Beatriz L. Molinari

Imbalance of antioxidant enzymes in tumor cells and inhibition of proliferation and malignant features by scavenging hydrogen peroxide

The aim of this study was to evaluate the endogenous alterations of the antioxidant enzymes in tumor cells and to specifically compensate the resulting changes in the levels of reactive oxygen species (ROS) to control the malignant growth. We determined and compared the activities of antioxidant enzymes and the levels of superoxide anion (

Induction and rejoining of DNA double strand breaks assessed by H2AX phosphorylation in melanoma cells irradiated with proton and lithium beams.

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H2O2 scavenging inhibits G1/S transition by increasing nuclear levels of p27KIP1

) and hydrogen peroxide (H2O2) in tumor cell lines with different degrees of malignancy, paired with regard to their origin (PB/CH72T4, PDV/PDVC57, and HBL‐100/MCF‐7). An increase in superoxide dismutase activity and a decrease in the activities of H2O2‐detoxifying enzymes, as a function of malignancy, coupled with a rise in H2O2 and a decrease in

Induction and Persistence of Large γH2AX Foci by High Linear Energy Transfer Radiation in DNA-Dependent protein kinase–Deficient Cells

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Increased secretory activity and estradiol receptor expression are among other relevant aspects of MCF-7 human breast tumor cell growth which are expressed only in the absence of serum

were demonstrated. Treatment of cells with exogenous catalase showed a dose‐dependent inhibition of proliferation. This inhibition was also demonstrated in several cell lines of different tissue origin and species, suggesting a general role of H2O2 in cell proliferation. Moreover, stable expression of human catalase in MCF‐7 cells inhibited proliferation and also reverted malignant features. We conclude that H2O2 played a crucial and general role in the regulation of proliferation and that an endogenous imbalance in antioxidant enzymes could be a relevant event in the carcinogenesis process.

Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells

PURPOSE The aim of this study was to evaluate the induction and rejoining of DNA double strand breaks (DSBs) in melanoma cells exposed to low and high linear energy transfer (LET) radiation. METHODS AND MATERIALS DSBs and survival were determined as a function of dose in melanoma cells (B16-F0) irradiated with monoenergetic proton and lithium beams and with a gamma source. Survival curves were obtained by clonogenic assay and fitted to the linear-quadratic model. DSBs were evaluated by the detection of phosphorylated histone H2AX (gammaH2AX) foci at 30 min and 6 h post-irradiation. RESULTS Survival curves showed the increasing effectiveness of radiation as a function of LET. gammaH2AX labeling showed an increase in the number of foci vs. dose for all the radiations evaluated. A decrease in the number of foci was found at 6 h post-irradiation for low LET radiation, revealing the repair capacity of DSBs. An increase in the size of gammaH2AX foci in cells irradiated with lithium beams was found, as compared with gamma and proton irradiations, which could be attributed to the clusters of DSBs induced by high LET radiation. Foci size increased at 6 h post-irradiation for lithium and proton irradiations in relation with persistent DSBs, showing a correlation with surviving fraction. CONCLUSIONS Our results showed the response of B16-F0 cells to charged particle beams evaluated by the detection of gammaH2AX foci. We conclude that gammaH2AX foci size is an accurate parameter to correlate the rejoining of DSBs induced by different LET radiations and radiosensitivity.

Kinetics of MTT-formazan exocytosis in phagocytic and non-phagocytic cells.

The aim of the present study was to evaluate cell cycle regulation by scavenging H(2)O(2) in tumor cells. A significant arrest in the G1 phase of the cell cycle was demonstrated in CH72-T4 carcinoma cells exposed to catalase, associated with a decrease in cyclin D1 and an increase in the CDK inhibitory protein p27(KIP1). Moreover, we found a differential intracellular distribution of p27(KIP1), which remained in the nucleus after catalase treatment. In vivo experiments showed an increase in nuclear levels of p27(KIP1) associated with the inhibition of tumor growth by H(2)O(2) scavenging, confirming in vitro results. To conclude, H(2)O(2) scavenging may induce cell cycle arrest through the modulation of cyclin D1 and p27(KIP1) levels and nuclear localization of p27(KIP1). To our knowledge, this is the first report that demonstrates that the modulation of ROS alters the intracellular localization of a key regulatory protein of G1/S transition.

Cell-based quantitative evaluation of the MTT assay.

PURPOSE To evaluate the cell response to DNA double-strand breaks induced by low and high linear energy transfer (LET) radiations when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an essential protein of the nonhomologous end-joining repair pathway, lacks kinase activity. METHODS AND MATERIALS CHO10B2, a Chinese hamster ovary cell line, and its derived radiosensitive mutant cell line, irs-20, lacking DNA-PKcs activity, were evaluated after 0 to 3 Gy of γ-rays, plateau and Bragg peak protons, and lithium beams by clonogenic assay, and as a measurement of double-strand breaks, phosphorylated H2AX (γH2AX) foci number and size were quantified by immunocytofluorescence. RESULTS Irs-20 exhibited greater radiosensitivity and a higher amount of γH2AX foci than CHO10B2 at 6 hours after irradiation for all types of radiations. Remarkably, CHO10B2 and irs-20 maintained their difference in radiosensitivity after high-LET radiation. Six hours after low-LET radiations, irs-20 did not reach basal levels of γH2AX at high doses, whereas CHO10B2 recovered basal levels for all doses. After high-LET radiation, only CHO10B2 exhibited a reduction in γH2AX foci, but it never reached basal levels. Persistent foci in irs-20 confirmed a repair deficiency. Interestingly, after 30 minutes of high-LET radiation both cell lines exhibited large foci (size>0.9 μm2) related to the damage nature, whereas at 6 hours irs-20 showed a higher amount of large foci than CHO10B2, with a 7-fold increase at 3 Gy, that could also be associated to radiosensitivity. CONCLUSIONS We demonstrated, for the first time, an association between deficient DNA-PKcs activity and not only high levels of H2AX phosphorylation but also persistence and size increase of γH2AX foci after high-LET irradiation.

Automated image analysis for monitoring oxidative burst in macrophages.

We compared the morphology, clonogenic ability, Percoll gradient distribution, estrogen receptor proteins, and interactions with mesenchymal cells in MCF-7 breast tumor cells grown in medium containing fetal calf serum and insulin (FCS-I) or in a defined medium with insulin (ID) as the only growth factor. In the absence of serum and at densities below 5000-8000 cells/cm2, MCF-7 cells required epidermal growth factor, insulin, and thrombin. When cells reached a density of 23,000-26,000 cells/cm2, only insulin was necessary for optimal growth. In ID medium cells showed an enlarged Golgi apparatus and marked plasma membrane modifications, suggesting increased secretory activity. Moreover there was an increase in the release of protein products to the culture medium and a time-dependent ability of these cells to form macrocolonies in soft agar. On the contrary, cells in FCS-I showed no Golgi complex and few plasma membrane modifications. In both culture media tight junctions, desmosomes, and tonofilaments were present. We investigated the effect of conditioned media from MCF-7 cells growing in FCS-I or ID on the growth of primary rat vaginal fibroblasts. The growth of these mesenchymal cells was stimulated by FCS-I medium and inhibited by ID medium. By contrast, the embryonic fibroblast (preadipocyte) line CHEF/18 was also stimulated by FCS-I for the first 48 h, but thereafter ceased growth and acquired lipid droplets and a differentiated morphology. With ID medium, CHEF/18 cells were only partially inhibited with no changes in morphology. The Percoll gradient profiles of ID cells showed the same six fractions of increasing density as recently described. However, there was a progressive increase in subpopulations with higher growth rates and a decrease in the relative amount of the most differentiated cells. A unique feature of the growth analysis of MCF-7 cells in the absence of serum is the increased expression of the estradiol receptor gene. These studies show that the growth and differentiated properties of tumor cells can depend upon the cellular environment and offer a model system in which to further study this modulation.

Oxidative metabolism of lung macrophages exposed to sodium arsenite

The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1.