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. Author manuscript; available in PMC: 2011 Feb 15.
Published in final edited form as: Clin Cancer Res. 2010 Feb 9;16(4):1179. doi: 10.1158/1078-0432.CCR-09-1787

Figure 4. LMW but not full-length cyclin E overexpressing MCF-7/Ac1 cells could partially override the letrozole inhibition of AD-induced G1 exit and AD-induced CDK2 protein levels.

Figure 4

Figure 4

(A) Cyclin E overexpression overrides the letrozole inhibition of AD-induced proliferation. MCF-7/Ac1 cells were cultured in IMEM with 10% charcoal-stripped serum medium (CSSM) without phenol red and with 600 µg/ml of G418 for 4 days before plating. Triplicate wells of 6-well plates were then infected with the indicated adenoviruses (at 4000 m.o.i.) 24 hours before drug treatment. Cells were then left untreated (E2W, estrogen withdrawal) or treated with 25 nM AD (AD) or treated with 25 nM AD and 1 µM letrozole (AD + Let) and collected 3 days later for cell number. Cell growth is expressed as the percentage of the cells compared with the control cells (25 nM AD treated cells). (B) LMW cyclin E overexpressing MCF-7/Ac1 cells could partially override the letrozole inhibition of AD-induced G1 exit. MCF-7/Ac1 cells were treated as described in A, and collected for flow cytometry analysis. Histograms represent the S-phase fraction expressed as the percentage of the cells in S-phase compared with the control cells (25 nM AD treated cells). (C) Cyclin E overexpression prevented the block by letrozole of AD-induced CDK2 protein levels. The same cell lysates as in A and B were subjected to western blot analysis (50 µg of protein) with cyclin E and CDK2 antibodies. The bar graph represents the densitometric values of the phosphorylated CDK2 bands. (D) LMW cyclin E overexpressing MCF-7/Ac1 cells cannot bypass the block by roscovitine of AD-induced increase in cell number. Left, MCF-7/Ac1 cells were cultured in IMEM with 10% charcoal-stripped serum medium (CSSM) without phenol red and with 600 µg/ml of G418 for 4 days before plating. Cells were then infected with the indicated adenoviruses (at 4000 m.o.i.) 24 hours before drug treatment. Cells were then treated with 25 nM AD and 1 µM letrozole and collected 3 days later for cell number. Right, cells were treated as in A except that letrozole was replaced by 20 µM of roscovitine. Columns, mean of two to three experiments, each in triplicates; bars, S.D.