Mónica A. Palmieri

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

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.

Bioevaluation of 32P patch designed for the treatment of skin diseases

OBJECTIVE The objective of this study was to design and evaluate a 32P patch for the treatment of skin diseases. MATERIALS AND METHODS The patch was prepared from chromic phosphate 32P and silicone. Bioelimination and biodistribution in healthy and treated animals, and the therapeutic efficacy of two treatment schemes (single dose and fractionated dose) in an animal model of skin cancer were studied. RESULTS Based on the bioelimination and biodistribution studies, no leakage of 32P from the patch was observed. The treated tumors reduced their mean diameter compared to controls. The single-dose therapeutic scheme showed a higher number of complete and partial remissions compared to the fractionated scheme. These results were confirmed by histopathological analysis of the samples. CONCLUSION The 32P patch was designed and produced according to specifications for the treatment of superficial lesions of the skin. Although the 32P patch is an open source, it behaves like a sealed one for use in brachytherapy treatments.

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

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.

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

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.

Inhibition of benzoyl peroxide-induced tumor promotion and progression by copper(II)(3,5-diisopropylsalicylate)2.

The ability of a biomimetic superoxide dismutase agent, copper(II)(3,5-diisopropylsalicylate)2 (CuDIPS), to modulate benzoyl peroxide (BzPo)-induced tumor promotion and progression in mouse skin multistage carcinogenesis was evaluated. The results showed a significant inhibition of tumor incidence by CuDIPS pretreatment during promotion-progression. Different types of tumors were developed: papillomas, keratoacanthomas and squamous cell carcinomas. There was a significant increase in the keratoacanthoma-papilloma ratio when the period of treatment with BzPo was prolonged, which was inhibited by CuDIPS pretreatment. CuDIPS induced a significant inhibition of malignant conversion. Our results suggest that reactive oxygen species could be important in BzPo-induced promotion and progression.

Effect of Sodium Arsenite on Mouse Skin Carcinogenesis

Arsenic is carcinogenic in human beings, and environmental exposure to arsenic is a public health issue that affects large populations worldwide. Thus, studies are needed to determine the mode of action of arsenic and prevent harmful effects arising from arsenic intake. The present study assessed the influence of sodium arsenite (As3+) on potentially carcinogenic processes that are either pre-existing or concomitant with chronic intake of water containing As3+. Experiments using SenCar mice were designed to evaluate the effect of chronic administration of As3+ (2, 20, or 200 mg of As3+/L) in drinking water that overlapped to varying degrees with a 2-stage carcinogenesis protocol carried out over 9 months. The results showed a time-dependent pattern. During early stages of carcinogenesis (6–12 weeks), animals exposed to As3+ and the carcinogenesis protocol showed increased numbers of tumors compared to control animals. During late carcinogenesis (16–30 weeks), the number of tumors stabilized to below control values, but the tumors showed increased malignancy. These findings indicate that the outcomes of the 2-stage skin carcinogenesis protocol are modified by the presence of arsenite in drinking water, which increases the rate of carcinoma development.

Biological effects of brachytherapy using a 32P-patch on the skin of Sencar mice ☆

In recent years, specially designed patches containing beta emitters have been developed for contact brachytherapy of skin lesions. The aim of the present work was to evaluate the biological effects of the (32)P-patch on the skin of Sencar mice as a result of a brachytherapy treatment. For this purpose, a (32)P-patch was prepared with Chromic (32)P-phosphate and silicone and the classical model of two-stage skin carcinogenesis was reproduced in Sencar mice. Animals were divided in six groups. Four groups received the contact brachytherapy treatments using a scheme of a single session of 40 and 60Gy (SD40 and SD60) and a scheme of two sessions of 40 and 60Gy each (FD40 and FD60). The other two groups were used as controls of the single (CSD) and the fractionated (CFD) treatments. Radiation doses were estimated with equations derived from the MIRD DOSE scheme, and biologically effective doses (BED) were calculated according to equations derived from the linear-quadratic model. The endpoint to evaluate the treatments effects was tumor size after a follow-up period of 44 days. Finally, animals were sacrificed in order to get samples of all tumors for histological analysis and PCNA staining. Erythema, dermatitis and skin ulceration developed in almost all treated animals, but they gradually healed with regeneration of tissue during the follow-up period. Radiation effects on the skin of SD40, SD60, FD40 and FD60 showed a significant reduction of the tumor size with regard to controls, independently of the scheme and the radiation dose considered. PCNA staining scores of control groups were higher than for treated groups, independently of the scheme and the radiation dose considered. This radioactive (32)P-silicone-patch which is easy to prepare and use in the treatment of skin diseases, seems promising as a radioactive device for clinical use.