Investigation of discordant sibling pairs from hereditary breast cancer families and analysis of a rare PMS1 variant.

BACKGROUND: It is likely that additional genes for hereditary breast cancer can be identified using a discordant sib pair design. Using this design we identified individuals harboring a rare PMS1 c.605G>A variant previously predicted to result in loss of function. OBJECTIVES: A family-based design and predictive algorithms were used to prioritize candidate variants possibly associated with an increased risk of hereditary breast cancer. Functional analyses were performed for one of the candidate variants, PMS1 c.605G>A. METHODS: 1) 14 discordant sister-pairs from hereditary breast cancer families were identified. 2) Whole exome sequencing was performed and candidate risk variants identified. 3) A rare PMS variant was identified in 2 unrelated affected sisters but no unaffected siblings. 4) Functional analysis of this variant was carried out using targeted mRNA sequencing. RESULTS: Genotype-phenotype correlation did not demonstrate tracking of the variant with cancer in the family. Functional analysis revealed no difference in exon 6 incorporation, which was validated by analyzing PMS1 allele specific expression. CONCLUSIONS: The PMS1 c.605G>A variant did not segregate with disease, and there was no variant-dependent impact on PMS1 exon 6 splicing, supporting this variant is likely benign. Functional analyses are imperative to understanding the clinical significance of predictive algorithms.

Periprosthetic fractures of the distal femur after total knee arthroplasty : Plate versus nail fixation.

The incidence of periprosthetic fractures about a TKA is increasing. Traditionally, these fractures are classified by their location and prosthesis integrity. In the setting of a supracondyar fracture about a well-fixed prosthesis, both plate and nail fixation of the fracture present themselves as options, each with unique benefits and pitfalls. Through review and discussion of the literature, we aim to describe some of the patient, fracture, and implant related factors that should be considered when planning fixation of periprosthetic fractures about a TKA. Additionally, we present several technical pearls that may be useful in the successful treatment of these difficult injuries. LEVEL OF EVIDENCE: 4.

Human cells contain a factor that facilitates the DNA glycosylase-mediated excision of oxidized bases from occluded sites in nucleosomes.

Reactive oxygen species generate some 20,000 base lesions per human cell per day. The vast majority of these potentially mutagenic or cytotoxic lesions are subject to base excision repair (BER). Although chromatin remodelers have been shown to enhance the excision of oxidized bases from nucleosomes in vitro, it is not clear that they are recruited to and act at sites of BER in vivo. To test the hypothesis that cells possess factors that enhance BER in chromatin, we assessed the capacity of nuclear extracts from human cells to excise thymine glycol (Tg) lesions from exogenously added, model nucleosomes. The DNA glycosylase NTHL1 in these extracts was able to excise Tg from both naked DNA and sites in nucleosomes that earlier studies had shown to be sterically accessible. However, the same extracts were able to excise lesions from sterically-occluded sites in nucleosomes only after the addition of Mg2+/ATP. Gel mobility shift assays indicated that nucleosomes remain largely intact following the Mg2+/ATP -dependent excision reaction. Size exclusion chromatography indicated that the NTHL1-stimulating activity has a relatively low molecular weight, close to that of NTHL1 and other BER glycosylases; column fractions that contained the very large chromatin remodeling complexes did not exhibit this same stimulatory activity. These results indicate that cells possess a factor(s) that promotes the initiation of BER in chromatin, but differs from most known chromatin remodeling complexes.

The genes encoding endonuclease VIII and endonuclease III in Escherichia coli are transcribed as the terminal genes in operons.

Escherichia coli endonuclease VIII and endo-nuclease III are oxidative base excision repair DNA glycosylases that remove oxidized pyrimidines from DNA. The genes encoding these proteins, nei and nth, are both co-transcribed as the terminal genes in operons. nei is the terminal gene in an operon with four open reading frames that encode proteins of unknown function. This operon has two confirmed transcription initiation sites upstream of the first open reading frame and two transcript termination sites downstream of nei. nth is the terminal gene in an operon with seven open reading frames that encode proteins of unknown function. The six open reading frames immediately upstream of nth show homology to the genes rnfA, rnfB, rnfC, rnfD, rnfG and rnfE from Rhodobacter capsulatis. The rnf genes are required for nitrogen fixation in R.capsulatis and have been predicted to make up a membrane complex involved in electron transport to nitrogenase. The nth operon has transcription initiation sites upstream of the first and second open reading frames and a single transcript termination site downstream of nth. The order of genes in these operons has been conserved or partially conserved in other bacteria, although it is not known whether the genes are co-transcribed in these other organisms.

SOS processing of unique oxidative DNA damages in Escherichia coli.

phi X174 replicative form (RF) I transfecting DNA containing thymine glycols (5,6-dihydroxy-5,6-dihydrothymine), urea glycosides or apurinic (AP) sites was used to study SOS processing of unique DNA damages in Escherichia coli. All three lesions can be found in DNA damaged by chemical oxidants or radiation and are representative of several common structural modifications of DNA bases. When phi X DNA containing thymine glycols was transfected into host cells that were ultraviolet-irradiated to induce the SOS response, a substantial increase in survival was observed compared to transfection into uninduced hosts. Studies with mutants demonstrated that both the activated form of RecA and UmuDC proteins were required for this reactivation. In contrast, no increase in survival was observed when DNA containing urea glycosides or AP sites was transfected into ultraviolet-induced hosts. These data suggest that SOS-induced reactivation does not reflect a generalized repair system for all replication-blocking, lethal lesions but rather that the efficiency of reactivation is damage dependent. Further, we found that a significant fraction of potentially lethal thymine glycols could be ultraviolet-reactivated in an umuC lexA recA-independent manner, suggesting the existence of an as yet uncharacterized damage-inducible SOS-independent mode of thymine glycol repair.

In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites.

When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS). In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed. The single strand break, a nucleotide gap containing 3' and 5' phosphate groups, was positioned one, three or six nucleotides 5' or 3' to the damage in the complementary DNA strand. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5' or 3' on the opposing strand. When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg. Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5' or 3' to the rAP site. To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3' phosphate termini, insert the "missing" nucleotide, and ligate the nicks, respectively. In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand. However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG. Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced. Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away. Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break.

Pyrimidine ring fragmentation products. Effects of lesion structure and sequence context on mutagenesis.

Free radicals produce a broad spectrum of damages to DNA, a major proportion of which includes ring fragmentation and contraction products of DNA bases as well as abasic sites. In this study, the mutagenic potential fo two pyrimidine ring fragmentation products, urea and beta-ureidoisobutyric acid (UBA), was analyzed using the i-d region of the Escherichia coli lacI gene contained in a single-stranded f1-K12 phage hybrid vector. Single-stranded DNA was used so that the in vivo interactions between the damage and the DNA polymerase could be assessed in the absence of excision repair. The i-d region contains 20 mutable thymine sites so that 20 separate sequence contexts containing a unique lesion at a position of thymine can be analyzed simultaneously. Urea and UBA residues were uniquely introduced into f1-K12 DNA by chemical and enzymatic methods and primer extension and piperidine analysis of the damage-containing template molecules demonstrated that the potential mutable thymine sites contained randomly distributed lesions. Both fragmentation products were poorly bypassed by DNA polymerases in vitro and in the cell; although in the presence of SOS-induction, UBA was bypassed more efficiently than urea. UBA was a potent premutagenic lesion with a rate of mutation induction more than sixfold above that observed with abasic sites derived from purines. Urea residues were about as mutagenic as abasic sites derived from purines, which in turn were more mutagenic than abasic sites derived from thymine. Mutations derived from urea, UBA and abasic sites were all dependent on SOS-induction of the host cells. Since both urea and UBA were derived from DNA thymine, these data demonstrate that adenine is not routinely inserted opposite products that no longer retain the structural integrity of the pyrimidine ring. Sequence analysis showed that the mutations were targeted at thymine with 62% of the urea-derived mutations being T to C transitions and 62% of the UBA derived mutations being T to A transversions. Thus, the two fragmentation products appeared to direct specific misinsertions. The mutations were not randomly distributed over the i-d region for either fragmentation product and hotspots were observed for both damages. The presence of hotspots suggests that in addition to lesion structure, sequence context plays an important role in base selectivity by DNA polymerases opposite DNA lesions. Energy minimization calculations were used to model the urea and UBA lesions at two contrasting hotspot sites. In both cases, there was significant agreement between the computational and biological data sets.

In vitro selection of sequence contexts which enhance bypass of abasic sites and tetrahydrofuran by T4 DNA polymerase holoenzyme.

The influence of sequence context on the ability of DNA polymerase to bypass sites of base loss was addressed using an in vitro selection system. Oligonucleotides containing either an aldehydic abasic site or tetrahydrofuran surrounded by four randomized bases on both the 5' and 3' sides were used as templates for synthesis by phage T4 DNA polymerase holoenzyme proficient or deficient in the 3'-->5' proofreading exonuclease activity. Successful bypass products were purified, subcloned and the sequences of approximately 100 subclones were determined for each of the four polymerase/lesion combinations tested. Between 7 and 19 % of the bypass products contained deletions of one to three nucleotides in the randomized region. In bypass products not containing deletions, biases for and against certain nucleotides were readily noticeable across the entire randomized region. Template strands from successful bypass products of abasic sites had a high frequency of T in most of the randomized positions, while those from bypass products of tetrahydrofuran had a high frequency of G at the positions immediately to the 3' and 5' side of the lesion. Consensus sequences were shared by successful bypass products of the same lesion but not between bypass products of the two lesions. The consensus sequence for efficient bypass of tetrahydrofuran was over-represented in several frames relative to the lesion. T4 DNA polymerase inserted A opposite abasic sites 63 % of the time in the presence of proofreading and 79 % of the time in its absence, followed by G>T>C, while the insertion of A opposite tetrahydrofuran ranged between 93 % and 100 % in the presence and absence of proofreading, respectively. Finally, sequence context influenced the choice of nucleotide inserted opposite abasic sites and consensus sequences which favored the incorporation of nucleotides other than A were defined.

Fabs specific for 8-oxoguanine: control of DNA binding.

Free radicals produce a broad spectrum of DNA base modifications including 7,8-dihydro-8-oxoguanine (8-oxoG). Since free radicals have been implicated in many pathologies and in aging, 8-oxoG has become a benchmark for factors that influence free radical production. Fab g37 is a monoclonal antibody that was isolated by phage display in an effort to create a reagent for detecting 8-oxoG in DNA. Although this antibody exhibited a high degree of specificity for the 8-oxoG base, it did not appear to recognize 8-oxoG when present in DNA. Fab g37 was modified using HCDR1 and HCDR2 segment shuffling and light chain shuffling. Fab 166 and Fab 366 which bound to 8-oxoG in single-stranded DNA were isolated. Fab 166 binds more selectively to single-stranded oligonucleotides containing 8-oxoG versus control oligonucleotides than does Fab 366 which binds DNA with reduced dependency on 8-oxoG. Numerous other clones were also isolated and characterized that contained a spectrum of specificities for 8-oxoG and for DNA. Analysis of the primary sequences of these clones and comparison with their binding properties suggested the importance of different complementarity determining regions and residues in determining the observed binding phenotypes. Subsequent chain shuffling experiments demonstrated that mutation of SerH53 to ArgH53 in the Fab g37 heavy chain slightly decreased the Fab's affinity for 8-oxoG but significantly improved its binding to DNA in an 8-oxoG-dependent manner. The light chain shuffling experiments also demonstrated that numerous promiscuous light chains could enhance DNA binding when paired with either the Fab g37 or Fab 166 heavy chains; however, only the Fab 166 light chain did so in an additive manner when combined with the Fab 166 heavy chain that contains ArgH53. A three-point model for Fab 166 binding to oligonucleotides containing 8-oxoG is proposed. We describe a successful attempt to generate a desired antibody specificity, which was not present in the animal's original immune response.

Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions.

Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is a base damage common to oxidative mutagens and the major stable radiolysis product of thymine in DNA. We assessed the mutagenic potential of thymine glycols in single-stranded bacteriophage DNA during transfection of Escherichia coli wild-type and umuC strains. cis-Thymine glycols were induced in DNA by reaction with the chemical oxidant, osmium tetroxide (OsO4); modification of thymines was quantitated by using anti-thymine glycol antibody. Inactivation of transfecting molecules showed that one lethal hit corresponded to 1.5 to 2.1 thymine glycols per phage DNA in normal cells, whereas conditions of W-reactivation (SOS induction) reversed 60 to 80% of inactivating events. Forward mutations in the lacI and lacZ' (alpha) genes of f1 and M13 hybrid phage DNAs were induced in OsO4-treated DNA in a dose-dependent manner, in both wild-type and umuC cells. Sequence analysis of hybrid phage mutants revealed that mutations occurred preferentially at cytosine sites rather than thymine sites, indicating that thymine glycols were not the principal pre-mutagenic lesions in the single-stranded DNA. A mutagenic specificity for C----T transitions was confirmed by OsO4-induced reversion of mutant lac phage. Pathways for mutagenesis at derivatives of oxidized cytosine are discussed.

Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy.

The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. We describe a multifluorescence technique to detect the localization of 8-oxoG in both nuclear and mitochondrial DNA using a mouse recombinant Fab 166. The Fab was generated by repertoire cloning and combinatorial phage display, and specifically recognized 8-oxoG in DNA, as determined by competitive enzyme-linked immunosorbent assays (ELISAs). In situ detection of 8-oxoG was accomplished using rat lung epithelial (RLE) cells and human B lymphoblastoid (TK6) cells treated with hydrogen peroxide (H(2)O(2)) or ionizing radiation, respectively. Using confocal scanning laser microscopy, we observed nuclear and perinuclear immunoreactivity of 8-oxoG in control cultures. The simultaneous use of a nuclear DNA stain, propidium iodide, or the mitochondrial dye, MitoTracker (Molecular Probes, Eugene, OR, USA), confirmed that 8-oxoG immunofluorescence occurred in nuclear and mitochondrial DNA. Marked increases in the presence of 8-oxoG in nuclear DNA were apparent after treatment with H(2)O(2) or ionizing radiation. In control experiments, Fab 166 was incubated with 200 microM purified 8-oxodG or with formamidopyrimidine DNA-glycosylase (Fpg) to remove 8-oxoG lesions in DNA. These protocols attenuated both nuclear and mitochondrial staining. We conclude that both nuclear and mitochondrial oxidative DNA damages can be simultaneously detected in situ using immunofluorescence labeling with Fab 166 and confocal microscopy.

Enzymatic processing of radiation-induced free radical damage in DNA.

A significant fraction of DNA damage produced by ionizing radiation comes from free radicals generated during the radiolysis of water, that is, by indirect effects. The hydroxyl radical, the principal damaging species, produces single-strand breaks and a plethora of base and sugar lesions that can be cytotoxic or mutagenic. Free radical-induced DNA damage is repaired by an efficient and ubiquitous process called "base excision repair" which is composed of either three or four enzymatic steps, depending on the initial lesion. The result is an intact DNA molecule with a short repair patch size. If, however, multiply damaged sites similar to those produced by ionizing radiation are processed by base excision repair, a double-strand break can result if the opposing lesions are more than three nucleotides apart. Because base excision repair evolved to protect the genome from endogenous damages, the proteins involved are highly conserved from bacteria to humans, not only at the functional level, but at the level of amino acid sequence.

Radiation-induced and transposon-induced chromosome damage in Drosophila: translocations and transmission distortion.

The possible interaction between X-ray- and transposon-induced chromosome damage was monitored in the P-M system of hybrid dysgenesis in Drosophila melanogaster. One- to two-day-old F1 dysgenic males originating from a cross between M strain females and P strain males were irradiated with 5.5 Gy (550 rad) or used as controls to monitor X-Y translocations and transmission ratio distortion. Two 3-day sperm broods were sampled for the former and two 4-day broods for the latter to detect damage induced in the most radiosensitive cells. F1 nondysgenic males derived from M female to M male crosses (controls) were treated identically. X-Y chromosome translocations induced by P element mobility alone declined sharply with a decrease in temperature (18 versus 21 degrees C) and they were significantly reduced with aging of hybrid males from brood 2, 4-8 days of age, to brood 3, 7-11 days of age. No significant increase in translocations was observed when X irradiation and P-M dysgenesis were combined, showing no interaction between damages induced by the two mutator systems. In contrast, interaction was observed in transmission ratio distortion which was significantly increased by X irradiation of hybrid males derived from both reciprocal M X P and P x M crosses. The preferential elimination of P element-bearing autosomes occurred when either spermatocytes or spermatids were irradiated. An aging effect was also observed, resulting in less distortion in 9- to 14-day-old dysgenic males compared to 5- to 10-day-old hybrids.

Processing of model single-strand breaks in phi X-174 RF transfecting DNA by Escherichia coli.

The inactivation efficiency and repair of single-strand breaks was investigated using model strand breaks created by endonucleolytic incision of damaged DNA. Phi X-174 duplex transfecting DNA containing either thymine glycols, urea residues, or abasic (AP) sites was incubated with AP endonucleases that produce breaks on the 3' side, the 5' side, or both sides of the lesion. For each lesion, incubation with Escherichia coli endonuclease III results in a single-strand break containing a 3' alpha, beta-unsaturated aldehyde (4-hydroxy-2-pentenal), while treatment of AP- or urea-containing DNA with E. coli endonuclease IV results in a single-strand break containing a 5' deoxyribose or a 5' deoxyribosylurea moiety, respectively. Incubation of lesion-containing DNA with both enzymes results in a base gap. Ligatable nicks containing 3' hydroxyl and 5' phosphate moieties were produced by subjecting undamaged DNA to DNase I. When the biological activity of these DNAs was assessed in wild-type cells, ligatable nicks were not lethal, but each of the other strand breaks tested was lethal, having inactivation efficiencies between 0.12 and 0.14. These inactivation efficiencies are similar to those of the base lesions from which the strand breaks were derived. In keeping with the current model of base excision repair, when phi X duplex DNA containing strand breaks with a blocked 3' terminus was transfected into an E. coli double mutant lacking the major 5' cellular AP endonucleases, a greater than twofold decrease in survival was observed. Moreover, when this DNA was treated with a 5' AP endonuclease prior to transfection, the survival returned to that of wild type. As expected, when DNA containing strand breaks with a 5' blocked terminus or DNA containing base gaps was transfected into the double mutant lacking 5' AP endonucleases, the survival was the same as in wild-type cells. The decreased survival of transfecting DNA containing thymine glycols, urea, or AP sites observed in appropriate base excision repair-defective mutants was also obviated if the DNA was incubated with the homologous enzyme prior to transfection. Thus, in every case, with both base lesions and single-strand breaks, the lesion was repaired in the cell by the enzyme that recognizes it in vitro. Furthermore, the repair step in the cell could be eliminated if the appropriate enzyme was added in vitro prior to transfection.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiation-induced DNA base damage detected in individual aerobic and hypoxic cells with endonuclease III and formamidopyrimidine-glycosylase.

X-ray-induced DNA base damage can be detected using endonuclease III and formamidopyrimidine-glycosylase, which create DNA strand breaks at enzyme-sensitive sites. Strand breaks can then be measured with excellent sensitivity using the alkaline comet assay, a single-cell gel electrophoresis method that detects DNA damage in individual cells. In using this approach to measure the oxygen enhancement ratio (OER) for radiation-induced base damage, we observed that the number of enzyme-sensitive sites increased with dose up to 4 Gy in air and 12 Gy in hypoxic WIL2NS cells. After rejoining of radiation-induced strand breaks, base damage was detected more easily after higher doses. The number of radiation-induced enzyme-sensitive sites was similar under both air and nitrogen. Base damage produced by hydrogen peroxide and 4-nitroquinoline-N-oxide (4NQO) was also measured. Results with hydrogen peroxide (20 min at 4 degrees C) were similar to those observed for X rays, indicating that enzyme-sensitive sites could be detected most efficiently when few direct strand breaks were present. Removing DNA-associated proteins before irradiation did not affect the ability to detect base damage. Base damage produced by 4NQO (30 min at 37 degrees C) was readily apparent after treatment with low concentrations of the drug when few 4NQO-induced strand breaks were present, but the detection sensitivity decreased rapidly as direct strand breaks increased after treatment with higher concentrations. We conclude that: (1) the OER for base damage is approximately 1.0, and (2) the presence of direct DNA strand breaks (>2000-4000 per cell) prevents accurate detection of base damage measured as enzyme-sensitive sites with the alkaline comet method.

Expression of the oxidative base excision repair enzymes is not induced in TK6 human lymphoblastoid cells after low doses of ionizing radiation.

Most of the DNA damage produced by ionizing radiation is repaired by the base excision repair (BER) pathway. To determine whether the BER genes were up-regulated by low doses of ionizing radiation, we investigated their expression in TK6 human lymphoblastoid cells by measuring mRNA levels using real-time quantitative PCR. No induction at the transcriptional level of any of the base excision repair genes, NTH1 (NTHL1), OGG1, NEIL1, NEIL2, NEIL3, APE1, POLB, or accessory protein genes, LIG3, XRCC1 or XPG, was found at gamma-radiation doses ranging from 1 cGy to 2 Gy in a 24-h period. As has been measured in other cell lines, a dose-dependent induction of CDKN1A (WAF1) mRNA levels was observed in TK6 cells in the dose range of 0.5 to 2.0 Gy. We also examined BER enzyme activity on 8-oxoguanine-, dihydrouracil- and furan-containing oligonucleotide substrates and found no increase in extracts of TK6 cells after gamma-ray doses of 0.5-2.0 Gy. These data were corroborated by Western blot analysis of APE1 and NTH1, suggesting that the BER enzymes are also not up-regulated at the post-transcriptional level after ionizing radiation exposure.

Immunochemical quantitation of thymine glycol in oxidized and X-irradiated DNA.

The present study demonstrates the usefulness of immunochemical assays for quantitating modified bases in oxidized and X-irradiated DNA. Escherichia coli, phi X174 RF I, PM2, and M13 DNA containing thymine glycols introduced by OsO4 oxidation were used as antigens in a direct enzyme-linked immunosorbent assay (ELISA). The number of thymine glycols per DNA molecule was determined by reactivity with antithymine glycol antibody standardized either to the acetol fragment assay or to the number of Escherichia coli endonuclease III-sensitive sites. The number of thymine glycols was also determined in phi X174 RF I DNA X-irradiated in either phosphate or Tris buffer under air. Using a direct ELISA with phi X174 RF I DNA irradiated in a phosphate buffer solution, the anti-thymine glycol antibody detected damage at the level of 40 Gy. The immunochemical assay was sensitive, specific, quantitative, and independent of DNA structure.

Molecular Biology to Radiation Oncology: A Model for Translational Research? Opportunities in basic and translational research. From a workshop sponsored by the National Cancer Institute, Radiation Research Program, January 26-28, 1997, Bethesda, Maryland.

Many exciting discoveries are being made that are providing new insights into how molecules, cells and tissues respond to ionizing radiation. There remains a need, however, to translate these findings into more effective treatments for cancer patients, including those treated with radiation therapy. This complex task will require the collaboration of scientists studying molecular, cellular and tissue responses, and those performing clinical trials of emerging therapies. The Radiation Research Program of the National Cancer Institute sponsored a workshop entitled "Molecular Biology to Radiation Oncology: A Model for Translational Research?" to bring together basic scientists and clinicians to exchange ideas and fundamental concepts and to identify opportunities for future research and collaboration. Four broad topics were addressed: signal transduction and apoptosis, the cell cycle, repair of radiation damage, and the microenvironment. The development, selection and use of appropriate experimental models is crucial to finding and developing new therapies, and opportunities exist in this area as well. This paper and the accompanying paper by Coleman and Harris that provides the viewpoint of radiation oncologists (Radiat. Res. 150, 134-147, 1998) summarize the background concepts and opportunities for translational research identified by the workshop participants.

Characterization of a monoclonal antibody to thymidine glycol monophosphate.

A monoclonal antibody specific for thymine glycol (TG) in irradiated or OsO4-treated DNA was obtained by immunizing with thymidine glycol monophosphate (TMP-glycol) conjugated to bovine serum albumin by a carbodiimide procedure. Screening by dot-immunobinding and enzyme-linked immunosorbant assay (ELISA) procedures gave eight clones that bound OsO4- treated DNA. One of them, 2.6F.6B.6C, an IgG2a kappa, was characterized further. Hapten inhibition studies with OsO4-treated DNA showed that the antibody was specific for TMP-glycol. Among the various inhibitors tested, inhibition was in the order TMP-glycol greater than 5,6-dihydrothymidine phosphate greater than TMP greater than thymidine glycol greater than TG. Inhibition by 5,6-dihydrothymidine, thymidine, thymine, AMP, and CMP was negligible. In OsO4-treated DNA, as few as 0.5 TG per 10,000 bp were detectable by direct ELISA. Inhibition assays could detect as few as 1.5 TG per 10,000 bp. The antibody was equally reactive with native or denatured DNA containing TG. Among the X-irradiated homopolymers dC, dA, dG, and dT, only dT reacted with the antibody. Using an ELISA, the antibody could detect damage in irradiated DNA at the level of 20 Gy. Thus the antibody is of potential use in assays for DNA damage caused by X rays or other agents that damage DNA by free radical interactions.

Properties of antibodies to thymine glycol, a product of the radiolysis of DNA.

Anti-thymine glycol antibodies were elicited by immunizing rabbits with thymine glycol monophosphate (TMP-glycol) conjugated by carbodiimide to BSA. The antibodies produced are specific for thymine glycol as measured by immunoprecipitation of TMP-glycol-RSA conjugates and hapten inhibition of reactivity with OsO4-treated DNA in an enzyme immunoassay. Using the enzyme immunoassay, the antibody is capable of detecting femtomole and picomole levels of thymine glycol in direct and competitive assays, respectively. This immunochemical assay is potentially suitable for measuring the production and repair of thymine glycol damage in cellular DNA.