Continuous activation of ER-K-ras^G12D by 500 µg tamoxifen treatment induced lung hyperplasia and adenoma formation in P53 L/L, LSL-ER-K-ras mice.

<p>A) Continuous activation of ER-K-ras^G12D by 500 µg tamoxifen I.P. daily injection for 12 weeks is sufficient to drive lung adenoma formation in P53 L/L, LSL-ER-K-ras mice after adeno-Cre treatment. The adenoma were positive for Ki67 immunostaining. B) The incidence of lung hyperplasia and/or adenoma formation in <i>P53L/L</i>, <i>LSL-ER-K-ras^G12D</i> mice was shown after adeno-Cre treatment in the absence or presence of tamoxifen treatment.</p

Sustained activation of ER-K-ras^G12D induced by tamoxifen treatment is important for cell proliferation, anchorage-independent cell growth, cell invasiveness and tumor maintenance.

<p>A) Cell number counting of indicated MEFs after withdrawal of tamoxifen after 6 passages in the presence of tamoxifen. The PEK-C MEFs always without tamoxifen treatment and the PEK-C MEFs always kept in tamoxifen were used as control. B) Tamoxifen were withdrawn from the PEK-C MEFs at day 5, 10 and 28 after continuous tamoxifen treatment in soft agar. C) Tamoxifen withdrawal from the PEK-C MEFs after indicated days of tamoxifen treatment in cell invasiveness assay in matrigel. D) Tamoxifen withdrawal for indicated time in the <i>P53L/L</i>, <i>LSL-ER-K-ras^G12D</i> mice previously treated for 12 weeks of tamoxifen. The typical lung pathology and cleaved caspase-3 immunostaining were shown.</p

Treatment of tamoxifen at an optimal dose induced the activation of ER-K- ras^G12D and downstream signaling.

<p>A) The <i>P53−/−</i>, <i>ER-K-ras^G12D</i> MEFs (PEK-C) derived from male embryo was counted for cell number after indicated dosage of tamoxifen treatment after 5 passages. B) Relative expression level of <i>K-ras</i> in indicated MEFs with or without tamoxifen treatment. PEK: <i>P53L/L</i>, <i>Loxp-Stop-Loxp ER-K-ras^G12D</i>; PEK-C: <i>P53−/−</i>, <i>ER-K-ras^G12D</i>; PK: <i>P53L/L</i>, <i>Loxp-Stop-Loxp-K- ras^G12D</i>; PKC: <i>P53−/−</i>, <i>K- ras^G12D</i>. (C) Detection of the ER-K-ras^G12D expression in PEK-C cells with or without 0.05 µM tamoxifen treatment. D) Detection of K-ras and ER-K-ras protein level in MEFs infected with or without adeno-Cre in the presence or absence of 0.05 µM tamoxifen treatment in indicated MEFs. β-actin serves as internal control. E) The activation of Ras effector PI3K pathway signaling was confirmed by western blot.</p