Abstract 4689: A Cyclin E fragment enhances radiation-induced cell death by modulating autophagy

Abstract

Abstract Autophagy is a type II programmed cell death pathway that is observed in various cellular systems. Our earlier studies have shown that Cyclin E is proteolytically cleaved to generate p18-Cyclin E (p18CycE) in hematopoietic tumor cells upon treatment with genotoxic agents, such as ionizing radiation and regulates apoptosis by displacing Bax from Ku70 and thus providing the first step of Bax activation. To investigate its possible role in regulating autophagy, p18CycE was stably expressed in H1299 lung carcinoma cells with expressions of proteins known to be involved in autophagy being examined following irradiation. Protein levels of LC3 I and II, classical markers of autophagy, were reduced in p18CycE-expressing, as compared to the parental H1299 cells. Radiation treatment further reduced LC3 expression in both cell lines, suggesting degradation or down regulation of LC3 at either protein or mRNA levels. Inhibition of lysosomal fusion with autophagosomes by chloroquine treatment resulted in accumulation of LC3 II in both parental and p18CycE-expressing cells. Irradiation, combined with chloroquine resulted in increased LC3 II levels compared to chloroquine treatment alone in p18CycE-expressing cells suggesting a higher autophagic influx. Beclin 1, another key regulator of autophagy, was expressed at comparable levels in both cell lines before as well as following irradiation. Levels of ATG3, known to be involved in LC3 I lipid modification were increased upon irradiation in p18CycE-expressing cells as compared to a low expression found in the parental cells. The expression of proteins participating in the ATG12-5 complex, which is required for autophagy, were diminished in p18CycE-expressing H1299 cells, however their expression was not changed upon irradiation. Moreover, levels of free ATG5 (not found in complex with ATG12) were reduced in p18CycE-expressing cells upon irradiation. Levels of ATG7, that is known to be required for ATG5-12 conjugation were increased after 6 hours following irradiation and then reduced to the expression levels found in H1299 cells while they decreased in p18CycE-expressing cells. Interestingly, levels of p62/SQSTM1, which is known to be involved in the autophagic degradation of poly-ubiquitinated protein aggregates, were diminished in p18CycE-expressing as compared to the parental cells. Taken together, these observations suggest that p18CycE-expressing cells exhibit a differential autophagic response upon irradiation compared to parental cells. Finally, PARP1 cleavage, as a marker of caspase-mediated apoptotic cell death, was enhanced in p18CycE-expressing cells following irradiation. Further investigation of the role of p18CycE in the regulation of autophagy following genotoxic stress could reveal an important crosstalk between apoptosis and autophagy, with p18CycE emerging as a possible candidate that links these two programmed cell death pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4689.