Tumor induction by an Lck-MyrAkt transgene is delayed by mechanisms controlling the size of the thymus

Abstract

Transgenic mice expressing MyrAkt from a proximal Lck promoter construct develop thymomas at an early age, whereas transgenic mice expressing constitutively active Lck-AktE40K develop primarily tumors of the peripheral lymphoid organs later in life. The thymus of 6- to 8-week-old MyrAkt transgenic mice is normal in size but contains fewer, larger cells than the thymus of nontransgenic control and AktE40K transgenic mice. Earlier studies had shown that cell size and cell cycle are coordinately regulated. On the basis of this finding, and our observations that the oncogenic potential of Akt correlates with its effect on cell size, we hypothesized that mechanisms aimed at maintaining the size of the thymus dissociate cell size and cell cycle regulation by blocking MyrAkt-promoted G(1) progression and that failure of these mechanisms may promote cell proliferation resulting in an enlarged neoplastic thymus. To address this hypothesis, we examined the cell cycle distribution of freshly isolated and cultured thymocytes from transgenic and nontransgenic control mice. The results showed that although neither transgene alters cell cycle distribution in situ, the MyrAkt transgene promotes G(1) progression in culture. Freshly isolated MyrAkt thymocytes express high levels of cyclins D2 and E and cdk4 but lower than normal levels of cyclin D3 and cdk2. Cultured thymocytes from MyrAkt transgenic mice, on the other hand, express high levels of cyclin D3, suggesting that the hypothesized organ size control mechanisms may down-regulate the expression of this molecule. Primary tumor cells, similar to MyrAkt thymocytes in culture, express high levels of cyclin D3. These findings support the hypothesis that tumor induction is caused by the failure of organ size control mechanisms to down-regulate cyclin D3 and to block MyrAkt-promoted G(1) progression.

Figure 1

Biological consequences of constitutively active Akt transgenes in mouse thymocytes. (A) (Left) Expression of MyrAkt and AktE40K transgenes in the thymus of two Lck-MyrAkt HA (M-1 and M-9) and two Lck HA AktE40K (E-3 and E-4) transgenic mouse lines. Ntg, nontransgenic control. (Right) Western blot of cell lysates derived from sorted mouse thymocytes probed with the anti-HA antibody shows that the transgene is expressed in CD4⁺, CD8⁺, and CD4⁺/CD8⁺ double-positive (DP) but not CD4⁻/CD8⁻ double-negative (DN) cells. (B) Mortality curves of transgenic mouse lines.

Figure 2

MyrAkt transgenic thymocytes are larger than AktE40K transgenic and nontransgenic control thymocytes. (A) The weight of the thymus of AktE40K and MyrAkt transgenic and nontransgenic (NTg) mice is the same (six age- and sex-matched mice from each group). (B) Thymuses of MyrAkt1 transgenic mice contain fewer cells than thymuses of nontransgenic or AktE40K transgenic mice. (C) Cell size (forward scatter, FS) comparisons between nontransgenic (Non-Tg) control and AktE40K, nontransgenic control and MyrAkt, and AktE40K and MyrAkt thymocytes. MyrAkt thymocytes are larger. (D) The effect of MyrAkt on the cell size is cell autonomous. CD4 and CD8 SP and CD4/CD8 DP MyrAkt thymocytes are larger than their counterparts derived from nontransgenic control mice. DN, double-negative.

Figure 3

Cell cycle distribution and expression of cell cycle regulators in freshly isolated transgenic and nontransgenic control thymocytes. (Left) Cell cycle distribution of freshly isolated thymocytes from nontransgenic, MyrAkt, and AktE40K transgenic mice. There were four 6- to 8-week-old, age- and sex-matched mice per group. (Right) Expression of cell cycle regulators in freshly isolated thymocytes from nontransgenic control and transgenic mouse lines. Lanes: 1, nontransgenic; 2, MyrAkt1 M-1; 3, MyrAkt1 M-9; 4, Akt1-E40K E-3; and 5, Akt1-E40K E-4. For cyclin D3 and cyclin D2, the results of two independent experiments are shown. The same blots were probed with the antibodies to cyclin D2 and cyclin D3. The intensity of individual bands was measured by densitometry. Band intensity ratios were as follows: cyclin D3, nontransgenic vs. MyrAkt M-1 transgenic thymocytes = 2.23 and 1.86 in the two experiments shown; cyclin D2, nontransgenic vs. MyrAkt M-1 transgenic thymocytes = 0.42 and 0.45; cyclin E, nontransgenic vs. MyrAkt M-1 transgenic thymocytes = 0.25; cdk2, nontransgenic vs. MyrAkt M-1 transgenic thymocytes = 2.89.

Figure 4

Cell cycle distribution and expression of cell cycle regulators in cultured thymocytes from nontransgenic control, MyrAkt, and AktE40K transgenic mice. (A) Cell cycle distribution of cultured thymocytes from nontransgenic, MyrAkt, and AktE40K transgenic mice. Cells were grown in complete media; eight independent cultures from four mice per group were compared. (B) Expression of cyclin D3 and cyclin D2 in freshly isolated nontransgenic and MyrAkt transgenic thymocytes, as well as in cultured MyrAkt and AktE40K thymocytes. The same blot was probed with both antibodies. (C) Percent of cells in S phase. Prestarved (ps) are cells growing exponentially in complete medium. Data are combined from cultures of thymocytes derived from four mice per group and two independent cultures per mouse. (D) Expression of cell cycle regulators in cultured thymocytes from nontransgenic, MyrAkt, and AktE40K transgenic mice.

Figure 5

Primary thymomas from MyrAkt transgenic mice express high levels of cyclin D3. (A) Western blots of cell lysates from freshly isolated and cultured MyrAkt transgenic thymocytes, as well as from freshly isolated tumor cells from six thymomas (three in each of two separate experiments) derived from MyrAkt transgenic mice were probed with an antibody to cyclin D3. (B) Equal loading was confirmed in the second experiment by reprobing with an anti β-actin antibody (Sigma).