Cimica et al. BMC Cancer 2010, 10:634 http://www.biomedcentral.com/1471-2407/10/634 Page 7 of 13 activities at both 6 and 12 hours (Figure 4C). These results suggest that combination of flavopiridol and 4OH-Tam could increase apoptosis by inducing and prolonging caspase 3/7 activities. Mechanism of G2 arrest mediated by flavopiridol and 4OH-Tam To determine the mechanism of cell cycle arrest induced by flavopiridol and 4OH-Tam, we examined the expression levels of several cell cycle regulatory and tumor suppressor proteins in the presence and absence of drugs. 100 nM flavopiridol induced p53 and p21Waf-1 and repressed cyclin B1, consistent with the observed G2 arrest (Figure 5A). We found that the majority of cyclins and cdks, including cyclins D1, A and E, and cdks 2, 4 and 6 were unaffected by concentrations of fla- vopiridol ≤ 100 nM (Figure 5A). Interestingly, the pre- sence of 4OH-Tam did not significantly alter the effects of flavopiridol on cyclin and cdk expression, but further decreased the level of pRb protein and phosphorylation (Figure 5A compare lanes 4 and 8). We also observed differential effects on pRb phosphorylation. Concentra- tions of flavopiridol ≤ 25 nM increased the level of phosphorylated pRb (Figure 5A lanes 2 and 3), but 100 nM dramatically decreased the phosphorylated form of pRb (Figure 5A lane 4). Addition of 4OH-Tam Figure 5 Effect of flavopiridol and 4OH-Tam on expression of cell cycle regulatory proteins: A. Immunoblots of various cell cycle proteins in the presence and absence of flavopiridol and 4OH-Tam, after two days of treatment. B. ap21 immunocytochemistry of MON cells treated with: no drug (panel 1), 100 nM flavopiridol (panel 2), 5 μM 4OH-Tam (panel 3), or both 100 nM flavopiridol and 5 μM 4OH-Tam (panel 4). Note the strong nuclear staining in the drug treated cells (panels 2-4). C. Quantitation of MON cells with nuclear p21 using a-p21 immunocytochemistry after treatment with no drug, 100 nM flavopiridol, 5μM 4OH-Tam, or 100 nM flavopiridol and 5μM 4OH-Tam. Average values represent counting of more than 200 cells in four random fields for each treatment group.