CBF-BETA-SMMHC, EXPRESSED IN M4EO AML, REDUCED CBF DNA-BINDING AND INHIBITED THE G1 TO S-CELL-CYCLE TRANSITION AT THE RESTRICTION POINT IN MYELOID AND LYMPHOID-CELLS

CBF beta-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. M ice lacking CBF subunits or expressing the CBF beta-SMMHC or AML1-ETO onco-proteins failed to develop definitive hematopoiesis. To investiga te these effects on hematopoiesis, we expressed CBF beta-SMMHC from th e metallothionein promoter, in both 32D c13 myeloid cells and Ba/F3 B- lymphoid cells. Addition of zinc increased CBF beta-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtain ed in 32D c13 cells were similar to those of endogenous CBF beta. Indi rect immunofluorescence revealed zinc-inducible speckled, nuclear stai ning in Ba/F3 cells and diffuse nuclear staining in 32D c13 cells. CBF beta-SMMHC reduced endogenous CBF DNA-binding fivefold in both cell t ypes, increased cell generation time 1.9-fold, on average, in 32D c13 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine i ncorporation into DNA correspondingly. CBFP beta-SMMHC increased the p roportion of cells in G1 1.7-fold, on average, in 32D c13 and Ba/F3 ce lls, and decreased the proportion of cells in S phase by a similar deg ree. CBF beta-SMMHC induced a marked increase in hypophosphorylated Rb , but did not alter IL-3 Receptor alpha or beta subunit levels. Neithe r apoptosis nor 32D differentiation was induced by zinc in IL-3 in the se lines. Induction of CBF beta-SMMHC WC in 32D c13 cells did not inhi bit their differentiation to neutrophils or their expression of myelop eroxidase mRNA in GCSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentia te inhibition of differentiation by CBF beta-SMMHC by allowing its inc reased expression.