c-myc upregulation occurs in late premalignancy and coincides with increased Akt signaling and Ki67 expression. (A) Forward scatter (FSC) and side scatter (SSC) analysis depicting increased blasting during premalignancy in the thymus, but not the lymph nodes, of tPTEN^−/− mice compared with littermate WT controls. Blasting is more pronounced in late premalignancy. (B) Flow cytometric analysis of Akt phosphorylation at Ser473 (P-Akt) and Ki67 expression in DP and CD4 SP thymocytes and CD3⁺ lymph node T cells in littermate tPTEN^−/− and WT mice. Late premalignancy is defined by a marked increase in both P-Akt and Ki67 in DP and CD4 SP thymocytes, but low expression of both markers in lymph node T cells. (C) Quantitative RT-PCR analysis showing the correlation of c-myc upregulation with late premalignancy in total thymocytes from littermate 13.5-week-old tPTEN^−/− mice. Early premalignant, late premalignant, and malignant populations were distinguished by the extent of blasting and Ki67 expression, as described in (A) and (B), and peripheral tumor formation. Fold change is relative to WT thymocytes. Data are presented as mean ± SD and are representative of 3 or more independent experiments. Statistics were calculated by Student t test (**P < .01, n.s., not significant).