Differential sensitivity of RIP3-proficient and deficient murine fibroblasts to camptothecin anticancer drugs

Abstract

Receptor-interacting protein 3 (RIP3) is a serine/threonine protein kinase, which has extensive substrates including its cognate kinase RIP1 and multiple metabolic enzymes involving oxidative phosphorylation [1,2] . RIP3 has been shown to be essential for development, immunity and some physiological or pathophysiological responses to exogenous and endogenous stimuli [3][4][5] . In 2009, three groups independently reported that RIP3 acted as a molecular switch between apoptosis and necrosis (also called as necroptosis) [6][7][8] . Specifically, RIP3 could turn tumor necrosis factor (TNF)-induced cell death from apoptosis to necrosis [6] . Most of small-molecule anticancer drugs elicit their anticancer effects via apoptotic induction [9] . However, it is unclear whether RIP3 affects the cellular sensitivity to small-molecule anticancer drugs.We treated A cells (NIH 3T3 cells, murine fibroblasts, RIP3 -/-; typically undergoing apoptosis in response to TNF stimulation) and N cells (NIH 3T3 cells, murine fibroblasts, RIP3 +/+ ; undergoing necroptosis in response to TNF stimulation) [6] with small-molecule anticancer drugs of different mechanisms of action. Detection by sulforhodamine B assays showed that A cells and N cells displayed differential sensitivity only to the examined topoisomerase I (Top1) inhibitors camptothecins. RIP3-deficient A cells revealed 32.6-and 40.2-fold higher sensitivity than RIP3-proficient N cells to SN38 and chimmitecan [10] , respectively ( Figure 1A). Such differential sensitivity was reflected as higher apoptosis rates ( Figure 1B) and more rapid G 2 /M arrest ( Figure 1C) in A cells than in N cells treated with chimmitecan. Consistently, exposure to chimmitecan caused faster reduction of the target protein Top1 in A cells than in N cells ( Figure 1D); and the treatment with chimmitecan or SN38 drove more γ-H2AX formation (a molecular marker for DNA double-strand breaks; Figure 1E) and produced more foci of phosphorylated ataxia telangiectasia mutated kinase (p-ATM) at Ser 1981 (sensing DNA double-strand breaks; Figures 1F and 1G) in A cells than in N cells. These results collectively indicate that the differential sensitivity of the RIP3-deficient A cells and the RIP3-proficient N cells to the examined Top1 inhibitors in all the tested events from inducing DNA double-strand breaks through sensing the damage signals to eliciting the final biological effects including cell cycle arrest, apoptosis and proliferation/growth inhibition.Both A cells and N cells were kindly gifted from Prof Jiahuai HAN, who used them to successfully demonstrate the role of RIP3 in regulating TNF-inducing cell death [6] . The differential sensitivity of the RIP3-deficient and proficient murine fibroblasts to the Top1 inhibitors suggests a role of RIP3 in determining the cellular sensitivity to those agents. In this case, however, RIP3 does not seem to directly affect the choice of cell death between apoptosis and necrosis, because no significant difference of the cellular sensitivity to the other examined anti...

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