Correlation between Rap sensitivity and p27^Kip1 levels (A) and the abrogation of antiproliferative effect of Rap by introduction of antisense cDNA into ED-40515(−) cells (B). (A) Left, the expression of p27^Kip1 after 48 hours culture with or without 10 nmol/L Rap in ED-40515(−) cells and HPB-ALL cells were examined. A total of 40 μg of whole cell lysates were subjected to immunoblotting with anti-p27^Kip1antibody. The relative proportion (%) of cells at S/G2M was also determined by a flow cytometric analysis after staining with acridine orange. Right: the antiproliferative effect of Rap on ED-40515(−) cells and HPB-ALL cells were examined by [³H]thymidine uptake for the last 6 hours of the 48-hour culture with or without 10 nmol/L Rap. Each column represents the means ± SD of triplicate samples. (B) Left: 5 μg of antisense p27^Kip1 cDNA, 5 μg of frame shifted-sense together with 5 μg of antisense p27^Kip1 cDNA, or 10 μg of empty vector were transfected into ED-40515(−) cells by electroporation method. The total amount of transfected plasmids was adjusted to 10 μg by adding a empty vector. The transfected cells were cultured in RPMI 1640 plus 10% FCS with 1 mg/mL G418 for 3 weeks. A total of 20 μg of protein from the transfected cells was subjected to immunoblotting with anti-p27^Kip1 antibody. Right: the transfected cells were cultured with or without 10 nmol/L Rap for 48 hours. The [³H]thymidine uptake by the cells for the last 6 hours was assayed. Each column represents the means ± SD of triplicate samples.