A major goal of molecular oncology is to identify means to kill cells lacking p53 function. Most current cancer therapy is based on damaging cellular DNA by irradiation or chemicals. Recent reports support the notion that, in the event of DNA damage, the p53 tumour-suppressor protein is able to prevent cell death by sustaining an arrest of the cell cycle at the G2 phase. We report here that adeno-associated virus (AAV) selectively induces apoptosis in cells that lack active p53. Cells with intact p53 activity are not killed but undergo arrest in the G2 phase of the cell cycle. This arrest is characterized by an increase in p53 activity and p21 levels and by the targeted destruction of CDC25C. Neither cell killing nor arrest depends upon AAV-encoded proteins. Rather, AAV DNA, which is single-stranded with hairpin structures at both ends, elicits in cells a DNA damage response that, in the absence of active p53, leads to cell death. AAV inhibits tumour growth in mice. Thus viruses can be used to deliver DNA of unusual structure into cells to trigger a DNA damage response without damaging cellular DNA and to selectively eliminate those cells lacking p53 activity.

Phosphorylation of H2AX (␥H2AX) is an early sign of DNA damage induced by replication stalling. However, the role of H2AX in the repair of this type of DNA damage is still unclear. In this study, we used an inactivated adeno-associated virus (AAV) to induce a stalled replication fork signal and investigate the function of ␥H2AX. The cellular response to AAV provides a unique model to study ␥H2AX function, because the infection causes pannuclear H2AX phosphorylation without any signs of damage to the host genome. We found that pannuclear ␥H2AX formation is a result of ATR overactivation and diffusion but is independent of ATM. The inhibition of H2AX with RNA interference or the use of H2AX-deficient cells showed that ␥H2AX is dispensable for the formation and maintenance of DNA repair foci induced by stalled replication. However, in the absence of H2AX, the AAV-containing cells showed proteosome-dependent degradation of p21, followed by caspase-dependent mitotic catastrophe. In contrast, H2AX-proficient cells as well as H2AX-complemented H2AX؊/؊ cells reacted by increasing p21 levels and arresting the cell cycle. The results establish a new role for H2AX in the p53/p21 pathway and indicate that H2AX is required for p21-induced cell cycle arrest after replication stalling.

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G 2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G 2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.The human adeno-associated virus type 2 (AAV2) can perturb cell cycle progression (51, 71) and mediate specific killing of p53-deficient cells (51). Cells with intact p53 activity were able to arrest with 4N DNA content, whereas cells without functional p53 were not able to sustain this arrest and died. This effect was shown to depend not on the viral capsid proteins or other virus-encoded proteins but on the presence of the viral DNA. The AAV2 particle contains a single-stranded DNA molecule of 4.7 kb flanked by identical inverted terminal repeats which form T-shaped hairpin structures (5). The inverted terminal repeats are thought to function as primers for viral DNA replication. The hairpin structures of AAV2 DNA together with its single strandedness were hypothesized (51) to induce DNA damage signaling after AAV2 infection. In the work presented here, we set out to test this hypothesis, to identify proteins that recognize AAV DNA, and to elucidate how these proteins then activate the pathway that leads to G 2 arrest.An appropriate cellular response to DNA damage is crucial for maintenance of normal cell fate. Ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia-and Rad3-related (ATR) proteins are the two major signaling kinases that respond to DNA damage in cells.

Adeno-associated virus Rep78 protein has antiproliferative effects on cells. It inhibits cell cycle progression, and, in particular, Rep78 induces a complete arrest within S phase, a response rarely seen after cell DNA damage. We examined how Rep78 achieves such an efficient S phase block. Rep78 inhibits Cdc25A activity by a novel means in which binding between the two proteins stabilizes Cdc25A, thus increasing its abundance, while at the same time preventing access to its substrates cyclin-dependent kinase (Cdk) 2 and Cdk1. This effect alone does not induce a complete S phase block. In addition, Rep78, as well as Rep68, produces nicks in the cellular chromatin, inducing a DNA damage response mediated by ataxia telangiectasia mutated (ATM) leading to G1 and G2 blocks. Mutational analysis shows that the zinc finger domain and nuclease activity of Rep78 are both required for the S phase block. The results suggest that a true S phase block cannot be achieved through a single pathway, and that adeno-associated virus Rep78 protein arrests cells within S phase by interfering with two pathways that would normally lead to an S phase slow-down.Cdc25A ͉ cell cycle ͉ DNA damage

Adeno-associated virus (AAV) has an antiproliferative action on cells. We investigated the effect of the AAV replication proteins (Rep) on the cell division cycle using retroviral vectors. Rep78 and Rep68 inhibited the growth of primary, immortalized and transformed cells, while Rep52 and Rep40 did not. Rep68 induced cell cycle arrest in phases G 1 and G 2 , with elevated CDK inhibitor p21 and reduced cyclin E-, A-and B1-associated kinase activity. Rep78-expressing cells were also impaired in S-phase progression and accumulated almost exclusively with hypophosphorylated retinoblastoma protein (pRb). The differences between Rep78 and Rep68 were mapped to the C-terminal zinc ®nger domain of Rep78. Rep78-induced S-phase arrest could be bypassed by adenoviral E1A or papillomaviral E7 proteins but not by E1A or E7 mutants unable to bind pRb. Rb ±/± primary mouse embryonic ®broblasts displayed a strongly reduced S-phase arrest when challenged with Rep78, compared with matched Rb +/+ controls. These results suggest that physiological levels of active pRb can interfere with S-phase progression. We propose that the AAV Rep78 protein arrests cells within S-phase by a novel mechanism involving the ectopic accumulation of active pRb.