Differential regulation of caspase-9 by ionizing radiation- and UV-induced apoptotic pathways in thymic cells

Abstract

In mouse thymic lymphoma 3SB cells bearing wild type p53, ionizing radiation (IR) and UV light are potent triggers of caspase-3-dependent apoptosis. Although cytochrome c was released from mitochondria as expected, caspase-9 activation was not observed in UV-exposed cells. Laser scanning confocal microscopy analysis showed that caspase-9 is localized in an unusual punctuated pattern in UV-induced apoptotic cells. In agreement with differences in the status of caspase-9 activation between IR and UV, subcellular protein fractionation experiments showed that pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1), normally a part of the apoptosome assembled in response to the release of cytochrome c from mitochondria, and B-cell lymphoma extra long (Bcl-xL), an inhibitor of the change in mitochondrial membrane permeability, were redistributed by the IR-exposure but not by the UV-exposure. Instead of the sequestration of the capase-9/apoptosome activation in UV-induced apoptotic cells, the extrinsic apoptotic signaling generated by caspase-8 activation and consequent activation of B-cell lymphoma extra long (Bid) to release cytochrome c from mitochondria was observed. Thus, the post-mitochondrial apoptotic pathway downstream of cytochrome c release cannot operate the apoptosome function in UV-induced apoptosis in thymic 3SB cells. The intracellular redistribution and sequestration of apoptosis-related proteins upon mitochondrion-based apoptotic signaling was identified as a novel cellular mechanism to respond to DNA damage in an agent type-specific manner. This finding suggests that the kind of the critical ultimate apoptosis-inducing DNA lesion complex form resulting from the agent-specific DNA damage responses is important to determine which of apoptosis signals would be activated.

Introduction

Various genotoxic agents including carcinogens and mutagens are present in our environment. The most typical genotoxicities are ionizing radiation (IR) and non-ionizing UV light. Through evolution, organisms have developed multiple mechanisms to protect themselves from DNA damage produced by both types of radiation. With respect to the mechanism of genome integrity maintenance, differences between IR and UV are well recognized regarding DNA repair and cell cycle checkpoints [1], [2]. An additional defense system in which damaged cells are removed, namely apoptosis or programmed cell death, is also important for protecting multicellular organisms from genotoxicities, but a distinctive apoptotic signaling pathway generated by DNA damage caused by IR and UV in various cells or tissues remains to be elucidated [3], [4], [5], [6].

Thymic apoptosis typifies the defense system used to remove DNA-damaged cells, showing cellular response to relatively small doses of ionizing radiation (IR) [7], [8], [9]. This response is p53-dependent with a rapid time course that is typically concluded within a day after the IR exposure [10], [11], [12], [13]. Similarly to highly penetrating IR, thymocytes or thymic cells cultured in vitro are also susceptible to UV-induced apoptosis [13], [14]. In caspase-9-null/null mice, however, apoptosis of thymocytes is sensitive to UV despite an apparent resistance to IR [15]. Thus, caspase-9 is likely to be essential for IR-induced thymic apoptosis but is dispensable for UV-induced thymic apoptosis in mice. In addition, a marginal role of caspase-9 for apoptosis was also reported in fibroblasts using serum starvation [16].

Mitochondria play a central role in the initiation of apoptosis [17]. The cytochrome c initiates caspase-9 processing by formation of the apoptosome, a multimeric complex assembled in response to the release of cytochrome c from mitochondria [18]. The apoptosome formed by pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1), cytochorome c, and caspase-9, activates the downstream executioners such as caspase-3 [19], [20], [21]. It has been reported that this post-mitochondrial pathway is conserved even in UV-induced rapid apoptosis in thymic cells [22], [23], [24]. This hypothesis is inconsistent with the observed differences in caspase-9 requirement between IR- and UV-induced thymic apoptosis in caspase-9-null/null mouse [15]. However, it is possible that the apoptotic signaling pathways generated by IR and UV partly overlap, similar to the situation with DNA repair systems and DNA damage-induced cell cycle checkpoints.

Here, we investigated the processing of caspase-9 after IR- or UV-exposure in mouse thymic 3SB cells. IR induced the mitochondrion-based intrinsic apoptotic-signaling pathway involving caspase-9 activation. On the other hand, despite rapid cytochrome c release and caspase-3 activation, caspase-9 remained inactive in UV-exposed cells. However, caspase-8 and its downstream mediator, pro-apoptotic BH3 interacting domain death agonist (Bid) that promotes cytochrome c release from mitochondria, were activated in UV-exposed cells. In contrast to IR-exposed cells, the subcellular distribution of procaspase-9 and its related apoptotic proteins such as Apaf-1 and anti-apoptotic B-cell lymphoma extra long (Bcl-xL) were unaltered after UV-exposure. Apoptotic cells caused by UV-exposure had an inclusion body-like localization of procaspase-9 within the cytoplasmic region. The DNA-damaging agent-specific sequestration of post-mitochondrial apoptotic signaling identified here suggests that cells exposed to the genotoxic stimuli might respond differently to various DNA-damaging agents not only in DNA repair and cell cycle checkpoint(s) but also in apoptotic response. The critical ultimate apoptosis-inducing DNA lesion complex form may be different between IR- and UV-exposed cells.