Abstract
The mechanism of bystander effect was considered to be due to the transfer of ganciclovir-metabolites derived from thymidine kinase (tk)-positive cells to the adjacent tk-negative cells via gap-junctional intercellular communication (GJIC). The purpose of this study is to clarify whether the compounds which are found to regulate the GJIC, promote or inhibit the bystander effect. GJIC is composed of 6 connexin 43s on astrocyte. However, the connexin 43 quantity is much smaller in human malignant glioma cell than that in rat normal fibroblast (NRF) as shown by western immunoblot. Connexin 43 was identified in NRF on the cell surface even In the state of semi-confluent by immunolight microscopic studies using anti-connexin 43 monoclonal antibody. Contrary, connexin 43 was not detected on the cell surface of malignant glioma. Connexin 43 In the PG13, human glioma cells are mainly localized intra-cytoplasmic rather than at the boundaries of cells. Result of GJIC by Lucifer yellow fluorescent dye microinjection method indicate that GJIC was evident on NRF. On the contrary, GJICs on RBRI17T and HBR96T were one-third and one-tenths of NRF respectively. GJIC on PG13 was not visible. Down-regulation of GJICs by phorbol were noted only in GJIC positive NRF and RBRI17T. Despite the absence or presence of small amount of GJICs in PG13 and glioma cell, we confirmed the bystander effect between human glioma cells and tk positive autologous glioma cells or PG13 (that hold HSVtk gene, but not the retroviral vector-producing cells) when these cells were treated with ganciclovir. Therefore, GJIC does not play a major role in the development of the bystander effect of PG13 or HSVtk gene transduced glioma cell on HSVtk negative glioma cell. We examined the effects of chemicals or anti-connexin 43 monoclonal antibody that modulate GJIC on the bystander effect. When dexamethasone or β-carotene was added during the coculture of glioma cell with autologous HSVtk positive glioma cell or PG13, dexamethasone and β-carotene had no consistent promoting effect on bystander effect. When anti-connexin 43 monoclonal antibody was added during the coculture of glioma cell with autologous tk positive glioma cell, anti-connexin 43 monoclonal antibody did not inhibit bystander effect.
