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Copyright by Eunyoung Park 2007 - The University of Texas at Austin

Copyright by Eunyoung Park 2007 - The University of Texas at Austin

the 1 and 10 μM retinol

the 1 and 10 μM retinol treatments in either cell line, indicating that maximal PI3K inhibition may have been achieved following treatment with 1 μM retinol. These data indicate that treatment of intact ATRA-resistant colon cancer cells with retinol rapidly decreases PI3K activity. Retinol does not decrease p110 and p85 protein levels or heterodimerization. Western blot analysis and immunoprecipitation were used to determine if retinol decreased PI3K activity by reducing PI3K protein levels or if retinol affected the heterodimerization of the p85 and p110 subunits of PI3K. As shown Fig. 1, PI3K activity was decreased after as little as 30 min of retinol treatment. In addition, we showed previously that the ability of retinol to inhibit cell invasion was independent of transcription and translation (144). Cells were treated with retinol from 30 to 240 min to determine the effect of retinol on p110 and p85 protein levels. Retinol treatment did not alter either p110 or p85 protein levels at any time point examined (Fig. 4.2 A, B, D and E). Heterodimerization of p85 and p110 activates PI3K (16). To determine retinol’s effect on the interaction of p85 and p110, cells were treated with retinol for 5 to 120 min. p110 antibody was used for immunoprecipitation and p85 antibody was selected for immunoblotting to detect the binding of p110 to p85. Retinol treatment did not change p85 and p110 heterodimerization at any time point examined (Fig. 4.2 C and F). These data indicate that retinol does not exert its inhibitory effect on PI3K activity by decreasing p85 or p110 protein levels or their heterodimerization. 79

Retinol decreases PI3K activity by inhibiting PI/PI3K interaction. To elucidate whether retinol decreased PI3K activity by inhibiting the ability of the PI3K substrate, PI, to interact with PI3K, we immunoprecipitated PI3K from non- retinol-treated cell lysates and measured the activity of this purified PI3K in the presence of increasing amounts of retinol. As can be seen in Fig. 4.3, the activity of PI3K was decreased by retinol in dose-dependent manner. The activity of PI3K in HCT-116 and SW620 cells was significantly decreased to 21.3 ± 10.9 and 41.8 ± 19.7% of vehicle control, when incubated with 10 μg retinol (0.6 μM retinol) and 10 μg PI, respectively. In addition, the activity of PI3K was further reduced to 10.3 ± 4.3, 6 ± 3.6, and 6.4 ± 3.4% of vehicle control by incubation with 20, 50, and 100 μg retinol, respectively (corresponding to 1.2, 2.9, and 5.8 μM retinol), in the HCT-116 cell line. Similarly, in SW620 cells, the activity of PI3K was decreased to 14.6 ± 6.6, 11.6 ± 8.8, and 11.5 ± 9.1% of vehicle control by incubation with 20, 50, and 100 μg retinol, respectively (corresponding to 1.2, 2.9, and 5.8 μM retinol). Retinol inhibits PI3K activity to a greater extent when PI3K is purified (Fig. 4.3) than when whole cells are treated with retinol (Fig. 4.1). Thus, it is conceivable that the metabolism of retinol or the sequestration of retinol by binding proteins, such as CRBP-I, may modulate the ability of retinol to inhibit PI3K activity in intact cells. Importantly, the ability of retinol to more significantly inhibit the activity of immunoprecipitated PI3K demonstrates that retinol is directly affecting the enzyme-substrate interaction. 80

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