Figure 10.

Loss of C/EBPα function blocks the ability of prolonged MAPK activation to reduce DNA synthesis and to increase protein levels of p21 in wild-type hepatocytes. Loss of C/EBPβ function abolishes the ability of prolonged MAPK activation to increase DNA synthesis in p21 null hepatocytes. (A) Antisense Ets2 modulation abolishes MAPK-induced proliferation in hepatocytes. (B) Antisense C/EBPα modulation alters the ability of MAPK signaling to cause cell cycle arrest and proliferation in wild-type and p21 null cells, respectively. (C) Antisense C/EBPβ modulation alters the ability of MAPK signaling to cause cell cycle arrest and proliferation in wild-type and p21 null cells, respectively. Hepatocytes were infected with kinase active ΔB-Raf:ER poly-L-lysine adenovirus (250 m.o.i.), followed by culture, as in Methods. In addition to ΔB-Raf:ER infection, cells were also infected with plasmids to express either antisense C/EBPα or antisense C/EBPβ. (A) Cells were transfected with an antisense oligonucleotide to Ets2. After 24 h to allow protein expression, hepatocytes were treated with either vehicle control or with 100 nM 4-hydroxytamoxifen (TAM) for 36 h (total time in primary culture, 60 h). In some experiments, before 4-hydroxytamoxifen addition, cells were treated with 50 μM PD98059. Hepatocytes under all conditions were continually incubated with 2 μCi ³H-thymidine throughout the final 36 h of culture, after which they were lysed and ³H-thymidine incorporation into DNA determined as in Methods. Addition of 4-hydroxytamoxifen alone did not alter the rate of DNA synthesis (our unpublished results). Data are the means (cpm ³H-thymidine incorporated into DNA ± SEM) of sextuplicate experiments from 4 separate animals. **p < 0.001 less than corresponding non-TAM treated control value; *p < 0.05 less than corresponding non-TAM treated control value. ^#p < 0.05 greater than corresponding value without antisense C/EBPα. ^$p < 0.05 greater than corresponding control value. ^&p < 0.05 less than corresponding control infected ΔB-Raf:ER + TAM value.