Joel S. Bedford

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.

A quantitative comparison of potentially lethal damage repair and the rejoining of interphase chromosome breaks in low passage normal human fibroblasts

After long postirradiation incubation periods, the residual frequency of prematurely condensed chromosome fragments following X-ray exposure of noncycling diploid human fibroblasts was found to be correlated with the frequency of chromosome aberrations observed under identical treatment conditions when the cells were subcultured and scored after they reached mitosis. Over a wide range of doses, the proportion of such cells without aberrations at their first metaphase was not significantly different from the proportion able to form macroscopic colonies. Further, the rate of rejoining of interphase chromosome breaks was the same as the rate of increase in survival due to the repair of potentially lethal damage (PLD). These results suggest that there is a one-to-one correspondence between the initial breakage and rejoining of G0 chromosomes and the induction and repair of PLD measured by delayed plating from plateau-phase cultures of these cells.

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.

DOSE RATE: ITS EFFECT ON THE SURVIVAL OF HELA CELLS IRRADIATED WITH GAMMA RAYS.

Ionizing radiations produce their effect on living material owing to the processes of ionization and excitation which follow the absorption of radiant energy. For a given type of radiation, the energy absorbed per gram (that is, the dose in rads) is the most important single factor affecting the biological response. In the case of relatively sparsely ionizing radiation, however, such as Xand y-rays, the rate of energy absorption (the dose rate) is also of fundamental importance. Since about 1920 there have been reports in the literature that dose rate modifies such radiation effects as division delays in chick tissue cultures (1), chromosome aberrations in Tradescantia (2), cleavage delay in Arbacia eggs (3) and growth inhibition of roots of Vicia faba (4). During the past decade, many extensive investigations have been reported concerning the influence of dose rate on the LD50(30o) for small mammals exposed to whole-body irradiation (5-12). The present communication describes a series of experiments in which HeLa cells, cultured in vitro, were irradiated with cobalt-60 7-rays at three different dose rates. The aim of the investigation was to determine whether the dose-rate effect commonly observed with small mammals could be fully accounted for at the cellular level, or whether a systemic effect on the whole animal was involved.

Dose-Rate Effects in Synchronous Mammalian Cells in Culture

The life cycle and survival of synchronized HeLa and Chinese hamster cells were examined during continuous irradiation at dose rates of 38 or 90 rads per hour. The results indicate that over a give...

Radiation-induced cellular reproductive death and chromosome aberrations.

If a major mode of cell killing by ionizing radiation is the death of cells containing visible chromosomal aberrations, as for example from anaphase-bridge formation at mitosis, then cells bearing such aberrations should be selectively eliminated from the population, resulting in an increased survival potential for the population remaining at each succeeding cell generation. Using synchronized V79B Chinese hamster cells, we measured the aberration frequency and the colony-forming ability of mitotic cells at each of the first three generations following irradiation in G1. Cells were resynchronized by mechanical harvest at each succeeding mitosis after irradiation in order to avoid mixing of generations in the cell population at later sampling times. As anticipated, the chromosome aberration frequencies decreased markedly from the first to the second and from the second to the third mitosis. The surviving fraction, however, was virtually the same for plating assays carried out immediately after irradiation, at the first, or at the second mitosis. The surviving fraction was significantly higher for cells reaching the third postirradiation mitosis. Survival and aberration frequencies were assayed again at approximately the fourteenth postirradiation division, by which time the irradiated and control populations were not significantly different.

The effect of hypoxia on the growth and radiation response of mammalian cells in culture

Abstract During prolonged hypoxia, a progressive change occurs in the radiosensitivity of cultured mammalian cells. An attempt was made to determine whether this could be explained on the basis of a progressive change in the life cycle or age distribution of cells during hypoxia. Cell population growth kinetics and the cell life cycle were studied by two conventional methods of analysis. Both indicated an appreciable heterogeneity in the cell populations with regard to the ability of individual cells to negotiate their life cycle during hypoxia. This heterogeneity was confirmed by studying the development of individual microcolonies under hypoxia. From this study, the following conclusions were reached regarding changes which occurred during hypoxia: (1) the growth rate was decreased and the life cycle lengthened; (2) the overall proportion of cells in DNA synthesis decreased with time; (3) on average, a disproportionate lengthening of the G1 phase of the life cycle was implicated; and (4) these changes w...

Incidence of acute myeloid leukemia and hepatocellular carcinoma in mice irradiated with 1 GeV/nucleon 56Fe Ions

Abstract Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the leukemogenic efficacy of one such HZE species, 1 GeV 56Fe ions, a component of space radiation, in a mouse model for radiation-induced acute myeloid leukemia. CBA/CaJ mice were irradiated with 1 GeV/nucleon 56Fe ions or 137Cs γ rays and followed until they were moribund or to 800 days of age. We found that 1 GeV/nucleon 56Fe ions do not appear to be substantially more effective than γ rays for the induction of acute myeloid leukemia (AML). However, 56Fe-ion-irradiated mice had a much higher incidence of hepatocellular carcinoma (HCC) than γ-irradiated mice, with an estimated RBE of approximately 50. These data suggest a difference in the effects of HZE iron ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis.

Repression of mutagenesis by Rad51D-mediated homologous recombination

Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute in a nonessential but critical manner to HRR proficiency. We constructed and characterized a knockout of the paralog Rad51D in widely studied CHO cells. The rad51d mutant (clone 51D1) displays sensitivity to a diverse spectrum of induced DNA damage including γ-rays, ultraviolet (UV)-C radiation, and methyl methanesulfonate (MMS), indicating the broad relevance of HRR to genotoxicity. Spontaneous chromatid breaks/gaps and isochromatid breaks are elevated 3- to 12-fold, but the chromosome number distribution remains unchanged. Most importantly, 51D1 cells exhibit a 12-fold-increased rate of hprt mutation, as well as 4- to 10-fold increased rates of gene amplification at the dhfr and CAD loci, respectively. Xrcc3 irs1SF cells from the same parental CHO line show similarly elevated mutagenesis at these three loci. Collectively, these results confirm the a priori expectation that HRR acts in an error-free manner to repress three classes of genetic alterations (chromosomal aberrations, loss of gene function and increased gene expression), all of which are associated with carcinogenesis.

Relationship between the Recovery from Sublethal X-Ray Damage and the Rejoining of Chromosome Breaks in Normal Human Fibroblasts

Using plateau-phase cultures of AG1522 normal human fibroblasts, we examined relationships between the breakage and rejoining of chromosomes and the induction and repair of sublethal damage (SLD) following fractionated doses of X rays. The rate constant for the rejoining of breaks in prematurely condensed interphase chromosomes, measured previously, accurately predicts both the rate of change in survival due to potentially lethal damage (PLD) repair and the rate of change in survival for dose fractionation due to SLD repair. Further, changes in the frequency of chromosome-type deletions and asymmetrical exchange aberrations measured in the first postirradiation mitosis corresponded closely with changes in cell killing when doses were fractionated, and a dose-fractionation- or dose-rate-independent alpha component of damage was similar for aberration and cell killing end points. These results substantiate the hypothesis that sublethal damage repair results from the rejoining of breaks in interphase chromatin produced by a first dose so they no longer are capable of interacting with those produced by a second dose. The fact that the repair of potentially lethal damage is also readily explained on the basis of chromosome break rejoining (M. N. Cornforth and J. S. Bedford, Radiat. Res. 111, 385-405 (1987)) strongly suggests that PLD and SLD repair are different manifestations of the same basic process operating on the same basic lesions.