The effect of AMD3100 on PARP cleavage and phosphorylation of Akt in MM cells cocultured with BMSCs. (A) MM.1S cells treated with AMD3100 (50 μM), bortezomib (5 nM), or their combination for 6 or 24 hours and expression of CXCR4 compared with nontreated cells were tested by flow cytometry. AMD3100 inhibited CXCR4 expression at 6 hours, but the expression recovered at 24 hours, whereas bortezomib had no effect on CXCR4 expression at either 6 or 24 hours. (B) MM.1S cells were cultured with BMSCs and treated with AMD3100 (50 μM), bortezomib (0, 2.5, and 5 nM), or their combination overnight in PARP cleavage experiments or for 24 hours in pAkt and pS6R experiments. Immunoblotting with anti-PARP antibody showed that AMD3100 alone did not induce PARP cleavage in MM cells, but it increased the PARP cleavage induced by bortezomib especially at low doses. This shows that the increase of the apoptotic effect of bortezomib induced AMD3100. Moreover, immunoblotting for pAkt and pS6R showed that AMD3100 abolished the phosphorylation of Akt and S6R in MM cells in coculture with BMSCs, which was shown to be a resistance mechanism to bortezomib.