Abstract

The murine embryonic teratocarcinoma cell line, P19, was genetically manipulated in order to provide preliminary information on compounds that induce differentiation. Without chemical induction, P19 cells remain in an undifferentiated state, but can be induced to differentiate into specific cell types. For example, dimethyl sulphoxide (DMSO) induces cardiac and skeletal muscle differentiation, whereas retinoic acid stimulates neuronal differentiation. P19 cells were transfected with a construct containing a segment of the mTert promoter sequence combined with the green fluorescent protein (GFP) gene, which acts as a reporter gene. Murine Tert (mTert) expression, the reverse transcriptase component of murine telomerase, is closely linked to telomerase activity and is down-regulated during differentiation. Three retinoids and DMSO induced the differentiation of P19 cells, which was determined by a reduction in mTert_GFP expression, detected by flow cytometry and confocal microscopy as independent methods of detection. A test substance, ethanol, and a control substance, saccharin, did not cause a decrease in mTert_GFP expression. In addition, it could be demonstrated that the mTert_GFP test detects developmentally relevant effects at non-cytotoxic concentrations. The ID50 values derived for the reduction of mTert_GFP expression were lower than the IC50 values detected with the MTT test, by a factor of 21.4 for all-trans retinoic acid, 12.7 for 9-cis retinoic acid, 29.6 for 13-cis retinoic acid, and 8.7 for DMSO. In comparison to the IC50 value for the P19 cell line, a similar IC50 value was obtained with 3T3 cells were equal for ethanol, but there was a 2-fold increase for DMSO. The retinoids were not cytotoxic to 3T3 cells at the concentrations tested This newly developed test is capable of detecting differentiation-inducing compounds at non-cytotoxic concentrations within 4 days. It offers a method for detecting chemicals with specific toxicological mechanisms, such as the retinoids, which could provide additional information in embryotoxicity testing as different promoters could be employed. Here, we report the use of this novel test system for the successful analysis of DMSO and three retinoids with different in vivo teratogenic potentials.

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2.2. Introduction

The establishment of alternative methods in the area of embryotoxicity requires testing strategies designed according to the users’ needs. Detailed information on target tissue-specific effects of drug candidates and their structural derivatives during organogenesis is often needed by the pharmaceutical industry. On the contrary, the chemicals policy of the European Union (Anon, 2001c) requires high throughput test systems to provide for the classification of chemicals with regard to their developmental toxic potential.

The P19 teratocarcinoma cell line was derived from an embryonic carcinoma induced in a C3H/He mouse (McBurney and Rogers, 1982), and shares some characteristics with embryonic stem cells. Both these cell types are pluripotent, and can differentiate in vitro into cell types representative of all three germ layers (van der Heyden and Defize, 2003). P19 cells express several early embryonic markers, such as the stage-specific embryonic antigen 1 (Solter and Knowles, 1978; Knowles et al., 1978). In comparison to embryonic stem cells, which show a high similarity to the in vivo situation, the embryotoxic relevance of results obtained from tumour-derived cells remains controversial.

P19 cells do not have a normal karyotype, and remain undifferentiated without chemical induction. Undifferentiated P19 cells do not depend on feeder cells or the addition of leukaemia inhibitory factor (LIF). This makes them attractive for large-scale biochemical approaches and well-defined culture conditions (van der Heyden and Defize, 2003). In addition, this characteristic gives the cell line an advantage, as it can be used to detect slight differences in differentiation levels in comparison with untreated or negative controls.

In order to induce their differentiation, P19 cells need to be stimulated with chemicals such as dimethyl sulphoxide (DMSO), which induces differentiation into cardiac and skeletal muscle cells (Stewart et al., 1994), or retinoic acid, which stimulates neuronal differentiation (McBurney et al., 1982; Jones-Villeneuve et al., 1982). P19 cells were one of the first examples described of cells able to differentiate into cardiac muscle in vitro, and have been the extensively characterised (van der Heyden and Defize, 2003). They were also previously employed as a reproducible cell

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system to the evaluate potential of agents to disrupt neuronal differentiation (Seeley and Faustman, 1998).

Telomerase, the enzyme that maintains the ends of linear eukaryotic chromosomes, has been demonstrated to be active in germ cells, embryonic stem cells, and cancer cells, as well as in immortal and tumour-derived cell lines, including P19 cells (Wright et al., 1996). Murine Tert (mTert) expression, which is the reverse transcriptase component of murine telomerase, is very closely linked to telomerase activity, and both are substantially reduced as a result of the differentiation of pluripotent cells (Sharma et al., 1995; Albanell et al., 1996; Holt et al., 1996;

Armstrong et al., 2000).

The diverse effects of retinoids on embryonic development in vivo and on cell differentiation in vitro are due to their receptor-mediated influence on gene expression. In vivo exposure to retinoids during embryogenesis results in severe malformations, but some retinoids have a higher teratogenic potential than others. For example, in mice, 13-cis retinoic acid (13-CRA) is 20–25 times less potent a teratogenic than all-trans retinoic acid (RA; (Kamm, 1982; Kamm et al., 1984;

Soprano and Soprano, 1995)), while 9-cis retinoic acid (9-CRA) is only half as potent a teratogen as RA (Kochhar et al., 1995). A study with the zebrafish model showed that RA has a greater or equal teratogenic potential than 9-CRA, which has a greater teratogenic potential than 13-CRA (Herrmann, 1995). It has previously been shown that 13-CRA binds to the retinoic acid receptors, RARα and RARβ, with a lower affinity than RA (Crettaz et al., 1990; Matsushima et al., 1992). In addition, 13-CRA shows very limited competition for binding to the retinoic acid receptors, RAR and RXR (Allenby et al., 1993).

Another explanation for the different teratogenic potentials was the transfer of substances to the embryo (Tzimas et al., 1994a). 13-CRA is transported through the placenta only to a very limited extent, while RA has been reported to reach concentrations in the embryo similar to those found in maternal plasma (Creech et al., 1989; Creech et al., 1991).

DMSO is an amphiphilic compound, which initiates a coordinated differentiation programme in various cell types. The PLC/PKC signalling cascade seems to be involved in the mechanism by

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which DMSO induces differentiation (Arcangeli et al., 1987; Wang, 1990; Chakravarthy et al., 1992; Morley and Whitfield, 1993; Cocco et al., 1999).

We have previously demonstrated opportunities for using genetically engineered pluripotent cells for detecting the potential embryotoxic hazard of chemicals (Bremer et al., 1999; Bremer et al., 2001; Paparella et al., 2002). In this study, P19 cells were transfected with a construct containing a segment of the mTert gene promoter sequence combined with the coding sequence of the green fluorescent protein (GFP), in order to provide a new in vitro test system to permit the detection of substances which affect differentiation. Such a test system would provide additional information to that provided by the validated embryonic stem cell test (Spielmann et al., 1997;

Scholz et al., 1999), which focuses on the detection of differentiation-inhibiting substances.

DMSO was used in the development of the test system, while three retinoids (RA, 9-CRA and 13-CRA) were used to evaluate its reliability.

The mTert_P19/GFP assay is not suitable for use as a broadly-applicable screening system for many chemical classes. However, about 17 relevant signalling pathways have been described during embryonic development, including such nuclear hormone receptor pathways as the retinoic acid signalling pathway, the wnt signalling pathway, and the receptor tyrosine kinase pathway (Anon, 2000b). Therefore, it would be promising to extend the method described here to promoters of genes known to be involved in these developmental pathways. A list of already genetically engineered murine ES cell lines for the major target tissues of embryotoxic compounds has recently been published (Bremer and Hartung, 2004).

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