| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1995: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
To improve the efficacy of HSVtk/ganciclovir system gene therapy, modification of proliferating pool and cell cycle, and bystander effect are studied. We confirmed the proleferating activity correlated to the transduction rate of recombinant retrovirus from producer cell to glioma cell.
Ganciclovir were more cytotoxic to the cells their doubling time is short and high ratio of PCNA positive cell. Trichostatin A has a cell cycle regulation activity. Removing of trichostatin from culture media increased the S-phase glioma cell.
Bystander effect also influenced the cytotoxicity of ganciclovir. 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. If the GJIC has a main
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role on bystander effect, these chemicals might have the influence on the bystander effect.
GJIC is composed of 6 connexin43s on astrocyte. However, the connexin43 quantity was much smaller in human malignant glioma cell than that in rat normal fibroblast (NRF) by western immunoblot.
Connexin43 was identified in mouse fibroblast NRF on the cell surface even in the state of semiconfluent by immunolight microscopic studies using anti-connexin43 monoclonal antibody. Contrary, connexin43 was not detected on the cell surface of malignant glioma.Connexin43 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, GJIC was apparent on NRF.On the contrary, GJIC on RBR17T and HBR96T wss one-third and one-tenths of NRF respectively. GJIC on PG13 was not apparent.Down-regulation of GJIC by phorbol was noted only GJIC positive NRF and RBR17T significantly.
Despite the absence or small amount of GJIC 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-connexin43 monoclonal antibody that modulate GJIC on the bystander effect. When dexamethasone or beta-carotene was added during the coculture of glioma cell with autologous HSVtk positive glioma cell or PG13, dexamethasone and beta-carotene had no consistent promoting effect on bystander effect. When anti-connexin43 monoclonal antibody was added during the coculture of glioma cell with autologous tk positive glioma cell, anti-connexin43 monoclonal antibody did not inhibit bystander effect.
Our results suggest : 1st.There is bystander effect between glioma cell and tk positive cell. This mechanism was noted even between cells from different species. 2nd.Cx 43 was mainly intracytoplasmic in glioma cell. Because the small amount of GJIC on glioma cells, functionally the GJIC does not play a major role in the development of the bystander effect. 3rd.Dexamethasone and beta-carotene have a cytostatic effect.However dexamethasone and beta-carotene had no consistent promoting effect on the bystander effect. We therefore conclude that enhancers of GJIC such as dexamethasone and beta-carotene have no benefit on the HSVtk/GCV gene therapy.
This is the first report to directly demonstrate the effects of chemicals and anti-connexin43 monoclonal antibody that modulate GJIC on the bystander effect. Less
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