Caenorhabditis elegans BUB-3 and SAN-1/MAD3 Spindle Assembly Checkpoint Components Are Required for Genome Stability in Response to Treatment with Ionizing Radiation.

Relatively little is known about the cross-talk between the spindle assembly checkpoint and the DNA damage response, especially in multicellular organisms. We performed a Caenorhabditis elegans forward genetic screen to uncover new genes involved in the repair of DNA damage induced by ionizing radiation. We isolated a mutation, gt2000, which confers hypersensitivity to ionizing radiation and showed that gt2000 introduces a premature stop in bub-3 BUB-3 is a key component of the spindle assembly checkpoint. We provide evidence that BUB-3 acts during development and in the germline; irradiated bub-3(gt2000) larvae are developmentally retarded and form abnormal vulvae. Moreover, bub-3(gt2000) embryos sired from irradiated worms show increased levels of lethality. Both bub-3 and san-1 (the C. elegans homolog of MAD3) deletion alleles confer hypersensitivity to ionizing radiation, consistent with the notion that the spindle assembly checkpoint pathway is required for the DNA damage response. bub-3(gt2000) is moderately sensitive to the cross-linking drug cisplatin but not to ultraviolet light or methyl methanesulfonate. This is consistent with a role in dealing with DNA double-strand breaks and not with base damage. Double mutant analysis revealed that bub-3 does not act within any of the three major pathways involved in the repair of double-strand breaks. Finally, the cdc-20 gain-of-function mutant cdc-20/fzy-1(av15), which is refractory to the cell cycle delay conferred by the spindle checkpoint, showed phenotypes similar to bub-3 and san-1 mutants. We speculate that BUB-3 is involved in the DNA damage response through regulation of cell cycle timing.

Chemopreventive mechanism of polypeptides from Chlamy Farreri (PCF) against UVB-induced malignant transformation of HaCaT cells.

To investigate polypeptide from Chlamy Farreri (PCF)'s protective effect against skin cancer, we used a cellular model of ultraviolet B (UVB)-induced malignant transformation. The human keratinocyte cell line HaCaT was repeatly exposed to UVB (10 mJ/cm(2), 20 times) and malignant transformation was confirmed by Gimesa staining, cell cycle analysis and various assays [anchorage independent growth, matrix metalloproteinase-9 (MMP9) activity, plating efficiency]. The malignant transformation was found to be effectively prevented by PCF pretreatment (2.84mM for 2h prior to each UVB exposure). We investigated the mechanism of PCF-mediated action by determining its effect on DNA methylation status of the tumour suppressor genes [P16 and ras association domain family 1 A (RASSF1A)] in the UVB-transformed cells. Both genes were found to be hypermethylated by chronic UVB exposure. The expression levels of P16, RASSF1A, DNA methyltransferases (DNMTs) and DNA damage inducible protein a (GADD45a) were measured by reverse transcriptase-polymerase chain reaction and western blotting. While chronic UVB exposure was found to suppress the expression of P16 and RASSF1A, it enhanced the expression of DNMT3b. In the early phase of UVB-induced malignant transformation, the GADD45a expression was increased, however, it declined with a continued irradiation of the cells. The UVB-induced DNA hypermethylation of P16 and RASSF1A and subsequent gene silencing was reversed by PCF treatment. The inhibition of DNMTs expression suggested that PCF blocked DNA methylation and thereby the silencing of tumour suppressor genes. Furthermore, the PCF-mediated substantial increase in GADD45a expression indicated that PCF promoted demethylation of tumour suppressor genes via GADD45a induction.

Non-equilibrium atmospheric pressure plasmas modulate cell cycle-related gene expressions in melanocytic tumors of RET-transgenic mice.

The incidence of cutaneous malignant melanoma is increasing at a greater rate than that of any other cancer in the world. However, an effective therapy for malignant melanoma has not been established. Recently, some studies have shown an antitumor effect of non-equilibrium atmospheric pressure plasmas (NEAPPs) in vitro. Here, we examined the in vivo effect of NEAPP on cell cycle regulators, key elements for malignant transformation, in spontaneously developed benign melanocytic tumors in a hairless animal model. NEAPP irradiation decreased expression levels of cell cycle promoters, Cyclin D1, E1 and E2, and increased expression level of a cell cycle repressor, p27(KIP) (1) . Cyclin D1, E1 and E2 and p27(KIP) expression levels were associated with malignant transformation of the benign tumor in the animal model. Our results suggest that NEAPP irradiation suppresses malignant transformation of a benign melanocytic tumor via control of the expression levels of cell cycle regulators.

Molecular imaging of the ATM kinase activity.

PURPOSE: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. METHODS AND MATERIALS: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. RESULTS: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. CONCLUSIONS: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice.

DNA damage induced by ionizing radiation activates the ATM kinase, which subsequently stabilizes and activates the p53 tumor suppressor protein. Although phosphorylation of p53 by ATM was found previously to modulate p53 levels and transcriptional activities in vivo, it does not appear to be a major regulator of p53 stability. We have utilized mice bearing altered Mdm2 alleles to demonstrate that ATM phosphorylation of Mdm2 serine 394 is required for robust p53 stabilization and activation after DNA damage. In addition, we demonstrate that dephosphorylation of Mdm2 Ser394 regulates attenuation of the p53-mediated response to DNA damage. Therefore, the phosphorylation status of Mdm2 Ser394 governs p53 protein levels and functions in cells undergoing DNA damage.

Ataxia-telangiectasia mutated and the Mre11-Rad50-NBS1 complex: promising targets for radiosensitization.

Radiotherapy plays a central part in cancer treatment, and use of radiosensitizing agents can greatly enhance this modality. Although studies have shown that several chemotherapeutic agents have the potential to increase the radiosensitivity of tumor cells, investigators have also studied a number of molecularly targeted agents as radiosensitizers in clinical trials based on reasonably promising preclinical data. Recent intense research into the DNA damage-signaling pathway revealed that ataxia-telangiectasia mutated (ATM) and the Mre11-Rad50-NBS1 (MRN) complex play central roles in DNA repair and cell cycle checkpoints and that these molecules are promising targets for radiosensitization. Researchers recently developed three ATM inhibitors (KU-55933, CGK733, and CP466722) and an MRN complex inhibitor (mirin) and showed that they have great potential as radiosensitizers of tumors in preclinical studies. Additionally, we showed that a telomerase-dependent oncolytic adenovirus that we developed (OBP-301 [telomelysin]) produces profound radiosensitizing effects by inhibiting the MRN complex via the adenoviral E1B55kDa protein. A recent Phase I trial in the United States determined that telomelysin was safe and well tolerated in humans, and this agent is about to be tested in combination with radiotherapy in a clinical trial based on intriguing preclinical data demonstrating that telomelysin and ionizing radiation can potentiate each other. In this review, we highlight the great potential of ATM and MRN complex inhibitors, including telomelysin, as radiosensitizing agents.

DNA double-strand break - induced pro-survival signaling.

Radiation and other types of DNA damaging agents induce a plethora of signaling events simultaneously originating from the nucleus, cytoplasm, and plasma membrane. As a result, this presents a dilemma when seeking to determine causal relationships and better insight into the intricacies of stress signaling. ATM plays critical roles in both nuclear and cytoplasmic signaling, of which, the DNA damage response (DDR) is the best characterized. We have recently created experimental conditions where the DNA damage signal alone can be studied while minimizing the influence from the extranuclear compartment. We have been able to document pro-survival and growth promoting signaling (via ATM-AKT-ERK) resulting from low levels of DSBs (equivalent to ≤2 Gy). More extensive DSBs (>2 Gy eq.) result in phosphatase-mediated ERK dephosphorylation, and thus shutdown of ERK signaling. In contrast, radiation does not result in such dephosphorylation even at very high doses. We propose that phosphatases are inactivated perhaps as a result of reactive oxygen species, which does not occur in response to 'pure' DNA damage. Our findings suggest that clinically relevant radiation doses, which are intended to halt tumor growth and induce cell death, are unable to inhibit tumor pro-survival signaling via ERK dephosphorylation.

Association between single nucleotide polymorphisms in the DNA repair gene LIG3 and acute adverse skin reactions following radiotherapy.

Many genes have been associated with radiotherapy toxicity, but most have only been found in a single study. Using our cohort of 480 breast cancer patients, we provide replicated evidence that a polymorphism near the LIG3 gene is associated with acute skin toxicity following radiotherapy.

Induction of MET by ionizing radiation and its role in radioresistance and invasive growth of cancer.

BACKGROUND: Ionizing radiation (IR) is effectively used in cancer therapy. However, in subsets of patients, a few radioresistant cancer cells survive and cause disease relapse with metastatic progression. The MET oncogene encodes the hepatocyte growth factor (HGF) receptor and is known to drive "invasive growth", a regenerative and prosurvival program unduly activated in metastasis. METHODS: Human tumor cell lines (MDA-MB-231, MDA-MB-435S, U251) were subjected to therapeutic doses of IR. MET mRNA, and protein expression and signal transduction were compared in treated and untreated cells, and the involvement of the DNA-damage sensor ataxia telangiectasia mutated (ATM) and the transcription factor nuclear factor kappa B (NF-κB) in activating MET transcription were analyzed by immunoblotting, chromatin immunoprecipitation, and use of NF-κB silencing RNA (siRNA). Cell invasiveness was measured in wound healing and transwell assays, and cell survival was measured in viability and clonogenic assays. MET was inhibited by siRNA or small-molecule kinase inhibitors (PHA665752 or JNJ-38877605). Combinations of MET-targeted therapy and radiotherapy were assessed in MDA-MB-231 and U251 xenografts (n = 5-6 mice per group). All P values were from two-sided tests. RESULTS: After irradiation, MET expression in cell lines was increased up to fivefold via activation of ATM and NF-κB. MET overexpression increased ligand-independent MET phosphorylation and signal transduction, and rendered cells more sensitive to HGF. Irradiated cells became more invasive via a MET-dependent mechanism that was further enhanced in the presence of HGF. MET silencing by siRNA or inhibition of its kinase activity by treatment with PHA665752 or JNJ-38877605 counteracted radiation-induced invasiveness, promoted apoptosis, and prevented cells from resuming proliferation after irradiation in vitro. Treatment with MET inhibitors enhanced the efficacy of IR to stop the growth of or to induce the regression of xenografts (eg, at day 13, U251 xenografts, mean volume increase relative to mean tumor volume at day 0: vehicle = 438%, 5 Gy IR = 151%, 5 Gy IR + JNJ-38877605 = 76%; difference, IR vs JNJ-38877604 + IR = 75%, 95% CI = 59% to 91%, P = .01). CONCLUSION: IR induces overexpression and activity of the MET oncogene through the ATM-NF-κB signaling pathway; MET, in turn, promotes cell invasion and protects cells from apoptosis, thus supporting radioresistance. Drugs targeting MET increase tumor cell radiosensitivity and prevent radiation-induced invasiveness.

The ATM kinase signaling induced by the low-energy ß-particles emitted by (33)P is essential for the suppression of chromosome aberrations and is greater than that induced by the energetic ß-particles emitted by (32)P.

Ataxia-telangiectasia mutated (ATM) encodes a nuclear serine/threonine protein kinase whose activity is increased in cells exposed to low doses of ionizing radiation (IR). Here we examine ATM kinase activation in cells exposed to either (32)P- or (33)P-orthophosphate under conditions typically employed in metabolic labelling experiments. We calculate that the absorbed dose of IR delivered to a 5cm×5cm monolayer of cells incubated in 2ml media containing 1mCi of the high-energy (1.70MeV) ß-particle emitter (32)P-orthophosphate for 30min is ∼1Gy IR. The absorbed dose of IR following an otherwise identical exposure to the low-energy (0.24MeV) ß-particle emitter (33)P-orthophosphate is ∼0.18Gy IR. We show that low-energy ß-particles emitted by (33)P induce a greater number of ionizing radiation-induced foci (IRIF) and greater ATM kinase signaling than energetic ß-particles emitted by (32)P. Hence, we demonstrate that it is inappropriate to use (33)P-orthophosphate as a negative control for (32)P-orthophosphate in experiments investigating DNA damage responses to DNA double-strand breaks (DSBs). Significantly, we show that ATM accumulates in the chromatin fraction when ATM kinase activity is inhibited during exposure to either radionuclide. Finally, we also show that chromosome aberrations accumulate in cells when ATM kinase activity is inhibited during exposure to ∼0.36Gy ß-particles emitted by (33)P. We therefore propose that direct cellular exposure to (33)P-orthophosphate is an excellent means to induce and label the IR-induced, ATM kinase-dependent phosphoproteome.

Protective effects of selenocystine against γ-radiation-induced genotoxicity in Swiss albino mice.

Selenocystine (CysSeSeCys), a diselenide aminoacid exhibiting glutathione peroxidase-like activity and selective antitumor effects, was examined for in vivo antigenotoxic and antioxidant activity in Swiss albino mice after exposure to a sublethal dose (5 Gy) of γ-radiation. For this, CysSeSeCys was administered intraperitoneally (i.p.) to mice at a dosage of 0.5 mg/kg body weight for 5 consecutive days prior to whole-body γ-irradiation. When examined in the hepatic tissue, CysSeSeCys administration reduced the DNA damage at 30 min after radiation exposure by increasing the rate of DNA repair. Since antigenotoxic agents could alter the expression of genes involved in cell cycle arrest and DNA repair, the transcriptional changes in p53, p21 and GADD45α were monitored in the hepatic tissue by real-time PCR. The results show that CysSeSeCys alone causes moderate induction of these three genes. However, CysSeSeCys pretreatment resulted in a suppression of radiation-induced enhancement of p21 and GADD45α expression, but did not affect p53 expression. Further analysis of radiation-induced oxidative stress markers in the same tissue indicated that CysSeSeCys significantly inhibits lipid peroxidation and prevents the depletion of antioxidant enzymes and glutathione (GSH) levels. Additionally, it also prevents radiation-induced DNA damage in other radiation sensitive cellular systems like peripheral leukocytes and bone marrow, which was evident by a decrease in comet parameters and micronucleated polychromatic erythrocytes (mn-PCEs) frequency, respectively. Based on these observations, it is concluded that CysSeSeCys exhibits antigenotoxic effects, reduces radiation-induced oxidative stress, and is a promising candidate for future exploration as a radioprotector.

Light exposure of Arabidopsis seedlings causes rapid de-stabilization as well as selective post-translational inactivation of the repressor of photomorphogenesis SPA2.

The COP1/SPA complex acts as an E3 ubiquitin ligase to repress photomorphogenesis by targeting activators of the light response for degradation. Genetic analysis has shown that the four members of the SPA gene family (SPA1-SPA4) have overlapping but distinct functions. In particular, SPA1 and SPA2 differ in that SPA1 encodes a potent repressor in light- and dark-grown seedlings, but SPA2 fully loses its function when seedlings are exposed to light, indicating that SPA2 function is hyper-inactivated by light. Here, we have used chimeric SPA1/SPA2 constructs to show that the distinct functions of SPA1 and SPA2 genes in light-grown seedlings are due to the SPA protein sequences and independent of the SPA promoter sequences. Biochemical analysis of SPA1 and SPA2 protein levels shows that light exposure leads to rapid proteasomal degradation of SPA2, and, more weakly, of SPA1, but not of COP1. This suggests that light inactivates the COP1/SPA complex partly by reducing SPA protein levels. Although SPA2 was more strongly degraded than SPA1, this was not the sole reason for the lack of SPA2 function in the light. We found that the SPA2 protein is inherently incapable of repressing photomorphogenesis in light-grown seedlings. The data therefore indicate that light inactivates the function of SPA2 through a post-translational mechanism that eliminates the activity of the remaining SPA2 protein in the cell.

UV-induced histone H2AX phosphorylation and DNA damage related proteins accumulate and persist in nucleotide excision repair-deficient XP-B cells.

DNA double strand breaks (DSB) may be caused by ionizing radiation. In contrast, UV exposure forms dipyrimidine photoproducts and is not considered an inducer of DSB. We found that uniform or localized UV treatment induced phosphorylation of the DNA damage related (DDR) proteins H2AX, ATM and NBS1 and co-localization of γ-H2AX with the DDR proteins p-ATM, p-NBS1, Rad51 and FANCD2 that persisted for about 6h in normal human fibroblasts. This post-UV phosphorylation was observed in the absence of nucleotide excision repair (NER), since NER deficient XP-B cells (lacking functional XPB DNA repair helicase) and global genome repair-deficient rodent cells also showed phosphorylation and localization of these DDR proteins. Resolution of the DDR proteins was dependent on NER, since they persisted for 24h in the XP-B cells. In the normal and XP-B cells p53 and p21 was detected at 6h and 24h but Mdm2 was not induced in the XP-B cells. Post-UV induction of Wip1 phosphatase was detected in the normal cells but not in the XP-B cells. DNA DSB were detected with a neutral comet assay at 6h and 24h post-UV in the normal and XP-B cells. These results indicate that UV damage can activate the DDR pathway in the absence of NER. However, a later step in DNA damage processing involving induction of Wip1 and resolution of DDR proteins was not observed in the absence of NER.

Inducible response required for repair of low-dose radiation damage in human fibroblasts.

Ionizing radiation (IR) induces a variety of DNA lesions among which DNA double-strand breaks (DSBs) are the biologically most significant. It is currently unclear if DSB repair is equally efficient after low and high doses. Here, we use gamma-H2AX, phospho-ATM (pATM), and 53BP1 foci analysis to monitor DSB repair. We show, consistent with a previous study, that the kinetics of gamma-H2AX and pATM foci loss in confluent primary human fibroblasts are substantially compromised after doses of 10 mGy and lower. Following 2.5 mGy, cells fail to show any foci loss. Strikingly, cells pretreated with 10 microM H(2)O(2) efficiently remove all gamma-H2AX foci induced by 10 mGy. At the concentration used, H(2)O(2) produces single-strand breaks and base damages via the generation of oxygen radicals but no DSBs. Moreover, 10 microM H(2)O(2) up-regulates a set of genes that is also up-regulated after high (200 mGy) but not after low (10 mGy) radiation doses. This suggests that low radical levels induce a response that is required for the repair of radiation-induced DSBs when the radiation damage is too low to cause the induction itself. To address the in vivo significance of this finding, we established gamma-H2AX and 53BP1 foci analysis in various mouse tissues. Although mice irradiated with 100 mGy or 1 Gy show efficient gamma-H2AX and 53BP1 foci removal during 24 h post-IR, barely any foci loss was observed after 10 mGy. Our data suggest that the cellular response to DSBs is substantially different for low vs. high radiation doses.

Oxidative stress induces mainly human centrin 2 polymerisation.

PURPOSE: To determine the human centrin 2 (Hscen 2) protein response to oxidising radicals in vitro and to evaluate the consequences on its biological functions. MATERIALS AND METHODS: Hscen 2 was submitted to hydroxyl and azide radicals produced by radiolysis in the absence of oxygen. The resulting products were characterised by biochemical, spectroscopic and mass spectrometry techniques. Their thermodynamics parameters of complexation with C-terminal fragment of Xeroderma pigmentosum C protein (C-XPC), one of the Hscen 2 cellular partners, were quantified by isothermal titration calorimetry (ITC). RESULTS: Both hydroxyl and azide radicals induce centrin 2 polymerisation as we characterised several intermolecular cross-links generating dimers, trimers, tetramers and higher molecular mass species. These cross-links result from the formation of a covalent bond between the only tyrosine residue (Tyr 172) located in the C-terminal region of each monomer. Remarkably, dimerisation occurs for doses as low as a few grays. Moreover, this Hscen2 dimer has a lower affinity and stoechiometry binding to C-XPC. CONCLUSIONS: These results show that as oxidative radicals induce high proportions of irreversible damages (polymerisation) centrin 2 is highly sensitive to ionising radiation. This could have important consequences on its biological functions.

Hemizygosity for Atm and Brca1 influence the balance between cell transformation and apoptosis.

BACKGROUND: In recent years data from both mouse models and human tumors suggest that loss of one allele of genes involved in DNA repair pathways may play a central role in genomic instability and carcinogenesis. Additionally several examples in mouse models confirmed that loss of one allele of two functionally related genes may have an additive effect on tumor development. To understand some of the mechanisms involved, we examined the role of monoallelic loss or Atm and Brca1 on cell transformation and apoptosis induced by radiation. METHODS: Cell transformation and apoptosis were measured in mouse embryo fibroblasts (MEF) and thymocytes respectively. Combinations of wild type and hemizygous genotypes for ATM and BRCA1 were tested in various comparisons. RESULTS: Haploinsufficiency of either ATM or BRCA1 resulted in an increase in the incidence of radiation-induced transformation of MEF and a corresponding decrease in the proportion of thymocytes dying an apoptotic death, compared with cells from wild-type animals. Combined haploinsufficiency for both genes resulted in an even larger effect on apoptosis. CONCLUSIONS: Under stress, the efficiency and capacity for DNA repair mediated by the ATM/BRCA1 cell signalling network depends on the expression levels of both proteins.

Dose response and kinetics of foci disappearance following exposure to high- and low-LET ionizing radiation.

PURPOSE: The effect of different radiation qualities on (i) 53BP1 (p53 Binding Protein 1) and p-ATM (phosphorylated ataxia telangiectasia mutated) foci induction, and (ii) on the kinetics of foci disappearance was analysed. MATERIAL AND METHODS: Normal human skin fibroblasts were exposed to 240 kV broad-field X-rays or targeted with individually counted helium ((3)He) particles or protons ((1)H) from a Charged Particle Microbeam. Anti-p-ATM and anti-53BP1 antibodies were used for foci visualisation via immunocytochemistry. RESULTS: 1 Gy of X-rays yielded approximately 33 53BP1-positive foci/cell. The ratio between the number of delivered particles and yielded tracks was found to be 1:1 and 3:1 after targeted (3)He and (1)H irradiation, respectively. It was determined that approximately 50% of radiation-induced damage was repaired as measured by loss of foci during the first 2, 6, and 10 hours following X-ray, protons, and (3)He irradiation, respectively. CONCLUSIONS: There was significant radiation quality dependence for 53BP1- and p-ATM-positive foci induction observed. Foci disappearance was radiation dose-independent in the samples irradiated with X-rays. Our results confirm that kinetics of foci disappearance depends on radiation quality, even when individual ions are targeted to cells.

The involvement of HER2 and p53 status in the regulation of telomerase in irradiated breast cancer cells.

Cancer cell characteristics may play a pivotal role in the response to therapy by activating or deactivating different molecular pathways. In the present study, we investigated the implication of breast cancer cell features, such as HER2 and p53 in the activation of telomerase upon exposure to ionizing radiation. Telomerase is among the most important cancer biomarkers, conferring to tumor cells unlimited proliferative capacity, increased survival potential and resistance to several types of cellular stress. We investigated possible mechanisms regulating telomerase in six irradiated breast cancer cell lines (MCF-7, MCF-7/HER2, MDA-MB-231, SK-BR-3, BT-474 and HBL-100) differing in their HER2, p53 and ERalpha status. hTERT mRNA expression was evaluated by real-time PCR and telomerase activity by the TRAP assay. HER2, c-myc, p53 and p21 protein levels were evaluated by Western blotting. Silencing of hTERT and HER2 was achieved by small interfering RNA technology. Chromatin immunoprecipitation was used to evaluate H3 histone acetylation status, as well as myc/mad/max and p53 transcription factors interaction with the hTERT promoter. Our results showed for the first time, that only HER2-positive cells, independently of their p53 status, upregulated hTERT/telomerase, while knockdown of hTERT increased radio-sensitivity. Knockdown of HER2 also led to increased radio-sensitivity and downregulation of hTERT/telomerase. We also demonstrated that c-myc and mad1 regulate hTERT expression in all irradiated breast cancer cells. We conclude, for the first time, that HER2 phenotype upregulates hTERT through c-myc activation and confers radio-resistance to breast cancer cells.

Development of the light sensitivity of the clock genes Period1 and Period2, and immediate-early gene c-fos within the rat suprachiasmatic nucleus.

The molecular mechanism underlying circadian rhythmicity within the suprachiasmatic nuclei (SCN) of the hypothalamus has two light-sensitive components, namely the clock genes Per1 and Per2. Besides, light induces the immediate-early gene c-fos. In adult rats, expression of all three genes is induced by light administered during the subjective night but not subjective day. The aim of the present study was to ascertain when and where within the SCN the photic sensitivity of Per1, Per2 and c-fos develops during early postnatal ontogenesis. The specific aim was to find out when the circadian clock starts to gate photic sensitivity. The effect of a light pulse administered during either the subjective day or the first or second part of the subjective night on gene expression within the rat SCN was determined at postnatal days (P) 1, 3, 5 and 10. Per1, Per2 and c-fos mRNA levels were assessed 30 min, 1 and 2 h after the start of each light pulse by in situ hybridization histochemistry. Expression of Per1 and c-fos was light responsive from P1, and the responses began to be gated by the circadian clock at P3 and P10, respectively. Expression of Per2 was only slightly light responsive at P3, and the response was not fully gated until P5. These data demonstrate that the light sensitivity of the circadian clock develops gradually during postnatal ontogenesis before the circadian clock starts to control the response. The photoinduction of the clock gene Per2 develops later than that of Per1.