrographs of cultured (a) ACC-MESO-1 cells, (b) ACC-MESO-4 cells, (c) Y-MESO-8A cells, and (d) Y-MESO-8D cells (original magnifications ×100).

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Mutation and expression analyses

To determine whether the four new cell lines harbored genetic changes of tumor suppresser genes, reported as frequently detected in MPM, we carried out mutation and expression analyses along with six other MPM cell lines. The tumor suppressor genes studied were TP53, NF2 and p16INK4A/p14ARF. We also analyzed protooncogenes, including KRAS, NRAS, BRAF, EGFR and HER2. The results are summarized in Table 1. Among the four new cell lines, homozygous deletions of p16INK4A/p14ARF were detected in all four, whereas NF2 was shown to be inactivated by a nonsense mutation (Q389X) only in ACC-MESO-1. Neither mutation nor homozygous deletion of TP53 was found.

Table 1. Genetic alterations in 10 mesothelioma cell lines
Cell lineACC-MESO-1ACC-MESO-4Y-MESO-8AY-MESO-8DH28H290H513H2052H2373MSTO-211H
  • Refer to Sekido et al. 1995. +, Undetectable mutation for target regions; HD, homozygous deletion; ND, not determined; Nt, nucleotide.

TP53++++++Nt717insA+++
NF2Q389X++++HDNDR341XHD+
p16INK4A/p14ARFHDHDHDHDHDHDHDHDHDHD
KRAS++++++++++
NRAS++++++++++
BRAF++++++++++
EGFR++++++++ND+
HER2++++++++ND+

To determine whether the SV40 large T antigen was involved in the pathogenesis of our new MPM cell lines, we carried out PCR analysis to detect the DNA of large T antigen. However, we found no evidence to indicate implication of the SV40 (data not shown).

Furthermore, we also tested expression of cell adhesion molecules, E-cadherin and N-cadherin, with western blot analysis, as these have been reported to be expressed aberrantly in MPM as well as being useful for differential diagnosis from poorly differentiated adenocarcinoma. E-cadherin expression was detected in an adenocarcinoma cell line, NCI-H358, and two mesothelioma cell lines, ACC-MESO-1 and ACC-MESO-4, but not in the other mesothelioma cell lines, Y-MESO-8A, Y-MESO-8D, NCI-H2373, NCI-H2052, NCI-H290, NCI-H513, MSTO-211H and NCI-H28. However, N-cadherin expression was detected in Y-MESO-8A, Y-MESO-8D, NCI-H2373, NCI-H2052, NCI-H290, NCI-H513, MSTO-211H and NCI-H28 (Fig. 2). Finally, we tested the expression of ERC/mesothelin, which has been reported as being expressed in MPM, using western blot analysis. An expected strong 41-kDa band was observed for ACC-MESO-1, ACC-MESO-4, Y-MESO-8A, NCI-H2052 and NCI-H2373 (data not shown).

image

Figure 2. Western blot analysis of E-cadherin and N-cadherin. Each lane was loaded with 15 µg of total cell lysate from Y-MESO-8A (lane 1), Y-MESO-8D (lane 2), NCI-H2373 (lane 3), NCI-H2052 (lane 4), NCI-H290 (lane 5), NCI-H513 (lane 6), MSTO-211H (lane 7), ACC-MESO-1 (lane 8), ACC-MESO-4 (lane 9), NCI-H28 (lane 10) and NCI-H358 (lane 11). β-Actin is shown as an internal control at the bottom.

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Genetic and biological differences between Y-MESO-8A and Y-MESO-8D

Y-MESO-8A and Y-MESO-8D were established from pleural effusion at the same time and showed distinct morphological patterns. As MPM are frequently composed from two separate subpopulations of cell groups (epithelial and sarcomatous types, which are diagnosed as biphasic MPM), we speculated that these two cell lines may represent both phenotypes, respectively.

First, we confirmed that Y-MESO-8A and Y-MESO-8D were from the same patient using a multiplex amplification and typing system for 16 STR with DNA derived from the white blood cells of the patient (data not shown). To determine the differences in tumorigenicity between Y-MESO-8A and Y-MESO-8D cells, we inoculated the cells into athymic nude mice. After subcutaneous injection of the Y-MESO-8A cells, a visible subcutaneous tumor developed in only one of the 10 nude mice at the site of inoculation, with histological examination of the xenotransplanted nodules showing papillary proliferation consisting of atypical epithelioid cells (Fig. 3). Meanwhile, the Y-MESO-8D cells showed no visible subcutaneous tumors in any of the 10 inoculated nude mice. Lastly, we performed cDNA microarray analysis to determine the differences in expression profiles. The 43 genes showing over a 5-fold difference of expression between the two cell lines are listed in Table 2. With selected cDNA probes synthesized, we carried out northern blot analyses for genes including CRIP1 and VCAM1 and confirmed the differences of expression between Y-MESO-8A and Y-MESO-8D (data not shown).

image

Figure 3. (a) Xenografted tumor of Y-MESO-8A. (b) Growth curve of the xenografted tumor in nude mice. (c) Histology of xenografted Y-MESO-8A tumor (haematoxylin and eosin, original magnification ×100 and ×400). As in epithelioid mesothelioma, the tumor shows papillary proliferation consisting of atypical epithelioid cells.

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Table 2. Genes showing over a five-fold difference in expression between Y-MESO-8A and Y-MESO-8D
Gene nameAccession no.Molecular functionRatio
Y-MESO-8A/Y-MESO-8D ≥ 5
Potassium large conductance calcium-activated channel, subfamily M, α member 1 (KCNMA1)NM_002247.2Unknown19.2
Microfibril-associated glycoprotein-2 (MAGP2)NM_003480.1Structural molecule activity15.4
Filaggrin (FLG)XM_048104.1Structural molecule activity14.5
Cysteine-rich protein 1 (intestinal) (CRIP1)NM_001311.2Binding13.9
UDP-N-acetyl-α-d-galactosamine : polypeptide N-acetylgalactosaminyltransferase 5 (GALNT5)XM_050509.6Unknown11.8
Hypothetical protein FLJ14834 (FLJ14834)NM_032849.2Unknown11.1
Glutathione peroxidase 6 (GPX6)NM_015696.2Unknown10.7
Decorin (DCN), transcript variant A1NM_001920.2Unknown10.5
KIAA0193 gene product (KIAA0193)NM_014766.2Catalytic activity10.4
LumicanNM_002345.2Morphogenesis 9.24
Selenium binding protein 1NM_003944.2Unknown 8.99
ATP-binding cassette, subfamily B (MDR/TAP)NM_000927.2Cell growth 8.63
S100 calcium binding protein A4NM_002961.2Cell growth 7.06
Plasminogen activatorNM_000930.2Unknown 7.01
Adenylate cyclase activating polypeptide 1NM_001117.2Cell communication 6.88
Serine protease inhibitorNM_021102.1Cell motility 6.77
Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the rad21p familyNM_005132.1Unknown 6.58
Adipose specific 2NM_006829.1Unknown 6.52
Tissue inhibitor of metalloproteinase 3NM_000362.3Unknown 6.38
EphA3NM_005233.2Cell communication 6.34
Sodium channel, non-voltage-gated 1αNM_001038.1Cell growth 6.31
Podocalyxin-likeNM_005397.1Unknown 6.21
Cut-like 1, CCAAT displacement proteinNM_001913.1Unknown 5.98
Ocular albinism 1NM_000273.1Cell communication 5.90
Paternally expressed 10NM_015068.1Unknown 5.89
Cytochrome P450, family 26, subfamily A, polypeptide 1 (CYP26A1)NM_057157.1Unknown 5.84
DesmoplakinNM_004415.1Morphogenesis 5.64
Complement component 4BNM_000592.3Unknown 5.60
Y-MESO-8D/Y-MESO-8A ≥ 5
Kynureninase (l-kynurenine hydrolase) (KYNU)NM_003937.1Unknown69.4
Aminopeptidase (LOC64167)NM_022350.1Unknown32.8
Aldo-keto reductase family 1, member B10 (aldose reductase) (AKR1B10)NM_020299.3Unknown17.8
Annexin A10 (ANXA10)NM_007193.2Unknown15.3
Vascular cell adhesion molecule 1 (VCAM1), transcript variant 1NM_001078.2Cell communication14.2
Hypothetical protein FLJ30834 (FLJ30834)NM_152399.1Unknown10.9
Hypothetical protein FLJ33957 (FLJ33957)NM_152322.1Unknown 9.47
Protease inhibitor 3, skin-derived (SKALP) (PI3)NM_002638.1Unknown 9.16
Interleukin 8 (IL8)NM_000584.2Cell growth 9.00
Interleukin 1, α (IL1A)NM_000575.3Cell growth 7.50
Aldo-keto reductase family 1, member C3 (AKR1C3)NM_003739.4Cell growth 6.35
Transmembrane 4 superfamily member 2 (TM4SF2)NM_004615.2Unknown 6.14
Glutathione S-transferase theta 2 (GSTT2)NM_000854.2Unknown 5.84
Hypothetical protein FLJ22761 (FLJ22761)NM_025130.1Unknown 5.74
Solute carrier family 21 (organic anion transporter), member 9 (SLC21A9)NM_007256.1Cell growth 5.04

In addition, we also carried out cytogenetic analysis of the Y-MESO-8A cells at passages 11 (data not shown). The modal chromosome number from the 12-karyotype analysis ranged from 41 to 91 with a median of 57. Extra copies of chromosomes 1, 3, 8, 11, 12, 15, 16, 17, 20 and X were noted in some metaphases, whereas loss of chromosomes 9, 13, 14 and 22 was noted in others. Homogeneous staining regions or double minutes were not detected.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The development of tumor cell lines has been an important tool in establishing suitable in vitro models for studying the biological properties of many tumors. Various types of human tumors have been selected for establishment of cell cultures, and in lung cancer, for example, there are several hundred cell lines that have been developed in many laboratories worldwide during recent decades.(17–20) In contrast, the number of other specific tumor cell lines is still small because of the rare incidence of diseases, infrequent availability of fresh specimens, and technical difficulties in cell culture development, all of which result in a large impediment for studying tumors aimed at basic and preclinical research. MPM is one such example, and MPM cell lines, which are available from public bioresource banks including ATCC, are very few.(4–8) Furthermore, only a few cell lines have been established from Japanese patients with MPM,(9) and there are only several abstracts from the Japan Medical Abstracts Society reporting the establishment of a single MPM cell line. In the present study, we established four MPM cell lines (ACC-MESO-1, ACC-MESO-4, Y-MESO-8A and Y-MESO-8D), derived from Japanese patients, characterized their genetic abnormalities and detected genes differentially expressed between Y-MESO-8A and Y-MESO-8D, which were derived from the same patient.

Traditional cytogenetic and loss of heterozygosity analyses, followed by recent comparative genomic hybridization techniques, identified common chromosomal abnormalities in MPM cells, including deletions on chromosomes 1, 3, 4, 9, 11, 14 and 22, some of which have already been shown to harbor target TSG for MPM.(21–26)NF2, which is located on chromosome 22q12 and is known to be one of the most frequently mutated TSG in MPM,(10) was mutated in ACC-MESO-1, although we found no NF2 mutation in the other three new MPM cell lines. p16INK4A, which is located on chromosome 9p21 and is involved in the development of many other types of cancers, has also been shown to be a target gene for MPM with frequent homozygous deletions being identified.(27) In the present study, we found homozygous deletions of p16INK4A in all four cell lines using primers of exons 1, 2 and 3 (Table 1), indicating that in the p14ARF gene, a second coding frame using another exon 1 (exon1β), along with exons 2 and 3, is also completely inactivated. However, a detailed analysis of 9p21 homozygous deletions in lung cancer have also identified that the deletion regions extend beyond the p16INK4A/p14ARF gene locus and affect other genes in the vicinity, including p15.(28) Thus, further analyses concerning these homozygous deletions in 9p21 should determine whether genes other than p16INK4A/p14ARF are also targeted in MPM.

In contrast, although TP53 is known to be one of the most frequently mutated TSG, previous analyses have shown that only a small subset of MPM have a TP53 mutation.(21) Consistent with this, we also did not find any activating mutation of the TP53 gene in any of the four new cell lines. Taken together, although the number of cell lines analyzed was small, the frequencies of genetic alterations in our new MPM cell lines, including mutation of NF2 and TP53 and homozygous deletion of p16INK4A/p14ARF, seem to be similar to previous reports, which may suggest that MPM from Japanese patients share common genetic abnormalities with Caucasians.

Meanwhile, SV40, which encodes two tumor antigens (large T and small t), has a well-characterized ability to trigger transformation of cells in culture. Recently, several studies suggested that SV40 is involved in the development of human mesothelioma, which was shown by detection of DNA sequences encoding the SV40 large T antigen and/or its protein expression.(29–32) However, some reported findings are strongly against a role for SV40 in the development of human MPM, and thus the implication of SV40 remains controversial.(33) To determine whether the SV40 large T antigen is involved, we also carried out PCR analysis to detect the DNA of large T antigen in our new MPM cell lines, as well as in six other MPM cell lines. However, we did not find any evidence for implication of the SV40 large T antigen.

Differential diagnosis of MPM from other thoracic malignancies, including poorly differentiated lung adenocarcinoma, is often difficult. Several molecular markers, including carletinin, Wilms’ tumor 1, cytokeratin 5/6 and mesothelin, have been suggested to be useful in distinguishing them.(34) E-cadherin and N-cadherin expression has also been used to distinguish MPM from adenocarcinoma, which is related to tumor invasion or progression.(35) We also confirmed the expression of these cadherins in our newly established cell lines. E-cadherin is expressed in epithelial cells, and reduction in the expression of E-cadherin has been associated with higher malignancy potential and invasiveness in epithelial neoplasms of the colon, ovary, stomach, pancreas, lung, breast, and head and neck, due to lack of cell–cell adhesion. Meanwhile, N-cadherin is restricted to tissues of nerve cells, developing skeletal muscle, embryonic and mature cardiac muscle cells, and mesothelial cells. Because the mesoderm-derived mesothelial cells that form the pleura express N-cadherin during embryonic development, it is suggested that N-cadherin plays an important role in the development and differentiation of mesothelial cells. Thus, the expression of N-cadherin in malignant mesothelioma has been thought to reflect its cell lineage and phenotype. Although it is not clearly understood how N-cadherin expression affects MPM cells pathologically, cell adhesion molecules including N-cadherin should be reconsidered in terms of the uniqueness of MPM progression, such as highly aggressive invasion of the thoracic region but only rare metastasis to distant organs.

Malignant pleural mesothelioma is usually classified into three pathological subtypes, epithelioid, sarcomatous and biphasic, but it is not clearly understood why MPM shows these variations in morphology, even in a single tumor. To our knowledge, this is the first report of two morphologically distinct MPM cell lines being established from the same patient. During many passages of Y-MESO-8A and Y-MESO-8D, both cell lines maintained consistent phenotypes, even under tissue culture condition, which is thought to be useful for in vitro models of MPM biphasic type. In the present study, we tried to determine the underlying mechanisms that affect the morphological differences in Y-MESO-8A and Y-MESO-8D. As described above, because we did not find any differences in the genetic abnormalities of major cancer-associated genes between the two cell lines, we suspect other genetic alterations or epigenetic alterations may account for the differences. To study this in more detail, we are currently carrying out further analyses, including comparative genomic hybridization, and preliminary data suggest that there are some differences in chromosomal gains and losses among most shared genotypes (data not shown). However, we also carried out expression profiling and found 15 genes with over a 10-fold difference in mRNA expression between the two cell lines. Among these genes, vascular cell adhesion molecule 1, microfibril-associated glycoprotein-2 and filaggrin, which are related to morphology, were found to be expressed in one cell line but not the other, which also needs to be analyzed for genetic or epigenetic changes to discover underlying mechanisms. It is also interesting to note that IL8 expression was different between the two cell lines. Whereas IL8 has been suggested to be involved in cell growth of MPM,(36) the expression might also be related to morphological differences between these cell lines.

In conclusion, we have established and characterized new human malignant mesothelioma cell lines, designated ACC-MESO-1, ACC-MESO-4, Y-MESO-8A and Y-MESO-8D, from Japanese patients. These cell lines will provide us with a new experimental system to study pathogenesis and biological behavior, as well as to test new therapeutic reagents of MPM.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We would like to thank Dr Adi F. Gazdar for the cell lines, Ms Yumie Narita and Ms Hiroko Kaga for their skillful technical assistance, and Dr Yuichi Ueda, Dr Hiromu Yoshioka and Dr Toshimichi Yamamoto for special encouragement and support. This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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