Ataxia telangiectasia: more variation at clinical and cellular levels.

Ataxia telangiectasia (A-T) is a rare recessively inherited disorder resulting in a progressive neurological decline. It is caused by biallelic mutation of the ATM gene that encodes a 370 kDa serine/threonine protein kinase responsible for phosphorylating many target proteins. ATM is activated by auto(trans)phosphorylation in response to DNA double strand breaks and leads to the activation of cell cycle checkpoints and either DNA repair or apoptosis as part of the cellular response to DNA damage. The allelic heterogeneity in A-T is striking. While the majority of mutations are truncating, leading to instability and loss of the ATM protein from the allele, a significant proportion of patients carry one of a small number of mutations that are either missense or leaky splice site mutations resulting in retention of some ATM with activity. The allelic heterogeneity in ATM, therefore, results in an equally striking clinical heterogeneity. There is also locus heterogeneity because mutation of the MRE11 gene can cause an obvious A-T like disorder both clinically and also at the cellular level and mutation of the RNF168 gene results in a much milder clinical phenotype, neurologically, with the major clinical feature being an immunological defect.

Accelerated telomere shortening in ataxia telangiectasia.

Ataxia telangiectasia (AT) is characterized by neurological deterioration, immunodeficiency, spontaneous chromosomal instability, hypersensitivity to ionizing radiation, predisposition to cancer, particularly T cell leukaemia and lymphoma, and premature ageing. The most commonly observed defect affecting telomeres in humans is telomeric fusions, particularly in T lymphocytes in AT patients. Rarely, some tumour cells, like senescent cells, have dicentric chromosomes that may arise as a result of telomeric sequence loss. We show that the AT mutation in the homozygous state confers a predisposition to accelerated telomere shortening with increasing age in peripheral blood lymphocytes (PBLs), which may be linked to premature senescence. We also show that telomeric fusions are associated with large (> 90%) preleukaemic translocation clones in T cells. We propose that these fusions may result from a compound effect of accelerated telomere shortening, together with a growth advantage of cells in large clones which leads to further telomere loss. Fusions are not observed in leukaemic cells in these patients. There is no evidence that either accelerated telomere loss per se or telomeric fusions are important in tumourigenesis. Telomerase is present in both normal and AT lymphocytes and so neither telomere shortening nor telomeric fusions can be explained by the absence of telomerase.

Severe reaction to radiotherapy for breast cancer as the presenting feature of ataxia telangiectasia.

BACKGROUND: Severe early and late radiation reaction to radiotherapy is extremely rare in breast cancer patients. Such a reaction prompted an investigation into a 44-year-old mother (patient A-T213). METHODS: A neurological examination was performed and blood lymphocytes and skin fibroblasts were assessed for radiosensitivity chromosomally and by colony-forming assay. The ATM gene was sequenced and ATM mutations modelled by site-directed mutagenesis. The ATM kinase activity was also assessed. RESULTS: Patient A-T213 was normally ambulant with no ataxia and minimal other neurological features. T lymphocytes and skin fibroblasts were unusually radiosensitive, although less sensitive than in classical ataxia telangiectasia (A-T). A lymphoblastoid cell line and skin fibroblasts expressed ATM protein with some retained kinase activity. One missense ATM mutation c.8672G>A (p.Gly2891Asp) and a c.1A>G substitution were identified. In the modelling system, the p.Gly2891Asp mutant protein was expressed and shown to have residual ATM kinase activity. CONCLUSION: Patient A-T213 has a milder form of A-T with biallelic ATM mutations, which may have contributed to breast cancer development, and certainly caused the severe radiation reaction. Ataxia telangiectasia should be investigated as a potential cause of untoward severe early and late radiation reactions in breast cancer patients.

Lymphoid tumours and breast cancer in ataxia telangiectasia; substantial protective effect of residual ATM kinase activity against childhood tumours.

BACKGROUND: Immunodeficiency in ataxia telangiectasia (A-T) is less severe in patients expressing some mutant or normal ATM kinase activity. We, therefore, determined whether expression of residual ATM kinase activity also protected against tumour development in A-T. METHODS: From a total of 296 consecutive genetically confirmed A-T patients from the British Isles and the Netherlands, we identified 66 patients who developed a malignant tumour; 47 lymphoid tumours and 19 non-lymphoid tumours were diagnosed. We determined their ATM mutations, and whether cells from these patients expressed any ATM with residual ATM kinase activity. RESULTS: In childhood, total absence of ATM kinase activity was associated, almost exclusively, with development of lymphoid tumours. There was an overwhelming preponderance of tumours in patients <16 years without kinase activity compared with those with some residual activity, consistent with a substantial protective effect of residual ATM kinase activity against tumour development in childhood. In addition, the presence of eight breast cancers in A-T patients, a 30-fold increased risk, establishes breast cancer as part of the A-T phenotype. CONCLUSION: Overall, a spectrum of tumour types is associated with A-T, consistent with involvement of ATM in different mechanisms of tumour formation. Tumour type was influenced by ATM allelic heterogeneity, residual ATM kinase activity and age.

Immunodeficiency in ataxia telangiectasia is correlated strongly with the presence of two null mutations in the ataxia telangiectasia mutated gene.

Immunodeficiency affects over half of all patients with ataxia telangiectasia (A-T) and when present can contribute significantly to morbidity and mortality. A retrospective review of clinical history, immunological findings, ataxia telangiectasia mutated (ATM) enzyme activity and ATM mutation type was conducted on 80 consecutive patients attending the National Clinic for Ataxia Telangiectasia, Nottingham, UK between 1994 and 2006. The aim was to characterize the immunodeficiency in A-T and determine its relationship to the ATM mutations present. Sixty-one patients had mutations resulting in complete loss of ATM kinase activity (group A) and 19 patients had leaky splice or missense mutations resulting in residual kinase activity (group B). There was a significantly higher proportion of patients with recurrent sinopulmonary infections in group A compared with group B (31 of 61 versus four of 19 P = 0.03) and a greater need for prophylactic antibiotics (30 of 61 versus one of 19 P = 0.001). Comparing group A with group B patients, 25 of 46 had undetectable/low immunoglobulin A (IgA) levels compared with none of 19; T cell lymphopenia was found in 28 of 56 compared with one of 18 and B cell lymphopenia in 35 of 55 compared with four of 18 patients (P = 0.00004, 0.001 and 0.003 respectively). Low IgG2 subclass levels and low levels of antibodies to pneumococcal polysaccharide were more common in group A than group B (16 of 27 versus one of 11 P = 0.01; 34/43 versus six of 17 P = 0.002) patients. Ig replacement therapy was required in 10 (12.5%) of the whole cohort, all in group A. In conclusion, A-T patients with no ATM kinase activity had a markedly more severe immunological phenotype than those expressing low levels of ATM activity.

Isolation and characterization of four adenovirus type 12-transformed human embryo kidney cell lines.

Four transformed cell lines were established from cultures of human embryo kidney (HEK) cells microinjected or transfected with cloned adenovirus 12 (Ad12) EcoRI-C DNA (0 through 16.5 map units of the left-hand end of the viral genome). Each cell line showed a different growth pattern. Southern blotting demonstrated that all of the cell lines contained Ad12-specific DNA sequences, but in the microinjected isolates these were at a much lower copy number than in the transfected isolate. Two cell lines (Ad12 HEK 1 and 3) appeared to contain tandemly repeated Ad12 EcoRI-C DNA fragments. Immunoprecipitation and Western blotting confirmed that Ad12 early region 1 (E1) proteins were being expressed by all four of the transformed cell lines, but indicated that E1A polypeptide expression was considerably less than E1B polypeptide expression. All of the Ad12-transformed HEK cell lines were tumorigenic when inoculated intracranially into athymic nude mice.

Properties of rat cells transformed by DNA plasmids containing adenovirus type 12 E1 DNA or specific fragments of the E1 region: comparison of transforming frequencies.

In this paper, we describe for the first time the transformation of normal rat cells by DNA equivalent to adenovirus type 12 Early Region 1 (E1A). This DNA was 30-fold less efficient at transformation than DNA encoding the entire E1 region. Those established lines expressing a full complement of adenovirus type 12 E1 proteins were phenotypically indistinguishable from adenovirus type 12 virus-transformed cell lines. However, cell lines produced by plasmids carrying subgenomic fragments of E1 DNA and therefore not expressing E1B Mr 52,000 protein took longer to establish and produced tumors only after a protracted latent period. A Giemsa-banding study showed that adenovirus transformation can occur without disruption of the normal rat karyotype.

Differential transformation of primary human embryo retinal cells by adenovirus E1 regions and combinations of E1A + ras.

The efficiency of transformation of primary human embryo retinal (HER) cells by the adenovirus E1 region (E1A + E1B) depended on the virus serotype whereas transformation by E1A alone was a rare event regardless of serotype. Activated human c-Ha-ras and N-ras genes co-operated differentially with different E1As for HER transformation but were ineffective without E1A. Ras + E1A co-transformants containing Ad 12 E1A established directly from foci, in contrast to those containing Ad 2 or Ad 5 E1A. A spectrum of activated ras gene expression was found in stable co-transformants with mRNA and protein levels being lower in Ad 12 E1A + N-ras than Ad 2 E1A + N-ras cell lines. Down regulation of E1A transcription in the absence of E1B was found in Ad 2 E1A + Ha-ras transformants only but E1A protein levels were similar to those in Ad 2 E1A + N-ras or Ad 5 E1A + E1B cell lines. HER cell transformants which contained Ad 12 E1A were more tumourigenic than those which contained the Ad 2 or Ad 5 E1A. This unique transformation system shows that stable malignant transformation of primary human cells in vitro is a complex process requiring the combined activities of two or more types of genes.

The binding of guanine nucleotide to N-ras p21--a phosphorous and proton magnetic resonance study.

One dimensional [1H] and [31P] nuclear magnetic resonance (NMR) studies have been carried out on purified wild type and mutant (Gly-12----Asp) N-ras protein expressed at high level in E. coli. Both proteins were isolated as stable 1:1 molar complexes with GDP with the upper limit for the first order rate constant for nucleotide dissociation 3 x 10(-4)s-1. From observation of the [31P] NMR spectrum after the addition of GTP it was concluded that the rate of nucleotide hydrolysis is appreciably greater than that of nucleotide exchange. Differences in the [31P] spectra of mutant and wild type proteins suggest that the mutation has a direct influence on the catalytic step. [1H] NMR spectra obtained for both mutant and wild type p21 were consistent with proteins of considerable stability and the addition of urea to concentrations of 4M appeared to cause little disruption in secondary structure. Additionally, the protein environment of the bound nucleotide remained well defined in the presence of a number of added reagents and over the pH range 5.8-9.5. The data are discussed in the light of the known crystal structure for H-ras p21 and indicate that the transforming mutation of aspartate for glycine-12 results in structural perturbations near the nucleotide binding site.

The expression of the retinoblastoma gene product Rb1 in primary and adenovirus-transformed human cells.

Polyclonal antibodies to the human retinoblastoma gene product (Rb1) have been produced in rats by immunisation with a fusion protein comprising part of Rb1 together with the E. coli beta-Gal sequence. We have used these antibodies in Western blotting studies to screen a number of human foetal tissues and organs and found approximately similar levels of expression of Rb1 in all of them. The protein seems to be somewhat more abundant in some cell lines produced by transfection of human embryo retinal (HER) cells with adenovirus 12 early region 1 (Ad 12 E1), Ad 5 E1, Ad 2 E1A + mutant N-ras or SV40 DNA. Using co-immunoprecipitation followed by Western blotting we have shown that the Rb1 protein binds to Ad 12 E1A 266 and 235 amino acid proteins. This interaction is ionic strength dependent but is unaffected by non-ionic detergent up to a concentration of at least 1%. In Ad 12 infected human cells it appears that less E1A is bound to Rb1 than in the transformants. These results are discussed in view of the known similarities and differences between the amino acid sequences of Ad 12 and Ad 5 E1A proteins.

Sensitivity to ionising radiation of transformed human cells containing mutant ras genes.

Measurement of colony forming ability following exposure to gamma-rays has been performed on human retinoblasts transformed with either adenovirus 5 or 12 early region 1 DNA, adenovirus early region 1A plus activated N- or H-ras DNA or SV40 DNA. In contrast to recently reported results (M.D. Sklar, 1988, Science, 239, 645-647), we found no general correlation between transformation with activated ras and increased radiation resistance. Similarly, there was no correlation between D0 values and the level of expression of ras p21 in transformed human retinoblasts as determined by liquid competition assay. Indeed, cell lines with very similar D0 values had ras contents varying by up to one hundred fold. Cell lines transformed with SV40 DNA were generally less sensitive to ionising radiation than adenovirus and/or ras transformants, but even so the variation in sensitivity within these encompassed the whole spectrum of values obtained for the ras transformants. It may be interesting to note, however, that two out of the three ras transformants which were least sensitive to gamma-rays were cell lines expressing the highest levels of p21.

Ataxia-telangiectasia-like disorder (ATLD)-its clinical presentation and molecular basis.

Comparison of the clinical and cellular phenotypes of different genomic instability syndromes provides new insights into functional links in the complex network of the DNA damage response. A prominent example of this principle is provided by examination of three such disorders: ataxia-telangiectasia (A-T) caused by lack or inactivation of the ATM protein kinase, which mobilises the cellular response to double strand breaks in the DNA; ataxia-telangiectasia-like disease (ATLD), a result of deficiency of the human Mre11 protein; and the Nijmegen breakage syndrome (NBS), which represents defective Nbs1 protein. Mre11 and Nbs1 are members of the Mre11/Rad50/Nbs1 (MRN) protein complex. MRN and its individual components are involved in different responses to cellular damage induced by ionising radiation and radiomimetic chemicals, including complexing with chromatin and with other damage response proteins, formation of radiation-induced foci, and the induction of different cell cycle checkpoints. The phosphorylation of Nbs1 by ATM would indicate that ATM acts upstream of the MRN complex. Consistent with this were the suggestions that ATM could be activated in the absence of fully functional Nbs1 protein. In contrast, the regulation of some ATM target proteins, e.g. Smc1 requires the MRN complex as well as ATM. Nbs1 may, therefore, be both a substrate for ATM and a mediator of ATM function. Recent studies that indicate a requirement of the MRN complex for proper ATM activation suggest that the relationship between ATM and the MRN complex in the DNA damage response is yet to be fully determined. Despite the fact that both Mre11 and Nbs1 are part of the same MRN complex, deficiency in either protein in humans does not lead to the same clinical picture. This suggests that components of the complex may also act separately.

Molecular pathology of ataxia telangiectasia.

Ataxia telangiectasia (A-T) is one of a group of autosomal recessive cerebellar ataxias. Presentation is usually by the age of 2 years and ataxia of both upper and lower limbs develops, such that by early teenage most patients require a wheelchair for mobility. Speech and eye movement are also affected. Other important features are t(7;14) translocations, immunodeficiency, a high serum alpha fetoprotein concentration, growth retardation, telangiectasia-most noticeably on the bulbar conjunctiva-and a very high risk of developing a lymphoid tumour. Patients also show an increased sensitivity to ionising radiation. The classic form of A-T results from the presence of two truncating ATM mutations, leading to total loss of the ATM protein, a protein kinase. Importantly, A-T shows clinical heterogeneity, including milder forms where neurological progression may be slower or of later onset. In these cases there is a correlation between the preservation of neurological function, decreased radiosensitivity, and the degree of retained ATM protein kinase activity. Considerable scope remains for understanding the progress of the disorder in relation to the types of ATM mutation present.

Transformation of human embryo retinoblasts with simian virus 40, adenovirus and ras oncogenes.

Few human cell systems have been described in which a number of different genes induce transformation. The present investigation reports on our studies using primary human embryo retinoblasts as a model system to monitor transformation and the subsequent behaviour of individual transformants in terms of establishment, the frequency of immortalization and tumourigenic potential. SV40, Adenovirus E1 and E1A, and combinations of Adenovirus E1A and activated H-ras or N-ras were examined as transforming agents. Considerable differences were observed in the ability of these genes to transform human cells, to induce immortal lines and to produce cell lines with a tumourigenic phenotype. Activated ras genes were non-transforming in this system and the degree of complementation with adenovirus E1As in transformation experiments was dependent on both the adenovirus serotype and the ras gene used. The development of tumourigenic cell lines required the expression of more than one oncogene and additional genetic events were required in some in some instances before immortal cell lines were obtained. These findings contribute to the concept that the development of cancer is a multi-step process.

Genetic and cellular features of ataxia telangiectasia.

Ataxia telangiectasia (AT) is a developmental disorder in which many organ systems are affected. The children are recognized by a progressive cerebellar deterioration. The gene for AT has now been localized to a region of chromosome 11q22-23 of no more than 3Mb in size and its product appears to be involved directly or indirectly in some form of DNA recombination. Patients and their cells are unusually sensitive to ionizing radiation and various radiometric drugs. Observations on the progressive nature of the disorder, with loss of selected cells or failure to develop normally, might be compatible with the pathological effect of an inability to correctly regulate apoptosis in some cell lineages. While this is an intriguing speculation there is, at present, no evidence for such a defect in AT.

A haplotype common to intermediate radiosensitivity variants of ataxia-telangiectasia in the UK.

In a study of ataxia-telangiectasia (A-T) in the UK, patients in 10 out of 60 families were shown to have a much lower level of chromosomal radiosensitivity compared with the majority of patients. In some patients the level of radiosensitivity was hardly distinguishable from normal. Patients in this group, however, could be distinguished clinically from the majority either by the later onset of severe cerebellar features or the slower rate of progress of the disorder. By using highly polymorphic microsatellite repeat markers a chromosome 11q22-23 haplotype common to the majority of these patients, and not occurring in any non-A-T chromosome in 60 families, was identified on one chromosome. The haplotype probably defines the region of the A-T gene in these families and the mutation associated with this haplotype may be much less severe than the second mutation thereby producing the slightly milder phenotype.

Genetic and physical mapping of the ataxia-telangiectasia locus on chromosome 11q22-q23.

The identification of A-T gene(s) using both positional and functional cloning techniques has been a major objective in A-T research over the past 10 years. Functional cloning, using complementation of the radiosensitivity phenotype, has met with some success, although technical problems remain to be overcome. Recent progress, however, in both genetic and physical mapping of the A-T locus on chromosome 11q22-q23, described in this review, suggests that the positional cloning of candidate genes should be achieved in the very near future. The region of the chromosome containing the gene(s) has been identified, and is no more than 1.6 Mb in size. The detailed physical characterization of this region, as a preliminary to candidate gene isolation, is now underway. There are, however, still some unresolved issues, most notably the existence of four A-T complementation groups, with the resulting supposition that these equate to a number of different genes. Although genetic linkage evidence does not support the hypothesis of genetic heterogeneity, the possibility of a cluster of genes at the 11q22-23 locus cannot be ruled out. It is likely that the explanations for this and other problems such as discrepancies in expected levels of consanguinity, and difficulties in the classification of atypical phenotypes will become much more obvious once a gene or genes have been cloned.

Cancer and DNA processing disorders.

Defects in cloned DNA repair genes are now associated with particular human disorders in which an important feature is a predisposition to cancer. Recently some repair genes have been implicated in other aspects of DNA metabolism such as transcription initiation. In addition mutations in a single gene can give rise to phenotypes recognised clinically as different disorders. These newly appreciated complexities, amongst others, will eventually help us to understand the development of the complete clinical phenotype in a range of 'DNA processing disorders'.

Differentiation of normal and adenovirus-12 E1 transformed human embryo retinal cells.

Following the treatment with dibutyryl cyclic AMP (dbcAMP) in the absence of serum, a proportion of cultured human embryo retinoblasts will differentiate rapidly. This is characterized by cell rounding and the extension of neuritic-type processes. A transformed cell line (Ad 12 HER 10), developed by transfection of human embryo retinoblasts with adenovirus 12 (Ad 12) early region 1 (E1) DNA, forms retinoblastoma-like tumours in athymic nude mice and retains the ability to extend neuritic-like processes after treatment with dbcAMP in the absence of serum. This response, which occurs in almost all cells (over 98%), is accompanied by growth arrest and reverses rapidly after re-exposure to serum. Other agents known to increase intracellular cAMP also mediate differentiation. Ad 12 HER 10 cells were shown to contain the neuronal markers, neurone-specific enolase and protein gene product 9.5, but not the glial cell marker glial fibrillary acidic protein (GFAP). Activity of the neurotransmitter enzyme acetylcholinesterase was also detected and found to increase after differentiation. Interestingly, the expression of the Ad 12 E1 transforming proteins did not change throughout differentiation. These results show first, that the Ad 12 HER 10 cell line can differentiate in a similar manner to that observed in a proportion of primary retinoblasts and second, that the expression of transforming proteins does not preclude at least part of that differentiative capacity. This is the first demonstration of in vitro differentiation in an adenovirus-transformed human cell line, as well as offering a useful system for the study of factors controlling growth and differentiation of tumorigenic human retinoblasts.

Host range mutants of adenovirus type 12 E1 defective for lytic infection, transformation, and oncogenicity.

The human adenovirus type 12 (H12) E1A region encodes two early proteins of 266 amino acid residues (266R) and 235R whilst the H12 E1B promoter directs the synthesis of two major proteins of 163R and 482R. To determine the functions of E1A and E1B in lytic infection and oncogenic transformation we have isolated and characterized a series of H12 E1 mutants. Mutant H12 hr 700 contains a point mutation in exon 1 that alters a single amino acid common to both the 266 and 235R proteins. This mutant synthesized reduced levels of E1 and structural proteins at delayed times in HEK cells, transformed BRK cells, and induced tumors in newborn rats at reduced efficiency compared to wild-type virus. The mutation in H12 in 600 truncates the 266R protein in its unique sequences but this mutant synthesized the 235R, E1B, and structural proteins at delayed times in HEK cells. H12 in 600 was nontransforming but induced rare tumors in newborn rats. A third E1A mutant H12 in 601 synthesized no E1A proteins, reduced levels of E1B and structural proteins at delayed times in lytic infections, and was not a transforming or oncogenic virus. Three E1B mutants were studied in detail. Both H12 hr 703 and H12 in 602 encode N-terminal truncated 482R proteins whereas H12 del 620 encodes an in-frame internally deleted 482R protein. All three synthesized reduced amounts of E1A proteins and the E1B 163R protein, identifying a regulatory function for the 482R protein. None of the E1B mutants could transform and only H12 del 620 could induce rare tumors in newborn rats. These results show that H12 oncogenesis requires the coordinated expression of the E1 proteins.