Fig. 3.
Overexpression of Six1 induces cyclin A1 expression and cellular proliferation. (A) MCF7-SIX1 transfectants have increased levels of cyclin A1 mRNA as compared with control transfectants. Upper shows a representative immunoprecipitation of Six1 in MCF7-SIX1 and MCF7-Control transfectants, whereas Lower demonstrates by qRT-PCR that cyclin A1 levels are increased in the Six1-transfectants, whereas cyclin A2 levels remain unchanged. (B) Immune-complex kinase assays demonstrate an increase in cyclin A1- and cdk2-associated kinase activities in Six1-overexpressing MCF7 cells, whereas cyclin A2-associated kinase activity remains unchanged. Histone H1 was used as a substrate for all immune complexes. (C and D) Assays measuring cell growth (C) and BrdUrd incorporation (D) demonstrate that Six1 overexpressing cell lines have a statistically significant increase in proliferation as compared with control cells. Each time point in the cell growth curve represents the mean of nine counts for each cell strain and is expressed as the mean ± SD of three cell lines per group. (E) Representative flow cytometry of one Six1-overexpressing (MCF7-SIX1–3) and one control (MCF7-Control 3) transfectant demonstrates that overexpression of Six1 accelerates cell cycle progression in MCF7 cells (similar results were obtained for the other Six1-overexpressing clones). The numbers on the bottom represent hours after release from serum starvation. (F) Expression of Six1 significantly increases tumor burden in nude mice. MCF7 cells (control and Six1-overexpressing) were injected into the flank of 8-week-old nude mice (previously implanted with estrogen pellets, four or five mice per group) at 10 × 106 cells per mouse in 100 μl of medium without serum, and tumor size was measured over a 6-week time period. Data are shown as mean ± SEM.