Proton-irradiated breast cells: molecular points of view.

Breast cancer (BC) is the most common cancer in women, highly heterogeneous at both the clinical and molecular level. Radiation therapy (RT) represents an efficient modality to treat localized tumor in BC care, although the choice of a unique treatment plan for all BC patients, including RT, may not be the best option. Technological advances in RT are evolving with the use of charged particle beams (i.e. protons) which, due to a more localized delivery of the radiation dose, reduce the dose administered to the heart compared with conventional RT. However, few data regarding proton-induced molecular changes are currently available. The aim of this study was to investigate and describe the production of immunological molecules and gene expression profiles induced by proton irradiation. We performed Luminex assay and cDNA microarray analyses to study the biological processes activated following irradiation with proton beams, both in the non-tumorigenic MCF10A cell line and in two tumorigenic BC cell lines, MCF7 and MDA-MB-231. The immunological signatures were dose dependent in MCF10A and MCF7 cell lines, whereas MDA-MB-231 cells show a strong pro-inflammatory profile regardless of the dose delivered. Clonogenic assay revealed different surviving fractions according to the breast cell lines analyzed. We found the involvement of genes related to cell response to proton irradiation and reported specific cell line- and dose-dependent gene signatures, able to drive cell fate after radiation exposure. Our data could represent a useful tool to better understand the molecular mechanisms elicited by proton irradiation and to predict treatment outcome.

Gene expression analysis of oxidative stress and apoptosis in proton-irradiated rat retina.

UNLABELLED: The aim of this study was to examine the induction of oxidative stress and apoptosis-associated gene expression profiles in retina after proton irradiation exposure at 0.5 to 4 Gy. MATERIALS AND METHODS: One eye of each Sprague-Dawley rat (6 per group) was irradiated with a conformal proton beam to total doses of 0, 0.5, 1 and 4 Gy. Retinal tissues were isolated for characterization of gene expression profiles 6 hours after proton radiation. RESULTS: For oxidative stress, many genes responsible for regulating the production of reactive oxygen species (ROS) were significantly up-regulated (Fmo2, Gpx2, Noxa1 and Sod3) compared to controls. Several important genes involved in the initiation or activation of apoptotic signaling pathways were significantly up-regulated following irradiation (Fas, Faslg, Trp63 and Trp73). TUNEL assay and caspase-3 immunocytochemical analysis revealed increased apoptotic immunoreactivity following irradiation. CONCLUSION: The data revealed that exposure to proton radiation induced oxidative stress-associated apoptosis. In response to ionizing radiation, the expression of genes involved in pathways mediating apoptosis may be differentially regulated in different dose regimens.

Testicular steroidogenesis is not altered by 137 cesium Chernobyl fallout, following in utero or post-natal chronic exposure.

The testis is especially sensitive to pollutants, including radionuclides. Following the Chernobyl nuclear power plant accident, several of these radionuclides were emitted and spread in the environment. Subsequently, children presented some disruptions of the endocrine system. To determine whether these disruptions were due to 137 cesium ((137)Cs) exposure, the effects of chronic contamination with low doses of (137)Cs in utero or from birth on testicular steroidogenesis in rats were studied. Contamination was continued for 9 months. No modification was observed in circulating level of hormones (17beta-estradiol, testosterone, follicle-stimulating hormone, luteinizing hormone) following in utero or post-natal contamination. Expression of several genes involved in testicular steroidogenesis was affected (cyp19a1, fxr, sf-1), without modification of protein expression or activity. Our results suggest that growing organisms may be affected at the molecular level by (137)Cs contamination at this post-accidental dose.

Interstrand cross-links: a new type of gamma-ray damage in bromodeoxyuridine-substituted DNA.

Interstrand cross-links (ICL) represent one of the most toxic types of DNA damage for dividing cells. They are induced both by natural products (e.g., psoralens + UVA) and by several chemical agents, some of which are used in chemotherapy (e.g., carboplatin and mitomycin C). Although repair mechanisms exist for interstrand cross-links, these lesions can induce mutations, chromosomal rearrangements, and cell death. Here, we report, for the first time, the formation of ICL by gamma-rays in brominated DNA. It is well established that the radiosensitization properties of bromodeoxyuridine (BrdUrd) result primarily from the electrophilic nature of the bromine, making it a good leaving group and leading to the irreversible formation of a uridinyl radical (dUrd(*)) or uridinyl anion (dUrd-) upon addition of an electron. We observe that the radiolytic loss of the bromine atom is greatly suppressed in double-stranded compared to single-stranded DNA. We have used a model DNA containing a bulge, formed by five mismatched bases, and have observed a linear dose-response for the formation of strand breaks on the single-stranded regions of both the brominated strand and the opposite nonbrominated strand. Surprisingly, we have observed the formation of interstrand cross-links exclusively in the mismatched region. Thus, we propose that the radiosensitization effects of bromodeoxyuridine in vivo will almost certainly be limited to single strand regions such as found in transcription bubbles, replication forks, mismatched DNA, and possibly the loop region of telomeres. Our results suggest that interstrand cross-links may contribute to the radiosensitization effects of BrdUrd. These findings may have profound implications for the clinical use of bromodeoxyuridine as a radiosensitizer, as well as for the development of targeted radiosensitizers.

Drug release from functionalized oligodeoxynucleotide by photo-induced electron transfer.

We synthesized a photoreactive hairpin-type oligodeoxynucleotide (P-ODN) possessing an o-nitrobenzyl chromophore and a triplet quencher of 1-aminonaphthalene. Photoirradiation of the hybrid of H-ODN with its complementary DNA led to release of drug in an efficient amount, while the photo-induced drug release was remarkably suppressed in the absence of complementary DNA.

Excitonic heterodimer formation in an HIV-1 oligonucleotide labeled with a donor-acceptor pair used for fluorescence resonance energy transfer.

In this study, we investigated the absorbance and fluorescence properties of cTAR, the complementary DNA sequence of the transactivation response element of the HIV-1 genome, doubly end-labeled by different dyes, 5(and 6)-carboxyfluorescein (Fl) and 5(and 6)-carboxytetramethylrhodamine (TMR), frequently used in fluorescence resonance energy transfer (FRET) studies. This oligonucleotide forms a stable stem-loop structure. The absorption spectrum of this species clearly differed from that of a doubly labeled cTAR derivative in which the terminal part of the stem is melted and from an equimolecular mixture of singly labeled species. Moreover, no significant TMR fluorescence change accompanies the dramatic Fl intensity increase when the doubly labeled native cTAR was melted by temperature or annealed with its complementary sequence. Both elements suggest the formation of an H-type ground-state heterodimer between Fl and TMR that may be described by the molecular exciton model. Moreover, time-resolved fluorescence further suggests that the nonfluorescent heterodimer is in equilibrium with a small population of partially melted species showing FRET. Based on the spectral shifts associated with heterodimer formation, an interchromophore distance of 7.7 A was calculated. Both the excitonic signal and the Fl fluorescence were used as sensitive tools to monitor the temperature-mediated and HIV nucleocapsid protein-mediated annealing of cTAR with its complementary sequence.

Radiation-inducible hSNK gene is transcriptionally regulated by p53 binding homology element in human thyroid cells.

We identified a species relevant to polo-like kinase family, a human homologue of mouse serum-inducible kinase, hSNK gene, whose mRNA expression was rapidly increased in cultured human thyroid cells after X-ray irradiation. The cDNA cloning and genomic analysis of the hSNK gene showed the presence of 14 exons spanning over 6 kb of genomic DNA that encodes a 2.9-kb mRNA product. Promoter analysis demonstrated possible existence of a radiation-responsive element in the p53 binding homology element (p53RE) localized to near upstream of basal promoter of the hSNK gene. Nuclear protein extracts from HeLa and various human thyroid carcinoma cell lines bound selectively to p53RE. Anti-p53 or anti-p73 antibodies, however, failed to recognize the p53RE-protein complex formed in the presence of such nuclear extracts. These results suggest that radiation-responsive transcription factor(s) directly participates in the regulation of hSNK gene expression via the binding to p53RE in promoter region.

Analysis of gene transcription in cells lacking DNA-PK activity.

The DNA-dependent protein kinase (DNA-PK), comprised of the Ku70/Ku80 (now known as G22p1/Xrcc5) heterodimer and the catalytic subunit DNA-PKcs (now known as Prkdc), is required for the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair. The mechanism of action of DNA-PK remains unclear. We have investigated whether DNA-PK regulates gene transcription in vivo after DNA damage using the subtractive hybridization technique of cDNA representational difference analysis (cDNA RDA). Differential transcription, both radiation-dependent and independent, was detected and confirmed in primary mouse embryo fibroblasts from DNA-PKcs(-/-) and DNA-PKcs(+/+) mice. We present evidence that transcription of the extracellular matrix gene laminin alpha 4 (Lama4) is regulated by DNA-PK in a radiation-independent manner. However, screening of both primary and immortalized DNA-PKcs-deficient cell lines demonstrates that the majority of differences were not consistently dependent on DNA-PK status. Similar results were obtained in experiments using KU mutant hamster cell lines, indicating heterogeneity of transcription between closely related cell lines. Our results suggest that while DNA-PK may be involved in limited gene-specific transcription, it does not play a major role in the transcriptional response to DNA damage.

Molecular cloning and characterization of the human KIN17 cDNA encoding a component of the UVC response that is conserved among metazoans.

We describe the cloning and characterization of the human KIN17 cDNA encoding a 45 kDa zinc finger nuclear protein. Previous reports indicated that mouse kin17 protein may play a role in illegitimate recombination and in gene regulation. Furthermore, overproduction of mouse kin17 protein inhibits the growth of mammalian cells, particularly the proliferation of human tumour-derived cells. We show here that the KIN17 gene is remarkably conserved during evolution. Indeed, the human and mouse kin17 proteins are 92.4% identical. Furthermore, DNA sequences from fruit fly and filaria code for proteins that are 60% identical to the mammalian kin17 proteins, indicating conservation of the KIN17 gene among metazoans. The human KIN17 gene, named (HSA)KIN17, is located on human chromosome 10 at p15-p14. The (HSA)KIN17 RNA is ubiquitously expressed in all the tissues and organs examined, although muscle, heart and testis display the highest levels. UVC irradiation of quiescent human primary fibroblasts increases (HSA)KIN17 RNA with kinetics similar to those observed in mouse cells, suggesting that up-regulation of the (HSA)KIN17 gene after UVC irradiation is a conserved response in mammalian cells. (HSA)kin17 protein is concentrated in intranuclear focal structures in proliferating cells as judged by indirect immunofluorescence. UVC irradiation disassembles (HSA)kin17 foci in cycling cells, indicating a link between the intranuclear distribution of (HSA)kin17 protein and the DNA damage response.

BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields.

We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

Monitoring the hybridization of the components of oligonucleotide mixtures to immobilized DNA via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Reported here is how the hybridization of individual components of oligonucleotide mixtures to solid-phase bound complementary strands can be monitored simultaneously by quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Three oligonucleotides, a DNA heptamer, a DNA octamer and a DNA octamer with a terminal cholic acid appendage were used as the test mixture. Upon cooling in the presence of a complementary undecamer on controlled pore glass, depletion of the components from the solution was observed. The resulting hybridization curves show the same relative affinities as traditional UV melting curves with single components and their complement. Assays of the kind described here may be used to select high affinity binders from combinatorial libraries of modified antisense oligonucleotides.

Ultraviolet-light induced p53 mutational spectrum in yeast is indistinguishable from p53 mutations in human skin cancer.

Ultraviolet (UV) light has been associated with the development of human non-melanoma skin cancers (NMSC). Such cancers often exhibit mutations in the p53 tumour suppressor gene. In order to determine the UV-induced p53 mutation spectrum, a yeast expression vector that harbours a human wild-type p53 cDNA was UV-irradiated in vitro and transfected into a yeast strain that contained the ADE2 gene regulated by a p53-responsive promoter. Forty-five mutant clones contained 51 mutations. Seven mutations were tandem base pair substitutions, four of which being CC-->TT, hallmark mutations of UV mutagenesis. Eighty percent (41/51) of the mutations were single or non-tandem base pair substitutions, the majority of which (27/41) were C-->T transitions. Ninety-five percent of such mutations occurred at dipyrimidine sites. Through a rigorous statistical test, the UV-induced p53 mutation spectrum appears to differ significantly (P < 0.008) from the one induced by the antineoplastic drug chloroethyl-cyclohexyl-nitrosourea, and to be indistinguishable from the one observed in NMSC (P = 0.4). These results demonstrate that the assay allows the determination of carcinogen-specific p53 mutation fingerprints and represents a new tool for molecular epidemiology.

DNA damage-dependent inactivation of complementary strand synthesis in Xenopus laevis egg or HeLa cell lysates.

Genotoxic lesions frequently arrest DNA synthesis and, as a consequence, result in the accumulation of incompletely replicated chromosomal segments containing long single-stranded regions of parental DNA. Here, we exploited complementary strand synthesis in Xenopus laevis egg or HeLa cell lysates to test how the eukaryotic replication machinery responds to such damaged single-stranded intermediates. Although both cell lysates promoted efficient conversion of M13 or phi X174 single-stranded templates to double-stranded products, their replication activity was inhibited by DNA damage arising from ultraviolet (UV) radiation or exposure to the alkylating agent N-methyl-N-nitrosourea (MNU). When M13 single-stranded DNA containing UV-or MNU-induced lesions was coincubated with single-stranded substrates containing no lesions, we observed suppression of DNA synthesis on both damaged and undamaged templates. In contrast, complementary strand synthesis remained unaffected in coincubation reactions containing damaged DNA in the double-stranded form. Effective inhibition of complementary strand synthesis on undamaged templates required the presence of at least stoichiometric amounts of UV-or MNU-treated single-stranded DNA, indicating that a DNA polymerase or accessory protein is excluded from DNA synthesis by immobilization at or near the lesion sites. In support of this competitive mode of inhibition, we found that inactivation of DNA synthesis by coincubation with damaged single-stranded DNA was relieved by the addition of an exogenous DNA polymerase that catalyzes processive strand elongation. In summary, this study reveals sequestration of critical components of the eukaryotic replication machinery in a DNA damage-dependent and single-strand-specific manner, thereby providing a potential mechanism to sense arrested replication intermediates during an early recognition step of S phase checkpoint responses.

Light induces accumulation of isocitrate lyase mRNA in a carotenoid-deficient mutant of Chlamydomonas reinhardtii.

A cDNA with sequence similarity to isocitrate lyase (ICL) genes was isolated from the unicellular eukaryotic green alga Chlamydomonas reinhardtii as a light-induced mRNA in the carotenoid biosynthetic mutant strain FN68. The 416 amino acid open reading frame shows significant sequence similarity to isocitrate lyases of bacteria (70%), molds (48%), yeasts (45%), and plants (47%). Expression of the Chlamydomonas ICL gene was tested in the mutant strain FN68, which when grown in the dark fails to accumulate carotenoids and is deficient in chlorophyll, and in CC400G, a strain that accumulates wild-type levels of carotenoids and chlorophyll. In vegetative CC400G cells, ICL mRNA accumulated to a high level in the dark and declined to a barely detectable level within 30 min of exposure to light. This response was more sensitive to white (tungsten filament) or red light than green or blue light, excluding cryptochrome and rhodopsin as the photoreceptor. These results are consistent with excitation by chlorophyll and/or a phytochrome-related photoreceptor. In vegetative FN68 cells, ICL mRNA abundance was very low in the dark, but increased dramatically in response to light. At intensities above threshold, excitation by far-red or red light-induced ICL mRNA accumulation to the highest levels. The threshold of the response was lowest for far-red and blue light. These results are consistent with excitation of a photochromic far-red-responsive pigment.

Roles of XPG and XPF/ERCC1 endonucleases in UV-induced immunostaining of PCNA in fibroblasts.

To investigate the relationship between proliferating cell nuclear antigen (PCNA) complex formation and dual incisions in the nucleotide excision repair (NER) process, xeroderma pigmentosum group G (XP-G), XP-F, and XP-G equivalent mouse UV-sensitive mutant ERCC group 5 cells were utilized as a model in this study. These cells are deficient in endonucleases related to 3' (XP-G and ERCC group 5) or 5' (XP-F) incision of the DNA lesions in the NER process. PCNA complex formation was detected by an indirect immunofluorescence method after the cells were fixed in methanol. When Sps1 (XP-G) and XL216-7 (ERCC group 5) cells were UV irradiated, neither of them showed PCNA staining. In contrast, SFN4 (a human normal strain) and heterokaryons of Sps1 and XP96TO (XP-A) cells fused by polyethylene glycol treatment showed PCNA staining following UV irradiation. Furthermore, XLgfPAneo1 cells, derived from XL216-7 cells transfected with a plasmid containing mouse ERCC5 (xpg) cDNA, also restored staining and UV sensitivity. On the other hand, we observed a very faint PCNA staining in XP2YO (XP-F) cells, expressing no detectable ERCC1 or XPF protein, after UV irradiation. X rays induced PCNA staining in all cell lines with a similar staining pattern, and radiosensitivity was exactly the same between XL216-7 and XLgfPAneo1 cells. These results may have implications for the NER process in vivo in that coordinately occurring dual incisions by XPG and XPF/ERCC1 proteins play an important role in inducing PCNA complex formation, but the step may not be required for PCNA-dependent repair of X-ray-induced DNA damage.

Expression of dominant negative CREB reduces resistance to radiation of human melanoma cells.

u.v.-responsive element (URE)-binding proteins were found to include members of the AP1 and ATF transcription factor families. To elucidate the functional contribution of URE-bound proteins to the characteristics of human melanoma, we have used a dominant negative CREB cDNA which is mutated within the DNA-binding domain and cloned into a mammalian expression vector driven by the RSV promoter (KCREB). As such, KCREB is still capable of heterodimerizing with its associated proteins, yet, due to its poor binding affinity to DNA it out competes transcriptional activity mediated by those proteins. Human melanoma cells (MeWo) were transfected with KCREB and three clones, designated K1, K2, and K10 which express KCREB transcripts were then selected for further characterization. When tested for binding activities in gel shift assays, proteins prepared from the three clones exhibited a different set of complexes than the parent MeWo and control MeWo(neo) cells (transfected with empty expression vector) under normal growth conditions, and after u.v.-irradiation. Using CAT vector, driven by a tetramer URE construct, revealed a striking decrease in transcriptional activity in each of the three clones before as well as after u.v.-irradiation. When tested for radiation resistance MeWo cells were found to exhibit 42% survival to a u.v.-dose of 16 J/m2, whereas, K1, K2 and K10 exhibited only 10.2, 3.9 and 4.2% survival, respectively. Exposure to 2 Gy of X-radiation led to 62.1% survival of MeWo as compared with 18.5% of K1 and 7.7% and 6.5% of K2 and K10, respectively. While no significant differences were noticed in their growth rate, all three clones exhibited fewer, and smaller colonies in soft agar, when compared with parent cells. These findings indicate that through their transcriptional activities, CREB and its associated proteins play an important role in the acquisition of characteristic phenotypes of human melanoma cells including resistance to u.v.-irradiation.

Mutational spectrum of X-ray induced TK- human cell mutants.

Mutations induced by ionizing radiation have historically elicited significant public concern. However, only a limited database of ionizing radiation-induced point mutations is available, particularly at endogenous human cell loci. Here, we report the mutational spectrum for 184 X-ray induced TK- mutants derived from TK6 human lymphoblasts. This report represents the first large scale utilization of the tk locus for investigation of mutational specificity at the DNA sequence level. Rapid, single nucleotide sequencing assays at frameshift polymorphism sites in tk exons 4 and 7 were used to partition TK- mutants into two groups: 126 were attributed to either partial gene deletion or to loss of heterozygosity, and DNA sequence alterations were identified for 51. X-ray-induced point mutations included all classes of transitions and transversions, tandem base substitutions, frameshifts, small deletions and a small duplication. The distribution within tk was characterized by clustering at some sites. Twelve TK- point mutations, including five entirely within the coding sequence in exons 3 and 4, resulted in aberrant splicing of the tk transcript. The spectrum of X-ray-induced point mutations was found to be highly reproducible when TK- mutations were compared with HPRT- mutations in TK6. A statistically significant decrease in transitions (P = 0.04) was observed in the combined data set as compared to the spontaneous background. These findings suggest a reproducible pattern which may be utilized in recognizing radiation-induced mutations at other loci of interest.

Functional complementation studies with X-ray-sensitive mutants of Chinese hamster cells closely resembling ataxia-telangiectasia cells.

In order to isolate a human gene complementing the defect in A-T-like hamster cell mutants, the mutants were used as recipients for genomic DNA transfection, using either HeLa chromosomal DNA or DNA from a human cosmid library. Three primary transformants with an intermediate X-ray sensitivity and almost normal sensitivity to MMS, but retaining radioresistant DNA synthesis (RDS), were obtained. To identify the human chromosome that complements the defect in the A-T-like mutants, and to assess the degree of complementation for survival and RDS, microcell-mediated chromosome transfer was used. At least 20 independent hybrid clones between the mutant and each one of the human chromosomes 1, 2, 4, 5, 15, 17 or 18 were isolated. All hybrid clones remained X-ray sensitive, except one with chromosome 4, and another with chromosome 15, both showing an intermediate X-ray sensitivity. By using in situ hybridization we found that this partial correction was due to the presence of a mouse chromosome. In these two hybrids containing the mouse chromosome together with human chromosome 4 or 15, RDS was fully complemented only in the hybrid with chromosome 4 but not in the one containing chromosome 15, suggesting that RDS and X-ray sensitivity may be complemented independently.

Molecular analysis of mutagenesis by high LET radiation.

Mutation induction by high linear energy transfer [LET] alpha particles and gamma-rays was scored in the human hamster hybrid [AL] cells. Southern blotting technique was used to analyse the molecular changes in the DNA from both the HGPRT- and S1- mutants. Dose dependent mutagenesis in the AL cells irradiated with the charged particles was higher by almost 20 fold at the S1 than the corresponding HGPRT locus. Southern analysis of the mutants induced by the high LET particles showed mostly multilocus deletion at both the HGPRT and S1 genes.