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ATLA 25, 625–639, 1997
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Genetically Engineered Cell Lines:
Characterisation and Applications in
Toxicity Testing
The Report and Recommendations of ECVAM Workshop 261,2
Friedrich J. Wiebel,3 Tommy B. Andersson,4 Daniel A. Casciano,5 Maurice
Dickins,6 Volker Fischer,7 Hansruedi Glatt,8 Jean Horbach,9 Robert J.
Langenbach,10 Walter Luyten,11 Gino Turchi12 and Alain Vandewalle13
3
Institute of Toxicology, GSF — National Research Centre for Environment and Health,
85758 Neuherberg, Germany; 4Department of Pharmacokinetics & Metabolism, Astra-Hässle,
Kärragatan 5, 43183 Molndal, Sweden; 5Division of Genetic Toxicology, NCTR, NCTR
Drive, Jefferson, AR 70279, USA; 6BIOMET Department, GlaxoWellcome, Building 2, Park
Road, Ware SG12 0DP, UK; 7Drug Metabolism & Pharmacokinetics, Novartis
Pharmaceutical Corporation, 59 route 10, East Hanover, NJ 07936, USA; 8Deutsches Institut
für Ernährungsforschung Abtl. Ernährungstoxikologie, Arthur-Scheunert-Allee 114–116,
14558 Bergholz-Rehbrücke, Germany; 9RITOX, Utrecht University, 3508 TD Utrecht, The
Netherlands; 10Laboratory of Carcinogenesis/Mutagenesis, NIEHS, Research Triangle Park,
NC 27709, USA; 11Department of Biochemical Pharmacology, Janssen Pharmaceutica NV,
Turnhoutsebaan 30, 2340 Beerse, Belgium; 12Dipartimento di Scienze dell’Ambiente e del
Territorio, Università di Pisa, Via S. Giuseppe 22, 56100 Pisa, Italy; 13INSERM U246,
Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France
Preface
This is the report of the twenty-sixth of a
series of workshops organised by the European Centre for the Validation of Alternative
Methods (ECVAM). ECVAM’s main goal, as
defined in 1993 by its Scientific Advisory
Committee, is to promote the scientific and
regulatory acceptance of alternative methods
which are of importance to the biosciences
and which reduce, refine or replace the use of
laboratory animals. One of the first priorities
set by ECVAM was the implementation of
procedures which would enable it to become
well-informed about the state-of-the-art of
non-animal test development and validation,
and the potential for the possible incorpora-
tion of alternative tests into regulatory procedures. It was decided that this would be
best achieved by the organisation of ECVAM
workshops on specific topics, at which small
groups of invited experts would review the
current status of various types of in vitro
tests and their potential uses, and make recommendations about the best ways forward
(1).
The workshop on Genetically Engineered
Cell Lines: Characterisation and Applications in Toxicity Testing was held in Angera,
Italy, on 26–27 February 1996, under the
chairmanship of Friedrich Wiebel (Institute
of Toxicology, GSF, Neuherberg, Germany).
One of the reasons for holding the workshop
was the recognition that genetically engi-
Address for correspondence: Professor Friedrich J. Wiebel, Institute of Toxicology, GSF — National Research
Centre for Environment and Health, 85758 Neuherberg, Germany.
Address for reprints: ECVAM, TP 580, JRC Environment Institute, 21020 Ispra (VA), Italy.
1
ECVAM — European Centre for the Validation of Alternative Methods. 2This document represents the agreed
report of the participants as individual scientists.
F.J. Wiebel et al.
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neered cell lines, particularly those expressing xenobiotic metabolising enzymes, are
being used increasingly in toxicological and
pharmacological studies, yet few of these cell
lines have been properly characterised,
which is of critical importance if they are to
be used for routine testing purposes.
The objectives of the workshop were to: a)
review the current status of genetically
engineered cell lines, with an emphasis on
mammalian cell lines expressing xenobiotic
metabolising enzymes; b) discuss the construction of genetically engineered cell
lines, including those derived from transgenic animals; c) review current applications of genetically engineered cell lines in
the drug development process; d) make recommendations for the further development
of genetically engineered cell lines; and e)
propose initiatives for: i) standardising the
nomenclature of recombinant cell systems;
ii) standardising, validating and ensuring
the quality control of the systems; and iii)
making them more readily available to the
scientific community.
Introduction
Established cell lines represent useful alternative test systems for toxicological and
pharmacological studies. A number of features favour their use, particularly in industrial and regulatory toxicology: a) they are
well-defined and stable; b) they are readily
available; c) they can be standardised and
exchanged between laboratories; and d) they
are suitable for the determination of cytotoxicity and genotoxicity.
Formerly, the usefulness of most established cell lines was severely restricted by
their low content of xenobiotic metabolising
enzymes. Attempts were made to overcome
this shortcoming by supplying the cell lines
with an exogenous source of these enzymes,
such as the post-mitochondrial supernatant
of the liver. However, this approach was only
partially successful. The exogenous enzyme
preparations were difficult to standardise,
the balance of the various xenobiotic
metabolising enzymes was distorted, and
there was always the risk that short-lived,
reactive metabolites formed outside the cells
would not reach their intracellular targets,
yielding false negative results. The prospects
for using established cell lines became much
brighter with the advent of genetic engineering techniques, which now enable substantial levels of xenobiotic metabolising
enzymes to be attained, as required, in established cell lines.
Genetically engineered cells (also termed
“cDNA expression systems” or “recombinant
cell systems”) expressing xenobiotic
metabolising enzymes have opened up new
fields of application in toxicology. They can
be used for determining the contributions of
specific enzymes to the overall metabolic
profile of a given xenobiotic, or for studying
the role of metabolism in its toxic effects.
Genetically engineered cells can also be used
to screen chemicals for toxic potential and to
analyse the molecular mechanisms by which
xenobiotics exert their biological effects.
Construction of Cell Lines Expressing
Xenobiotic Metabolising Enzymes
There are three main steps in the construction of genetically engineered cell lines: a)
the generation of a full-length cDNA for the
protein to be expressed; b) the selection of an
expression vector which is compatible with
the host cell; and c) the transfer of the
expression vector containing the cDNA into
the host cell.
The cDNAs for many of the xenobiotic
metabolising enzymes have already been
cloned and are becoming available for commercial purposes. This applies in particular
to the cytochromes P450 which, playing a
pivotal role in xenobiotic metabolism, are the
subjects of intense study. If the cDNAs of
specific isoforms of xenobiotic metabolising
enzymes are not available, full-length cDNAs
can be synthesised by applying standard
reverse transcriptase polymerase chain reaction procedures (2).
Choice of expression system
Several types of systems are available for the
heterologous expression of xenobiotic
metabolising enzymes (Table I). The advantages and disadvantages of these systems
have been described (2, 3). The choice of
expression system is governed by a variety of
criteria.
Of primary importance is the overall purpose of the proposed study. It governs the
decision as to whether the expression of the
xenobiotic metabolising enzymes should be
ECVAM Workshop 26: genetically engineered cell lines
Table I: Types of heterologous expression systems
Duration
transient
permanent
System
bacteria
yeast
insect cells
mammalian cells
transgenic animals
transient or permanent, in prokaryotic or
eukaryotic cells, and as monolayer or suspension cultures. For example, the most relevant systems for determining the biological
effects of xenobiotics are continuous cell
lines, which are capable of permanently
expressing the heterologous enzyme(s). In
contrast, for studies of xenobiotic metabolism, transient expression systems containing higher expression levels may be more
appropriate. Also, if large quantities of the
enzyme are required, cells capable of growing in suspension are preferable to anchorage-dependent cells, because they are easier
to scale-up to large cultures with high cell
yields.
Another important criterion concerns the
nature of the endpoint to be measured. For
example, while macromolecular binding can
be measured in virtually any cell system,
measurements of gene locus mutations or
observations of malignant transformation
are only possible in a restricted array of cells.
A consideration which is often overlooked
when selecting expression systems is the
“background” of xenobiotic enzymes
against which the heterologous enzyme is to
be expressed. Not only do bacteria, yeast,
insect cells and mammalian cells differ
greatly in their endogenous expression of
xenobiotic metabolising enzymes, but mammalian cell lines derived from various tissues and species also exhibit widely
different patterns of these enzymes (4).
Knowledge of the metabolic profile of the
host cells is of paramount importance if the
proposed study is aimed at analysing complex pathways of xenobiotic metabolism,
and at evaluating the biological effects of
the various metabolites.
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Finally, the expression system should possess the following properties: a) a high transfection frequency; b) a capacity for
expressing the enzyme of interest; c) stability; and d) maintenance of the catalytic
fidelity of expressed enzymes.
Choice of expression vector
The choice of vector partially determines
whether transient or stable expression of the
cDNA-encoded enzyme is achieved in the
host cell. Transient expression systems are
generally based on a viral vector. The host
cells are infected with a cDNA-bearing virus,
and the cDNA-derived protein is harvested
as soon as maximal expression has been
achieved. Since viral vectors usually cause
lysis, the host cells are generally not suitable
for studying metabolism-dependent toxicity.
Stable expression systems can be based on
either integrating vectors or extrachromosomal vectors (5). The use of an integrating
vector is advantageous since it enables cDNA
expression to be maintained in the absence
of selection for the vector, but is disadvantageous in that the level and stability of
expression are affected by the site of integration in the host genome and by the number
of integration events. In contrast, when an
extrachromosomal vector is used, selection is
necessary for long-term stability, but there is
little variability in expression between isolated clones of transfected cells.
Extrachromosomal vectors do not need to
integrate in the host genome to achieve
expression because they carry their own origin of replication. For example, the extrachromosomal vector derived from the
Epstein–Barr virus carries OriP sequences
which act with the viral EBNA-1 gene product to stably transform human lymphoblastoid AHH-1 cells (6). In contrast, integrating
vectors do not contain a eukaryotic origin of
replication, so they have to integrate in the
host genome to achieve stable transformation. In the case of plasmid DNA, integration
can be improved by linearisation of the plasmid before transfection (7).
Alternatively, efficient integration in the
host genome can be achieved by using retroviral vectors (8). This has enabled the stable
expression of several human cytochrome
P450 cDNAs (9–13).
Another factor influencing the choice of
vector is the promoter required for expression of the cDNA of interest. The most com-
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monly used promoters are the SV40 late promoter, the cytomegalovirus promoter, the
thymidine kinase (TK) promoter from the
herpes simplex virus, and the promoter from
the long terminal repeat of the murine
moloney leukemia virus. The most suitable
promoter will depend on the nature of the
host cell.
In summary, there is no general rule for
choosing a vector applicable to all cell lines
and suitable for all applications. At present,
retroviral vectors are the most versatile gene
carriers in that they can be used with a wide
range of host cells. However, they do not necessarily provide the highest levels of expression.
Introduction of vectors into cells
Vectors encoding cDNAs can be transfected
into host cells in a variety of ways (14). The
usual method is calcium phosphate precipitation, but other methods include retroviral
infection, electroporation, and direct
microinjection of the vector into the nucleus.
Another method, lipofection, uses commercially available cationic liposomes and may
result in a higher efficiency, depending on
the cell type (15).
In the case of retroviral infection, the
retroviral vector carrying the cDNA is first
introduced into a helper cell line, which produces the viral proteins necessary for packaging the retroviral RNA (16). Upon
infection of a cell, the retroviral RNA
released into the cytoplasm is transcribed by
reverse transcriptase to DNA, which
migrates to the nucleus. This strategy
results in the efficient integration of the
cDNA into the host genome. No spreading
infection can occur because the viral proteins
needed for replication of the integrated virus
are not available in the host cell (17).
Selection of cell clones
To select for cell clones which have taken up
the expression vector, co-transfection can be
carried out with a plasmid carrying a selectable marker gene, such as the neomycin
phosphotransferase gene or the hygromycin
B resistancy gene. The likelihood of the
selection plasmid being taken up along with
the expression vector can be improved by
carrying out the transfection with plasmid
mixtures containing only 5% or less of the
selection plasmid. An even better strategy is
F.J. Wiebel et al.
to use expression vectors which carry both
the selection marker and the cDNA of interest (10, 11, 18).
Validation of cDNA-expressed enzymes
To validate an expression system, the kinetic
parameters of the expressed enzyme need to
be verified, taking into account the effects of
inducers and co-factors. Verification of
kinetic parameters is particularly important
if the primary sequence of the enzyme has
been modified to achieve expression, for
example, in bacteria.
It is suggested that comparisons are
undertaken between the levels and activities
of the human xenobiotic metabolising
enzymes expressed in genetically engineered
cell lines and those expressed in human liver
microsomes. Ideally, such a comparison
would include a consideration of: a) the
apparent Km; b) the apparent Vmax; and c) the
rank order of metabolism rates of different
substrates.
Expression of Xenobiotic Metabolising
Enzymes in Bacteria
The heterologous expression of human xenobiotic metabolising enzymes in bacteria,
such as Escherichia coli, is a fast and inexpensive way of producing large quantities of
the polypeptide. It is therefore the method of
choice when attempting to crystallise a given
enzyme for use in structural studies. However, bacterial systems are only useful when
the intended application does not require
post-translational modifications of the
enzyme, such as glycosylation. Furthermore,
modifications have to be made to the cDNA
sequence to achieve efficient expression (19).
Another disadvantage is that a given human
cytochrome P450 can only be characterised
when it has been purified and reconstituted
in the presence of lipids, cytochrome P450
reductase, and other co-factors. It may therefore be difficult to obtain the authentic molecular structure and substrate specificity of
the human cytochrome in question.
Expression of Xenobiotic Metabolising
Enzymes in Yeast
Several properties make yeast an attractive
system for heterologous protein expression:
ECVAM Workshop 26: genetically engineered cell lines
629
a) it is a eukaryote with a similar metabolic
profile to that of mammalian cells; b) it can
be grown quickly and cheaply to high cell
densities, allowing for large-scale fermentation; c) a variety of different host strains and
expression vectors are available, offering an
abundant choice of promoters, selection
markers and plasmid copy numbers; d) it
enables either constitutive or inducible
expression, and even gene replacement; and
e) making constructs and transforming yeast
is straightforward.
The yeast Saccharomyces cerevisiae has
been used extensively for the expression of
mammalian (including human) xenobiotic
metabolising enzymes, particularly the
cytochromes P450 (20). It is an attractive
host for cytochrome P450 expression because
the low levels of endogenous cytochrome
P450 are combined with the presence of
yeast NADPH-P450 oxidoreductase and
cytochrome b5. Most of the cytochromes
P450 heterologously expressed in yeast have
been functional, enabling the metabolism of
xenobiotics to be studied by using either
intact cells or microsomal preparations. In
general, the level of expression has been sufficiently high for enzyme kinetic and spectroscopic studies to be performed.
The S. cerevisiae expression system has
been improved by co-expressing human
cytochromes P450 with NADPH-P450 oxidoreductase and cytochrome b5 (21, 22).
Phase II enzymes have also been coexpressed (23). By fine-tuning the relative
levels of these enzymes, the in vivo situation
can be mimicked without losing the advantage of working with well-defined and pure
components. Another development has been
the combined use of yeast expression and
computer simulations of enzyme kinetics
(23).
A disadvantage of using yeast cells is the
rigid cell wall, which complicates the preparation of microsomes, and may pose a barrier
for the uptake of test chemicals. This disadvantage could be overcome by developing cell
wall mutants. Also, drug–drug interactions
cannot be studied in yeast, unless inducible
transcription factors are also incorporated.
Another disadvantage is that heterologous
cytochrome P450 does not always interact
well with endogenous NADPH-cytochrome
P450 reductase and cytochrome b5.
Despite its present shortcomings, genetically engineered yeast is a potentially useful
system for: a) the study and prediction of
xenobiotic metabolism; b) the identification
and manufacture of metabolites; c) genotoxicity testing; d) achieving a better understanding of structure–function relationships
in P450 enzymes; and e) the production of
recombinant cytochrome P450 for therapeutic and biosensor applications.
Expression of Xenobiotic Metabolising
Enzymes in Insect Cells
A number of xenobiotic metabolising
enzymes, including cytochromes P450 and
flavin-dependent mono-oxygenases, have
been expressed in insect cells infected with
baculovirus constructs containing the relevant cDNA sequences (24). The baculovirus
expression system is suitable for the expression of human enzymes because the infected
insect cells are eukaryotic and therefore contain the cell organelles which target the
recombinant proteins to their appropriate
subcellular compartments. This enables a
preparation analogous to human liver microsomes to be generated for use as an in vitro
metabolising system. The advantages and
disadvantages of baculovirus expression are
summarised in Table II.
A detailed protocol for constructing recombinant baculovirus was developed by Summers & Smith (25). The baculovirus used for
cDNA expression, AcMNPV, has a genome of
about 130,000 base pairs, so the cDNA cannot be directly inserted by using standard
enzymatic procedures (26). Instead, the
cDNA is first cloned into a shuttle vector,
which then inserts the cDNA into the baculovirus genome. Typically, the cDNA is
inserted at the site of the polyhedrin gene.
The capsule protein specified by this gene
protects the virus during its life cycle in
insects, but is not essential for viral replication or infection. The polyhedrin protein can
represent up to 50% of the total cellular protein in the “late” stages (> 20 hours) of
infection. Following infection, the synthesis
of other insect cell proteins is down-regulated in a time-dependent manner, so the
proteins of interest are the dominant ones at
the time of harvest. The baculovirus life
cycle results in the lysis of insect cells at late
stages of infection. Thus, this expression system can be used as a lysed cell or membrane
preparation, but not as a continuous cell line.
F.J. Wiebel et al.
630
Table II: Advantages and disadvantages of baculovirus expression systems
Advantages
dispensable gene products — polyhedrin, p10
genome can accommodate large cDNA fragments
strong promoter for high level expression
eukaryotic system — appropriate membrane environment
— post-translational processing
safe system — low risk to user
easy to scale-up to large volume bioreactors
Disadvantages
transient system — virus-mediated lysis of host cells
insect cells take longer to culture than bacteria or yeast
A dual expression vector has been developed which uses both the polyhedrin site and
the site for another capsule protein, p10,
which is under the control of a strong promoter. This makes it possible to insert, for
example, cytochrome P450 cDNA at the
polyhedrin site and cDNA for human
cytochrome P450 reductase at the p10 site,
with the promoters being aligned in opposite
directions. Insect cells are infected with the
recombinant baculovirus and are harvested
72 hours later for the preparation of a membrane fraction. In the presence of an appropriate substrate, a co-factor (NADPH) and a
source of haem precursors, cytochrome P450
activity is readily reconstituted and is comparable to that obtained when liver microsomes are used as the enzyme source (27).
Expression of Xenobiotic Metabolising
Enzymes in Mammalian Cells
Compared with bacterial and yeast cells,
mammalian cells are expensive and difficult
to cultivate. However, they are more relevant for the detection of certain toxicological endpoints, such as cytotoxicity,
mutagenicity, chromosomal abberations,
micronucleus formation, and cellular transformation. Mammalian cells are particularly well-suited for the expression of
cytochromes P450, because the activities of
these enzymes are dependent on the recipient cells containing adequate amounts of
endoplasmic reticulum, NADPH-P450 oxidoreductase and cytochrome b5, all of which
are required for optimal functioning of
cytochromes P450.
The first mammalian system used for the
heterologous expression of a xenobiotic
metabolising enzyme, a cytochrome P450,
was the African Green Monkey kidney cell
line, COS (28). This system constitutively
expresses the SV40 large T antigen, which
maintains incoming extrachromosomal vectors carrying the SV40 origin of replication.
The system is only suitable when transient
expression not exceeding 4 days is sufficient
for the study in question. Alternatively,
cytochrome P450 genes can be transferred
into COS cells by infection with vaccinia
virus (29) or RNA tumour virus (30).
In the last decade, at least 20 cell lines
have been genetically engineered for the
expression of xenobiotic metabolising
enzymes (Table III). Most of these cell lines
are derived from human and hamster tissues; relatively few are derived from mice or
rats.
Human cells
Human cells expressing human xenobiotic
metabolising enzymes are more likely to be
predictive of human susceptibility to the biological effects of chemicals than non-human
cells. The use of human cells in toxicological
and pharmacological research has been
reviewed by Crespi (5).
A range of cell constructs engineered for
the single or multiple expression of human
cytochromes P450 has been developed from
the human B-lymphoblastoid cell line,
AHH-1 TK+/– (18, 31, 32). The cells have
the following characteristics: a) they grow
in suspension culture; b) they form colonies
with an efficiency of 30–50%; c) they contain low constitutive cytochrome P4501A1
ECVAM Workshop 26: genetically engineered cell lines
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Table III: Cell lines used for the heterologous expression of xenobiotic metabolising
enzymesa
Species
Recipient cell lineb
Human
MCF-7
AHH-1 TK+/–
T47D
Hep G2
Chinese hamster
V79MZ
V79
V79NH
CHO
CHO-UV5
CHL
8
4
1
9
2
2
Syrian hamster
BHK21 Cl.13
1
Mouse
NIH/3T3
CH3 10T1/2
NS20Y
BTG9
5
4
1
1
Rat
L
MatB
H4IIE
2
1
1
a
Number of constructs
9
11
2
1
From the Catalogue of Cell Lines in Toxicology & Pharmacology (4).
b
Cell lines expressing the transfected enzymes permanently (for at least ten passages).
activity, which is inducible by polycyclic
aromatic hydrocarbons; and d) they can be
used for measuring gene mutations at the
hypoxanthine guanine phosphoribosyl
transferase (HGPRT) and TK loci. The heterologous genes are contained on extrachromosomal plasmid vectors, of which 5–40
copies per cell are present, depending on the
selection system used. A subclone of AHH-1
TK+/– cells has been used to construct a
series of cell lines expressing up to five different xenobiotic metabolising enyzmes
(31).
Whereas AHH-1 TK+/– cells are largely
employed as the recipients of cytochrome
P450 cDNAs, the human mammary tumour
cell line, MCF-7, is used as an expression system for enzymes such as aldehyde dehydro-
genase, aromatase, glutathione peroxidase
and glutathione S-transferase (4).
The partially differentiated human
hepatoma line, Hep G2, has been genetically
engineered to transiently express human
cytochrome P450 cDNAs by using a vaccinia
virus vector (33). The enzyme levels in cell
lysates, 3–4 days after infection, are sufficient for conducting spectroscopic and
enzyme kinetic studies (34).
Hamster cells
Two Chinese hamster cell lines have been
widely used for the heterologous expression
of xenobiotic metabolising enzymes: V79
lung cells and Chinese hamster ovary (CHO)
cells. The two lines are of interest for different reasons. V79 cells are distinguished by
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their fast growth rate and high cloning efficiency, making them ideal for determining
the mutagenic potentials of chemicals. In
addition, they are unusual in that they lack
major types of xenobiotic metabolising
enzymes, such as cytochromes P450, glucuronosyl transferase, and phenol sulphotransferase (4, 35). For example, only
minimal activities of cytochrome P4501A1
have been detected in these cells (36), at
amounts too low to be of concern in toxicological studies. Stable expression of several
isoforms of cytochrome P450 was achieved in
V79 cells by integrating the SV40 early promoter vector into chromosomal DNA (37).
Recently, V79 cells were constructed for the
stable co-expression of a cytochrome P450
and NADPH-cytochrome P450 reductase, to
compensate for the low endogenous activity
of the latter enzyme (38).
CHO cells share many properties in common with V79 cells. Variants of CHO cells
deficient in DNA repair have been particularly useful in toxicological studies. Constructs of these cells expressing, for example,
cytochromes P450 (39), are the systems of
choice for analysing the mechanisms by
which chemicals are metabolically activated
and by which DNA is subsequently damaged
and repaired.
Mouse cells
Two closely related mouse cell lines,
NIH/3T3 and CH3 10T1/2, derived from
embryos of the Swiss NIH mouse and the
CH3 mouse, respectively, have been used as
heterologous expression systems. Their use
has been instrumental in elucidating mechanisms of growth control and neoplastic
transformation. Although both cell lines contain low levels of cytochromes P450 (4), the
expression of these enzymes can be increased
10–50 fold by retroviral transfection of the
cells (10, 13).
Establishment of Cell Lines from
Transgenic Mice
The development of the techniques of transgenesis, by which foreign genes are introduced into the germ line, represents a major
technological advance in biological research.
Various models of carcinogenesis have been
established by using transgenic mice (40). In
these models, the transgene consists of an
F.J. Wiebel et al.
oncogene, generally the SV40 large T antigen or small t antigen, placed under the control of the regulatory sequences of a
tissue-specific gene. The transgene will only
be activated in the cell type expressing the
corresponding endogenous gene and will be
regulated accordingly. Cells carrying such
transgenes and the appropriate sets of transcription factors combine the proliferative
capacity of transformed cells with the maintenance of cell-specific functions.
A large variety of epithelial and nonepithelial cell lines, exhibiting hepatic,
intestinal, renal or neuronal properties, have
been established by transgenesis. For example, proximal tubule cell lines have been
derived from transgenic mice carrying the
SV40 large T or small t antigen, placed under
the control of the 5´-regulatory regions of
the L-pyruvate kinase gene. The cell lines
maintain the major specialised functions of
proximal tubule cells (41). They form monolayers of polarised cells, express microvilliassociated hydrolases and villin, are sensitive
to parathormone, and express the transcription factors which regulate the expression of
glycolytic and neoglucogenic enzymes. In
addition, they exhibit a high capacity for
fluid-phase endocytosis and express basolaterally-located receptors for peptide YY (42).
Since the proximal tubule represents the
preferential site of action of many xenobiotics, proximal tubule cell lines offer a useful
model for studying the toxic effects of drugs
(43).
In the near future, new types of transimmortalised cell lines are likely to be
derived from transgenic mice carrying other
types of transgenes. Hopefully, new cell lines
will also be derived from transgenic animals
other than the mouse, such as rats and rabbits.
Applications of Genetically
Engineered Cell Lines in Drug
Development
Pharmaceutical companies are required by
regulatory agencies to study the metabolism
of drug candidates. Traditionally, metabolism studies were performed in vivo by using
animal models such as the rat, dog and monkey. In recent years, however, there has been
a considerable increase in the use of in vitro
systems (44). Genetically engineered cell
ECVAM Workshop 26: genetically engineered cell lines
633
lines have made a significant contribution to
this trend.
The potential uses of heterologously
expressed enzymes in drug development
have been reviewed extensively by Remmell
& Burchell (45). Current applications
include: a) the generation and identification
of drug metabolites; b) the prediction of in
vivo clearance; c) the study of drug–drug
interactions; and d) the study of enzyme
induction. The induction of drug metabolising enzymes by the drug candidate can cause
serious clinical problems, since the metabolism of both the drug candidate and other coadministered drugs may be increased. At
present, only the induction of CYP1A can be
studied in human cell lines.
In the drug development process, genetically engineered cell lines are generally used
in combination with other in vitro systems,
forming an integrated programme of tests.
At an early stage in the process, emphasis is
often placed on finding compounds which are
metabolically stable and on avoiding metabolism by “disadvantageous” enzymes. At a
later stage, routine investigations are carried
out into metabolic profiles and potential
drug–drug interactions, and the specific
enzymes which metabolise the drug candidate in humans are identified. Most studies
have focused on the human cytochrome P450
isoforms considered to play clinically relevant roles. The interest in cytochromes P450
for drug development is reflected in the
plethora of cell lines genetically engineered
for the expression of these enzymes (Table
IV). Increasing numbers of cell lines are also
being established for the heterologous
expression of other xenobiotic metabolising
enzymes; this is particularly true for various
types of transferases, such as the sulphotransferases (46, 47).
In the future, the process of drug discovery and development would benefit from: a)
the immortalisation of functional human
hepatocytes and, if applicable, the stable
integration into cell lines of DNA-responsive elements fused to reporter genes; b) the
automation of efficient systems for selecting
metabolically advantageous compounds in
early screening programmes; and c) the
development of systems expressing all types
of human drug metabolising enzymes. For
those enzymes in which human polymorphisms have been observed (48), for example, N-acetyltransferases and glutathione
Table IV: Heterologous expression of xenobiotic metabolising enzymes in
mammalian cell linesa
Enzyme
Aldehyde dehydrogenase
Catalase
DT-Diaphorase
Epoxide hydrolase
Sulphotransferase
Aromatase
Glutathione-peroxidase
UDP-Glucuronosyltransferases
Superoxide dismutases
N-Acetyltransferases
Glutathione S-transferases
Cytochromes P450
a
b
Number
of constructs
Number
of cell lines
Number
of speciesb
1
1
1
1
1
2
1
1
1
1
1
2
1
1
1
1
1
1
2
4
4
6
11
31
2
1
3
1
6
14
2
1
1
2
3
5
From the Catalogue of Cell Lines in Toxicology & Pharmacology (4).
Species from which the enzyme is derived.
F.J. Wiebel et al.
634
S-transferases, the availability of cell lines
expressing the different isoforms would be
useful.
Catalogue of Cell Lines in Toxicology &
Pharmacology
A Catalogue of Cell Lines in Toxicology &
Pharmacology (CCLTOP) has been produced
by Sabine Hornhardt and Friedrich Wiebel
(GSF, Neuherberg, Germany), with the support of ECVAM (4). The catalogue provides
information on: a) parent cell lines with
regard to their characteristics and their
endogenous expression of xenobiotic
metabolising enzymes; and b) genetically
engineered cell lines with regard to the types
of enzymes expressed, the standard substrates used, enzyme activities, and appropriate references. An indication of the type of
information provided is given in Tables III
and IV. Copies of the CCLTOP are available
from ECVAM.
The information on the expression of
xenobiotic metabolising enzymes in established cell lines was retrieved from a variety
of data banks and from a wide range of literature sources. Typically, the data given in
CCLTOP are taken from peer-reviewed publications. The cell lines included in the catalogue are all considered to be “permanent”,
that is, stable for at least ten passages. They
are supposed to exist in frozen stocks and to
be available to researchers either commercially or from the scientists who established
or used the cells. The wealth of information
included in CCLTOP should form the basis
for a rational selection of carrier cells in the
future.
Conclusions and Recommendations
Standardisation of nomenclature
1. The nomenclature of recombinant cell
systems/lines should be standardised.
The workshop participants suggest that
the following nomenclature is adopted:
cell line/species (abbreviated) — enzyme
(abbreviated)
For example, Chinese hamster ovary cells
(CHO) genetically engineered to express
human cytochrome P4501A2 would be
referred to as: CHO/hCYP1A2.
Quality control of recombinant cell systems
2. The developers of recombinant systems
should characterise their properties,
including: a) their profile of xenobiotic
metabolising enzymes; b) their growth
characteristics (for example, cloning efficiency and division time); c) their karyotype; and d) their stability. In addition,
the following should be documented: a)
the cDNA; b) the vector; c) the optimal
culture conditions; d) the presence of contaminants (for example, mycoplasma);
and e) the presence of inducers of the
recombinant enzyme. The recombinant
enzyme should be characterised with
respect to: a) the total protein expressed;
b) its kinetics (that is, the Km and Vmax
values for marker substrates); and c) its
lack of metabolism of marker substrates
for other enzymes. Ideally, the kinetic
parameters obtained for recombinant
enzymes should be compared with those
obtained with human liver microsomes or
cytosolic fractions.
Use of recombinant cell systems
3. The users of recombinant systems should
be responsible for: a) providing information on the passage number of the cells
being studied; b) providing information
on the actual activity of the enzyme
toward marker substrates; and c) checking for any potential effects of recombination on the genetic endpoint of
interest.
4. Host cells should be selected according to
the objective of the toxicity testing. If the
aim is to determine the toxic potentials of
organic chemicals, the host cell should
contain as few xenobiotic metabolising
enzymes as possible. If the aim is to evaluate the toxic potency, the host cells
should mimic the in vivo situation as
closely as possible.
Further development of recombinant cell
systems
5. There is a need to develop systems
which express xenobiotic metabolising
enzymes, other than the cytochromes
P450, which are potentially involved in
bioactivation and/or deactivation, for
example, 1-electron oxidoreductases,
sulphotransferases, prostaglandin syn-
ECVAM Workshop 26: genetically engineered cell lines
635
thase, flavin-dependent mono-oxygenases, β-lyase and conjugating enzymes.
Research on xenobiotic metabolising
enzymes which are poorly characterised
at present (for example, esterases)
should be supported. Priority should be
given to enzymes with known genetic
polymorphisms, such as N-acetyltransferase and glutathione S-transferase. In
those cases where polymorphic enzymes
differ in their substrate specificities, cell
systems which express the major variants should be developed.
production, should be established, so that
these can be made readily available to the
scientific community. The repository
should be supported by a comprehensive
database. An outline of the proposed purpose, structure and contents of the repository and database is given in Appendix 1.
It is recommended that ECVAM establishes a working group to define more
precisely the objectives and requirements
of such a repository and supporting database.
6. For risk assessment purposes, xenobiotic
metabolising enzymes of commonly used
species of laboratory animals need to be
expressed in recombinant cell systems.
Priority should be given to rat enzymes,
since many pharmacological and toxicological data are derived from this species.
Also, systems expressing cytochrome
P450 isoforms of key food-producing animals (for example, cattle and swine)
should be constructed.
7. For studies on mechanisms of metabolic
activation, enzymes acting in concert,
such as cytochrome P4501A2 and Nacetyltransferases or sulphotransferases,
need to be co-expressed in recombinant
systems. This also applies to enzymes
involved in competing metabolic pathways. In general, greater effort must be
directed toward developing recombinant
systems which express more than one
xenobiotic metabolising enzyme and
which are tailored to the mechanisms of
activation and inactivation of specific
groups/classes of chemicals.
8. It is essential to establish mammalian
cell lines which exhibit high levels of
enzyme activities (for example, of the
magnitude achievable in bacterial recombinant systems). Preferably, the host
cells should be of human origin, since this
should aid extrapolation of the results
obtained to the human situation.
9. Possibilities for using transgenic animals
as a source of cell lines exhibiting specific
genotypes should be further investigated.
11. The Catalogue of Cell Lines in Toxicology
& Pharmacology (CCLTOP) is a valuable
source of information on mammalian
host cells and constructs. It is recommended that efforts are made to make it
available on the Internet.
Acknowledgements
This report was compiled and edited by
Andrew Worth and Julia Fentem (ECVAM).
The workshop was attended by Sarah Bull
(ECVAM), who has also contributed to the
report, and by Dietmar Pettauer (European
Chemicals Bureau, Joint Research Centre,
Ispra, Italy), both as observers.
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Appendix 1
Proposal for a European Repository of Recombinant Cell Systems
(EURECS)
The proposed repository could have an
autonomous structure or belong to an academic or government institution, such as a university or research centre. A possible
alternative name (and abbreviation) for the
repository is the European Repository of
Genetically
Engineered
Cell
Lines
(ERGENIC).
Management
The management would have overall control
of the repository. It would be responsible for:
a) ensuring the quality and stability of the
biological materials held in the repository; b)
making them available to all users; and c)
checking the accuracy of data inserted into
the database (see below). The management
would be supported by a scientific committee, which would discuss and approve proposed improvements and/or expansions of
the system. It would be able to delegate tasks
to experts in specific fields.
Since the repository would be a non-profit
making organisation, it would need to gain
financial support from a variety sources, for
example: a) ECVAM and/or other services of
the European Commission; b) national centres for the validation of alternative methods
(for example, the Netherlands Centre Alternatives to Animal Use, Utrecht, The Netherlands, and ZEBET, BgVV, Germany); and c)
industrial companies. Some additional support could be derived from sales and the provision of technical services.
Database
A database should be set up for informing
potential users on the materials maintained
in the repository and on their availability.
The database should be readily accessible,
that is, provide for a flexible data retrieval
system. The structure and content of the
database should be devised by a group of
experts, paying particular attention to the
requirements of users and to the availability
of existing databases in the field. For example, data could be collated by collaborating
with the European Node of Hybridoma Data
Bank, the European Collection of Animal
Cell Cultures (ECACC), the European Collection for Biomedical Research, the Microbiological Strain Data Network, and the
Catalogue of Cell Lines in Toxicology &
Pharmacology.
The following information on the biological materials should be available: a) identification (name, code, etc.); b) origin
(species, strain, sex, tissue, clone, etc.); c)
specific function(s) (organ of origin, primary, established, tumour-derived, etc.); d)
preservation and culture characteristics
(culture medium, serum medium, serum
freezing, antibiotics, specific additive[s],
split ratio, growth factors, mycoplasma,
etc.); e) retrieval sources (references); f)
specific function(s) after genetic manipulation (gene expressed, level of expression,
polymorphic variants, etc.); and g) constructs, vectors (method of production,
characteristics, etc.).
Repository
Cell cultures, micro-organisms, vectors and
constructs should be stored in liquid nitrogen tanks safeguarded by appropriate monitoring systems. The repository should
carry out basic quality controls to ensure
that the material is authentic and free from
contamination. If necessary, materials
could be returned to the depositor for verification. Biological materials (micro-organisms, vectors, cell lines and constructs)
would be made available to all potential
users, subject to a number of conditions
elaborated by the scientific committee. The
precise conditions would differ for users
affiliated with non-profit institutions or
projects and for users having commercial
interests. In the latter case, biological
materials would not be offered for sale or
ECVAM Workshop 26: genetically engineered cell lines
used for commercial purposes without the
prior agreement of the original depositor.
The main conditions envisaged are the following.
1. The biological material should not be distributed to third parties.
2. Publications relating to the biological
material should refer to the work of the
original depositor. No alteration should
be made to the original name or code.
3. Any innovative product or derivative
developed from biological material supplied by the repository must be deposited
into the collection.
4. Data derived from the use of repository
biological material should be submitted
for entry into the repository databank.
The European Repository as an
International Depositary Authority
To protect an invention on an international
basis, the inventor must secure a patent
from either: a) a national patent office in a
country/state which is party to the appropriate international treaty; or b) a regional
patent office, such as the European Patent
Office (EPO). Where an invention involves a
micro-organism or the use of a micro-organism, disclosure is not possible in writing; it
can only be effected by the deposit of a sample of the micro-organism with an appropriate institution.
639
To avoid the need to deposit a sample in
each country in which protection is sought,
the Budapest Treaty on the International
Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure
(the Budapest Treaty; 1) provides that the
deposit of a micro-organism with any international depositary authority suffices for the
purposes of patent protection throughout all
of the contracting states. A so-called “international depositary authority” (IDA) is any
scientific institution capable of storing
micro-organisms which has acquired the status of IDA through the provision (by the contracting state in which it is located) of
assurances to the Director General of the
World Intellectual Property Organization to
the effect that the institution complies with,
and will continue to comply with, certain
requirements of the Budapest Treaty.
The European Repository could therefore
seek to become an IDA for genetically engineered cell lines, micro-organisms, vectors
and constructs, under the terms and conditions of the Budapest Treaty, by applying to
the EPO, or to the patent office of the Member State in which it is located.
Reference
1.
Anon. (1977). Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure, 12 pp.
Geneva: World Intellectual Property Organization.