Exogenous cdk4 overcomes reducedcdk4 RNA and inhibition of G1 progression in hematopoietic cells expressing a dominant-negative CBF – a model for overcoming inhibition of proliferation by CBF oncoproteins

Abstract

Core Binding Factor (CBF) is required for the development of de®nitive hematopoiesis, and the CBF oncoproteins AML1-ETO, TEL-AML1, and CBFb-SMMHC are commonly expressed in subsets of acute leukemia. CBFb-SMMHC slows the G1 to S cell cycle transition in hematopoietic cells, but the mechanism of this e ect is uncertain. We have sought to determine whether inhibition of CBF-mediated trans-activation is su cient to slow 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 ligand binding domain, also slows G1, if its DNA-binding domain is intact. Also, exogenous AML1 overcame CBFb-SMMHC-induced inhibition of proliferation. Representational di erence analysis (RDA) identi®ed cdk4 RNA expression as an early target of KRAB-AML1 activation. Inhibition of CBF activities by KRAB-AML1-ER or CBFb-SMMHC rapidly reduced endogenous cdk4 mRNA levels, even in cells proliferating at or near control rates as a result of exogenous cdk4 expression. Over-expression of cdk4, especially a variant which cannot bind p16 INK4a , overcame cell cycle inhibition resulting from activation of KRAB-AML1-ER, although cdk4 did not accelerate proliferation when expressed alone. These ®ndings indicate that mutations which alter the expression of G1 regulatory proteins can overcome inhibition of proliferation by CBF oncoproteins.

Potential involvement of the AML1-MTG8 fusion protein in the granulocytic maturation characteristic of the t(8;21) acute myelogenous leukemia revealed by microarray analysis

“…This result appears to be inconsistent with the finding by Lou et al, 76 that inhibition of AML1 function by KRAB-AML1-ER in mouse 32Dcl3 myeloid cells prevents Mpo induction in response to G-CSF. This may result from the different cell lines used.…”

“…The other explanation is that the effect of AML1-MTG8 on cell proliferation may result in the differentiated phenotypes. Recently, it was reported that inhibition of AML1 activity in mouse Ba/F3 B-lymphoid cells retards cell proliferation by down-regulating the Cdk4 gene, 76 and induced expression of AML1-MTG8 in human U937 leukemia cells cause growth arrest with decreased expression of CDK4, MYC and BCL2. 77 L-G cells expressing AML1-MTG8 also show slight growth retardation in the presence of IL-3, 38 although with the microarray analysis we could not find any alteration in expression of Cdk4, Myc and Bcl2 (data not shown).…”

The AML1-ETO fusion protein promotes the expansion of human hematopoietic stem cells

“…AML1 represses expression of p21 waf1/cip1 and shortens the G 1 phase of the cell cycle. 31,32 Also, in studies using a dominant-negative inducible AML1 protein, KRAB-AML1-ER, Lou et al 33 observed an inhibition of cell cycle progression with a concomitant decrease in cdk4 levels, again arguing for the involvement of AML1, and by extension AML1-ETO, in the G 1 -to-S transition. Whether an inhibitory effect of AML1-ETO on cell cycle progression contributes to the negative effects on committed progenitor cells requires further investigation.…”

CCAAT/enhancer-binding proteins are required for granulopoiesis independent of their induction of the granulocyte colony-stimulating factor receptor

“…On the other hand, alterations that stimulate 32D cl3 proliferation during G 1 , such as expression of exogenous c-Myb, cdk4, or cyclin D2, prevent their differentiation in response to G-CSF. 62,63 Thus, inhibition of G 1 progression by K␣ER does not account for its ability to block 32D cl3 granulopoiesis, as slowed proliferation is expected to accelerate their differentiation.…”