mesothelioma cancer

Primary Mesothelial Cell Lines

Primary cultures of mesothelial cells have been established from rats, rabbits,
mice, and humans (Table 5.1). Mesothelial cell lines provide several advantages
for experimental studies: they provide a large number of cells isolated from a
single donor, cell lines can be isolated from genetically engineered mice, and
primary cell lines limit the number of animals required for experiments.
However, cell lines have several disadvantages: variability among donors,
variability in culture conditions in different laboratories, potential
phenotypic and genetic instability, and a limited life span in vitro (reviewed
in ref. 1). Some of these disadvantages can be overcome by quality control
procedures. For example, cell lines should not be passaged indefinitely; frozen
stocks should be maintained and thawed at regular intervals to prevent
phenotypic and genetic instability (reviewed in ref. 2). As in all cell cul¬ture
models, precautions are required to prevent cross-contamination and
contamination with bacteria or viruses. DNA profiles could be use¬ful to
identify cell lines; for example Manning et al (3) established initial genetic
profiles for their panel of human malignant mesothelioma cell lines. All
cultures should be screened for Mycoplasma and other pathogens (2).

Technical details regarding primary human mesothelial cell cultures have been
summarized by Versnel et al (1) and Gerwin (4). Briefly, primary human
mesothelial cells require enriched culture media sup¬plemented with 10% to 20%
fetal bovine serum, exogenous growth factors [usually epidermal growth factor
(EGF)], insulin, transferrin, and hydrocortisone. Rabbit, mouse, and rat primary
mesothelial cells require similar growth conditions, with the important
exception that growth of rat pleural mesothelial cells is inhibited by EGF. As
reviewed by Walker et al (5), there are additional differences in expression of
growth factors and their receptors between human and rat mesothelial cells (6).
Differences in growth factor responses have been described in primary human
mesothelial cell cultures derived from different donors (7).

Mesothelial cell cultures have been characterized by morphology, electron
microscopy, immunocytochemistry, and cytogenetics (1,8). Although mesothelial
cells can form monolayers with epithelial mor¬phology, this growth pattern can
be altered in vitro as described below. At the ultrastructural level,
mesothelial cells typically show surface microvilli, abundant mitochondria,
extensive rough endoplasmic retic-ulum, perinuclear intermediate filaments,
desmosomes, and tight junc¬tions (9). Immunocytochemistry is useful to confirm
expression of markers specific for mesothelial cells, especially coexpression of
inter¬mediate filaments, keratin, and vimentin (10) and expression of the Wilms’
tumor suppressor gene, WT1 (11). These markers are also useful for the
immunohistochemical diagnosis of human malignant mesothe-liomas (12,13).
Cytogenetic studies of human mesothelial cell lines reveal a normal karyotype
that may acquire abnormalities after several passages (1). One primary murine
mesothelial cell line has been reported that spontaneously acquired a point
mutation in exon 5 of the p53 tumor suppressor gene. This mutation increased
growth rate in vitro; however, it did not confer tumorigenicity (14).
Primary cell lines provide a valuable model to study the cell biology and
differentiation of normal mesothelial cells. Primary cultures have also been
used to investigate the toxicologic effects of asbestos and man-made mineral
fibers (15). The responses of mesothelial cells to various growth factors and
cytokines are discussed in Chapter 7.
The mesothelium is derived embryologically from the mesoderm. At approximately
embryonic day 7.5 in the mouse, epithelial cells undergo mesenchymal
differentiation to form the mesoderm cell layer. This morphologic
differentiation is governed by transcription factors snail and slug that
modulate expression of cadherins and cytoskeletal pro¬teins characteristic of
mature mesothelial cells (16,17). In response to mechanical injury, peritoneal
dialysis, or chronic inflammation, mesothelial cells also revert from an
epithelial to a mesenchymal phenotype. This transdifferentiation is termed the
epithelial-mesenchymal transition (18) and has been investigated in primary
cultures of human mesothelial cells isolated from reactive peritoneal effusions
or dialysis effluent (Table 5.1). In these pathologic conditions, human
mesothelial cells detach from the mesothelial monolayer and survive in
suspension. When these reactive mesothelial cells are placed in monolayer
culture, they express epithelial or mesenchymal phenotypes (18,19). Wu et al
(20) first characterized the expression of cytoskeletal proteins includ¬ing
actin, vimentin, and several cytokeratins by mesothelial cells iso¬lated from
ascitic fluid. Modulation of the epithelial phenotype in vitro depended on
culture conditions: serum, EGF, and hydrocortisone induced a mesenchymal
phenotype (21), while supplementation with retinoic acid induced an epithelial
phenotype (22). The epithelial– mesenchymal transition of reactive human
mesothelial cells in vitro is characterized by reduced expression of some cell
surface proteoglycans (syndecan-4, glypican-1), the WT1 tumor suppressor gene
(19), and decreased expression of E cadherin in parellel with expression of the
transcription factor snail (18). Transdifferentiation of omental meso-thelial
cells in vitro was also induced by mechanical wounding of mesothelial monolayers
or by exposure to the inflammatory mediators, transforming growth factor-b1
(TGF-b1) or interleukin-1b (IL-1b) (18). Mesothelial cells are sensitive target
for transformation by asbestos fibers. The biologic basis for this increased
sensitivity is unknown. Studies conducted with cell culture models have provided
evidence that the iron-catalyzed generation of reactive oxygen species is a
plau¬sible mechanism for asbestos carcinogenicity (23). Reactive oxygen species
have been implicated in asbestos-induced apoptosis (24,25), chromosomal damage
(26), oxidative DNA damage (27), and DNA strand breaks (28) in human and rat
pleural mesothelial cells. Varia¬tions in antioxidant defense mechanisms have
been hypothesized to contribute to pulmonary disease induced by fibers and
particulates (29). The antioxidant defense pathways of primary rat pleural
mesothe-lial cells have been characterized in detail; these cultures have low
catalase activity and depend primarily on the glutathione pathway for protection
against oxidant stress (30). These mechanistic studies suggest that mesothelial
cells are highly susceptible to DNA and chro¬mosomal damage in response to
asbestos exposure. Mesothelial cells with asbestos-induced DNA damage that
escape apoptosis may be pre¬cursors for the development of malignant
mesothelioma (24).

In Vitro Transformation of Mesothelial Cells

In vitro models of cell transformation have been developed to assess the ability
of viral, physical, or chemical agents to induce immortal¬ization and
transformation of target cells (reviewed in ref. 31). Primary cultures of rat
pleural mesothelial cells have been used to investigate the ability of
chrysotile asbestos fibers to induce colony formation and tumorigenicity (Table
5.2). Unfortunately, rodent cells, including mesothelial cells, become
spontaneously immortalized at late passages.

In two rat mesothelial models, these spontaneously immortalized cultures
showed disorganized growth, loss of contact inhibition, decreased doubling
times, and growth in soft agar. In both models, immortalized cell populations
became aneuploid with trisomy of chro¬mosome 1 (32,33). Late passages of
spontaneously immortalized rat pleural mesothelial cells were tumorigenic after
subcutaneous injection in nude mice (32). Single or repeated exposures of rat
pleural mesothe-lial cells to chrysotile asbestos fibers induced in vitro
transformation and tumorigenicity at earlier passages (32). In contrast to
rodent meso-thelial cells, human mesothelial cells stop dividing after 15 (34)
to 55 (35) population doublings and do not immortalize spontaneously. Exposure
of human pleural mesothelial cells to crocidolite asbestos fibers is toxic and
does not induce transformation in vitro (36). Human peritoneal cells can be
induced to proliferate indefinitely (greater than 100 population doublings) by
transfection with hTERT, the catalytic subunit of telomerase. These immortalized
cultures are still dependent on EGF, hydrocortisone, or serum for growth and do
not show mor¬phologic characteristics of transformed cells (35).
Cell transformation models are valuable tools to identify specific genes
responsible for immortalization and tumorigenicity. Initial studies using rodent
fibroblasts identified at least two collaborating oncogenes (e.g., ras and myc)
that were required for in vitro transfor¬mation (37). In contrast, human cells
cannot be fully transformed by these collaborating oncogenes unless exposed to
chemical or physical carcinogenic agents (reviewed in ref. 38). Human peritoneal
mesothe-lial cells transfected with activated H-ras oncogene show
characteris¬tics of transformed cells; however, they are not tumorigenic when
injected subcutaneously in nude mice (39). A human pleural mesothe-lial line
(Met5A) was stably immortalized by simian virus 40 (SV40) early region that
encoded large-tumor antigen; these cells formed colonies in vitro and were
hypodiploid with multiple chromosomal abnormalities but they are not tumorigenic
(34). However, Met5A cells transfected with activated H-ras oncogene were
tumorigenic in nude mice (40).
With the recent discovery of SV40 viral DNA sequences in human malignant
mesotheliomas (see Chapter 3), additional assays have been conducted to
investigate specific genes required for in vitro trans¬formation of human
pleural mesothelial cells (Table 5.2). Human peritoneal mesothelial cells appear
to be more susceptible to transfor¬mation by SV40 large-tumor antigen (41) than
pleural mesothelial cells (36). Human pleural mesothelial cells immortalized by
transfection of hTERT show in vitro transformation by SV40 large-T and small-t
anti¬gens or by SV40 small-t antigen plus activated H-ras oncogene (42).
Crocidolite asbestos in combination with SV40 large-tumor or large-tumor and
small-tumor antigens induced in vitro transformation and clonal chromosomal
aberrations (36).
Most tumor cells, including human malignant mesotheliomas, show a common set of
characteristics: autonomous cell growth, resistance to growth-inhibitory
signals, evasion of apoptosis, indefinite proliferation potential, angiogenesis,
and invasion and metastasis (reviewed in ref.
43). Most tumor cell lines, including rodent and human mesothelioma cell lines
(reviewed below and in Chapter 6) show multiple genetic alterations and genetic
instability that may have enabled tumor cells to acquire these characteristics.
Numerous in vitro studies using rodent or human mesothelial cell lines exposed
to asbestos fibers have demon¬strated their genotoxic and clastogenic activities
(reviewed in ref. 44). In these in vitro cell transformation models, rat and
human mesothe-lial cells exposed to asbestos fibers also show chromosomal damage
(32,34,36). It is hypothesized that reactive oxygen species generated in
response to asbestos fibers induce chromosomal or genetic instability that
enables subsequent immortalization and transformation by re-expression of hTERT,
SV40 large-tumor and small-tumor antigens, and activation of intracellular
signaling pathways (reviewed in Chapter 2). Most of these genetic changes that
have been defined as the minimal requirements for transformation of human cells
(38) are also found in human malignant mesotheliomas. For example, telomerase
activity has been detected in human pleural mesotheliomas (45,46). Inactivation
of the pRB growth inhibitory pathway by SV40 large-tumor antigen or deletion of
the p16 tumor suppressor gene has been described in human and murine
mesothelioma cell lines. The p53 tumor suppressor gene that controls cell cycle
checkpoints and apoptosis is inactivated by SV40 large-tumor antigen. Silencing
and deletion of the p14 or p19ARF tumor suppressor genes have been found in both
human and murine mesothelioma cell lines (reviewed in Chapter 6). Finally,
exposure of mesothelial cells to asbestos fibers in vitro activates multiple
cell sig¬naling pathways leading to sustained cell proliferation (as described
in Chapter 2).

Malignant Mesothelioma Cell Lines

Cell lines have been derived from rodent (Table 5.3) or human malig¬nant
mesotheliomas (Table 5.4). Most of the rodent cell lines were derived after
direct intrapleural or intraperitoneal injection of asbestos fibers; no cell
lines have been isolated from rodents exposed to fibers by inhalation. Despite
the unnatural route of delivery, these rodent malignant mesotheliomas produced
by direct injection of fibers closely resemble human malignant mesotheliomas
with respect to their mor¬phology, pattern of growth, and natural history
(reviewed in ref. 47). A limited number of molecular studies have been carried
out with

rodent malignant mesothelioma cell lines; in general, the murine cell
lines resemble human mesothelioma cell lines with respect to common alterations
in tumor suppressor genes, especially deletions of p16 and p19ARF (reviewed in
ref. 48). Rodent and human malignant mesothe-lioma cell lines are important for
testing of novel therapeutic strategies. There are several caveats in using
mesothelioma cell lines to inves¬tigate the pathogenesis of diffuse malignant
mesothelioma. First, most of the available rodent and human malignant cell lines
have been derived from malignant tumors. These malignant cell lines represent
the end point in the neoplastic process and provide limited informa-tion about
the molecular events involved in earlier stages of tumor development and
progression (47). Second, rodent cells are more easily immortalized than human
cells. Immortalized human cell lines and most malignant cell lines have acquired
expression of telomerase. In contrast, in most adult mouse tissues, telomerase
is expressed consti-tutively. Third, fewer genetic changes are required to
induce cancer in murine models in comparison to human cancers (reviewed in 38).
Newer genetically engineered mice may be developed to replicate more closely the
molecular alterations found in human malignant mesothe-liomas. Finally, cell
lines propagated as monolayers in vitro do not rep¬resent the complex tumor
microenvironment in vivo (49,50). Growth of mesothelioma cell lines as spheroids
or cocultured with stromal cells would more accurately model tumors in situ. We
have developed an in-vivo, ex-vivo approach to study the molecular pathogenesis
of malignant mesothelioma induced by direct intraperitoneal injection of
crocidolite asbestos fibers in mice. Mesothelial cell lines were derived from
mice at various intervals in the development of these tumors rep¬resenting
reactive, preneoplastic, or neoplastic mesothelial cells (51). We used
complementary DNA (cDNA) microarrays (52) to develop gene expression profiles of
these murine mesothelial cell lines. Upreg-ulation of genes involved in signal
transduction and cell proliferation was found in preneoplastic mesothelial cell
lines. Neoplastic mesothe-lial cell overexpressed genes involved in cell
proliferation, altered cell cycle regulation, and resistance to apoptosis. It is
hypothesized that additional genes are upregulated at later stages in the
development of malignant mesothelioma that allow these tumors to induce
angiogen-esis and invade (Fig. 5.1). The importance of these genetic alterations
in the pathogenesis of mesotheliomas can be assessed using genetically
engineered mice as described in Chapter 4.

Figure 5.1. Molecular pathogenesis of malignant mesothelioma. CDK, cyclin-dependent
kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase;
PKC, protein kinase C; VEGF, vascular endothelial growth factor.

Acknowledgment

The research conducted in the author’s laboratory was supported by National
Institutes of Health (NIH) grant R01 ES03721 from the National Institute of
Environmental Health Sciences.

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