Endocycling cells do not apoptose in response to DNA rereplication genotoxic stress

Abstract

Initiation of DNA replication at origins more than once per cell cycle results in rereplication and has been implicated in cancer. Here we use Drosophila to examine the checkpoint responses to rereplication in a developmental context. We find that increased Double-parked (Dup), the Drosophila ortholog of Cdt1, results in rereplication and DNA damage. In most cells, this rereplication triggers caspase activation and apoptotic cell death mediated by both p53-dependent and -independent pathways. Elevated Dup also caused DNA damage in endocycling cells, which switch to a G/S cycle during normal development, indicating that rereplication and the endocycling DNA reduplication program are distinct processes. Unexpectedly, however, endocycling cells do not apoptose regardless of tissue type. Our combined evidence suggests that endocycling apoptosis is repressed in part because proapoptotic gene promoters are silenced. Normal endocycling cells had DNA lesions near heterochromatin, which increased after rereplication, explaining why endocycling cells must constantly repress the genotoxic apoptotic response. Our results reveal a novel regulation of apoptosis in development and new insights into the little-understood endocycle. Similar mechanisms may operate during vertebrate development, with implications for cancer predisposition in certain tissues.[Keywords: DNA replication; DNA damage; endocycle; checkpoint; apoptosis] Supplemental material is available at http://www.genesdev.org. The timely duplication of the genome during S phase of every cell division cycle requires that DNA replication initiate from thousands of origins. If too few origins initiate, replication forks can collapse, resulting in DNA damage and incomplete replication of the genome. Initiation of DNA replication from origins more than once per cell cycle, however, results in "rereplication" and subsequent DNA damage (Arias and Walter 2007). In recent years, it has become increasingly apparent that problems with DNA replication are common in premalignant cells, with subsequent checkpoint defects leading to genome instability and cancer (Dutta 2007). It remains unclear, however, whether all cells in development are equivalent with respect to their regulation of DNA replication and checkpoint responses. Here, we use Drosophila to investigate the checkpoint responses to rereplication in a developmental context. Two important steps in the cell cycle regulation of DNA replication are the assembly and activation of a prereplicative complex (pre-RC) (Sivaprasad et al. 2006). The pre-RC assembles onto origins in early G1 and is subsequently activated in S phase. During pre-RC assembly, the hexameric origin recognition complex (ORC) serves as a scaffold for origin association of Cdc6 and Cdt1, which are both required to load the hexameric minichromosome maintenance complex (MCM), the replicative helicase (Randell et al. 2006;Sivaprasad et al. 2006). Once the MCM complex is tightly bound, the origins are considered to be "licensed" and competent to initiate replic...

Fundamental differences in endoreplication in mammals and Drosophila revealed by analysis of endocycling and endomitotic cells

“…The enriched expression of genes involved in ATP biosynthesis and the robust expression of mitochondrial genes are consistent with a recently proposed role of mitochondria in polyploidization of maternal decidual cells (40). GO terms unique to the mitotic cell cycle and apoptosis were less common, in agreement with recent studies showing polyploid cells to have antiapoptotic properties, important in avoiding cell cycle checkpoints (22,41).…”

The function of Drosophila p53 isoforms in apoptosis

“…Mid-late third instar larvae and ovaries were dissected in either 1 × PBS or Grace's solution, and fixed in 6% formaldehyde as previously described, 76,77 and immunolabeled using either anti-cleavedCaspase-3, 1 : 50 (Cell Signaling, Danvers, MA, USA) or anti-cleaved DCP-1 antibody, 1 : 100 (Cell Signaling). Secondary antibodies were anti-rabbit Alexa 488 and anti-mouse Alexa 568 at 1 : 500 dilutions, and DNA was counterstained with DAPI.…”

TCTP directly regulates ATM activity to control genome stability and organ development in Drosophila melanogaster

“…Thus, in parallel with the peptide screen, we have also examined the role of dTCTP for growth regulation in salivary glands, a well-known model organ for studying cell growth 32 . In this study, we unexpectedly found that considerable levels of dTCTP protein are located in the cell nucleus of the salivary glands and fat bodies (Fig.…”

“…Thus, we checked whether dATM activity is impaired in dTCTP mutants by assaying g-H2Av level of endocycling larval salivary gland cells. In wild-type, these cells have a large number of DSBs in their nuclei due to repeated replication 32 , resulting in high levels of H2Av phosphorylation. Immunohistochemistry and western blot analysis showed that g-H2Av levels in homozygous dTCTP EY09182 (dTCTP EY/EY ) and dTCTP EY/h59 mutants are considerably reduced to about half of the wild-type level (Fig.…”