Defective DNA Repair Mechanisms Research Articles

Congenital DNA repair deficiency results in protection against renal ischemia reperfusion injury in mice

Cockayne syndrome and other segmental progerias with inborn defects in DNA repair mechanisms are thought to be due in part to hypersensitivity to endogenous oxidative DNA damage. The accelerated aging-like symptoms of this disorder include dysmyelination within the central nervous system, progressive sensineuronal hearing loss and retinal degeneration. We tested the effects of congenital nucleotide excision DNA repair deficiency on acute oxidative stress sensitivity in vivo. Surprisingly, we found mouse models of Cockayne syndrome less susceptible than wild type animals to surgically induced renal ischemia reperfusion injury, a multifactorial injury mediated in part by oxidative damage. Renal failure-related mortality was significantly reduced in Csb(-/-) mice, kidney function was improved and proliferation was significantly higher in the regenerative phase following ischemic injury. Protection from ischemic damage correlated with improved baseline glucose tolerance and insulin sensitivity and a reduced inflammatory response following injury. Protection was further associated with genetic ablation of a different Cockayne syndrome-associated gene, Csa. Our data provide the first functional in vivo evidence that congenital DNA repair deficiency can induce protection from acute stress in at least one organ. This suggests that while specific types of unrepaired endogenous DNA damage may lead to detrimental effects in certain tissues, they may at the same time elicit beneficial adaptive changes in others and thus contribute to the tissue specificity of disease symptoms.

Genetic instability in the human population: A working hypothesis1,2

In several rare syndromes, genetic instability, expressed as chromosome instability, is known. Patients with these syndromes are predisposed to cancer. Evidence has been accumulating to show that chromosome instability occurs in some other diseases also, though the frequency was not as high. Genetic instability may induce mutational events in a chromosome (second step) whose homolog has a congenital defect (primary step) towards neoplasia in a target cell. Genetic instability may also induce the initial step. It is highly probable that genetic instability in the human population is not an all-or-none situation. Gradations of genetic instability may exist with a variety of underlying causes. Persons may have latent instability which normally cannot be detected by cytogenetic methods, e.g. defective DNA repair mechanisms. With DNA damage, there will be little or no repair in the cells of persons with defective repair systems, hence more chromosomal aberrations. When challenged with clastogens, the cells of persons with mildly defective repair systems may show a higher rate of chromosome aberrations than those of stable persons. This paper presents a working hypothesis on genetic instability in the human population and methods to detect the masked instability.

Higher Prevalence of Prior Prostate Cancer in Patients Diagnosed with Chronic Myelogenous Leukemia.

If defective DNA repair mechanisms result in the development of the Philadelphia chromosome and chronic myelogenous leukemia (CML), then individuals with CML are at higher risk in general to develop genetic mutations. These mutations can then result in other cancers. A retrospective review of patients treated with interferon and then imatinib for CML did not demonstrate an overall increased risk of cancer, but did report a 4-fold increased incidence of prostate cancer among men receiving imatinib compared with the general population1. The increased risk of prostate cancer might be associated with the use of imatinib. Alternatively, it may be due to a predisposition to prostate cancer among men with CML.Using the United Kingdom's General Practice Research Database (GPRD) we conducted a nested case-control study to examine whether men with CML have a higher prevalence of prostate cancer compared to men without CML. The GPRD contains extensive clinical information on a population of approximately 3.5 million patients, about 5% of the UK population. The collected data include demographics, medical diagnoses, prescriptions, referrals to hospitals, lifestyle variables, hospital discharge reports, as well as patient care information2.One-hundred ninety seven males with CML were identified in the database from 1995 to 2005. Of these, 12 were excluded because they had not participated in the database for at least 180 days, no controls could be found, or the medical history was inadequate. For the remaining 185 patients, 1844 controls were matched for age, general practice attended, and the year of start in the practice.In total, 8 cases and 52 controls had a history of prostate cancer. The mean age among the cases was 63.2 years (range 18 to 89 years); the mean age among the controls was 63.1 years (range 8 to 97 years).In a conditional logistic regression model, the relative risk of having been previously diagnosed with prostate cancer among patients with CML was 2.65 (95% confidence interval 1.18 to 5.94, p=0.018).The mean time between prostate cancer diagnosis and first diagnosis of CML was 1033.5 days (range 61 to 2996 days). The mean time from the diagnosis of prostate cancer and identification as a control was 1391 days (range 2 to 2951 days). These times and ranges are not disparate enough to suggest that an ascertainment bias could account for a higher incidence of CML among patients with prostate cancer.Thus, the relative risk of developing CML is higher among patients with a prior diagnosis of prostate cancer, and factors inherent to the host may predispose them to develop prostate cancer and CML. Furthermore, this study provides support for the theory that either exposure to imatinib may not predispose to second malignancy at all, or that the oncogenic effect of imatinib may be augmented by a pre-existing risk for second malignancy among these patients because the prostate cancer predated the diagnosis of CML.This database will also be used to determine whether the association between CML and prostate cancer can be extended to CML and other cancers. The data presented here will allow for a greater understanding of any future prospective studies examining the association between exposure to imatinib and the development of prostate cancer and other malignancies.

Primary immune deficiencies − improving our understanding of their role in immunological disease

Primary immune deficiencies − improving our understanding of their role in immunological disease

Mutation of the mouse Rad17 gene leads to embryonic lethality and reveals a role in DNA damage-dependent recombination.

Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents.

Immunodeficiency associated with DNA repair defects.

Cellular DNA is subjected to an onslaught of damaging insults which threaten cellular control and replication. It is therefore unsurprising that a plethora of damage response mechanisms operate to maintain genomic stability. These include processes that recognize and repair the damage, cell cycle checkpoints that prevent cell cycle progression in the presence of damage, and mechanisms, such as apoptosis, that remove damaged cells. In contrast, the development of effective immune responses is totally dependent on the generation of some 1012 genetically diverse cells, each bearing a unique receptor capable of recognizing a unique antigen/MHC combination. Only in this way can the immune system recognize the vast array of antigens that may be encountered. Higher organisms create this huge number of genetically diverse cells by breaking, randomly re-sorting and then joining the DNA sequences coding for antigen receptors by adapting the DNA repair mechanisms normally utilized to maintain genome stability. Individuals with defective DNA repair mechanisms have pleiotropic phenotypes including a predisposition to cancer, neurodegeneration and developmental abnormalities. Increasingly, immunodeficiency is recognized as a feature of some of these syndromes. Further evidence for the overlap between DNA repair and immune development is the finding that some individuals with poorly understood immunodeficiencies exhibit cellular ionizing radiation sensitivity, probably as a consequence of defective repair of radiation-induced DNA damage. This review discusses the common molecular defects identified in DNA repair-defective syndromes associated with immunodeficiency, as well as detailing the clinical features seen in affected individuals.

Cisplatin-modification of DNA repair and ionizing radiation lethality in yeast, Saccharomyces cerevisiae

Cis-diamminedichloroplatinum II (cisplatin) is a DNA inter- and intrastrand crosslinking agent which can sensitize prokaryotic and eukaryotic cells to killing by ionizing radiation. The mechanism of radiosensitization is unknown but may involve cisplatin inhibition of repair of DNA damage caused by radiation. Repair proficient wild type and repair deficient (rad52, recombinational repair or rad3, excision repair) strains of the yeast Saccharomyces cerevisiae were used to determine whether defects in DNA repair mechanisms would modify the radiosensitizing effect of cisplatin. We report that cisplatin exposure could sensitize yeast cells with a competent recombinational repair mechanism (wild type or rad3), but could not sensitize cells defective in recombinational repair (rad52), indicating that the radiosensitizing effect of cisplatin was due to inhibition of DNA repair processes involving error free RAD52-dependent recombinational repair. The presence or absence of oxygen during irradiation did not alter this radiosensitization. Consistent with this result, cisplatin did not sensitize cells to mutation that results from lesion processing by an error prone DNA repair system. However, under certain circumstances, cisplatin exposure did not cause radiosensitization to killing by radiation in repair competent wild type cells. Within 2 h after a sublethal cisplatin treatment, wild type yeast cells became both thermally tolerant and radiation resistant. Cisplatin pretreatment also suppressed mutations caused by exposure to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a response previously shown in wild type yeast cells following radiation pretreatment. Like radiation, the cisplatin-induced stress response did not confer radiation resistance or suppress MNNG mutations in a recombinational repair deficient mutant (rad52), although thermal tolerance was still induced. These results support the idea that cisplatin adducts in DNA interfere with RAD52-dependent recombinational repair and thereby sensitize cells to killing by radiation. However, the lesions can subsequently induce a general stress response, part of which is induction of RAD52-dependent error free recombinational repair. This stress response confers radiation resistance, thermal tolerance, and mutation resistance in yeast.

Genetic predisposition to skin cancer.

Here we review recent insights in the genetics of skin cancer susceptibility as gleaned from studies of three hereditary syndromes: basal cell nevus syndrome, familial melanoma/dysplastic nevus syndrome, and xeroderma pigmentosum. We provide a brief synopsis of the recent findings related to these syndromes in an attempt to illustrate several emerging themes in the genetics of skin cancer. These themes include 1) the recent identification of multiple cancer susceptibility genes that occur in a myriad of cellular regulatory pathways; 2) the relative specificity of certain regulatory pathways to the development of specific types of cancer; and 3) the important role of DNA damage caused by ultraviolet radiation and defective DNA repair mechanisms in the development of skin cancer. We also review the implications of this knowledge to clinical practice relative to risk assessment, primary prevention, and therapy.

Irradiation and second cancers. The thyroid as a case in point

The thyroid gland is highly sensitive to radiation during childhood: the risk of thyroid tumours is increased for mean doses as low as 100 mGy and for higher doses, the risk increases linearly with the dose. Excess relative risk is important, being 7.7 for 1 Gy delivered to the thyroid gland during childhood. The risk of thyroid tumours is modified by several factors: a) age at exposure: in childhood, the risk decreases with increasing age at exposure and is not significant after 20 years; b) gender: females are two times more likely than males to develop thyroid tumours; c) genetic predisposition due to a defect in DNA repair mechanisms, and dietary and hormonal factors may modify the risk; d) the influence of fractionation and dose rate is not well established. Radioiodine 131 (1311) used for medical purposes has almost no tumourigenic effect on the adult thyroid gland. The consequences of the Chernobyl accident have clearly shown that the risk of thyroid cancer after exposure to 1311 in childhood is important, and that such exposure should be prevented by potassium iodine prophylaxis. RET/PTC rearrangements are found in 60–80 % of papillary carcinomas and in 45 % of adenomas occurring after radiation exposure. They are found in 5–15 % of papillary carcinoma and in no follicular adenomas that occurred in the absence of radiation exposure.

Erratum: Molecular genetic features of myelodysplastic syndromes (MDS)

Recent developments in molecular genetics have provided insights on the molecular mechanisms that lead to myelodysplasias (MDS), secondary acute myelogenous leukemia (AML), therapy-induced AML, and elderly AML. These disorders are characterized by dysregulation of growth and differentiation of multilineage stem cells, a genetic profile characterized by unbalanced abnormalities that result in "unfavorable cytogenetics," and an increased frequency of intrinsic multidrug resistance. The unfavorable cytogenetics associated with this group of disorders include chromosome 5 and 7 monosomy, deletions of the long arm of chromosomes 5 and 7, inversions of chromosome 3, translocations, deletions and trisomies involving several other chromosomes. Presumably, these unbalanced chromosomal aberrations result in hemizygosity and unmasking of oncogenes or inactivation of tumor suppressor genes. In addition, polymorphisms in genes encoding metabolic detoxification enzymes, defective DNA repair mechanisms, and intrinsic chromosomal instability have been implicated in the etiology of the myelodysplastic syndromes. It is evident that the cytogenetics associated with MDS are highly complex and heterogeneous. This review summarizes the most recent developments in the understanding of the molecular changes associated with the development of myelodysplasias and related leukemic disorders.

The application of microsatellites in molecular pathology.

Present within the genome are large numbers of seemingly unimportant DNA segments arranged in repetitive units. Furthermore, these stretches of DNA contain variations or polymorphisms that are characteristic for an individual and results in a unique DNA fingerprint. Approximately 30% of the DNA repeat sequences are arranged as short tandem repeat sequences, which are called microsatellites. Microsatellites may consist of 1, 2 or 3 nucleotides; dinucleotides being the commonest. Microsatellites are characterised by being: stably inherited and hence highly conserved from one generation to the next, and unique to an individual and the same in different cells from the same individual. As a result of the above features, microsatellites can be used for personal identification, population genetic analysis and construction of evolutionary trees. In addition, they are located in several important gene loci and this allows microsatellites to be used as markers of disease and to provide information about individual gene status, especially in tumors. This can be accomplished by assessing allelic imbalance or loss of heterozygosity of a particular gene by analysing microsatellites located at specific loci in the gene. Recently, mutations within microsatellites have been described as a result of defective DNA repair mechanisms, resulting in the phenomenon of microsatellite instability. This has been implicated in the aetiopathogenesis of several hereditary and non-hereditary conditions. There are several ways of analysing microsatellites, the popular using radioactively-labelled primers and autoradiography. This method has several drawbacks, especially the use of radioactivity and interpretative/technical problems. The use of fluorescently-labelled primers, automated DNA sequencing coupled with a computer software package obviates these problems. This technique has the added advantage of analysing several microsatellites in large numbers of cases, simultaneously. Thus, microsatellite analysis has become an important investigative tool for the molecular biologist and has provided new information in many diseases.

Incidence of DNA replication errors in patients with multiple primary cancers.

Multiple primary cancers are a feature of hereditary nonpolyposis colorectal cancer in which defects in DNA repair mechanisms result in accumulation of replication errors within tumor DNA. We assessed replication error incidence in multiple primary cancer patients who may have similar genetic defects. DNA was obtained from 69 patients from the Yorkshire region who had developed colorectal cancer and one other primary tumor from the hereditary nonpolyposis colorectal cancer tumor spectrum (28 colorectal, 12 stomach, 15 ovary, and 14 uterus). DNA was also obtained from 86 sporadic, single primary cancer patients attending a colorectal cancer clinic. Replication error status was assessed at five microsatellite loci using fluorescent polymerase chain reaction and computer-assisted analysis. The replication error phenotype was observed in 7 of 86 (8 percent) of the sporadic single primary patients. This compared with 23 of 69 (33 percent) of the multiple primary group (P < 0.001). Replication error was also observed more frequently in each subgroup. Even excluding patients from families meeting the Amsterdam criteria (likely to be hereditary nonpolyposis colorectal cancer and have the replication error phenotype), this increased frequency remained in both the multiple primary group (P < 0.005) and multiple colorectal and colorectal/uterine subgroups (P < 0.001). Results suggest that genetic instability plays an important role in development of multiple primary cancers, particularly from certain cancer subsets. Testing for replication errors may be an appropriate way of identifying individuals at risk of multiple primary cancers.

Cerebellar neurodegeneration in human hereditary DNA repair disorders

Recent findings have focused attention on the role of apoptosis in neurodegenerative diseases, however, the apoptotic process in child-onset brain disorders has been little investigated. Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are hereditary disorders characterized by impaired DNA repair and neurodegeneration. We investigated apoptotic cell death in the cerebellum of five cases of XP group A (XPA), four cases of CS, and twelve controls, using TdT-mediated DIG-dUTP nick-end labeling (TUNEL) and immunohistochemical staining for bcl-2, bcl-x, p53, bax, BDNF and Trk B. The TUNEL-positive cells were found in the granule cells of the cerebellar cortex of two patients with XPA and two patients with CS, whereas such cells were not detected in the cerebellar cortex in controls. Upregulation of bcl-2 or BDNF was not observed, and bcl-x expression was not altered. Some patients showed nuclear expression of p53 in the granule cells and/or molecular layer, bax-positive glial cells in the cerebellar white matter, and a few Trk B-positive cells in the granular layer. These findings suggest that apoptotic cell death can be involved in the cerebellar degeneration in patients with hereditary defects in DNA repair mechanisms.

The role of DNA repair processes in determining response to cancer therapy

INTRODUCTION THE MOLECULAR basis of the radiosensitivity and chemosensitivity of human cells both normal and malignant, remains one of the most important areas of research in cancer biology. The field of mammalian DNA repair has undergone major developments over the last decade as a result of progress in somatic cell genetics and molecular biology. The aim of this paper is to review the contribution made by these areas to our current understanding of the role of DNA repair processes in determining the response of human cells to cancer therapy. Variations in DNA repair proficiency in an individual may determine susceptibility to environmental mutagens, carcinogens, or cancer therapy. Evidence for the importance of DNA repair pathways comes most directly from the rare, inherited human disorders, in which defects in DNA repair mechanisms are associated with hypersensitivity to radiation and DNA damaging drugs. These disorders are pleiotropic in phenotype, but one feature is an associated cancer-proneness. See Table 1. Although these syndromes are themselves rare in the general population, heterozygote carriers have been estimated at frequencies of O. l-10%. It is conceivable that heteroxygous individuals may also have some impairment of DNA repair, and indeed relatives of ataxia telangectasia homozygotes have an increased incidence of some common solid tumours [l]. Thus a defect in DNA repair processes may be involved in susceptibility to carcinogenesis and such individuals may be more prevalent in the cancer treatment population than the population generally. The complexity of DNA repair processes in mammalian cells is well exemplified by our current level of knowledge of the ultraviolet radiation-DNA repair process. A large number of different cell lines have been established exhibiting hypersensitivity to ultraviolet radiation in nitlo, both from rodents, and patients with xeroderma pigmentosum [2, 31. Using cell fusion techniques, it is possible to demonstrate that such lines represent different genetic defects leading to expression of the ultraviolet hypersensitive phenotype, i.e. they belong to different ‘genetic complementation’ groups. One interpretation of this would be that a large number of genes are involved in repair of ultraviolet induced DNA damage.

NEUROLOGICAL DISEASE IN XERODERMA PIGMENTOSUM: DOCUMENTATION OF A LATE ONSET TYPE OF THE JUVENILE ONSET FORM

Xeroderma pigmentosum (XP) is an autosomal recessive, neurocutaneous disorder characterized by sunlight-induced skin cancers and defective DNA repair. Many XP children develop a primary neuronal degeneration. We describe 2 unusual XP patients who had a delayed onset of XP neurological disease. Somatic cell genetic studies indicated that they have the same defective DNA repair gene and are both in XP complementation group A. These 2 patients, together with a group A patient previously reported from London, establish as a distinct clinical entity the late onset type of the juvenile onset form of XP neurological disease. The functional capacity of these patients' cultured fibroblast strains to survive after treatment with ultraviolet radiation indicates that their DNA repair defect is less severe than that of typical group A patients who have a more severe neurodegeneration with an earlier symptomatic onset. The premature death of nerve cells in XP patients (which is presumably due to their inherited defects in DNA repair mechanisms) suggests that normal repair of damaged DNA in neurons is required to maintain integrity of the human nervous system.

Leiomyoma of the Suprarenal Gland in a Child with Ataxia-Telangiectasia

We report the occurrence of a leiomyoma of the suprarenal gland in a 10-year-old girl with ataxia-telangiectasia (A-T). Muscle cell tumors are very uncommon in this gland as they are in A-T. Possible reasons for developing nonhematologic tumors in this syndrome are reviewed. A defect in DNA repair mechanisms probably favors, in young children, the expression of tumors normally expected in the aged.

Duchenne muscular dystrophy: recent concepts.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease, affecting only males and transmitted by females. Recent proposed pathogeneses are defective DNA repair mechanism and cell-mediated cytotoxicity. Serum CPK estimation is most frequently high (30–300) and is also reliable for detecting carriers. It is important to recognise the affected boys as early as possible. Diagnosis of DMD is made from clinical features, CPK estimation, EMG and ECG abnormalities. The use of agents to reduce influx of intracellular calcium in patients with DMD are emerging for therapeutic consideration. New advance in genetic counselling is the localisation of the gene for DMD using DNA polymorphisms.

Alzheimer disease fibroblasts are hypersensitive to the lethal effects of a DNA-damaging chemical

A group of fibroblats lines from three patients with the sporadic form of Alzheimer disease (AD) showed a small but statistically significant hypersensitivity to the lethal effects of the DNA-damaging chemical N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) when compared with lines from eight normal control subjects. A fibroblast line from a patient with a dominantly inherited form of familial AD had a hypersensitivity similar to that of the three sporadic AD lines. However, fibroblast lines from a group of five patients with spinal muscular atrophy (SMA) were not hypersensitive to the chemical, demonstrating that not every primary neuronal degeneration manifests hypersensitivity to this chemical. These findings are consistent with possibility that a defect in DNA-repair mechanisms may be the cause of the in vitro hypersensitivity, as well as the premature death of neurons in vivo, in both the sporadic and familial forms of AD.

Hypersensitivity to DNA-damaging agents in cultured cells from patients with Usher's syndrome and Duchenne muscular dystrophy.

Lymphoblastoid lines from nine Usher's syndrome (recessively inherited retinitis pigmentosa and congenital sensorineural deafness) patients (representing eight kindreds) and from ten Duchenne muscular dystrophy patients (representing seven kindreds) showed a small but statistically significant hypersensitivity to the lethal effects of X-rays, as measured by the cellular ability to exclude the vital dye trypan blue, when compared with lines from 26 normal control subjects. Fibroblast lines from the Usher's syndrome patients, treated with X-rays or the radiomimetic, DNA-damaging chemical N-methyl-N'-nitro-N-nitrosoguanidine, also showed a statistically significant hypersensitivity when compared to normal fibroblast lines. These findings are consistent with the possibility that defective DNA repair mechanisms may be involved in the pathogenesis of these degenerative diseases.

Recombination in Saccharomyces cerevisiae: a DNA repair mutation associated with elevated mitotic gene conversion.

A mutant haploid strain of Saccharomyces cerevisiae has been isolated that is sensitive to the alkylating agent methyl methanesulfonate at a concentration of 0.01% (vol/vol). The strain also shows sensitivities to x-rays and ultra-violet light, which cosegregate with sensitivity to methyl methanesulfonate as a single gene defect. An analysis of the sensitivity to ultraviolet light indicates that the mutation interferes with the excision of pyrimidine dimers. Diploids homozygous for the mutant gene exhibit elevated frequencies of spontaneous mitotic recombination at the ade6 locus. The results indicate that all the events are due to gene conversion. Mitotic recombination was also found to be elevated for three loci other than ade6. Thus, the recombinational effect seems not to be locus specific. Linkage and allelism tests indicate that the mutation is an allele of the known radiation-sensitive gene rad18. The various effects of this new rad18 allele (rad18-3) are discussed in terms of a defect in DNA repair mechanisms.