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. Mice lacking CBF subunits or expressing the CBF beta-SMMHC or AML1-ETO oncoproteins failed to develop definitive hematopoiesis. To investigate these effects on hematopoiesis, we expressed CBF beta-SMMHC from the metallothionein promoter, in both 32D cl3 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 obtained in 32D cl3 cells were similar to those of endogenous CBF beta. Indirect immunofluorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and diffuse nuclear staining in 32D cl3 cells. CBF beta-SMMHC reduced endogenous CBF DNA-binding fivefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBF beta-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBF beta-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor alpha or beta subunit levels. Neither apoptosis nor 32D differentiation was induced by zinc in IL-3 in these lines. Induction of CBF beta-SMMHC in 32D cl3 cells did not inhibit their differentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of differentiation by CBF beta-SMMHC by allowing its increased expression.
Cao
W
W
Johns Hopkins Oncology Center, Division of Pediatric Oncology, Baltimore, Maryland 21287, USA.
Britos-Bray
M
M
Claxton
D F
DF
Kelley
C A
CA
Speck
N A
NA
Liu
P P
PP
Friedman
A D
AD
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D016193:G1 Phase / Q000235:genetics*; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D015479:Leukemia, Myelomonocytic, Acute / Q000235:genetics* / Q000378:metabolism; D008223:Lymphoma / Q000235:genetics* / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009218:Myosins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009195:Peroxidase / Q000235:genetics / Q000378:metabolism; D016185:Receptors, Interleukin-3 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000235:genetics; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D004268:DNA-Binding Proteins; D016185:Receptors, Interleukin-3; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D016179:Granulocyte Colony-Stimulating Factor; D008668:Metallothionein; D009195:Peroxidase; D009218:Myosins; D015032:Zinc