Yuanlin Peng

Caspase 3–mediated stimulation of tumor cell repopulation during cancer radiotherapy

In 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. Furthermore, activated caspase 3, a key executioner in apoptosis, is involved in the growth stimulation. One downstream effector that caspase 3 regulates is prostaglandin E2 (PGE2), which can potently stimulate growth of surviving tumor cells. Deficiency of caspase 3 either in tumor cells or in tumor stroma caused substantial tumor sensitivity to radiotherapy in xenograft or mouse tumors. In human subjects with cancer, higher amounts of activated caspase 3 in tumor tissues are correlated with markedly increased rate of recurrence and death. We propose the existence of a cell death–induced tumor repopulation pathway in which caspase 3 has a major role.

Apoptotic Cells Activate the “Phoenix Rising” Pathway to Promote Wound Healing and Tissue Regeneration

Caspases in dying cells trigger the release of growth signals that promote tissue repair. Rising from the Dead Tissue injury causes cell death. Apoptotic cell death enables the elimination of damaged cells, clearing the way for tissue regeneration. Enzymes called caspases are activated in apoptotic cells, and Li et al. show that in addition to helping to execute the death process, caspases 3 and 7 stimulate the release of proliferation signals from dying cells. The authors found that skin wound healing and liver regeneration were compromised in mice deficient in caspase 3 or 7. Furthermore, stem or progenitor cells injected with or without irradiated cells or injected into irradiated or undamaged tissue proliferated more in the presence of irradiated cells or tissue. The authors dub the molecular events associated with death-induced proliferation the “phoenix rising” pathway, which involves the caspase-mediated activation of phospholipase A2 and the subsequent production and release of the lipid signal prostaglandin E2, a stimulator of cell proliferation. 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 E2, 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.

Deficiency in the catalytic subunit of DNA-dependent protein kinase causes down-regulation of ATM.

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 end-joining 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.

Radiation Leukemogenesis in Mice : Loss of PU. 1 on Chromosome 2 in CBA and C57BL/6 Mice after Irradiation with 1 GeV/nucleon 56Fe Ions, X Rays or γ Rays. Part I. Experimental Observations

Abstract Peng, Y., Brown, N., Finnon, R., Warner, C. L., Liu, X., Genik, P. C., Callan, M. A., Ray, F. A., Borak, T. B., Badie, C., Bouffler, S. D., Ullrich, R. L., Bedford, J. S. and Weil, M. M. Radiation Leukemogenesis in Mice: Loss of PU.1 on Chromosome 2 in CBA and C57BL/6 Mice after Irradiation with 1 GeV/nucleon 56Fe Ions, X Rays or γ Rays. Part I. Experimental Observations. Radiat. Res. 171, 474–483 (2009). Since deletion of the PU.1 gene on chromosome 2 is a crucial acute myeloid leukemia (AML) initiating step in the mouse model, we quantified PU.1 deleted cells in the bone marrow of γ-, X- and 56Fe-ion-irradiated mice at various times postirradiation. Although 56Fe ions were initially some two to three times more effective than X or γ rays in inducing PU.1 deletions, by 1 month postirradiation, the proportions of cells with PU.1 deletions were similar for the HZE particles and the sparsely ionizing radiations. These results indicate that while 56Fe ions are more effective in inducing PU.1 deletions, they are also more effective in causing collateral damage that removes hit cells from the bone marrow. After X, γ or 56Fe-ion irradiation, AML-resistant C57BL/6 mice have fewer cells with PU.1 deletions than CBA mice, and those cells do not persist in the bone marrow of the C57B6/6 mice. Our findings suggest that quantification of PU.1 deleted bone marrow cells 1 month postirradiation can be used as surrogate for the incidence of radiation-induced AML measured in large-scale mouse studies. If so, PU.1 loss could be used to systematically assess the potential leukemogenic effects of other ions and energies in the space radiation environment.

Role of Homologous Recombination in the Alpha-Particle-Induced Bystander Effect for Sister Chromatid Exchanges and Chromosomal Aberrations

Abstract Nagasawa, H., Peng, Y., Wilson, P. F., Lio, Y-C., Chen, D. J., Bedford, J. S. and Little, J. B. Role of Homologous Recombination in the Alpha-Particle-Induced Bystander Effect for Sister Chromatid Exchanges and Chromosomal Aberrations. Radiat. Res. 164, 141–147 (2005). The bystander effect for sister chromatid exchanges (SCEs) and chromosomal aberrations was examined in hamster cell lines deficient in either DNA-PKcs (V3 cells, deficient in nonhomologous end joining, NHEJ) or RAD51C (irs3 cells, deficient in homologous recombination, HR). Cells synchronized in G0/G1 phase were irradiated with very low fluences of α particles such that <1% of the nuclei were traversed by an α particle. Wild-type cells showed a prominent bystander response for SCE induction; an even greater effect was observed in V3 cells. On the other hand, no significant induction of SCE was observed in the irs3 RAD51C-deficient bystander cells irradiated at various stages in the cell cycle. Whereas a marked bystander effect for chromosomal aberrations occurred in V3 cells, the induction of chromosomal aberrations in irs3 bystander cells was minimal and similar to that of wild-type cells. Based on these findings, we hypothesize that HR is essential for the induction of SCE in bystander cells; however, HR is unable to repair the DNA damage induced in NHEJ-deficient bystander cells that leads to either SCE or chromosomal aberrations.

A unique role of the DNA fragmentation factor in maintaining genomic stability

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 DFF/CAD-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 DFF/CAD-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 mutations/deletions in the DFF/CAD 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.

Differential role of DNA-PKcs phosphorylations and kinase activity in radiosensitivity and chromosomal instability.

Abstract The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is the key functional element in the DNA-PK complex that drives nonhomologous end joining (NHEJ), the predominant DNA double-strand break (DSB) repair mechanism operating to rejoin such breaks in mammalian cells after exposure to ionizing radiation. It has been reported that DNA-PKcs phosphorylation and kinase activity are critical determinants of radiosensitivity, based on responses reported after irradiation of asynchronously dividing populations of various mutant cell lines. In the present study, the relative radiosensitivity to cell killing as well as chromosomal instability of 13 DNA-PKcs site-directed mutant cell lines (defective at phosphorylation sites or kinase activity) were examined after exposure of synchronized G1 cells to 137Cs γ rays. DNA-PKcs mutant cells defective in phosphorylation at multiple sites within the T2609 cluster or within the PI3K domain displayed extreme radiosensitivity. Cells defective at the S2056 cluster or T2609 single site alone were only mildly radiosensitive, but cells defective at even one site in both the S2056 and T2609 clusters were maximally radiosensitive. Thus a synergism between the capacity for phosphorylation at the S2056 and T2609 clusters was found to be critical for induction of radiosensitivity.

VARIATIONS IN RADIOSENSITIVITY AMONG INDIVIDUALS: A POTENTIAL IMPACT ON RISK ASSESSMENT?

To have an impact on risk assessment for purposes of radiation protection recommendations, significantly broad variations in carcinogenic radiosensitivity would have to exist in significant proportions in the human population. Even if we knew all the genes where mutations would have major effects, individual genome sequencing does not seem useful, since we do not know all these genes, nor can we be certain of the phenotypic effect of polymorphisms discovered. Further, sequencing would not reveal epigenetic changes in gene expression. Another approach to develop phenotypic biomarkers for cells or tissues for which variations in radiation response may reflect the variations in carcinogenic sensitivity. To be useful, experimental evidence for such a correlation would be crucial, and it is also evident that correlations may be tissue or tumor specific. Some cellular markers are discussed that have shown promise in this regard. They include chromosome aberration induction and DNA repair assays that are sufficiently sensitive to measure after modest or low doses or dose rates. To this end we summarize here some of these assays and review the results of a number of experiments from our laboratory that show clear differences in DNA repair capacity reflected by gamma-H2AX foci formation in cells from a high proportion (perhaps 1/3) of apparently normal individuals. A low dose-rate assay was used to amplify such differences. Another promising assay combines G(2) chromosomal radiosensitivity with the above gamma-H2AX foci on mitotic chromosomes. There are other potentially useful assays as well.

Suppression of DNA-PK by RNAi has Different Quantitative Effects on Telomere Dysfunction and Mutagenesis in Human Lymphoblasts Treated with γ Rays or HZE Particles

Abstract Zhang, Q., Williams, E. S., Askin, K. F., Peng, Y., Bedford, J. S., Liber, H. L. and Bailey, S. M. Suppression of DNA-PK by RNAi has Different Quantitative Effects on Telomere Dysfunction and Mutagenesis in Human Lymphoblasts Treated with γ Rays or HZE Particles. Radiat. Res. 164, 497–504 (2005). Basic to virtually all relevant biological effects of ionizing radiation is the underlying damage produced in DNA and the subsequent cellular processing of such damage. The damage can be qualitatively different for different kinds of radiations, and the genetics of the biological systems exposed can greatly affect damage processing and ultimate outcome—the biological effect of concern. The accurate repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and function. Incorrect repair of such lesions results in chromosomal rearrangements and mutations that can lead to cancer and heritable defects in the progeny of irradiated parents. We have focused on the consequent phenotypic effects of faulty repair by examining connections between cellular radiosensitivity phenotypes relevant for carcinogenesis after exposure to ionizing radiation, and deficiencies in various components of the non-homologous end-joining (NHEJ) system. Here we produced deficiencies of individual components of the DNA-dependent protein kinase (DNA-PK) holoenzyme (Ku86 and the catalytic subunit, DNA-PKcs), both singly and in combination, using RNA interference (RNAi) in human lymphoblastoid cell lines. Exposure of cells exhibiting reduced protein expression to either γ rays or 1 GeV/nucleon iron particles demonstrated differential effects on telomere dysfunction and mutation frequency as well as differential effects between radiation qualities.

Comparison of Several Radiation Effects in Human MCF10A Mammary Epithelial Cells Cultured as 2D Monolayers or 3D Acinar Stuctures in Matrigel

Abstract Lin, Y., Nagasawa, H., Peng, Y., Chuang, E. Y. and Bedford, J. S. Comparison of Several Radiation Effects in Human MCF10A Mammary Epithelial Cells Cultured as 2D Monolayers or 3D Acinar Stuctures in Matrigel. Radiat. Res. 171, 708–715 (2009). It has been argued that the cell-cell and cell-matrix interaction networks in normal tissues are disrupted by radiation and that this largely controls many of the most important cellular radiation responses. This has led to the broader assertion that individual cells in normal tissue or a 3D normal-tissue-like culture will respond to radiation very differently than the same cells in a 2D monolayer culture. While many studies have shown that, in some cases, cell-cell contact in spheroids of transformed or tumor cell lines can alter radiation responses relative to those for the same cells in monolayer cultures, a question remains regarding the possible effect of the above-mentioned disruption of signaling networks that operate more specifically for cells in normal tissues or in a 3D tissue-like context. To test the generality of this notion, we used human MCF-10A cells, an immortalized mammary epithelial cell line that produces acinar structures in culture with many properties of human mammary ducts. We compared the dose responses for these cells in the 2D monolayer and in 3D ductal or acinar structures. The responses examined were reproductive cell death, induction of chromosomal aberrations, and the levels of γ-H2AX foci in cells after single acute γ-ray doses and immediately after 20 h of irradiation at a dose rate of 0.0017 Gy/min. We found no significant differences in the dose responses of these cells in 2D or 3D growth conditions. While this does not mean that such differences cannot occur in other situations, it does mean that they do not generally or necessarily occur.