Download Examination of the role of signal transduction and oxidative stress in

HSE
Health & Safety
Executive
Examination of the role of signal
transduction and oxidative stress in
mineral fibre-induced cell proliferation
Prepared by
MRC Toxicology Unit
for the Health and Safety Executive
CONTRACT RESEARCH REPORT
401/2002
HSE
Health & Safety
Executive
Examination of the role of signal
transduction and oxidative stress in
mineral fibre-induced cell proliferation
Stephen P Faux
MRC Toxicology Unit
Hodgkin Building
University of Leicester
Lancaster Road
Leicester LE1 9HN
United Kingdom
Cell signalling pathways mediated by oxidative stress leading to cell proliferation has been implicated as
a mechanism of carcinogenesis. This study was designed to investigate whether asbestos-mediated
oxidative stress activates signalling pathways important in cell proliferation.
We compared the responses of asbestos with man-made fibres where the pathogenicity is unclear.
Induction of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) were examined in rat lung
fibroblast and mesothelial cell lines. Crocidolite asbestos, but not MMVF-21, MMVF-10 or RCF-4,
caused a dose-dependent significant increase in AP-1 and NF-κB. RCF-1 increased NF-κB, but this was
not statistically significant. Asbestos increases in AP-1 and NF-κB were due to asbestos-mediated lipid
peroxidation as vitamin E ameliorated the effects.
To further compare the fibre responses in target cells, a range of fibres were examined on their ability to
upregulate epidermal growth factor-receptor (EGF-R) protein expression in rat pleural mesothelial
(RPM) cells. Crocidolite and erionite induced intense staining patterns of EGF-R protein, whereas milled
crocidolite and chrysotile did not. Patterns of EGF-R correlated with fibre carcinogenicity in the
mesothelium and the ability of fibres to induce RPM cell proliferation. Cell proliferation has been
observed in the pleura in vivo in the absence of fibres. The responses observed might be due to
macrophage-derived soluble mediators that initiate this proliferative response.
Using a co-culture system we have assessed the role of macrophages in signalling events leading to
mesothelial cell proliferation. Asbestos exposed macrophages caused an activation of AP-1 and NF-κB,
which was attributable to arachidonic acid metabolites, or TNFα being secreted from the macrophages.
Our study suggests that a range of in vitro assays, including the measurement of EGF-R protein in RPM
cells and measurement of AP-1 and NF-κB DNA binding activities in target cells are good predictors of
carcinogenicity. These assays could be used for human risk assessment in evaluating fibre toxicity and
potential carcinogenicity.
This report and the work it describes were funded by the Health and Safety Executive. Its contents,
including any opinions and/or conclusions expressed, are those of the author and do not necessarily
reflect HSE policy.
HSE BOOKS
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First published 2002
ISBN 0 7176 2248 7
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ii
Contents
Page
1.
INDUCTION OF ACTIVATOR PROTEIN-1 (AP-1) AND NUCLEAR
FACTOR-κB (NF-κB) DNA BINDING ACTIVITY BY ASBESTOS
1
1.1
1.2
1.3
1.4
1
14
16
24
BACKGROUND
MATERIALS AND METHODS
RESULTS
DISCUSSION
2.
DEVELOPMENT OF AN IN VITRO CO-CULTURE SYSTEM TO
ASSESS THE ROLE OF MACROPHAGES IN CELLULAR SIGNALLING
LEADING TO AP-1 and NF-κB INDUCTION BY ASBESTOS
28
2.1
2.2
2.3
2.4
28
33
34
39
BACKGROUND
MATERIALS AND METHODS
RESULTS
DISCUSSION
3.
POTENTIAL ROLE OF THE ARACHIDONIC ACID CASCADE IN
THE PERTURBATION OF AP-1 AND NF-κB DNA BINDING BY
ASBESTOS
43
3.1
3.2
3.3
3.4
43
52
52
58
BACKGROUND
MATERIALS AND METHODS
RESULTS
DISCUSSION
4.
EXAMINATION OF THE ROLE OF CELL SURFACE
RECEPTORS, IN PARTICULAR THE EPIDERMAL GROWTH FACTORRECEPTOR (EGF-R), IN THE CELL SIGNALLING RESPONSE OF RAT
PLEURAL MESOTHELIAL (RPM) CELLS TO CARCINOGENIC AND
NON-CARCINOGENIC FIBRES
62
4.1
4.2
4.3
4.4
BACKGROUND
MATERIALS AND METHODS
RESULTS
DISCUSSION
62
65
67
74
5.
REFERENCES
78
iii
iv
Rationale and aims of study
Although asbestos and other naturally-occurring mineral fibres have been shown to
cause intrathoracic cancers, the mechanisms by which they do so are poorly understood.
This is somewhat disturbing as an increasing large range of manufactured inorganic and
organic fibres are appearing in the workplace without a clear understanding of their
pathogenicity. It is therefore important to study any possible molecular mechanisms that
allow any risk prediction for established or novel fibrous materials. A number of
researchers have looked at the mechanistic aspects of fibre toxicity, but as yet, no clear
consensus has emerged. One of the key conclusions of the WHO Consultation meeting
(May 1992) on the "Validity for Methods for Assessing the Carcinogenicity of ManMade Fibres" was that "The inhalation model is appropriate for hazard identification.
For quantitative risk assessment, the inhalation model is used, but there is concern that
mathematical modelling within the risk assessment procedure could be misleading. This
is because of the absence of knowledge concerning the mechanisms of fibre- induced
fibrogenesis and carcinogenicity." Furthermore, they state that "In vitro tests are not yet
adequately developed to provide predictive power for human risk assessment or hazard
identification evaluation. The development of improved in vitro procedures for
evaluating fibre toxicity and potential carcinogenicity is encouraged, including
validation relative to laboratory and human exposure."
Oxidative stress has been widely implicated as a mechanism of carcinogenesis. The
study was designed to investigate if oxygen radicals play a role in signal transduction
pathways and cell proliferation induced by asbestos. Many synthetic fibres have been
and continue to be developed as replacements for asbestos. There are conflicting reports
v
regarding the toxicity of some of these fibres. It is necessary to assess and compare the
cytotoxic and proliferative properties of these newer fibres with asbestiform fibres and
comparisons have been made throughout the studies detailed below.
The report is divided into a number of discreet sections where a programme of work has
been undertaken.
vi
1. INDUCTION OF ACTIVATOR PROTEIN-1 (AP-1) AND
NUCLEAR FACTOR-κB (NF-κB) DNA BINDING ACTIVITY BY
ASBESTOS
1.1 BACKGROUND
Asbestos displays a variety of biological effects in different cell types. Mesothelial
cells are particularly sensitive to the cytotoxic effects of asbestos (Lechner et al, 1985).
In human (Lechner et al, 1985) and rodent (Jaurand et al, 1986) mesothelial cells
asbestos causes both chromosomal aberrations and morphological transformation.
Bronchial epithelial cells appear to be less sensitive than mesothelial cells to the
genotoxic action of asbestos (Lechner et al, 1985; Mossman et al, 1983). Asbestos has
also been shown to enhance cell transformation by other agents such as polycyclic
aromatic hydrocarbons in fibroblasts (Hei et al, 1985) and epithelial cells (Reiss et al,
1983) but not in mesothelial cells (Paterour et al, 1985). In light of the fact that there is
no correlation between smoking history and mesothelioma occurrence in humans
(Mossman et al, 1990), these observations suggest that asbestos is a complete
carcinogen in the development of mesothelioma.
Carcinogenesis is conventionally regarded as a series of events in a multistage process
and is conveniently divided into three stages, initiation, promotion and progression. The
initiation phase is the introduction of an inheritable genetic change or mutation resulting
from carcinogen- induced DNA damage or as a result of an error during DNA
replication or repair. Asbestos may initiate cells in certain cases. Both crocidolite and
1
chrysotile asbestos cause chromosomal alterations in rat and human mesothelial cells
(Lechner et al, 1985), although not in bronchial epithelial cells (Lechner et al, 1985;
Mossman et al, 1983). Cells containing mutations in key proto-oncogenes or tumour
suppressor genes may exhibit a ‘response modification’, freeing them from normal
growth controls or changing their response to external signals such as growth factors
(Cerutti, 1988; Cerutti, 1989). Promotion and progression may involve the abnormal
expression of proto-oncogenes which control cellular function and proliferation.
Chronic cell proliferation is strongly implicated in the process of clonal expansion of
initiaited cells during the promotion phase and appears to be an important factor in the
development of human cancers by many carcinogens (Preston-Martin et al, 1990). In
these cases, cells possessing genetic errors clonally expand increasing the risk of
multiple genetic defects eventually leading to a fully neoplastic cell. Oxygen radicals
and subsequent oxidant stress are known to play a role in carcinogenesis (Cerutti, 1985).
Peristant oxidative stress in tissues can result in modulation of growth and
differentiation involving the reprogramming of entire families of genes. Importantly,
oxidative stress induces the transcription of the immediate early response genes, such as
c-fos, c-jun and c-myc (Crawford et al, 1988). Asbestos can elicit an oxidative stress by
itself (Mossman and Landesman, 1983) or via the activity of resident phagocytic cells
(Donaldson et al, 1985).
Compared with cigarette smoke, asbestos is weakly carcinogenic in epithelial cells of
the respiratory tract. In experimental models using rodent tracheal grafts, asbestos acts
as a cocarcinogen (Mossman and Craighead, 1986) or tumour promotor (Topping and
Nettesheim, 1980) possibly by facilitating the uptake, metabolism and/or DNA binding
of chemical carcinogens (Eastman et al, 1983). Alternatively, asbestos is known to
2
cause chronic inflammation at the site of deposition and may act as a tumour promotor
by chronically stimulating cell proliferation. At sublethal concentrations, chrysotile and
crocidolite asbestos cause proliferation of epithelial (Marsh and Mossman, 1988) and
mesothelial (Rajan et al, 1972) cells. The precise mechanism of stimulation of cell
proliferation by asbestos is not known but evidence suggests the involvement of
secondary messenger pathways. Studies in hamster tracheal epithelial (HTE) cells have
shown that crocidolite causes increased accumulation of diacylglycerol, hydrolysis of
phosphatidylinositol and stimulation of protein kinase C (PKC) (Sesko et al, 1990;
Perderiset et al, 1991).
PKC plays a key role in cell signal transduction involving activation of oncogenes,
cellular growth and tumour promotion. The classical tumour promoter 12-Otetradecanoylphorbol-13-acetate (TPA) binds directly to PKC to activate the enzyme.
However, it is more likely that asbestos activates PKC indirectly possibly by activation
of phospholipases (Roney and Holian, 1989), such as phospholipase C, with subsequent
release of diacylglycerol and inositol 1,4,5-triphosphate (IP3 ) from the plasma
membrane (Sesko et al, 1990), which activates PKC (Kishimoto et al, 1980) and release
calcium respectively (Streb et al, 1983). Both diacylglycerol and calcium are required
by PKC for maximal activity (N ishizuka, 1984; Nishizuka, 1986). PKC stimulation
increases the levels of ornithine decarboxylase (ODC), a rate limiting enzyme in the
biosynthesis of polyamines necessary for the initiation of cell division. Crocidolite and
chysotile induce ODC activity in hamster trachael epithelial (HTE) cells and this
activity is significantly reducd by calcium entry antagonists and PKC inhibitors (Marsh
and Mossman, 1988; Marsh and Mossman, 1991). These results suggest that asbestos
provides an extracellular signal which leads to a cascade of cellular events resulting in
3
alteration of normal cellular function. In addition, it has been reported that PKC
phosphorylates a NADPH oxidase on the plasma membrane that catalyses the reduction
of water to the superoxide anion. (Cox et al, 1985). In this way the asbestos- induced
activation of PKC may also exacerbate the oxidative stress elicited by asbestos fibres.
One consequence of the activation of PKC is the induction of accessory transcription
factors such as activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). These
transcription factors are centrally involved in cell proliferation and their activation by
asbestos may provide a persistent growth signal to the target cells and may constitute a
promotional activity of asbestos.
AP-1 refers to a family of accessory transcription factors that interact with the
consensus sequence 5’-TGA G/C TCA-3’ (Angel et al, 1987). AP-1 has been identified
as the transcriptional factor that binds the TPA (12-O-tetradecanoylphorbol-13-acetate)
response element of several cellular genes whose transcription is induced in response to
treatment of cultured cells with this phorbol ester tumour promoter (Angel et al 1987).
TPA is a potent activator of protein kinase C (PKC; Nishizuka, 1984) and other agents
which lead to PKC activation such as serum and growth factors also induce the
expression these genes. Inhibitors of PKC block the induction of these genes (Imbra and
Karin, 1987; Brenner et al, 1989). In addition, insertion of synthetic
oligodeoxynucleotides that form efficient AP-1 binding sites in front of a thymidine
kinase promoter renders it TPA inducible, whereas sequences not conforming to this
consensus do not confer inducibility (Angel et al, 1987). Hence, AP-1 binding is
essential for the transcriptional activation of AP-1-dependent genes following PKC
4
activation. In addition, AP-1 activation is controlled by a complex hierarchy of other
proto-oncogenes (Figure 1). As evident from the multiple levels at which AP-1 is
regulated various proto-oncogenes may employ different mechanisms to modulate the
activation of AP-1. The AP-1 complex appears to play a general role in signal
transduction from the membrane to the nucleus. It is likely that specific cellular
decisions in response to external signals is made by other transcription factors higher up
the signalling pathway.
Growth factor
fms
neu
src
abl
Receptor
fes
plasma-membrane
associated
Ras
raf mos
cytoplasm
Fos
Jun
nucleus
AP-1
Transformation
Figure 1. Hierarchical order of proto-oncogenes affecting AP-1 activity. The cellular
locations of the corresponding gene products are shown on the right (modified from
Angel and Karin, 1991).
AP-1 exists as a protein dimer composed of two distinct proto-oncogenes. AP-1 can be
a homodimeric complex of two members of the Jun family or a heterodimeric complex
5
consisting of Jun and Fos proteins. The formation of homo- or hetero-dimeric forms has
important functional consequences. Fos alone does not bind to any known sequence of
DNA and cannot form dimers with itself (Angel and Karin, 1991). Therefore Fos alone
has no transcriptional activating ability. Fos does however form heterodimers with other
proteins such as Jun (Angel and Karin, 1991), JunB (Nakabeppu et al, 1988), JunD
(Hirai and Yaniv, 1989) and FosB (Zerial et al, 1989). Fos forms stable heterodimers
with any of the Jun proteins via hydrophobic interactions between their ‘leucine-zipper’
regions (Landschultz et al, 1990). The resulting dimer has higher DNA binding activity
than Jun homodimers. In this scheme, cFos participates directly in regulating gene
expression by using the Jun proteins as an anchor to bind to TREs (Angel and Karin,
1991). All of these complexes seem to have very similar recognition properties and
interact with the consensus TRE and slight deviations from it (Nakabeppu et al, 1988;
Nakabeppu and Nathans, 1989), although their DNA binding activities are markedly
different in vitro (Ryseck and Bravo, 1991).
The cellular regulation of Fos and Jun proteins is complex. Transcription of both c-fos
and c-jun is induced by TPA and other activators of PKC (Table 1) resulting in
increased production of Fos and Jun proteins. This induction occurs rapidly and does
not require de novo protein synthesis (Kruijer et al, 1984; Angel et al, 1988). The c-jun
promoter contains an almost identical sequence to the consensus TRE (Hattori et al,
1988) which is recognised by AP-1 (Angel et al, 1988). The presence of these sites
within the c-jun promoter suggests that the transcription of c-jun is the subject of a
positive autoregulatory loop whereby c-jun transcription is stimulated by it’s own gene
product. The requirement for the presence of preformed cJun homodimers for activation
of c-jun transcription may seem paradoxical but can be explained by assuming that a
6
signal generated by activation of PKC stimulates the activity of pre-existing Jun
homodimers and thereby increases their ability to activate c-jun transcription. The most
common post-translational modification known to modulate protein activity is
phosphorylation (Cohen, 1982). Indeed, examination of cJun from a number of sources
have indicated that it is a phosphoprotein with phosphorylation sites on between five
and seven serine and threonine residues (Boyle et al, 1991). Stimulation of HeLa cells
or human fibroblasts with TPA leads to a rapid decrease in phosphorylation of several
residues on cJun (Boyle et al, 1991). The observed net dephosphorylation is a specific
response to PKC (Boyle et al, 1991; Seamon and Daly, 1986). Some of the
phosphorylation sites are in close proximity to the part of the protein responsible for
binding DNA suggesting that phosphoryla tion of cJun alters it’s DNA-binding
properties. The phosphorylation sites are also highly conserved suggesting that they
play an important regulatory role (Boyle et al, 1991; Vogt & Bos, 1990). Interestingly,
purified cJun is not a substrate for PKC indicating that PKC activation by TPA or other
agents does not directly lead to modification of cJun (Hai et al, 1988), and therefore
must involve another protein kinase. It is not currently known whether
dephosphorylation of Jun is effected by inhibition of the protein kinase which maintains
the sites in their phosphorylated state or to activation of a specific protein phosphatase.
Incidentally, in response to PKC activation there is a rapid increase in the level of Fos
phosphorylation (Barber and Verma, 1987), although the functional significance of this
modification has not been studied in detail. The positive feedback regulation of Jun is
likely to be responsible for signal amplification and conversion of transient signals by
external stimuli or PKC activation into long term transcriptional responses (Angel and
Karin, 1991).
7
Transcription of c-fos is induced by various stimuli (Table 1) and occurs within minutes
without the need for new protein synthesis (Angel and Karin, 1991). Multiple protein
binding sites have been identified within the c-fos promoter (Verma and Sassone-Corsi,
1987). For induction of c-fos by serum, TPA and certain other growth factors including
PDGF and EGF, binding of a 20 base pair transcriptional enhancer element known as
the dyad symmetry element (DSE) is required. This palindromic sequence is sufficient
to confer serum responsiveness upon heterologous promoters (Büscher et al, 1988;
Treisman, 1985). The transcription factor that binds to the DSE is the serum response
factor (SRF). SRF binds the DSE as a dimer (Norman et al, 1988) and requires an
additional protein, p62TCF, for optimal induction of c-fos by serum (Shaw et al, 1989).
The precise mechanism of activation of transcription by the SRF-p62TCF complex is
not well understood. The ability of cJun to positively regulate c-jun transcription is in
contrast to the negative effect of cFos and cJun on c-fos transcription (Sassone-Corsi et
al, 1988; Schönthal et al, 1989). The transcription of c-fos is down-regulated by it’s
own gene product. Over-expression of cFos leads to a rapid decrease in both basal and
serum induced levels of c-fos transcription (Sassone-Corsi et al, 1988; Schönthal et al,
1989). The mechanism of this repression is only poorly understood. The c-fos mRNA
and protein product have shorter half- lives than c-jun mRNA and protein product
(Angel et al, 1988; Schönthal et al, 1988). Therefore, the composition of the AP-1
complex changes from predominantly Jun based homodimers before induction, to
mostly Fos-Jun heterodimers immediately following induction by a suitable stimuli. On
removal of the stimulus, Fos proteins decay, and existing AP-1 complexes revert back
to predominantly Jun homo-dimers. Thus, activators of PKC and possibly other protein
kinases stimulate the formation of DNA binding Fos-Jun dimers which activates the
transcription of genes containing TRE promoters. On removal of the stimulus, these
8
AP-1 heterodimers decay to be replaced by less active Jun homodimers with a gradual
decrease in TRE occupancy. In this way, the TRE occupancy by Jun dimers is
responsible for the maintenance of background levels of AP-1 mediated gene expression
and Fos-Jun dimers for mediating gene transcription in response to external stimuli and
to maintain an increased rate of transcription of AP-1 dependent genes for a sufficient
length of time to allow progression through the G1 phase of the cell cycle, beyond the
restriction point and entry into S-phase (Angel and Karin, 1991).
Stimulus
c-fos
c-jun
Serum
+++
++
Platelet-Derived Growth
+++
++
TNF-α
++
++
Interleukin-1
++
+
TPA
++
++
Factor
+= induced.
Table 1. Transcriptional regulation of c-jun and c-fos by external stimuli (from Angel
and Karin, 1991).
NF-κB is a ubiquitously expressed primary transcription factor that has an active role in
cytoplasmic/nuclear signalling and pathogenic stimulation of cells. NF-κB activity is
highly regulated and is linked to the activation of a number of genes containing the κB
cis-regulatory elements in their promoter region. Genes induced by NF-κB include
9
genes encoding various interleukins, cytokines, nitric oxide synthase and c-myc (Duyao
et al, 1990; Matsushima et al, 1985; Sung, S-S et al, 1988).
The active forms of human NF-κB are dimers, most frequently composed of the two
DNA binding subunits p50 and RelA (formerly p65). This heterodimer binds with high
affinity to decameric DNA sequences of the consensus 5’-GGGPuNNPyPyCC-3’
(Grimm and Baeuerle, 1993). p50 is considered a ‘helper’ subunit allowing the
transactivating RelA subunit to bind as a heterodimer with high affinity to κB sites. At
present, it is unclear to what extent homodimers of RelA or p50 control gene expression
under physiological conditions. Although p50 homodimers are frequently observed as a
constitutive activity in nuclear extracts, RelA homodimers have not yet been
unambiguously identified in nuclear extracts and the co-existence of p50 and RelA
homodimers in cells is unlikely since the two subunits have a much higher tendency to
heterodimerise than to homodimerise (Baeuerle and Henkel, 1994).
The activation of NF-κB is a complex process involving proteolysis, phosphorylation
and nuclear translocation of the protein complex. In the cytoplasm the activity of NF-κB
is controlled primarily by specific inhibitory IκB subunits which bind to the RelA
subunit (Schmitz et al, 1991; Lewin, 1991). Activation of PKC and other protein
kinases leads to an active form of NF-κB, apparently due to phosphorylation and
subsequent release of IκB proteins (Figure 2; Link et al, 1992; Ghosh and Baltimore,
1990). IκB proteins serve two main functions, preventing the binding of NF-κB to DNA
(Baeuerle and Baltimore, 1988) and preventing the nuclear uptake of NF-κB proteins.
Once NF-κB is liberated from these inhibitory units it can move freely into the nucleus
10
and bind to specific DNA regulatory sequences. Virutally coincident with the
appearance of active NF-κB is the depletion of I? B (Brown et al, 1993) probably
following degradation by proteases (Baeuerle and Henkel, 1994). Degradation of the
inhibitor in this way means that NF-κB can only be inactivated through it’s decay or by
newly synthesised IκB (Brown et al, 1993). In addition to IκB proteins, p105, the
precursor protein of p50 can also bind to p50 and RelA, inhibiting their DNA binding
activity and nuclear translocation (Rice et al, 1993).
IkB phosphorylation
NF-kB •IkB
IkB
Protease
IkB
Degradation
mRNA
GGGGAATTCC
Nucleus
Figure 2. Post-translational control of NF-κB activity.
NF-κB is activated by a large range of diverse stimuli. A common mechanism through
which these diverse agents influence NF-κB activity may be the cellular redox status. A
11
large number of anti-oxidant agents, including N-acetylcysteine and vitamin E, have
been reported to potently suppress the activation of NF-κB (Schreck et al, 1992). The
diversity of these inhibitors suggests that it is the ir common anti-oxidant activity that is
responsible for suppressing activation of NF-κB rather than a particular structure-related
activity (Baeuerle and Henkel, 1994).
NF-κB is involved in the control of cell proliferation, particularly in cells of the immune
system. It responds to many T and B cell activating signals leading to differentiation
and proliferation. In quiescent fibroblasts, NF-κB is transiently activated in the G0-G1
transition upon stimulation with serum (Baldwin et al, 1991). The κB site found in the
c-myc upstream promoter (Duyao et al., 1990) confers serum inducibility on reporter
genes suggesting a potential involvement of NF-κB in the immediate-early growth
response. NF-κB is also extensively exploited in cells of the immune system and acts as
a transcriptional activator for many genes encoding immunologically relevant proteins.
For instance, stimulation of macrophages leads to the rapid expression of many
immunomodulatory proteins including macrophage colony stimulating factor, tumour
necrosis factor, interleukins, chemotactic factors, leukotriene B4 and nitric oxide
synthase (Müller et al, 1993). With this large number of genes under it’s control, NF-κB
plays a central role in many cellular responses including proliferation and inflammation.
1.1.1 Activation of AP-1 and NF-κB by asbestos
One consequence of the activation of PKC by asbestos is the induction of accessory
transcription factors such as AP-1 and NF-κB. Recent studies have shown that both
12
crocidolite and chrysotile cause a dose-dependent and persistent increase in the
expression of c-fos and c-jun in rat pleural mesothelial cells and c-jun in HTE cells
(Heintz et al, 1993; Janssen et al, 1994; Janssen et al, 1995a). This is in contrast to the
classical tumour promotor TPA which gives a transient increase in these gene products,
with maximum induction at 1h (Heintz et al, 1993). Both c-fos and c-jun are immediate
early response genes associated with the transition of the cells from the G1 stage of the
cell cycle to S phase (Angel and Karin, 1991) and their induction may provide the
molecular switch for cell proliferation. In addition, recent studies have shown that
crocidolite asbestos induces an increase in NF-κB DNA binding and NF-κB-dependent
genes such as c-myc (Janssen et al, 1995b).
Transcription factors couple events at the cellular surface to changes in gene expression
that modulate cell type specific responses including cell proliferation, changes in
phenotype and apoptosis (Angel and Karin, 1991; Demoly et al, 1992; Ransone and
Verma, 1990). Both AP-1 and NF-κB are primary transcription factors that can alter the
expression of many genes in response to growth factors, cytokines, and tumour
promoters. Aberrant or persistent activation of these factors may lead to loss of growth
control and neoplastic transformation (Nishizuka, 1984; Herrlich and Ponta, 1989;
Rozengurt, 1986; Bishop, 1987) and their chronic activation may be important in
asbestos pathogenicity.
This part of the study investigated the ability of asbestos to increase the DNA binding
activity of AP-1 and NF-κB transcription factors in RFL-6 and 4/4 RM-4 cells. To
investigate the possible role of the cellular redox status in the perturbation of AP-1 and
NF-κB DNA binding activity, the intracellular GSH level was modulated using BSO
13
and NAC. In addition, the influence of the lipid peroxidation inhibitor α-tocopherol
(vitamin E) was investigated.
1.2 MATERIALS AND METHODS
1.2.1 Mineral fibres and cell culture
Reference samples of UICC crocidolite asbestos were a kind gift from Dr R.C. Brown
(MRC Toxicology Unit, Leicester, UK). The man- made vitreous fibres (MMVF)
MMVF-21, MMVF-10, MMVF-22 and refractory ceramic fibres (RCF) RCF-1 and
RCF-4 were obtained from the Thermal Insulation Manufacturers Association Fibre
Repository (Littleton, CO, USA). A rat lung fibroblast cell line (RFL-6; obtained from
the European Collection of Animal Cell Cultures, Porton Down, Wiltshire, UK) were
cultured in Hams F-12 medium containing L- glutamine (Gibco) supplemented with
penicillin (50 units/ml), streptomycin (100mg/ml), non-essential amino acids (1%;
Gibco) and fetal bovine serum (10%; Gibco). Cells were grown to confluence in culture
dishes (100mm2 ; Corning), and 24 hours prior to treatment the growth medium was
replaced with medium containing 2% growth serum. BSO (1mM) and/or NAC (5mM)
were added to the media 24 hours prior to treatment if appropriate. α-tocopherol (2mM)
was added concommitantly with asbestos fibres if appropriate. A rat mesothelial cell
line (4/4 RM-4; obtained from the European Collection of Animal Cell Cultures, Porton
Down, Wiltshire, UK) were cultured in Hams F-12 medium containing L-glutamine
(Gibco) supplemented with penicillin (50 units/ml), streptomycin (100mg/ml and fetal
bovine serum (15%; Gibco). Cells were grown to confluence in culture dishes (100mm2 ;
14
Corning), and 24 hours prior to treatment the growth medium was replaced with
medium containing 2% growth serum. BSO (1mM) and/or NAC (5mM) was added to
the media 24 hours prior to treatment with fibres if appropriate. α-tocopherol (2mM)
was added concommitantly with asbestos fibres if appropriate.
1.2.2 Preparation of nuclear protein extracts
At the end of the incubation period with fibres, cells were harvested and nuclear
proteins were prepared as described previously (Staal et al, 1990). Briefly, cells were
scraped into phosphate buffered saline (PBS) and pelleted by centrifugation. The pellet
was resuspended in buffer A (10mM HEPES, 10mM KCl, 2mM MgCl2 , 100µM EDTA,
200µM NaF, 200µM Na3 VO4 , 1mM DTT, 400µM PMSF, 0.3mg/ml leupeptin; 400µl)
and incubated on ice for 15min. Buffer B (10% triton X 100 in UHQ water; 25µl) was
then added and the mixture vortexed for 15sec. The solution was centrifuged and the
pellet resuspended in buffer C (50mM HEPES, 50mM KCl, 0.3M NaCl, 100µM EDTA,
0.1ml/ml glycerol, 200µM NaF, 200µM Na3 VO4 , 1mM DTT, 644µM PMSF; 50µl) and
incubated at 4°C for 20min. The solution was then centrifuged and the supernatant
stored at -20°C. An aliquot (5µl) was analysed for protein content using the Biorad
protein assay reagent.
1.2.3 Gel mobility shift assay for AP-1 and NF-κB DNA binding activities
Protein extracts (4µg) were incubated in DNA binding buffer (40mM Hepes buffer, 4%
Ficoll, 200ng poly (dI-dC) per µl, 1mM MgCl2 , 0.1mM dithiothreitol and 0.175 pmol
15
32
P-end labelled double stranded AP-1 or NF-κB oligonucleotide as appropriate) for
40min at room temperature. The protein- DNA complexes were resolved by
electrophoresis in non-denaturing 4% polyacrlyamide gels. Gels were blotted onto
paper, dried at 80°C and visualised by autoradiography. The retarded bands were
quantitated using a β-imaging system (Molecular Dynamics, USA).
1.3 RESULTS
1.3.1 Time - and dose-dependent increases in AP-1 and NF-κB DNA binding
activity following treatment with crocidolite
The present study found a dose-dependent increase in both AP-1 and NF-κB DNA
binding in RFL-6 and 4/4 RM-4 cells following 24h incubation with sub-cytotoxic
doses of crocidolite (Janssen et al, 1994; S.Faux, unpublished observations). In RFL-6
cells, crocidolite significantly enhanced AP-1 DNA binding at all concentrations above
2.5 µg/cm2 (Figure 3; P<0.05) with maximum induction at 10 µg/cm2 (P<0.02).
Similarly, NF-κB DNA binding activity was also significantly enhanced at the lowest
asbestos concentration (2.5 µg/cm2 ; P<0.05) with maximal induction at 10 µg/cm2
(Figure 3; P<0.001).
16
DNA binding (arbitrary units)
15
10
AP-1
NF-kB
5
0
0
2.5
5
10
Crocidolite (ug/cm2)
Figure 3. Dose-dependent induction of the transcription factors AP-1 and NF-κB in
RFL-6 cells following treatment with crocidolite (2.5 - 10 µg/cm2 ). The results are the
mean ± SD of at least four independent determinations.
In 4/4 RM-4 cells, AP-1 DNA binding was only significantly increased over basal
levels at the highest fibre concentration tested (5 µg/cm2 ; Figure 4; P<0.05). NF-κB
DNA binding was increased slightly above basal levels at 2.5 µg/cm2 (P<0.05) and to a
greater extent at 5 µg/cm2 (Figure 4; P<0.001). The induction of the transcription
factors AP-1 and NF-κB was found to be time-dependent in both RFL-6 and 4/4 RM-4
cells. In RFL-6 cells, 10 µg/cm2 of crocidolite gave maximal induction of AP-1 at 8 and
24 hours and NF-κB at 24 hours (Figure 5; P<0.05).
17
DNA binding (arbitrary units)
15
10
AP-1
NF-kB
5
0
0
2.5
5
Crocidolite (ug/cm2)
Figure 4. Dose-dependent induction of the transcription factors AP-1 and NF-κB in 4/4
RM-4 cells following treatment with crocidolite (5 µg/cm2 ). The results are the mean ±
SD of at least four independent determinations.
DNA binding (arbitrary units)
20
15
AP-1
10
NF-kB
5
0
4
8
24
Time (hrs)
Figure 5. Time-dependent induction of the transcription factors AP-1 and NF-κB in
RFL-6 cells. The results are the mean ± SD of at least four independent determinations.
18
In 4/4 RM-4 cells, 5 µg/cm2 of crocidolite induced AP-1 and NF-κB to maximal levels
after 8 hours (Figure 6; P<0.05). The levels of AP-1 and NF-κB DNA binding were not
found to be significantly different between the 8 and 24h incubations.
DNA binding (arbitrary units)
15
10
AP-1
NF-kB
5
0
4
8
24
Time (hrs)
Figure 6. Time-dependent induction of the transcription factos AP-1 and NF-κB in 4/4
RM-4 cells. The results are the mean ± SD of at least four independent determinations.
Preliminary experiments described above indicated an optimal treatment protocol for
the cells used in this study. Subsequent experiments were carried out for either 8 or 24
hours using the maximum non-cytotoxic dose of 10 µg/cm2 crocidolite in RFL-6 cells
and 5 µg/cm2 crocidolite in 4/4 RM-4 cells.
1.3.2 Effect of other mineral fibres on AP-1 DNA binding in 4/4 RM-4 cells
To examine the effects of other mineral fibres on AP-1 DNA binding in mesothelial
cells (4/4 RM-4 cells) we chose the TIMA bank of fibres, some of which had
19
demonstrated pathogenicity in animal experiments. The fibrous preparations that we
used were RCF-1, RCF-4, MMVF-10, MMVF-21 and MMVF-22. 4/4 RM-4 cells were
treated at equal mass and time- frame as for crocidolite asbestos. None of the fibres
induced a statistically significant increase in AP-1 DNA binding activitiy in EMSAs
above control levels of AP-1 DNA binding in any of our experiments. No further work
was undertaken with these fibres.
1.3.3 Influence of anti-oxidants on induction of AP-1 and NF-κB DNA binding by
crocidolite
To examine the potential protective role of intracellular glutathione, RFL-6 and 4/4
RM-4 cells were incubated with crocidolite following pre-treatment of cells with BSO
(1mM) and/or NAC (5mM). BSO, an inhibitor of γ-glutamyl cysteine synthetase
(Griffith and Meister, 1979) was used to deplete the cells of glutathione prior to
treatment and NAC used to replenish intracellular thiol levels.
Treatment of RFL-6 cells with crocidolite (10 µg/cm2 ) for 24h resulted in a significant
enhancement in AP-1 and NF-κB DNA binding activity (Figure 7; P<0.05). Treatment
of RFL-6 cells with crocidolite (10 µg/cm2 ) pre-treated with BSO (1mM) resulted in
significantly greater DNA binding for both transcription factors (Figure 7; P<0.05).
Treatment of RFL-6 cells with crocidolite (10 µg/cm2 ) pre-treated with BSO (1mM) and
NAC (5mM) resulted in a complete amelioration of this effect, reducing the activity of
AP-1 and NF-κB to basal levels. (Figure 7). Pre-treatment with BSO (1mM) alone was
not found to significantly alter AP-1 or NF-κB activity from basal activity. However co-
20
culture with NAC (5mM) was found to depress levels of AP-1 and NF-κB DNA
DNA binding (arbitrary units)
binding activity to below basal levels (Figure 7; P<0.05).
15
AP-1
10
NF-kB
5
0
None
BSO (1mM)
NAC(5mM)
10ug/cm2
Croc (C)
10ug/cm2
C+BSO
(1mM)
10ug/cm2
C+BSO
(1mM)+NAC
(5mM)
Treatment
Figure 7. Induction of AP-1 and NF-κB transcription factor DNA binding activities in
RFL-6 cells following 24 hour incubation with crocidolite (10 µg/cm2 ). RFL-6 cells
were pre-treated overnight with BSO (1mM) or NAC (5mM) prior to exposure to fibres.
The results are the mean ± SD of at least four independent determinations.
Treatment of 4/4 RM-4 cells with crocidolite (5 µg/cm2 ) for 24 hours resulted in a
significant enhancement in AP-1 and NF-κB DNA binding activity (Figure 8; P<0.05).
Treatment of 4/4 RM-4 cells pre-treated with BSO (1mM) before exposure to
crocidolite (5 µg/cm2 ) resulted in a significantly greater induction of both transcription
factors (P<0.05). Treatment of 4/4 RM-4 cells pre-treated with both BSO (1mM) and
NAC (5mM) before exposure to crocidolite (5 µg/cm2 ) resulted in a complete
amelioration of this effect, reducing the activity of AP-1 and NF-κB to basal levels.
21
(Figure 8; P<0.05). Treatment of cells with BSO (1mM) or NAC (5mM) alone was not
DNA binding (arbitrary units)
found to significantly alter AP-1 or NF-κB activity from basal levels.
25
20
AP-1
15
NF-kB
10
5
0
None
BSO (1mM)
NAC(5mM)
10ug/cm2
Croc (C)
10ug/cm2
C+BSO
(1mM)
10ug/cm2
C+BSO
(1mM)+NAC
(5mM)
Treatment
Figure 8. Induction of AP-1 and NF-κB transcription factors in 4/4 RM-4 cells
following 24 hour incubation with crocidolite (5µg/cm2 ) following pre-treatment
overnight with BSO (1mM) and/or NAC (5mM). The results are the mean ± SD of at
least four independent determinations.
To investigate the possible role of lipid peroxidation in the activation of AP-1 and NFκB DNA binding activity, cells were treated with crocidolite in the presence of vitamin
E, an inhibitor of lipid peroxidation. In RFL-6 cells, vitamin E (0.5 and 2mM) was
found to be highly effective at preventing activation of AP-1 and NF-κB by crocidolite
(Figure 9). Vitamin E at 0.5mM was found to reduce both AP-1 and NF-κB activity
compared with crocidolite treatment in the absence of vitamin E, although this was not
statistically significant. However at 2mM, vitamin E was found to significantly reduce
both AP-1 and NF-κB activity (Figure 9; P<0.05). This level of vitamin E was not
22
completely protective however since the resulting AP-1 and NF-κB activity was still
DNA binding (arbitrary units)
significantly above basal levels (P<0.05).
20
15
AP-1
10
NF-kB
5
0
None
Vitamin E
(2mM)
10ug/cm2
Croc
10ug/cm2
Croc+vit E
(0.5mM)
10ug/cm2
Croc+vit E
(2mM)
Treatment
Figure 9. Induction of AP-1 and NF-κB transcription factors in RFL-6 cells following
24 hour incubation with crocidolite (5 µg/cm2 ) in the presence of vitamin E (0.5 and
2mM). The results are the mean ± SD of three independent determinations.
Similarly in 4/4 RM-4 cells, vitamin E was found to be highly effective at preventing
activation of AP-1 and NF-κB by crocidolite (Figure 10). Vitamin E at 0.5mM and
2mM was found to reduce both AP-1 and NF-κB activity compared with crocidolite
treatment in the absence of vitamin E (P<0.02).
23
DNA binding (arbitrary units)
20
15
AP-1
NF-kB
10
5
0
None
Vitamin E
(2mM)
10ug/cm2
Croc
10ug/cm2
Croc+vit E
(0.5mM)
10ug/cm2
Croc+vit E
(2mM)
Treatment
Figure 10. Induction of AP-1 and NF-κB transcription factors in 4/4 RM-4 cells
following 24 hour incubation with crocidolite (5 µg/cm2 ) in the presence of vitamin E
(0.5 and 2mM). The results are the mean ± SD of three independent determinations.
1.4 DISCUSSION
This part of the study showed that exposure of RFL-6 and 4/4 RM-4 cells to asbestos
results in enhanced levels of the transcription factors AP-1 and NF-κB. Previous studies
have shown a persistent increased expression of the c-jun and c-fos mRNA following
asbestos exposure. In these studies it was shown that in rat pleural mesothelial cells both
c-fos and c-jun products were enhanced by crocidolite and chrysotile, however in
hamster tracheal epithelial cells only c-jun was expressed (Heintz et al, 1993). These
studies indicate cell specific differences in the regulation of c-fos gene expression in
relation to asbestos exposure. In the RFL-6 and 4/4 RM-4 cell types used in this study
however, the end-response of DNA binding activity of AP-1 and NF-κB appears to be
comparable in time course and extent. In contrast to asbestos fibres, none of the TIMA
bank of fibres used in these studies produced significant increases in either AP-1 or NF-
24
κB suggesting from our studies that these fibres may not induce cell proliferation in
target cell of disease and therefore may have little or no pathogenicity.
Glutathione levels were shown in the present study to control the activity of the
transcription factors AP-1 and NF-κB as induced by asbestos. Previous work has
indicated that the cellular thiol status is influential in controlling the expression of c-fos
and c-jun in response to asbestos (Janssen et al, 1995a) and other external stimuli such
as γ-irradiation (Manome et al, 1993) or chemical oxidants (Bergelson et al, 1994). In
this scheme glutathione does not necessarily control the expression of these genes
through its antioxidant activity but rather by maintaining intracellular thiol levels
(Janssen et al, 1995a; Meister, 1994). The redox status of the cell is also likely to affect
the activity of the AP-1 gene product since the DNA binding activity of this complex is
under redox control (Angel and Karin, 1991).
The results presented here support an increasing body of evidence to suggest a
protective role for glutathione against the effects of asbestos and other oxidant stress
(Janssen et al, 1995a; Kinnula et al, 1992). Although little is known about glutathione
pools in the cells of the lung, it is known that rat alveolar macrophages exposed in vitro
to asbestos release glutathione into the extracellular medium (Boehme et al, 1992).
Such elevations in extracellular glutathione concentration correspond with
concentration-dependent decreases in intracellular glutathione and are not seen after
exposure to non-toxic particles. These observations suggest that depletion of glutathione
may occur in the target cells of asbestos-induced disease. Pre-treatment of cells with
BSO alone did not significantly alter AP-1 or NF-κB binding. This indicates that
glutathione depletion per se is not causally related to transcription factor activity.
25
However glutathione depletion did make the cells more sensitive to asbestos suggesting
that intracellular thiol pools may act as a buffer against asbestos- mediated induction of
the early response genes.
The ability of vitamin E to ameliorate asbestos- induced AP-1 and NF-κB in this study
and other data suggests that initiation of these signalling events occurs at the plasma
membrane (Devary et al, 1992). Vitamin E could be active via several mechanisms.
Vitamin E is a well characterised inhibitor of lipid peroxidation (Packer and Landvick,
1989; Packer, 1991) with additional roles that include membrane stabilisation,
regulation of membrane fluidity (Packer and Landvick, 1989) and vitamin E has been
shown to prevent asbestos-induced lipid peroxidation (Howden and Faux, 1996).
Vitamin E may inhibit the activation of AP-1 and NF-κB DNA binding by preventing
the breakdown of the plasma membranes by peroxidation or via it’s activity as an
inhibitor of 5- lipoxygenase activity (Redanna et al, 1989) thus preventing the
breakdown of arachidonic acid into active metabolites.
The results from this part of the study propose a non- genotoxic mechanism of action of
asbestos fibres in the cell types studied (Figure 11). Through persistent activation of the
early response gene pathway, asbestos may produce chronic cell proliferation and
inflammation at the site of deposition with subsequent initiation and promotion of
altered cells in the lung and pleura (Preston-Martin et al, 1990). Persistent induction of
NF-κB may modulate the immune response to asbestos through the increased
production of various cytokines, interleukins and cytokines resulting in inflammation
and increased immune cell activity. This hypothesis is consistent with the observation
that durable fibres are more pathogenic than fibres which dissolve or are removed from
26
the lung (Mossman et al, 1990; Doll, 1989). Similarly riebeckite, the non- fibrous
equivalent of crocidolite and other non- fibrous materials do not induce c-fos or c-jun
(Heintz et al, 1993) indicating that the induction of cell proliferation may be an inherent
property of the fibre and thus not requiring the activity of ancillary cells such as those of
the immune system. The persistence of AP-1 and NF-κB binding is significant because
induction of gene transcription by these transcription factors requires their sustained
expression (Trejo et al, 1992). Thus, biopersistent fibres may provide a constant growth
signal during the long latency periods of tumour formation and therefore contribute to
the introduction of genetic changes by asbestos or other agents leading to neoplastic
alteration. In addition, the persistent activation of these factors in lung fibroblasts may
account for the incidence of fibrosis in asbestos exposed lung as a result of over-active
fibroblast activity.
S-Phase
Proliferation
Protein
kinases
Asbestos
Fibres
(eg PKC)
AP-1
(cytoplasm)
Signal
transduction
NF-kB
(nucleus)
Chronic induction of
early response genes
Cell
Proliferation
Cancer
Figure 11. Possible mechanism of asbestos- induced chronic cell proliferation and
carcinogenesis.
27
2. DEVELOPMENT OF AN IN VITRO CO-CULTURE SYSTEM TO
ASSESS THE ROLE OF MACROPHAGES IN CELLULAR
SIGNALLING LEADING TO AP-1 and NF-κB INDUCTION BY
ASBESTOS
2.1 BACKGROUND
The primary route of exposure to asbestos is via inhalation. Fibres in the respirable
range are drawn into the lung where they deposit throughout the bronchial tree,
particularly at duct bifurcations and terminal bronchioles (Brody et al., 1981; Coin et
al., 1992; Warheit et al., 1984). Once in the lung, fibres exert direct effects on a number
of pulmonary cell types, and these interactions may contribute to the pathogenic process
(Rom et al., 1987). Persistent exposure of tissues to asbestos fibres results in chronic
inflammation, which precedes the excessive deposition of collagen by fibroblasts and
formation of scar tissue. This remodelling within the lung leads to a loss of lung
elasticity and impaired gaseous exchange and contributes to a progressive fibrotic
condition known as asbestosis. Although not a malignant disease, asbestosis is a
condition reflecting abnormal proliferation and activity of fibroblasts in the lung. The
association of the excess of lung cancers in smoking asbestos workers with either
clinically or pathologically diagnosed asbestosis has lead some to suggest that fibrosis is
causally linked to the development of tumours (Mossman et al., 1990). It has also been
suggested that pleural fibrosis may play a role in the initiation or promotion of pleural
mesothelioma (Mossman et al., 1990).
28
The inflammatory response in relation to asbestos deposition is characterised by an
influx of immune cells into the vicinity of the fibre, allowing defensive agents to be
concentrated at this site. In particular, there is an accumulation of alveolar macrophages
and polymorphonuclear neutrophils (Sibille and Reynolds, 1990). In experimental
animal models of asbestos exposure (Mossman et al., 1990; Petruska et al., 1990) and in
humans diagnosed with asbestosis (Garcia et al., 1989; Rom et al., 1987), alveolar
macrophages (AM) have been shown to accumulate at the sites of fibre deposition. AMs
are the resident phagocytes of the distal airspaces and are the primary facilitator of
particle removal from the distal areas of the lung which do not possess a mucociliary
escalator. AMs produce reactive oxygen species (ROS) such as the superoxide anion,
nitric oxide and peroxynitrite (Cross et al., 1994) as part of their normal phagocytic
activity (Sibille and Reynolds, 1990). Crocidolite and chrysotile have both been shown
to induce the production of superoxide radicals in rat (Hansen and Mossman, 1987),
guinea pig (Roney and Holian, 1989) and human AMs, and this activity is significantly
enhanced when fibres are coated in components of lung lining fluid such as opsonin and
IgG (Hill et al., 1995; Hill et al., 1996). It is known that ROS derived from AMs plays
a critical role in host defence but accumulating evidence indicates that inappropriate
release of ROS can contribute to many types of lung disease (Freeman and Crapo, 1982;
Hansen and Mossman, 1987; Mossman and Marsh, 1989). AMs are also capable of
modulating and amplifying the inflammatory process through their extensive range of
secretory products (Nathan, 1987; Perez-Arellano et al., 1990). The effects of AM
secretory products are varied and include the regulation of polymorphonuclear
neutrophil (PMN) migration (Sibille and Reynolds, 1990), degranulation and production
of superoxide anions (Donaldson et al., 1993). One particularly important group of
macrophage secretory products are the cytokines, a group of polypeptides involved in
29
inter-cell signalling during inflammation and immune responses. The interleukins and
tumour necrosis factor (TNF) are a particularly important subgroup of cytokines that
have varied functions including the recruitment and activation of neutrophils, upregulation of PMN adhesion to the endothelial surface and the activation of neutrophil
bactericidal activity (Donaldson et al., 1993).
2.1.1 Asbestos and the inflammatory response
Alveolar macrophages and polymorphonuclear neutrophils are the primary cells
involved in the pulmonary response to inhaled fibres. Their activation and persistence in
the lower respiratory tract is thought to be centrally involved in the processes leading to
lung injury and fibrosis associated with asbestos exposure (Barnes, 1990). These cells
remove fibres by phagocytosis, engulfing and translocating them to the lymph nodes
(Harmsen et al., 1985) or muco-ciliary escalator (Lehnert and Morrow, 1985).
Phagocytic cells utilise reactive oxygen species to uptake particles (Doelman and Bast,
1990). This ‘respiratory burst’ produces a group of highly reactive microbicidal agents
which may causes functional and morphological alterations in local tissues.
Increased amounts of superoxide anions have been shown to be produced by AMs
exposed fibrous materials (Hansen and Mossman, 1987; Roney and Holian, 1989) and
results indicated that the fibrous nature of the phagocytosed particle was essential to
elicit a response, with long thin fibres stimulating significantly more superoxide release
than non-fibrous equivalents (Mossman et al., 1990). This indicates that the
dimensional properties of the fibres dictates the magnitiude of the AM response,
30
possibly due to the inability of AMs to completely phagocytose long particles, leading
to incomplete or ‘frustrated’ phagocytosis (Archer, 1979). In addition, work by Hill et
al. (1995) has demonstrated a differential affinity for opsonin by long and short fibre
amosite. These fibres were previously thought to be indentical apart from length and
therefore illustrates that other factors may be important in the biological activity of
fibres.
The sequelae of events involving ROS and substances released by activated AMs may
be important in the functional impairment of fibroblasts, epithelial and mesothelial cells
by asbestos. The direct oxidative stress caused by the ROS released by AMs could
affect the local tissues directly. Recent studies have shown that the oxidative stress
induced by asbestos can initiate a series of celullar signalling events which lead to the
activation of the oxidative stress sensitive transcription factors AP-1 (Heintz et al.,
1993) and NF-κB (Janssen et al., 1995b). AP-1 and NF-κB are involved in cell
proliferation and the inflammatory response and may be important in the development
of asbestosis and malignant disease (Mossman et al., 1990). In this scheme, the source
of ROS may be unimportant. Asbestos may produce ROS directly via it’s inherent
radical producing surface (Weitzman and Graceffa, 1984) or via the action of resident
phagocytic cells. Either way, asbestos may elicit an oxidative stress in local tissues with
subsequent modulation of cellular function, ultimately leading to the development of
overt pulmonary disease.
In addition to ROS, asbestos-activated AMs release a wide range of secretory products,
including injurious proteases, cytokines and growth factors (Donaldson et al., 1993). A
large body of evidence has accumulated to implicate these agents in asbestos and other
31
fibre- induced pathogenicity (Mossman et al., 1990; Dona ldson et al., 1993).
Macrophages exposed in vitro to crocidolite and chrysotile asbestos release increased
amounts of various cytokines including interleukin-1 (Donaldson et al., 1993) and
tumour necrosis factor-α (TNF-α) (Dubois et al., 1989). The levels of these substances
is also found to be enhanced following inhalation of crocidolite and chrysotile by rats
(Donaldson et al., 1993). IL-1 and TNF-α possess a wide spectrum of of immunological
and pro- inflammatory activites and may act together in initiating and maintaining lung
inflammatory reactions (Donaldson et al., 1993).
In this part of the study a novel in vitro co-culture system was developed to assess the
role of asbestos-exposed macrophage secretory products in cellular signalling events
leading to AP-1 and NF-κB induction in RFL-6 and 4/4 RM-4 cells. RFL-6 and 4/4
RM-4 cells were co-cultured in the presence of a human macrophage (Mono Mac 6) cell
line. The cells types were physically seperated by a semi-permeable membrane allowing
the sole effects of diffusible macrophage-derived products to be evaluated. Direct
interaction of asbestos fibres with RFL-6 and 4/4 RM-4 cells is known to increase the
activity of the AP-1 and NF-κB transcription factors (Heintz et al., 1993; Janssen et al.,
1995b). It was postulated that macrophages exposed to asbestos may produce
substances which can also stimulate the pathways leading to AP-1 and NF-κB
induction. In this scheme, macrophage products could induce a proliferative response in
target cells, which could constitute a non-genotoxic mechanism of carcinogenesis. In
addition, constant stimulation of fibroblasts in this way could explain the observed
fibrosis in asbestos-exposed lung.
32
2.2 MATERIALS AND METHODS
2.2.1 Co-culture of rat lung fibroblasts (RFL-6) and rat mesothelial (4/4 RM-4)
cells with macrophages (Mono Mac 6)
RFL-6 and 4/4 RM-4 were cultured to confluency, as described previously in Part 1,
and 24 hours prior to treatment, media was replaced with medium containing 2% FBS.
A macrophage cell line with characteristics of mature monocytes, Mono Mac 6 (a kind
gift from Professor K. Donaldson, Department of Biological Sciences, Napier
University, Edinburgh, UK) were grown in suspension in RPMI 1640 medium
containing L- glutamine (1%) supplemented with penicillin (50 U/ml), streptomycin
(100 µg/ml), non-essential amino acids (1%), transferrin (10 µg/ml), sodium pyruvate
(1mM), oxaloacetate (1mM) and foetal bovine serum (10%).
When RFL-6 or 4/4 RM-4 cells were confluent, a Mono Mac 6 cell suspension (1.2 x
106 cells/ml complete medium) was prepared in fresh medium and 1ml of the MM-6
suspension added to a Millicell-PCF culture plate insert (0.4 µm, 30mm diameter,
Millipore, Watford, UK). The insert was placed in the centre of the RFL-6 or 4/4 RM-4
culture dish. Crocidolite (12.5, 25 and 50 µg/cm2 ) was added to the Mono Mac 6 insert
for 24 hours at 37°C. Additional incubations were performed with crocidolite (50
µg/cm2 ) in the presence or absence of 5,8,11,14-eicosatetraynoic acid (ETYA; 5µM) or
nordihydroguaiaretic acid (NDGA; 50µM), inhibitors of arachidonic acid metabolism,
in the Mono Mac 6 well insert.
33
2.2.2 Isolation of nuclear proteins and gel mobility shift analyses of AP-1 and NFκB DNA binding activity
Nuclear extracts were isolated from RFL-6 and 4/4 RM-4 cells as previously described
in Part 1. AP-1 and NF-κB DNA binding activities were analysed by the gel mobility
shift assay, as described previously in sections 1.2.2 and 1.2.3
2.3 RESULTS
2.3.1 Induction of AP-1 and NF-κB in RFL-6 and 4/4 RM-4 cells following coculture with Mono Mac 6 cells exposed to crocidolite
Mono Mac 6 cells were co-cultured with RFL-6 and 4/4 RM-4 cells. Mono Mac 6 cells
were treated with crocidolite (12.5, 25 and 50 µg/cm2 ) whereas no exogenous agents
were added to the RFL-6 and 4/4 RM-4 media. Despite the two cell types being
physically seperated by a semi-permeable membrane and the target cells receiving no
external stimuli, increases in the activity of the transcription factors AP-1 and NF-κB
were observed.
In the Mono Mac 6 and RFL-6 co-culture, treatment of Mono Mac 6 cells with
crocidolite was found to significantly enhance both AP-1 and NF-κB DNA binding
activities in RFL-6 cells at the highest concentration tested (Figure 12; P<0.002 and
P<0.01 respectively). In Mono Mac 6 and 4/4 RM-4 co-culture, treatment of Mono Mac
6 cells with crocidolite was found to significantly enhance AP-1 DNA binding activity
34
in 4/4 RM-4 cells at crocidolite concentrations above 12.5 µg/cm2 , with maximum
induction at 50 µg/cm2 (Figure 13; P<0.05). NF-κB DNA binding activity was found to
be significantly enhanced at all concentrations tested with maximal induction at 25
µg/cm2 .
DNA binding activity
(relative units)
20
15
10
AP-1
NF-kB
5
0
None
MM-6 (M)
M+Croc
(12.5ug/cm2)
M+Croc
(25ug/cm2)
M+Croc
(50ug/cm2)
Treatment
Figure 12. Induction of AP-1 and NF-κB transcription factors in RFL-6 cells following
24 hour co-culture with Mono Mac 6 cells exposed to crocidolite (12.5, 25 and 50
µg/cm2 ). The results are the mean ± SD of three independent determinations.
35
DNA binding activity
(relative units)
15
AP-1
10
NF-kB
5
0
None
MM-6 (M)
M+Croc
(12.5ug/cm2)
M+Croc
(25ug/cm2)
M+Croc
(50ug/cm2)
Treatment
Figure 13. Induction of AP-1 and NF-κB transcription factors in 4/4 RM-4 cells
following 24 hour co-culture with Mono Mac 6 cells exposed to crocidolite (12.5, 25
and 50 µg/cm2 ). The results are the mean ± SD of three independent determinations.
2.3.2 Modulation of Mono Mac 6 response to asbestos by inhibitors of arachidonic
acid metabolism
Mono Mac 6 cells, in co-culture with RFL-6 and 4/4 RM-4 cells, were treated with
crocidolite (50 µg/cm2 ) in the presence of ETYA (5 µM) and NDGA (10 µM) to
investigate the possible involvement of the arachidonic acid cascade in the Mono Mac 6
cell response.
In Mono Mac 6 and RFL-6 co-culture, AP-1 DNA binding in RFL-6 cells was reduced
significantly by both NDGA (P<0.001) and ETYA (P<0.001) compared to levels
obtained with crocidolite alone and reduced to levels not significantly different to basal
36
levels (Figure 14). The reduction by ETYA was found to be greater than that of NDGA
but this difference was not found to be statistically significant. Similarly, NF-κB DNA
binding was reduced significantly by both NDGA (P<0.002) and ETYA (P<0.05)
compared to levels obtained with crocidolite alone and reduced to levels not
significantly different to basal levels (Figure 14).
To eliminate the possibility that NDGA and ETYA were diffusing into the RFL-6 media
and acting directly on the RFL-6 cells rather than on the Mono Mac 6 cells, NDGA and
ETYA were added directly to the RFL-6 media. This did not ameliorate the induction of
DNA binding activity
(relative units)
AP-1 and NF-κB by crocidolite-treated Mono Mac 6 cells (Figure 14).
20
15
AP-1
NF-kB
10
5
0
0 ug/cm2
Croc (C)
50 ug/cm2
C
50 ug/cm2
C+NDGA
(10uM)
50 ug/cm2
C+ETYA
(5uM)
NDGA
(10uM) /
ETYA
(5uM)
Treatment
Figure 14. Induction of AP-1 and NF-κB transcription factors in RFL-6 cells following
24 hour co-culture with Mon Mac 6 cells exposed to crocidolite (50 µg/cm2 ) in the
presence of NDGA (10 µM) and ETYA (5 µM). The results are the mean ± SD of three
independent determinations.
37
In Mono Mac 6 and 4/4 RM-4 co-culture, AP-1 and NF-κB DNA binding in 4/4 RM-4
cells was not found to be significantly reduced by NDGA compared to levels obtained
with crocidolite alone (Figure 15). However, ETYA was found to significantly reduce
AP-1 and NF-κB DNA binding (Figure 15; P<0.05 and P<0.01 respectively), altho ugh
the levels obtained were still found to be significantly higher than basal levels indicating
only a partial inhibition.
To eliminate the possibility that NDGA and ETYA were diffusing into the 4/4 RM-4
media and acting directly on the 4/4 RM-4 cells rather than on the Mono Mac 6 cells,
NDGA and ETYA were added directly to the 4/4 RM-4 media. This did not ameliorate
the induction of AP-1 and NF-κB by crocidolite-treated Mono Mac 6 cells (Figure 15).
DNA binding activity
(relative units)
15
AP-1
Series2
10
5
0
0 ug/cm2
Croc (C)
50
ug/cm2 C
50
ug/cm2
C+NDGA
(10uM)
50
ug/cm2
C+ETYA
(5uM)
NDGA
(10uM) /
ETYA
(5uM)
Treatment
Figure 15. Induction of AP-1 and NF-κB transcription factors in 4/4 RM-4 cells
following 24 hour co-culture with Mono Mac 6 cells exposed to crocidolite (50 µg/cm2 )
in the presence of NDGA (10 µM) and ETYA (5 µM). The results are the mean ± SD of
three independent determinations.
38
2.4 DISCUSSION
Inflammatio n is a universal observation in asbestos-exposed lung and there is increasing
evidence that the influx of inflammatory cells to the sites of asbestos deposition plays a
central role in asbestos pathogenicity (Craighead, 1982). During the phagocytic process,
macrophages produce ROS and a range of biological mediators which serve to
perpetuate the inflammatory process and may contribute to the fibrotic and neoplastic
changes observed following asbestos exposure. To investigate this possibility, a coculture system consisting of progenitor cells of pulmonary diseases (RFL-6 and 4/4
RM-4 cells) with a macrophage cell line was developed. RFL-6 cells are a fibroblast
cell line which if inappropriately activated in vivo may lead to fibrosis and 4/4 RM-4
cells are a mesothelial cell line which are the precursors of mesothelioma tumours.
The co-culture system used here physically seperates the target cells, RFL-6 or 4/4 RM4 cells, from the Mono Mac 6 cells. Thus, any observed effect in the target cells must be
due to diffusible factors secreted by the activated macrophages. The results presented
here show that Mono Mac 6 cells exposed to asbestos secrete an as yet unidentified
substance(s) which can diffuse through a semi-permeable membrane (0.4 µm) and
effect a response in RFL-6 and 4/4 RM-4 cells as evidenced by the increase in the DNA
binding activity of AP-1 and NF-κB transcription factors. Asbestos-stimulated
macrophages release a range of substances, including prostaglandins (Dubois et al.,
1989), ROS (Hansen and Mossman, 1987; Roney and Holian, 1989), 1991), growth
factors and cytokines (Donaldson et al., 1993). It is unlikely that ROS will be
responsible for the observed activity since most ROS have very short half lives. Most
likely, cytokines or other second messenger species are responsible. For instance, TNF-
39
α, a potent cytokine which acts via a specific receptor, is released by activated
macrophages and has been shown to rapidly induce the transcription of c-fos and c-jun
(Haliday et al., 1991). Direct interaction of asbestos with various cell types also
activates these genes (Janssen et al., 1995a) and this has been suggested as a potential
pathogenic mechanism.
Previous work in our laboratory has shown that arachidonic acid metabolism is involved
in the induction of AP-1 and NF-κB in RFL-6 and 4/4 RM-4 cells following treatment
with crocidolite (Faux and Howden, 1997). This part of the study investigated the
potential role of arachidonic acid metabolism in the observed activity of macrophages.
Previous studies have shown that AMs release arachidonic acid metabolites in response
to asbestos. In particular AMs have been shown to release leukotriene B4 and
prostaglandin E2 (Dubois et al., 1989).
The results clearly suggest a role for arachidonic acid in the activity of macrophages in
this co-culture system. Inhibition of the entire arachidonic acid cascade with the
competitive inhibitor ETYA, completely eliminated the Mono Mac 6 influence on RFL6 cells and partially ameliorated it in 4/4 RM-4 cells. However, the identity of the
arachidonic acid metabolites responsible was not determined. There are two possible
functions of arachidonic acid (Figure 16). Asbestos may stimulate the arachidonic acid
cascade in macrophages with subsequent release of these metabolites into the
extracellular medium where they diffuse into the target cell media. AMs are known to
release a range of eicosanoids including prostaglandin E2 and leukotriene B4 (Dubois et
al., 1989) and previous work from our laboratory has strongly implicated these
substances in the induction of AP-1 and NF-κB by asbestos (Faux and Howden, 1997).
40
Thus it is possible that eicosanoids produced by Mono Mac 6 cells may interact with
eicosanoid receptors on the target cells to initiate a signalling pathway leading to the
activation of AP-1 and NF-κB DNA binding (Figure 16, mechanism A).
(A)
Fibre
phagocytosis
Receptor for AA
metabolite
AA
AA
Metabolite
Nucleus
AP-1 and NF-kB
activation
Nucleus
Diffusible
factor
(B)
Receptor
Mono Mac-6 cell
RFL-6 or
4/4 RM-4 cell
Semi-permeable
membrane
Figure 16. Possible mechanisms of induction of AP-1 and NF-κB in target cells by
crocidolite-exposed macrophages.
A second possible mechanism is that the diffusible factor may not be an eicosanoid, but
rather another extracellular messenger whose transcription or activation is under the
control of arachidonic acid metabolites (Figure 16, mechanism B). A candidate
messenger is TNF-α. Exposure of guinea-pig macrophages to chrysotile asbestos results
in a significant increase in phospholipase A2 activity (Dubois et al., 1989), and
subsequent synthesis of eicosanoids. The eicosanoids prostaglandin E2 and leukotriene
B4 are known to regulate TNF-α at the transcriptional level. Another consequence of
phospholipase A2 activity is the release of diacylglycerol, a required activator of PKC,
41
an enzyme implicated in stimulating TNF release in macrophages in response to
asbestos (Donaldson et al., 1993). TNF-α has been shown to induce c-fos and c-jun in
an adipogenic TA1 cell line (Haliday et al., 1991). It has been shown that c-fos
induction by TNF-α is mediated by hydroperoxyeicosatetranoic acid (HPETE) products
of the lipoxygenase pathway. This pathway is inhibited by NDGA. In RFL-6, NDGA
treatment in Mono Mac 6 cells reduced the induction of AP-1 and NF-κB to basal
levels, although in 4/4 RM-4 NDGA did not appear to be inhibitory, perhaps suggesting
different responses between the two cell types.
In summary, this part of the study has shown that soluble factors released from
crocidolite-treated macrophages induce signalling pathways leading to AP-1 and NF-κB
induction in untreated RFL-6 and 4/4 RM-4 cells. The results also suggest that
arachidonic acid metabolism in the macrophage is important in this activity. The precise
mechanism and the identity of the substances involved is not known and more research
is needed in this important area.
42
3. POTENTIAL ROLE OF THE ARACHIDONIC ACID CASCADE
IN THE PERTURBATION OF AP-1 AND NF-κB DNA BINDING BY
ASBESTOS
3.1 BACKGROUND
Cells exposed to asbestos often exhibit altered differentiation and growth
characteristics. Studies have indicated that this proliferative response may be mediated
by activation of protein kinase C (PKC) (Sesko et al, 1990). Rat fibroblasts which overproduce a transfected isoform of PKC display altered morphology (Housey et al, 1988)
and inhibitors of PKC reduce the induction of ornithine decarboxylase, a key enzyme in
cell proliferation, by asbestos (Marsh and Mossman, 1988). For optimum activity, PKC
requires the presence of calcium and phospholipid (Nishizuka, 1984; Nishizuka, 1986).
The endogenous phospholipid activator of PKC is diacylglycerol which is released from
the cellular membrane by the action of phospholipases (Figure 17; Majerus et al, 1986),
and this action is mimicked by many soluble tumour promoters such as phorbol esters
(Castagna et al, 1982). Since it is unlikely that insoluble asbestos fibres can bind
directly to PKC to activate it, other mechanisms have been investigated.
Exposure to crocidolite, but not non- fibrous particles, has been shown to alter the
metabolism of phospholipids (Marsh and Mossman, 1988; Sesko et al, 1990). Following
exposure to asbestos there is an increase in the products of inositol phospholipid
hydrolysis, namely diacylglycerol and inositol phosphate(s), strongly suggesting the
activation of phospholipase C (Sesko et al, 1990) in response to the fibre.
43
Phosphatidylcholine /
phosphoinositides
Phospholipase A2
Phosphatidylcholine /
phosphoinositides
Phospholipase C
Phosphorylcholine /
inositol polyphosphate
Diacylglycerol
Lysophosphatidylcholine
Lipase
Arachidonic acid
Arachidonic acid
Figure 17. The pathways for the release of arachidonic acid from membrane
phospholipids by phospholipase A2 and phospholipase C.
The observation that crocidolite can perturb the normal homeostasis of membrane
metabolism may play a central role in asbestos pathogenicity. Stimulation of
phospholipid hydrolysis produces diacyglycerol and inositol phosphates. Diacylglycerol
is an activator of PKC, which is known to be involved in the proliferative response.
Inositol phosphates, such as inositol triphosphate (IP3 ) are triggers for the release of
stored intracellular calcium that is also a required co-activator of PKC as well as an
important intracellular messenger. In addition, diacylglycerol can be metabolised further
to give arachidonic acid for which an increasing body of evidence suggests both an
extra- and intra-cellular signalling role (Liscovitch and Cantley, 1994). In this scheme,
asbestos may act via secondary lipid messengers. Asbestos may activate phospholipases
within the membrane with subsequent release of protein kinase activators such as PKC
or stimulate the release of arachidonic acid that may be metabolised further to
biologically active end-products.
44
3.1.1 Arachidonic acid metabolism
Arachidonic acid (5,8,11,14-eicosatetraenoic acid) is a naturally occurring C20
polyunsaturated fatty acid. It is the main precursor of the eicosanoids, a family of
oxygenated C20 fatty acids with varied and often potent physiological activity including
the control of platelet aggregation, macrophage recruitment, smooth muscle contraction
and sodium reabsorption. The eicosanoids are the end-products of the arachidonic acid
cascade, a series of enzymic and non-enzymic reactions and consist broadly of 3 subgroups; the prostanoids (prostaglandins and thromboxanes), leukotrienes and lipoxins.
Before arachidonic acid can be converted into metabolites it must first be released from
the phospholipids present in the plasma membrane. Under normal physiological
conditions the intracellular concentration of arachidonic acid is low and is only
increased in response to extracellular signals. Such signals can be mediated by growth
factors, eicosanoids themselves or by membrane damage which activates membrane
bound phospholipases (van Kuijk, 1987). The most active phospholipases with respect
to arachidonic acid release are A2 and C (Liscovitch and Cantley, 1994). Phospholipase
A2 has been shown to protect membranes against lipid peroxidation by preferentially
acting on peroxidized fatty acids, cleaving them from the membrane and allowing
metabolism and detoxification by glutathione peroxidase (van Kuijk, 1987).
Phospholipase C catalyses the formation of arachidonic acid via diacylglycerol derived
from membrane phosphatidylcholine and phosphoinositides.
45
Free arachidonic acid is turned over rapidly in cells by either being reincorporated into
membrane phospholipid or through metabolism. In the absence of an agonist
arachidonic acid is metabolised by arachidonyl-CoA synthetase and
lysophosphoglycerate acyl transferase resulting in it’s reincorporation into phospholipid
(Figure 18). However, in the presence of an agonist the reincorporation mechanism is
saturated allowing the intracellular concentration of arachidonic acid to increase and
make it available for conversion to it’s metabolites. Thus arachidonic acid metabolites
may be produced for the duration of the agonist signal.
Lysophosphatidylcholine
Lysophosphoglycerate
acyl transferase
Phosphatidylcholine
CoA-SH
Arachidonic acid
Arachidonyl-CoA
Arachidonyl-CoA
synthetase
arachidonic acid
metabolites
Figure 18. Arachidonic acid release and reincorporation into membrane phospholipids.
Once free arachidonic acid is present in the cell, there begins a cascade of metabolic
events which yields all enzymatic and non-enzymatic products. The spectrum of
products formed depends on the relative distribution of enzymes present in the
particular cell type since not all enzymes of the arachidonic acid cascade are present in
46
any one cell type. For example thromboxane A2 (TXA2 ; an aggregation factor) is the
predominant metabolite formed in platelets, prostaglandin I2 (PGI2 ) in smooth muscle,
prostaglandin E2 (PGE2 ) in kidney collecting tubules and prostaglandin F2? (PGF 2α) in
uterine endometrium.
The main enzymes which determine the metabolism of arachidonic acid are PGG2 /H2
synthetase (prostaglandin endoperoxide synthetase), the first enzyme in the pathway for
the formation of prostaglandins and thromboxanes and the lipoxygenases, a group of
enzymes leading to leukotrienes, lipoxins and 12-HPETE (Figure 19). These enzymes
are associated with intracellular membranes, such as the endoplasmic reticulum, or the
cytoplasm allowing the formation of arachidonic acid metabolites in the interior of the
cell contrary to many other intracellular messengers which are formed at the plasma
membrane. In addition, an important characteristic of arachidonic acid metabolites is
that many of them are lipid soluble allowing them to diffuse out of cells into the
extracellular medium without the aid of carrier proteins allowing them to act as
autocrine or paracrine hormones and chemotactic agents etc (Barritt, 1992).
COOH
15’-lipoxygenase
PGG/H synthetase
Arachidonic Acid
5’-lipoxygenase
PGH2
12’-lipoxygenase
15-HPETE
5-HPETE
12-HPETE
Prostaglandins
Lipoxins
Thromboxanes
Leukotrienes
Figure 19. The arachidonic acid cascade - the main metabolic pathways for arachidonic
acid.
47
The conversion of arachidonic acid to prostaglandin H2 (PGH2 ) involves two enzymecatalysed reactions, and both sites reside on the same enzyme (PGG2 /H2 ; prostaglandin
endoperoxide synthetase). The first reaction, catalysed by cyclooxygenase, converts
arachidonic acid to PGG2 followed by the subsequent conversion of PGG2 to PGH2 by a
peroxidase (Figure 20). PGH2 can then be converted enzymically to a range of
prostaglandins and thromboxanes including PGE2 , PGD2 , PGI2 , PGF 2α and TXA2 . A
range of additional compounds are also formed by non-enzymatic reactions.
Arachidonic Acid
PGG2 /H2 synthetase
(cyclooxygenase component)
PGG 2
PGG2 /H2 synthetase
(peroxidase component)
PGE 2
PGF 2α
PGH 2
PGI 2
PGD 2
TXA 2
Figure 20. Formation of prostaglandins and thromboxanes from arachidonic acid via the
PGG2 /H2 synthetase pathway
Arachidonic acid can also be metabolised by lipoxygenases to give a range of
leukotrienes (Figure 21). 5’- and 12’- lipoxygenase convert arachidonic acid to 5- and
12-hydroperoxyeicosatetraenoic acid (5- and 12-HPETE) respectively. 5-HPETE is
further metabolised to a highly unstable 5,6-epoxide derivative, leukotriene A4 (LTA4 )
which is either hydrolysed to give leukotriene B4 (LTB4 ) or reacts with glutathione
48
(catalysed by glutathione-S-trasnferase) to form leukotriene C4 (LTC4 ). This 5-hydroxy6-glutathionyl derivative is metabolised successively by γ-glutamyl transpeptidase and
cysteinyl- glycine dipeptidase to form the further products leukotriene D4 (LTD4 ) and
leukotriene E4 (LTE4 ). Other products of lipoxygenases include the lipoxins which are
the product of the sequential actions of 15’- lipoxygenase and 5’-lipoxygenase on
arachidonic acid to give lipoxin A and B.
Arachidonic Acid
5’-Lipoxygenase
5-HPETE
Glutathione
LTA 4
Glutathione
S-transferase
Glutamate
LTC4
γ -Glutamyl
transpeptidase
LTD 4
Glycine
LTB 4
LTE 4
Figure 21. Formation of leukotrienes from arachidonic acid via the lipoxygenase
enzymatic pathway.
Once formed, the biologically active metabolites of arachidonic acid are rapidly
converted to inactive metabolites by both enzymatic and non-enzymatic reactions. The
degradation of the prostaglandins and thromboxanes is via oxidation of the 15- hydroxyl
group by a variety of 15- hydroxyprostaglandin dehydrogenases. Leukotrienes are
hydroxylated at carbon atom positions 19 or 20 to initiate degradation.
49
Prostaglandins, thromboxanes and leukotrienes have been implicated as both intra- and
extra-cellular messengers, eliciting signals to specialised cell types (Barritt, 1992).
These messages are often specific to each particular chemical and also to the cell type
receiving the signal. Since the metabolites are lipid soluble they readily diffuse out of
the initiating cell through the plasma membrane into the extracellular medium where
they can act as agonists for receptors on surrounding cells, activating intracellular
messengers and protein kinases (Barritt, 1992). Receptors for PGE2 , PGF 2 , PGI2 , TXA2 ,
LTB4 and LTD4 have already been discovered and it is likely that others await
detection.
It has also been suggested that arachidonic acid and it’s metabolites may act as
intracellular messengers in the same way as cyclic nucleotides and inositol 1,4,5triphosphate. For instance, arachidonic acid metabolites have been implicated in the
opening of K+ channels by acetylcholine (Kim et al, 1989). In addition, arachidonic acid
and some of it’s metabolites have also been shown to activate protein kinase C and
guanylate cyclase as well as releasing Ca2+ from the endoplasmic reticulum (Barritt,
1992).
3.1.2 Arachidonic acid release following asbestos exposure
Previous studies from our laboratory have shown that mineral fibres cause time- and
dose-dependent induction of lipid peroxidation, as evidenced by an increase in
thiobarbituric acid reactive substances, in RFL-6 cells (Howden and Faux, 1996). This
is potentially important since there is now considerable evidence that the products of
50
lipid peroxidation are involved in carcinogenesis (Ames and Gold, 1991). Additionally,
certain end-products of this process may act as cellular messengers, conveying the
oxidative signal from the plasma membrane to the nucleus. Recent studies have shown
that H2 O2-induced c-fos expression in rodent smooth muscle cells is mediated by
products of lipid peroxidation, in particular from the metabolism of arachidonic acid via
the lipoxygenase pathway (Rao et al, 1993), as is the induction of c-fos by TNF
(Haliday et al, 1991). Similarly, fecapentaene-12, a potent colon mutagen which causes
oxidative stress in HeLa cells (Plummer and Faux, 1994) and induces c-jun via the
cyclooxygenase pathway of the arachidonic acid cascade (S.M.Plummer, personal
communication). The involvement arachidonic acid metabolism in signalling pathways
leading to the induction of transcription factors by asbestos has not previously been
assessed.
This part of the study investigated the potential role of leukotriene and prostaglandin
biosynthesis in asbestos- mediated induction of the transcription factors AP-1 and NFκB in RFL-6 and 4/4 RM-4 cells. The role of the arachidonic acid cascade was
investigated using inhibitors of the enzymes involved. 5,8,11,14-eicosatetraynoic acid
(ETYA) was used to competitively inhibit arachidonic acid metabolism (Ondrey et al,
1989), indomethacin was used to inhibit the cycooxygenase activity of the PGG2 /H2
pathway (Lands and Hanel, 1983) and nordihydroguaiaretic acid (NDGA) used to
inhibit the lipoxygenase pathways (Dubois et al, 1989).
The data presented here suggests that in RFL-6 and 4/4 RM-4 cells, the induction of
AP-1 and NF-κB DNA binding activity by asbestos is mediated by the metabolism of
51
arachidonic acid. Additional data suggests that lipoxygenase products are important
mediators of this response.
3.2 MATERIALS AND METHODS
3.2.1 Cell Culture
RFL-6 and 4/4 RM-4 cells were cultured as described previously (section 1.2.1). Cells
were incubated with crocidolite (10 µg/cm2 RFL-6; 5 µg/cm2 4/4 RM-4) in the presence
or absence of indomethacin (50, 100 and 150 µM), NDGA (5, 10 and 25 µM) or ETYA
(5 µM) for 8 hours at 37°C. At the end of the culture period, nuclear extracts were
prepared as described previously (section 1.2.2).
3.2.2 Measurement of AP-1 and NF-κB DNA binding activity
AP-1 and NF-κB binding activity was measured using the gel mobility shift assay as
described previously (section 1.2.3).
3.3 RESULTS
To investigate the potential involvement of arachidonic acid metabolism in AP-1 and
NF-κB transcription factor induction by asbestos, RFL-6 and 4/4 RM-4 cells were
52
incubated with crocidolite in the presence or absence of various inhibitors of the
arachidonic acid cascade.
To investigate whether arachidonic acid metabolism is involved, cells were incubated
with crocidolite in the presence or absence of ETYA (5 µM), an arachidonic acid
analogue and competitive inhibitor of arachidonic acid metabolism. Following this
treatment in RFL-6 cells, the level of AP-1 DNA binding was reduced significantly
(P<0.05) to a level not significantly different to basal levels (Figure 22). Similarly, the
level of NF-κB DNA binding following treatment with ETYA was also significantly
DNA binding (arbitrary units)
reduced (P<0.001) to basal levels (Figure 22).
10
AP-1
NF-kB
5
0
None
ETYA (5uM)
10ug/cm2
Croc
10ug/cm2
Croc+ETYA
(5uM)
Treatment
Figure 22. Induction of AP-1 and NF-κB transcription factors in RFL-6 cells following
8 hour incubation with crocidolite (10 µg/cm2 ) in the presence or absence of ETYA.
The results are the mean ± SD of three independent determinations.
53
Following treatment of 4/4 RM-4 cells with crocidolite in the presence of ETYA (5
µM), the level of AP-1 DNA binding was reduced significantly (P<0.005) to a level not
significantly different to basal levels (Figure 23). Similarly, the level of NF-κB DNA
binding following treatment with ETYA was also significantly reduced (P<0.05) to
DNA binding (arbitrary units)
basal levels (Figure 23).
15
AP-1
10
NF-kB
5
0
None
ETYA (5uM)
5ug/cm2
Croc
5ug/cm2
Croc+ETYA
(5uM)
Treatment
Figure 23. Induction of AP-1 and NF-κB transcription factors in 4/4 RM-4 cells
following 8 hour incubation with crocidolite (5 µg/cm2 ) in the presence or absence of
ETYA. The results are the mean ± SD of three independent determinations.
To further elucidate the signalling pathway, the metabolic fate of arachidonic acid
produced following crocidolite exposure was investigated. Indomethacin (IC 50 =0.1
µM), an inhibitor of the cyclooxygenase component of the arachidonic acid cascade was
used to examine the potential role of prostaglandin and thromboxane products.
54
Treatment of RFL-6 cells with crocidolite in the presence of indomethacin did not
significantly alter AP-1 or NF-κB DNA binding compared with crocidolite alone at any
of the concentrations tested (50-150 µM; Figure 24).
DNA binding (arbitrary units)
15
10
AP-1
NF-kB
5
0
None
Indo (100uM)
10ug/cm2 Croc (C)
10ug/cm2 C+Indo(50uM)
10ug/cm2 C+Indo(100uM)
10ug/cm2 C+Indo(150uM)
Treatment
Figure 24. Induction of AP-1 and NF-kB transcription factors in RFL-6 cells following
8 hour incubation with crocidolite (10 µg/cm2 ) in the presence or absence of
indomethacin. The results are the mean ± SD of three independent determinations.
Treatment of 4/4 RM-4 cells with crocidolite in the presence of indomethacin
significantly reduced the induction of both AP-1 and NF-κB at 50 µM (P<0.01 and
P<0.02 respectively; Figure 25). However at both higher concentrations tested (100 and
150 µM) indomethacin did not significantly alter AP-1 or NF-κB DNA binding activity
from that obtained with crocidolite alone (Figure 25).
NDGA (IC 50 =0.2 µM for 5- lipoxygenase and 30 µM for 12- and 15- lipoxygenases), an
inhibitor of the lipoxygenase component of the arachidonic acid cascade was used to
55
investigate the possible role of leukotrienes in the induction of AP-1 and NF-κB by
asbestos.
DNA binding (arbitrary units)
15
10
AP-1
NF-kB
5
0
None
Indo (100uM)
10ug/cm2 Croc (C)
10ug/cm2 C+Indo(50uM)
10ug/cm2 C+Indo(100uM)
10ug/cm2 C+Indo(150uM)
Treatment
Figure 25. Induction of AP-1 and NF-κB transcription factors in 4/4 RM-4 cells
following 8 hour incubation with crocidolite (5 µg/cm2 ) in the presence or absence of
indomethacin. The results are the mean ± SD of three independent determinations.
Treatment of RFL-6 cells with crocidolite in the presence of NDGA reduced the
induction of AP-1 at all the concentrations tested (5-25 µM) but this was only found to
be significant at 10 µM (P<0.001; Figure 26). Similarly, NF-κB DNA binding activity
was reduced at all concentrations, with the reduction being significant only at 10 µM
(P<0.02; Figure 26).
56
DNA binding (arbitrary units)
15
10
AP-1
NF-kB
5
0
None
NDGA
(5uM)
10ug/cm2
Croc (C)
10ug/cm2
C+NDGA
(5uM)
10ug/cm2
C+NDGA
(10uM)
10ug/cm2
C+NDGA
(25uM)
Treatment
Figure 26. Induction of AP-1 and NF-κB transcription factors in RFL-6 cells following
8 hour incubation with crocidolite (10 µg/cm2 ) in the presence or absence of
nordihydroguaiaretic acid (NDGA). The results are the mean ± SD of three independent
determinations.
Treatment of 4/4 RM-4 cells with crocidolite in the presence of NDGA significantly
reduced the induction of AP-1 at 10 and 25 µM (P<0.05 and P<0.05 respectively; Fig.
6.11). NF-? B DNA binding activity was reduced significantly at 5? M (P<0.05), and
reduced, but not significantly at 10 µM (Figure 27). NDGA at 25 µM was not found to
significantly alter NF-κB activity from that obtained with crocidolite alone.
57
DNA binding (arbitrary units)
15
10
AP-1
NF-kB
5
0
None
NDGA
(5uM)
10ug/cm2
Croc (C)
10ug/cm2
C+NDGA
(5uM)
10ug/cm2
C+NDGA
(10uM)
10ug/cm2
C+NDGA
(25uM)
Treatment
Figure 27. Induction of AP-1 and NF-κB transcription factors in 4/4 RM-4 cells
following 8 hour incubation with crocidolite (5 µg/cm2 ) in the presence or absence of
nordihydroguaiaretic acid (NDGA). The results are the mean ± SD of three independent
determinations.
3.4 DISCUSSION
There is strong evidence that signalling pathways are involved in the enhanced
expression of oxidative stress inducible genes such as c-fos and c-jun and the
transcription factors AP-1 and NF-κB (Schreck et al, 1991). There is also evidence to
suggest that the initiation of these signalling events occurs at the plasma membrane
(Devary et al, 1992; Devary et al, 1993), and involve protein kinase cascades (Devary et
al, 1992) and redox regulation (Meyer et al, 1993). Previous work in this study and by
others has indicated that cellular membranes are an important target for asbestosderived reactive oxygen species manifesting as lipid peroxidation. Previous work from
our laboratory has shown that crocidolite causes lipid peroxidation in RFL-6 cells
58
(Howden and Faux, 1996). It is known that in response to lipid peroxidation,
phospholipase A2 (van Kuijk et al, 1987) may be activated to detoxify the damaged
lipids, resulting in the release of various species including arachidonic acid.
Arachidonic acid and it’s metabolites possess potent biological activity and this part of
the study investigated their possible role in the induction of the AP-1 and NF-? B
transcription factors by asbestos.
Arachidonic acid may be highly relevant to signal transduction within the cell because it
is a substrate for the synthesis of eicosanoids, namely prostaglandins and leukotrienes,
by cyclooxygenases and lipoxygenases respectively. Little is currently known about the
role of arachidonic acid or its metabolites in asbestos-mediated gene transcription, but
in recent studies the induction of c-fos gene expression with agents such as TNF
(Haliday et al, 1991) or H2 O2 (Rao et al, 1993) have been shown to be mediated by the
conversion of arachidonic acid to a metabolite(s) via the lipoxygenase pathway. In these
studies, cyclooxygenase inhibitors had no effect on the modulation of mRNA levels of
c-fos.
This study has shown for the first time that arachidonic acid metabolites are involved in
the cellular signalling events leading to AP-1 and NF-κB transcription factor induction
by asbestos. Work with ETYA, a competitive inhibitor of arachidonic acid, clearly
showed that arachidonic acid is important in the induction of these factors. However
results with indomethacin and NDGA were inconclusive. In RFL-6 cells, indomethacin
did not appear to have any inhibitory activity suggesting that the cyclooxygenase
pathway leading to prostaglandins and thromboxanes is inactive. NDGA reduced AP-1
and NF-κB to basal levels at 10 µM but had no significant inhibitory activity at higher
59
concentrations. In 4/4 RM-4 cells, indomethacin was found to have variable activity
with 50 µM being significantly inhibitory whereas higher concentrations were not found
to have any inhibitory activity at all. NDGA was only found to be inhibitory at the
lowest concentration tested.
In summary, the results from this part of the study show that arachidonic acid or
products of arachidonic acid metabolism are important in the induction of AP-1 and NFκB activity by asbestos. In this scheme, asbestos may initiate the release of arachidonic
acid from the plasma membrane by lipid peroxidation or via the activation of
phospholipases. The released arachidonic acid may then be metabolised to a range of
biologically active species which possibly act as intra-cellular messengers or diffuse out
of the cell into the extra-cellular medium to interact with other cells, possibly via
specific receptors (Figure 28).
Receptor for AA
metabolite
Asbestos-induced
lipid peroxidation
AA
Intracellular
Signal
AA
Metabolite
Nucleus
Receptor for AA
metabolite
Figure 28. Postulated role of arachidonic acid metabolites as intra- and inter-cellular
messengers.
60
It is clear from these results that arachidonic acid may play an important role in
asbestos-pathogenicity by transducing a signal elicited by asbestos at the plasma
membrane into alterations in gene expression in the nucleus, leading to proliferation and
possibly other cellular changes. However, further work is required to establish the
relative importance of the various metabolic products of arachidonic acid in this
process.
61
4. EXAMINATION OF THE ROLE OF CELL SURFACE
RECEPTORS, IN PARTICULAR THE EPIDERMAL GROWTH
FACTOR-RECEPTOR (EGF-R), IN THE CELL SIGNALLING
RESPONSE OF RAT PLEURAL MESOTHELIAL (RPM) CELLS
TO CARCINOGENIC AND NON-CARCINOGENIC FIBRES
4.1 BACKGROUND
Occupational and environmental exposure to asbestos, a family of fibrous crystalline
silicates, is the causative agent associated with the development of diffuse malignant
mesothelioma, a fatal tumour arising from the mesothelial lining in the pleura and
peritoneal cavities (Wagner et al., 1965). Although mechanisms of fibre carcinogenicity
are not well understood, several parameters of fibres such as physical dimensions
(length and diameter), biopersistence and surface reactivity are thought to play an
important role, contributing to chronic inflammation and unregulated proliferation of
the target cells of the pleura (Mossman et al., 1990).
Previous in vivo studies by Stanton et al. have shown a relationship between fibre length
and pathogenicity (Stanton et al., 1977). These studies, with intrapleurally implanted
fibres, showed an increasing ability to cause mesothelioma for fibres above 8µm in
length and below 0.2µm in diameter. In rodent inhalation and intraperitoneal instillation
studies using two amosite asbestos samples, differing only in length, Davis et al. (4)
demonstrated that the short and long samples had dramatically differing pathogenic
potential. In these studies the long fibre amosite produced fibrosis, lung tumours and
62
mesotheliomas, whilst the short fibre sample was without pathogenic activity. Many
fibres in the long fibre amosite preparations in studies by Davis et al. (1984) were in the
"Stanton range", whilst there were no "Stanton" fibres in the short fibre preparation.
Complementary studies by Dona ldson et al. (1989) have shown that the long fibre
sample is many times more active than the short fibre sample in producing
inflammation, a characteristic response to carcinogenic mineral dusts.
Cell signalling events elicited by asbestos may lead to mesothelial cell proliferation
producing tumour initiation, promotion and progression. In this scheme of events,
alterations in gene expression by asbestos initiated at the plasma membrane may
facilitate chronic cell proliferation. Sustained cell proliferatio n appears to be a universal
factor in human cancers (Preston-Martin et al., 1990) and in this process cancer appears
to result from genetic errors induced or fixed during the process of cell division, as
increased mitogenesis increases the risk of multiple genetic defects including mutations,
translocations and amplification of oncogenes. Recent studies in mesothelial cells have
shown that asbestos fibres activate the "early response" protooncogenes c-fos and c-jun
(Heintz et al., 1993). Unlike the classical tumour promoter TPA that induces transient
increases in c-fos and c-jun mRNA peaking at l h, crocidolite causes persistent
induction of these protooncogenes and related transcription factors for at least 24h and
may serve as a chronic stimulation for cell proliferation after deposition of fibres in the
lung or pleura. The induction of gene expression is dose-dependent and is not observed
with polystyrene beads or riebeckite, which is chemically similar to crocidolite but nonfibrous, and suggests that fibre geometry is critical in protooncogene induction (Janssen
et al., 1994).
63
Interaction of fibres with cell surface receptors prior to the initiation of the signalling
cascade in the regulation of immediate early genes may be important with asbestos.
Mitogen activated protein (MAP) kinases are members of a family of enzymes involved
in regulating the response of eukaryotic cells to extracellular signals (Boulton et al.,
1990). Activation of MAP kinase is involved in the upstream events leading to c-fos and
c-jun transcription because following phosphorylation, MAP kinases, specifically
extracellular signal regulated kinases (ERKs), translocate to the nucleus
phosphorylating substrates that are involved in the transactivation of the
protooncogenes (Zinck et al., 1993). Recently, asbestos has been shown to cause
stimulation of the ERK 1 MAP kinase cascade after phosphorylation of the epidermal
growth factor-receptor (EGF-R) (Zanella et al., 1996). In these studies, crocidolite, but
not its non-fibrous analogue riebeckite, induced MAP kinase activity that could be
blocked by suramin, shown to inhibit growth factor interactions, and by tyrphostin AG
1478, a specific inhibitor of the EGF-R tyrosine kinase (LevitzLi and Gazit, 1995). It is
possible that the EGF-R plays a critical role in the carcinogenic process by asbestos.
Studies have shown that the upregulation of EGF-R expression in NIH 3T3 cells leads
to tumorigenicity (Hudziak et al., 1987) and elevated expression of the EGF-R has been
found in several types of human malignancy, including mesothelioma (Dazzi et al.,
1990). Whether asbestos fibres cause upregulation of the EGF-R in normal mesothelial
cells is of considerable importance.
The present studies were designed to determine if we can discriminate between
carcinogenic fibres, such as crocidolite asbestos and erionite fibres, and noncarcinogenic preparations, such as milled crocidolite and chrysotile, by their ability to
induce EGF-R protein expression in normal rat pleural mesothelial cells.
64
4.2 MATERIALS AND METHODS
4.2.1 Cell culture of rat pleural mesothelial cells and exposure to test agents
Male Fischer 344 rats aged 12-20 weeks were killed by exsanguination under halothane
anaesthesia and the thorax opened. Rat pleural mesothelial (RPM) cells were isolated by
gentle scraping of the parietal pleura as described by Jaurand et al. (1983). Cells were
maintained in DMEM/F-12, supplemented with penicillin (50 units/ml), streptomycin
(100 µg/ml), containing 15% foetal bovine serum (FBS), hydrocortisone (100 ng/ml),
insulin (2.5 µg/ml), transferrin (2.5 µg/ml) and selenium (2.5 ng/ml) as described by
Heintz et al. (1993). Cells were grown to confluency and 24 hours before exposure to
mineral dusts, TNF-α or TPA, cells were switched to medium containing reduced serum
(0.5 % FBS). Mineral dusts were suspended in DMEM/F-12 at 1 mg/ml, sonicated for
4min in a water bath, triturated 8 times with a 22-gauge needle and then added to the
medium for final concentrations of 2.5 and 5.0 µg/cm2 . Reference samples of Union
Internationale Contre le Cancer (UICC) processed crocidolite, chrysotile B and milled
crocidolite asbestos fibres were used. Erionite was obtained from Rome, Oregon, USA
and a respirable sample prepared as described previously (Poole et al., 1983). TNF-α
(Calbiochem, Nottingham, UK) and TPA (Sigma, Dorset, UK) were added to the
culture medium at final concentrations of 10 and 100 ng/ml, respectively.
4.2.2 lmmunofluorescence detection of EGF-R by laser scanning confocal
microscopy
RPM cells were grown on coverslips (No 1, 19mm diameter; Chance Proper Ltd.,
Warley, UK) and when 70% confluent the growth medium was replaced with medium
containing reduced serum (0.5% FBS). Cells were then treated with mineral fibres (5
µg/cm2 ) or TNF-α (10 ng/ml in calcium and magnesium- free phosphate buffered saline
(CMFPBS)/1% BSA) for 2, 8, 24, 48 and 72 hours in the presence and absence of a
30min pre-treatment with cycloheximide (10 µg/ml, Sigma, Dorset, UK). Cells were
65
fixed in methanol for 20 minutes and permeabilised in 0.1% Triton X-100/4% FBS for
20 minutes. The cells were then blocked with CMFPBS containing 10% goat serum for
15 minutes to prevent non-specific binding of the antibodies. Cells were incubated with
an EGF-R rabbit polyclonal antibody (2.5 µg/ml in CMFPBS/10% goat serum, Santa
Cruz Biotechnology Inc, Wiltshire, UK), a mouse monoclonal antibody to the active
EGF-R (Chemicon International Ltd., Harrow, UK) and/or a proliferating cell nuclear
antigen (PCNA) mouse monoclonal antibody (2.5 µg/ml in CMFPBS/10% goat serum;
Novocastra Laboratories Ltd, Newcastle upon Tyne, UK) for 12 hours at room
temperature. The cells were then labelled with a rhodamine- linked (TRITC) goat antirabbit immunoglobulin G (25 µg/ml in CMFPBS/10% goat serum) and/or a fluoresceinlinked (FITC) goat anti- mouse immunoglobulin G (100 µg/ml in CMFPBS/10% goat
serum) for 1 hour at 37°C in 5% CO2. The coverslips were mounted on microscope
slides using Fluoromount (BDH, Lutterworth, UK) and kept at 4°C until analysis by
confocal microscopy.
A Leica True Confocal Scanner 4D was used to visualise and locate the EGF-R in RPM
cells. The microscope was used to scan simultaneously in two channels, at 488 and 568
nm, to excite fluorescein and rhodamine respectively. Throughout the experiments
levels of laser emission were kept the same for all the samples and both photomultiplier
tube settings were kept constant. The respective emissions were detected and cell
preparations were optically sectioned. Optical data stacks were combined into a
maximum projection and stored as true colour 24-bit digital images. The digital images
were imported into Adobe Photoshop software and colour output was obtained using a
Kodak dye sublimation printer.
66
4.2.3 Isolation of nuclear proteins and electrophoretic mobility shift analyses
(EMSAs) of AP-1 DNA binding
After various time periods of exposure to fibres (2.5 and 5 µg/cm2 ) or TPA (100 ng/ml),
in the presence or absence of suramin (20 µM; Calbiochem, Nottingham, UK) or
tyrphostin AG 1478 (10 µM; Calbiochem, Nottingham, UK), a selective inhibitor of
EGF-R, nuclear protein extracts were isolated as described in section 1.2.2 and EMSAs
performed as described in section 1.2.3.
4.3 RESULTS
4.3.1 Immunofluorescence detection of EGF-R protein in RPM cells by laser
confocal scanning microscopy
To determine if mineral dusts increase EGF-R protein expression in RPM cells, we have
used a combination of fluorescence and reflectance laser scanning confocal microscopy
to examine the immunolocalisation of EGF-R in these cells. Crocidolite and erionite,
two fibrous preparations known to induce mesothelioma, showed intense patterns of
EGF-R protein expression in RPM cells. In contrast, milled crocidolite (non- fibrous)
and chrysotile asbestos, two preparations with much less or no ability to induce
mesothelioma, did not show increases in EGF-R protein expression over the sham
control (Figure 29; Table 2). By comparing the confocal fluorescence image, indicating
EGF-R immunoreactivity (Figure 30; panel C), with a phase constrast image
demonstrating fibre morphology (Figure 30; panel B), we have shown that cells
attempting to phagocytose long crocidolite fibres showed increased staining for EGF-R
67
acquired a fusiform shape, whereas cells in contact with a number of shorter fibres
showed negligible immunostaining for EGF-R and remained polygonal in morphology.
To investigate whether crocidolite asbestos increases new EGF-R protein levels rather
than redistributing protein within RPM cells, we incubated cells with crocidolite for 8h
following pre-treatment for 30min with cycloheximide, an inhibitor of protein synthesis,
and with crocidolite for 2h. In these experiments, cycloheximide abrogated crocidolite
mediated EGF-R protein induction and at 2h, EGF-R protein expression was not
different from the sham controls (Figure 30; panels D and E, respecively). Upregulation
of EGF-R protein expression was only observed after 8h fibre exposure and continued
for at least 72h. To determine if crocidolite upregulates phosphorylated EGF-R (pEGFR), we incubated with an antibody to this active form of the receptor. In these studies at
24h, crocidolite increased pEGF-R protein over sham controls (Figure 31).
To determine if cell proliferation is the phenotypic end-point for asbestos- mediated
EGF-R protein expression, as EGF is a potent mitogen in mesothelial cells, we assessed
the immunoreactivity of antibodies to EGF-R and PCNA, a marker of cell
proliferation, in mesothelial cells exposed to crocidolite, by dual fluorescence labelling
with secondary antibodies linked to rhodamine (red) and fluorescein (green),
respectively. Crocidolite increased both EGF-R protein expression and nuclear PCNA
at both 48 and 72h exposure, with TNF-α as a positive control for PCNA at both timepoints (Figure 32; data for 72h not shown). No increase in PCNA expression was
observed at 8h and 24h time-points.
68
Figure 29. Expression of EGF-R protein in RPM cells treated with crocidolite, erionite,
milled crocidolite and chrysotile aas shown by immunofluorescence detected by laser
scanning confocal microscopy. The arrows indicate cells showing increases in
immunoreactive protein
69
Figure 30. Distribution of the external domain of the EGF-R protein in RPM cells
exposed to crocidolite asbestos for 8h as indicated by the arrows in the top panel (A).
The middle panel shows both phase microscopy (B) and immunofluorescence (C)
illustrating the fusiform shape of cells encompassing long crocidolite fibres and
expressing intense patterns of EGF-R protein. Cells exposed to a number of shorter
fibres remain polygonal with no increase in EGF-R protein. The bottom panel shows
inhibition of crocidolite- mediated EGF-R protein with cycloheximde at 8h (D) and no
increase in EGF-R protein following 2h exposur e with crocidolite (E)
70
Figure 31. Expression of the phosphorylated form of the EGF-R protein in RPM cells
treated with crocidolite for 8h as shown by immunofluorescence detected by laser
scanning confocal microscopy
71
Figure 32. Crocidolite induces EGF-R expression (red) at 24, 48, 72h and PCNA
expression (green) at 48 and 72h in RPM cells as shown by co- immunofluorescence
detected by laser scanning confocal microscopy. TNF-α was used as a positive control
for PCNA expression at 48h
72
Table 2. Quantification of numbers of RPM cells showing mild or intense patterns of
EGF-R protein expression as detected by immunofluorescence with laser scanning
confocal microscopy
Mineral fibre preparation
control
crocidolite
milled crocidolite
erionite
chrysotile
% cells showing mild
staining for EGF-R protein
10 ± 4
11.54 ± 6.31
11.41 ± 3.17
10 ± 7
16.66 ± 8.66
% cells showing intense
staining for EGF-R protein
0
45.46 ± 4.54
0
40 ± 7.62
0
The results are the mean ± S.D of 10 random fields.
4.3.2 EMSA analyses of AP-1 DNA binding in mesothelial cells
To determine if crocidolite asbestos induces AP-1 DNA binding in RPM cells via a
mechanism dependent on expression of EGF-R protein, we incubated RPM cells in the
presence of suramin or tyrphostin AG 1478, inhibitors of membrane receptor binding
and EGF-R, respectively. In RPM cells treated in the presence of suramin, crocidolitemediated increases in AP-1 DNA binding activity were significantly reduced (P<0.05,
Figure 33A). A similar effect was observed when suramin was replaced by tyrphostin
AG 1478 in the incubation (P<0.05, Figure 33B).
73
4.4 DISCUSSION
The pathogenicity of mineral fibres has been attributed to both chemical and physical
properties of these fibres. The high iron content of the asbestiform mineral fibres may
drive redox reactions on the fibre surface producing reactive oxygen species (ROS)
which are both mutagenic and mitogenic (Faux et al., 1994; Howden and Faux, 1996).
Oxidant-dependent and independent mechanisms may explain why asbestos fibres over
a certain length (8 µm) are more potent in inducing mesothelioma than shorter
fibres (Davis et al., 1984). Larger asbestos fibres are able to induce greater ROS release
from alveolar and peritoneal macrophages than shorter fibres, which is thought to be
due to incomplete phagocytosis (Donaldson et al., 1988). Asbestos fibres, but not their
non- fibrous analogues, can directly trigger cell signalling pathways in mesothelial cells
that are initiated at the cell surface (Zanella et al., 1996; Jimenez et al., 1997). These
recent studies show that activation of the MAP kinase cascade in mesothelial cells
(Zanella et al., 1996) occurs following a number of phosphorylation events, including
autophosphorylation of the EGF-R. Subsequently, there is activation of transcription
factors such as nuclear factor-κB (Janssen et al., 1997) and activator protein-1 (AP-1)
(Heintz et al., 1993), which are associated with the transactivation of intermediate early
response genes important in cell proliferation and carcinogenesis.
In the present studies we have shown for the first time in normal RPM cells that
crocidolite asbestos and erionite, two chemically different fibres both known to induce
mesothelioma in rats and humans, increase the expression of a growth factor receptor
protein, EGF-R, whereas fibres such as chrysotile asbestos and non-fibrous milled
crocidolite, which do not cause mesothelioma, did not increase the expression of EGF-R
74
A
7.5
no crocidolite
-2
2.5µ gcm crocidolite
Relative Units
6.0
-2
5.0µ gcm crocidolite
4.5
3.0
1.5
0.0
control
crocidolite
crocidolite +
suramin (20µ M )
TPA
(100ng/ml)
B
2.4
no crocidolite
-2
2.5µ gcm crocidolite
2.0
-2
Relative Units
5.0µ gcm crocidolite
1.6
1.2
0.8
0.4
0
control
crocidolite
crocidolite +
Tyrphostin AG 1478 (10µ M )
Figure 33. Effect of suramin (A) and EGF-R inhibitor tyrophostin AG 1478 (B) on crocidolitemediated AP-1 DNA binding activity in RPM cells
75
protein in RPM cells. Evidence that new protein is expressed following asbestos
exposure is provided by the experiments using cycloheximide, an inhibitor of protein
synthesis (Obrig et al., 1971). Because crocidolite-mediated receptor expression is
abrogated in the presence of cycloheximide, we conclude that crocidolite asbestos fibres
cause increased expression of EGF-R protein, and not just altered distribution of the
growth factor receptor protein.
Our observations in normal RPM cells differ in the time-frame of induction of EGF-R
protein from previous observations in Met5A cells, a human mesothelial cell line (Pache
et al., 1998). In the present studies, upregulation of EGF-R protein was only observed
after 8 hours exposure, compared to 2 hours in Met5A cells. A factor that may be
important in accounting for this difference is that Met5A cells are transformed with the
SV-40 T antigen which is known to alter in vitro cellular function and cause
transformation (Sontag et al., 1997). The mechanisms by which this viral antigen
enhances the transformation of resting cells may be related to its ability to stimulate or
repress the transcription of certain genes. Thus, a wide variety of protooncogenes
encoding growth factors and growth factor receptors could be affected by SV40 in
combination with asbestos and the effect of SV40 on EGF-R expression by asbestos
cannot be ruled out in the investigations using the Met5A cell line (Pache et al., 1998).
It has been demonstrated in a number of cell types that the addition of epidermal growth
factor (EGF) causes autophosphorylation of the EGF-R that in turn activates the MAP
kinase cascade linked to transactivation of protooncogenes important in mitogenesis
(Cobb and Goldsmith, 1995). In the present studies we have shown that crocidolite
asbestos upregulates the phosphorylated form of the EGF-R by immunofluorescence
76
using a monoclonal antibody directed against the active form of this growth factor
receptor. Mesothelial cell fibre interactions lead to a number of phosphorylation events
associated with the EGF-R signal transduction pathway, involving MAP kinase. The
functional implication of EGF-R protein expression has not previously been determined
with crocidolite asbestos. In contrast to a number of other growth factors, addition of
EGF to mesothelial cells induces cell proliferation (Gabrielson et al., 1988). In the
present work, we have shown by co- immunofluorescence that the expression of EGF-R
protein in mesothelial cells preceeds the expression of PCNA, a marker of cell
proliferation, following exposure to crocidolite asbestos.
Downstream events following the initiation of cellular signalling events at the plasma
membrane by asbestos involve the activation of transcription factors, such as AP-1, that
are important in cell proliferation (Angel and Karin, 1991). Using both suramin, a
compound that uncouples G-proteins from receptors and prevents receptor function
(Kopp and Pfeiffer, 1990) and tyrphostin AG 1478, a potent and selective inhibitor of
EGF-R itself (Levitzki and Gazit, 1995), we have shown that the activation of growth
factor receptors, notably EGF-R, by asbestos is crucial to the initiation of signalling
events leading to AP-1 induction in mesothelial cells by asbestos.
In summary, we have shown that the ability of mineral fibres to upregulate the
expression and activation of EGF-R protein in RPM cells correlates with their
carcinogenicity. This suggests that chronic expression of EGF-R protein and subsequent
proliferation in RPM cells could act as a tumour promoting event fixing any DNA
damage induced after asbestos exposure, ultimately leading to neoplastic progression.
77
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