PMC

Heterotrimeric Gi-protein-dependent signalling drives proliferation of CRC cell lines. A 10 μM lysophosphatidic acid (LPA), 100 μM Isoproterenol, 100 ng/ml PTX and 10 nM PGE2 were administered alone or in combination to serum-starved HT-29 cells. 96 h later [³H]-thymidine incorporation into cellular DNA was scored as described before. Data are mean ± S.E.M. of counts per minute (normalized values to average of control) of tetraplicates. B Effect of PTX and LPA on PKA substrate phosphorylation. Serum-deprived HT-29 cells were treated as indicated in the legend and PKA-substrate phosphorylation was assessed as before.

Effect of EP3 receptor selective agonists on Lovo cell proliferation and cAMP signalling. A HT-29 cells were deprived of serum and stimulated with 10 nM PGE2, 10 μM butaprost, 10 μM sulprostone or 10 μM 11-deoxy-PGE1. DNA synthesis was monitored 5 days later. Data are mean ± S.E.M. of cells/well of 3 experiments with triplicate measurements. B Serum-starved HT-29 cells were stimulated for 20 min with the same agonists as in A followed by measurement of cAMP levels. Student's t test: P values ***P < 0,001, **P < 0,01, *P < 0,05.

Effect of EP3 receptor selective agonists and antagonists on HT-29 cell proliferation. A HT-29 cells were deprived of serum overnight and challenged with 10 nM PGE2, 10 μM butaprost, 10 μM sulprostone or 10 μM 11-deoxy-PGE1. DNA synthesis was assessed 5 days later. Data are mean ± S.E.M. of cells/well of 3 experiments with triplicate measurements.B Serum-starved HT-29 cells were stimulated for 20 min with the same agonists as in A and cAMP levels were determined as before. C Serum-starved HT-29 cells were challenged with 10 nM PGE2 alone or in the presence of 250 nM each of L-818638 (EP1 antagonist), L-826266 (EP3 antagonist) or MF-191 (EP4 antagonist). Proliferation was measured 4 days later by [³H]-thymidine incorporation. Two-sample comparisons (all vs. control) were performed with Student's t test. P values ***P < 0,001, **P < 0,01, *P < 0,05.

PGE2 does not ostensibly affect apoptosis of HT-29 cells. A HT-29 were cultured for varying lengths of time in the absence of serum or in the presence of 10% FCS, 10 nM PGE2 or 10 μM PGE2. At the indicated time points cells were lysed and PARP cleavage was assessed by western blotting. Vinculin levels were determined to ascertain equal protein loading. Staurosporine was administered at 1 μM as a positive control to induce apoptosis. Quantification is shown as the densitometrically-determined ratio of cleaved to non-cleaved PARP levels. Shown here is one representative experiment out of three. B PGE2 does not affect the number of Annexin V positive cells. HT-29 cells were treated with staurosporine, FCS or the indicated PGE2 concentrations followed by AnnexinV-FITC and propidium iodide staining and FACS analysis. The mean percentage of cells gated in the Annexin+/PI- (early apoptotic) and Annexin+/PI+ area (late apoptotic) ± S.E.M. (n = 3) is indicated below each panel.

Concentration-dependent effect of PGE2 on cAMP levels in CRC cell lines. A Serum-starved cells were exposed to 100 μM Isoproterenol or the indicated concentrations of PGE2 for 15 min. Cyclic AMP levels were determined as described in the experimental section. B Cyclic AMP-raising agents induce phosphorylation of PKA-substrates in CRC cells. Cells were challenged with 100 μM Isoproterenol or 10 μM forskolin for 20 min and phosphorylation of PKA substrates was determined by western blotting with an antibody directed against the phosphorylated PKA-consensus site. Vinculin levels were determined to ascertain equal loading. C CRC cells were deprived of serum overnight and challenged with varying doses of PGE2 for 15 min. Phosphorylation of PKA substrates was assessed as in B. A densitometric quantification of the signal for the 70–100 kD region is shown. Two additional experiments produced essentially the same results. Two-sample comparisons (all vs. control) were performed with Student's t test. P values ***P < 0,001, **P < 0,01, *P < 0,05.

Expression of prostanoid receptors in HT-29 cells. A RT-PCR analysis of EP1-4 receptor expression in serum-fed HT-29 cells. See methods section and table 1 for details. Note that the primer pair employed in this experiment detects all known EP3 splice variants. B Serum-starvation induces EP3 mRNA levels. HT-29 cells were either kept in serum or serum was removed for the indicated number of days. EP3 expression was determined as in A. RT-PCR for GAPDH was run in parallel to control for equal loading. C Expression and regulation of EP3 splice variants. HT-29 cells were either kept in serum or deprived of serum for 24 h prior to performing RT-PCR on total RNA preparations. Primer pairs for individual EP3 splicing isoforms are listed in table 1. RT-PCR on total RNA from K562 cells was run as a control for EP3 isoform expression. As a further control HA-tagged EP3 subtype 3 was transfected into HT-29 cells prior to RT-PCR analysis. Note that EP3 subtypes 1–3 cannot be discriminated by use of different primer pairs but on the basis of the different size of the amplified fragments. A fragment amplified by the EP3 type 1/2/3 primer set of about 180 bp size that cannot be attributed to any known splice variant is marked by an asterisk. Control lane indicates an RT-PCR run using water as a template.

Concentration-dependent induction of DNA synthesis by PGE2 in CRC cell lines. A HT-29, Caco-2, Lovo and SW480 cells were seeded in 24-well plates, deprived of serum overnight and challenged with 10% FCS or the indicated doses of PGE2 for further 3 days. Proliferation was scored by [³H]-thymidine incorporation into cellular DNA. Data are mean ± S.E.M. of counts per minute (normalized values to average of control) of tetraplicates of three independent experiments. B Time response of PGE2-induced DNA synthesis. Serum-starved cells were exposed to 10 nM PGE2 for various days and [³H]-thymidine incorporation was monitored and plotted as in A. A more prolonged time response is shown for HT-29 cells in the right panel. Two further experiments yielded similar results. C PGE2-dependent cell proliferation scored by automatic cell counting. Serum-starved HT-29 cells were challenged with varying doses of PGE2 for 7 days and subjected to automatic cell counting as described in the experimental section. Data are mean ± S.E.M. of cells/well of 4 experiments with triplicate measurements. D COX-2 inhibition does not affect PGE2 induced cell proliferation. Serum-starved HT-29 cells were challenged with 10 nM PGE2 or 10% FCS alone or in combination with 10 μM NS398. 4 days later DNA synthesis was assessed as before. Two-sample comparisons (all vs. control) were performed with Student's t test. P values ***P < 0,001, **P < 0,01, *P < 0,05.