Limiting amounts of p27(Kip1) correlates with constitutive activation of cyclin E-CDK2 complex in HTLV-I-transformed T-cells

Human T-cells immortalized (interleukin-2 [IL-2] dependent) by the human T- cell lymphotropic/leukemia virus type I (HTLV-I), in time, become transform ed (IL-2 independent). To understand the biochemical basis of this transiti on, we have used the sibling HTLV-I-infected T-cell lines, N1186 (IL-2 depe ndent) and N1186-94 (IL-2 independent), as models to assess the responses t o antiproliferative signals. In N1186 cells arrested in G1 after serum/inte rleukin-2 (IL-2) deprivation, downregulation of the cyclin E-CDK2 kinase ac tivity correlated with decreased phosphorylation of CDK2 and accumulation o f p27(Kip1) bound to the cyclin E-CDK2 complex, as seen in normal activated PBMCs (peripheral blood mononuclear cells). In contrast, N1186-94 cells fa iled to arrest in G1 upon serum starvation, displayed constitutive cyclin E -associated kinase activity, and, although CDK2 was partially dephosphoryla ted, the amount of p27(Kip1) bound to the complex did not increase. This ob servation, extended to two other IL-2-dependent as well as to three IL-2-in dependent HTLV-I-infected T-cell lines, suggests that the lack of cyclin E- CDK2 kinase downregulation found in the late phase of HTLV-I transformation may correlate with insufficient amounts of p27(Kip1) associated with the c yclin E-CDK2 complex. Reconstitution experiments demonstrated that the addi tion of p27(Kip1) to lysates from N1186-94 starved cells resulted in the do wnregulation of cyclin E-associated kinase activity supporting the notion t hat the unresponsiveness of the cyclin E-CDK2 complex to growth inhibitory signals may be due to inadequate amounts of p27(Kip1) assembled with the co mplex in HTLV-I-transformed T-cells, In fact, the amount of p27(Kip1) prote in was lower in most HTLV-I-transformed (IL-2-independent) than in the immo rtalized (IL-2-dependent) HTLV-I-infected T-cells, Furthermore, specific in hibitors of the phosphatidylinositol 3-kinase (PI3K) induced an increase of p27(Kip1) protein levels, which correlated,vith G1 arrest, in both IL-2-de pendent and IL-2-independent HTLV-I-infected T-cells, Altogether, these res ults suggest that maintaining a low level of expression of p27(Kip1) is a k ey event in HTLV-I transformation.