Caffeine-induced apoptosis reveals a persistent lesion after treatment with bromodeoxyuridine and ultraviolet-B light.

Ultraviolet-B (UVB) light treatment of synchronized V79 Chinese hamster cells after pulse-labeling with bromodeoxyuridine (BrdUrd) reveals a marked age response for cell killing. Incubation after treatment in growth medium containing caffeine increases cell killing during the resistant portions of the cell cycle, resulting in a much less marked age response to UVB irradiation. Examination of the split-dose survival curves for BrdUrd and UVB light in the presence or absence of caffeine indicates that sensitization by caffeine is completely independent of the sparing effect of dose fractionation. Further, sensitization by caffeine is nearly complete after the first mitosis after the UVB exposure. With delayed addition of caffeine, however, nearly full sensitization can be elicited as late as two cell cycles after the treatment with BrdUrd and UVB light. Also, synchronized cells exposed to caffeine after treatment with BrdUrd and UVB in the S phase cease to incorporate radiolabeled thymidine and undergo apoptosis after the second mitosis. Thus exposure to caffeine reveals a persistent sensitivity lasting for at least two cell cycles, after the injury induced by treatment with BrdUrd and UVB.

Role of Fenton chemistry in thiol-induced toxicity and apoptosis.

Under certain conditions, many radioprotective thiols can be toxic, causing loss of colony-forming ability in cultured mammalian cells in a biphasic fashion whereby the thiols are not toxic at high or low concentrations of the drug, but cause decreased clonogenicity at intermediate (0.2-1.0 mM) drug levels. This symposium paper summarizes our studies using dithiothreitol (DTT) as a model thiol to demonstrate the role of Fenton chemistry in thiol toxicity. The toxicity of DTT in V79 cells has several characteristics: it is dependent on the medium used during exposure of cells to the drug; the toxicity is decreased or prevented by addition of catalase exogenously, but superoxide dismutase has no effect; the toxicity is increased by addition of copper, either free or derived from ceruloplasmin in serum; and the toxicity can be modified intracellularly by altering glucose availability or pentose cycle activity. Thus the data are consistent with a mechanism whereby DTT oxidation produces H2O2 in a reaction catalyzed by metals, predominantly copper, followed by reaction of H2O2 in a metal-catalyzed Fenton reaction to produce the ultimate toxic species, .OH. Studies comparing 12 thiols have shown that the magnitude of cell killing and pattern of dependence on thiol concentration vary among the different agents, with the toxicity depending on the interplay between the rates of two reactions: thiol oxidation and the reaction between the thiol and the H2O2 produced during the thiol oxidation. The addition of other metals, e.g. Zn2+, and metal chelators, e.g. EDTA, can also alter DTT toxicity by altering the rates of thiol oxidation or the Fenton reaction. Recent studies have shown that in certain cell lines thiols can also cause apoptosis in a biphasic pattern, with little apoptosis at low or high drug concentrations but greatly increased apoptosis levels at intermediate (approximately 3 mM) thiol concentrations. There appears to be a good correlation between those thiols that cause loss of clonogenicity and those that induce apoptosis, suggesting similar mechanisms may be involved in both end points. However, thiol-induced apoptosis is not prevented by addition of exogenous catalase. These observations are discussed in relation to the possible role of Fenton chemistry in induction of apoptosis by thiols.

Radiation-induced apoptosis in HL60 cells: oxygen effect, relationship between apoptosis and loss of clonogenicity, and dependence of time to apoptosis on radiation dose.

Apoptosis in HL60 human leukemia cells irradiated in vitro was quantified using a DNA fragmentation assay. Dose-response curves for induction of apoptosis in HL60 cells 6 h after irradiation with 280 kVp X rays in air and hypoxia give an oxygen enhancement ratio (OER) of 2.7. This is similar to the OER of 2.8 obtained from survival curves for HL60 cells using a soft agar clonogenic assay. However, HL60 cells are much more sensitive to radiation-induced loss of clonogenicity than to induction of apoptosis at 6 h. For example, 12 Gy in air reduces the surviving fraction to about 0.002 in a clonogenic assay, but 12 Gy does not cause any significant increase in the percentage of apoptosis-like DNA fragmentation 6 h after irradiation compared to unirradiated controls. However, if apoptosis is assayed 2-4 days after irradiation, the HL60 cells show greater sensitivity, with 5 Gy in air causing 45-50% apoptosis at 3 days. When apoptosis is measured 3 days after irradiation, the OER is similar to that obtained for survival and for apoptosis at 6 h. Although the HL60 cells exhibit radiation-induced apoptosis if one waits 2-4 days after low doses of radiation, rather than just 6 h, to conduct the assay, the amount of cells undergoing apoptosis is still not sufficient to account for all the loss of clonogenicity seen when HL60 cells are exposed to ionizing radiation.