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To benchmark the psychological state and physical rehabilitation of patients who have sustained limb loss as a result of terrorist activity in Northern Ireland and to determine their satisfaction with the period of primary prosthetic rehabilitation and the artificial limb.All patients who sustained limb loss as a result of the Troubles and were referred to our rehabilitation centre were sent a questionnaire. The main outcome measures were the SIGAM mobility grades, the General Health Questionnaire (GHQ12) and three screening questions for Post Traumatic Stress Disorder (PTSD).Out of a 66% response rate, 52 (69%) patients felt that the period of primary prosthetic rehabilitation was adequate; 32 (54%) lower limb amputees graded themselves SIGAM C or D; 45 (60%) patients stated that they were still having significant stump pain. Significant stump pain was associated with poorer mobility. Nine (56%) upper limb amputees used their prosthetic limb in a functional way; 33 (44%) patients showed "psychiatric caseness" on the GHQ 12 and 50 (67%) had symptoms of PTSD.Most patients felt that the period of physical rehabilitation had been adequate; those who did not were more likely to be having ongoing psychological problems. A high percentage of patients continue to have psychological problems and stump pain.

Altered splicing of parkin under cellular stress could lead to changes in gene expression and altered protein activity. The causative role of parkin in sporadic Parkinson's disease (PD) is unknown. We described a parkin splice variant (SV) in the substantia nigra and leukocytes of sporadic PD patients. Using a case control methodology, we investigated the exon 4 SV (E4SV) and wild-type parkin expression in the leukocytes of sporadic PD patients and healthy individuals. We identified a parkin E4SV in the substantia nigra and leukocytes of sporadic PD patients and controls by reverse transcriptase–polymerase chain reaction (PCR). The exon 4 (122 bp) deletion resulted in a reading frame shift over the junction of exons 3–5 and a stop codon (tga) 17 bp downstream from exon 3. The translated truncated protein was associated with a total loss of the two-RING finger functional domain. Utilizing TaqMan real-time PCR with probes located across the junction of exons 3–4 or 3–5, we demonstrated an over-expression of E4SV/wild-type parkin ratio in the leukocytes of sporadic PD patients compared to age-, gender-, and race-matched controls (p

Loss of chromosomes 1, 2, 6, 10, 13, 17, and 21 is a characteristic finding in chromophobe renal-cell carcinoma (ChRCC). Previously, cytogenetic and molecular genetic techniques were used in demonstrating the chromosomal monosomies in ChRCCs. We performed interphase fluorescent in situ hybridization (FISH) using centromeric probes for chromosomes 1, 2, 6, and 10 on touch imprint smears from six histologically proven ChRCCs. All six ChRCC tumors showed one FISH signal corresponding to one copy number for each of these chromosomes. The percent cells with one FISH signal ranged from 48-88% (chromosome 1), 36-89% (chromosome 2), 26-98% (chromosome 6), and 64-99% (chromosome 10). In addition, 3 of the 6 cases were further studied with centromeric probes for chromosomes 13, 17, and 21. All three revealed monosomy of these three chromosomes. We conclude that interphase FISH performed on touch imprint smears is a relatively simple, rapid, and reliable method for detecting chromosome abnormalities which are specific for ChRCCs.

Tumour cell invasion and metastasis is a multistep process that involves the degradation of extracellular matrix proteins by matrix metalloproteinases (MMPs). Tissue inhibitors of metalloproteinases (TIMPs) act as negative regulators of MMPs and thus prevent tumour cell invasion and metastasis by preserving extracellular matrix (ECM) integrity. In the present study we examined the expression of one member of TIMPs, TIMP-1, in 39 thyroid tumour specimens and two thyroid carcinoma cell lines (NPA and SW579). We also investigated the effect of high TIMP-1 expression on the invasive potential of NPA cells. Northern blot analysis showed that TIMP-1 mRNA levels correlated directly with tumour aggressiveness: the highest number of TIMP-1 transcripts was found in stages III and IV vs benign goitre (P < 0.0001). However, TIMP-1 expression was not increased in NPA and SW579 cells, both of which are derived from poorly differentiated thyroid tumours. Immunohistochemical study showed strong TIMP-1 staining in the stroma cells of advanced stages of carcinomas. Overexpression of TIMP-1 by gene transfer resulted in a significant suppression of the malignant phenotype of NPA cells as judged by an in vitro tumour invasion assay. These results suggest that high levels of TIMP-1 transcripts in advanced stages of thyroid carcinoma likely come from stroma rather than thyroid cancer cells, and TIMP-1 may function as a thyroid tumour invasion/metastasis suppressor. © 1999 Cancer Research Campaign

A point mutation in the mRNA of NADP(H): quinone oxidoreductase 1 (NQO1, DT-diaphorase) is believed to be responsible for reduced enzyme activity in the adenocarcinoma BE cell line. The present study examined nine cultured human non-cancerous fibroblast cell strains, five of which were from members of a single cancer-prone family, which demonstrated widely varying activity levels of DT-diaphorase (41 - 3462 nmol min-1 mg-1 protein), to determine if genetic alteration of the NQO1 or NOQ2 gene was involved in determining enzyme activity. All cell strains expressed NQO1 and NQO2 mRNA as measured by a quantitative polymerase chain reaction amplification technique. No relationship was found between the level of mRNA expressed and the enzyme activity in the cells. Sequencing of the entire complementary DNA from the cell strains revealed only a single base substitution at nucleotide 609 in one allele encoding NQO1 in every cell strain from members of the cancer-prone family, except for one cell strain which expressed only the T at nucleotide 609 in both alleles. Subsequent examination of genomic DNA from 44 individuals revealed that this base substitution is present in approximately 50% of the population. The presence of the T at nucleotide 609 in the NQO1 locus does not appear to be directly causal for altered DT-diaphorase activity. Images Figure 1 Figure 2 Figure 3

The quantitative appraisal of the number of foreign gene copies integrated within the genomes of stably transfected cells is most conveniently performed using the strategy known as Southern blotting (1,2). First introduced by E. M. Southern in 1975 (2) the basic protocol involves the following steps: Size-fractionated DNA is first transferred from a gel matrix to a solid support under conditions that prevent self-annealing. The DNA molecules are then immobilized (covalently linked to the support) and processed for hybridization to a radiolabeled probe that has a nucleotide sequence complementing that of the target sequence to be detected. The blot is then washed extensively to remove unreacted probe molecules, and the hybrids formed between the probe and target sequences are revealed by autoradiography. The relative intensity of each autoradiographic signal will reflect the amount of hybridized material present. Quantification may be performed visually, by scanning laser densitometry, or, if the hybrids are sufficiently radioactive, by liquid scintillation counting.

Efforts to expand the current repertory of cell types amenable to transfection have often been thwarted by a common obstacle-namely, the low tolerance displayed by the recipient cells toward the gene-transfer regimen itself. As a result, several laboratories have turned to the use of synthetic polycations for delivering copious amounts of exogenous DNA to target cells without compromising their viability or clonogenicity. One of these compounds, polybrene, is mostly known for its ability to enhance the infectivity of retroviruses in culture by serving as an electrostatic bridge between the negatively charged viral particles and the anionic components residing on the surface of recipient cell membranes. Recently we, as well as others (1-4), have demonstrated that introduction of naked foreign DNA can be accomplished with high efficiency in a variety of mammalian cell types by exploiting a two-stage gene-transfer schedule in which polybrene is used first to promote the binding of DNA molecules to the target cell population, and dimethyl sulfoxide (DMSO) is then employed to permeabilize the DNA-coated cells. The method is simple to perform and has proven very effective, producing stable transfection frequencies on the order of 0.0l-0.l % with only nanogram quantities of input DNA. In a typical experiment, the process is initiated by bathing the recipient cell population in growth medium supplemented with polybrene and the transfecting DNA. Following a period of incubation during which polybrene-DNA complexes are allowed to form and attach to the cell surface, the cells are permeabilized by a brief exposure to growth medium containing DMSO in order to facilitate the uptake of the adsorbed complexes. The cells are then rinsed to remove the DMSO, and the transfected cell population is allowed to recover in fresh growth medium before being submitted to a dominant selection schedule or assayed for the "transient" expression of the transfected gene.

We have determined the gene sequence of a temperature-sensitive allele of the cell cycle-related ubiquitin-conjugating enzyme CDC34 (UBC 3) from Saccharomyces cerevisiae. The basis of temperature sensitivity is a missense mutation resulting in a proline to serine substitution at a residue that is conserved in all ubiquitin-conjugating enzymes identified thus far. This observation raised the possibility that other temperature-sensitive ubiquitin-conjugating enzymes could be generated in the same way. We therefore created the corresponding substitution in the DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), and examined the effect of temperature on the cell proliferation and DNA repair-related functions of this altered polypeptide. Yeast strains carrying this mutation proved to be temperature-sensitive with respect to cell proliferation but not with respect to the DNA damage-processing phenotypes exhibited by other rad6 mutants. Upon further investigation of the proliferation defect exhibited by this mutant, we discovered that other rad6 gene mutants deleted for the gene undergo cell cycle arrest at the nonpermissive temperature, whereas the engineered temperature-sensitive allele showed no evidence of a cell cycle defect. From these findings, we conclude that the proliferation function of RAD6 can be subdivided into a growth component and a cell division cycle component and that the growth component is unrelated to the DNA repair functions of RAD6. A reasonable interpretation of these results is that different proteins are targeted for ubiquitination in each case. The conserved proline residue of RAD6 and CDC34 is part of a turn motif common to all ubiquitin-conjugating enzymes. It is therefore likely that site-directed substitution of prolines located in turns can be generally applied for the creation of other temperature-sensitive ubiquitin-conjugating enzymes and possibly other proteins as well.

Cytogenetic and molecular genetic techniques have been used in demonstrating the chromosomal abnormalities which characterize specific subtypes of renal cell carcinoma (RCC). The aim of this study was to determine the efficiency of fluorescent in situ hybridization (FISH) technique in characterizing various subtypes of RCC based on the presence of specific chromosome abnormalities found in each RCC subtype.FISH was performed on touch imprint smears from eight renal cell carcinomas histologically confirmed by established criteria.In four tumors with histologic features of chromophobe renal cell carcinoma (ChRCC), interphase FISH was performed using centromeric probes for chromosomes 1, 2, 6, 10, 12, 17 and 21. All four ChRCC tumors showed one FISH signal corresponding to one copy number for each of these chromosomes. Two papillary RCCs included in this study showed trisomy 7 and 17, and loss of chromosome Y, using the corresponding chromosome centromeric probes. Similarly, we tested two clear cell RCCs for chromosome 3 short arm deletion with DNA probe 3p21.3. Both tumors showed loss of 3p21.3 signal.We conclude that interphase FISH performed on touch imprint smears is a relatively simple, rapid and reliable method for detecting chromosome abnormalities which are specific for various subtypes of RCC.

Measurement of domiciliary nasal peak inspiratory flow rate (PIFR) may have a role in the objective assessment of treatment response in seasonal allergic rhinitis (SAR).We wished to evaluate the relationship between domiciliary measurement of nasal PIFR and a variety of symptoms associated with rhinitis.Thirty-eight nonasthmatic patients, mean age (SEM) 30 years (1.4), with symptomatic SAR were evaluated in a placebo-controlled, single-blind, double-dummy, three way parallel group study. Patients received oral cetirizine 10 mg once daily and were randomized to receive, in addition, either: (i) intranasal mometasone furoate 200 microgram (n = 14); (ii) oral montelukast 10 mg (n = 11); or (iii) placebo (n = 13). All treatments were given once daily for 4 weeks and were preceded by a 1 week placebo period. Domiciliary diary cards were used to record morning (am) and evening (pm) domiciliary nasal PIFR and symptom (nasal, eye, throat) scores and impact on daily activity. A total daily symptom score was then calculated from the sum of these separate symptom scores.Baseline values for symptom scores and PIFR after placebo run-in were not significantly different when comparing the three groups. After 4 weeks of active treatment, there were significant (P0.05) improvements in nasal symptoms, total daily symptoms and PIFR with all treatments, with there being no significant confounding effect of pollen count, when analysed as a covariate. There were significant (P0.01) correlations for nasal symptom scores vs PIFRam (r = - 0.51) and PIFRpm (r = - 0.56), and similarly for daily activity vs PIFRam (r = - 0.42) and PIFRpm (r = - 0.48).These results suggest that domiciliary measurements of nasal peak flow correlate significantly with symptoms of seasonal allergic rhinitis and may therefore be a potentially useful objective short-term marker of treatment response.

Loss of chromosomes 1, 2, 6, 10, 13, 17, and 21 is a characteristic finding in chromophobe renal-cell carcinoma (ChRCC). Previously, cytogenetic and molecular genetic techniques were used in demonstrating the chromosomal monosomies in ChRCCs. We performed interphase fluorescent in situ hybridization (FISH) using centromeric probes for chromosomes 1, 2, 6, and 10 on touch imprint smears from six histologically proven ChRCCs. All six ChRCC tumors showed one FISH signal corresponding to one copy number for each of these chromosomes. The percent cells with one FISH signal ranged from 48-88% (chromosome 1), 36-89% (chromosome 2), 26-98% (chromosome 6), and 64-99% (chromosome 10). In addition, 3 of the 6 cases were further studied with centromeric probes for chromosomes 13, 17, and 21. All three revealed monosomy of these three chromosomes. We conclude that interphase FISH performed on touch imprint smears is a relatively simple, rapid, and reliable method for detecting chromosome abnormalities which are specific for ChRCCs.

FHIT, a candidate tumour suppressor gene, has recently been identified at chromosomal region 3p14.2, and deletions of the gene have been reported in many types of human cancers. Loss of heterozygosity (LOH) at this region has also been found frequently in follicular thyroid carcinoma (FTC). To investigate the potential role of FHIT in thyroid tumorigenesis, we examined 57 thyroid tumour specimens (eight benign adenomas, 40 papillary, four follicular and five anaplastic carcinomas), and two thyroid carcinoma cell lines (NPA, SW579) for genetic alterations by using reverse transcription–polymerase chain reaction (RT–PCR), PCR product sequencing, single-strand conformation polymorphism (SSCP) and Southern blot analysis. Two cervical carcinoma cell lines (C-33A, HeLa) were included as positive controls. We detected truncated FHIT transcripts in three of eight (38%) benign adenomas, nine of 40 (23%) papillary, and two of five (40%) anaplastic carcinomas, and in three cell lines (SW579, C-33A, HeLa). Most of the truncated transcripts lacked exons 4 or 5 to 7 or 8 of the gene and were presumably non-functional as the translation start site is located in exon 5. SSCP analysis of the coding exons failed to detect any point mutations among the samples without abnormal FHIT transcripts. Southern blot analysis demonstrated either loss or reduced intensity of major Bam HI restriction fragments in the three cell lines found to have abnormal FHIT transcripts, indicating, respectively, either intragenic homozygous or heterozygous deletions of the FHIT gene. Intragenic homozygous deletions were also found in two papillary thyroid carcinoma specimens: one was missing a 13 kb Bam HI fragment which contains exon 4, the other had deletions of 15.5, 13 and 4.2 kb fragments which contain exons 2 and 9, 4, and 5, respectively. The absence of a defective FHIT gene in FTC indicates that an additional tumour suppressor gene may reside in this region and be involved in the development of FTC. Given that defective FHIT genes were found in both benign and malignant thyroid tumours, the inactivation of this putative tumour suppressor gene is likely to be an early event in the pathogenesis of some forms of thyroid neoplasms.

Ataxia telangiectasia (AT) is an autosomal recessive human disorder featuring diverse clinical abnormalities including proneness to cancer and extreme sensitivity to ionizing radiation. Although cells from AT patients exhibit faulty activation of the p53 signal transduction pathway at early times after radiation exposure, it has been proposed that high levels of DNA damage persisting in AT cells may up-regulate p53 through an ATM-independent mechanism at late times after irradiation, leading to cell death by apoptosis. In this study we demonstrate that diploid skin fibroblast strains homozygous for the AT mutation fail to up-regulate p53 protein at late times (or = 48 h) after irradiation with 60Co gamma rays. Moreover, exposure of normal and AT fibroblasts to a dose of 8 Gy does not result in a significant increase in the fraction of apoptotic cells. Since this treatment reduces the clonogenic potential of human cells by at least two orders of magnitude, we conclude that apoptosis is not the primary mechanism of cell death induced by ionizing radiation in human normal and AT fibroblast cultures. Therefore, our results are not in accordance with the current hypothesis suggesting that increased radiosensitivity of AT cells is associated with deregulated apoptosis.

Proliferating human cells exposed to ionizing radiation show complex cellular responses including a delay in progression through various phases in the cell cycle. These cell cycle checkpoints are regulated by mitogenic signaling pathways which transduce the extracellular signals to the cell cycle control machinery. In this study we demonstrate that microinjection of a cellular extract, prepared from gamma-irradiated (40 Gy) HeLa cells, into the cytoplasm of normal human fibroblasts results in suppression of DNA replicative synthesis, indicating the presence of a trans-acting DNA synthesis-inhibiting factor(s). The addition of this same extract to the culture medium for a short time (or = 2 h) also inhibits DNA synthesis in human fibroblasts, affecting both replicon initiation and DNA chain elongation processes. Moreover, a 2-h incubation of the fibroblast cultures with the extract causes a transient delay in cell progression from G1 to S phase coupled with up-regulation of the p53 tumor suppressor protein. Both the DNA synthesis-inhibiting and G1-phase-blocking activities are reduced markedly when the extract is heated (80 degrees C; 10 min) prior to its addition to the culture medium. On the other hand, pretreatment of the fibroblast cultures with KN62, an inhibitor of calmodulin-dependent kinase II (CaMKII), serves to abrogate the inhibitory effect of the extract on DNA synthesis without influencing its ability to induce the G1-phase block. These results are compatible with the presence in HeLa cell extracts of a heat-labile trans-acting factor that triggers, in normal human cells, the activation of (1) a CaMKII-dependent signal transduction pathway mediating suppression of DNA synthesis and (2) a p53-dependent pathway mediating G1-phase checkpoint control.

Upper limb surgeons have little in the way of training in the identification or management of the psychological aspects of limb injury. Surgeons in training tend to see their speciality in technical terms, only slowly appreciating that the psychological state of the patient profoundly affects outcome in many cases.The case report that follows charts the psychological progress of a patient following severe upper limb injury. Surgeons usually view rehabilitation in physical terms. This report emphasizes the psychological aspects of a major limb injury to a policeman (MP) who subsequently studied psychology at PhD level.

Cultured cells from patients inheriting the rare cancer-prone and radiotherapy-sensitive disorder ataxia-telangiectasia (A-T) exhibit anomalies in cell cycle control and protein kinase C (PKC)-mediated upregulation of p53 protein following exposure to ionizing radiation. It remains unclear, however, as to whether this irregularity in a p53-dependent signal transduction pathway controlling the G1/S checkpoint is causally linked to the most consistent molecular hallmark of A-T-namely, marked attenuation in the inhibition of replicative DNA synthesis at early times (or = 2 h) after irradiation [radioresistant DNA synthesis (RDS)]. We report here that treatment of normal human fibroblast strains with inhibitors of calmodulin (CaM) (i.e. W7 and W13) and CaM-dependent protein kinases II and IV (i.e. KN62) prior to radiation exposure elicits an 'A-T-like' RDS phenotype, whereas treatment with PKC inhibitors (e.g. staurosporine) does not produce this response. Moreover, at 1 h post-gamma irradiation A-T fibroblasts undergo normal induction of p53 protein while exhibiting the RDS trait. At later times (e.g. 4 h) following irradiation, however, these A-T cells contain abnormally low levels of p53 protein, as do their lymphoblastoid cell line counterparts during the entire post-gamma ray incubation period. On the other hand, human cells which either lack the p53 gene completely (i.e. HL60 leukemia cells) or harbor a germline mutation in the gene (i.e. Li-Fraumeni syndrome cells) shut down their DNA replication machinery normally upon sustaining radiation damage. We thus conclude that the transitory delay in DNA synthesis routinely experienced by human cells in the face of radiation injury is mediated through a CaM-dependent regulatory cascade which involves neither PKC nor p53 protein. Accordingly, A-T cells appear to be malfunctional in at least two distinct radiation-responsive signalling pathways, one regulating the G1/S checkpoint and governed by p53 and PKC and another controlling passage through S phase and requiring CaM.

We have prospectively analyzed Wilms' tumors from 232 patients registered on the National Wilms' Tumor Study for loss of heterozygosity (LOH) on chromosomes 11p, 16q, and 1p. These chromosomal aberrations were found in 70 (33%), 35 (17%), and 21 (12%) of the informative cases, respectively. LOH for two of these regions occurred in only 25 cases, and only one tumor harbored LOH at all three sites. There was no statistically significant association between LOH at any of the three regions and either the stage or histological classification of the tumor. Patients with tumor-specific LOH for chromosome 16q had relapse rates 3.3 times higher (P = 0.01) and mortality rates 12 times higher (P0.01) than patients without LOH for chromosome 16q. These differences remained when adjusted for histology or for stage. Patients with LOH for chromosome 1p had relapse and mortality rates three times higher than those for patients without LOH for chromosome 1p, but these results were not statistically significant. In contrast, LOH for chromosome 11p had no effect on measures of outcome. These molecular markers may serve to further stratify Wilms' tumor patients into biologically favorable and unfavorable subgroups, allowing continued use of the clinical trial mechanism in the study of Wilms' tumor.

A new nuclease digestion assay was developed to elucidate the human excision-repair system operating on cyclobutyl pyrimidine dimers and (6-4) photoproducts. We analyzed lesions that accumulated in excised oligonucleotide fragments during incubation of UV-treated cultured fibroblasts. (6-4) photoproducts were removed intact, whereas excised cyclobutyl dimers often contained ruptured interpyrimidine phosphodiester bonds, raising the possibility that the intradimer backbone-cleavage reaction may help promote the bypass of unexcised dimers by the DNA replication or RNA transcription machinery. Cell strains representing eight different inherited forms of the cancer-prone skin disease xeroderma pigmentosum (XP) were generally found to exhibit characteristic abilities to excise the two classes of photolesions, ranging from total deficiency in groups A and G to normal proficiency in the variant. The capacity of any given XP group to act on one class of photoproducts in no way predicted its ability to act on the other. Finally, in those XP strains displaying significant levels of dimer removal, the ratio of intact-versus-modified dimers was normal, implying that rupture of the intradimer backbone linkage occurs independently of subsequent excision-repair reactions. Our data indicate that cyclobutyl dimers and (6-4) photoproducts are processed by distinct nucleotide-excision-repair pathways in human cells.

We have developed preparative in situ hybridization (Prep-I.S.H.) of complex DNA populations to mitotic chromosomes as a means of generating chromosome region-specific cDNA subpopulations. Prep-I.S.H. is a combination of two cytogenetic techniques: hybridization of DNA molecules to mitotic chromosomes, and chromosome microdissection. Here we provide technical details of the procedure and describe its application to the human chromosome 11q22–23 region containing the ataxia telangiectasia genes. Prep-I.S.H. has a number of applications in studies of gene expression and genome organization, including efficient cytogenetic sorting of tissue-specific cDNAs and in dramatically reducing the number of candidate genes to aid in gene discovery. Prep-I. S.H. provides a technical approach to a “positional candidate” strategy for gene discovery that differs from positional cloning by focusing on analysis of the genes in a chromosomal region of interest rather than on detailed analysis of physical and genetic maps.

Ataxia-telangiectasia (A-T) is a rare human recessively-inherited disorder characterized by, among other symptoms, a devastating and sometimes fatal reaction to conventional radiotherapy (Boder, 1985; Sedgwick and Boder, 1991). Radiation intolerance in vitro, as manifested by impaired colony-forming ability and exessive chromosomal instability, is universally displayed by cultured dermal fibroblasts and peripheral blood lymphocytes derived from A-T donors (Lehmann, 1982; Taylor, 1982; Paterson et al, 1984). As an extension of these radiobiological studies on A-T, we have recently conducted a detailed study on the deleterious effects of 4-nitroquinoline 1-oxide (4NQO), a partially radiomimetic carcinogen, on A-T fibroblast strains and have demonstrated that representative strains belonging to complementation groups A (AT2BE and AT3BI) and C (AT4BI) are defective in removal of a class of alkali-stable 4NQO-DNA adducts, whereas a group D strain (AT5BI) exhibits normal repair capacity (Mirzayans et al, 1989; Mirzayans and Paterson, 1991a). In this investigation, repair was monitored with the aid of 1-β-D-arabinofuranosylcytosine (araC), a potent inhibitor of DNA polymerases α and δ (Wist, 1979; Cleaver, 1984; Keeney and Linn, 1990). In this widely used approach, the extent of DNA strand breaks accumulating in cultures incubated with araC, following carcinogen treatment, becomes a measure of the efficiency to perform long-patch excision repair (Snyder et al., 1984; Cleaver, 1989; Mirzayans and Paterson, 1991b).

Our recent studies indicate that enzymatic hydrolysis of the intradimer phosphodiester linkage constitutes an early reaction in processing UV light-induced cis-syn-cyclobutane pyrimidine dimers in cultured human fibroblasts. Before characterizing the resultant modified dimer sites in cellular DNA, it is necessary to establish experimental conditions that can distinguish backbone-nicked from intact dimers. We thus constructed a model substrate, i.e. p(dT) 10p(dT)10 containing a dimer with a ruptured sugar-phosphate bond, and determined the products of its reaction with snake venom phosphodiesterase and alkaline phosphatase, an enzymatic digestion mixture known to release dimers from UV-treated poly(dA).poly(dT) within trinucleotides with the photoproduct intact at the 3'-end (d-TpTpT). The model substrate was prepared by (i) end labeling p(dT)9 using terminal deoxynucleotidyltransferase and [3H]thymine-labeled TTP; and (ii) annealing the chromatographically purified p(dT)10 oligomers to poly(dA) followed by UV (290 nm)-induced ligation. Photoligated 20-mers with one radioactive and modified internal dimer were isolated and enzymatically digested. High performance liquid chromatographic analysis of the reaction products revealed a novel trithymidylate with its backbone severed at the 3'-terminus (d-TpTdT), demonstrating that this procedure could discriminate between intact and modified dimers. The procedure was then exploited to show that (i) Escherichia coli DNA photolyase can monomerize, albeit inefficiently, backbone-ruptured dimers; and (ii) phage T4 polynucleotide kinase can catalyze the phosphorylation of d-TpTdT, thus facilitating the development of a sensitive postlabeling assay suitable for modified dimer detection under biologically relevant conditions.

Loss of heterozygosity studies have been used to identify chromosomal regions which are frequently deleted and thus indicate areas which may harbor tumor suppressor genes. As a result, both the WT1 gene located in chromosome 11p13 and an unidentified gene(s) within chromosome 11p15 have been implicated in Wilms' tumorigenesis. Cytogenetic and linkage studies suggest that additional non-chromosome 11 sites are involved in Wilms' tumor. Because these sites may also involve loss of heterozygosity, loci on 33 autosomal arms were screened for allele loss in a series of Wilms' tumors. We found that in addition to loss on chromosome 11p (11 of 25 informative tumors) there was significant loss on chromosome 16q (9 of 45 informative tumors), while the total frequency of allele loss excluding these loci was low (9 of 426 total informative loci). These data indicate that losses of both chromosome 11p and 16q alleles are nonrandom events and suggest that 16q is the location of a third tumor suppressor gene underlying Wilms' tumorigenesis. The parental origin of the lost chromosome 16q allele was determined in eight sporadic tumors. Alleles of paternal and of maternal origin were each lost in four sporadic tumors indicating that, unlike chromosome 11p, alleles of either parental origin are lost on 16q.

We have determined the gene sequence of a temperature-sensitive allele of the cell cycle-related ubiquitin-conjugating enzyme CDC34 (UBC 3) from Saccharomyces cerevisiae. The basis of temperature sensitivity is a missense mutation resulting in a proline to serine substitution at a residue that is conserved in all ubiquitin-conjugating enzymes identified thus far. This observation raised the possibility that other temperature-sensitive ubiquitin-conjugating enzymes could be generated in the same way. We therefore created the corresponding substitution in the DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), and examined the effect of temperature on the cell proliferation and DNA repair-related functions of this altered polypeptide. Yeast strains carrying this mutation proved to be temperature-sensitive with respect to cell proliferation but not with respect to the DNA damage-processing phenotypes exhibited by other rad6 mutants. Upon further investigation of the proliferation defect exhibited by this mutant, we discovered that other rad6 gene mutants deleted for the gene undergo cell cycle arrest at the nonpermissive temperature, whereas the engineered temperature-sensitive allele showed no evidence of a cell cycle defect. From these findings, we conclude that the proliferation function of RAD6 can be subdivided into a growth component and a cell division cycle component and that the growth component is unrelated to the DNA repair functions of RAD6. A reasonable interpretation of these results is that different proteins are targeted for ubiquitination in each case. The conserved proline residue of RAD6 and CDC34 is part of a turn motif common to all ubiquitin-conjugating enzymes. It is therefore likely that site-directed substitution of prolines located in turns can be generally applied for the creation of other temperature-sensitive ubiquitin-conjugating enzymes and possibly other proteins as well.

Drawing upon the comprehensive population-based Northern Alberta Breast Cancer Registry containing 704 patients with histologically negative axillary lymph nodes who have been followed for 5-16 years, we have undertaken a retrospective case-control study to evaluate the utility of genomic amplification of specific protooncogenes [c-erbB-2 (nee HER-2/neu), c-erbA, c-myc, int-2, and hst-1] as predictive indicators of clinical outcome in node-negative disease. To this end, 115 women with node-negative breast cancer who had recurred at any time up to 16 years posttreatment (cases) were matched pairwise for appropriate clinicopathological variables (size of primary tumor, menopausal state, estrogen receptor status, anniversary year of treatment, and patient age) with a second group of 115 women (controls) selected from a cohort of 502 node-negative patients who had not relapsed during long-term follow-up. Tumor DNA extracted from archival formalin-fixed, paraffin-embedded tissue blocks were analyzed for protooncogene copy number by slot-blot hybridization. Taking a gene copy number of 3 as the cutoff, 27 of the 230 tumor samples examined contained from 3- to 22-fold elevation in c-erbB-2 genomic equivalents. Twenty-one of the 27 tumors amplified for c-erbB-2 were derived from cases and 6 from controls, signifying that 18% of the node-negative patients who had relapsed harbored excessive copies of the protooncogene in their malignant tissue compared to only 5% for the patients who had remained in remission. Accordingly, the occurrence of amplification of c-erbB-2 proved to be a statistically significant predictor of poor prognosis, especially disease-free interval (P = 0.006). Moreover, this genetic alteration appeared to be independent of and to have greater predictive power than most commonly used prognostic factors. Our findings also indicated that as a clinical test, measurement of c-erbB-2 amplification suffers from low sensitivity; however, when greater than 6 gene copies are present, the test has a positive predictive value for recurrence of 70%. Concurrent analysis of tumor DNA blots with probes for the other four protooncogenes examined revealed that their amplification, which others have reported to arise often, especially in node-positive disease, was seldom found even in our high-risk case group (2-3%). In short, our data strongly suggest that amplification of c-erbB-2 may contribute to the pathogenesis of some forms of node-negative breast cancer and thus may serve as a useful genetic marker to identify a subset of high-risk patients.

Cells cultured from most patients suffering from the sunlight-sensitive hereditary disorder xeroderma pigmentosum are defective in the ability to excise ultraviolet light (UV)-induced pyrimidine dimers from their DNA. There is, however, one class of these patients whose cells are completely normal in this excision repair process. We have found that these cells have an abnormality in the manner in which DNA is synthesized after UV-irradiation. The time taken to convert initially low-molecular-weight DNA synthesized in UV-irradiated cells into high-molecular-weight DNA similar in size to that in untreated cells is much greater in these variants than in normal cells. Furthermore, this slow conversion of low to high-molecular-weight newly synthesized DNA is drastically inhibited by caffeine, which has no effect in normal cells. Two cell lines from classes of xeroderma pigmentosum that are defective in excision-repair show intermediate effects, with regard to both the time taken to convert newly synthesized DNA to high molecular weight and the inhibition of this process by caffeine.

Cultured human and embryonic chick fibroblasts possess different enzyme-mediated processes to repair cyclobutyl pyrimidine dimers induced in their deoxyribonucleic acid (DNA) by ultraviolet (UV) radiation. While dimers are corrected in human cells by excision repair, a photoenzymatic repair process exists in embryonic chick cells for the removal of these potentially deleterious UV photoproducts. We have utilized a sensitive enzymatic assay to monitor the disappearance, i.e. repair, of dimer-containing sites in fused populations of human and chick cells primarily consisting of multinucleate human/chick heterokaryons. Fused cultures were constructed such that UV photoproducts were present only in chick DNA when evaluating excision repair and only in human DNA when evaluating photoenzymatic repair. Based on the kinetics of site removal observed in these cultures we are led to conclude the following: Within heterokaryons per se the photoreactivating enzyme derived from chick nuclei and at least one excision-repair enzyme (presumably a UV endonuclease) derived from human nuclei act on UV-damaged DNA in foreign nuclei with an efficiency equal to that displayed toward their own nuclear DNA. Hence, after cell fusion these chick and human repair enzymes are apparently able to diffuse into foreign nuclei and once therein competently attack UV-irradiated DNA independently of its origin. In harmony with the situation in nonfused parental cultures, in heterokaryons the chick photoenzymatic repair process rapidly removed all dimer-containing sites from human DNA including the residual fraction normally acted upon slowly by the human excision-repair process.

The properties of minicell producing mutants of Escherichia coli deficient in gentic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC- phenotypes are unaffected by min+/- genotypes in whole cells. In contrast to minicells produced by rec+ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transfereed DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permits unrestricted Hfr DNA breakdown in minicells produced by a recA1 strain. This results in an increase in TCA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R 64-11 or lambdadv, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and [32P]-labelled linear DNA from bacteriophage T7 for substrate, we found that the ATP-dependent exonuclease of the recBC complex (exo V) is present in rec+ and recA- minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC- minicells results in isolation of larger than average Hfr DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products.

Two non-transformed human skin fibroblast strains, GM38 and 3437T, were found to be more sensitive to the bioreductive alkylating agents mitomycin C (MMC) and porfiromycin (PM) under hypoxic compared to aerobic conditions. One of these strains, 3437T, was 6-7 times more resistant to these agents under aerobic exposure conditions, but was identical in sensitivity to the normal strain, GM38, under hypoxic conditions. Aerobic 3437T cells demonstrated no increased resistance to cisplatin compared to the normal strain, arguing against enhanced ability to repair DNA interstrand cross-links as the underlying explanation for the mitomycin resistance. The aerobic resistance of 3437T was not altered by dicumarol, an inhibitor of the enzyme DT-diaphorase which is believed to be involved in aerobic activation of MMC and PM. Dicumarol did increase the resistance of GM38, but not to the same level of resistance demonstrated by 3437T. These results suggest that the aerobic MMC and PM resistance of 3437T may arise, in part, from a deficiency in DT-diaphorase activity. The identical sensitivities under hypoxic conditions indicate that drug activation pathways operative in the absence of oxygen are similar in both the normal and 3437T cells.

Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation. In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.

Escherichia coli K-12, polAl − is a mutant strain whose extracts are deficient in Kornberg deoxyribonucleic acid (DNA) polymerase activity. We have compared the mutant and parental strains on the basis of a number of responses to ultraviolet (UV) and X-irradiation. For both types of radiation, the mutant is more sensitive by approximately the same factor as measured by reduction in colony formation, depression of DNA synthesis, and enhancement of DNA degradation. The rate of repair of X-ray-induced single-strand breaks in the mutant is also slower, as is the repair of breaks after excision repair of UV damage. On the other hand, the mutant has a significant capability to reactivate UV-irradiated λ phage, although it is almost totally deficient in the ability to carry out UV reactivation. The data indicate that the polAl mutation leaves the cells with some ability to perform excision and strand-rejoining repair but that an exonuclease, whose identity remains obscure, is the agent responsible for the extensive breakdown of the DNA in polAl − cells after irradiation.

Auxotrophic, morphological, sensitive and suppressor mutant strains of Coniochaeta velutina are described. Of 1334 auxotrophic mutants isolated, all but 58 have been characterized as to their specific requirements. The mutation spectrum obtained on malt medium differs in several respects from that on complete medium. Arginine, adenine, cysteine, methionine, histidine and pyrimidine requiring mutants were most frequently isolated. Of 12 diauxotrophs tested genetically, 7 proved to be double mutants, but the remaining 5 behaved genetically as single mutants with a double requirement. Four of these required both adenine and histidine for growth and the other both phenylalanine and tyrosine. Cysteine-sensitive mutants and histidine-sensitive strains are described. Other mutants isolated included a variety of morphological, conidial and colour variants, UV-sensitive strains, canavanine-sensitive strains and arginine-suppressor mutations.

A partially purified extract from Micrococcus luteus contains endonucleolytic activity toward ultraviolet (UV)-irradiated deoxyribonucleic acid (DNA). We found that the same extract also acts on superhelical, covalently-closed circular λ dv DNA isolated from γ-irradiated minicells of E. coli . The introduction of nicks in isolated covalently-closed circular DNA by an endonuclease in the extract results in relaxed circles, and these two circular DNA species are easily distinguishable by their sedimentation properties in alkaline sucrose. The frequency with which the endonuclease-susceptible lesions are produced in superhelical DNA is only marginally enhanced when 60 Co γ-rays are administered to an aerobic rather than an anoxic minicell suspension. The ratio of endonuclease-sensitive defects to single-strand scissions, induced by γ-irradiation in air, is about 1:3. The nuclease-sensitive lesions disappear from γ-irradiated minicells during incubation after radiation presumably as a consequence of excision repair. Since the addition of UV-irradiated calf-thymus DNA depresses the ability of the M. luteus extract to attack not only UV-damaged E. coli DNA (a known substrate for the so-called UV-specific endonuclease that catalyzes the initial single-strand incision adjacent to the structural defect) but, also λ dv DNA injured by γ-rays, we conclude that physicochemical alterations induced by both types of radiation are recognized by one and the same endonuclease.

SummaryChromosomeless minicells are formed by misplaced cell fissions near the polar extremities of an Escherichia coli K12 mutant strain. Superhelical λdv DNA can be introduced into minicells by segregation from a λdv-containing derivative of the original mutant. One single-strand break changes the sedimentation constant of such a DNA and hence sedimentation of minicell lysates in neutral and alkaline sucrose gradients enables one accurately to quantitate the numbers of ‘clean’ chain breaks and alkali-labile bonds produced in DNA by γ-irradiation in vivo. Although the total number of single-strand breaks measured in alkali is enhanced three- to fourfold when 60Co γ-rays are delivered to an oxygenated minicell suspension, the fraction which requires an alkaline environment for expression is almost twice as great in minicell samples irradiated in the absence (∼ 35 per cent) as in the presence (∼ 21 per cent) of oxygen.In contrast to chain breaks, alkali-labile bonds do not disappear from DNA of minicells d...

Primary embryonic chick cells have been evaluated on the basis of their capacity to repair photochemical lesions produced in the deoxyribonucleic acid (DNA) by ultraviolet (UV) radiation. The fate of one prominent class of UV photoproducts, cyclobutane pyrimidine dimers, was monitored by an in vitro enzymatic assay. UV-irradiated cultures were incubated for prescribed times after which their damaged, radioactive-labeled DNA was extracted and exposed to a purified UV endonuclease selectively active toward sites altered by dimer formation. Single-strand scissions specifically introduced by the enzyme treatment and, therefore, the dimer-containing sites remaining in the DNA were quantified retrospectively by velocity sedimentation in alkaline sucrose. When the chick fibroblasts were incubated in black light, essentially all nuclease-susceptible sites rapidly disappeared from the UV-damaged DNA. In sharp contrast, incubation of the irradiated cultures in total darkness severely impeded the metabolic machinery responsible for site elimination. A substantial amount of UV-stimulated DNA repair synthesis was also detected in the chick cells by conventional techniques involving isopyknic centrifugation and autoradiography. However, the UV photoproducts triggering this indicator of excision repair were probably not dimers since incubation of the irradiated cultures in the light rather than in the dark did not lead to a diminution in the extent of repair synthesis. By these criteria of DNA repair, it appears that embryonic chick cells primarily rely on a highly proficient, light-requiring mechanism, presumably enzymatic photoreactivation, for dimer elimination but also possess a light-independent, excision-type process to cope with other, as yet unidentified, photochemical defects.

XERODERMA pigmentosum is a rare cutaneous disease of man displaying an autosomal recessive pattern of inheritance1. The most conspicuous clinical symptom exhibited by homozygously afflicted individuals is hypersensitivity of the skin to solar radiation. Typically, the disorder eventuates in a high incidence of multiple carcinomas and frequently becomes terminal after the onset of metastatic epithelioma2. Two clinical syndromes characterise the disease: (i) the more common classical form manifested solely by the cutaneous abnormalities and (ii) the De Sanctis-Cacchione (DSC) syndrome in which severe neurological complications, including mental retardation, accompany the skin malignancies2.

An Escherichia coli mutant, extracts of which show no DNA polymerase activity, can excise pyrimidine dimers induced by ultraviolet irradiation. The increased radiation sensitivity of this strain is related to the extensive degradation of irradiated DNA.

A sensitive enzymatic assay has been utilized to monitor repair of UV-induced damage to DNA in primary human and embryonic chick cells and in multinucleate heterokaryons artificially derived from both. The assay exploits the unique ability of a purified repair endonuclease to attack UV-irradiated DNA at sites containing pyrimidine dimers. These nuclease-susceptible sites are subsequently observed as single-strand scissions by velocity sedimentation in alkaline sucrose gradients. Incubation of UV-damaged cultures followed by extraction and enzymatic analysis of the radioactively labeled DNA enables one to trace the disappearance of such sites in vivo and hence to monitor endogenous repair activity. When UV-irradiated human cells are incubated in the dark, the curve for site removal exhibits a two-phase exponetial decline; i.e. there exists a fast component responsible for elimination of 60% of the initial damage and a second one approximately 7 times slower in rate. The removal of sites is not further enhanced by exposing cells to blacklight during post-UV incubation. Conversely, UV-damaged chick cells rid their DNA of all nuclease-susceptible sites rapidly (i.e. at an exponential rate approximately 13 times faster than the fast component of site removal in human cells) when incubated under blacklight but not when kept in the dark. These data indicate the presence in human and embryonic chick cells of distinct enzymatic mechanisms for the elimination of dimer-containing sites. Wheneras human fibroblasts rely heavily on a light-independent process, excision-repair, chick fibroblasts possess a light-dependent mechanism, presumably photoenzymatic repair. Advantage has been taken of the contrasting repair properties of the human and embryonic chick fibroblasts to evaluate the extent to which each can assist the other in the removal of UV-induced damage from its DNA. The two cell types were fused to form giant human/chick heterokaryons containing a number of intact nuclei from both strains. Experimental conditions were selected so that UV-induced damage resided only in DNA foreign to the repair enzymes under study. Our results strongly suggest that repair enzyme(s) coded for by either fusion partner can remove dimer-containing sites from the DNA of the other with an efficiency comparable to that attained when acting on its own DNA in unfused, parental cells. Further, the light-requiring repair process supplied by the chick is more proficient at operating on these sites in human DNA than is excision-repair, the parallel mechanism available to human cells for this purpose.

The effect of local nerve blockade on the relief of postoperative pain is reported in a series of 167 patients who underwent surgery of the arm, knee or foot. In 80% of cases analgesics were not required within 4 hours postoperatively, and in 39% analgesics were not required within 8 hours. Conventional methods of postoperative pain relief are not always effective. Local nerve blockade can be used to provide complete analgesia after limb surgery and is therefore of great value to postoperative care.

The deleterious effects of many environmental agents--ultraviolet (UV) light, ionizing radiation, and polycyclic aromatic hydrocarbons, to cite a few--are primarily attributed to their ability to react with, and thereby structurally modify the deoxyribonucleic acid (DNA) of the living cell1. Given the appreciable level of damage believed to occur naturally in DNA2 and the need to maintain its fidelity as the repository of the genetic script, it s understandable that all living organisms, humans included, should possess multiple cellular processes whose combined actions promote the repair of damage to DNA3. An increasing number of genetically transmitted disorders in man is being found to be associated with enhanced sensitivity to extrinsic DNA-damaging agents. Patients afflicted with any one of these particular Mendelian single-gene traits are typically cancer-prone and suffer from progressive neurodegeneration, and, at least for a few disorders, their cells when cultured in vitro exhibit anomalies in one or more DNA epair processes4–8. The majority of these hereditary diseases can be divided into two broad groups: (i) disorders associated with hypersensitivity to UV light and UV-mimetic chemicals; and (ii) disorders associated with hypersensitivity to ionizing radiation and radiomimetic chemicals. Exemplary diseases of the former group--namely, xeroderma pigmentosum (XP), Bloom syndrome (BS), and Cockayne syndrome--are reviewed elsewhere in this volume. Here we present an overview of the clinical and laboratory features of the latter group of disorders with emphasis on the prototype disease, ataxia telangiectasia (AT)7,8, and comment briefly on current insight into the pathogenesis of these disorders and its implications for theories on environmental carcinogenesis and neurodegenerative processes in man.

Our recent findings suggest that enzymatic hydrolysis of the intradimer phosphodiester bond may constitute the initial step in the repair of UV light-induced cyclobutane pyrimidine dimers in human cells. To examine the susceptibility of this phosphodiester linkage to enzyme-mediated hydrolysis, the trinucleotide d-Tp-TpT was UV-irradiated and the two isomeric compounds containing a cis-syn-cyclobutane dimer were isolated by high performance liquid chromatography and treated with various deoxyribonucleases. Snake venom phosphodiesterase hydrolyzed only the 3'-phosphodiester group in the 5'-isomer (d-T less than p greater than TpT) but was totally inactive toward the 3'-isomer (d-TpT less than p greater than T). In contrast, calf spleen phosphodiesterase only operated on the 3'-isomer by cleaving the 5'-internucleotide bond. Kinetic analysis revealed that (i) the activity of snake venom phosphodiesterase was unaffected by a dimer 5' to a phosphodiester linkage, (ii) the action of calf spleen phosphodiesterase was partially inhibited by a dimer 3' to a phosphodiester bond, and (iii) Escherichia coli phr B-encoded DNA photolyase reacted twice as fast with d-T less than p greater than TpT as with d-TpT less than p greater than T. Mung bean nuclease, nuclease S1, and nuclease P1 all cleaved the 5'-internucleotide linkage, but not the intradimer phosphodiester bond, in d-TpT less than p greater than T. Both phosphate groups in d-T less than p greater than TpT were refractory to mung bean nuclease or nuclease S1. Incubation of d-T less than p greater than TpT with nuclease P1, however, generated the novel compound dT less than greater than d-pTpT containing a severed intradimer phosphodiester linkage. Accordingly, nuclease P1 represents the first purified enzyme known to hydrolyze an intradimer phosphodiester linkage.

Ataxia telangiectasia (AT) is a rare autosomal recessive disorder in man characterized by progressive loss of muscular coordination (due to neurodegeneration) and permanent dilation of the small blood vessels of the eyes and skin1,2. AT patients also have repeated sinopulmonary infections, immune defects associated with thymus underdevelopment, and abnormal endocrine functions1,2. Affected patients are at high risk of developing malignancy, particularly lymphomas and lymphatic leukaemias1, and there are clinical indications3–5 that AT patients are hypersensitive to conventional radiotherapy administered for treatment of malignancy. Cultured diploid fibroblasts from AT donors are consistently hypersensitive to ionizing radiation2,6,7, apparently due to defective enzymatic repair of radiogenic DNA damage2,8. We have determined the survival responses and DNA repair abilities of AT cells exposed to 4-nitroquinoline-1-oxide (4NQO), a chemical carcinogen whose DNA-damaging properties partially mimic those of ionizing radiation9. We report here that certain AT cell strains show hypersensitivity to inactivation by 4NQO and defective repair of 4NQO-induced adducts in DNA.

We have compared the initial yield and subsequent enzymatic repair of DNA strand breaks and 1-beta-D-arabinofuranosylcytosine (araC)-detectable sites in cultured human cell lines after irradiation with soft (40-75 kVp) X rays and hard (60Co) gamma rays, two forms of low-LET radiation whose average ionization densities differ by 15-fold (i.e., approximately 4.5 keV/mu and approximately 0.3 keV/mu, respectively). Incubation of X- or gamma-irradiated cell cultures in the presence of araC resulted in the accumulation of DNA single-strand interruptions, as measured by conventional velocity sedimentation analysis in alkaline sucrose gradients. Since the drug is a selective inhibitor of DNA polymerase alpha, this observation implicates polymerase alpha in the repair of radiogenic damage. Specifically, DNA repair analysis in X-ray-treated cells suggested that polymerase alpha is a key participant in the repair of a distinct, albeit structurally undefined, class of radioproducts [presumably a combination of double-strand breaks and alkali-stable lesions (e.g., modified base/sugar residues and DNA crosslinks)], but takes little, if any, part in the restitution of single-strand breaks (whether frank scissions or alkali-labile bonds) induced by ionizing radiation. Accordingly, the drug did not significantly inhibit the repair of DNA damage in cultures exposed to less than or equal to 100 Gy of gamma radiation. A remarkable difference was evident with respect to the absolute yields of these two major classes of DNA injury--that is, araC-detectable sites and single-strand breaks--depending on the type of radiation administered. For the same dose, the level of araC-detectable sites found in cells treated with gamma rays (less than or equal to 150 Gy) was as much as 3.4 times lower than that observed in X-ray-exposed cells (i.e., one site per 1,155 and 335 eV, respectively). In contrast, the number of strand breaks induced by gamma rays was approximately 2.3-fold higher than that produced by X rays (one break per 27 and 61 eV, respectively). Assuming that, among other conditions, the relative biological effectiveness of the comparatively densely ionizing soft X rays is roughly twice that of the sparsely ionizing hard gamma rays, our data strongly imply that those radioproducts which are manifested as araC-detectable sites may be approximately 45 times more deleterious, on a per lesion basis, than those observed as single-strand breaks.

Retrospective studies of cancer incidence in AT families suggest that the major detrimental impact of the AT gene on the health of the general population stems from its disease-predisposing potential in heterozygotes. The absence of a definitive marker for routine identification of such carriers, however, has previously precluded direct validation of this predicted effect of AT heterozygosity. We demonstrate that chronic gamma-ray exposure, because it expands the difference in radiation cytotoxicity between normal and heterozygotic strains compared to acute dose delivery, may point the way to development of a reliable laboratory diagnostic procedure for identification of carriers of a defective AT gene.

Human xeroderma pigmentosum (XP) or Fanconi anemia (FA) fibroblasts displayed shouldered 45 degrees C heat survival curves not significantly different from normal fibroblasts, a result similar to that previously found for ataxia telangiectasia (AT) cells, indicating heat resistance is not linked to either uv or low-LET ionizing radiation resistance. Hyperthermia (45 degrees C) sensitized normal and XP fibroblasts to killing by gamma radiation but failed to sensitize the cells to the lethal effects of 254 nm uv radiation. Thermal inhibition of repair of ionizing radiation lesions but not uv-induced lesions appears to contribute synergistically to cell death. The thermal enhancement ratio (TER) for the synergistic interaction of hyperthermia (45 degrees C, 30 min) and gamma radiation was significantly lower in one FA and two strains (TER = 1.7-1.8) than that reported previously for three normal strains (TER = 2.5-3.0). These XP and FA strains may be more gamma sensitive than normal human fibroblasts. Since hyperthermia treatment only slightly increases the gamma-radiation sensitivity of ataxia telangiectasia (AT) fibroblasts compared to normal strains, it is possible that the degree of thermal enhancement attainable reflects the genetically inherent ionizing radiation repair capacity of the cells. The data indicate that both repair inhibition and particular lesion types are required for lethal synergism between heat and radiation. We therefore postulate that the transient thermal inhibition of repair results in the conversion of gamma-induced lesions to irrepairable lethal damage, while uv-type damage can remain unaltered during this period.

Phage T4 polynucleotide kinase (EC 2.7.1.78) proved incapable of catalyzing the phosphorylation of thymidylyl-(3'----5')-thymidine containing either a cis-syn-cyclobutane pyrimidine dimer (d-T less than p greater than T) or a 6-4'-[pyrimidin-2'-one]pyrimidine photoproduct (d-T[p]-T), and similarly the UV-modified compounds of (dT)3 bearing either photoproduct at their 5'-end (d-T less than p greater than TpT and d-T[p]TpT). In contrast, the 3'-structural isomers of these trinucleotides (d-TpT less than p greater than T and d-TpT[p]T) were phosphorylated at the same rate as the parent compound. These phosphorylatable lesion-containing oligonucleotides are quantitatively released from UV-irradiated poly(dA):poly(dT) by enzymatic hydrolysis with snake venom phosphodiesterase and alkaline phosphatase (Liuzzi, M., Weinfeld, M., and Paterson, M. C. (1989) J. Biol. Chem. 264, 6355-6363). By combining this digestion regimen with phosphorylation by polynucleotide kinase and [gamma-32P]ATP, pyrimidine dimers were quantitated at the fmol level following exposure of poly(dA):poly(dT) and herring sperm DNA to biologically relevant UV fluences. The rate of dimer induction in the synthetic polymer, approximately 10 dimers/10(6) nucleotides/Jm-2, was in close agreement with that obtained by conventional methods. Dimers were induced at one-fourth of this rate in the natural DNA. Further treatment of the phosphorylated oligonucleotides derived from irradiated herring sperm DNA with nuclease P1 released the labeled 5'-nucleotide, thus permitting analysis of the nearest-neighbor bases 5' to the lesions. We observed a ratio for pyrimidine-to-purine bases of almost 6:1, implicating tripyrimidine stretches as hotspots for UV-induced DNA damage.