The Effect of C/EBPalpha or C/EBPepsilon on 32Dcl3 Murine Myeloblasts and #1111 Acute Promyelocytic Leukemic Cells

Lee, Young Jin; Kogan, Scott

Abstract

BACKGROUND
The pathogenesis of acute myeloid leukemia (AML) is complicated by DNA damage, balanced or unbalanced translocation, deletion, inversion, abnormal transcription factors, receptors, and others. The CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBP epsilon (C/EBPepsilon), one of transcription factors, play important roles in normal granulopoiesis. We wished to assess whether increasing the activity of either C/EBPalpha or C/EBPepsilon could suppress the leukemic myeloblasts. METHODS: To make retrovirus, BOSC23 cells were transfected with retroviral constructs; mouse stem cell retrovirus-internal ribosomal entry site-green fluorescent protein (MIG), MIG-C/EBP alpha-estrogen receptor (ER) and MIG-C/EBP epsilon-ER. 32Dcl3 murine myeloblastic (32Dcl3) cells or #1111 acute promyelocytic leukemic (#1111 APL, #1111) cells were transduced with each retrovirus. Growth rate and differential cell count were examined, and granulocytic surface markers of Gr-1 and Mac-1 were checked. Transduced #1111 cells were injected into 20 sublethally irradiated (4.5 Gy) mice; at day 14, 4 groups of 5 mice each were input into subcutaneous tissue with placebo, 4-hydroxytamoxifen (4HT), all trans retinoic acid (ATRA), or 4HT & ATRA pellets; survival times were analysed when they died. RESULTS: The number of GFP (+) transduced 32Dcl3 cells with MIG (control group) at days 2, 4, and 6 were 684976, 1975965, and 2808244; 32Dcl3 cells with MIG-C/EBP alpha-ER were 77354, 53180, and 39460; and 32Dcl3 cell with MIG-C/EBP epsilon-ER were 328384, 698424, and 974850, respectively. The control group didn't express both Gr-1 and Mac-1, but C/EBP alpha expressed 56.1%, 55.6% and C/EBPepsilon expressed 31.3% and 32.6%, respectively. The differential counts of immature, intermediate, and mature forms in control group were 90.0%, 6.0%, and 4.0%; C/EBP 4.3%, 33.7%, and 62.0%; C/EBPepsilon 41.0%, 48.3%, and 10.7%, respectively. The mean survival time of transduced #1111 cells with MIG-C/EBP alpha-ER injected mice was 30.5 days in placebo group, 41.8 days in 4HT (C/EBP ) group, 69.0 days in ATRA group, and 97.8 days in 4HT (C/EBP ) & ATRA group. In case of MIG-C/EBP epsilon-ER, the survival time was 26.4 days in placebo group, 33.0 days in 4HT (C/EBP ) group, 49.6 days in ATRA group, and 52.5 days in 4HT (C/EBP ) & ATRA group. CONCLUSIONS: Both C/EBP and C/EBP suppressed cell growth and differentiation of 32Dcl3 cells, and they also suppressed cell growth of #1111 cells. The ATRA was more effective than C/EBP in APL, and C/EBP and ATRA had synergistic effects in APL. The growth arrest and differentiated action of C/EBPalpha was more powerful than that of C/EBPepsilon.