Modeling the repair of DNA strand breaks caused by γ-radiation in a minichromosome.

The objective of the studies described here was the development of a mathematical model which would fit experimental data for the repair of single and double strand breaks induced in DNA in living cells by exposure to ionizing radiation, and which would allow to better understand the processes of DNA repair. DNA breaks are believed to play the major role in radiation-induced lethality and formation of chromosome deletions, and are therefore crucial to the response of cells to radiotherapy. In an initial model which we reported on the basis of data for the repair of Epstein-Barr minichromosomes in irradiated Raji cells, we assumed that DNA breaks are induced only at the moment of irradiation and are later removed by repair systems. This work gives a development of that mathematical model which fits the experimental results more precisely and suggests strongly that DNA breaks are generated not only by direct irradiation but also later, probably by systems engaged in repair of oxidative damage.

Association between single-nucleotide polymorphisms of selected genes involved in the response to DNA damage and risk of colon, head and neck, and breast cancers in a Polish population.

Single-nucleotide polymorphisms in genes involved in DNA-damage-induced responses are reported frequently to be a risk factor in various cancer types. Here we analysed polymorphisms in 5 genes involved in DNA repair (XPD Asp312Asn and Lys751Gln, XRCC1 Arg399Gln, APE1 Asp148Glu, NBS1 Glu185Gln, and XPA G-4A) and in a gene involved in regulation of the cell-cycle (CCND1 A870G). We compared their frequencies in groups of colon, head and neck, and breast cancer patients, and 2 healthy control groups: (1) matched healthy Polish individuals and (2) a NCBI database control group. Highly significant differences in the distribution of genotypes of the APE1, XRCC1 and CCND1 genes were found between colon cancer patients and healthy individuals. The 148Asp APE1 allele and the 399Gln XRCC1 allele apparently increased the risk of colon cancer (OR = 1.9-2.3 and OR = 1.5-2.1, respectively). Additionally, frequencies of XPD genotypes differed between healthy controls and patients with colon or head and neck cancer. Importantly, no differences in the distribution of these polymorphisms were found between healthy controls and breast cancer patients. The data clearly indicate that the risk of colon cancer is associated with single-nucleotide polymorphism in genes involved in base-excision repair and DNA-damage-induced responses.

Transcription regulation in differential expression of the human GSTP1 gene in breast and choriocarcinoma cells.

Glutathione S-transferase P1 is a major phase II detoxification enzyme in most cell types. Aberrant expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. GSTP1 gene transcription is regulated by promoter methylation and by transcription factors. To elucidate the mechanisms responsible for the different levels of GSTP1 expression observed in Hbl-100 and BeWo cells we utilized truncated promoter constructs to compare the functional role of different promoter elements. We also identified transcription factors binding the responsive elements by electrophoretic mobility shift assay. The applied approaches provided the evidence that binding of transcription factors to ARE, CRE and NF-kappaB sites are responsible for the cell specific levels of GSTP1 expression in Hbl-100 and BeWo cells. It was also indicated that partial promoter methylation occurs in BeWo cells.

Relationships between acute reactions to radiotherapy in head and neck cancer patients and parameters of radiation-induced DNA damage and repair in their lymphocytes.

PURPOSE: To study the relationship between lymphocyte radiosensitivity measured in vitro and acute reactions to radiotherapy in patients with head and neck cancer. MATERIALS AND METHODS: Acute reactions were measured in 34 patients using the Dische scale. Lymphocyte radiosensitivity was measured using the alkaline comet assay, the micronucleus assay, the nuclear division index and morphological assessment of apoptosis. RESULTS: There was a weak, statistically significant correlation between in vitro radiosensitivity measured as the rate of DNA damage repair and the cumulative radiation dose exerting the maximum acute reaction scored (r = -0.366, p = 0.039, n = 34). Subgroup analyses showed that for patients with a low level of radiation-induced DNA damage there was a statistically significant relationship between lymphocyte radiosensitivity measured as inhibition of proliferation and acute toxicity (r = -0.621, p = 0.007, n = 18). For patients with a high level of residual DNA damage, there was a relationship between lymphocyte radiosensitivity measured using the micronucleus assay and acute toxicity (r = -0.597, p = 0.023, n = 14). CONCLUSIONS: Combining two measures of radiosensitivity improves the ability to correlate in vitro lymphocyte radiosensitivity and acute radiotherapy toxicity data.

[Some aspects of glutathione S-transferase P1-1 gene transcription regulation in human placenta].

Glutathione S-transferase P1-1 is the main phase II xenobiotic metabolism enzyme in human placenta. Low level of its gene expression and corresponding ineffective protection of fetus from toxic compounds is associated with pregnancy disorders such as preeclampsia and abnormalities of fetus development. It was previously reported that environmental radioactive contamination caused down-regulation of GSTP1 transcription in human placenta, but mechanisms responsible for such changes were unclear. In the present study we have found that observed changes in transcription of this gene are not caused by promoter methylation because GSTP1 promoter was not methylated in any of analyzed 91 placental samples. Regulation of GSTP1 by methylation or transcription factors was not previously studied in human placenta. Using "Gene Expression Atlas" online software the placental expression profile of transcription factors known to interact with GSTP1 promoter in other cell types, was identified. According to computer analysis the genes coding for GATA2, GATA3, Fos-B, Nrf3 and MafK transcription factors are highly expressed in human placenta, while genes coding for c-Fos, Juns, Mafs, ERbeta, RARalpha and NF-kappaB factors have moderate level of expression. Competitive EMSA provided the evidence that ARE and NF-kappaB-like sites specifically interacted with placental nuclear proteins. Among these proteins transcription factors AP-1 and NF-kappaB were identified using corresponding consensus oligonucleotides as competitors in EMSA.

Adaptor protein Ruk1 forms protein-protein complexes with endonuclease activity in HEK293 cells.

The structural and functional organization of the adaptor protein Ruk(1) is characterized by the presence of three SH3-domains at the N-terminus followed by Pro- and Ser-rich sequences and a C-terminal coiled-coil region. Multiple modules in the Ruk(1) structure involved in protein-protein interactions can provide for formation of ligand clusters with varied properties and subcellular location. To study the nature and biological role of such complexes, the recombinant protein Ruk(1) with a Glu-epitope at the C-terminus (Ruk(1) Glu-tagged) was purified from transfected HEK293 cells by affinity chromatography on protein G-Sepharose with covalently conjugated anti-Glu-tag antibodies. By SDS polyacrylamide gel electrophoresis with subsequent staining with silver, a set of minor bands in addition to the 85-kD Ruk(1) Glu-tagged was detected in the purified preparation of the recombinant protein. Proteins with affinity for nucleic acids were also revealed in the Ruk(1) Glu-tagged preparation by retardation of electrophoretic mobility of 32P-labeled oligodeoxyribonucleotides in gel. The Ruk(1) Glu-tagged preparation was also shown to hydrolyze both deoxyribonucleotides and plasmid DNA. ZnCl2 and heparin inhibited the DNAse activity. These findings suggest the presence of DNases associated with the Ruk(1) protein in HEK293 cells. Such complexes were isolated from lysates of HEK293 cells by chromatography on heparin-Sepharose. By elution with 0.5 and 1.0 M NaCl, two fractions with DNase activity and containing proteins with molecular weights of 83, 80, and 72 kD were obtained. The reaction was inhibited by ZnCl2 and heparin, and previous precipitation of Ruk-related proteins with anti-Ruk antibodies resulted in the exhaustion of nuclease activity. By immunoblotting with anti-Ruk antibodies, 83-kD protein immunologically related to the Ruk(1) protein was identified in the fractions. It was concluded that the adaptor protein Ruk(1) forms complexes with endonucleases in HEK293 cells.

Antioxidant vitamins C, E and beta-carotene reduce DNA damage before as well as after gamma-ray irradiation of human lymphocytes in vitro.

The protective effect of Vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in human lymphocytes in vitro was investigated. Cultured lymphocytes were exposed to increasing concentration of these vitamins either before or after irradiation with 2Gy of gamma-rays and DNA damage was estimated using micronucleus assay. A radioprotective effect was observed when antioxidant vitamins were added to cultured cells before as well after irradiation; the strongest effect was observed when they were added no later than 1h after irradiation. The radioprotective effect of vitamins also depended on their concentration; Vitamins C added at low concentration (1 microg/ml) before exposure of the cells to radiation prevented induction of micronuclei. Vitamin E at the concentration above 2 microg/ml decreased the level of radiation-induced micronuclei when compared to the cells irradiated without vitamin treatment. beta-Carotene was effective at all tested concentrations from 1 to 5 microg/ml and reduced the number of micronuclei in irradiated cells. The vitamins had no effect on radiation-induced cytotoxicity as measured by nuclear division index. The radioprotective action of antioxidant Vitamins C, E and beta-carotene was dependent upon their concentration as well as time and sequence of application.

Detection of damage-recognition proteins from human lymphocytes.

Proteins recognizing and binding to damaged DNA (DDB-proteins) were analyzed in human lymphocytes obtained from healthy donors. Using an electrophoretic mobility shift assay several complexes between nuclear extract proteins and damaged DNA were detected: a complex specific for DNA damaged by N-acetoxy-N-acetylaminofluorene, another complex specific for UV-irradiated DNA, and two complexes specific for DNA damaged by cis-dichlorodiammine platinum. All the detected complexes differed in electrophoretic mobility and possibly contained different proteins. Complexes specific for free DNA ends were also detected in protein extracts from lymphocytes.

Damaged DNA-binding proteins: recognition of N-acetoxy-acetylaminofluorene-induced DNA adducts.

Proteins which bind to the DNA damaged by genotoxic agents can be detected in all living organisms. Damage-recognition proteins are thought to be generally involved in DNA repair mechanisms. On the other hand, the relevance to DNA repair of some other proteins which show elevated affinity to damaged DNA (e.g. HMG-box containing proteins or histone H1) has not been established. Using the electrophoretic mobility-shift assay we have investigated damage-recognition proteins in nuclei from rat hepatocytes. We detected two different protein complexes which preferentially bound the DNA damaged by N-acetoxy-acetylaminofluorene. One of them also recognized the DNA damaged by benzo(a)pyrene diol epoxide (yet with much lower efficiency). The proteins which bind to damaged DNA are permanently present in rat cells and their level does not change after treatment of animals with the carcinogens. Differences in the affinity of the detected damage-recognition proteins to DNA lesion evoked by either carcinogen did not correlate with more efficient removal from hepatic DNA of 2-acetylaminofluorene-induced adducts than benzo(a)pyrene-induced ones.

Characterization of DNA sequences localized 11 to 17.5 kb upstream of the chicken embryonic pi-globin gene.

We have analyzed the DNA fragment localized about 11 to 17.5 kb upstream of the chicken alpha-globin gene domain (the fragment was designed as alpha-0). The nucleotide sequence of its 3.3 kb-long 5' part was established and interactions with nuclear matrix proteins were studied. The DNA region localized about 16 kb upstream of the embryonic pi-globin gene showed high affinity to nuclear matrices in vitro. Two palindromes and a cluster of inverted repeats were co-localized in the same region. The whole 6.6 kb alpha-0 fragment decreased the activity of linked CAT reporter gene when transfected into chicken erythroblastoid cells.

Modifying effect of vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in mouse cells.

The modifying effect of treatment with vitamins C, E and beta-carotene on the clastogenic activity of gamma rays was investigated in mice. Damage in vivo was measured by the micronucleus assay in bone marrow polychromatic erythrocytes and exfoliated bladder cells. The vitamins were administered orally, either for five consecutive days before or immediately after irradiation with 2 Gy of gamma rays. The results show that pretreatment with vitamin E (100-200 mg/kg/day) and beta-carotene (3-12 mg/kg/day) were effective in protecting against micronucleus induction by gamma rays. Vitamin C depending on its concentration enhanced the radiation effect (400 mg/kg/day), or reduced the number of micronucleated polychromatic erythrocytes (50-100 mg/kg/day). Such effect was weekly observed in exfoliated bladder cells. The most effective protection in both tissues was noted when a mixture of these vitamins was used as a pretreatment. Administration of the all antioxidant vitamins to mice immediately after irradiation was also effective in reducing the radiation-induced micronucleus frequency. The data from the in vitro experiments based on the comet assay show that the presence of the vitamins in culture medium influences the kinetic of repair of radiation-induced DNA damage in mouse leukocytes.

The non-random distribution of UV-induced photoproducts in the nuclear matrix and non-matrix DNA fractions.

The formation of UV-induced photoproducts in the chromatin fractions of human lymphocytes was studied by 32P-post-labeling. A higher level of DNA lesions was found in the matrix-attached DNA fraction as compared to non-matrix DNA of irradiated cells (about 150 and 110 adducts per 10(6) nucleotides, respectively, at a 500 J/m2 254 nm-UV dose). Formation of photoproducts in a MAR (matrix attached region) sequence from the mouse kappa immunoglobulin gene irradiated in vitro was examined as well. The MAR sequence showed a two-fold higher level of adducts as compared to non-MAR DNA. The effect of photoproducts on complex-formation between MAR DNA and proteins of the nuclear matrix was studied in vitro. The amount of UV-induced adducts was 1.5-fold higher in matrix-bound fraction as compared to non-fractionated DNA (and five-fold higher as compared to unbound fraction), which possibly resulted from preferential binding of lesion-containing DNA fragments to the nuclear matrix proteins.

Interactions of rat repetitive sequence MspI8 with nuclear matrix proteins during spermatogenesis.

Using the Southwestern blot analysis we have studied the interactions between rat repetitive sequence MspI8 and the nuclear matrix proteins of rat testis cells. Starting from 2 weeks the young to adult animals showed differences in type of testis nuclear matrix proteins recognizing the MspI8 sequence. The same sets of nuclear matrix proteins were detected in some fractions enriched in spermatocytes and spermatides and obtained after fractionation of testis cells of adult animals by the velocity sedimentation technique.

Interactions of the matrix attachment region of DNA with the matrix proteins from the copper preincubated liver nuclei.

Preincubation of rat liver nuclei with copper ions influenced the stability and protein composition of the nuclear matrices isolated by a "high salt" method. Also the specific interaction between matrix proteins and the kappa Ig matrix attachment region of DNA was affected.

DNA repair is less efficient in the nuclear matrix than in non-matrix nuclear fractions in the liver of rats treated with 2-aminofluorene.

The amount of DNA adducts and radioactive thymidine incorporation into DNA fractions attached and not attached to the nuclear matrix in the liver of rats treated with the carcinogen 2-aminofluorene (2-AF) were compared. The rate of [3H]thymidine incorporation was directly proportional to the amount of adducts in total hepatic DNA. Within the first 10 h after the carcinogen treatment, the level of adducts in the nuclear matrix DNA was higher than in the whole nuclei. The rate of [3H]thymidine incorporation into the nuclear matrix DNA was 5-30% lower than into DNA in whole nuclei at any time after 2-AF injection. We suggest that in rat liver cells, the 2-AF-induced DNA repair does not occur in close contact with the nuclear matrix.

MspI8, the repetitive sequence specifically interacting with nuclear matrix of rat testis cells.

The nuclear matrix bound DNA fraction of rat testis showed enrichment in repetitive sequences found in the 450 bp band after gel electrophoresis of the MspI digested rat DNA. DNA fragments isolated from this band were cloned. DNA of the clone pMspI8 showed homology to some representatives of rat LINE sequence family, and complexed in vitro more efficiently with testes nuclear matrix proteins than with yeast ARS1 sequence containing the matrix association region (MAR) or DNA from an other clone, MspI19. Western blot analysis showed that MspI8 sequence interacts with testes matrix protein of about 120 kDa.

Partial hepatectomy of rats 3 weeks before or simultaneously with 2-aminofluorene injection can affect the amounts of adducts induced in hepatic DNA.

The influence of partial hepatectomy on the level of 2-aminofluorene (2-AF) induced DNA adducts in rat liver was studied. We found that partial hepatectomy performed either 3 weeks before or simultaneously with the injection of 2-AF affected the amounts of adducts in rat hepatic DNA compared to controls. The level of DNA adducts in rats that were treated with 2-AF and simultaneously hepatectomized was higher (19.9 fmol/microgram DNA) than in non-hepatectomized ones (14.4 fmol/microgram DNA) when measured 48 h after 2-AF administration. In rats treated with the carcinogen 3 weeks after hepatectomy the level of DNA adducts was significantly higher than in nonhepatectomized rats when measured 15 days after the injection of 2-AF (10.9 fmol/microgram DNA and 5.9 fmol/microgram DNA respectively). The high level of DNA adducts in that group of hepatectomized animals was correlated with a relatively lower rate of 2-AF-induced radioactive thymidine incorporation into hepatic DNA (in comparison to non-hepatectomized rats).

DNA and proteins of the nuclear matrix are the main targets of benzo[a]pyrene's action in rat hepatocytes.

The binding of [14C]benzo[a]pyrene (B[a]P) to DNA and proteins in total nuclei and subnuclear fractions of cultured rat hepatocytes was compared. The main targets of B[a]P were non-histone high molecular weight proteins of the nuclear matrix and DNA sequences attached to this structure. Following 24 h exposure to B[a]P the amounts of adducts in the nuclear matrix DNA and proteins were twice as high as in total nuclei. After withdrawal of the carcinogen containing medium the level of B[a]P-induced adducts gradually decreased but always remained the highest in the nuclear matrix proteins. Removal of adducts from the nuclear matrix DNA was more efficient than from the other DNA fractions, and 72 h after exposure to the carcinogen the level of DNA adducts in this fraction was similar to that in total nuclei.

Formation and removal of DNA adducts in liver of rats treated with hepatocarcinogens 2-aminofluorene or 2-acetylaminofluorene.

The level of adducts in DNA of rats treated with 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) was compared at the times from 1 h till 28 days after injection. The highest amount of DNA adducts was observed 12 h after treatment with 2-AF and 24 h after treatment with 2-AAF, and reached values of about 18 and 21 fmol per micrograms DNA, respectively. Participation of the nonacetylated form, dG-C8-AF, in the total amount of DNA adducts was only slightly greater in rats treated with 2-AF then in those treated with 2-AAF.