SummaryIn cancer treatment, apoptosis is a well-recognized cell death mechanism through which cytotoxic agents kill tumor cells. Here we report that dying tumor cells use the apoptotic process to generate potent growth-stimulating signals to stimulate the repopulation of tumors undergoing radiotherapy. Surprisingly, activated caspase 3, a key executioner of apoptosis, plays key roles in the growth stimulation. One downstream effector that caspase 3 regulates is prostaglandin E2, which can potently stimulates growth of surviving tumor cells. Deficiency of caspase 3 either in tumor cells or in tumor stroma caused significant tumor sensitivity to radiotherapy in xenograft or mouse tumors. In human cancer patients, higher levels of activated caspase 3 in tumor tissues are correlated with significantly increased rate of recurrence and deaths. We propose the existence of a “Phoenix Rising” pathway of cell death-induced tumor repopulation in which caspase 3 plays key roles.

The ability to regenerate damaged tissues is a common characteristic of multicellular organisms. We report a role for apoptotic cell death in promoting wound healing and tissue regeneration in mice. Apoptotic cells released growth signals that stimulated the proliferation of progenitor or stem cells. Key players in this process were caspases 3 and 7, proteases activated during the execution phase of apoptosis that contribute to cell death. Mice lacking either of these caspases were deficient in skin wound healing and in liver regeneration. Prostaglandin E 2 , a promoter of stem or progenitor cell proliferation and tissue regeneration, acted downstream of the caspases. We propose to call the pathway by which executioner caspases in apoptotic cells promote wound healing and tissue regeneration in multicellular organisms the "Phoenix Rising" pathway.

Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKcs with small interfering RNA, Chinese hamster ovary V3 cells, lacking DNA-PKcs, had reduced levels of ATM and hSMG-1, but both were restored after transfection with PRKDC. Atm levels were also reduced in murine scid cells. Reduction of ATM in a human glioma cell line lacking DNA-PKcs was accompanied by defective signaling through downstream substrates, post-irradiation. A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences. This was accompanied by a reduction in ATM. These data were confirmed using immunocytochemical detection of the proteins. Within hours after transfection, a decline in PRKDC mRNA was seen, followed by a more gradual decline in DNA-PKcs protein beginning 1 day after transfection. No change in ATM mRNA was observed for 2 days post-transfection. Only after the DNA-PKcs reduction occurred was a reduction in ATM mRNA observed, beginning 2 days post-transfection. The amount of ATM began to decline, starting about 3 days post-treatment, then it declined to levels comparable to DNA-PKcs. Both proteins returned to normal levels at later times. These data illustrate a potentially important cross-regulation between the nonhomologous endjoining system for rejoining of DNA double-strand breaks and the ATM-dependent damage response network of pathways, both of which operate to maintain the integrity of the genome.

DNA fragmentation is a hallmark of apoptosis (programmed cell death). However, the biological function of apoptotic DNA fragmentation remains unclear. Here, we show that DNA fragmentation factor plays an important role for maintaining genomic stability. Inhibition or loss of the DNA fragmentation factor (DFF)͞ caspase-activated DNase (CAD), whose nuclease activity is responsible for digesting genomic DNA during apoptosis, led to significant increases in spontaneous or induced gene mutations, gene amplifications, and chromosomal instability in primary mouse cells and transformed human cell lines. The mechanism underlying genetic instability in DFF͞CAD-deficient cells, at least in part, involves a small but significant elevation in the survival of cells exposed to ionizing radiation, suggesting that apoptotic DNA fragmentation factor contributes to genomic stability by ensuring the removal of cells that have suffered DNA damage. In support of this hypothesis are the observations of increased cellular transformation of mouse embryonic cells from the DFF͞CAD-null mice and significantly enhanced susceptibility to radiation-induced carcinogenesis in these mice. These data, in combination with published reports on the existence of tumor-specific gene mutations͞dele-tions in the DFF͞CAD genes in human cancer samples, suggest that apoptotic DNA fragmentation factor is required for the maintenance of genetic stability and may play a role in tumor suppression.NA fragmentation is a hallmark of apoptosis (1, 2). It is now recognized that apoptotic DNA fragmentation is carried out by a heterodimeric protein complex called DNA fragmentation factor (DFF) (3, 4) or caspase-activated DNase (CAD) (5, 6). DFF is a heterodimeric protein complex composed of two subunits, DFF40͞CAD and DFF45͞inhibitor (I)CAD (3). DFF45͞ICAD is an inhibitor as well as a chaperone of DFF40͞CAD that ensures its proper folding. Expression of DFF40͞CAD in various systems in the absence of coexpressed DFF45 results in generation of DFF40-inactive aggregates. Under normal circumstances, DFF40͞CAD is complexed with DFF45͞ICAD, so its DNase activity is inhibited (3-5). When apoptosis is initiated, the activated caspase cleaves DFF45͞ICAD, and DFF40͞CAD is released into the nucleus to carry out DNA fragmentation.Recently, it was discovered that the genes encoding the nuclease that are responsible for the fragmentation of DNA during apoptosis, DFF40͞CAD and DFF45͞ICAD, are aberrantly expressed in many tumor types. In addition, the abnormalities in this gene are associated with poor prognosis in cancer patients (7-9). Most significantly, tumor-specific DFF45 gene mutations or deletions were identified in human germ cell tumors and neuroblastoma tumors from patients (10, 11), indicating the involvement of this gene in tumor development.The present study was initiated to explore the potential relationship between apoptotic DNA fragmentation and tumor development. We reasoned that the process involved in the destruction of genomic DNA might have a direct effect on g...