Exogenous cdk4 overcomes reduced cdk4 RNA and inhibition of G1 progressionin hematopoietic cells expressing a dominant-negative CBF - a model for overcoming inhibition of proliferation by CBF oncoproteins

Core Binding Factor (CBF) is required for the development of definitive hem atopoiesis, and the CBF oncoproteins AML1-ETO, TEL-AML1, and CBF beta-SMMHC are commonly expressed in subsets of acute leukemia. CBF beta-SMMHC slows the G1 to S cell cycle transition in hematopoietic cells, but the mechanism of this effect is uncertain, We have sought to determine whether inhibitio n of CBF-mediated trans-activation is sufficient to stow proliferation. We demonstrate that activation of KRAB-AML1-ER, a protein containing the AML1 DNA-binding domain, the KRAB repression domain, and the Estrogen receptor l igand binding domain, also slows G1, if its DNA-binding domain is intact, A lso, exogenous AML1 overcame CBF beta-SMMHC-induced inhibition of prolifera tion, Representational difference analysis (RDA) identified cdk4 RNA expres sion as an early target of KRAB-AML1 activation. Inhibition of CBF activiti es by KRAB-AML1-ER or CBF beta-SMMHC rapidly reduced endogenous cdk4 mRNA l evels, even in cells proliferating at or near control rates as a result of exogenous cdk4 expression, Over-expression of cdk4, especially a variant wh ich cannot bind p16(INK4a) overcame cell cycle inhibition resulting from ac tivation of KRAB-AML1-ER, although cdk4 did not accelerate proliferation wh en expressed alone. These findings indicate that mutations which alter the expression of G1 regulatory proteins can overcome inhibition of proliferati on by CBF oncoproteins.