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A simple RP-HPLC method based on fluorescence detection was developed for the quantitation of 7-amino-4-trifluoro methylcoumarin (AFC) in cell lysates from JEG-3 choriocarcinoma cells for determination of caspase-4 activity. In contrast to the established methods of AFC detection using a fluorescence microplate reader or using a fluorescence photometer, the separation of AFC-signals from interfering fluorescence signals by a reversed phase column affords more precise quantitation of released AFC. This can be important for analyses of cell lysates with low caspase activity or experimental series with marginal differences among samples. By applying this new method, a linear dynamic range of 40pmol/mL to 3nmol/mL with a correlation coefficient of 0.9996 was achieved. Due to the short retention time ( approximately 7min), the determination of AFC by RP-HPLC under isocratic conditions requires small amounts of samples (50microL injection volume), and allows increased sample throughput. This method should be easily applied with little or no modification to other caspase assays by using the same fluorophore.

Extravillous trophoblast cells resemble cancer cells with regard to their intrinsic invasiveness. They invade decidual tissue, but, unlike tumor cells, shut down their invasive properties, when they become inappropriate. Stimuli involved in the modulation of invasion, as well as their underlying signaling mechanisms require further clarification. We were especially interested in discovering signals capable of stimulating invasion in otherwise low-invasive cells involved in reproduction. Using the choriocarcinoma cell line Jeg-3 as a model, we have addressed the potential role of cytokine/growth factor-driven activation of signal transducer and activator of transcription 3 (STAT3) in this process. Jeg-3 cells were treated with various factors known to induce trophoblast proliferation, differentiation, migration, or invasiveness (insulin-like-growth-factor-II (IGF-II), hepatocyte growth factor (HGF), interleukin-6 (IL-6), and leukemia inhibitory factor (LIF)). Only LIF elicited strong tyrosine phosphorylation and specific DNA-binding activity of STAT3. It induced a significant acceleration of cell proliferation and promoted the capability of Jeg-3 cells to invade into an artificial extracellular matrix. Moreover, LIF influenced the expression pattern of proteases and protease inhibitors with potential relevance for invasiveness (downregulation of mRNA for tissue inhibitor of metalloproteinase 1 (TIMP-1) and upregulation of mRNA for caspase-4). In conjunction with earlier work, in which we found that STAT3 DNA-binding activity was increased in invasive cells (choriocarcinoma, first trimester trophoblasts) and absent in non-invasive cells (term trophoblasts), these findings suggest a connection between LIF-driven STAT3 activity and invasiveness of choriocarcinoma and trophoblast cells.

Aberrant activity of the signal transducer and activator of transcription 3 (Stat3) is believed to be essential for neoplastic cell behaviour and thus for the malignancy of tumor cells [Bowman T, Garcia R, Turkson J, Jove R. STATs in oncogenesis. Oncogene 2000;19:2474-88]. Extravillous trophoblast cells resemble malignancies in their invasive and destructive features, excluding the fact of sequential restriction to the first trimester of pregnancy. Trophoblast cells from term placentas have reduced invasive capacity [Hohn HP, Denker HW. Experimental modulation of cell-cell adhesion, invasiveness and differentiation in trophoblast cells. Cells Tissues Organs 2002;172:218-36]. Constitutively activated Stat3 DNA-binding activity in choriocarcinoma cells, carcinomatous derivates of trophoblast cells, have been reported to correlate with its invasiveness [Corvinus FM, Fitzgerald JS, Friedrich K, Markert UR. Evidence for a correlation between trophoblast invasiveness and STAT3 activity. Am J Reprod Immunol 2003;50:316-21]. Here we demonstrate using RNAi that Stat3 activation is necessary in the invasive phenotype of trophoblast cells and can be controlled via Leukemia Inhibitory Factor (LIF). LIF provides a soluble extracellular signal that stimulates invasion in trophoblast and Jeg-3 choriocarcinoma cells. Loss of LIF-mediated invasion in these cells subsequent to STAT3 knock-down strongly suggests that STAT3 plays a crucial role in mediating this invasion. © 2005 Published by IFPA and Elsevier Ltd.