DNA double strand break quantification in skin biopsies

Qvarnström, Olov Fredrik; Simonsson, Martin; Johansson, Karl-Axel; Nyman, Jan; Turesson, Ingela

doi:10.1016/j.radonc.2004.07.009

Cited by 97 publications

(86 citation statements)

References 13 publications

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“…The nuclear DAPI staining further revealed that a subset of ÁH2AX-stained apoptotic cells also displayed several morphological features of apoptosis such as nuclear condensation, rounding and formation of apoptotic bodies. The ÁH2AX apoptotic staining pattern was easily distinguished from previously described foci patterns corresponding to individual DSBs (16,36).…”

Section: Resultsmentioning

confidence: 77%

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γH2AX and cleaved PARP-1 as apoptotic markers in irradiated breast cancer BT474 cellular spheroids

Qvarnström

Simonsson²,

Eriksson³

et al. 2009

Int J Oncol

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Abstract. Chemo-and radiotherapy induce apoptosis in tumours and surrounding tissues. In a search for robust and reliable apoptosis markers, we have evaluated immunostaining patterns of ÁH2AX and cleaved PARP-1 in paraffin-embedded cellular spheroids. Breast cancer BT474 cells were grown as cell spheroids to diameters of 700-800 μm. The spheroids contained an outer cell layer with proliferative cells, a deeper region with quiescent cells and a central area with necrosis. They were irradiated with 5 Gy and the frequency of apoptotic cells was determined at several time points (0-144 h) and distances (0-150 μm) from the spheroids surface. ÁH2AX and cleaved PARP-1 were quantified independently. Apoptotic frequencies for the two markers agreed both temporally and spatially in the proliferative regions of the spheroids. The ÁH2AX signal was stronger and had lower background compared to cleaved PARP-1. The central necrotic region was intensely stained with cleaved PARP-1, whereas no ÁH2AX could be detected. The apoptotic frequency increased with distance from surface for all time points. However, apoptotic frequencies, above unirradiated control levels, could only be detected for the last time point, 144 h after irradiation. We have shown that the spheroid model is a practical system for evaluation of staining patterns and specificities of apoptosis markers. Also, the radial gradient provides the opportunity to study apoptosis under a range of physiological conditions within the same system. We have further shown that ÁH2AX and cleaved PARP-1 are applicable markers for apoptosis in the proliferative regions of the spheroids. However, the more intense and clear staining patterns of ÁH2AX suggests that this marker is preferable for quantification of apoptosis in spheroids and similar paraffin-embedded materials.

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Section: Resultsmentioning

confidence: 77%

“…The dose of 5 Gy, chosen to induce sufficient levels of apoptosis, is not optimal since this will result in a high degree of foci overlap (36,38).…”

Section: Apoptosis As a Function Of Time And Depth In The Spheroidsmentioning

confidence: 99%

γH2AX and cleaved PARP-1 as apoptotic markers in irradiated breast cancer BT474 cellular spheroids

Qvarnström

Simonsson²,

Eriksson³

et al. 2009

Int J Oncol

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“…Immunohistochemical stainings for HER2 were performed as described in several of our previous reports on clinical tumor samples (36,37). The digital image analyses of the autoradiographs were performed using modified versions of previously described procedures and software (12,38).…”

Section: Discussionmentioning

confidence: 99%

“…The stained area was determined by a series of digital image analysis algorithms implemented in Java™ and the Java Advanced Imaging (JAI) platform. The labeled structures in the images were extracted by binary segmentation using a fixed gray level threshold (38). The software was designed so that 10x30 µm-regions were defined along radial drawn lines from the surface of the spheroids towards the center, so the selected regions were aligned perpendicular to the surface (Fig.…”

Section: Growth Of Spheroidsmentioning

confidence: 99%

Effects of affinity on binding of HER2-targeting Affibody molecules: Model experiments in breast cancer spheroids

Tran

2011

Int J Oncol

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Abstract. Binding of a targeting agent in tumor tissue is influenced by many factors such as molecular weight, charge and affinity of the targeting agent and vascularization of the tumor. In this study, we analyzed tumor cell binding of three HER2-specific and radiolabeled Affibody molecules with different affinities. The Affibody molecules had affinities in the range of 0.12-3.8 nM. Cellular binding was analyzed, after 2 h of incubation, in tumor spheroids composed of BT474 breast cancer cells, which highly express HER2. Binding was, due to the binding-site barrier, limited to the outer 15±5 µm rim of the spheroids, independent of affinity when the concentration of the substances was low. When the concentration was high, the binding site barrier was overcome and the binding occurred approximately 35±5 µm into the spheroids for the two high affinity substances and 50±5 µm for the low affinity substance. The lower affinity might allow for penetration into deeper regions due to less firm binding. We conclude that there is a binding site barrier within tumor spheroids which can be overcome by increased concentration of substance and modified by affinity.

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“…This suggests that H2AX phosphorylation could be used to monitor patient responses to chemoand radiotherapies (for review, see Kao et al, 2006). In support of this possibility, the presence of H2AX foci was recently examined in skin biopsies of patients undergoing radiotherapy and found to reproducibly correspond with the dose given in a linear manner (Qvarnstrom et al, 2004).…”

Section: H2ax and Cancermentioning

confidence: 99%

Chromatin structure and DNA double-strand break responses in cancer progression and therapy

Downs

2007

Oncogene

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Defects in the detection and repair of DNA double-strand breaks (DSBs) have been causatively linked to tumourigenesis. Moreover, inhibition of DNA damage responses (DDR) can increase the efficacy of cancer therapies that rely on generation of damaged DNA. DDR must occur within the context of chromatin, and there have been significant advances in recent years in understanding how the modulation and manipulation of chromatin contribute to this activity. One particular covalent modification of a histone variant-the phosphorylation of H2AX-has been investigated in great detail and has been shown to have important roles in DNA DSB responses and in preventing tumourigenesis. These studies are reviewed here in the context of their relevance to cancer therapy and diagnostics. In addition, there is emerging evidence for contributions by proteins involved in mediating higher order structure to DNA DSB responses. The contributions of a subset of these proteins-linker histones and high-mobility group box (HMGB) proteins-to DDR and their potential significance in tumourigenesis are discussed.

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DNA double strand break quantification in skin biopsies

Cited by 97 publications

References 13 publications

γH2AX and cleaved PARP-1 as apoptotic markers in irradiated breast cancer BT474 cellular spheroids

γH2AX and cleaved PARP-1 as apoptotic markers in irradiated breast cancer BT474 cellular spheroids

Effects of affinity on binding of HER2-targeting Affibody molecules: Model experiments in breast cancer spheroids

Chromatin structure and DNA double-strand break responses in cancer progression and therapy

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