Wortmannin, an inhibitor of p110 PI 3-kinase, also inhibits DNA-dependent protein kinase, which is known to mediate DNA double strand break repair. It was recently demonstrated that wortmannin sensitized cells to ionizing radiation (IR) (Price and Youmell, Cancer Res., 56, 246-250, 1996). Wortmannin was used to determine if the potentiation of IR-induced cytotoxicity in Chinese hamster ovary cells could be accounted for by an inhibition of DNA double strand break (DSB) repair. Wortmannin, at concentrations which were non-toxic per se (5 and 20 microM), increased IR cytotoxicity with dose enhancement factors at 10% survival of 2.7+/-0.28 (5 microM) and 5.3+/-0.86 (20 microM). The effects of wortmannin on DSB levels were assessed by neutral elution. The effects of wortmannin on the kinetics of DSB repair were evaluated over a 3 h time course. Wortmannin (50 microM) completely inhibited DSB repair over this period, without having any effect on DSB levels itself. The concentration-dependent effects of wortmannin on DSB levels showed that inhibition of DSB repair was significant at 1 microM, and near-maximal at 20 microM. In marked contrast, it exerted no effect on the kinetics of single strand break (SSB) repair as assessed by alkaline elution, even at concentrations as high as 50 microM. There was an excellent correlation between the concentration-dependence and exposure time of wortmannin required to enhance IR cytotoxicity and inhibit DSB repair. These data implicate inhibition of DNA-dependent protein kinase, and the consequent inhibition of DSB repair, as the mechanism whereby wortmannin potentiates the cytotoxicity of IR.
A fusion protein linking a truncated form of diphtheria toxin (DT 388 ) to human interleukin-3 (DT 388 IL3) kills malignant progenitors from some patients with acute myeloid leukemia (AML) while sparing normal progenitors. This study evaluated two variants of DT 388 IL3 with increased affinity for the IL-3 receptor (IL-3R) for their cytotoxicity to AML progenitors and determined the ability of quantitative reverse transcription-PCR assessment of expression of the IL-3R subunits to predict the effectiveness of wild-type DT 388 IL3 and its variants. Both the IL-3 deletion variant (D125-133) and the amino acid substitution variant (K116W) showed enhanced toxicity against AML colony-forming cells (AML-CFC; but not normal CFC) compared with wild-type DT 388 IL3 with the K116W variant achieving >90% AML-CFC kill with 17 of 23 patient samples. This variant was also more effective against AML cells engrafting in nonobese diabetic severe combined immunodeficient mice. There was a significant correlation between the expression of the a and, particularly, the common h subunit of the IL-3R on AML blasts detected by quantitative reverse transcription-PCR and AML-CFC kill. Thus, the combined use of IL-3R expression to select patients most likely to respond to DT 388 IL3 and the improved cytotoxicity of the K116W DT 388 IL3 variant against leukemic progenitors may enhance the clinical usefulness of these fusion proteins.
An increased expression of CD38 which may be due to action of proinflammatory cytokines produced in tumor-host reactions appears to account for the elevations in erythrocyte CD38-associated enzyme activities in cancer patients. The changes in these enzyme activities may provide a prognostic outlook in view of their apparently close correlation to tumor progressions.
Increased intracellular iron levels can both promote cell proliferation and death, as such; iron has a "two-sided effect" in the delicate balance of human health. Though the role of iron in the development of cancer remains unclear, investigations of iron chelators as anti-tumor agents have revealed promising results. Here, we investigated the influence of iron and desferrioxamine (DFO), the iron chelating agent on intracellular calcium in a human leukemia cell line, K562. Iron uptake is associated with increased reactive oxygen species (ROS) generation. Therefore, we showed that iron also caused dose-dependent ROS generation in K562 cells. The measurement of intracellular calcium was determined using Furo-2 with a fluorescence spectrophotometer. The iron delivery process to the cytoplasmic iron pool was examined by monitoring the fluorescence of cells loaded with calcein-acetoxymethyl. Our data showed that iron increased intracellular calcium, and this response was 8 times higher when cells were incubated with DFO. K562 cells with DFO caused a 3.5 times increase of intracellular calcium in the presence of doxorubicin (DOX). In conclusion, DFO induces intracellular calcium and increases their sensitivity to DOX, a chemotherapeutic agent.
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