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  • 1
    Electronic Resource
    Electronic Resource
    Doxorubicin and γ rays increase the level of DNA topoisomerase IIα in nuclei of normal and xeroderma pigmentosum fibroblasts (1998)
    Springer
    Journal of cancer research and clinical oncology 124 (1998), S. 355-366 
    Details
    ISSN: 1432-1335
    Keywords: Key words Immunohistochemistry ; DNA repair ; Radiation-inducible response ; Apoptosis ; p16 ; Bax protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract DNA topoisomerase IIα was monitored with the monoclonal antibody Ki-S1 in human fibroblasts after irradiation of cells with γ rays from a 137Cs source or treatment with the DNA topoisomerase II inhibitor doxorubicin. DNA topoisomerase IIα was localized immunohistochemically as bright fluorescent dots in the karyoplasm. The fibroblasts investigated originated from normal human donors and a xeroderma pigmentosum (XP) patient (XP12BE). All cell lines examined showed a time- and dose-dependent increase in DNA topoisomerase IIα abundance after irradiation or treatment with doxorubicin. No principal difference in response was seen between normal and XP fibroblasts towards either treatment alone. After irradiation with 9 Gy, the effect was detectable after as little as 30 min and lasted for at least 6 h. After doxorubicin treatment, topoisomerase II overexpression occurred within less than 2 h. It passed through a maximum and began to decrease after approximately 6 h. In principle, the increase in DNA topoisomerase IIα may result from (i) architectural changes of interphase chromatin leading to enhanced accessibility of preformed enzyme to the antibody, (ii) enhanced gene expression, or (iii) enhanced stabilization of mRNA or protein molecules. The increase in enzyme levels may be part of the well-known DNA damage responses that operate in cell-protective or DNA-reparative pathways. Thus, the action of DNA topoisomerase II would serve to catalyze preparatory steps in DNA repair. We also found overexpression of the Bax protein and p16 predominantly in treated XP cells, suggesting that the DNA-damaging protocols elicited signals for apoptosis and cell-cycle arrest. From the simultaneous increase in DNA topoisomerase IIα and Bax, one may conclude that DNA topoisomerase IIα also plays role in apoptosis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004320050184
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  • 2
    Electronic Resource
    Electronic Resource
    Subnuclear distribution of DNA topoisomerase I and Bax protein in normal and xeroderma pigmentosum fibroblasts after irradiation with UV light and c rays or treatment with topotecan (1999)
    Springer
    Journal of cancer research and clinical oncology 125 (1999), S. 193-208 
    Details
    ISSN: 1432-1335
    Keywords: Key words Immunohistochemistry ; DNA repair ; Radiation-inducible response ; Apoptosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Immunohistochemical methods were used to determine abundance and subnuclear distribution of DNA topoisomerase I and the Bax protein in normal and excision-repair-deficient xeroderma pigmentosum (XP) fibroblasts after irradiation of cells with γ rays or UV light, or exposure to the topoisomerase I inhibitor topotecan. DNA topoisomerase I and Bax were monitored using antisera raised against the human proteins. In addition, topoisomerases IIα and IIβ were made visible with specific antibodies. In untreated cells, DNA topoisomerase I was found to occur in the cytoplasm and in nucleoli. Irradiation with γ rays (2–12 Gy) or UV light (0.3–1.2 mW/cm2) changed the staining pattern in nuclei such that a multitude of small topoisomerase-I-rich centers occurred, which were evenly distributed over the karyoplasm. Simultaneously nucleoli disintegrated. Treatment of fibroblasts with topotecan (6–100 μM concentrations) resulted in similar alterations although the changes were much more pronounced. Combinations of topotecan and γ irradiation caused additive effects. We conclude that the increase in the number of topoisomerase-I-positive spots and the high fluorescence intensity of the latter may reflect three biological processes: (i) enhanced transcriptional activity (e.g. of DNA damage response genes), (ii) tagging of damaged DNA sites for repair, or (iii) initiation of apoptosis. In separate assays using normal and XP cells, a dose-dependent increase in protein reacting with Bax antibody was observed in nuclei, following treatment with γ rays or topotecan. In addition, topotecan induced a netlike arrangement of this Bax protein in nuclei. The meshes of the net structure resembled vesicles. DNA staining with 4′,6-diamidino-2-phenylindole dihydrochloride revealed that the vesicle-type structures contained DNA. Upon further incubation with topotecan, cells showing the netlike Bax arrangement eventually died. We conclude that topotecan-induced changes made visible by nuclear Bax protein are associated with apoptosis. XP cells, when treated with topotecan, responded more readily than normal cells with both an increase in nuclear Bax protein and rearrangement of Bax, indicating that UV repair functions may be required to process DNA damage inflicted by topotecan. Monitoring of DNA topoisomerases IIα and IIβ in γ-irradiated cells with antibodies revealed a dramatic increase in the IIα form and a redistribution of the IIβ form representing fragmentation of nucleoli.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004320050263
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  • 3
    Electronic Resource
    Electronic Resource
    Xeroderma pigmentosum patients from the federal Republic of Germany: Decrease in post-UV colony-forming ability in 30 xeroderma pigmentosum fibroblast strains is quantitatively correlated with a decrease in DNA-incising capacity (1985)
    Springer
    Journal of cancer research and clinical oncology 109 (1985), S. 227-240 
    Details
    ISSN: 1432-1335
    Keywords: DNA repair ; Xeroderma pigmentosum ; Colony-forming ability ; Alkaline elution ; DNA ; incising capacity ; UV light ; N-acetoxy-2-acetylaminofluorene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A total of 16 normal and 46 XP fibroblast strains from the Mannheim Collection were investigated for colony-forming ability following exposure to both UV light and the “UV-like” carcinogen (Ac)2ONFln. The dose-response experiments included up to 13 dose levels. The exponential segments of the curves were analysed by linear regression and the negative reciprocal of the regression coefficient (D0) was calculated for each cell strain. For quantitating the DNA-incising capacity, DNA elution curves were determined at several UV dose levels. Plotting the initial velocities of the elution curves versus the UV dose yielded a regression line, the slope of which was used to obtain the characteristic value E0. Comparing D0 with E0 values showed that cell strains in which colony-forming ability was reduced suffered a reduction of DNA-incising capacity of the same magnitude. There were only 3 exceptional strains in which reduction of DNA-incising capacity was less pronounced than reduction of colony-forming ability. We have previouly shown (Fischer et al. 1982) that D0 values from 27 XP strains of the Mannheim Collection were correlated with clinical symptoms. This correlation is now being extended by relating colony-forming ability to the magnitude of the DNA incision defect. From our data we conclude that the best quantitative biochemical denominator to explain the sun sensitivity of XP is that of a defective incision of UV-damaged DNA. A considerable similarity in sensitivity towards both UV light and (Ac)2ONFln was found in 16 normal and 46 XP strains. This seems to indicate that UV-and (Ac)2ONFln-induced DNA damage are removed to a large extent by the same pathways in human fibroblasts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00390362
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  • 4
    Electronic Resource
    Electronic Resource
    Expression of mitochondrial genes and DNA-repair-related nuclear genes is altered in xeroderma pigmentosum fibroblasts (1994)
    Springer
    Journal of cancer research and clinical oncology 120 (1994), S. 454-464 
    Details
    ISSN: 1432-1335
    Keywords: Xeroderma pigmentosum fibroblasts ; DNA repair ; Recombinant DNA ; cDNA libraries ; Phage λgt10 ; pBluescript vector ; In vitro transcripts ; Differential hybridization ; Mitochondrial neuromyopathies ; Genetic defects ; Uracil-DNA glycosylase ; Glyceraldehyde-3-phosphate dehydrogenase ; Lactate dehydrogenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Differential hybridization was used to detect repair defects in xeroderma pigmentosum (XP) that are not amenable to current analyses. cDNA libraries were constructed from cytoplasmic RNA of normal and XP fibroblast strains (complementation groups A and D) and analyzed for differential gene expression. More than 40000 λgt10 cDNA clones were differentially screened with in vitro transcripts made from cDNA in the pBluescript vector. Six differential clones were detected in the libraries of the XP group A and D strains which caused stronger or weaker signals when probed with transcripts from XP strains than with those from the normal strains. Two clones coded for mitochondrial genes: mitochondrial 16 S rRNA and ATPase 6L. Overexpression of mitochondrial genes in XP may indicate that functions of the ATP-generating system are impaired since such functions are intensified whenever they become insufficient, for example as a consequence of DNA damage. It is tempting to assume that abnormal mitochondria are one of the causes for the neurological malfunctions in XP. Furthermore, densitometric analysis of Northern blots revealed that mRNA of lactate dehydrogenase, chain M, was less abundant in four XP group A strains (extent of reduction: 70%) and in two XP group D strains (extent of reduction: 58%). Enzyme activity was also diminished. In addition, mRNA of the gene for glyceraldehyde-3-phosphate dehydrogenase was less expressed in the same XP group A and D fibroblast strains investigated (reduction in both complementation groups: 50%). Both glycolytic enzymes have nuclear functions apart from their role in sugar metabolism. Lactate dehydrogenase, chain M, is identical to a helix-destabilizing protein; it is closely associated with chromatin and unfolded DNA, suggesting a role in DNA synthesis and transcription. The 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase is involved in transcription and was shown to be identical to uracil-DNA glycosylase, a base-excision repair enzyme. We presume that the nuclear functions of these glycolytic enzymes may be thwarted in the XP strains investigated and may account for malfunctions in XP, particularly for neurological disturbances.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01191798
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