Primordial germ cell-derived embryonic germ cells of the mouse—in vitro model for cytotoxicity studies with chemical mutagens

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Pergamon

Toxicology in Vitro 10 (1996) 755-763

Primordial Germ Cell-derived Embryonic Germ Cells of the Mouse-In Vitro Model for Cytotoxicity Studies with Chemical Mutagens U. SEHLMEYER, J. ROHWEDEL and A. M. WOBUS* Institut für Ptlanzengenetik und Kulturptlanzenforschung, In Vitro Oifferentiation Group, 0-06466, Gatersleben, Germany (Accepted 24 July 1996)

Abstract-Oifferent screening methods to detect the toxic effects of xenobiotics using cells from vertebrates and invertebrates in cytotoxicity and viability assays have been developed, but up to now appropriate in vitro methods with mammalian germ cells have not been available. In the present study the primordial germ (PG) cell-derived permanent embryonic germ (EG) cell line EG-I was used as in vitro model in toxicity studies with chemical mutagens. EG-I cells and embryonic stem cells of line 03 were comparatively investigated for their cell survival in response to N-ethyl-N-nitrosourea (ENV), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and mitomycin C (MMC) and the results compared with those obtained for undifferentiated embryonic carcinoma cells of line PI9 and differentiated epithelioid EPI-7 cells. As a prerequisite for in vitro toxicity and viability studies the cultivation conditions for EG-I and 03 cells in the absence of a feeder layer were improved by a conditioned medium, increasing the plating efficiency from 0.08% to 17.5% and from 21.1% to 25.1% for EG-I and 03 cells, respectively. The resulting mean generation time (MGT) of 16.9 hr for EG-I cells was identical to the generation time of PG cells in vivo, and was not significantly different from the MGT of 03 (15.6 hr) and EPI-7 (13.7 hr) cells, but significantly longer than the MGT of PI9 cells (9.3 hr). Calculations of the concentrations resulting in vitro in a 50% decrease in cell survival demonstrated that EG-I cells were more sensitive to the toxic effects of ENV, MNNG and MMC than 03 and PI9 cells and, with the exception of MNNG, also more sensitive than EPI-7 cells. It is proposed that EG cells are used as a model system to screen for toxic effects of teratogenic and embryotoxic chemical agents in vitro. © 1997 Elsevier Science Ltd. Ali rights reserved.

INTRODUCTION

Because cytotoxic effects of chemicals are thought to be at least partiany responsible for the embryotoxic and/or teratogenic capacity of xenobiotics, several in vitro methods using cytotoxicity and viability tests with cells from vertebrates and invertebrates have been deve10ped for the estimation of the embryotoxic and/or teratogenic potential of chemical substances (Kimmel et al., 1982; Schwetz et al., 1991). The present study was do ne to answer the question

*Author for correspondence at: Institut für Ptlanzengenetik und Kulturptlanzenforschung, Corrensstraf3e 3, 0-06466 Gatersleben, Germany. Abbreviations: dpc = daypostcoitum;OMEM = Oulbecco's modified minimal essential medium; EC = embryonic carcinoma; EG = embryonic germ; ENV = N-ethyl-Nnitrosourea; ES = embryonic stem; FCS = foetal calf serum; IC so = concentration resulting in vitro in a 50% decrease in cell survival; IC90 = concentration resuIting in vitro in a 90% decrease in cell survival; LIF = leukaemia inhibitory factor; MGT = mean generation time; MMC = mitomycin C; MNNG = Nmethyl-N'-nitro-N-nitrosoguanidine; PBS = phosphate buffered saline; PE = plating efficiency; PG = primordial germ.

whether primordial germ (PG) cell-derived embryonic germ (EG) cells may be used to analyse toxic effects on germ cens in vitro. Mouse PG cens are first identified within the proximal margin of the mouse epiblast 6.5 days post coitum (dpc) (Lawson and Rage, 1994). PG cells then migrate from the posterior of the primitive streak near the base of the allant ois through the gut mesentery and begin to arrive at the genital ridges by 10.5 dpc, proliferating from about 150 cens at 8.5 dpc to approximately 25,000 at 13.5 dpc (Ginsburg et al., 1990; Tarn and Snow, 1981). By 13.5 dpc PG cells within the genital ridge cease di vi ding; those in the male undergo mitotic arrest and those in the female enter meiosis (Ginsburg et al., 1990). In a few mouse strains, for example 129/Sv, this normal programme can be disrupted if the male genital ridge from an 11.5 to 12.5 dpc embryo is grafted to an ectopie site such as the testis or kidney capsule. Under these conditions PG cells may give rise to teratomas or teratocarcinomas containing pluripotent embryonic carcinoma (EC) stem cells (Noguchi and Stevens, 1982; Stevens and Makinson, 1961) resembling the undifferentiated cens of embryonic

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ectoderm with respect to morphological, biochemical and immunological characteristics (for example, Martin, 1980; Martin and Evans, 1975; Solter and Knowles, 1978; Fig. 1). This observation suggested that normal mitotic arrest of PG cells might be prevented in vitro, and that conditions could be found in which they could be reprogrammed to behave as pluripotent cells of the early embryo (Matsui et al., 1992). There were many attempts to isolate and purify mouse PG cells from various stages (for example, De Felici and McLaren, 1982 and 1983; Donovan et al., 1986; McCarrey et al., 1987), resulting in permanent lines of undifferentiated EG cells (Labosky et al., 1994b; Matsui et al., 1992; Resnick et al., 1992; Stewart et al., 1994). EG cells showed characteristics of undifferentiated embryonic cells as reported earlier for embryonic stem (ES) cel\s, totipotent celllines directly established from the inner cell mass of preimplantational mouse blastocysts (Evans and Kaufmann, 1981; Martin, 1981; Wobus et al., 1984), and for EC cells (Martin, 1980; Martin and Evans, 1975; Stevens, 1983): (i) activity of endogenous alkaline phosphata se (Bernstine et al., 1973; Resnick et al., 1992); (ii) expression of the stage-specific embryonic antigen SSEA-l (Resnick et al., 1992; Solter and Knowles, 1978); (iii) expression of the germline-specific transcription factor OCT-4 (Sch6ler et al., 1990); and (iv) deve\opmental

Oocyte .... Sperm ....

n

~

pluripotency Itotipotency in vivo and in vitro (for example, Maltsev et al., 1993; Rohwedel et al., 1994 and 1996; Strübing et al., 1995; Wobus et al., 1991). When injected into embryos, ES (Bradley et al., 1984) and EG (Labosky et al., 1994a; Stewart et al., 1994) cells gave rise to ail somatic lineages as wel\ as functional gametes. Laschinski et al. (1991) used blastocyst-derived ES cens and the tetrazolium (MTT) cytotoxicity test (Borenfreund et al., 1988; Denizot and Lang, 1986) as an in vitro approach to screen for teratogenic compounds. This method is based on the metabolic activity of cens, but does not re\y on the reproductive integrity of cens after chemical treatment (Wilson, 1992). To develop further screening systems for embryotoxicity, the establishment of in vitro assays with PG cell-derived EG cells is of particular importance. The present study investigated whether EG cel\s of line EG-I (Stewart et al., 1994) may be used as a cellular model for PG cells to analyse toxic effects of chemical mutagens in vitro resulting in cell death (Wilson, 1992). The improvement of cultivation conditions increasing the plating efficiency of EG-l cells in the absence of feeder cells was a prerequisite for their use in the se studies. Because of the close relationship between the three types of undifferentiated embryonic cells which are available as

Zygote Teratocarcinoma

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Fig. 1. Interrelationship of EG cells (EGC), PG cells (PGC), ES cells (ESC) and EC cells (ECC); END-2, EPI-7 and MES-I cens were characterized as endoderm-, ectoderm- and mesoderm-like cells (Mummery et al., 1985). The figure is based on data from Mummery et al, (\985), Schôler et al. (1990), Evans and Kaufmann (1981), and Stewart et al. (\994).

757

Cytotoxicity of embryonic germ cells in vitro Table 1. Additives to OMEM used for the cultivation of EG-I and 03 cells in the absence of a feeder layer and of PI9 and EPI-7 cells Additives FCS (%) NEAA (x 100) L-Glutamine (mM) Penicillin (1 U) Streptomycin (/lg!ml) 5637-Conditioned medium (%) p-Mercaptoethanol (!lM) MTG (nM) Transferrin (!lgjml) BSA (%) Na-Selenite (nM) L1F (ng/ml)

EG-I!03 'medium A'

PI9 'PI9-medium'

EPI-7

15 1:100 2 50 50 30

15 1:100 2 50 50

15 1:100 2 50 50

10 150

10 0.2 20 10

BSA = bovine serum albumin MTG = monothioglycerol NEAA = non-essential amino acids

permanent cell lines (Fig. 1), EG-I cells were compared with other undifferentiated cell types: ES cells of hne D3 (Doetschman et al., 1985) and EC cells of line P 19 established from a mouse teratocarcinoma (McBurney and Rogers, 1982). Ali three stem cell lines showed the characteristics of undifferentiated cells (see earlier and Resnick et al., 1992; Sch61er et al., 1990) including similar cell cycle distribution with a short G, phase (Rohwedel et al., 1996). The three undifferentiated stem cell hnes were compared with a differentiated cell hne, epithelioid ectoderm-like EPI -7 cells isolated as a clonalline after retinoic acid treatment of undifferentiated EC PI9 cells (Mummery et al., 1985; Fig. 1). EPI-7 cells where chosen because no permanent differentiated D3- or EG-I-derived cell lines are available and because EPI -7 cells express biochemical markers and growth characteristics typical for ectodermal cells (Mummery et al., 1985). In addition, EPI-7 cells were found to have a longer G, phase (x 2) of the cell cycle characteristic for differentiating cells (Rosenstrauss et al., 1982) than the undifferentiated stem cell hnes D3, PI9 and EG-I (Rohwedel et al., 1996). Here, it is shown that EG-I cells are more sensitive in response to the toxic effects of the chemical agents N-ethyl-N-nitrosourea (ENU), mitomycin C (MMC) andN -methyl- N' -nitro- N -nitrosoguanidine(MNNG), resulting in decreased cellular survival compared with that for undifferentiated D3 ES and PI9 EC cells, and, with the exception of MNNG, have higher sensitivity th an differentiated EPI-7 cells. MATERIALS AND METHODS

EG-I and D3 cells were grown in an undifferentiated state on a MMC-inactivated feeder layer of primary cultures of mouse embryonic fibroblasts (Wobus et al., 1984) and cultivated on gelatin (0.1 %)-coated plastic petri dishes (Nunc, Wiesbaden, Germany) in Dulbecco's modified minimal essential (DMEM) (Gibco-BRL, Eggenstein, medium Germany) supplemented with heat-inactivated foetal calf serum (FCS, se1ected batches from Boehringer, Mannheim, Germany), L-glutamine (Serva, Heidelberg, Germany), non-essential amino acids, penicillin and streptomycin (Gibco), and p-mercaptoethanol or

monothioglycerol (Serva) as shown in Table 1. PI9 and EPI-7 cells were cultivated as previously described (Sehlmeyer and Wobus, 1994). For preparation of conditioned medium the 5637 cellline derived from a human bladder carcinoma was used according to Wiles (1993): cells were grown to confluence in Iscove medium (Gibco) supplemented with 20% FCS. Subsequently, the medium was replaced by Iscove medium containing 2% FCS. After 3-4 days the conditioned medium was collected, centrifuged, filter sterilized (0.2 Ilm) and stored at - 2OUe. This procedure was repeated three to four times with the same monolayer, and the conditioned medium was used as additive at a concentration of 30% (Table 1). Feeder-independent growth of EG-I and D3 cells was established by culturing cells for three to five passages in 'Medium A' (Table 1) in the presence of leukaemia inhibitory factor (LIF) prepared from Escherichia coli strain JM 109 transformed with plasmid pGEX2T-LIF-58 and isolated according to the methods described by Smith and Johnson (1988) and Gearing et al. (1989). Because high alkaline phosphatase activity is a pro minent characteristic of undifferentiated stem cells (Bernstine et al., 1973; Resnick et al., 1992; Schôler et al., 1990), all stem cell lines were routinely tested for this specific enzymatic activity (Cooke et al., 1993) and only cell populations containing at least 90% of alkahne phosphatase-positive stem cells were used. The plating efficiency (PE) was determined by plating 100 (PI9) or 200 (EG-I, D3, EPI-7) cells per 60-mm dish. After cultivation for 6 (PI9) or 7 (EG-I, D3, EPI-7) days colonies were fixed with methanol, stained with 0.01 % crystal violet and scored for the ca\culation of PE (Sehlmeyer and Wobus, 1994). The me an generation time (MGT) was determined in the logarithmic growth phase by seeding 5 x 104 cells/60mm dish and daily cell counting during 1 wk of incubation (Freshney, 1987). To evaluate cellular survival, 5 x 10 5 cells/60-mm plate were seeded and after an 18 hr preincubation time treated with different concentrations of either, ENU (CAS No. 759-73-9; Sigma, Deisenhofen, Germany), MNNG (CAS No. 70-25-7; Sigma) or MMC (CAS No. 50-07-7; Serva) in serum-free

U. Sehlmeyer et al.

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Table 2. PE of mouse EG-I and 03 cells in vitro

A

± SEM) 17.5 ± 2.9 0.08 ± 0.1 25.1 ± 07 21.1 ± 2.4

Cell line (medium)

100

ENU

PE (%

EG-I ('medium A') EG-I ('PI9-medium')' D3 ('medium A') 03 ('PI9-medium')'

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'Supplemented with 10 ng/ml L1F. Values are means ± SEM (n = 18). 4

medium for 5 hr. ENU was dissolved in DMEM by vigorous shaking and immediately used for treatment after sterilization through 0.2 !lm cellulose acetate filters (Sehlmeyer and Wobus, 1994). A stock solution of MNNG was prepared by dilution in dimethyl sulfoxide (Merck, Darmstadt, Germany) and sodium phosphate buffer (pH 6.0) to a final concentration of 1 mM. Aliquots were stored at - 80°C and diluted with buffer to appropriate concentrations immediately before use (Matic et al., 1991; Sehlmeyer and Wobus, 1995). MMC stock solution of 600 pM in water was diluted with phosphate buffered saline (PBS) to appropriate concentrations immediately before use (Sehlmeyer et al., 1996). Cells treated only with the diluted solvent were included as controls. Cells were washed three times with PBS after treatment, and the PE determined as described earlier. Toxic activity was determined as per cent survival after treatment of the cel1s with the test substances and cultivation for clonai growth. Cel1ular survival was calculated according to the equation: Survival [% 1= (PET/PEe) x 100; PET = plating efficiency of the treated sample and PEe = plating efficiency of the control sample). Percent survival was logarithmically plotted v. the concentrations of the chemicals tested. All experiments were performed at least in triplicate and the mean values ± SE are given. Statistical analysis was done with the Mann-Whitney's rank sum test using the computer software Sigma/Stat (landel Scientific, Erkrath, Germany). RESULTS

EG and ES cells were routinely cultivated on a feeder layer to prevent spontaneous differentiation (Stewart et al., 1994; Wobus et al., 1984). However, cells have to be grown in the absence of a feeder layer for cytotoxicity and viability studies. Determination of the PE indicated that conditioning of the medium is required for feeder layer-independent growth of undifferentiated EG-l and D3 cells. EG-I cells Table 3. MGTofundifferentiated mouse EG-I, 03, PI9 cens and epithelial EPI-7 cens in vitro Cenline

MGT (hr)

EG-I ('medium A') 03 ('medium A') PI9' EPI-7'

16.9 ± l.1 15.6±1.2 9.3 ± 0.5""

13.7 ± 2.1

'Sehlmeyer and Wobus (1994). Values are means ± SEM (n = 3).

Concentration ofENU [mM] 100

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Fig. 2. Toxicity of 5 hr treatment with ENU (A), MNNG (B) and MMe (C) on EG-l, D3, Pl9 and EPI-7 cells measured as per cent survival. Values are means ± SEM (n = 3). Values for Pl9 and EPI-7 cells are from Sehlmeyer and Wobus (1994), Sehlmeyer and Wobus (1995) and Sehlmeyer et al. (1996).

cultivated in unconditioned 'PI9-Medium' (see Table 1) supplemented with 10 ng/ml LlF showed a very low PE of 0.08 ± 0.1% compared with EG- 1 cells grown in conditioned 'Medium A' with a PE of 17.5 ± 2.9% (Table 2). In the same way, the PE of 2l.I ± 2.4% obtained for D3 cells could be improved to 25.1 ± 0.7% (Table 2). Colony staining with crystal violet of EG-I and D3 cells after cultivation in conditioned and unconditioned media, respectively, revealed (Plate 1) that both EG-l and D3 cells showed typical compact morphology of undifferentiated ES cell clones, which in the case of line D3 were surrounded by outgrowths of differentiated cells. Clones of differentiated EPI -7 cells (Plate 1F) were less compact and stained less intensively than undifferentiated cells ofline P19 (Plate lE). To prove that EG cells resemble undifferentiated stem cells with respect to MGT, the generation times of EG-I and D3 cells were determined under improved cultivation conditions. They amounted to similar values of 16.9 ± l.l hr for EG-I cells and 15.6 ± 1.2 hr for D3 cells respectively (Table 3). These results demonstrate that application of conditioned medium allowed in vitro culture of

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Plate 1. Clone morphology of EG-l (A and B), D3 (C and D), Pl9 (E) and EPI-7 (F) cens, EG-l and D3 cens were plated using conditioned 'medium A' (B and D) (see Materials and Methods) and for comparison with unconditioned 'P19 medium' (A and C) supplemented with JO ng/ml LIF, Clones ofPl9 and EPI-7 cens were grown in normal growth medium (see Materials and Methods), 759

761

Cytotoxicity of embryonic germ cells in vitro Table 4. Cytotoxicity of ENU, MNNG and MMC in mouse EG-I, D3, PI9 and EPI-7 cells in vitro (IC" and EG-I Substance ENU MNNG MMC

PI9

D3

IC"

IC,.

892 ± 46 0.67 ± 0.09 0.14 ± 0.01

3058 ± 207 5.5 ± 0.01 0.61 ±O.II

IC"

IC~)

2861 ± 367t 5581 ± 195t 5.6 ± 1.6t 22.5 ± 3.4t 0.48 ± 0.05t 1.3±0.19'

IC~)

in jlmolflitre)

EPI-7

IC"

IC"

5100 ± 232't 10.6 ± 0.7t bt 1.3 ± O.q

8342 ± 221t 28.3 ± 5.8; 10.0 ± I.2t

IC"

IC,.

770 ± 60' 4026 ± 74' 0.3 ± 0.005 b, 0.8 ± 0.18t 0.8 ± 0.29 0.25 ± 0.08"

'Sehlmeyer and Wobus (1994); bSehlmeyer and Wobus (1995): 'Sehlmeyer et al. (1996)*P';; 0.05: tP,;; 0.01: tP,;; 0.001 v. EG-I cells: Mann-Whitney's rank sum test.

undifferentiated EG and ES cells without a feeder layer and their use in comparative toxicity studies. By comparing the toxic effects of ENU, MNNG and MMC in undifferentiated and differentiated cells a higher sensitivity was detected in EG-I cells th an in 03 cells (Fig. 2). ENU treatment of EG-I cells resulted in a linear decrease in the logarithmic survival curve which was below the values of 03 cells (Fig. 2A). Extrapolation of the concentrations resulting in 50% survival of the cells (IC so ) supported this observation with IC so values of 892 ± 46 and 2861 ± 367 f.lM ENU for EG-I and 03 cells, respectively (Table 4). MNNG induced higher toxic effects in EG-I cells than in 03 cells (Fig. 2B). Whereas the survival curve ofEG-1 cells initially showed a drastic decline, 03 ES cells showed a lower sensitivity. The IC so value of 0.67 ± 0.09 f.lM for EG-I cells was significantly lower than the value of 5.6 ± 1.6 f.lM for 03 cells (Table 4). Incubation of EG cells with MMC resulted in a dramatic decrease in cellular survival (Fig. 2C). In EG-l cells a concentration of 0.14 ± 0.01 f.lM MMC decreased the survival rate to 50% (Table 4), whereas the sensitivity of 03 cells (Fig. 2C) was lower resulting in an IC so value of 0.48 ± 0.05 f.lM MMC (Table 4). DISCUSSION

The present study shows for the first time that PG cell-derived EG-I cells, when cultivated under improved conditions, offer the opportunity to study toxic effects of chemical agents influencing cell survival of mouse EG cells in vitro. The MGT of EG-I cells was not significantly different from the MGT of 03 ES cells in vitro and of PG cells in vivo (Lawson and Hage, 1994; Tarn and Snow, 1981). As shown in this study, the sensitivity of EG-I cells to toxic effects of ENU, MNNG and MMC resulting in decreased cell survival was higher than in undifferentiated cells of line 03. The sa me is true when the values are compared with those previously reported for PI9 cells (Sehlmeyer et al., 1996; Sehlmeyer and Wobus, 1994 and Sehlmeyer and Wobus, 1995). Compared with differentiated EPI-7 cells EG-I cells were more sensitive in response to ENU and MMC, but less sensitive with respect to the alkylating agent MNNG. CaIculation of the IC so values resulted in the sensitivity order: EG-I ~ EPI-7 > 03 > PI9 (ENU), EG-I > EPI-7 > D3 > PI9 (MMC), EPI-7> EG-

1 > 03 > PI9 (MNNG). The higher sensltlVlty of EG cells compared with that of other undifferentiated cells is also supported by the concentration resulting in vitro in a 90% decrease in cell survival (IC 90 ) showing a similar sensitivity order and is in accordance with high IC so values of another ES cell line (Laschinski et al., 1991). Oifferentiated cell lines were less sensitive than EG-I cells to the cytotoxic effects of MMC (Oavies et al., 1993a,b; Laschinski et al., 1991) and MNNG (Gosh and Bhattacharjee, 1989; Goth-Goldstein and Hughes, 1987). However, with respect to the cytotoxicity of ENU, differentiated cell lines showed similar (Godfrey et al., 1992; Heflich et al., 1982) or higher sensitivity (Jensen and Thilly, 1986) than to EG-I cells. Although at present it is impossible to draw any correlation between the effects of ENU, MNNG and MMC resulting in decreased cell survival on EG cells in vitro and the embryotoxic effects ofthese chemicals on early embryos in vivo, the results may reflect the in vivo situation. Oeath of early embryonic cells may result in embryonic loss and dead implantations as determined by the dominant lethal test (Ehling, 1971 and 1974; Ehling et al., 1972). A correlation was proposed to exist between cell killing and induction of genetic damage in vivo (Oe Rooij et al., 1990). PG cells appeared to be more sensitive than stem cell spermatogonia (Russel et al., 1990; Shibuya et al., 1993). But, it has also been demonstrated with a variety of chemical mutagens that the response of individual germ cell stages was characteristic for a given chemical and could differ from compound to compound (Ehling, 1974; Ehling et al., 1972): Oespite the observation that several chemicals and X-rays induced more mutations in post-gonial cells than in spermatogonia (Meistrich, 1993), the spermatogonial stage was the most sensitive with respect to MMC as shown in the dominant lethal (Oobrzynska and Gajewski, 1994) and mouse-specific locus tests (Ehling, 1971), whereas no mutations were induced in post-spermatogonial germ cell stages (Ehling, 1974). The authors studies to establish a toxicity test on the basis of cellular survival with EG cells in vitro are the proposition for further cytotoxicity and genotoxicity studies on germ cells in vitro. They showed that PG cell-derived EG-I cells offer the opportunity to study embryotoxic effects of chemical agents on germ cells in vitro, as proposed for many years by Spielmann and Eibs (1978) and Brusick and Kilbey (1983). Further experiments with a broad range of embryotoxic, teratogenic and mutagenic substances

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in combination with different toxicity assays will have to show the suitability of the undifferentiated EG cells in vitro as an alternative to in vivo tests. AcknOldedgement.l'-The skilful technical assistance of Brigit Adam, Helga Berthold and Sabine Sommerfeld is gratefuUy acknowledged. The authors wish to thank Ors Colin L. Stewart (Nutley, USA) for kindly providing EG-l ceUs; Christine L. Mummery (Utrecht, The Netherlands) for EPI-7 ceUs, Michael V. Wiles (Basel, Switzerland) for kindly providing 5637 bladder carcinoma ceUs, and for information and helpful discussions regarding conditioned media, and M. Strauss (Berlin, Germany) for providing plasmid pGEXZT2T-LIF-58. The results were obtained with financial support of the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF, No. 0310299A) and Fonds der Chemischen Industrie, Germany.

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