Further characterisation of polyclonal antiserum for DNA photoproducts: the use of different labelled antigens to control its specificity

Mitchell, David L.; Haipek, Carrie A.; Clarkson, Judith M.

doi:10.1016/0167-8817(85)90002-1

Cited by 28 publications

(19 citation statements)

References 11 publications

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“…This property may reflect the occurrence of multiple photoproduct-specific excision enzymes, of which only one has reverted, or broad specificity in an excision enzyme complex (38) that has partially reverted allowing detection of only a few important lesions. The high immunogenicity of the (6-4) photoproduct supports the notion that this may be a more readily detectable lesion (20).…”

supporting

confidence: 57%

“…On the basis of this assay, the XP129 revertant and the parental XP12RO cell line both appear to be defective in the removal of cyclobutane dimers (Table 1), whereas the two normal cell lines removed about 60% of the initial number of dimers in 24 h. When repair of cyclobutane and (6-4)pyrimidinepyrimidone dimers was monitored by radioimmunoassays that specifically detect these lesions in DNA (18,20,21,36), slightly different results were obtained. Normal cells removed 50% of the cyclobutane dimers within 6 h of irradiation compared with none for XP12RO cells (Fig.…”

mentioning

confidence: 96%

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Unique DNA repair properties of a xeroderma pigmentosum revertant.

Cleaver¹,

Cortés²,

Lutze³

et al. 1987

Mol. Cell. Biol.

118

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A group A xeroderma pigmentosum revertant with normal sensitivity was created by chemical mutagenesis. It repaired (6-4) photoproducts normally but not pyrimidine dimers and had near normal levels of repair replication, sister chromatid exchange, and mutagenesis from UV light. The rate of UV-induced mutation in a shuttle vector, however, was as high as the rate in the parental xeroderma pigmentosum cell line.Xeroderma pigmentosum (XP) is a human recessive disorder that in the homozygote exhibits a large increase in skin cancer induced by sunlight (5). The sensitivity of XP cells in culture to UV light (254 nm) is 5-to 10-fold greater than nQrmal (1,15), and the cells fail to repair a wide range of radiation-and chemically induced DNA damage (5, 10, 27), including (5-5,6-6)cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts, measured by radioimmunoassay (4, 19-21) and enzymatic methods (7,13,14,44). The relative proportions of cyclobutane dimers to other photoproducts determined from their photoreactivation suggest that (6-4) photoproducts may account for as much as 30% of the total lesions (24), and they are highly mutagenic (2). One of the problems encountered in attempts to clone the XP gene results from the reversion of the XP phenotype to UV resistance (16,28,29); therefore, we decided to fully characterize revertants themselves.XP12RO (a simian virus 40-transformed group A cell line), GM637 (a simian virus 40-transformed normal cell line), and normal fibroblasts (HS27 and AG) were grown in Eagle minimal essential medium with fetal calf serum, penicillin, and streptomycin. The XP12RO cell line was cloned by choosing a single colony from among those that grew from a culture of 100 to 200 cells. Large cultures (107 to 108 cells) of XP12RO were exposed to ethyl methanesulfonate at a concentration of 1, 3, or 6 mM in culture medium for 16 h, after which the medium was replaced and the cultures were grown for 1 week. The cultures were subsequently transferred on regular occasions (approximately twice per week), based on the cell density. They were irradiated in phosphate-buffered saline with 1 to 3 J of UV light per m2 (254 nm; 1.3 J/m2 per s) at each transfer. After an accumulated dose of approximately 60 J/m2, the cultures were given a dose of 6.5 J/m2 and allowed to grow for 3 weeks to produce colonies. One colony was chosen for each ethyl methanesulfonate dose and grown into a large culture for subsequent analysis. Cell survival after UV irradiation was determined from the number of macroscopic colonies (>50 cells) formed in 21 days (J. E. Cleaver and G. H. Thomas, J. Invest. Dermatol., in press). Each of the cell lines derived from XP12RO proved to be resistant to UV light (Fig. 1A), and, accordingly, they were defined as revertants (designated XP129, XP322, and * Corresponding author. XP644). When similar-size cultures were not exposed to ethyl methanesulfonate, no surviving colonies were recovered. Revertants showed intermediate sensitivities to 4-nitroquinoline-1-oxide (4NQO) (Fig....

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supporting

confidence: 57%

mentioning

confidence: 96%

Unique DNA repair properties of a xeroderma pigmentosum revertant.

Cleaver¹,

Cortés²,

Lutze³

et al. 1987

Mol. Cell. Biol.

118

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“…Since Levine et al (17) first raised an antiserum against UV-irradiated single-stranded DNA and developed an immunological method for measuring DNA photoproducts using complement fixation in 1966, a number of other antisera have been prepared in many laboratories for DNA photoproduct measurement with some modifications (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). A polyvalent antiserum, however, may contain a heterogeneous population of antibodies that recognize various kinds of DNA damage (25,32,33). An immunological assay is an indirect method in which selective binding of an antibody to the corresponding antigen is essential and monoclonal antibodies are the most specific probes.…”

Section: Introductionmentioning

confidence: 99%

Quantitation and Visualization of Ultraviolet‐Induced DNA Damage Using Specific Antibodies: Application to Pigment Cell Biology

Kobayashi

Katsumi

Imoto

et al. 2001

Pigment Cell Research

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The major types of DNA damage induced by sunlight in the scope. This latter method allows us to reconstruct three-diskin are DNA photoproducts, such as cyclobutane pyrimidine mensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and hisdimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isotology in multilayered epidermis. Using those techniques, one mers of 6-4PPs. A sensitive method for quantitating and can determine the induction and repair of these three distinct visualizing each type of DNA photoproduct induced by biologtypes of DNA photoproducts in cultured cells and in the skin ically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological efexposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techfects. We have established monoclonal antibodies specific for niques and antibodies, we describe the DNA repair kinetics CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodpigmented cells and in human epidermis. ies with in situ laser cytometry to visualize and measure DNA

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“…However, the very rapid removal of these binding sites and the lack of correlation with the kinetics of CPD‐specific UV endonuclease‐sensitive sites (14) were not consistent with what was known about the behavior of the CPD. RIA of pyrimidine homopolymers and alternating copolymers irradiated under different UV conditions eventually showed that the predominant binding site of polyclonal antisera raised against UV‐DNA was, in all probability, the (6‐4) PD (6,16,17). This immune response is probably predicated on the fact that the helical distortion of the (6‐4) PD elicited a significantly greater immune response in rabbits compared to the CPD and dominated the affinity of most antisera (see below).…”

Section: Antibody Production and Characterizationmentioning

confidence: 99%

“…These results were not consistent with the known repair kinetics using biochemical and enzymatic techniques. Subsequently, it was discovered that polyclonal antisera raised against heavily UV‐irradiated DNA contained multiple antibody populations predominated by the pyrimidine (6‐4) pyrimidone dimer [(6‐4) PD] (6). Combined with immunoassays created specifically for the cyclobutane pyrimidine dimer (CPD) it was shown that the (6‐4) PD was repaired much more rapidly than the CPD in “repair‐deficient” rodent cells (7).…”

mentioning

confidence: 99%

Antibodies and DNA Photoproducts: Applications, Milestones and Reference Guide

Mitchell¹,

Brooks

2010

Photochem & Photobiology

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Polyclonal and monoclonal antibodies recognizing the primary DNA photoproducts induced by ultraviolet radiation (UVR) have proven to be essential tools in the study of photochemical and photobiological phenomena. As specific ''DNA damage binding proteins'' these reagents have been used to develop a diverse array of technical procedures applied to a plethora of important problems in DNA photochemistry and the biological effects of UVR at the molecular, developmental, organism and population levels. This survey attempts to cover this science from an historical perspective and to reveal the great breadth of discovery and contribution associated with the development and application of DNA damage antibodies to the current body of science.Three papers published in the 1960s were seminal to the development of immunoassays for the study of photochemical and photobiological phenomena. In 1960 Rosalyn Yalow and Solomon Berson published a paper in the Journal of Clinical Investigation entitled ''Immunoassay of endogenous plasma insulin in man'' (1), which described the development of a sensitive radioimmunoassay (RIA) to measure minute quantities of hormones in blood samples. This technique played a seminal role in the exponential expansion of research in medicine and the biological sciences over the past half century. In 1977 Dr. Yalow was awarded the Nobel Prize in Medicine for her work. In 1964 Otto Plescia and colleagues published a paper in the Proceedings of the National Academy of Sciences entitled ''Production of antibodies to denatured deoxyribonucleic acid (DNA)'' (2). Using a technique whereby singlestranded DNA was electrostatically coupled to methylated bovine serum albumin they were able to elicit an immune response in rabbits. In the wake of this paper it was not long before a group led by Lawrence Levine and Helen van Vanukis published an article in Science in 1966 entitled ''Antibodies to photoproducts of deoxyribonucleic acids irradiated with ultraviolet light'' (3). Using the technique established by Plescia, this group was able to produce a rabbit antiserum using DNA irradiated with 270 nm light with antigenic determinants that were reversible by 235 nm light and responsive to UV-irradiated thymidine nucleotides. It appeared that antibodies had been created in response to thymine dimers.Over the course of the following two decades a number of polyclonal and monoclonal antibodies to various UV-induced DNA lesions were produced and characterized. The technology that developed blossomed and has been applied to a broad spectrum of problems in photochemistry, the biological response to UV radiation (UVR), photomedicine and environmental photobiology. Early immunoassays generated DNA repair curves showing rapid removal of most antibody binding sites leaving behind a significant remnant (4,5). These results were not consistent with the known repair kinetics using biochemical and enzymatic techniques. Subsequently, it was discovered that polyclonal antisera raised against heavily UVirradiated DNA contai...

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Further characterisation of polyclonal antiserum for DNA photoproducts: the use of different labelled antigens to control its specificity

Cited by 28 publications

References 11 publications

Unique DNA repair properties of a xeroderma pigmentosum revertant.

Unique DNA repair properties of a xeroderma pigmentosum revertant.

Quantitation and Visualization of Ultraviolet‐Induced DNA Damage Using Specific Antibodies: Application to Pigment Cell Biology

Antibodies and DNA Photoproducts: Applications, Milestones and Reference Guide

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