Embryonal carcinoma (EC) cells provide a caricature of pluripotent embryonic stem (ES) cells and may be used as surrogates for investigating the mechanisms that regulate cell differentiation during embryonic development. NTERA-2 is a human EC cell line that differentiates in response to retinoic acid yielding cells that include terminally differentiated neurons. The expression of genes known to be involved in the formation of the vertebrate nervous system was examined during retinoic acid-induced NTERA-2 differentiation. Differentiation of these human EC cells into neurons could be divided into three sequential phases. During phase 1, in the first week of differentiation, hath1 mRNA showed a small transient increase that correlated with the rapid accumulation of nestin message, a marker of neuroprogenitors. Transcripts of nestin were quickly downregulated during phase 2 as expression of neuroD1, characteristic of neuroprogenitors exiting the cell cycle, was induced. A neural cell surface antigen, detected by the monoclonal antibody A2B5, was expressed by cells exiting the cell cycle, correlating with the expression of neuroD1 as the cells became post-mitotic. Markers of mature neural cells (e.g. synaptophysin and neuron-specific enolase) were subsequently increased during phase 3 and were maintained. This regulated pattern of gene expression and commitment to the neural lineage indicates that differentiation of NTERA-2 neurons in vitro follows a similar pathway to that observed by neural ectodermal precursors during vertebrate neurogenesis in vivo.

In Wilson's disease and Indian childhood cirrhosis (ICC) copper accumulates in the liver resulting in poor hepatocyte regeneration and fibrosis. An inhibition of hepatocyte proliferation and an increase in cell death could account for these outcomes. To establish how the toxicity of this metal ion impacts upon the proliferation and viability ofthe HepG2 cells they were cultured in 4-32 jiM copper(II) sulphate (CuS04)). These levels were comparable to the circulatory and tissue concentrations of copper recorded for these two diseases. Specific uptake comparable to levels of copper recorded in the livers of patients with Wilson's disease and ICC was measured in the HepG2 cells. After 48 h acid vesicle function increased from 4 to 32 jiM Cu2 + but significantly declined at 64 MM compared to the controls. Lysosomal acid phosphatase showed a concen-tration dependent decline in activity at 72 h. Introduction Copper is a potent catalyst of processes generating reactive oxygen intermediates and its cellular up-take, transport and excretion are therefore tightly controlled. Elevated hepatic copper concentrations are associated with liver damage in Wilson's disease2, in Indian childhood cirrhosis3 (ICC), and in other copper-related cirrhoses of infancy. Nevertheless, the cellular consequences of copper toxicity remain to be understood in the context of the disease pathology. In Wilson's disease, biliary excretion of copper is impaired by mutations in a trans-Golgi P-type ATPase4 resulting in intracellular hepatic copper accumulation5 with progressive dysfunction, fibrosis, and liver failure. However, clinical variability of Wilson's disease and the lack of a clear genotype/ Cellls exposed to 64 MM Cu2 + had a potential doubling time (Tpot) 21 h longer than the control cells due to a prolonged DNA synthesis phase. At 64 jiM Cu2 +, increases of necrosis up to 18% were seen whereas comparable levels ofapoptotic and necrotic cells (< 5%) were seen below this concentration. Chronic exposure over 8 weeks impaired colony-forming effi-ciency at concentrations of 16 MM Cu2 + and above. This study suggests that when liver cells sequester large amounts of copper, the toxic effects include delayed cell-cycle progression, a gradual loss of replicative capacity, and an increased incidence of cell death.

Testosterone-induced vasodilatation is proposed to contribute to the beneficial effects associated with testosterone replacement therapy in men with cardiovascular disease, and is postulated to occur via either direct calcium channel blockade, or through potassium channel activation via increased production of cyclic nucleotides. We utilised flow cytometry to investigate whether testosterone inhibits the increase in cellular fluorescence induced by prostaglandin F 2 in A7r5 smooth muscle cells loaded with the calcium fluorescent probe indo-1-AM, and to study the cellular mechanisms involved. Two-minute incubation with testosterone (1 µM) significantly inhibited the change in cellular fluorescence in response to prostaglandin F 2 (10 µM) (3·6 0·6 vs 7·6 1·0 arbitrary units, P=0·001). The change in cellular fluorescence in response to prostaglandin F 2 (10 µM) was also significantly attenuated in the absence of extracellular calcium (3·6 0·3 vs 15·6 0·7 arbitrary units, P=0·0000002), and by a 2-min incubation with the store-operated calcium channel blocker SK&F 96365 (50 µM) (4·7 0·8 vs 8·1 0·4 arbitrary units, P=0·003). The response was insensitive to similar incubation with the voltage-operated calcium channel blockers verapamil (10 µM) (12·6 1·2 vs 11·9 0·2 arbitrary units, P=0·7) or nifedipine (10 µM) (13·9 1·3 vs 13·3 0·5 arbitrary units, P=0·7). Forskolin (1 µM) and sodium nitroprusside (100 µM) significantly increased the cellular concentration of cyclic adenosine monophosphate and cyclic guanosine monophosphate respectively, but testosterone (100 nM-100 µM) had no effect. These data indicate that the increase in intracellular calcium in response to prostaglandin F 2 occurs primarily via extracellular calcium entry through store-operated calcium channels. Testosterone inhibits the response, suggesting an antagonistic action upon these channels.

Spermatozoa from the sperm-rich fractions of the semen of 6 beagle dogs were capacitated and the effect of both zona pellucida (ZP) proteins and progesterone on calcium flux and the acrosome reaction measured. Sperm calcium flux was determined using the dual wavelength calcium probe indo-1/AM (6 microM) in a flow cytometric assay (one ejaculate from each dog examined; n = 6). No calcium flux was observed in the negative control treatments (RPMI medium or DMSO). Both heat-solubilized bitch ZP proteins and progesterone caused a similar response characterized by a gradual but marked influx of calcium ions which was sustained over 2 min. Acrosomal status was assessed by indirect immunofluorescence using a specific monoclonal antibody following 1 hr incubation for each treatment (four ejaculates from each dog examined; n = 24). The level of acrosomal exocytosis was very high for samples treated with ZP proteins (70.3 +/- 2.1%) and progesterone (84.6 +/- 1.5%) and was significantly different from the respective controls (P < 0.001). Interestingly the patterns of calcium flux in response to both ZP proteins and progesterone were in contrast to the situation in other species studied to date raising the possibility that the mechanism for triggering the acrosome reaction may be different in dog spermatozoa. In addition the high degree of progesterone-induced acrosomal exocytosis compared to other species raises the probability that the majority of dog spermatozoa are already undergoing the acrosome reaction before they reach the egg ZP.

To investigate whether the failure of human EC cells that do not differentiate is due to the loss of key differentiationpermissive functions or the acquisition of specific inhibitory functions, we tested the ability to differentiate of 2 hybrids produced between a relatively nullipotent human EC cell line, 2102Ep, and a pluripotent human EC cell line NTERA2. Both hybrids, which exhibited an EC phenotype, were able to differentiate readily in response to retinoic acid. Furthermore, 1 hybrid produced a well-differentiated xenograft tumor, which contained, like the NTERA2 tumors, glandular structures, loose mesenchymal tissues and nodules of cartilage, after injection into a SCID mouse. Thus, the failure of 2102Ep EC cells to differentiate is recessive and due to the loss of a key gene function or functions. Nevertheless, the hybrids differed from the pluripotent NTERA2 line by failing to differentiate in neurons, indicating that 2102Ep cells also had acquired a specific, dominantly-acting, inhibitory mutation specific to the neural lineage. Furthermore, the expression of collagen II by one hybrid before and after induction with retinoic suggested a propensity for spontaneous differentiation not evident in the parental NTERA2 cells. Thus, the mechanisms that restrict the differentiation capacity of the nullipotent 2102Ep line are complex and include both recessive and dominant acting factors.