INTRODUCTION: 9-hydroxystearic acid (9HSA) induces cell proliferation arrest in G0/G1 by inhibiting HDAC1 and inducing p21 expression. Serum-starved cells are known to exit G1 and enter the G0 phase, but treatment with either serum or specific growth factors induces them to reenter the cycle. On the contrary, 9HSA treated cells, if stimulated with EGF, are not responsive and undergo a further arrest. In the effort of understanding the possible mechanisms of this effect we analysed the degree of internalisation of the EGF/EGFR complex and the interactions between the complex and other proteins, such as HDAC1 and pRb. The phosphorilation level of EGFR was indicative of its activity in the cells. Cyclin D1 promotor being one of the most important EGF/EGFR target involved in cell proliferation, we also analysed its expression, complexation, and localization with HDAC1. MATERIALS AND METHODS: HT29 colon adenocarcinoma cells, serum starved (SS) were treated with 9HSA, EGF, or both. By citofluorimetry we analysed the levels of EGF/EGFR internalisation and cyclin D1 expression. Cyclin D1 expression, HDAC1/cyclin D1 and HDAC1/EGFR complexes were analysed by confocal microscopy. Furthermore EGFR activity and interactions with HDAC1 and pRb were evaluated by co-immunoprecipitation and WB. RESULTS: In response to EGF treatment, SS cells exhibited an increase in 3H-thymidine incorporation, whereas 9HSA treated cells were uresponsive to EGF. There was no quantitative difference in EGF/EGFR internalisation as seen in citofluorimetry. There was no quantitative difference in cyclin D1 expression in 9HSA treated- and EGF stimulated-HT29. The interaction between cyclin D1 and HDAC1 was visible in cells that respond to EGF after serum starvation but not after 9HSA treatment. EGFR/HDAC1 complex quantitatively increased in 9HSA treated but not in SS cells after EGF stimulation and localised outside the nucleus: this does not allow HDAC1 to interacts with cyclin D1. PRb was increased in its low phosphorilation condition by EGF stimulation in 9HSA treated cells, justifing the further arrest of the cell proliferation. EGFR was phosphorilated in cells treated with 9HSA and stimulated with EGF, as if the mitogen signal was blocked. This work was supported by grants from COFIN 2004
9-HYDROXYSTEARIC ACID INTERFERES WITH EGF SIGNALLING IN A HUMAN COLON ADENOCARCINOMA
CALONGHI, NATALIA;PAGNOTTA, ELEONORA;BOGA, CARLA;MASOTTI, LANFRANCO
2006
Abstract
INTRODUCTION: 9-hydroxystearic acid (9HSA) induces cell proliferation arrest in G0/G1 by inhibiting HDAC1 and inducing p21 expression. Serum-starved cells are known to exit G1 and enter the G0 phase, but treatment with either serum or specific growth factors induces them to reenter the cycle. On the contrary, 9HSA treated cells, if stimulated with EGF, are not responsive and undergo a further arrest. In the effort of understanding the possible mechanisms of this effect we analysed the degree of internalisation of the EGF/EGFR complex and the interactions between the complex and other proteins, such as HDAC1 and pRb. The phosphorilation level of EGFR was indicative of its activity in the cells. Cyclin D1 promotor being one of the most important EGF/EGFR target involved in cell proliferation, we also analysed its expression, complexation, and localization with HDAC1. MATERIALS AND METHODS: HT29 colon adenocarcinoma cells, serum starved (SS) were treated with 9HSA, EGF, or both. By citofluorimetry we analysed the levels of EGF/EGFR internalisation and cyclin D1 expression. Cyclin D1 expression, HDAC1/cyclin D1 and HDAC1/EGFR complexes were analysed by confocal microscopy. Furthermore EGFR activity and interactions with HDAC1 and pRb were evaluated by co-immunoprecipitation and WB. RESULTS: In response to EGF treatment, SS cells exhibited an increase in 3H-thymidine incorporation, whereas 9HSA treated cells were uresponsive to EGF. There was no quantitative difference in EGF/EGFR internalisation as seen in citofluorimetry. There was no quantitative difference in cyclin D1 expression in 9HSA treated- and EGF stimulated-HT29. The interaction between cyclin D1 and HDAC1 was visible in cells that respond to EGF after serum starvation but not after 9HSA treatment. EGFR/HDAC1 complex quantitatively increased in 9HSA treated but not in SS cells after EGF stimulation and localised outside the nucleus: this does not allow HDAC1 to interacts with cyclin D1. PRb was increased in its low phosphorilation condition by EGF stimulation in 9HSA treated cells, justifing the further arrest of the cell proliferation. EGFR was phosphorilated in cells treated with 9HSA and stimulated with EGF, as if the mitogen signal was blocked. This work was supported by grants from COFIN 2004I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
