Virus-mediated killing of cells that lack p53 activity

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.

Chk1 instability is coupled to mitotic cell death of p53-deficient cells in response to virus-induced DNA damage signaling

Peter Martin Beard, Michail Fragkos

Adeno-associated virus (AAV) DNA, by mimicking a stalled replication fork, provokes a DNA damage response that can arrest cells in the G2/M phase of the cell-cycle. This response depends strictly on DNA damage signaling kinases ATR and Chk1. Here, we used AAV to study long-term effects of DNA damage signaling in cells with altered p53 status. In HCT116 cells, in response to damage signaling, p53 represses transcription of the genes encoding mitotic regulators Cdc25C, cyclin B1, and Plk1 to establish a firm G2 arrest. Isogenic cells lacking p53 maintain these three proteins at constant levels yet can still arrest initially in G2 because Chk1 signaling inhibits their enzymatic activities. Unexpectedly, the levels of Chk1 fall abruptly in a proteasome-dependent manner after two days of arrest in G2. In p53-deficient cells, this Chk1 instability is coupled to recovery of the phosphatase activity of Cdc25C and in the kinase activities of Plk1 and Cdk1/cyclin B1. Consequently, the p53-deficient cells enter lethal mitosis. Thus, the Chk1-mediated arrest is transient: it initially causes cells to accumulate in G2 until p53-dependent transcriptional repression of mitotic proteins takes over. p53-deficient cells cannot maintain the DNA damage signaling-induced G2 arrest after Chk1 has disappeared, and continue into catastrophic mitosis. Restoring Chk1 prevents the cells from entering such mitosis. These results reveal a mechanism based on Chk1 stability that regulates mitotic entry after DNA damage and elucidate the controversial phenomenon of p53-promoted cell survival in the face of damage signaling.

2007

Cells with defective p53-p21-pRb pathway are susceptible to apoptosis induced by p84N5 via caspase-6

Peter Martin Beard

Adeno-associated virus (AAV) infection triggers a DNA damage response in the cell. This response is not induced by viral proteins but by virtue of the structure of AAV ssDNA being recognized by the cell as damaged DNA. The consequence of this is the killing of cells lacking p53 activity. We have observed that cells that lack p21 or pRb activity are also sensitive to AAV-induced cell death. We report that cells respond to AAV infection by activating two DNA damage signaling cascades. The first activates the p84N5 protein, which in turn activates caspase-6, leading to cell death. The second cascade activates the p53-21-pRb pathway, which inhibits activation of the p84N5 protein and thus prevents cell death. The result of the antagonistic interaction between these two pathways is that cells that do not exhibit functional p53-p21-pRb signaling undergo apoptosis as a consequence of AAV infection. Cells with a functional p53-21-pRb pathway are refractory to AAV-induced cell death. These results show that p53, although a proapoptotic protein, together with pRb and p21 proteins, is a member of an antiapoptotic cellular mechanism. As such, these experiments reveal features that may be exploited to specifically kill cells that lack the p53-p21-pRb pathway, such as cancer cells. The use of AAV to expose these subtle characteristics of intracellular signaling further highlights the advantages of using viruses as precision tools with which to address questions of cell biology.

2007

H2AX is required for cell cycle arrest via the p53/p21 pathway

Peter Martin Beard, Michail Fragkos

Phosphorylation of H2AX (gamma 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 gamma H2AX. The cellular response to AAV provides a unique model to study gamma H2AX function, because the infection causes pannuclear H2AX phosphorylation without any signs of damage to the host genome. We found that pannuclear gamma 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 gamma 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.

2009