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A list ofendogenaus DNA·damaging agents and processes is given. Endogenaus e/ectrophiles are found with the cosubstrates of physiological transfer reactions (S-adenosylrnethionine for methylation, A TP for phosphorylation, NAD\(^+\) for ADP-ribosylation, acetyl CoA for acetylation). Aldehyde groups (glyceraldehyde- 3-phosphate, formaldehyde, open forms of reducing sugars, degradation products of peroxidation) or alkylating degradation products derived from endogenaus nitrose compounds represent additional possibilities. Radical-forming reactions include leakage of the superoxide anion radical from terminal cytochromes and redox cycles, hydroxyl radical formation by the Fenton reaction from endogenaus hydrogen peroxide, and the formation of lipid peroxides. Genetic instability by spontaneaus deaminations and depurinations as well as replicative instability by tautomer errors andin the presence of mutagenic metal ions represent a third important dass of endogenaus genotoxic processes. The postulated endogenaus genotoxicity could form the mechanistic basis for what is called 'spontaneous' tumor incidence and explain the possibility of an increased tumor incidence after treatment of animals with non-genotoxic compounds exhibiting tumor-promoting activity only. Individual differences are expected to be seen also with endogenaus DNA damage. The presence of endogenaus DNA darnage implies that exogenaus DNAcarcinogen adducts give rise to an incremental darnage which is expected to be proportional to the carcinogen dose at lowest Ievels. An increased tumor risk due to exposure to exogenaus genotoxic carcinogens could therefore be assessed in terms of the background DNA damage~ for instance in multiples of the mean Ievel or of the interindividual variability in a population.
1.2-Dioxetanes, very reactive and high energy molecules. are involved as labile intermediates in dioxygenase- activated aerobic metabolism and in physiological processes. Various toxico1ogica1 tests reveal that dioxetanes are indeed genotoxic. In supercoiled DNA of bacteriophage PM2 they induce endonucleasesensitive sites, most of them are FPG protein-sensitive base modifications (8-hydroxyguanine, fonnamidopyrimidines). Pyrimidinedimersand sites ofbase loss (AP sites) which were probed by UV endonuclease and exonuclease 111 are minor lesions in this system. While the alky1-substituted dioxetanes do not show any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetanes such as benzofuran and furocoumarin dioxetanes are strongly mutagenic in S. typhimurium strain TA I 00. DNA adducts formed with an intermediary alkyJating agent appear to be responsible for the mutagenic activity of benzofuran dioxetane. We assume that the benzofuran epoxides, generated in situ from benzofuran dioxetanes by deoxygenation are the ultimate mutagens of the latter. since benzofuran epoxides are highly mutagenic in the S. typhimurium strain TAIOO and they form DNA adducts. as detected by the 212Ppostlabelling technique. Our results imply that the type of D NA darnage promoted by dioxetanes is dependent on the structural feature of dioxetanes. Furthermore, the direct photochemical DNA darnage by energy transfer. i.e., pyrimidine dimers, plays a minor role in the genotoxicity of dioxetanes. Instead, photooxidation dominates in isolated DNA. while radical darnage and alkylation prevail in the cellular system.
Pigment cells and neuronal cells both are derived from the neural crest. Here, we describe the Pit-Oct-Unc (POU) domain transcription factor Brn3a, normally involved in neuronal development, to be frequently expressed in melanoma, but not in melanocytes and nevi. RNAi-mediated silencing of Brn3a strongly reduced the viability of melanoma cell lines and decreased tumour growth in vivo. In melanoma cell lines, inhibition of Brn3a caused DNA double-strand breaks as evidenced by Mre11/Rad50-containing nuclear foci. Activated DNA damage signalling caused stabilization of the tumour suppressor p53, which resulted in cell cycle arrest and apoptosis. When Brn3a was ectopically expressed in primary melanocytes and fibroblasts, anchorage-independent growth was increased. In tumourigenic melanocytes and fibroblasts, Brn3a accelerated tumour growth in vivo. Furthermore, Brn3a cooperated with proliferation pathways such as oncogenic BRAF, by reducing oncogene-induced senescence in non-malignant melanocytes. Together, these results identify Brn3a as a new factor in melanoma that is essential for melanoma cell survival and that promotes melanocytic transformation and tumourigenesis.
Background
High expression of constitutive histone γ-H2AX, a sensitive marker of DNA damage, might be indicative of defective DNA repair pathway or genomic instability. 53BP1 (p53-binding protein 1) is a conserved checkpoint protein with properties of a DNA double-strand breaks sensor. This study explores the relationship between the clinical radiosensitivity of tumor patients and the expression/induction of γ-H2AX and 53BP1 in vitro.
Methods
Using immunostaining, we assessed spontaneous and radiation-induced foci of γ-H2AX and 53 BP1 in peripheral blood mononuclear cells derived from unselected breast cancer (BC) patients (n=57) undergoing radiotherapy (RT). Cells from apparently healthy donors (n=12) served as references.
Results
Non-irradiated cells from controls and unselected BC patients exhibited similar baseline levels of DNA damage assessed by γ-H2AX and 53BP1 foci. At the same time, the γ-H2AX assay of in vitro irradiated cells revealed significant differences between the control group and the group of unselected BC patients with respect to the initial (0.5 Gy, 30 min) and residual (2 Gy, 24 h post-radiation) DNA damage. The numbers of 53BP1 foci analyzed in 35 BC patients were significantly higher than in controls only in case of residual DNA damage. A weak correlation was found between residual foci of both proteins tested. In addition, cells from cancer patients with an adverse acute skin reaction (grade 3) to RT showed significantly increased radiation-induced γ-H2AX foci and their protracted disappearance compared to the group of BC patients with normal skin reaction (grade 0–1). The mean number of γ-H2AX foci after 5 clinical fractions was significantly higher than that before RT, especially in clinically radiosensitive patients.
Conclusions
The γ-H2AX assay may have potential for screening individual radiosensitivity of breast cancer patients.
INTRODUCTION: Recently, we could show that angiotensin II, the reactive peptide of the blood pressure-regulating renin-angiotensin-aldosterone-system, causes the formation of reactive oxygen species and DNA damage in kidneys and hearts of hypertensive mice. To further investigate on the one hand the mechanism of DNA damage caused by angiotensin II, and on the other hand possible intervention strategies against end-organ damage, the effects of substances interfering with the renin-angiotensin-aldosterone-system on angiotensin II-induced genomic damage were studied.
METHODS: In C57BL/6-mice, hypertension was induced by infusion of 600 ng/kg • min angiotensin II. The animals were additionally treated with the angiotensin II type 1 receptor blocker candesartan, the mineralocorticoid receptor blocker eplerenone and the antioxidant tempol. DNA damage and the activation of transcription factors were studied by immunohistochemistry and protein expression analysis.
RESULTS: Administration of angiotensin II led to a significant increase of blood pressure, decreased only by candesartan. In kidneys and hearts of angiotensin II-treated animals, significant oxidative stress could be detected (1.5-fold over control). The redox-sensitive transcription factors Nrf2 and NF-κB were activated in the kidney by angiotensin II-treatment (4- and 3-fold over control, respectively) and reduced by all interventions. In kidneys and hearts an increase of DNA damage (3- and 2-fold over control, respectively) and of DNA repair (3-fold over control) was found. These effects were ameliorated by all interventions in both organs. Consistently, candesartan and tempol were more effective than eplerenone.
CONCLUSION: Angiotensin II-induced DNA damage is caused by angiotensin II type 1 receptor-mediated formation of oxidative stress in vivo. The angiotensin II-mediated physiological increase of aldosterone adds to the DNA-damaging effects. Blocking angiotensin II and mineralocorticoid receptors therefore has beneficial effects on end-organ damage independent of blood pressure normalization.
Background
The prognostic value of histone γ-H2AX and 53BP1 proteins to predict the radiotherapy (RT) outcome of patients with rectal carcinoma (RC) was evaluated in a prospective study. High expression of the constitutive histone γ-H2AX is indicative of defective DNA repair pathway and/or genomic instability, whereas 53BP1 (p53-binding protein 1) is a conserved checkpoint protein with properties of a DNA double-strand breaks sensor.
Methods
Using fluorescence microscopy, we assessed spontaneous and radiation-induced foci of γ-H2AX and 53BP1 in peripheral blood mononuclear cells derived from unselected RC patients (n = 53) undergoing neoadjuvant chemo- and RT. Cells from apparently healthy donors (n = 12) served as references.
Results
The γ-H2AX assay of in vitro irradiated lymphocytes revealed significantly higher degree of DNA damage in the group of unselected RC patients with respect to the background, initial (0.5 Gy, 30 min) and residual (0.5 Gy and 2 Gy, 24 h post-radiation) damage compared to the control group. Likewise, the numbers of 53BP1 foci analyzed in the samples from 46 RC patients were significantly higher than in controls except for the background DNA damage. However, both markers were not able to predict tumor stage, gastrointestinal toxicity or tumor regression after curative RT. Interestingly, the mean baseline and induced DNA damage was found to be lower in the group of RC patients with tumor stage IV (n = 7) as compared with the stage III (n = 35). The difference, however, did not reach statistical significance, apparently, because of the limited number of patients.
Conclusions
The study shows higher expression of γ-H2AX and 53BP1 foci in rectal cancer patients compared with healthy individuals. Yet the data in vitro were not predictive in regard to the radiotherapy outcome.
Background:
In recent years attention has focused on \(\gamma\)H2AX as a very sensitive double strand break indicator. It has been suggested that \(\gamma\)H2AX might be able to predict individual radiosensitivity. Our aim was to study the induction and repair of DNA double strand breaks labelled by \(\gamma\)H2AX in a large cohort.
Methods:
In a prospective study lymphocytes of 136 rectal cancer (RC) patients and 59 healthy individuals were ex vivo irradiated (IR) and initial DNA damage was compared to remaining DNA damage after 2 Gy and 24 hours repair time and preexisting DNA damage in unirradiated lymphocytes. Lymphocytes were immunostained with anti-\(\gamma\)H2AX antibodies and microscopic images with an extended depth of field were acquired. \(\gamma\)H2AX foci counting was performed using a semi-automatic image analysis software.
Results:
Distinct increased values of preexisting and remaining \(\gamma\)H2AX foci in the group of RC patients were found compared to the healthy individuals. Additionally there are clear differences within the groups and there are outliers in about 12% of the RC patients after ex vivo IR.
Conclusions:
The \(\gamma\)H2AX assay has the capability to identify a group of outliers which are most probably patients with increased radiosensitivity having the highest risk of suffering radiotherapy-related late sequelae.
In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells
(2015)
Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi-or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS\(^{61K}\) in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation.
Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing.
Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells.