Lysines 3241 and 3260 of DNA-PKcs are important for genomic stability and radioresistance.

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) in the non-homologous end-joining (NHEJ) pathway. The DNA-PK holoenzyme consists of a catalytic subunit (DNA-PKcs) and DNA-binding subunit (Ku70/80, Ku). Ku is a molecular sensor for double-stranded DNA and once bound to DSB ends it recruits DNA-PKcs to the DSB site. Subsequently, DNA-PKcs is activated and heavily phosphorylated, with these phosphorylations modulating DNA-PKcs. Although phosphorylation of DNA-PKcs is well studied, other post-translational modifications of DNA-PKcs are not. In this study, we aimed to determine if acetylation of DNA-PKcs regulates DNA-PKcs-dependent DSB repair. We report that DNA-PKcs is acetylated in vivo and identified two putative acetylation sites, lysine residues 3241 and 3260. Mutating these sites to block potential acetylation results in increased radiosensitive, a slight decrease in DSB repair capacity as assessed by γH2AX resolution, and increased chromosomal aberrations, especially quadriradial chromosomes. Together, our results provide evidence that acetylation potentially regulates DNA-PKcs.

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.

How reliable is immunohistochemical staining for DNA mismatch repair proteins performed after neoadjuvant chemoradiation?

Immunohistochemistry (IHC) testing for mismatch repair proteins (MMRP) is currently being used primarily in colorectal cancer resection specimens. We aimed to compare the results of IHC staining performed on biopsy specimens obtained at endoscopy with that performed on surgical specimens after neoadjuvant therapy. Thirty-two rectal cancer subjects had paired preneoadjuvant and postneoadjuvant tissue available for IHC staining (MLH1, MSH2, MSH6, and PMS2), whereas 39 rectosigmoid cancer patients who did not receive neoadjuvant treatment served as controls. Each slide received a qualitative (absent, focal, and strong) and quantitative score (immunoreactivity [0-3] × percent positivity [0-4]). The quantitative scores of MMRP from the operative material were significantly lower in the neoadjuvant group than in the control (P < .05 for all).The scores of all MMRP from endoscopic biopsies were not significantly different between the neoadjuvant and the control groups. Disagreement between the endoscopic biopsy and the operative material was evident in 23 of 128 stains (18.5%) in the neoadjuvant group and in 12 of 156 stains (7.7%) in the control group (P = .009). In the neoadjuvant group, a disagreement pattern of "endoscopic strong operative focal" was observed in 28.1% for PMS2, 12.5% for MSH6, 12.5% for MLH1, and 6.3% for MSH2, and in the control group, this same disagreement pattern was found in 12.8% for PMS2, 7.7% for MSH6, 7.7% for MLH1, and 0% for MSH2. Based on our findings, we suggest that for rectal cancer, the endoscopic material rather than the operative material should serve as the primary material for IHC staining.

Light-dependent destabilization of PHL in Arabidopsis.

Plants sense environmental stimuli such as light to regulate their flowering time. In Arabidopsis, phytochrome B (phyB) is the major photoreceptor that perceives red and far-red light, and destabilizes transcriptional regulator CONSTANS (CO) protein. However the mechanism that links photoreceptor and CO protein degradation is largely unknown. We recently showed that PHYTOCHROME-DEPENDENT LATE-FLOWERING (PHL) protein inhibits phyB signaling through direct protein-protein interaction. Here, we report that light exposure destabilizes PHL protein as is the case with CO. Fluorescence from PHL-YFP fusion protein expressed under the control of Cauliflower Mosaic Virus (CaMV) 35S promoter (35S::PHL-YFP) almost disappeared after four-hour treatment of white light. Furthermore, the similar results were also obtained from the analysis of PHL-GUS fusion protein expressed by PHL promoter (PHLpro::PHL-GUS phl-1). These results highlight the importance of post-transcriptional regulation in phyB-mediated flowering regulation and will give us hints how phyB regulates CO protein amount.

DNA-damage response in chromatin of ribosomal genes and the surrounding genome.

DNA repair events have functional significance especially for genome stability. Although the DNA damage response within the whole genome has been extensively studied, the region-specific characteristics of nuclear sub-compartments such as the nucleolus or fragile sites have not been fully elucidated. Here, we show that the heterochromatin protein HP1 and PML protein recognize spontaneously occurring 53BP1- or γ-H2AX-positive DNA lesions throughout the genome. Moreover, 53BP1 nuclear bodies, which co-localize with PML bodies, also occur within the nucleoli compartments. Irradiation of the human osteosarcoma cell line U2OS with γ-rays increases the degree of co-localization between 53BP1 and PML bodies throughout the genome; however, the 53BP1 protein is less abundant in chromatin of ribosomal genes and fragile sites (FRA3B and FRA16D) in γ-irradiated cells. Most epigenomic marks on ribosomal genes and fragile sites are relatively stable in both non-irradiated and γ-irradiated cells. However, H3K4me2, H3K9me3, H3K27me3 and H3K79me1 were significantly changed in promoter and coding regions of ribosomal genes after exposure of cells to γ-rays. In fragile sites, γ-irradiation induces a decrease in H3K4me3, changes the levels of HP1ß, and modifies the levels of H3K9 acetylation, while the level of H3K9me3 was relatively stable. In these studies, we confirm a specific DNA-damage response that differs between the ribosomal genes and fragile sites, which indicates the region-specificity of DNA repair.

Studying the protein expression in human B lymphoblastoid cells exposed to 1.8-GHz (GSM) radiofrequency radiation (RFR) with protein microarray.

In the present study, the protein microarray was used to investigate the protein expression in human B-cell lymphoblastoid cells intermittently exposed to 1.8-GHz GSM radiofrequency radiation (RFR) at the specific absorption rate (SAR) of 2.0 W/kg for 24 h. The differential expression of 27 proteins was found, which were related to DNA damage repair, apoptosis, oncogenesis, cell cycle and proliferation (ratio >1.5-fold, P<0.05). The results validated with Western blot assay indicated that the expression of RPA32 was significantly down-regulated (P<0.05) while the expression of p73 was significantly up-regulated in RFR exposure group (P<0.05). Because of the crucial roles of those proteins in DNA repair and cell apoptosis, the results of present investigation may explain the biological effects of RFR on DNA damage/repair and cell apoptosis.

[Radiation biology of structurally different Drosophila genes. Report 2. The vestigial gene: molecular characteristics of chromosome mutations].

The results of the PCR-assay of mutation lesions at each of 16 fragments overlapping the entire vestigial (vg) gene of Drosophila melanogaster in 52 gamma-ray-, neutron- and neutron + gamma-ray-induced vg mutants having the inversion or translocation breakpoint within the vg microregion are presented. 4 from 52 mutants studied were found to have large deletions of about 200 kb covering the entire vg gene and adjacent to sca and l(2)C gene-markers as well. 23 mutants from 48 (47.9%) were found to have a wild-type gene structure showing that the exchange breakpoints are located outside of the vg gene. 25 others display the intragenic lesions of different complexity detected by PCR as the absence of(i) either one fragment or (ii) two or more (6-7) adjacent fragments and (iii) simultaneously several (i) or (i) and (ii) types separated by normal gene regions. It is important that 6 from 25 mutants have the breakpoint inside the vg gene and display the (i) or (ii) type of lesions at the gene regions containing the putative break whereas 5 others from 25 with the above lesions have the exchange breakpoint outside the vg gene. Therefore, the breakpoints underlying either inversions or translocations induced by low- and high-LET radiation are likely to be located within and outside the gene under study. Thereby, the formation of exchanges is accompanied by DNA deletions of various sizes at the exchange breakpoints. The molecular model of formation of such exchange-deletion rearrangements is elaborated and presented. Also, conception of the predominately clustered action of both low- and high-LET radiation on the germ cell genome is suggested as the summing-up of the presented results. The ability of ionizing radiation to induce the clusters of genetic alterations in the form of hidden DNA damages as well as gene/chromosome mutations is determined by the track structure and hierarchical organization of the genome. To detect the quality and frequency patterns of all components of the cluster, joint molecular, genetic and cytological techniques need to be used.

Three-dimensional imaging reveals the spatial separation of γH2AX-MDC1-53BP1 and RNF8-RNF168-BRCA1-A complexes at ionizing radiation-induced foci.

BACKGROUND AND PURPOSE: Ionizing radiation (IR)-induced DNA damage causes the accumulation of DNA damage response (DDR) proteins as visible foci in cell nuclei. Despite the identified functional roles in DNA repair, the spatial relationships of different DDR proteins at foci have not been explicitly examined. This study aims to systematically compare the distribution of DDR proteins at IR-induced foci. MATERIALS AND METHODS: MCF-7 cells were treated with IR, stained for γH2AX, MDC1, RNF8, RNF168, 53BP1, Abraxas (CCDC98), BRCA1, BRCC36, Merit40 (NBA1) and RAP80, and then imaged using high-resolution three-dimensional (3-D) confocal microscopy to assess the relative localization of proteins at foci. RESULTS: All BRCA1-A complex components displayed strong co-localization, which overlapped significantly with RNF8 and RNF168, but not with γH2AX and MDC1. Intriguingly, 53BP1 co-located well with γH2AX and MDC1, but remained separate from RNF8 and RNF168. These co-localization patterns were consistent for at least 3h after IR. CONCLUSIONS: The foci formations of γH2AX-MDC1-53BP1 and RNF8-RNF168-BRCA1-A complexes are spatially independent. Such divergence was not anticipated from prior studies on the recruitment of these proteins to foci. This information indicates that individual foci may represent distinct sites of DNA repair facilitated by a specific subset of DDR proteins.

Function of erbB receptors and DNA-PKcs on phosphorylation of cytoplasmic and nuclear Akt at S473 induced by erbB1 ligand and ionizing radiation.

BACKGROUND AND PURPOSE: In the present study effect of erbB2 as well as DNA-PKcs on ionizing radiation (IR)- and erbB1 ligand-induced phosphorylation of Akt at S473 in cytoplasmic and nuclear fractions was investigated. MATERIALS AND METHODS: DNA-PKcs proficient and deficient syngeneic colon carcinoma sublines of HCT116 and the glioblastoma cell lines MO59K and MO59J as well as the lung carcinoma cell line A549 were used. Akt-S473 phosphorylation was investigated in cells pre-treated with pharmacological inhibitors or transfected with siRNA by immunoprecipitation, Western blotting and confocal microscopy after different stimuli, i.e., ligands and IR. RESULTS: IR-induced phosphorylation of Akt in both MO59K and MO59J cell lines but not in HCT116 cells was DNA-PKcs dependent. In A549 cells, IR-induced phosphorylation of nuclear Akt-S473 was dependent on erbB1, erbB2, and DNA-PKcs. EGF induced phosphorylation of nuclear Akt-S473 in a DNA-PKcs and erbB2 independent manner. CONCLUSION: Data indicate that the function of DNA-PKcs on IR-induced Akt-S473 phosphorylation is cell line specific. IR-induced, but not EGF-induced phosphorylation of cytoplasmic and/or nuclear Akt-S473 is erbB2 dependent.

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.

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.

Loss of neuronal protein expression in mouse hippocampus after irradiation.

The effects of radiation on neurons are incompletely characterized. We evaluated changes in the expression of neuronal nuclear and other proteins in the mouse hippocampus after 17-Gy whole-brain irradiation. Expression of neuronal nuclei (NeuN), neuron-specific enolas, prospero-related homeobox 1 (Prox1), calbindin D28k, and synaptophysin 1 in the CA1, CA3, and dentate gyrus of the hippocampus was determined by immunohistochemistry; neuronal numbers were estimated by design-based stereology. At 7 days after irradiation, there was a marked reduction of NeuN neurons in CA3. Stereologic estimates confirmed a significant reduction in NeuN neurons in CA3 at 7 days, in the dentate gyrus at 7 days, 3 weeks and 2 months, and in CA1 at 2 months compared with controls; neuron-specific enolase and prospero-related homeobox 1-positive neurons in the CA3 subregion were also decreased at 7 days. The numbers of granule and pyramidal cells identified by 4'6-diamidino-2-phenylindole nuclear staining, however, remained unchanged, and there were no changes in calbindin D28k or synaptophysin 1 immunoreactivity after irradiation. We conclude that irradiation may result in a temporary loss of neuronal protein expression in mouse hippocampus. These changes do not necessarily indicate loss of neurons and indicate the need for caution regarding the use of phenotypic markers such as NeuN to estimate changes in neuronal numbers after irradiation.

Light-induced COP9 signalosome expression in the Indian false vampire bat Megaderma lyra.

The COP9 signalosome (CSN) is a multi-subunit protein complex conserved in plants and animals. CSN subunits have been identified as light-mediated master regulators of eukaryotic circadian clocks from fungi to animals. The Indian false vampire bat Megaderma lyra is completely adapted to an anthropic biotope and behavioral studies have reported that M. lyra exhibits light-sampling behavior to assess environmental light. LC-MS-MS results for a 36 kDa protein were analyzed using the Sequest search engine, and COP9 signalosome subunit 5 (CSN5) was pinpointed as having the highest score with 6 matching peptides. To confirm the presence of CSN5, up-regulated cDNA was amplified, sequenced, and identified as CSN5. Furthermore, semi-quantitative RT-PCR analysis demonstrated that the level of induction of CSN5 was regulated by environmental light. We estimated the level of expression across a light-dark cycle and observed a higher level of expression at the end of the light phase. Similarly, when the animal was shifted from continuous dark to light, CSN5 expression was induced. Correspondingly, we detected the similar pattern of translated protein with JAB1 antibody. Knowledge about the circadian rhythm and its molecular mechanism in Chiroptera is very limited and this study suggests that CSN5 might be involved in the M. lyra light-signaling process.

[Changes in the antigenic properties of proteins of laboratory mice during oxidative stress].

Changes in the level of oxidative damage to proteins in CD1 outbred mice gamma irradiated with a dose of 3 Gy have been studied. The changes were estimated from the amount of carbonyl groups (CG) in the proteins. It was found that two hours after exposure to gamma radiation, the amount of CG in the cytoplasmic and nuclear fractions of the liver, heart, brain, and spleen sharply increased. Two months after irradiation, the level of CG in the cytoplasmic and nuclear subcellular fractions of the liver and brain decreased to the level of CG in the control animals, which were not exposed to radiation. In the subcellular fractions of the heart and spleen, the increase in the degree of damage was more significant and a high level of damage was observed even two months after irradiation. An enhancement of the antigenic properties of proteins from the liver, heart, and spleen in the postirradiation period was found. Spleen proteins were most immunogenic. A comparison of the antigenic properties of proteins isolated from the tissues 60 days after irradiation revealed a correlation between the level of oxidative damage and the immunogenicity of the total protein fraction.

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.

Interaction of light and hormone signals in germinating seeds.

Seed germination is regulated by several environmental factors, such as moisture, oxygen, temperature, light, and nutrients. Light is a critical regulator of seed germination in small-seeded plants, including Arabidopsis and lettuce. Phytochromes, a class of photoreceptors, play a major role in perceiving light to induce seed germination. Classical physiological studies have long suggested the involvement of gibberellin (GA) and abscisic acid (ABA) in the phytochrome-mediated germination response. Recent studies have demonstrated that phytochromes modulate endogenous levels of GA and ABA, as well as GA responsiveness. Several key components that link the perception of light and the modulation of hormone levels and responsiveness have been identified. Complex regulatory loops between light, GA and ABA signaling pathways have been uncovered.

RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage.

Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or poleta only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In the absence of damage these proteins are not physically associated within the nucleoplasm.

Effect of somatostatin on repair of ionizing radiation-induced DNA damage in pituitary adenoma cells GH3.

Ionizing radiation and somatostatin analogues are used for acromegaly treatment to achieve normalization or reduction of growth hormone hypersecretion and tumor shrinkage. In this study, we investigated a combination of somatostatin (SS14) with ionizing radiation of (60)Co and its effect on reparation of radiation-induced damage and cell death of somatomammotroph pituitary cells GH3. Doses of gamma-radiation 20-50 Gy were shown to inhibit proliferation and induce apoptosis in GH3 cells regardless of somatostatin presence. It has been found that the D(0) value for GH3 cells was 2.5 Gy. Somatostatin treatment increased radiosensitivity of GH3 cells, so that D(0) value decreased to 2.2 Gy. We detected quick phosphorylation of histone H2A.X upon irradiation by the dose 20 Gy and its colocalization with phosphorylated protein Nbs-1 in the site of double strand break of DNA (DSB). Number of DSB decreased significantly 24 h after irradiation, however, clearly distinguished foci persisted, indicating non repaired DSB, after irradiation alone or after combined treatment by irradiation and SS14. We found that SS14 alone triggers phosphorylation of Nbs1 (p-Nbs1), which correlates with antiproliferative effect of SS14. Irradiation also increased the presence of p-Nbs1. Most intensive phosphorylation of Nbs1 was detected after combined treatment of irradiation and SS14. The decrease of the number of the DSB foci 24 h after treatment shows a significant capacity of repair systems of GH3 cells. In spite of this, large number of unrepaired DSB persists for 24 h after the treatment. We conclude that SS14 does not have a radioprotective effect on somatomammotroph GH3 cells.

Loss of xeroderma pigmentosum C (Xpc) enhances melanoma photocarcinogenesis in Ink4a-Arf-deficient mice.

Despite an extensive body of evidence linking UV radiation and melanoma tumorigenesis, a clear mechanistic understanding of this process is still lacking. Because heritable mutations in both INK4a and the nucleotide excision repair (NER) pathway predispose individuals to melanoma development, we set out to test the hypothesis that abrogation of NER, by deletion of the xeroderma pigmentosum C (Xpc) gene, will heighten melanoma photocarcinogenesis in an Ink4a-Arf-deficient background. Experimentally, we generated a strain of mice doubly deficient in Xpc and Ink4a-Arf and subjected wild-type, Xpc-/-Ink4a-Arf+/+, Xpc-/-Ink4a-Arf-/-, and Xpc+/+Ink4a-Arf-/- mice to a single neonatal (day P3) dose of UVB without additional chemical promotion. Indeed, there was a significant increase in the development of dermal spindle/epithelioid cell melanomas in Xpc-/-Ink4a-Arf-/- mice when compared with Xpc+/+Ink4a-Arf-/- mice (P = 0.005); wild-type and Xpc-/-Ink4a-Arf+/+ mice failed to develop tumors. These neoplasms bore a striking histologic resemblance to melanomas that arise in the Tyr-vHRAS/Ink4a-Arf-/- context and often expressed melanocyte differentiation marker Tyrp1, thus supporting their melanocytic origination. All strains, except wild-type mice, developed pigmented and non-pigmented epidermal-derived keratinocytic cysts, whereas Xpc+/+Ink4a-Arf-/- mice exhibited the greatest propensity for squamous cell carcinoma development. We then screened for NRas, HRas, Kras, and BRaf mutations in tumor tissue and detected a higher frequency of rare Kras(Q61) alterations in tumors from Xpc-/-Ink4a-Arf-/- mice compared with Xpc+/+Ink4a-Arf-/- mice (50% versus 7%, P = 0.033). Taken together, results from this novel UV-inducible melanoma model suggest that NER loss, in conjunction with Ink4a-Arf inactivation, can drive melanoma photocarcinogenesis possibly through signature Kras mutagenesis.