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- Materials and Methods
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)
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.
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- Materials and Methods
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.