Abstract
In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-alpha-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-alpha resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications.
Highlights
It is well established that an appropriate response to stress stimuli is crucial for preventing cellular transformation as well as for maintaining normal tissue function
In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway
Granzyme B-mediated Target Cell Death Involves p53 Phosphorylation—Because p53 phosphorylation is essential in the regulation of its activity and to further investigate how p53 activity impacts on granzyme B (GrB)-induced killing of T1 cells, we examined the relationship between p53 phosphorylation and GrBinduced apoptotic cell death
Summary
GrB, granzyme B; wt, wild type; CTL, cytotoxic T-lymphocyte; Ab, antibody; Dioc6(3), 3,3Ј-dihexyloxacarbocyanine; CMA, concanamycin A; PFT-␣, pifithrin-␣; ROS, reactive oxygen species; siRNA, small interference RNA; PBS, phosphate-buffered saline; SLO, streptolysin-O; ATM, ataxia telangiectasia mutated. The p53 protein has a short half-life and is often undetectable in normal cells It is activated as a transcription factor through numerous post-translational modifications that allow its stabilization and accumulation in the nucleus to regulate target gene expression [11]. The role of this tumor suppressor protein in the control of apoptosis mediated by cytotoxic T-lymphocyte (CTL) is not well documented In this regard we have previously shown that p53 is a key determinant in anti-tumor CTL response as it regulates induction of Fas receptor expression, cellular FLICE/ caspase-8 inhibitory protein (cFLIP) short protein degradation, and CD95-induced activation of mitochondrial pathway in tumor cells [22, 23]. We first demonstrated that CTL-tumor target cell interaction resulted in p53 accumulation and activation Such activation is mediated by GrB and contributes at least in part to GrB-induced apoptosis. The current study emphasizes that in addition to its role in controlling irradiation and drug responses, p53 plays a key role in the regulation of CTLmediated apoptosis of tumor cells
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