Efficient apoptotic cell clearance prevents the release of potentially immunogenic intracellular contents into the surrounding environment to maintain tissue homeostasis; however, the importance of membrane blebbing for apoptotic cell recognition and clearance has not been established. To address fundamental questions regarding the influence of ROCK1 cleavage and consequent apoptotic membrane blebbing in cellular corpse clearance and the maintenance of tissue homeostasis, mice were generated with a single amino acid substitution in the caspase cleavage site (D1113A) that converts ROCK1 to a non-cleavable (ROCK1nc) form. We previously showed that during apoptosis, caspase cleavage of ROCK1 removes an auto-inhibitory carboxy-terminal region to yield a constitutively active kinase fragment that leads to phosphorylation of downstream targets which promote contractile force generation leading to shrinkage, blebbing and nuclear disintegration. When apoptosis was induced in mouse embryonic fibroblasts, the biochemical apoptotic programme itself was unaffected, although cells had significantly impaired morphological alterations. Furthermore, by imaging apoptosis of GFP labelled melanoblasts in ex vivo embryonic skin explants, the effect of the ROCK1nc point mutation was to impair typical apoptotic morphological changes. To understand the biological role of membrane blebbing in the maintenance of tissue homeostasis, we used the liver-selective genotoxic compound diethylnitrosamine (DEN) to induce acute tissue damage. Following DEN treatment, we found that TUNEL positive apoptotic cells were inefficiently cleared in ROCK1nc mice, which resulted in increased neutrophil infiltration in response to the accumulating apoptotic debris. Histologically, the ROCK1nc livers appeared more damaged with evidence of steatohepatitis and perivenular damage, which was accompanied by higher serum alanine transferase levels. We then asked whether defects in tissue damage responses in ROCK1nc mice would affect tumor development in a model of DEN-induced hepatocellular carcinoma. Interestingly, ROCK1nc mice had reduced tumor volume and numbers, suggesting that defective blebbing may be beneficial for reducing tumor initiation and growth. Taken together, our results indicate that efficiency of phagocytic clearance may contribute to cancer susceptibility.
Citation Format: Linda Julian, Michael F. Olson. The role of apoptotic blebbing in tissue homeostasis and cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2956. doi:10.1158/1538-7445.AM2014-2956