Metabolic labeling of human cells with tritiated nucleosides results in activation of the ATM-dependent p53 signaling pathway and acceleration of DNA repair

Mirzayans, Razmik; Pollock, Scott; Scott, April; Gao, Cindy Q.; Murray, David

doi:10.1038/sj.onc.1206514

Cited by 21 publications

(15 citation statements)

References 28 publications

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“…Global levels of specific proteins were evaluated by immunoblotting as described [50]. Protein immunofluorescence and BrdUrd immunofluorescence assays were performed as described [51].…”

Section: Methodsmentioning

confidence: 99%

Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome

Mirzayans

Andrais

Scott

et al. 2017

IJMS

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Loss of wild-type p53 function is widely accepted to be permissive for the development of multinucleated giant cells. However, whether therapy-induced multinucleation is associated with cancer cell death or survival remains controversial. Herein, we demonstrate that exposure of p53-deficient or p21WAF1 (p21)-deficient solid tumor-derived cell lines to ionizing radiation (between 2 and 8 Gy) results in the development of multinucleated giant cells that remain adherent to the culture dish for long times post-irradiation. Somewhat surprisingly, single-cell observations revealed that virtually all multinucleated giant cells that remain adherent for the duration of the experiments (up to three weeks post-irradiation) retain viability and metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and the majority (>60%) exhibit DNA synthesis. We further report that treatment of multinucleated giant cells with pharmacological activators of apoptosis (e.g., sodium salicylate) triggers their demise. Our observations reinforce the notion that radiation-induced multinucleation may reflect a survival mechanism for p53/p21-deficient cancer cells. With respect to evaluating radiosensitivity, our observations underscore the importance of single-cell experimental approaches (e.g., single-cell MTT) as the creation of viable multinucleated giant cells complicates the interpretation of the experimental data obtained by commonly-used multi-well plate colorimetric assays.

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“…Global levels of specific proteins were evaluated by immunoblotting as described [50]. Protein immunofluorescence and BrdUrd immunofluorescence assays were performed as described [51].…”

Section: Methodsmentioning

confidence: 99%

Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome

Mirzayans

Andrais

Scott

et al. 2017

IJMS

Self Cite

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“…Many studies have been done to study transcriptional activation of p53-dependent genes considering that it appears to be mutated in almost 50% of human cancer [13]. Luker et al have studied protein interactions, developing a reporter gene by fusing the mutant HSV1-sr39tk linked to green fluorescent protein (GFP), allowing the visualisation of the interaction of the p53 gene and the large T antigen of simian virus 40 using 8-[ 18 F]fluoroganciclovir (FGCV), 5'-iodo-2'-fluoro-2'-deoxy-1-ß-D-arabinofuranosyl-5'-iodouracil (FIAU) and fluoro-hydroxymethylbutyl-guanine (FHBG) as substrates [22,23].…”

Section: Gene Expression Studiesmentioning

confidence: 99%

“…The techniques in Table 1 are used to study, among other things, different features of tumoral cells: gene expression [12][13][14][15][16][17][18], DNA repair mechanisms [13] and signalling pathways and receptor expression [12,19]; and biological events associated to cancer: cell proliferation [12][13][14], invasiveness and apoptosis [15] and identification of tumour markers (RFLP, AFLP) [12]. On the other hand, in vitro techniques are also used to confirm data that have been obtained in vivo; PET signal in vivo can be quantitatively linked with gene expression levels directly obtained in vitro [14,[20][21][22][23].…”

Section: In Vitro Radioactive Techniquesmentioning

confidence: 99%

Use of radionuclides in cancer research and treatment

Macias

2009

Clin Transl Oncol

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Cancer occurs as a result of misregulation of cell growth, which appears to be a consequence of alteration in the function of oncogenes and tumour suppressor genes. Ionising radiation has been used, since the discovery of X-rays in 1896 by Roentgen, both in cancer research and treatment of the disease. The main purpose of cancer research is to understand the molecular alterations involved in the development and progression of the disease in order to improve diagnosis and develop personalised therapies, by focusing on the features of the tumoral cell and the biological events associated to carcinogenesis. Radioisotopic techniques have been used routinely for in vitro research in the molecular and cellular biology of cancer for more than 20 years and are in the process of being substituted by alternative non-radioactive techniques. However in vivo techniques such as irradiation of cells in culture and/or experimental animal models and radioactive labelling are in development, due in part to advances in molecular imaging technologies. The objective of this review is to analyse in an integrative way the applications of ionising radiation in cancer research and therapy. It had been divided into two parts. The first one will approach the techniques applied to cancer research and the second will summarise how ionising radiation is applied to the treatment of neoplastic disease.

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“…Analysis of p53 stress response according to p53 gene dosage 3 H-methyl-thymidine is a radioactive DNA-labelling reagent, incorporated into DNA during replication, which can cause cell stress in the form of DNA damage (Yanokura et al, 2000;Hu et al, 2001;Mirzayans et al, 2003). UV irradiation also induces stress by generating DNA damage such as cyclobutane pyrimidine dimers and 6-4-photoproducts (Latonen and Laiho, 2005).…”

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confidence: 99%

Loss of one p53 allele results in four-fold reduction of p53 mRNA and protein: a basis for p53 haplo-insufficiency

Lynch

Milner

2006

Oncogene

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A haploid genotype may be insufficient to support normal wild-type function. Such haplo-insufficiency has recently been documented for numerous tumour suppressor genes. p53 is a crucial tumour suppressor governing DNA repair, cell cycle arrest and apoptosis via its role as a stressresponsive transcription factor. p53 haplo-insufficiency has been observed in vivo with human familial cancer in Li-Fraumeni Syndrome (LFS) and in mouse p53-knockout models of LFS. The increased tumorigenesis associated with loss of one p53 allele has been attributed to reduced p53-dependent stress responses. However, the underlying biochemical basis for such attenuated responses in p53 þ /À cells remains unclear. Here we have determined basal p53 messenger RNA (mRNA) and protein levels, and compared the p53 stress response in p53 þ / þ , p53 þ /À and p53À/À isogenic clones derived from HCT116 cells. Basal expression of p53 in p53 þ /À cells was 25% relative to p53 þ / þ cells, and this differential was maintained following oncogenic stress. This deficiency was manifested at both p53 mRNA and protein levels and resulted in attenuated p53 stress responses, in particular for p21 waf1 upregulation and survivin downregulation, and reduced G1 arrest and apoptosis. These observations identify a molecular basis for wild-type p53 haplo-insufficiency, which may explain the attenuated tumour-suppressive phenotype observed in cells with a single wild-type p53 allele and in humans with LFS.

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Metabolic labeling of human cells with tritiated nucleosides results in activation of the ATM-dependent p53 signaling pathway and acceleration of DNA repair

Cited by 21 publications

References 28 publications

Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome

Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome

Use of radionuclides in cancer research and treatment

Loss of one p53 allele results in four-fold reduction of p53 mRNA and protein: a basis for p53 haplo-insufficiency

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