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ATLA 25, 625–639, 1997 625 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. 626 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. 627 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- 628 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 631 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 632 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. References 1. 2. 3. 4. 5. 6. Establishment of a repository and database 10. A repository of recombinant cell systems, and the micro-organisms, vectors, host cell lines and constructs used in their 7. Anon. (1994). ECVAM News & Views. ATLA 22, 7–11. Doehmer, J. & Greim, H. (1993). Cytochrome P450 in genetically engineered cell cultures: the gene technological approach. In Handbook of Experimental Pharmacology, Volume 105, Cytochrome P450 (ed. J.B. Schenkman & H. Greim), pp. 415–429. Berlin: Springer Verlag. Waterman, M.R. & Johnson, E.F. (1991). Methods in Enzymology, Vol. 206, Cytrochrome P450, 716 pp. New York: Academic Press. Hornhardt, S. & Wiebel, F.J. (1996). 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Validation and use of cloned, expressed human drug-metabolizing enzymes in heterologous cells for analysis of drug metabolism and drug–drug interactions. Biochemical Pharmacology 46, 559–566. 46. Glatt, H. (1997). Bioactivation of mutagens via sulfation. FASEB Journal 11, 314–321. 47. Glatt, H., Bartsch, I., Christoph, S., Coughtrie, M.W.H., Falany, C.N., Hagen, M., Landsiedel, R., Pabel, U., Phillips, D.H., Seidel, A. & Yamazoe, Y. (1997). Sulfotransferase-mediated activation of mutagens, studied using heterologous expression systems. Chemico-Biological Interactions, in press. 48. Daly, A.K. (1996). Molecular basis of polymorphic drug metabolism. Journal of Molecular Medicine 11, 539–553. 35. 36. 37. 38. 39. 40. F.J. Wiebel et al. 638 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.