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Apoptotic DNA Fragmentation Factor Maintains Chromosome Stability in a P53-Independent Manner

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Apoptotic DNA Fragmentation Factor Maintains Chromosome Stability in a P53-Independent Manner Oncogene (2006) 25, 5370–5376 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Apoptotic DNA fragmentation factor maintains chromosome stability in a P53-independent manner B Yan1, H Wang3, H Wang2, D Zhuo1,FLi1, T Kon1, M Dewhirst1 and C-Y Li1 1Department of Radiation Oncology, Duke University Medical Center, Durham, NC, USA; 2Department of Medicine, Duke University Medical Center, Durham, NC, USA and 3West China 2nd Affiliated Hospital, Sichuan University, Chengdu, China DNA fragmentation factor (DFF)/caspase-activated DFF45/ICAD (inhibitor of CAD). During apop- DNase (CAD) is responsible for DNA fragmentation, a tosis, activated caspases cleave DFF45/ICAD, allowing hallmark event during apoptosis. Although DNA frag- DFF40/CAD degrade chromosomal DNA (Liu et al., mentation is an evolutionarily conserved process across 1997; Enari et al., 1998; Lechardeur et al., 2000). Defects species, its biological function is not clearly understood. in either DFF40/CAD or DFF45/ICAD lead to In this study, we constructed cell lines expressing a deficiency in DNA fragmentation during apoptosis. mutant ICAD (inhibitor of CAD) protein that is resistant Deficiency of dff40/dff45 genes was revealed in a num- to caspase cleavage and therefore constantly binds to ber of neoplasms (Maris et al., 1995; Caron et al., 1996; DFF/CAD and inhibits DNA fragmentation. We found Schwab et al., 1996; Van Gele, 1998a, b; Judson et al., that irradiation of these cells led to increased chromosome 2000). Decreased DFF45 expression is shown to asso- aberrations and aneuploidy when compared with their ciate with higher-stage neuroblastoma (Abel et al., 2002) parental controls. The increased chromosome instability and a poorer prognosis in some cancer patients (Konishi is observed irrespective of cellular P53 status, suggesting et al., 2002). DFF40/45 is also a candidate tumor that the effect of DFF/CAD is independent of P53. suppressor for neuroblastomas. Deletion or tumor- Inhibition of apoptotic DNA fragmentation resulted in specific mutations of dff45 gene were found in neuro- increased clonogenic survival of irradiated cells and blastoma and other tumor specimen and neuroblastoma a delay in removal of cells with DNA damages induced cell lines (Ohira et al., 2000; Abel et al., 2004). However, by radiation, an effect similar to that in cells with p53 the molecular mechanism underlying the association mutations. Consistent with DFF/CAD’s effect on clono- between DFF deficiency and human malignancies has genic survival, tumors established from cells deficient in not been clarified. DNA fragmentation showed enhanced growth in nude Chromosome instability (CIN), involving gains mice. Therefore, our results suggest that DFF/CAD plays and losses of whole or sections of chromosomes, occurs an important and P53-independent role in maintaining frequently in most human malignancies. The molecular chromosome stability and suppressing tumor development. basis of CIN is not well understood. Functional Oncogene (2006) 25, 5370–5376. doi:10.1038/sj.onc.1209535; alterations in many genes have been implicated in published online 17 April 2006 the generation CIN. Some of these genes are directly involved in maintaining chromosome structures. Exam- Keywords: apoptosis; DNA fragmentation; chromo- ples of these include genes involved in chromosome some instability; tumor/cancer condensation, sister-chromatid cohesion, kinetochore structure and function, and centrosome/microtubule formation and dynamics (Hoyt et al., 1990; Spencer et al., 1990). One such gene is the tubulin-encoding Introduction tub gene. tub gene mutants displayed increased CIN (Hoyt et al., 1990). Other genes are indirectly involved. DNA fragmentation factor (DFF) or caspase-activated An example is checkpoint genes that monitor the deoxyribonuclease (CAD) is a heterodimeric protein proper progression through the cell cycle (Murray, complex that functions downstream of caspases to 1995; Elledge, 1996; Nasmyth, 1996; Paulovich et al., execute DNA fragmentation during apoptosis. The 1997). One of the most important checkpoint genes is smaller subunit DFF40/CAD is a DNase that is the DNA-damage checkpoint gene p53. p53 mutations normally complexed with its inhibitor and chaperon, are seen in over 50% of all human tumors and p53 deficiency is an important contributing factor for CIN. In this study, we show that apoptotic DNA fragmen- Correspondence: Dr C-Y Li, Department of Radiation Oncology, tation is an important step to maintain chromosome Duke University Medical Center, Box 3455, Durham, NC 27710, stability and to suppress tumor development. It achieves USA. E-mail: [email protected] this through elimination of cells that have already suf- Received 22 December 2005; revised 14 February 2006; accepted 14 fered extensive damage. In addition, it appears to February 2006; published online 17 April 2006 function independently of P53. Apoptotic DNA fragmentation BYanet al 5371 Results and discussion content. As shown in Figure 1b, DNA fragmentation was significantly inhibited in mICAD-transduced L929 Effective inhibition of apoptotic DNA fragmentation cells when apoptosis was induced by tumor necrosis in cells expressing mutant ICAD factor (TNF)-a. Reduced DNA fragmentation in mI- DFF45/ICAD is a chaperon as well as an inhibitor CAD cells was not owing to decreased apoptosis because of DFF40/CAD. ICAD is normally complexed with the control and mICAD-expressing cells underwent CAD and inhibits its DNase activity. When apoptosis apoptosis at about the same rate (Figure 1b). These is induced, ICAD is cleaved by caspase and CAD is data are consistent with a previous report (Sakahira released to carry out DNA fragmentation in the nucleus. et al., 1998). There are two caspase cleavage sites in ICAD, D117 Although mICAD expression inhibited apoptotic and D224. We engineered a mutant ICAD (mICAD) DNA fragmentation, it was not completely abolished according to a published method by introducing point by mICAD in either TNF-a or radiation-treated cells mutations at the two cleavage sites, D117E and D224E. (Figure 1b and c). There was still residual fragmenta- mICAD is resistant to caspase cleavage and inhibits tion, which may be owing to the presence of endonu- DNA fragmentation during apoptosis in both human clease G or endogenous ICAD expression. In mICAD- and mouse cells (Sakahira et al., 1998). We established expressing cells, a small amount of CAD may be com- three cell lines expressing mICAD, TK6, WTK1 and plexed with ICAD and was released and activated when L929, as shown in Figure 1a. apoptosis is induced. Endonuclease G, on the other DNA fragmentation was analysed by DNA histogram hand, is a nuclease in the mitochondria that is released and TdT-mediated dUTP nick-end labeling (TUNEL) and translocates to the nucleus in a caspase-independent assay in these stable cells. Small DNA fragments pro- manner during apoptosis (van Loo et al., 2001). It has duced by CAD digestion of the cellular chromosomes been shown that endonuclease G may be able to can leak out of the ethanol-fixed cells. Therefore, cells compensate partially for the loss of CAD activity (Li that undergo DNA fragmentation have o2N DNA et al., 2001). a TK6 WTK1 L929 TK6 WTK1 L929 CM C MCM CM C M C M Anti-ICAD Anti-HA Anti-Actin b 120 L929/control 35 L929/control L929/mICAD 30 100 25 L929/mICAD 80 20 60 15 40 10 (<2N DNA) 5 20 % of apoptotic cells % of SubG1 cells 0 0 0hr 15hrs 27hrs 48hrs 0hr 15hrs 27hrs 48hrs Time after TNFa treatment Time after TNFa treatment c 50 40 45 L929/Ctrl 35 L929/Ctrl 40 L929/mICAD 30 L929/mICAD 35 30 25 25 20 20 15 15 10 10 5 5 % of TUNEL-positive 0 % of cells with <2N DNA 0 0Gy 5Gy 0Gy 5Gy Radiation Dose Radiation Dose Figure 1 Inhibition of DNA fragmentation in mutant ICAD-expressing cells. (a) Expression of mutant (left panel) and endogenous (right panel) ICAD protein as analysed by Western blotting in three cell lines. C: Parental control cells with mock transfections; M: mutant ICAD-transduced cells. (b) DNA fragmentation and apoptosis rate in L929 cells transduced with mutant inhibitor of CAD (mICAD) and treated with tumor necrosis factor (TNF)-a. (Left panel) DNA fragmentation in treated cells was determined by fluorescence-activated cell sorter (FACS) analysis of cells with o2N DNA content by cell cycle analysis in L929 cells treated with TNF-a. (Right panel) The levels of apoptosis as determined by annexin V–propidium iodide staining at different points after TNF-a treatment. (c) DNA fragmentation after exposure to irradiation. Control and mICAD-transduced L929 cells were exposed ionizing radiation (5 Gy) and examined for DNA fragmentation by use of two independent methods, FACS analysis of sub-G1 fraction (left panel) and TdT-mediated dUTP nick-end labeling assay (right panel). Oncogene Apoptotic DNA fragmentation BYanet al 5372 of CIN in cancers. In order to study the effect of a 18 DNA fragmentation on aneuploidy, we analysed the 16 TK6/control TK6/mICAD chromosomal ploidy of TK6 and WTK1 cells stably 14 transfected with mICAD. Parental TK6 and WTK1 12 show basic diploid cell karyotypes (Skopek et al., 1978; 10 Liber and Thilly, 1982; Yandell and Little, 1986). Con- 8 sistent with previous reports, fluorescence-activated cell 6 sorter (FACS) analysis showed that by far the majority 4 (98%) of TK6/control cells were diploid. TK6/mICAD % of cells w/ aberrations 2 cells had significantly increased aneuploid cells (9%, 0 P 0.01). Similar finding was made in WTK1 cells. Cells 0Gy 1.5Gy 3Gy o that overexpress mICAD had significantly more aneu- Dose of Radiation ploid cells than the control (Po0.03) (Figure 3). It indi- b 35 cates that inhibition of apoptotic DNA fragmentation WTK1/Control leads to chromosome number instability regardless of 30 WTK1/mICAD P53 gene status. 25 20 Inhibition of DNA fragmentation during apoptosis 15 delayed removal of cells with DNA damages induced 10 by radiation 5 In order to understand the mechanism underlying % of cells w/ aberrations 0 increased CIN in DNA fragmentation-inhibited cells, 0Gy 1.5Gy 3Gy we examined the time course of chromosome aberra- Dose of Radiation tions in irradiated cells at different time points after radiation.
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