Primordial germ cell-derived mouse embryonic germ (EG) cells in vitro resemble undifferentiated stem cells with respect to differentiation capacity and cell cycle distribution.

Embryonic germ (EG) cells of line EG-1 derived from mouse primordial germ cells were investigated for their in vitro differentiation capacity. By cultivation as embryo-like aggregates EG-1 cells differentiated into cardiac, skeletal muscle and neuronal cells accompanied by the expression of tissue-specific genes and proteins as shown by RT-PCR analysis and indirect immunofluorescence. In comparison to embryonic stem (ES) cells of line D3 the efficiency of differentiation into cardiac and muscle cells was comparatively low, whereas spontaneous neuronal differentiation was more efficient than in D3 cells. Furthermore, the distribution of cell cycle phases as a parameter for the differentiation state was analysed in undifferentiated EG cells and ES cells and compared to data obtained for embryonic carcinoma (EC) cells of line P19 and differentiated, epithelioid EPI-7 cells. Flow cytometric analysis revealed similar cell cycle phase distributions in EG, EC and ES cells. In contrast, the somatic differentiated EPI-7 cells showed a longer G1-phase and shorter S- and G2/M-phases. Together, our results demonstrate that the differentiation state and capacity of EG cells in vitro resemble that of totipotent ES cells.

Low mutagenic effects of mitomycin C in undifferentiated embryonic P19 cells are correlated with efficient cell cycle control.

Pluripotent undifferentiated embryonic carcinoma cells of line P19 and their differentiated progeny, epithelioid ectoderm-like EPI-7 cells, showed different responses to mitomycin C (MMC) with respect to induction of micronuclei, mutations at the HPRT-locus and cell cycle control. Cytotoxic effects of MMC after a 5-h treatment were lower in undifferentiated P19 cells than in differentiated EPI-7 cells with IC50 values of 1.3 and 0.25 microM for P19 and EPI-7 cells, respectively. MMC did not induce 6-thioguanine-resistant mutants in P19 cells but significantly increased the mutation frequency in EPI-7 cells with concentrations of 0.25, 0.5 and 1.0 microM MMC. Micronuclei determined by flow-cytometry were induced by MMC in both cell lines at equitoxic concentrations of 4.5 (P19) and 0.75 (EPI-7) microM, reducing the viability in both cell lines to 10%. Whereas the induction of micronuclei in P19 cells was maximal 28 h after treatment and declined thereafter, micronucleus induction peaked 48 h post treatment in EPI-7 cells and remained significantly increased even 67 h after the treatment. Flow-cytometric determination of the distribution of MMC-treated P19 and EPI-7 within the cell cycle revealed a distinct G2/M-block in P19 cells, whereas EPI-7 cells showed normal progression through S-phase and a negligible G2/M-block. Therefore, we conclude that the lower effectivity of MMC to induce gene mutations and micronuclei in P19 cells seemed to be correlated with a more efficient cell cycle control in undifferentiated compared to differentiated EPI-7 cells.

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

Different 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-1 was used as in vitro model in toxicity studies with chemical mutagens. EG-1 cells and embryonic stem cells of line D3 were comparatively investigated for their cell survival in response to N-ethyl-N-nitrosourea (ENU), 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 P19 and differentiated epithelioid EPI-7 cells. As a prerequisite for in vitro toxicity and viability studies the cultivation conditions for EG-1 and D3 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-1 and D3 cells, respectively. The resulting mean generation time (MGT) of 16.9 hr for EG-1 cells was identical to the generation time of PG cells in vivo, and was not significantly different from the MGT of D3 (15.6 hr) and EPI-7 (13.7 hr) cells, but significantly longer than the MGT of P19 cells (9.3 hr). Calculations of the concentrations resulting in vitro in a 50% decrease in cell survival demonstrated that EG-1 cells were more sensitive to the toxic effects of ENU, MNNG and MMC than D3 and P19 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.

Lower mutation frequencies are induced by ENU in undifferentiated embryonic cells than in differentiated cells of the mouse in vitro.

The pluripotent embryonic carcinoma cells of line P19 established from undifferentiated cells of the early mouse embryo and their differentiated progeny, the epithelioid ectoderm-like EPI-7 cells, were investigated for the induction of mutations at the HPRT locus by the alkylating agent N-ethyl-N-nitrosourea (ENU). We showed that the cytotoxic effects of ENU after a 5-h treatment were lower in undifferentiated P19 cells than in differentiated EPI-7 cells. The IC50 values of ENU in the two cell lines amounted to 0.6 mg/ml and 0.09 mg/ml for P19 and EPI-7 cells, respectively. The induction of 6-thioguanine-resistant mutants by ENU (1.0 mg/ml) determined after an expression time of 8 days for both cell lines resulted in similar mutation frequencies. Using expression times of 8 days for P19 and 11.75 days for EPI-7 cells, taking into account the longer generation time of differentiated EPI-7 cells (13.7 +/- 3.6 h) in comparison to undifferentiated P19 cells (9.3 +/- 0.9 h), ENU induced significantly higher mutant frequencies in EPI-7 cells (4865 mutants/10(6) cells) than in P19 cells (282 mutants/10(6) cells). Our results and data from the literature on UV irradiation-induced repair support the idea that the induction of lower mutation frequencies in embryonic cells may correlate with different proliferation capacities, cell cycle parameters and/or different mechanisms of DNA repair in embryonic stem cells and differentiated cells, respectively.

Accumulation of chromium in Chinese hamster V79-cells and nuclei.

The bioavailability of carcinogenic compounds plays an important role in the process of oncogenesis. Chromium in its hexavalent oxidation state is classified as carcinogen to humans. Therefore we studied the uptake of chromate(VI) into Chinese hamster V79 cells and nuclei isolated after the incubation of the whole cells with chromate. The chromium content of cells and nuclei was determined by atomic absorption spectroscopy. Chromate is taken up in a concentration dependent manner and accumulates to about 30 fold over the extracellular concentration of 0.1 mmol/l. Incubating whole cells with the same concentration results in an intranuclear concentration of up to 6 mmol/l after 3 h. The kinetic parameters (KM = 0.34 mmol/l, Vmax = 0.12 fmol per cell and minute) are in the same order of magnitude as previously published data. The consequences of the high intracellular and intranuclear concentrations are discussed in terms of the genotoxic effects.