P-315

Mitomycin-C treatment protects islets from the progression of central damage during culture

J. Haga¹, T. Saitoh¹, T. Anazawa¹, T. Tsuchiya¹, A. Kenjo¹, T. Sato¹, N. Sato¹, T. Saito¹, K. Ise¹, M. Yamamoto², S. Waguri², M. Gotoh^1
1 Fukushima Medical University, Surgery, Fukushima, Japan; ² Fukushima Medical University, Anatomy and Histology, Fukushima, Japan

Objectives: Cultured islets were found to be less immunogenic in many studies, however, islet loss during culture prior to islet transplantation was frequently observed. We recently reported that mitomycin-C (MMC) treatment and subsequent culture of islets significantly prolongs graft survival in allo- and xenotransplantation models. The present study was performed to determine the changes in morphology and signal transduction in pancreatic islets after MMC treatment.

Methods: Freshly isolated rat islets were treated with 10 ?g/ml MMC for 30 min and then cultured for up to 3 days. The samples were processed for immunohistological studies and electron-microscopic examination at various times after treatment. A DNA fragmentation assay was performed to detect apoptotic cell death. Western blotting was performed to determine effects of MMC on signal transduction.

Results: As early as 4 h after culture, the islets showed central damage; the majority of cells were necrotic and stained with anti-HMGB1 antibody, and a few were apoptotic. The islet recovery rate after 3 days culture was improved by MMC treatment and the ratio of the damaged area to the whole area was significantly decreased following MMC treatment. Western blotting showed that MMC treatment increased the levels of activated forms of p53 and p21waf1 while the levels of the activated forms of Akt and caspase-3 were unchanged.

Conclusions: Our results indicated that the central damage during islet culture was mostly due to necrotic events being associated with the intracellular expression of HMGB1 rather than apoptotic events. MMC treatment significantly reduced these events, probably through a mechanism involving the p53–p21waf1 pathway.

P-315

Mitomycin-C treatment protects islets from the progression of central damage during culture

J. Haga¹, T. Saitoh¹, T. Anazawa¹, T. Tsuchiya¹, A. Kenjo¹, T. Sato¹, N. Sato¹, T. Saito¹, K. Ise¹, M. Yamamoto², S. Waguri², M. Gotoh^1
1 Fukushima Medical University, Surgery, Fukushima, Japan; ² Fukushima Medical University, Anatomy and Histology, Fukushima, Japan

Objectives: Cultured islets were found to be less immunogenic in many studies, however, islet loss during culture prior to islet transplantation was frequently observed. We recently reported that mitomycin-C (MMC) treatment and subsequent culture of islets significantly prolongs graft survival in allo- and xenotransplantation models. The present study was performed to determine the changes in morphology and signal transduction in pancreatic islets after MMC treatment.

Methods: Freshly isolated rat islets were treated with 10 ?g/ml MMC for 30 min and then cultured for up to 3 days. The samples were processed for immunohistological studies and electron-microscopic examination at various times after treatment. A DNA fragmentation assay was performed to detect apoptotic cell death. Western blotting was performed to determine effects of MMC on signal transduction.

Results: As early as 4 h after culture, the islets showed central damage; the majority of cells were necrotic and stained with anti-HMGB1 antibody, and a few were apoptotic. The islet recovery rate after 3 days culture was improved by MMC treatment and the ratio of the damaged area to the whole area was significantly decreased following MMC treatment. Western blotting showed that MMC treatment increased the levels of activated forms of p53 and p21waf1 while the levels of the activated forms of Akt and caspase-3 were unchanged.

Conclusions: Our results indicated that the central damage during islet culture was mostly due to necrotic events being associated with the intracellular expression of HMGB1 rather than apoptotic events. MMC treatment significantly reduced these events, probably through a mechanism involving the p53–p21waf1 pathway.