Reactive oxygen species (ROS) are involved in amplifying the radiation-induced damage to macromolecules as well as in downstream early responses leading to DNA repair or cell death and in delayed responses leading to tissue damage and/or tumorigenesis. We found in immortalized normal human bronchial epithelial cells (HBEC-3KT) following exposure to 1 Gy low (X-rays) or high (Fe ions) LET radiation that ROS levels persist elevated for up to eight population doublings (2 weeks) in the progeny of cells that repaired the radiation-induced damage and survived. To evaluate the function of persistently elevated ROS levels, we interrogated cells over the same period for genomic instability, proliferation and senescence biomarkers and evaluated whether ROS are functionally related to any of these phenotypes. We found that increased ROS production overlapped temporally with the persistence of reporters for genomic instability, increased micronucleus frequency and the presence of γH2AX-53BP1 positive ionizing radiation-induced foci. Although low LET radiation at high doses and high LET radiation induced a senescence-like phenotype dependent on ATM and p38 MAPK activity, this pathway was not a causative factor in ROS generation and did not affect cell proliferation rates at day 7 post-irradiation. On the contrary, when this pathway was inhibited, ROS levels increased further and micronucleus formation frequency was reduced. Because these results suggest a positive effect for increased ROS levels in the resolution of persistent micronucleus formation, their role was further confirmed by exposure to exogenous hydrogen peroxide, which resulted in a reduced micronucleus frequency. These results implicate ROS as an effector in the resolution of genomic instability and suggest that the senescence phenotype induced by high LET radiation or high low LET radiation dose promotes genomic instability by interfering with the cellular mechanism regulating ROS levels or the DNA repair machinery.
While evidence supporting the notion that exposures to heavy ion radiation increase the risk for cancer and other disease development is accumulating, the underlying biological mechanisms remain poorly understood. To identify novel phenotypes that persist over time that may be related to increased disease development risk, we performed a quantitative global proteome analysis of immortalized human bronchial epithelial cells (HBEC3-KT) at day 7 post exposure to 0.5 Gy Fe ion (600 MeV/nucleon, Linear Energy Transfer (LET) = 175 keV/μm). The analysis revealed a significant increase in the expression of 4 enzymes of the cholesterol biosynthesis pathway. Elevated expression of enzymes of the cholesterol pathway was associated with increased cholesterol levels in irradiated cells and in lung tissue measured by a biochemical method and by filipin staining of cell-bound cholesterol. While a 1 Gy dose of Fe ion was sufficient to induce a robust response, a dose of 5 Gy X-rays was necessary to induce a similar cholesterol accumulation in HBEC3-KT cells. Radiation-increased cholesterol levels were reduced by treatment with inhibitors affecting the activity of enzymes in the biosynthesis pathway. To examine the implications of this finding for radiotherapy exposures, we screened a panel of lung cancer cell lines for cholesterol levels following exposure to X-rays. We identified a subset of cell lines that increased cholesterol levels in response to 5 Gy X-rays. Survival studies revealed that statin treatment is radioprotective, suggesting that cholesterol increases are associated with cytotoxicity. In summary, our findings uncovered a novel radiation-induced response, which may modify radiation treatment outcomes and contribute to risk for radiation–induced cardiovascular disease and carcinogenesis.
Most cells do not undergo continuous cell division and DNA replication, yet they can still acquire novel RNA mutations that can result in the production of mutant proteins and induce a phenotypic change. All cells are frequently subjected to genotoxic insults that give rise to damaged nucleotides which, similarly to DNA replication, can undergo base mispairing during transcription. This mutagenic lesion bypass by RNA polymerase, transcriptional mutagenesis (TM), has been studied in a variety of systems and organisms, and may be involved in diverse pathogenic processes, such as tumorigenesis and the acquisition of bacterial antibiotic resistance. Tumor cells and bacteria within the human body are subject to especially high levels of oxidative stress, which can damage DNA and consequently drive TM. Mutagenesis at the level of transcription may allow cells to escape growth arrest and undergo replication that could permanently establish mutations in DNA in a process called retromutagenesis (RM). Here, we review the broad range of DNA damages which may result in TM including a variety of non-bulky lesions and some bulky lesions, which recent studies indicate may not completely block transcription, and emerging evidence supporting the RM concept in the context of tumorigenesis and antibiotic resistance.
MicroRNA-21 (miR-21) is an oncomir overexpressed in most human tumors where it promotes malignant growth and progression by acting on multiple targets. Here we broaden the impact of miR-21 in cancer by demonstrating that it regulates formation of reactive oxygen species (ROS) that promote tumorigenesis. Key targets of miR-21 in mediating this function were SOD3 and TNF-a. We found that miR-21 inhibited the metabolism of superoxide to hydrogen peroxide, produced either by endogenous basal activities or exposure to ionizing radiation, by directing attenuating SOD3 or by an indirect mechanism that limited TNF-a production, thereby reducing SOD2 levels. Importantly, both effects contributed to an elevation of IR-induced cell transformation. Our findings therefore establish that miR-21 promotes tumorigenesis to a large extent through its regulation of cellular ROS levels.
High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB repair via the HR DNA repair pathway. Here we found that expression of Bcl2 results in decreased HR activity and retards the repair of DSBs induced by HZE particles (i.e. 56iron and 28silicon) by inhibiting Mre11 complex activity. Exposure of cells to 56iron or 28silicon promotes Bcl2 to interact with Mre11 via the BH1 and BH4 domains. Purified Bcl2 protein directly suppresses Mre11 complex-mediated DNA resection in vitro. Expression of Bcl2 reduces the ability of Mre11 to bind DNA following exposure of cells to HZE particles. Our findings suggest that, after cellular exposure to HZE particles, Bcl2 may inhibit Mre11 complex-mediated DNA resection leading to suppression of the HR-mediated DSB repair in surviving cells, which may potentially contribute to tumor development.
8-Oxoguanine (8OG) is efficiently bypassed by RNA polymerases in vitro and in bacterial cells in vivo, leading to mutant transcripts by directing incorporation of an incorrect nucleotide during transcription. Such transcriptional mutagenesis (TM) may produce a pool of mutant proteins. In contrast, transcription-coupled repair safeguards against DNA damage, contingent upon the ability of lesions to arrest elongating RNA polymerase. In mammalian cells, the Cockayne syndrome B protein (Csb) mediates transcription-coupled repair, and its involvement in the repair of 8OG is controversial. The DNA glycosylase Ogg1 initiates base excision repair of 8OG, but its influence on TM is unknown. We have developed a mammalian system for TM in congenic mouse embryonic fibroblasts (MEFs), either WT or deficient in Ogg1 (ogg−/−), Csb (csb−/−), or both. This system uses expression of the Ras oncogene in which an 8OG replaces guanine in codon 61. Repair of 8OG restores the WT sequence; however, bypass and misinsertion opposite this lesion during transcription leads to a constitutively active mutant Ras protein and activation of downstream signaling events, including increased phosphorylation of ERK kinase. Upon transfection of MEFs with replication-incompetent 8OG constructs, we observed a marked increase in phospho-ERK in ogg−/− and csb−/−ogg−/− cells at 6 h, indicating persistence of the lesion and the occurrence of TM. This effect is absent in WT and csb−/− cells, suggesting rapid repair. These studies provide evidence that 8OG causes TM in mammalian cells, leading to a phenotypic change with important implications for the role of TM in tumorigenesis.
DNA damage occurs continuously, and faithful replication and transcription are essential for maintaining cell viability. Cells in nature are not dividing and replicating DNA often; therefore it is important to consider the outcome of RNA polymerase (RNAP) encounters with DNA damage. Base damage in the DNA can affect transcriptional fidelity, leading to production of mutant mRNA and protein in a process termed transcriptional mutagenesis (TM). Abasic (AP) sites and strand breaks are frequently occurring, spontaneous damages that are also base excision repair (BER) intermediates. In vitro studies have demonstrated that these lesions can be bypassed by RNAP; however this has never been assessed in vivo. This study demonstrates that RNAP is capable of bypassing AP sites and strand breaks in Escherichia coli and results in TM through adenine incorporation in nascent mRNA. Elimination of the enzymes that process these lesions further increases TM; however, such mutants can still complete repair by other downstream pathways. These results show that AP sites and strand breaks can result in mutagenic RNAP bypass and have important implications for the biologic endpoints of DNA damage.
In eukaryotic cells, proteins can occupy multiple intracellular compartments and move between compartments to fulfill critical biological functions. Unfortunately, no methods have been developed to robustly measure the distribution of a protein among compartments. To address this need, we have developed an automated method termed quantitative subcellular compartmentalization analysis (Q-SCAn). Q-SCAn is a quantitative analytical tool for providing broader and more detailed analysis of the localization of multicompartment proteins as compared to the currently available approaches. In eukaryotic cells, proteins can occupy multiple intracellular compartments and even move between compartments to fulfill critical biological functions or respond to cellular signals. Examples include transcription factors that reside in the cytoplasm but are mobilized to the nucleus as well as dual-purpose DNA repair proteins that are charged with simultaneously maintaining the integrity of both the nuclear and mitochondrial genomes. While numerous methods exist to study protein localization and dynamics, automated methods to quantify the relative amounts of proteins that occupy multiple subcellular compartments have not been extensively developed. To address this need, we present a rapid, automated method termed quantitative subcellular compartmentalization analysis (Q-SCAn). To develop this method, we exploited the facile molecular biology of the budding yeast, Saccharomyces cerevisiae. Individual subcellular compartments are defined by a fluorescent marker protein and the intensity of a target GFP-tagged protein is then quantified within each compartment. To validate Q-SCAn, we analyzed relocalization of the transcription factor Yap1 following oxidative stress and then extended the approach to multicompartment localization by examining two DNA repair proteins critical for the base excision repair pathway, Ntg1 and Ung1. Our findings demonstrate the utility of Q-SCAn for quantitative analysis of the subcellular distribution of multicompartment proteins.
Recent studies indicate that the transient receptor potential canonical 6 (TRPC6) channel is highly expressed in several types of cancer cells. However, it remains unclear whether TRPC6 contributes to the malignancy of human non-small cell lung cancer (NSCLC). We used a human NSCLC A549 cell line as a model and found that pharmacological blockade or molecular knockdown of TRPC6 channel inhibited A549 cell proliferation by arresting cell cycle at the S-G2M phase and caused a significant portion of cells detached and rounded-up, but did not induce any types of cell death. Western blot and cell cycle analysis show that the detached round cells at the S-G2M phase expressed more TRPC6 than the still attached polygon cells at the G1 phase. Patch-clamp data also show that TRPC whole-cell currents in the detached cells were significantly higher than in the still attached cells. Inhibition of Ca2+-permeable TRPC6 channels significantly reduced intracellular Ca2+ in A549 cells. Interestingly, either blockade or knockdown of TRPC6 strongly reduced the invasion of this NSCLC cell line and decreased the expression of an adherent protein, fibronectin, and a tight junction protein, zonula occluden protein-1 (ZO-1). These data suggest that TRPC6-mediated elevation of intracellular Ca2+ stimulates NSCLC cell proliferation by promoting cell cycle progression and that inhibition of TRPC6 attenuates cell proliferation and invasion. Therefore, further in vivo studies may lead to a consideration of using a specific TRPC6 blocker as a complement to treat NSCLC.