Characterization of nuclear structures containing superhelical DNA.
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Structures resembling nuclei but depleted of protein may be released by gently lysing cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids sediment in gradients containing intercalating agents in a manner characteristic of DNA that is intact, supercoiled and circular. The concentration of salt present during isolation of human nucleoids affects their protein content. When made in I-95 M NaCl they lack histones and most of the proteins characteristic of chromatin; in 1-0 M NaCl they contain variable amounts of histones. The effects of various treatments on nucleoid integrity were investigated. (+info)
Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage.
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A sensitive and rapid in situ method was developed to visualize sites of single-stranded (ss) DNA in cultured cells and in experimental test animals. Anti-bromodeoxyuridine antibody recognizes the halogenated base analog incorporated into chromosomal DNA only when substituted DNA is in the single strand form. After treatment of cells with DNA-damaging agents or gamma irradiation, ssDNA molecules form nuclear foci in a dose-dependent manner within 60 min. The mammalian recombination protein Rad51 and the replication protein A then accumulate at sites of ssDNA and form foci, suggesting that these are sites of recombinational DNA repair. (+info)
Exposure to indoor background radiation and urinary concentrations of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage.
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We investigated whether exposure to indoor [gamma]-radiation and radon might be associated with enough free radical formation to increase urinary concentrations of 8-hydroxydeoxyguanosine (8-OHdG), a sensitive marker of DNA damage, due to a hydroxyl radical attack at the C8 of guanine. Indoor radon and [gamma]-radiation levels were measured in 32 dwellings for 6 months by solid-state nuclear track detectors and thermoluminescent dosimeters, respectively. Urine samples for 8-OHdG determinations were obtained from 63 healthy adult subjects living in the measured dwellings. An overall tendency toward increasing levels of 8-OHdG with increasing levels of radon and [gamma]-radiation was seen in the females, presumably due to their estimated longer occupancy in the dwellings measured. Different models were considered for females, with the steepest slopes obtained for [gamma]-radiation with a coefficient of 0.500 (log nmol/l of 8-OHdG for each unit increase of [gamma]-radiation on a log scale) (p<0.01), and increasing to 0.632 (p = 0.035), but with larger variance, when radon was included in the model. In conclusion, there seems to be an effect of indoor radioactivity on the urinary excretion of 8-OHdG for females, who are estimated to have a higher occupancy in the dwellings measured than for males, for whom occupational and other agents may also influence 8-OHdG excretion. ree radicals; [gamma]-radiation; radon. (+info)
Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of transgenic mice.
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Little is known about the effects of ischemia-reperfusion on the inductive, commitment, or execution phases of apoptosis. We have created a genetically defined model to study the response of small intestinal epithelial cells to ischemia-reperfusion injury as a function of their proliferative status and differentiation. Occlusion of the superior mesenteric artery for 20 min in adult FVB/N or C57BL/6 mice results in the appearance of TUNEL-positive apoptotic cells in the jejunal epithelium within 4 h, with a maximum response occurring at 24 h. Stimulation of apoptosis is greater in postmitotic, differentiated epithelial cells located in the upper portions of villi compared with undifferentiated, proliferating cells in the crypts of Lieberkuhn (7-fold vs. 2-fold relative to sham-operated controls). Comparisons of p53(+/+) and p53(-/-) mice established that the apoptosis is p53 independent. To further characterize this response, we generated FVB/N transgenic mice that express human Bcl-2 in epithelial cells distributed from the base of crypts to the tips of their associated villi. The fivefold elevation in steady-state Bcl-2 concentration is not accompanied by detectable changes in the levels or cellular distributions of the related anti-apoptotic regulator Bcl-xL or of the proapoptotic regulators Bax and Bak and does not produce detectable effects on basal proliferation, differentiation, or death programs. The apoptotic response to ischemia-reperfusion is reduced twofold in the crypts and villi of transgenic mice compared with their normal littermates. These results suggest that both undifferentiated and differentiated cells undergo a commitment phase that is sensitive to Bcl-2. Forced expression of Bcl-2 also suppressed the p53-dependent death that occurs in proliferating crypt epithelial cells following gamma-irradiation. Thus suppressibility by Bcl-2 operationally defines a common feature of the apoptosis induced in the crypt epithelium by these two stimuli. (+info)
Cyclin D1 overexpression enhances radiation-induced apoptosis and radiosensitivity in a breast tumor cell line.
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Overexpression of cyclin D1, a G1 cell cycle regulator, is often found in many different tumor types, such as breast carcinoma and squamous cell carcinoma of the head and neck. The overexpression of this protein is, in several cases, associated with a poor prognosis. In this study, the effect of cyclin D1 on radiosensitivity was investigated in a breast tumor cell line, MCF7, containing a cyclin D1 gene construct under the control of a tetracycline-sensitive regulator. MCF7 cells cultured without tetracycline resulted in a 6-fold increase in the cyclin D1 protein. Cyclin D1-overexpressing MCF7 cells were more sensitive to ionizing radiation than the nonoverexpressing counterparts. The cyclin D1-overexpressing cells also exhibited a higher induction of apoptosis. Treatment with a dose of 5 Gy resulted in a rapid increase of p53 and p21 in the cyclin D1-overexpressing cells. Nonoverexpressing cells showed a more transient expression of these proteins after ionizing radiation. A pronounced G2-M block was observed in both cell lines. The cyclin D1-overexpressing cells were, however, released earlier from the block than the control cells. These data suggest that overexpression of cyclin D1 alters sensitivity toward ionizing radiation by modulating gamma-radiation-induced G2-M transition. (+info)
Functional interaction between retinoblastoma protein and stress-activated protein kinase in multiple myeloma cells.
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Previous studies have demonstrated that gamma-irradiation (IR)-induced apoptosis in multiple myeloma (MM) is associated with activation of stress-activated protein kinase (SAPK). In the present study, we examined the molecules downstream of SAPK/C-Jun N-terminal kinase (JNK), focusing on the role of retinoblastoma protein (Rb) during IR-induced MM cell apoptosis. The results demonstrate that IR activates SAPK/JNK, which associates with Rb both in vivo and in vitro. Far Western blot analysis confirms that SAPK/JNK binds directly to Rb. IR-activated SAPK/JNK phosphorylates Rb, and deletion of the phosphorylation site in the COOH terminus domain of Rb abrogates phosphorylation of Rb by SAPK/JNK. Taken together, our results suggest that Rb is a target protein of SAPK/JNK and that the association of SAPK/JNK and Rb mediates IR-induced apoptosis in MM cells. (+info)
Regulation of p53 expression by thymidylate synthase.
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Previous studies showed that thymidylate synthase (TS), as an RNA binding protein, regulates its own synthesis by impairing the translation of TS mRNA. In this report, we present evidence that p53 expression is affected in a similar manner by TS. For these studies, we used a TS-depleted human colon cancer HCT-C cell that had been transfected with either the human TS cDNA or the Escherichia coli TS gene. The level of p53 protein in transfected cells overexpressing human TS was significantly reduced when compared with its corresponding parent HCT-C cells. This suppression of p53 expression was the direct result of decreased translational efficiency of p53 mRNA. Similar results were obtained upon transfection of HCT-C cells with pcDNA 3.1 (+) containing the E. coli TS gene. These findings provide evidence that TS, from diverse species, specifically regulates p53 expression at the translational level. In addition, TS-overexpressing cells with suppressed levels of p53 are significantly impaired in their ability to arrest in G1 phase in response to exposure to a DNA-damaging agent such as gamma-irradiation. These studies provide support for the in vivo biological relevance of the interaction between TS and p53 mRNA and identify a molecular pathway for controlling p53 expression. (+info)
Phosphorylation of p53 protein in response to ionizing radiation occurs at multiple sites in both normal and DNA-PK deficient cells.
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The tumour suppressor gene product, p53, is involved in mediating cellular responses to DNA damage including growth arrest and/or apoptosis. The mechanism by which p53 protein senses the presence of damaged DNA is not understood. The possibility that p53 may be post-translationally modified by enzymes that are activated in response to DNA damage including DNA-dependent protein kinase (DNA-PK), poly(ADP-ribose) polymerase and stress activated protein kinase has received considerable attention. Recent studies have indicated that DNA-PK is not required for the transactivation or apoptosis-promoting activities of p53 protein. However, the possibility that other functions of p53 may be dependent on phosphorylation by DNA-PK has not been explored. Here we describe a series of experiments that compares the expression, function and phosphorylation status of p53 protein in normal and DNA-PK-deficient scid cells. While several novel p53 phosphoforms are generated in response to DNA damage in normal cells, the same phosphoforms are observed in scid cells. (+info)