BRCA1 and c-Myc Associate to Transcriptionally Repress Psoriasin, a DNA Damage-Inducible Gene

Evidence is accumulating to suggest that some of the diverse functions associated with BRCA1 may relate to its ability to transcriptionally regulate key downstream target genes. Here, we identify S100A7 (psoriasin), S100A8, and S100A9, members of the S100A family of calcium-binding proteins, as novel BRCA1-repressed targets. We show that functional BRCA1 is required for repression of these family members and that a BRCA1 disease–associated mutation abrogates BRCA1-mediated repression of psoriasin. Furthermore, we show that BRCA1 and c-Myc form a complex on the psoriasin promoter and that BRCA1-mediated repression of psoriasin is dependent on functional c-Myc. Finally, we show that psoriasin expression is induced by the topoisomerase IIA poison, etoposide, in the absence of functional BRCA1 and increased psoriasin expression enhances cellular sensitivity to this chemotherapeutic agent. Therefore, we identified a novel transcriptional mechanism that is likely to contribute to BRCA1-mediated resistance to etoposide.

What role for DNA damage and repair in the bystander response?

The radiation-induced bystander effect challenges the accepted paradigm of direct DNA damage in response to energy deposition driving the biological consequences of radiation exposure. With the bystander response, cells which have not been directly exposed to radiation respond to their neighbours being targeted. In our own studies we have used novel targeted microbeam approaches to specifically irradiate parts of individual cells within a population to quantify the bystander response and obtain mechanistic information. Using this approach it has become clear that energy deposited by radiation in nuclear DNA is not required to trigger the effect, with cytoplasmic irradiation required. Irradiated cells also trigger a bystander response regardless of whether they themselves live or die, suggesting that the phenotype of the targeted cell is not a determining factor. Despite this however, a range of evidence has shown that repair status is important for dealing with the consequences of a bystander signal. Importantly, repair processes involved in the processing of dsb appear to be involved suggesting that the bystander response involves the delayed or indirect production of dsb-type lesions in bystander cells. Whether these are infact true dsb or complexes of oxidised bases in combination with strand breaks and the mechanisms for their formation, remains to be elucidated.

Comet sensitivity in assessing DNA damage and repair in different cell cycle stages

The comet assay is a sensitive tool for estimation of DNA damage and repair at the cellular level, requiring only a very small number of cells. In comparing the levels of damage or repair in different cell samples, it is possible that small experimental effects could be confounded by different cell cycle states in the samples examined, if sensitivity to DNA damage, and repair capacity, varies with the cell cycle. We assessed this by arresting HeLa cells in various cell cycle stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand break levels measured by the comet assay (predominantly single-strand breaks) at different cell cycle stages, increasing from G1 into S and falling again in G2. Over and above this variation in endogenous strand break levels, a significant difference in susceptibility to breaks induced by 3.5 Gy ionizing radiation was also evident in different cell cycle phases. Levels of induced DNA damage fluctuate throughout the cycle, with cells in G1 showing slightly lower levels of damage than an asynchronous population. Damage increases as cells progress through S phase before falling again towards the end of S phase and reaching lowest levels in M phase. The results from repair experiments (where cells were allowed to repair for 10 min after exposure to ionizing radiation) also showed differences throughout the cell cycle with G1-phase cells apparently being the most efficient at repair and M-phase cells the least efficient. We suggest, therefore, that in experiments where small differences in DNA damage and repair are to be investigated with the comet assay, it may be desirable to arrest cells in a specific stage of the cell cycle or to allow for differential cycle distribution.

Cdk1 participates in BRCA1-dependent S phase checkpoint control in response to DNA damage.

Cdk2 and cdk1 are individually dispensable for cell-cycle progression in cancer cell lines because they are able to compensate for one another. However, shRNA-mediated depletion of cdk1 alone or small molecule cdk1 inhibition abrogated S phase cell-cycle arrest and the phosphorylation of a subset of ATR/ATM targets after DNA damage. Loss of DNA damage-induced checkpoint control was caused by a reduction in formation of BRCA1-containing foci. Mutation of BRCA1 at S1497 and S1189/S1191 resulted in loss of cdk1-mediated phosphorylation and also compromised formation of BRCA1-containing foci. Abrogation of checkpoint control after cdk1 depletion or inhibition in non-small-cell lung cancer cells sensitized them to DNA-damaging agents. Conversely, reduced cdk1 activity caused more potent G2/M arrest in nontransformed cells and antagonized the response to subsequent DNA damage. Cdk1 inhibition may therefore selectively sensitize BRCA1-proficient cancer cells to DNA-damaging treatments by disrupting BRCA1 function.

A comparison of the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells.

One of the important temporal stages of radiation action in cellular systems is the chemical phase, where oxygen fixation reactions compete with chemical repair reactions involving reducing agents such as GSH. Using the gas explosion technique it is possible to follow the kinetics of these fast (> 1 ms) reactions in intact cells. We have compared the chemical repair kinetics of the oxygen-dependent free radical precursors leading to DNA single-strand and double-strand breaks, measured using filter elution techniques, with those leading to cell killing in V79 cells. The chemical repair rates for DNA dsb (670s-1 at pH 7.2 and 380s-1 at pH 9.6) and cell killing (530s-1) were similar. This is in agreement with the important role of DNA dsb in radiation induced cell lethality. The rate for DNA ssb precursors was significantly slower (210s-1). The difference in rate between DNA ssb and dsb precursors may be explained on the basis of a dsb free radical precursor consisting of a paired radical, one radical on each strand. The instantaneous probability of one or other of these radicals being chemically repaired and not proceeding to form a dsb will be twice that of a ssb radical precursor. This agrees well with the concept of locally multiply damaged sites (LMDS) produced from clusters of ionizations in DNA (Ward 1985).

The role of non-protein sulphydryls in determining the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells. I

Chinese hamster V79 fibroblasts were irradiated in the gas explosion apparatus and the chemical repair rates of the oxygen-dependent free radical precursors of DNA double-strand breaks (dsb) and lethal lesions measured using filter elution (pH 9.6) and a clonogenic assay. Depletion of cellular GSH levels, from 4.16 fmol/cell to 0.05 fmol/cell, by treatment with buthionine sulphoximine (50 mumol dm-3; 18 h), led to sensitization as regards DNA dsb induction and cell killing. This was evident at all time settings but was particularly pronounced when the oxygen shot was given 1 ms after the irradiation pulse. A detailed analysis of the chemical repair kinetics showed that depletion of GSH led to a reduction in the first-order rate constant for dsb precursors from 385 s-1 to 144 s-1, and for lethal lesion precursors from 533 s-1 to 165 s-1. This is generally consistent with the role of GSH in the repair-fixation model of radiation damage at the critical DNA lesions. However, the reduction in chemical repair rate was not proportional to the severe thiol depletion (down to almost-equal-to 1% for GSH) and a residual repair capacity remained (almost-equal-to 30%). This was found not to be due to compartmentalization of residual GSH in the nucleus, as the repair rate for dsb precursors in isolated nuclei, washed virtually free of GSH, was identical to that found in GSH-depleted cells (144 s-1), also the OER remained substantially above unity. This suggests that other reducing agents may have a role to play in the chemical repair of oxygen-dependent damage. One possible candidate is the significant level of protein sulphydryls present in isolated nuclei.

BRCA1-BARD1 complexes are required for p53Ser-15 phosphorylation and a G1/S arrest following ionizing radiation-induced DNA damage

BRCA1 is a major player in the DNA damage response. This is evident from its loss, which causes cells to become sensitive to a wide variety of DNA damaging agents. The major BRCA1 binding partner, BARD1, is also implicated in the DNA damage response, and recent reports indicate that BRCA1 and BARD1 co-operate in this pathway. In this report, we utilized small interfering RNA to deplete BRCA1 and BARD1 to demonstrate that the BRCA1-BARD1 complex is required for ATM/ATR (ataxia-telangiectasia-mutated/ATM and Rad3-related)-mediated phosphorylation of p53(Ser-15) following IR- and UV radiation-induced DNA damage. In contrast, phosphorylation of a number of other ATM/ATR targets including H2AX, Chk2, Chk1, and c-jun does not depend on the presence of BRCA1-BARD1 complexes. Moreover, prior ATM/ATR-dependent phosphorylation of BRCA1 at Ser-1423 or Ser-1524 regulates the ability of ATM/ATR to phosphorylate p53(Ser-15) efficiently. Phosphorylation of p53(Ser-15) is necessary for an IR-induced G(1)/S arrest via transcriptional induction of the cyclin-dependent kinase inhibitor p21. Consistent with these data, repressing p53(Ser-15) phosphorylation by BRCA1-BARD1 depletion compromises p21 induction and the G(1)/S checkpoint arrest in response to IR but not UV radia-tion. These findings suggest that BRCA1-BARD1 complexes act as an adaptor to mediate ATM/ATR-directed phosphorylation of p53, influencing G(1)/S cell cycle progression after DNA damage.

Investigations of DNA damage induction and repair resulting from cellular exposure to high dose-rate pulsed proton beams

Studies regarding the radiobiological effects of low dose radiation, microbeam irradiation services have been developed in the world and today laser acceleration of protons and heavy ions may be used in radiation therapy. The application of different facilities is essential for studying bystander effects and relating signalling phenomena in different cells or tissues. In particular the use of ion beams results advantageous in cancer radiotherapy compared to more commonly used X-rays, since the ability of ions in delivering lethal amount of doses into the target tumour avoiding or limiting damage to the contiguous healthy tissues. At the INFN-LNS in Catania, a multidisciplinary radiobiology group is strategically structured aimed to develop radiobiological research, finalised to therapeutic applications, compatible with the use of high dose laser-driven ion beams. The characteristic non-continuous dose rates with several orders of magnitude of laser-driven ion beams makes this facility very interesting in the cellular systems' response to ultra-high dose rates with non-conventional pulse time intervals cellular studies. Our group have projected to examine the effect of high dose laser-driven ion beams on two cellular types: foetal fibroblasts (normal control cells) and DU145 (prostate cancer cells), studying the modulation of some different bio-molecular parameters, in particular cell proliferation and viability, DNA damage, redox cellular status, morphological alterations of both the cytoskeleton components and some cell organelles and the possible presence of apoptotic or necrotic cell death. Our group performed preliminary experiments with high energy (60 MeV), dose rate of 10 Gy/min, doses of 1, 2, 3 Gy and LET 1 keV/µm on human foetal fibroblasts (control cells). We observed that cell viability was not influenced by the characteristics of the beam, the irradiation conditions or the analysis time. Conversely, DNA damage was present at time 0, immediately following irradiation in a dose-dependent manner. The analysis of repair capability showed that the cells irradiated with 1 and 2 Gy almost completely recovered from the damage, but not, however, 3 Gy treated cells in which DNA damage was not recovered. In addition, the results indicate the importance of the use of an appropriate control in radiobiological in vitro analysis.

BRCA1, FANCD2 and Chk1 are potential molecular targets for the modulation of a radiation-induced DNA damage response in bystander cells

Radiotherapy is an important treatment option for many human cancers. Current research is investigating the use of molecular targeted drugs in order to improve responses to radiotherapy in various cancers. The cellular response to irradiation is driven by both direct DNA damage in the targeted cell and intercellular signalling leading to a broad range of bystander effects. This study aims to elucidate radiation-induced DNA damage response signalling in bystander cells and to identify potential molecular targets to modulate the radiation induced bystander response in a therapeutic setting. Stalled replication forks in T98G bystander cells were visualised via bromodeoxyuridine (BrdU) nuclear foci detection at sites of single stranded DNA. γH2AX co-localised with these BrdU foci. BRCA1 and FANCD2 foci formed in T98G bystander cells. Using ATR mutant F02-98 hTERT and ATM deficient GM05849 fibroblasts it could be shown that ATR but not ATM was required for the recruitment of FANCD2 to sites of replication associated DNA damage in bystander cells whereas BRCA1 bystander foci were ATM-dependent. Phospho-Chk1 foci formation was observed in T98G bystander cells. Clonogenic survival assays showed moderate radiosensitisation of directly irradiated cells by the Chk1 inhibitor UCN-01 but increased radioresistance of bystander cells. This study identifies BRCA1, FANCD2 and Chk1 as potential targets for the modulation of radiation response in bystander cells. It adds to our understanding of the key molecular events propagating out-of-field effects of radiation and provides a rationale for the development of novel molecular targeted drugs for radiotherapy optimisation.

ATM acts downstream of ATR in the DNA damage response signaling of bystander cells.

This study identifies ataxia-telangiectasia mutated (ATM) as a further component of the complex signaling network of radiation-induced DNA damage in nontargeted bystander cells downstream of ataxia-telangiectasia and Rad3-related (ATR) and provides a rationale for molecular targeted modulation of these effects. In directly irradiated cells, ATR, ATM, and DNA-dependent protein kinase (DNA-PK) deficiency resulted in reduced cell survival as predicted by the known important role of these proteins in sensing DNA damage. A decrease in clonogenic survival was also observed in ATR/ATM/DNA-PK–proficient, nonirradiated bystander cells, but this effect was completely abrogated in ATR and ATM but not DNA-PK–deficient bystander cells. ATM activation in bystander cells was found to be dependent on ATR function. Furthermore, the induction and colocalization of ATR, 53BP1, ATM-S1981P, p21, and BRCA1 foci in nontargeted cells was shown, suggesting their involvement in bystander DNA damage signaling and providing additional potential targets for its modulation. 53BP1 bystander foci were induced in an ATR-dependent manner predominantly in S-phase cells, similar to ?H2AX foci induction. In conclusion, these results provide a rationale for the differential modulation of targeted and nontargeted effects of radiation.

Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells

Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms.

Mechanisms of DNA Damage Response to Targeted Irradiation in Organotypic 3D Skin Cultures

DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2.

Mitochondrial DNA Damage Following Isolated Muscle Exercise: A Preliminary Investigation Into HO-1 Gene Expression

PURPOSE: This preliminary investigation was designed to test the hypothesis that high intensity single-leg exercise can cause extensive cell DNA damage, which subsequently may affect the expression of the HO-1 gene. METHODS: Six (n=6) apparently healthy male participants (age 27 + 7 yrs, stature 174 + 12 cm, body mass 79 + 4 kg and BMI 24 + 4 kg/m2) completed 100 isolated and continuous maximal concentric contractions (minimum force = 200 N, speed of contraction = 60°/sec) of the rectus femoris muscle. Using a spring-loaded and reusable Magnum biopsy gun with a 16-gauge core disposable biopsy needle, skeletal muscle micro biopsy tissue samples were extracted at rest and following exercise. mRNA gene expression was determined via two-step quantitative real-time PCR using GAPDH as a reference gene. RESULTS: The average muscle force production was 379 + 179 N. High intensity exercise increased mitochondrial 8-OHdG concentration (P < 0.05 vs. rest) with a concomitant decrease in total antioxidant capacity (P < 0.05 vs. rest). Exercise also increased protein oxidation as quantified by protein carbonyl concentration (P < 0.05 vs. rest). HO-1 expression increased (> 2-fold change vs. rest) following exercise, and it is postulated that this change was not significant due to low subject numbers (P > 0.05). CONCLUSION: These preliminary findings tentatively suggest that maximal concentric muscle contractions can cause intracellular DNA damage with no apparent disruption to the expression of the antioxidant stress protein HO-1. Moreover, it is likely that cell oxidant stress is required to activate the signal transduction cascade related to the expression of HO-1. A large-scale study incorporating a greater subject number is warranted to fully elucidate this relationship.