Clearance of apoptotic cells by macrophages induces HGF secretion. We examined the regulatory mechanisms of HGF mRNA and protein expression in macrophages upon exposure to apoptotic cells. The interaction of RAW 264.7 macrophages with apoptotic Jurkat cells, but not with viable cells, resulted in expression of HGF mRNA and protein. Exposure of RAW 264.7 cells to apoptotic cells induced activation of RhoA, the PI3K/Akt pathway, and MAPKs, including p38 MAPK, ERK, and JNK. Down-regulation of the RhoA/Rho kinase pathway by pharmacological inhibitors or a RhoA-specific siRNA suppressed HGF mRNA and protein expression by macrophages in response to apoptotic cells through the phosphorylation of Akt and the MAPKs. Inhibition of PI3K decreased phosphorylation of Akt and the MAPKs. Inhibition of JNK, but not p38 MAPK and ERK, reduced Akt phosphorylation. The pharmacological inhibitor of PI3K and the MAPKs blocked HGF mRNA and protein expression. Other types of apoptotic cells, such as HeLa cells and murine thymocytes, could also induce HGF mRNA through the RhoA-dependent pathway. Likely, the RhoA-dependent signaling pathway was required for HGF mRNA induction in primary cells of peritoneal macrophages in response to apoptotic cells. An HGFR-blocking antibody did not alter apoptotic cell-induced activation of RhoA, Akt, and the MAPKs, as well as HGF production. Overall, the data provide evidence that activation of the RhoA/Rho kinase pathway up-regulates transcriptional HGF production in response to apoptotic cells.

Gene therapy is used to induce immune responses, regulate tumor growth, or sensitize tumor cells to specific treatment. For sensitizing tumor cells to specific drug, we considered a prodrug-converting system using membrane-bound intestinal alkaline phosphatase (IAP) as the prodrug-activating genes. The IAP is capable of converting a relatively non-cytotoxic prodrug, etoposide phosphate (EP), into etoposide with a significant antitumor activity. We used the retroviral vector for transducing IAP gene into SNU638 gastric cancer cells and EP was prepared by phosphorylation of etoposide. To determine the chromosomal incorporation of membrane-bound IAP gene and AP activity in IAP gene-transduced cells (SNU638/IAP), we performed genomic PCR and AP activity analysis. In genomic DNA of SNU638/IAP cells, full cDNA fragment of a 2.5 kb IAP was detected, and AP activity was shown at most 15 approximately 18-fold increase compared with control cells. According to the in vitro cytotoxicity study, SNU638/IAP cells greatly enhanced the cytotoxic effect in proportion to the concentration of EP, while control cells didn't cause any cytotoxic effects after EPtreatment. Especially, the cell population of G2/M phase was increased in EP-treated SNU638/ IAP cells because P4 DNA unknotting activity of topoisomerase II was decreased by EP treatment such as the action mechanism of etoposide. Finally, a strong antitumor response was observed in SNU638/IAP cancer cells-bearing nude mice that were treated with EP. These results suggest that the prodrug-converting system by membrane-bound IAP gene and EP prodrug is useful as the strong strategy of gene therapy for cancer treatment.