WO2003020977A2 - Nucleotide repeats assay - Google Patents

Abstract

The present invention relates to a method for identifying a substance capable of modulating the number of nucleotide repeats in a cell displaying expanded nucleotide repeat instability. The present invention further provides the use of a substance or substances identified by the assay of the present invention in the treatment of inherited human or animal disease characterised by nucleotide repeat expansion, for example Huntington's disease and the like as well as cells and/or cell lines, which may be used in the method.

Description

Nucleotide Repeats Assay Field of the Invention

The present invention relates to a method for identifying a substance capable of modulating the number of nucleotide repeats in a cell displaying expanded nucleotide repeat instability. The present invention further provides the use of a substance or substances identified by the assay of the present invention in the treatment of inherited human or animal disease characterised by nucleotide repeat expansion, for example Huntington's disease and the like as well as cells and/or cell lines, which may be used in the method. Background of the Invention

The expansion of unstable trinucleotide sequences in the human genome is the primary genetic defect associated with a particular class of complex human diseases, including myotonic dystrophy type 1 (DM1), Huntington disease (HD), fragile X syndrome (FRAXA), Friedreich ataxia (FRDA) and an ever increasing number of spinocerebellar ataxias (SCAs) (1). Most of these disorders, such as DM1, HD and the SCAs are associated with the expansion of CAG'CTG repeats. In addition to a common genetic defect, these conditions also share an unusual pattern of inheritance known as genetic anticipation, that is, a decreasing age of onset and the worsening of symptoms through successive generations. Moreover, the repeats are also somatically unstable and inter- and intra-tissue differences in repeat length distributions are commonly observed.

Of all of the CAG'CTG expansion disorders, DM1 presents the widest range of expansion sizes between patients and is the disorder for which somatic mosaicism is most fully characterised. DM1 is an autosomal, dominantly inherited, disorder characterised by muscle weakness, wasting and myotonia, but also including a variety of additional symptoms with a wide range of ages of onset (2). DM1 is associated with the expansion of a CTG sequence within the 3'-untranslated region (UTR) of the DM protein kinase (DMPK gene on chromosome 19 (3-8). The expanded DM1 alleles exhibit extensive germline and somatic instability, biased towards expansion (5, 9-16). Interestingly, larger repeat lengths are consistently observed in the major affected tissue, muscle, relative to blood DNA (11-13,16). In addition, longer average repeat lengths and broader ranges of variability have been found in older patients (16-18). The size of the inherited expanded allele and the age of the patient have been identified as critical factors controlling the degree of somatic mosaicism (17,18). Given that expansion-biased germline instability accounts for the phenomenon of genetic anticipation (19), it seems likely that the characteristic expansion-biased, age-dependent, tissue-specific dynamics of the repeat in the soma accounts, at least in part, for the progressive nature and tissue specificity of the symptoms in DM1. Moreover, Kennedy and Shelbourne have recently revealed striking large expansions restricted to the affected tissue, striatum, in a knock-in mouse model that suggests that somatic mosaicism similarly contributes to pathogenesis in HD (20).

It is generally assumed that variability arises through DNA replication slippage during cell division (21,22). Such a mechanism might be facilitated by the propensity of these sequences to adopt unorthodox non-B-DNA structures, such as slipped strand DNA (S-DNA) (23), which could induce polymerase stalling and/or stabilise replication intermediates. Attractive as this suggestion is, there is as yet no direct evidence from a mammalian system to support the existence of such structures in vivo, nor the hypothesis that mutations arise during DNA replication. Indeed, there are no obvious relationships between tissues in which the repeats are prone to change and rates of cell turnover (11-13,16,20,24-26). However, it should be considered that such analyses have been primarily performed on whole tissues or organs comprising multiple cell types likely to have very differing dynamics in terms of both cell turnover and repeat metabolism. Such a complex scenario confounds attempts to make simple correlations and may have obscured some more subtle relationships.

In order to gain greater insight into the mechanisms underlying triplet repeat dynamics, trinucleotide tracts have been cloned into simple model organisms, such as Escherichia coli (27) and Saccharomyces cerevisiae (28). These systems have revealed some important insights into the factors affecting repeat dynamics, such as orientation (putatively with respect to replication origins), transcription and DNA repair gene mutations. However, the repeats are inherently biased toward contraction in such systems, in complete contrast to the predominantly expansion-biased behaviour observed at most loci in humans. So far the analyses of repeat stability in cell cultures of patient-derived cells have yielded mixed results. The DM1 repeat continued to expand in vitro in primary cells derived from a DM1 foetus (29). In contrast, Epstein-Barr virus (EBN)-transformed lymphoblastoid cell lines (LBCLs) derived from DM1 patients adopt an unusual pattern of repeat variability (30). In addition to the frequent small length change mutations that are biased toward expansion (54), rarer, but very large, deletion mutants are also observed at a frequency exceeding that detected in vivo. These results suggest that the EBV-transformation process results in altered cellular DΝA metabolism. Such an effect might be expected given that some EBN proteins alter the transcriptional activity of cellular genes involved in DΝA processing (31). Recently it has been reported that expanded GAA repeats at the FRDA locus in EBN-transformed LBCLs display contractions and expansions of similar magnitudes and frequencies (32). Thus, it would appear that although EBN-transformed LBCLs may be useful for modelling some aspects of repeat metabolism, their failure to faithfully recreate the in vivo dynamics limits their overall utility. Moreover, although primary human cell cultures may be good models, their availability from individuals with rare inherited disorders is severely limited.

In order to create additional model systems in which repeat biology may be assessed in vivo in a mammalian system, transgenic mice have been created containing unstable expanded CAG^«CTG arrays (26,33-38). One transgenic mouse line in particular, Dmt-D, which comprises a single copy of the construct carrying a portion of ~750bp of flanking DΝA from the Human DM1 locus, including most of the DMPK 3'-UTR and a repeat array of ~ 162 CTG repeats, derived from the human DM1 locus, has been shown to reproduce the dramatic tissue-specific, age-dependent and expansion-biased repeat instability associated with somatic mosaicism in DM1 patients (25). However, in the Dmt-D line there was evidence for significant instability in a number of tissues further confirming a major role for flanking DΝA sequences in modifying repeat dynamics (25). Similar mutational dynamics have been reported in transgenic models carrying large 45kb fragments of the human DM1 locus, incorporating ~300 repeats (26). Transgenic mouse models that mimic the trinucleotide somatic instability observed in patients represent a good tool to resolve the molecular mechanisms regulating repeat dynamics in a mammalian environment. However, a disadvantage in the use of transgenic mouse models in such studies include the high overheads involved.

The current art does not readily permit the analysis of repeat instability in cell lines derived from mammals modelling expanded repeat instability and recreating the expansion biased, tissue specific, age dependent somatic mosaicism characteristic of CAG'CTG expansion diseases. Indeed, such analysis of cell lines has yielded varying results, whose interpretation is hampered by the use of EBN and SN40 transformed cell lines, which affect the transcriptional activity of genes involved in DΝA processing. Furthermore, a method of screening for therapeutic agents for associated diseases using such cell lines has not been provided. It is amongst the objects of the present invention to obviate and/or mitigate the aforementioned disadvantages.

Summary of the Invention

Without wishing to be bound by theory, the present invention is based in part on the development of a cell culture system from animals that faithfully reproduce their in vivo nucleotide repeat dynamics in vitro. The cell culture system can be used to investigate the multiple factors affecting the dynamics of repetitive sequences under controlled conditions. Thus, the present inventors have established primary cell cultures from tissue samples harvested from Dmt-O transgenic mice and monitored the CTG nucleotide repeat over extensive time periods, with the aim of clarifying the effect of a large number of cell divisions on nucleotide repeat stability.

It is an object of the present invention to provide an assay for screening agents having an effect on nucleotide repeat number.

In a first aspect, the present invention provides an in vitro method for identifying a substance capable of modulating the number of nucleotide repeats in a cell, which method comprises: a) providing a test substance to a cell that displays expanded nucleotide repeat instability; and b) determining if said substance modulates the number of nucleotide repeats of the cell or cell line derived from said cell.

The cell according to the present invention is typically part of a cell line and may be, for example, an immortal cell or cell line and/or for example, a cell or cell line that displays late rapid cell growth characteristics. Alternatively, the cell may be obtained from a) a subject-derived cell or a transgenic animal displaying expanded nucleotide repeat instability immediately prior to use of the cell in the assay of the present invention or b) an established cell line displaying expanded nucleotide repeat instability. Preferably, the cell is a mammalian cell such as a mouse cell. Conveniently, the Dmt-O mouse (25), the entire contents of which are incorporated herein, may be used as a highly controlled renewable source of primary cells thus, not relying on the cells or cell lines being immortalised. Briefly, the Dmt-O transgenic mouse according to the present invention, herein referred to as the "Dmt-O mouse", carries a "Dmt-162 transgene". The Dmt-162 transgene according to the present invention comprises a portion of ~750bp of flanking DNA from the human DM1 locus, including most of the DMPK 3'-UTR, and an expanded CAG'CTG repeat array of 162 repeats also derived from the human DM1 locus (25). Advantageously, however, the cell according to the present invention may be spontaneously transformed and immortalised without transformation with viruses, such as Epstein-Barr virus (EBN) or SN40. For example, transformation of cells, including subject-derived cell lines, with viruses in the analyses of repeat instability is proposed to hamper the transcriptional activity of genes involved in DΝA processing and therefore, the failure of transformed cells to accurately recreate the repeat dynamics of the human disease would limit the cell's overall utility as discussed herein. Conveniently, the cell may be a dividing cell or a non-dividing cell.

If the cell used is a dividing cell, the method according to the first aspect of the present invention may further comprise the additional step before step b), of allowing the cell to divide for a number of generations, or providing a substance to the cell line that arrests the cell division of the cell. Conveniently, said substance used to arrest the cell division of the cell may be Mitomycin-C or apicidin.

Advantageously, said substance used to arrest cell division, for example, apicidin may enhance instability of said cell or cell line. Without wishing to be bound by theory, the inventors have observed that when cells have been arrested the cell or cell line still displays expanded nucleotide repeat instability. Conveniently, the arrest of cell division of a cell or cell line may reduce selection effects. Advantageously, the arrest of cell division of a cell or cell line may increase the throughput of the method of the present invention for the study of modulation of nucleotide repeats in a cell or cell line, as said arrested cells or cell lines will require less maintenance compared with non-arrested cells or cell lines.

In a further aspect of the present invention, there is provided a method of obtaining a cell for the study of the cellular metabolism of trinucleotide repeat expansion associated with inherited human disease comprising the steps of: a) harvesting a tissue sample from a Dmt-O transgenic mouse; b) growing cells obtained from the tissue sample; and c) identifying and isolating cells comprising a Dmt-162 transgene and displaying expanded trinucleotide repeat instability.

In a further aspect of the present invention there is provided a cell for the study of the cellular metabolism of trinucleotide repeat expansion associated with inherited human disease, the cell comprising a Dmt- 162 transgene and displaying: a) expanded trinucleotide repeat instability. The term expanded nucleotide repeat instability according to the present invention is understood to mean an increase in the number of nucleotide repeats in the repetitive part of a genome, which may form part of the coding sequence or untranslated region of a gene. When this increase in number of nucleotide repeats enters the well-understood expanded disease- associated range, the nucleotide repeats often become dramatically unstable in the germline and also throughout the soma. Instability may be expansion-biased, contributing towards the unusual genetics, and most likely the tissue-specificity and progressive nature of symptoms. The length of such repeats is frequently polymorphic and unstably amplified repeats appear to be the major cause of such genetic defects such as Huntington's Disease, fragile X syndrome, spinobulbar muscular atrophy and myotonic dystrophy. Typically, the nucleotide repeat refers to multiple copies of the same base sequence, for example a trinucleotide repeat or tetranucleotide repeat.

Typically, the cell according to the present invention is identified by measuring cell growth characteristics by determining the number of population doublings based on cell number according to methods known in the art. Subsequently, the determination of late rapid cell growth characteristics of the cell is consistent with spontaneous immortalisation of cells. Furthermore, the nature of the cultured cells may be confirmed immunocytochemically by staining with primary antibodies directed against vimentin and/or cytokeratins (41). For example, if the cell cultures are positive for vimentin and negative for cytokeratins, this is consistent with a fibroblastic rather than an epithelial phenotype. Advantageous^, the precise morphology of the cells may be clearly distinguished between the cultures derived from the different tissues, indicating a different absolute origin for each culture.

It is understood that the term "modulation" refers to both positive and negative modulation. "Positive modulation", as used herein refers to an increase in the repeat number of nucleotide repeats or rate of nucleotide repeat expansion in the cell or cell line and hence an increase in total array length, relative to the number of nucleotide repeats in the cell or cell line of the present invention before the provision of a test substance to the cell or cell line. "Negative modulation" as used herein refers to a decrease in the repeat number of nucleotide repeats in the cell or cell line or to the slowing of the rate of nucleotide repeat expansion , relative to the number of nucleotide repeats in the cell or cell line of the present invention before the provision of a test substance to the cell or cell line. Nucleotide repeat number of nucleotide repeats in the cell or cell line, according to the present invention after treatment with a test substance may be measured by conventional PCR amplification of 'bulk' DNA for high throughput analysis, using for example, 1,000+ cellular equivalents of DNA (as in a standard PCR) followed by resolution and detection for example, by polyacrylamide gel electrophoresis with radioactive or fluorescenfly labelled oligonucleotide primers. Preferably, the nucleotide repeat number is measured using small pool PCR (SP-PCR; 16, 43). Conveniently, nucleotide repeat number may be measured over a three month period by for example, harvesting batches of cells at regular intervals over the 3 month period and assessing repeat number using SP-PCR.

More preferably, modulation may be assessed by comparing treated cells with untreated cells cultured in parallel, including cultures that have been maintained for an equivalent time period and those maintained for the same number of cell divisions.

Typically, tissue samples are harvested from a Dmt-O mouse using the explant technique (e.g eye) or enzymatic dissociation procedure (e.g. lung and kidney). (48,55)

Preferably, the cell divides forming a cell line, or is within an existing cell line which enters a continuous exponential growth phase, proliferating at similar and constant rates. Conveniently, the cell line may display a late rapid cell growth, consistent with the spontaneous immortalisation of cells. Advantageously, the cell lines according to the present invention may be maintained in continuous passage for over 300 days. Conveniently, the cell isolated from the transgenic mouse retains the tissue-specific, expansion-biased instability observed in vivo. Conveniently, clones derived from said cell, by limited dilution, and that are grown for a further 20 passages, for example, exhibit repeat size heterogeneity, which corroborates the progression of somatic instability in culture.

Generally the assay of the present invention may be carried out in parallel cultures. Advantageously, this allows the investigation into the influence of stochastic variation on repeat dynamics in the culture system of the present invention. Typically, the assay may be carried out in multi-well tissue culture plates.

Typically, the assay of the present invention may use established primary cell lines as hereinbefore described. Advantageously, these established cell lines, being older, may have already have acquired multiple mutations advantageous for growth. In other words, these established cell lines may be more stable in terms of their ability to undergo additional selective sweeps. It is assumed that these selective sweeps arise due to the acquisition of advantageous mutations in single cells that quickly take over the culture by clonal expansion. Typically, such selective sweeps tend to reduce the overall level of variation within a culture thus compromising the ability to deteπnine a simple measure of the level of repeat stability within a culture over time. Thus, by overcoming such selective sweeps, the present invention enhances the measurement of any modulation in repeat dynamics on a cell line by a test substance. Alternatively/conveniently, the assay of the present invention may use primary cell lines derived from cultures during their early passages.

A method of providing a test substance to the cell may be performed by for example adding directly to the cell culture medium or injection into the cell. The test substance is thus contacted with the cell. Preferably, the test agent will be provided over a range of doses.

The term "test substance" according to the present invention is understood to include chemicals, nucleic acid analogues, peptides and/or proteins. For example these may include coumarin antibiotics such as novobiocin; DNA damaging agents such as hydrogen peroxide; nucleic acid analogues such as 5-azacytidine; nonsteroidal antiinflammatory drugs such as aspirin or other agents such as caffeine. Further details of exemplary substances referred to above are given as follows:

Novobiocin. Coumarin antibiotics such as novobiocin, for example, are potent inhibitors of DNA topoisomerase II (49). Topoisomerase II is an essential enzyme that interconverts different topological forms of DNA by passing one nucleic acid segment through a transient double-stranded break generated in a second segment. By virtue of its double-stranded DNA passage reaction, topoisomerase II is able to regulate DNA over- and underwinding, and can resolve knots and tangles in DNA. Novobiocin and related compounds have had wide use as anticancer agents. Given the propensity of large expanded triplet repeats to form unusual DNA secondary structures, and without wishing to be bound by theory, it is hypothesised that topoisomerases might play some role in their resolution and hence inhibition of topoisomerase activity might have an effect on repeat metabolism.

Hydrogen peroxide (H₂O₂). High levels of oxidative stress are well known to lead to the production of free radicals that are capable of damaging DNA directly. Indeed, treatment of DNA with hydrogen peroxide has been shown to directly induce in microsatellite instability in E. coli based model systems (50). Moreover, considerable evidence has accumulated to suggest the involvement of energy metabolism dysfunction, excitotoxic processes, and oxidative stress in the pathogenic pathway in Huntington disease. Indeed, without wishing to be bound by theory, it is possible that a vicious circle is established in HD neurons in which the expansion increases the levels of oxidative stress which itself further destabilises the repeat leading to further increases in the level of oxidative stress etc etc resulting in neuronal dysfunction and eventually death of the cell.

Caffeine. Caffeine is able to uncouple DNA repair and replication from the progression of the cell cycle. This is now known to be achieved by directly inhibiting ATM kinase activity (51). Progression through the G2/M DNA-damage checkpoint before replication and repair are completed inevitably leads to a general increase in the mutation rate. Thus, without wishing to be bound by theory, it is possible that 'forced' progression through the cell cycle may also destabilise difficult to replicate expanded repeats.

5-azacytidine. The inventors have previously observed striking correlations between repeat expandability, GC content and proximity to CpG islands (56). A potential way in which this effect could be mediated is by DNA methylation at CpG dinucleotides. The nucleotide analogue 5-azacytidine is a potent inhibitor of CpG methylation in a mammalian cells and results in global hypomethylation (52). Associated with global demethylation some previously inactivated genes may be come reactivated. In addition, treatment with 5- azacytidine also mediates changes in chromatin structure. Again, without wishing to be bound by theory, interlocus differences might also be mediated via chromatin structure, thus, this drug could well have an effect on repeat metabolism.

Aspirin. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and sulindac have previously been demonstrated to suppress the mutator phenotype associated with hereditary nonpolyposis colorectal cancer (HNPCC) (53). Treatment of cell lines with known mutations in the mismatch repair pathway with aspirin and sulindac reduced the microsatellite repeat instability normally associated with such cells. Aspirin is known to reduce the proliferative capacity of cell lines, produce a build up of cells at the G0/G1 boundary and induce apoptosis, although it is not clear which, if any, of these pathways through which instability is suppressed. Given the established link with microsatellite instability and NSAIDs, and without wishing to be bound by theory, aspirin would appear to be an excellent candidate chemical agent that might also effect the metabolism of expanded triplet sequences.

Indeed, the present inventors have observed a modulation in the number and array length of trinucleotide repeats in a cell according to the present invention using the substance novobiocin, caffeine, hydrogen peroxide, rhodamine 6-G, ethidium bromide, ara C, 5- azacytidine and aspirin. Thus, in a further aspect of the present invention there are provided substances capable of negatively modulating the number of nucleotide repeats in a cell, wherein the substances are selected from the group consisting of novobiocin, hydrogen peroxide, rhodamine 6-G, ethidium bromide, ara C, 5-azacytidine and aspirin. Typically, the nucleotide repeat is a trinucleotide repeat, as associated for example with Huntington's Disease.

In a further aspect of the present invention there is provided a substance capable of positively modulating the number of nucleotide repeats in a cell, wherein the substance is caffeine. Typically, the nucleotide repeat is a trinucleotide repeat as associated for example with Huntington's Disease.

In a further aspect, the present invention provides a substance(s) determined by the method according to the present invention and its/their use in the treatment of inherited human or animal disease characterised by nucleotide repeat expansion. For example this substance may be novobiocin, hydrogen peroxide, rhodamine 6-G, ethidium bromide, ara C, 5-azacytidine and aspirin. It should be understood that this may include any suitable pharmaceutical composition comprising, as active agent, any said substance, or any synthetic functional derivative thereof and a suitable carrier. Such substances may also be used in conjunction with known treatments of such disorders as well as, if necessary, one or more pharmaceutically current adjuvant. This composition may be administered by various means for example intravenously, orally or through an implant or patch.

In a yet further aspect, the present invention provides the use of a substance determined by the present invention for the manufacture of a medicament for the treatment or prophylaxis of human and/or animal disease characterised by nucleotide repeat expansion.

A further aspect of the present invention provides the use of a transgenic animal model displaying nucleotide repeat instability for testing substances determined by the method of the present invention. Typically, the animal model may be the Dmt-D mouse.

Thus, without being bound by theory the present invention is based on the inventors' observation that the cause of nucleotide repeat instability as found in various complex diseases, including Huntington's Disease, may be mediated by binding and aberrant repair of unusual secondary structures by the mismatch repair machinery and not by the generally assumed mechanism of polymerase slippage during DNA replication (46). The present inventors have proposed that by modifying the DNA topology, the levels of repeat instability can be modulated. Indeed, the present inventors have shown that treatment of a cell line that displays expanded trinucleotide repeat expansion with an inhibitor of an enzyme, involved in modifying DNA topology, such as Topoisomerase II, for example, causes a decrease in the expansion of trinucleotide repeats in the treated cells. Consequently, the suppression of somatic nucleotide repeat expansion is a valid therapeutic approach and the present primary cell line system can be used to identify therapeutic agents, which reduce the repeat number of nucleotide repeats in such a cell line.

Treatment of disease by chemogenetherapy will be achieved by modifying the nature of endogenous DNA sequences by the application of exogenous chemicals. Thus, the present invention has shown that treatment of cell lines with exogenous chemical agents can modify expanded repeat dynamics in vitro. Some of these effects may be mediated by cell selection in rapidly dividing cultures. Nonetheless, drugs that produced similar results in vivo may still have therapeutic benefits. The experiments described herein act as an important proof of principle and a basis for more extended screens. Promising agents identified in vitro for example novobiocin, hydrogen peroxide, rhodamine 6-G, ethidium bromide, ara C, 5- azacytidine and aspirin, may be further assessed for their utility in vivo in the many transgenic mouse models in which the unstable DNA phenotype is replicated 4,5,20. Given the technical problems associated with standard gene therapy, chemogenetherapy is very attractive since it may be achievable with small molecule drugs for which delivery methods would be less problematic.

The present invention will now be further described by way of example and with reference to the figures and table, which show:

Brief description of the drawings

Figure 1. Growth dynamics of the cell cultures. The graphs show the growth curves for three cell lines established from a 6-month old Dmt-D male mouse: lung (filled triangles), eye (filled circles) and kidney (filled squares). For each culture the cells were counted at each passage, the population doubling time calculated and the cumulative number of population doublings plotted as a function of days in culture.

Figure 2. Fibroblastic morphology of cultured cells. The pictures show the morphology of cultured lung, eye and kidney cells after 20 doublings derived from a 6-month old Dmt-D mouse: A) Light micrographs. Note the prevalent fibroblastic spindle morphology. B) Immunofluorescence detection of vimentin. Cells were stained with a primary mouse monoclonal antibody raised against human vimentin, and a secondary anti-mouse IgM-FITC conjugate. All cells stained positive. All cells stained negative for cytokeratins. All pictures are at 5 Ox magnification.

Figure 3. Repeat length variation in cultured cells. Small pool PCR analysis was used to monitor transgene repeat length variation in DNA samples extracted from cultured lung (A), eye (B) and kidney cells (C) harvested from a 6-month old Dmt-D male mouse. Five to 10 copies of genomic DNA, collected at different passages were amplified in 6 independent SP- PCR reactions. Representative SP-PCR reactions of DNA isolated from the progenitor tissues are shown on the left; each reaction containing around 20 copies of the transgene, except for the first kidney panel, in which around 50-100 molecules of DNA were amplified. The scale on the right displays the molecular weight markers converted into CTG repeat numbers. The repeat lengths of the major PCR products detected are shown on the left.

Figure 4. Repeat length variation in cultured kidney cells. The giaphs display the repeat length distributions observed in cultured kidney cells harvested from a 6-month old Dmt-D male mouse. For each time point at least 100 individually amplified transgene repeat tracts were sized by SP-PCR at low DNA concentration (1-3 DNA molecules per reaction). Allele lengths were grouped into 5 repeat size ranges.

Figure 5. Repeat length variation in single cell clones of cultured kidney cells. The autoradiographs shown are representative SP-PCR analysis of DNA samples extracted from three independent single cell clones derived from cultured kidney cells harvested from a 6- month old Dmt-D male mouse. An average of five DNA molecules were amplified in each reaction. The scale on the left displays the molecular weight markers converted into CTG repeat numbers.

Figure 6. Age of donor effects on repeat length variation in cultured kidney cells. The autoradiographs shown are representative SP-PCR analyses of DNA samples collected from cultured kidney cells harvested from 5-week, 3-month and 30 month old Dmt-D mice. Representative SP-PCR reactions of DNA isolated from the progenitor kidneys are also presented on the left, each reaction containing around 5 to 50 copies of the transgene. The molecular weight markers are shown on the right, after conversion into number of CTG repeats. The length sizes of the main PCR products detected are shown on the left.

Figure 7. SP-PCR amplifications of DNA samples extracted from a control and a novobiocin-treated culture.

Around 50 transgene molecules have been amplified per reaction.

Figure 8. Boxplots showing the repeat size of the longer alleles in each cell sample.

The line across the box displays the median repeat size, whereas the bottom and top sides of the box correspond to the first (Ql) and third quartiles (Q3), respectively. The lines extending from the top and the bottom of the box, include values that fall inside the lower and upper limits: Q1-1.5(Q3-Q1) and Q3+1.5(Q3-Q1), respectively. The asterisks represent repeat numbers that are outside these two limits. Samples 1-6: control cells. Samples 7-12: novobiocin-treated cells. For each sample ~15-25 molecules were sized using a standard SP- PCR protocol. The median repeat length in treated cells is consistently smaller than the median repeat length in control samples.

Figure 9: Novobiocin treatment and expanded CAG'CTG repeat dynamics. a The autoradiographs shown are representative SP-PCR analyses of DNA samples extracted from six replicate D2763 cultures treated with 60μM novobiocin for 73 days, control cultures maintained for approximately the same amount of time (77 days) and the progenitor culture (P) from which all cells were derived at day zero. The scale on the left displays the molecular weight markers converted into CTG repeat numbers. Note that the D2763 cultures spontaneously split into two main populations of cells labelled 'upper' (U, >300 repeats) and 'lower' (L, <300 repeats). An average of 5 to 50 DNA molecules were amplified in the reactions shown. For the quantitative analyses 50 to 150 alleles from each culture were sized by single molecule SP-PCR. b The box plots show the degree of variation observed in both the U and L populations in treated and control cells. The top and bottom of the boxes correspond to the third (Q3) and first quartiles (Ql), respectively and the line across the box displays the median. The lines extending from the top and the bottom of the boxes, include values that fall inside the lower and upper limits: Q1-1.5(Q3-Q1) and Q3+1.5(Q3-Q1), respectively. Figure 10: Chemical treatment and expanded CAG»CTG repeat dynamics. a The autoradiographs shown are representative SP-PCR analyses of DNA samples extracted from replicate D2763c2 cells cultured for 80 population doublings (PD) treated with 60μM novobiocin (99 days), 2mM caffeine (130 days), 250nM ethidium bromide (128 days, but only 66 PD), lOOμM hydrogen peroxide (126 days), 50nM rhodamine 6-G (116 days) and control cultures maintained for 82 (70 PD), 97 (82 PD) and 122 days (106 PD), and the progenitor culture from which all cells were derived at day zero. Evidence for clonal growth in the hydrogen peroxide and rhodamine 6-G treated cultures is indicated with arrows. For the quantitative analyses 20 to 80 alleles from each culture were sized by single molecule SP- PCR. b The box plots show the degree of variation observed in treated and control cultures as described in Fig. lb. Statistically significant differences (*, PO.05) in the median rates of expansion, corrected for time and population doublings, were observed for some treatments relative to both time and PD controls.

Figure 11: Ethidium bromide reduces cytochrome oxidase II RNA levels. a The autoradiographs show a northern blot analysis of mRNA levels for cytochrome oxidase II (COII) and β-actin transcripts in D2763c2 cells treated with 250nM ethidium bromide relative to control cultures, b The graph shows the quantitative analyses of expression levels of COII relative to the β-actin control. The observed reduction in COII levels in treated cells is highly significant (E=0.0002, two-tailed t-test).

Figure 12: Ethidium bromide increases the mobility of alternative expanded CAG»CTG conformers in agarose gels.

The autoradiographs show PCR products containing expanded CAG'CTG tracts (56 and 200 repeats) amplified with various combinations of oligonucleotide primers (DM-A, -H, -C, - BR, -DR and -ER; 16,33) resolved in 1.8% agarose gels with (left) and without (right) 200ngml-l ethidium bromide in both the gel and running buffer (0.5X TBE buffer (45mM Tris-borate, ImM EDTA)). The scale on the left shows the position of the molecular weight markers (M) in base pairs. Note the multiple alternative products observed in the absence of ethidium bromide. Materials and Methods

Mouse tissue samples

Dmt-D transgenic mice were sacrificed by cervical dislocation, and the kidneys, eyes and lungs removed and kept on ice for up to 1 hour until processed. All mice were hemizygous for the transgene on a pure FNB/N background.

Establishment and maintenance of cell cultures

Depending on the tissue, the primary cultures were established either by the explant technique (eye cultures) or enzymatic dissociation (lung and kidney). The eye balls were transferred to 100mm dishes, rinsed and dissected in PBS. The tissue was placed cell-side down on a 60mm dish and minced into small fragments (~lmm²). All pieces of tissue were flattened and dispersed on the dish. Culture medium was gently added and the culture was incubated at 37 ° C in a humidified 5% CO atmosphere. The standard growth medium consisted of Dulbeco's modified Eagle medium with high glucose (DMEM) supplied with 10% foetal bovine serum (FBS), 100 Urnl^"1 of penicillin and lOOμgml^"1 streptomycin (GibcoBRL Life Technologies, UK). Once cells became 60-80% confluent they were subsequently subcultured at a 1 :5 or 1 : 10 ratio during the first 5 passages and kept under the same conditions. The enzymatic dissociation of kidney and lung tissues was essentially performed as described previously (48). The organs were transferred to 100mm dishes containing 10ml of sterile PBS and minced into 1mm³ cubes. The minced tissues were transferred into 15ml Falcon tubes and washed twice with sterile PBS. The tissues were digested with 5ml of 0.25% trypsin solution in a 37 ° C humidified 5% CO₂ incubator for 30 minutes with limited shaking. After the incubation the pieces of tissue were allowed to settle down and the supernatants transferred into fresh 15ml tubes. The cells in suspension were collected by centrifugation at 800g for 5 minutes. The final pellets were resuspended in 5ml of standard culture medium, plated on 25cm² flasks and incubated at 37°C in a humidified 5% CO₂ atmosphere. The remaining portions of tissue were repeatedly digested with fresh trypsin for 6 times. After the first passage all the pellets derived from the same tissue were pooled together and an initial single culture was maintained for each tissue, as described above for the eye cell cultures. Clones derived from a single cell were established by seeding an average of 0.5 cells in each well of a 24-well cluster. All the cultures were grown under the same conditions and in the same medium. Once the cells reached 80-100%) confluency they were subcultured either at a 1 :40, 1:50 or 1:100 ratio. At every passage the number of population doublings was determined based on the cell number.

Immunocytochemistry

Vimentin and cytokeratins were detected immunocytochemically in cultured mouse cells using mouse monoclonal antibodies raised against human proteins, which cross react with mouse. Semi confluent cells growing on 8-well chamber slides were washed once with serum-free DMEM and fixed in 2% paraformaldehyde in DMEM for 20 minutes at room temperature. The cells were washed once with PBS, left in 0.1M glycine for 20 minutes and washed twice with PBS. Cells were permeabilised in 1% Triton-XlOO in PBS for 6 minutes, and washed twice with PBS. Monoclonal anti -vimentin (mouse IgM isotype; Sigma, catalogue number: N5255) or monoclonal anti-pan cytokeratins (mouse IgGl isotype; Sigma, catalogue number: C1801) were applied at 1/200 in 0.01% Triton-XlOO and incubated over night at 4°C with gentle shaking. The cells were washed 4 times in PBS for five minutes. Both anti-mouse IgM-FITC conjugate (Sigma, catalogue number: F9259) and anti-mouse IgG-TRITC conjugate (Sigma, catalogue number: T7657) were applied in a 1/100 dilution in 3% BSA in PBS with 0.01% Triton-XlOO and incubated for 2 hours at room temperature with gentle shaking. Cells were finally washed 4 times in PBS for five minutes and observed using fluorescence microscopy.

DΝA extraction from tissues and cultured cells

Mouse tissue DΝA was isolated following phenol/chloroform extraction, using standard procedures. Cell culture DΝA samples were extracted using a Νucleon DΝA extraction kit for blood and tissue culture (Νucleon), following the manufacturer's protocol.

PCR analysis

SP-PCR analyses were performed in a Biometra UNO thermoblock as described previously, with 0.1 μM of carrier primer DM-C, and using PCR primers DM-C and DM-BR (16). The PCR products were electrophoresed on a 1.25% multipurpose agarose gel (Boehringer Mannheim), transferred onto a nylon membrane (Osmonics), hybridised with a radio-labelled double stranded probe comprising 56 CTG repeats and detected by autoradiography. The PCR products were sized using Kodak Digital Science ID software (Kodak). Dmt-D mouse kidney cell cultures (D2763 line). The D2763 line was a previously described (57) spontaneously transformed kidney cell line derived from a 6-month old Dmt- D transgenic mouse (33,25). The D2763c2 line was cloned by limiting dilution from the D2763 line (clone 2 from Gomes-Pereira et al.(51)). Fresh culture medium, with or without additional tested drugs, was added to the cultures every 2-3 days and the cells were passaged weekly.

Assessment of trinucleotide repeat size variability. DNA samples were extracted from cultured cells and SP-PCR analysis performed as previously described (57). Median rates of expansion in treated cultures, corrected for time and population doublings, were compared with the control culture closest in terms of time and PD, using the two-tailed Mann- Whitney C/ test. Statistical analyses were performed using Microsoft Excel and MINITAB (vlθ.51, Minitab Inc.).

Northern blot hybridisation analysis. Approximately 6x10⁶ cells were collected after trypsin digestion, and RNA extracted with Tri Reagent™ (Sigma) following the manufacturer's protocol. Fifteen microgram RNA samples were resolved by electrophoresis through a 1.3% (w/v) multipurpose agarose gel (Boehringer Mannheim), in IX MOPS pH 8.0, containing 4% (w/v) formaldehyde for 4 hours at 150N. RΝA was transferred onto a Biodyne B nylon membrane (Flowgen) by capillary action, in 10X SSC. Double stranded DΝA probes, generated by PCR amplification of genomic DΝA, were used for northern blot hybridisation to detect COII and βactin mRΝA transcripts. COII mRΝA levels were quantified using Kodak Digital Science ID software (Kodak).

Detailed description of the invention

Examples

Growth dynamics of mouse cell cultures

In an attempt to establish primary mammalian cell cultures in which the dynamics of expanded CAG^»CTG repeat tracts could be monitored and investigated in vitro, we harvested lung, eye and kidney from a six-month old male Dmt-D transgenic mouse. Using either the explant technique (eye) or enzymatic dissociation procedure (lung and kidney), mouse cell cultures were successfully established. Following an initial period characterised by low growth rates, all the cell cultures entered a continuous exponential growth phase, proliferating at similar and constant rates (Figure 1). The cell proliferative capacity of each culture was estimated according to the observed population doubling time (PDT), which was calculated based on the cell counts determined at each passage (Table 1). The late rapid cell growth observed in vitro is consistent with the spontaneous immortalisation of cells, which is known to occur at a relatively high frequency with mouse cell cultures (39,40). The time taken to reach peak growth rate was longest for the kidney culture which took around 120 days, in contrast to the lung and eye cell cultures which took ~60 days.

Table 1. Population doubling times of cell lines.

The proliferative capacity of each cell culture was estimated based on the population doubling times (PDT), which was calculated as a function of the cumulative cell numbers determined at each passage.

Mouse age tissue PDT trinucleotide instability

6 months lung 32 hours low

eye 30 hours medium

kidney 31 hours high

3 months kidney 45 hours medium

5 weeks kidney 42 hours low

Characterisation of cultured cell types

Each culture consisted of an initial heterogeneous cell population, comprising cells with clearly different morphologies and probable distinct proliferative capacities. The cultures soon became more homogeneous, exhibiting prevalent spindle morphology, typical of fibroblasts, after less than 5 passages (Figure 2). The nature of the cultured cells was confirmed immunocytochemically by staining with primary antibodies directed against vimentin and cytokeratins (41). All the cultures were positive for vimentin and negative for cytokeratins, consistent with a fibroblastic rather than epithelial phenotype (Figure 2). Nonetheless, the precise morphology was clearly distinct between the cultures derived from the different tissues, indicating a different absolute origin for each culture.

Tissue-specific trinucleotide instability and selection for longer alleles in cultured mouse cells

Following the establishment of these tissue-specific Dmt-D murine cell lines, DNA samples were collected at every passage and transgene repeat length variability assessed by small pool PCR analysis (SP-PCR) (16,42). The length of the repeat in the cultures and the level of variation was compared with the progenitor allele length in the donor mouse (173 repeats, as determined by PCR length analysis of tail DNA at weaning) and the level of variability present in the tissue from which the culture was originally derived:

Variability in lung cell cultures.

The lung tissue from which the culture was established showed relatively low levels of variability with most alleles (>90%) remaining within +/- 10 repeats of the progenitor allele (173 repeats) (Figure 3 A). After 15 doublings the lung cell culture displayed an even lower level of variability with the vast majority of cells within +/- 5 repeats of the predominant allele (-175 repeats). The reduction in variability observed in the progression from in vivo to in vitro, suggests that only a very few cells grew in culture. Indeed, the degree of repeat length homogeneity observed in the culture and the relative increase in size detected, suggest that this culture may have very quickly been taken over by derivatives of a few, or possibly even only one, of the cells present in the original tissue carrying a slightly larger allele. Surprisingly, even after as many as 100 or 200 doublings in vitro (>300 days), the level of repeat variability remained very low with only a small increase in average allele length up to 177 repeats. The maintenance of such a low level of variability in vitro after so many doublings indicates that mammalian cells are capable of faithfully replicating large expanded CAG'CTG repeat tracts through many many cell divisions, even at a locus that is extremely unstable in other cells. Also of note was the relatively late appearance in the culture of a subset of cells carrying a deletion of ~30 repeats relative to the major allele in the culture. After 200 doublings these cells comprised approximately 10% of the cell population, but rose to -25% by 250 doublings. Presumably, this shift was mediated by drift and/or selection within the cell population rather than repeated mutations to the same length allele. This assertion is supported by the bi-directional nature and different sizes of similar shifts observed in other cultures (see below).

Variability in eye cell cultures.

Higher levels of instability were detected in cultured eye cells (Figure 3B). Once again, very early in the establishment of the culture, the range of variability observed was very different to that detected in the progenitor tissue. By as few as 5 doublings, the predominant cell population contained an average allele length of ~185 repeats, 12 repeats larger than the major allele in the original tissue (173 repeats). By 10 doublings a distinct sub-population of cells appeared, with an average size of -210 repeats. This population presumably represents clonal expansion of a rare cell carrying a large expanded allele, either present in the original tissue or having arisen as a spontaneous mutant in vitro during one of the earlier passages. Nonetheless, by 10 doublings each population showed moderate size variability, characterised by small changes mostly limited to fewer than +/-5 repeats around the average repeat length. The moderate instability was maintained, with the level of variability and the average allele length gradually increasing within each of the two main populations of cells. This effect was very clear up to 70 doublings, by which time the average allele size in each population had risen by a further 5 repeats relative to that observed at 10 doublings. After 110 doublings however, the proportion of cells in the population carrying the larger alleles started to decrease. By 240 doublings in vitro, the eye cell population initially carrying -185 repeats had increased in average allele length to -195 repeats, but had overgrown the culture causing a reduction in overall repeat length variability.

Variability in kidney cell cultures.

Kidney is the tissue which shows the highest levels of variability and the largest expansions in vivo. Repeat variability within the original tissue showed the typical trimodal distribution highly biased towards expansion (25) (Figure 3C). Most cells carried repeats within the first peak of variability with alleles within -5 to +10 repeats of the progenitor (173 repeats). Additional peaks of variability were observed most clearly at -200 and -230 repeats. Although a relatively high level of variability was retained after 5 doublings in vitro, the predominant alleles from the first peak of variability detected in the original kidney tissue were entirely absent. It appears as if only cells from the second and third peaks were able to grow in vitro. Indeed, by as few as 20 doublings, the second peak of cells had disappeared and only cells carrying the largest repeats were maintained. Single molecule analyses allowed us to quantitatively define more clearly the progression of repeat variability in cultured kidney cells (Figure 4). After 20 doublings a narrow range of repeat sizes was defined, with an average repeat length of -260 repeats, which not only corresponded to the largest repeats initially found in vivo, but also overlapped with the largest peak described above after 5 doublings in culture. After 50 doublings, a pattern of instability very reminiscent of the highly positively skewed distributions observed in vivo in both humans (16) and mice (25) was observed, with very few alleles being detected below the lower boundary of -250 repeats. The level of repeat variability peaked at this stage, with a mean allele length of 270 repeats and with ~5% of cells containing alleles >300 repeats in length. However, the repeat length heterogeneity was reduced dramatically and the mean allele length decreased to 260 repeats after 100 divisions in culture. A similarly low level of variability was retained even after 150 population doublings. The repeat dynamics in cultured kidney cells suggests the development of an expansion-biased mechanism in vitro, which may be disturbed by population fluctuations, such as the dramatic selective sweeps described at 20 and 100 doublings.

It is important to note that no obvious relationship was found between the repeat instability in the three tissue specific cell lines analysed and their proliferative capacity, as assessed by the population doubling times (Table 1). Taken together, the results reported above for the three different tissues, support the maintenance of in vivo tissue-specific trinucleotide instability in vitro, which is expansion-biased, particularly during the early stages of the eye and kidney cell cultures.

Accumulation of mutations in single cell-derived clones from kidney cell cultures

To discount the possibility that the variability detected in culture could be an artefact derived purely from an in vitro selection process for the cells harbouring longer alleles, rather than the outcome of the intrinsic instability of the transgene in vitro, clonal lines derived from single cells were established. The clones were isolated by limiting dilution from the kidney cell culture after 20 population doublings, grown for a further 20 doublings, and the CTG repeat variability determined by SP-PCR (Figure 5). All the clones exhibited repeat size heterogeneity, which corroborates the progression of somatic instability in culture. In addition, they showed different average repeat sizes and different ranges of repeat distribution. For one clone in particular (clone 2), a very broad range of repeat sizes was observed. Such a difference could not be accounted for by cell division rates, since no significant variations in the proliferative capacities were observed between the three clones studied here (data not shown).

Age-of-donor effect on trinucleotide instability observed in culture

To investigate the influence of the age of the donor mouse at sacrifice on the trinucleotide instability detected in culture, kidney cell lines were established from two younger Dmt-D male mice, aged 5 weeks and 3 months, and one very old mouse aged 30 months. The two cultures from the young mice showed similar growth rates, with population doubling times of 42 and 45 hours, respectively (Table 1). Consistent with the age-dependent accumulation of somatic mosaicism in vivo, the youngest mouse analysed in this study displayed very little somatic variation in the original tissue from which the cell line was derived, with most cells with an allele size of -160 to -175 CTG repeats (Figure 6A). As with previous cultures though, the level of variability was almost immediately reduced in the first few passages and by 5 doublings the average allele size was -165 repeats with most variants within +/- 5 repeats. The range of variation and average allele length increased up to 35 doublings, by which point two major populations of cells with -170 and 185 repeats were present. After an additional 30 doublings the cells containing the larger alleles predominated, accompanied by an overall reduction in the range of variability, reflective of a selective sweep. The repeat sizes ranged from -185 to -215 repeats in the original kidney tissue of the 3-month old mouse, intermediate between that observed in the 5 week and 6 month old mice (Figure 6B). Once again, variability was rapidly reduced in culture, most notably by 20 doublings the average allele length had increased to -195 repeats, but with a reduced overall range. By 35 doublings a second major population of cells carrying approximately 225 repeats appeared. This population took over the culture completely by 65 doublings and increased in length up to -240 repeats. The level of variation present in vivo in the very old mouse was, as expected, very high with a small subset of cells carrying alleles as large as 720 repeats (Figure 6C). Nonetheless, the most predominant cells contained expansions in the range of 160 to 180 repeats. This high level of variability though, was massively reduced very early in the establishment of the culture. By 5 passages only three major, but highly distinct, populations persisted suggesting that this culture was most likely derived from only 3 progenitor cells. Once again the allele sizes present in the cells that predominated in culture were much larger than present in the majority of cells in vivo. Overall, these data confirm the proliferative advantage in vitro of cells that contained large repeats in vivo. They also indicate that the expansion-biased progression of somatic instability in vitro is a reproducible phenomenon using kidneys from Dmt-D mice as a source material and that selective sweeps are a common occurrence. These data also appear to support an effect of the age of the mouse at sacrifice on the stability of the transgene in culture, with the repeat appearing to be less stable in cell lines derived from older mice than those from younger mice. However, this may be a result of the longer allele lengths that predominate in the cultures derived from older mice, rather than a true age-of-donor effect on repeat stability.

Novobiocin

Twelve parallel cell cultures were established from a single progenitor cell line: six cultures were used as controls and six were treated with 60 μM of novobiocin, an amount of drug that has little effect on the population doubling time. The cell growth medium was changed every other day, and the cells subcultured once a week for 73-75 days, during which period they underwent 60 population doublings. At the end of the treatment the repeat length variability in each culture was studied by SP-PCR.

Example 1 (Novobiocin)

The analysis revealed that all cultures consisted of two main subpopulations of cells: a major population carrying -250 CTG repeats, and a smaller one comprising longer alleles, up to -400 repeats (Figure 7). Since longer trinucleotide repeats are known to display higher levels of instability (18), this study has been focused on the second subpopulation of cells, as it would be easier to detect an effect of the drug on the repeat dynamics, either positive or negative. Preliminary SP-PCR analysis, performed at high DNA concentration, has revealed that novobiocin-treated cells carry shorter alleles, when compared to control cells, although they exhibit greater repeat size variability. To quantify this observation 10-25 long alleles were accurately sized by single molecule analysis, and the results summarised as boxplots in Figure 8. Assuming there is no statistical difference between the repeat size distributions within control and novobiocin-treated cells, the results obtained for the six replicates were pooled together and two different statistical tests performed. A upper-tailed Mann- Whitney W-test, has confirmed that the median repeat number carried by control cells is significantly higher than the median repeat number detected in novobiocin-treated cells: 391 versus 369 (W=\ 1781.0, p<0.0001). An E-test has revealed that the variance of repeat numbers is significantly higher for novobiocin-treated cells, than for control cells: 299 versus 177 (F- test, E=0.5918, p<0.001), confirming a higher repeat size heterogeneity in treated cells, compared to controls.

Example 2 (Novobiocin)

At the end of the exposure period the degree of repeat length variation in all cultures was assessed using SP-PCR and compared to the starting culture (Figure 9).

Analysis of the control cultures revealed that the during the experimental period the cell line had split into two sub-populations, here described as 'Lc' (lower control <300 repeats) and 'Uc' (upper control >300 repeats): in population Lc the repeat had expanded only slowly (median gain = 0.043 repeats per day); in population Uc though, the repeat expanded rapidly (median gain = 1.78 repeats per day). Both populations of cells (referred to as L_T and Up) were also observed in all the treated cultures in similar proportions to that observed in the control cells. Although most dramatic in Uχ_; the rate of expansion in both populations of treated cells was significantly reduced relative to the control cells (median gain = 1.65 repeats per day for Up, P=0.0082 and -0.017 repeats per day for Lp, E=0.0131) (Fig. 9b). These data indicate that inhibition of topoisomerase II resulted in a decrease in the rate of expansion, possibly mediated by destabilisation of mutation intermediates by increased superhelical tension.

Other Agents

For these studies a cloned derivative of the D2763 cell line was used in which the repeat expands rapidly with time (D2763c2, clone 2 from Gomes-Pereira et al., 57). In addition to repeating novobiocin treatment, D2763c2 cells were exposed for up to 130 days to other candidate compounds with known effects on various aspects of DNA metabolism: caffeine, which inhibits ATM kinase activity and thus uncouples DNA repair and replication from the progression of the cell cycle⁽⁵¹⁾ ethidium bromide, a DNA intercalating agent with well known mutagenic properties⁽⁶²⁾ hydrogen peroxide, a free radical producer that can damage DNA directly and has been shown to increase microsatellite mutation rates⁽⁵⁰⁾; rhodamine 6-G, which inhibits oxidative phosphorylation and also leads to an increase in the levels of oxidative stress⁽⁶³^ ; Ara C, a DNA polymerase inhibitor; 5-azacytidine, an inhibitor of CpG methylation in mammalian cells; and aspirin, an NSAID, which has been shown to suppress the mutator phenotype associated with hereditary nonpolyposis colorectal cancer. Unlike novobiocin, the additional treatments affected the proliferative capacity of the cells and resulted in a 24 to 66% increase in the population doubling time. Previous studies indicated that cell proliferation rates were not the most critical factor underlying differences in expansion rates between cell lines (18), nevertheless, treated cells were also compared to controls grown for the same population doublings (Fig. 10a). As expected, the transgenic repeat length continued to expand rapidly in the control cells (median rate of expansion = 0.887 repeats per day after 97 days). As in the previous experiment, novobiocin treatment did result in a decrease in the median rate of expansion (median rate of expansion = 0.792 repeats per day), although the measured reduction was not statistically significant. However, the other treatments resulted in statistically significant differences (EO.05) in the rate of expansion compared to both time and population doubling controls (Fig. 10b). Interestingly, high doses of caffeine resulted in an increase in the rate of expansion (median rate of expansion = 1.006 repeats per day, E=0.0202). These data indicate that forced progression through the G2/M DNA-damage checkpoint leads to increased repeat instability, consistent with the accumulation of non-repaired DNA replication slippage errors during mitosis. In contrast, hydrogen peroxide (median rate of expansion = 0.180 repeats per day, E=0.0202), rhodamine 6-G (median rate of expansion = 0.298 repeats per day, E=0.0051), ethidium bromide (median rate of expansion = 0.310 repeats per day, P=0.0051), Ara C (median rate of expansion = 0.219 repeats per day, P=0.0082), continuous 5-azacytidine (median rate of expansion = 0.461, E=0.0202), interrupted 5-azacytidine (median rate of expansion = 0.547, P=0.0453), and aspirin (median rate of expansion = 0.565, E=0.0453) treatment all resulted in decreases in the rate of expansion (see Table 1). Interestingly, the frequency of cells containing large deletions was dramatically increased with hydrogen peroxide. However, some of the hydrogen peroxide and rhodamine treated cell cultures showed evidence of reduced variability, suggesting that some of this effect may have been mediated by clonal growth of cells selected for enhanced viability under conditions of oxidative stress (Fig. 10a). Ethidium bromide also resulted in a suppression of the rate of repeat expansion, but was not associated with the accumulation of cells with large deletions and revealed no evidence for clonal growth. As ethidium bromide also affects mitochondrial metabolism, we investigated mRNA levels for the mitochondrial enzyme cytochrome oxidase II and found they were reduced by -2/3 in exposed cells (Fig. 11), consistent with ethidium bromide induced oxidative stress (58). Thus, three different treatments that increase the levels of oxidative stress in cells surprisingly resulted in a reduction in the rate of expansion. This effect might be mediated via alternative processing of mutation intermediates by components of the base excision repair pathway that is altered in cells exposed to reactive oxygen species (59). However, ethidium bromide also interacts directly with nuclear DNA and can also inhibit topoisomerase II activity (60). It has also previously been reported that the physical properties of expanded CGG repeats were specifically modified by the presence of ethidium bromide (61). Similarly, we have determined that expanded CAG'CTG repeats migrate as a single product in agarose gels containing ethidium bromide, but as multiple alternative products in its absence (Fig. 12). Gel extraction and re-annealing experiments (data not shown) have confirmed that these multiple products represent alternative slipped strand DNA structures (S-DNA) adopted by expanded repeat tracts (23). As these data indicate that ethidium bromide can interact with and modify the properties of S-DNA, it may alternatively exert its effect on repeat length dynamics via a direct interaction with unusual DNA structures adopted by expanded repeats.

Table 2 Summary of the effects of multiple chemical treatments on the dynamics of expanded trinucleotide repeats in D2763Kc2 cells.

The table shows the time and number of population doublings (PD) D2763Kc2 cell cultures were exposed to each chemical, and the consequent increase in population doubling time (PDT). The median repeat gain was determined by single molecule SP-PCR analysis, corrected for both time and population doublings, and compared with the closest control cultures in terms of days and PD in culture, respectively (two-tailed Mann- Whitney t/test).

Time PD Increase Median Two-tailed Media Two-tailed

(days) in PDT^b repeat Mann- n Mann- gain Whitney repeat Whitney per day Utest (p) gain 17 test (p) per PD

Control 82 70 - 0.890 - 1.043 -

Control 97 82 - 0.887 - 1.050 -

Control 122 106 - 0.615 - 0.709 -

Caffeine 130 80 38% 1.006 0.0202 1.634 0.0453

AraC 95 71 13% 0.219 0.0082 0.293 0.0082

Continuous³ 99 80 5% 0.461 0.0202 0.548 0.0202 5-azacytidine

Interrupted^b 97 80 2.5% 0.547 0.0306 0.663 0.0453 5-azacytidine

Aspirin 99 80 5% 0.565 0.0453 0.699 0.0453

Continuous 5-azacytidine: cells were treated with 10 μM 5-azacytidine for 80 PD. .

"Interrupted 5-azacytidine: cells were treated with 10 μM 5-azacytidine for 40 PD, followed for a further 40 PD in the absence of the chemical.

Trinucleotide repeat dynamics in Dmt-D cells growing at high density in low foetal bovine serum

In order to check for the possibility that non-dividing cells can still accumulate repeat length mutations, Dmt-D kidney cells were arrested by serum starvation. Four D2763K and D3111K cell cultures (results not shown) were maintained in standard growth medium, supplemented with 10% (v/v) foetal bovine serum (FBS), until they reached 80-90% confluency and the FBS levels were then decreased to 0.25% (v/v) to inhibit cell proliferation. Nevertheless, the accumulation of multiple cells layers over time were observed by phase contrast microscopy, suggesting that continuing cell proliferation is still possible even under conditions of low FBS. In addition, the culture medium became acidic very quickly, as revealed by the development of a yellow/orange colour, indicating a rapid cell metabolism in serum-starved cultures.

Cells maintained at high density in 0.25% (v/v) FBS, without subculturing, were harvested at different time points and the levels of repeat size variability assessed by high DNA input SP-PCR sensitive techniques, and compared to those detected in the progenitor cells and in proliferating control cultures, maintained in 10%> (v/v) FBS for similar periods of time (results not shown). None of the control cultures exhibited major trinucleotide repeat instability, with no major expansion or deletion mutants being detected. The repeat dynamics in serum-starved D2763K did not reveal major differences relative to the controls. However, a small subpopulation of shorter alleles, showing clear evidence of clonal expansion, was detected at 56 days. In contrast, equally short repeats were not detected in the corresponding control culture (results not shown). Similarly, D3111K confluent cultures, maintained in low FBS, did not exhibit major differences in the degree of repeat length variation relative to the controls, up to 70 days in culture. By this time point, a population of cells carrying longer repeats, and again showing clear signs of clonal proliferation, overgrew the culture (results not shown).

The accumulation of subpopulations of mutant alleles, showing little repeat length variation, in Dmt-D cells cultures grown in 0.25% (v/v) FBS, is indeed consistent with the clonal expansion of a few mutant cells exhibiting enhanced survival to conditions of high density, and rejects the hypothesis that these cultures were truly arrested by serum starvation. Nevertheless, it is not unreasonable to assume that serum-starved cells were indeed proliferating at lower rates than control cultures, as they were not passaged for at least 56 days.

Trinucleotide repeat dynamics in confluent Dmt-D cell cultures arrested by contact inhibition

Since cultured mouse cells undergo spontaneous immortalisation(40), primary kidney cultures arrested by contact inhibition were used as an alternative to monitor the dynamics of expanded CAG'CTG trinucleotide repeats in non-dividing cells. D4393K cells were collected by enzymatic dissociation from a kidney tissue sample harvested from a 6-month old male Dmt-D mouse. These cells showed a typical fibroblastic phenotype, as revealed by their spindle morphology, vimentin expression and lack of staining for a panel of cytokeratins (results not shown). Multiple parallel replicates were established after the first passage and expanded until they reached confluency. The cultures were maintained at high density, and fresh medium was added regularly. SP-PCR analyses were performed at different time points, to monitor repeat size variability in two arrested replicate cultures (results not shown). The analysis revealed minor differences over the first 63-65 days in culture, but a dramatic accumulation of longer alleles was detected in arrested cells at later stages. A similar subpopulation of cells carrying longer alleles was not detected in control cells, growing for 89 days and 22 population doublings, but a few expanded mutants were observed at high DNA concentrations. However, similar expanded alleles were later observed in the control culture grown for 118 days and 40 population doublings (results not shown). To quantify this observation, 20-50 transgene molecules from each culture were individually sized. The expanded alleles were on average -300 repeat longer than those initially detected in culture. Given the low degree of repeat length variability within the population of longer alleles (results not shown), they were considered to have resulted from the clonal expansion of mutant cells exhibiting greater survival under conditions of high cell density, and analysed separately. Single molecule analysis of the shorter repeat tracts (<240 repeats) carried by control cells revealed the progressive expansion of trinucleotide repeat tracts from day 65 to day 118 (median gain of 0.132 repeats per day). Arrested cells also showed some degree of repeat expansion, but the increase in repeat number was not always statistically significant (results not shown). Single molecule analysis of the longer alleles (>240 repeats) carried by arrested cells (results not shown) revealed a dramatic and statistically significant increase in the median repeat number from day 87 to day 123 (p=0.0001, two-tailed Mann- Whitney U test).

Despite evidence suggesting that the longer repeats resulted from the clonal expansion mutant cells, rather than the accumulation of repeat size variation in non-dividing cells, extensive development of multiple cell layers was not observed using phase contrast microscopy. Therefore, a 5'-bromo-2-deoxyuridine (BrdU) incorporation and detection analysis was performed on D4393K cells hypothetically arrested by contact inhibition. Two incubation periods with BrdU were used. A 15-minute BrdU incubation period should only label the proportion of cells that are actively undergoing DNA replication during S phase of the cell cycle (also known as the "labelling index"). On the other hand, a 30-hour incubation should label most, if not all, of the cells that are still capable of active proliferation (also known as the "growth fraction") . Despite the low plating efficiency of arrested cells onto an eight- well chamber slide, following BrdU incubation, BrdU staining was not detected in the nucleus of cells arrested by contact inhibition, in clear contrast with the extensive nuclear BrdU staining observed in proliferating cells (results not shown). An average of ~20-30%_> of the cells showed nuclear staining following a 15-minute incubation, whereas -90% of the cell population displayed nuclear BrdU incorporation under proliferating conditions (results not shown). In summary, although BrdU incorporation and immunodetection analysis failed to reveal DNA synthesis in the nuclei of D4393K cells arrested by contact inhibition, the accumulation of longer alleles most likely resulted from the proliferation and clonal expansion of a few mutant cells, which were resistant to contact inhibition and happened to carry longer repeats than the average repeat number. Alternatively, in those cultures where repeat size variability was detected amongst the new mutants (results not shown), the expanded alleles might have resulted from cell proliferation into the gaps left by dead cells, which tend to dissociate from the substrate, possibly allowing their neighbouring cells to divide.

Trinucleotide repeat dynamics in D t-D cells arrested by mitomycin C

As an alternative attempt to stop cellular proliferation in culture, spontaneously immortalised cell lines, in which the trinucleotide repeat dynamics had been previously described, were treated with mitomycin C. Replicate cultures derived from the D2763Kc2 clonal cell line, carrying a rapidly expanding trinucleotide repeat tract were treated with 30 μM mitomycin C for three hours, and maintained in standard growth medium thereafter. Mitomycin C exposure resulted in the death of ~50%> of the cells, as revealed by the levels of cell viability assessed by the acridine orange and ethidium bromide method , one week after the treatment. However, no further decrease in cell viability was detected 49, 77 and 91 days following mitomycin C exposure, with the total number of living cells remaining unaltered. The repeat size variability was monitored at different time points by SP-PCR techniques, not only in cells exposed to mitomycin C, but also in control cells, which were maintained under proliferating conditions. High DNA input SP-PCR analyses revealed clear differences between the mutation profiles in mitomycin C treated cultures and progenitor cells (results not shown). Twenty to 50 transgene molecules collected from each control and arrested cultures were accurately sized (results not shown), and the analysis revealed that not only the control cells exhibited a significant median repeat gain of 0.897 units per day (p<0.0001, two-tailed Mann- Whitney U test), but also mitomycin C treated cells, showed a significant median expansion of 0.341 repeats per day (p=0.0389, two-tailed Mann- Whitney Utest). One could argue that the differences observed in the median repeat number between mitomycin C treated cells and the progenitor culture are intimately associated with a selection for resistant cells, given the great cell death caused by exposure to this DNA cross linker agent. If that were the case, the median repeat size would be expected to stabilise as long as the total cell numbers remained constant. However, statistical analysis revealed significant differences in the median repeat length carried by mitomycin C treated cells between days 49 and 91 (p=0.0484, two-tailed Mann- Whitney U test), a period when a decrease in the number of viable cells was not detected (results not shown). Moreover, in addition to an increase in the median repeat length, the overall range of allele sizes also showed a shift towards longer repeat numbers, suggesting the appearance of new mutants in mitomycin C treated cultures.

BrdU incorporation and immunodetection techniques revealed very low levels of nuclear staining in mitomycin C treated cells per culture, even following a 30-hour incubation with BrdU (<5%>) (results not shown), indicative of some DNA synthesis, which in some cases is associated with distinctive nuclear foci (results not shown). In contrast, ~90-95%> of the proliferating cells showed positive labelling, following an identical incubation period.

The dynamics of expanded CAG^»CTG repeats was also studied in two additional Dmt-D kidney cell lines, D2967K and D3111K (results not shown), following cell cycle arrest by mitomycin C exposure, and SP-PCR analysis as described above. Both cell lines carried slowly expanding trinucleotide sequences, and therefore bulk DNA SP-PCR amplifications might have masked subtle differences in allele size heterogeneity between mitomycin C treated cells and the progenitor culture. Single molecule analysis was then performed, and at least 100 transgene molecules collected from each culture were individually sized. Indeed, the repeat distributions confirmed very low levels of somatic instability in the control cells of both Dmt-D kidney lines, and an expansion bias was not always observed (results not shown). However, some statistically significant differences were revealed, when the repeat distributions were compared between control cells and their progenitor cultures (p<0.05, two-tailed Mann- Whitney U test). More interestingly, similar differences were also observed between some cultures arrested by mitomycin C, and the original cells from which all cultures were derived (results not shown). To account for the hypothesis of mitomycin C-induced cell selection and preferential survival, as described previously, the repeat profiles in treated cultures were also compared to those detected in the first treated cells collected after cell arrest, and significant differences were still detected (results not shown).

Although the results presented here, particularly in regards to D2967K and D3111K cells lines are not totally conclusive, and do not represent the ultimate experimental evidence for the accumulation of repeat size mutations in cells arrested by mitomycin C, they do suggest that variation in trinucleotide repeat size may happen independently of cell division and DNA replication.

Trinucleotide repeat dynamics in Dmt-D cells arrested by apicidin

Although mitomycin C was able to inhibit cell proliferation of cultured Dmt-D cells, this chemical acts as a potent DNA crosslinker and is likely to affect multiple aspects of DNA metabolism apart from replication, including transcription and repair. Therefore, alternative ways of arresting cell proliferation in culture were explored.

Continuous exposure of six replicate D2763Kc2 and D979K cultures to 320 nM apicidin for a week resulted in a minor decrease (~9%>) in the number of viable cells, as assessed by the acridine orange and ethidium bromide method. However, the difference did not prove to be statistically significant (p>0.05, two-tailed t test). The total number of living cells appeared to remain constant one month later, when cell viability was assessed again by the same method. Multiple cell layers could not be observed by phase contrast microscopy, indicating that cells were not proliferating upon exposure to apicidin. The effects of apicidin on cell proliferation were associated with morphological changes in cell shape and cell-to-cell adhesion, suggesting that growth of Dmt-D kidney cells in apicidin restores contact inhibition (results not shown). To confirm cell cycle arrest, BrdU incorporation and detection analyses were performed on both cell lines following one-week exposure to apicidin. Both D2763Kc2 and D979K treatedcells exhibited very low levels of nuclear staining per culture (~5%>-10%>), indicative of low levels of DNA synthesis in the presence of this drug (results not shown).

Trinucleotide repeat length variability was assessed in two treated replicate cultures at different time points, and compared with control cells, proliferating under normal growth conditions for similar periods of time. High DNA input SP-PCR analyses revealed that D2763Kc2 treated cultures not only continued to accumulate expanded alleles, but they were also exhibited a higher expansion rate than control cells (results not shown). Single molecule analysis was performed to confirm these observations, and accurate sizing of 20 to 50 transgene molecules collected from each culture revealed that the median repeat number in apicidin treated cultures was in fact significantly higher than in the progenitor cells (p<0.0003, two-tailed Mann- Whitney U test) at all time points analysed (results not shown). More interestingly, while the median repeat number showed an increase of 0.426 units per day in the control cells, the apicidin treated cultures exhibited a significantly higher median repeat gain: 1.112 and 1.448 repeats per day (pO.OOOl, two-tailed Mann- Whitney [/test). A similar analysis was performed on D979K cells arrested by continuous exposure to 320 nM apicidin. However, the analysis proved more difficult, not only because the progenitor culture consisted of two subpopulations of cells as revealed by SP-PCR amplification, but also because the subpopulation carrying longer alleles soon overgrew the control cultures, with the concomitant loss of cells carrying shorter repeats (results not shown). Nevertheless, accurate sizing of 20 to 50 molecules revealed statistically significant differences, between D979K cultures exposed to apicidin for 98 days and the progenitor culture (p<0.05, two-tailed Mann- Whitney [/test) (results not shown).

As observed with mitomycin C treated cells, the increase in the median repeat size in apicidin treated cells, was accompanied by an expansion-biased trend in the repeat size range detected in both D2763Kc2 and D979K cultures (results not shown). This finding strongly suggests that changes in the mutation profiles are indeed the result of the occurrence of new mutants in culture exposed to apicidin.

References

1. Cummings, CJ. and Zoghbi, H.Y. (2000) Fourteen and counting: unraveling trinucleotide repeat diseases. Hum. Mol. Genet., 9, 909-916.

2. Harper, P.S. Myotonic dystrophy as a trinucleotide repeat disorder - a clinical perspective, in Genetic Instabilities and Hereditaiy Neurological Diseases, R.D. Wells and S.T. Warren, Editors. 1998, Academic Press: San Diego, p. 115-130.

3. Buxton, J., Shelbourne, P., Davies, J., Jones, C, Tongeren, T.N., Aslanidis, C, de Jong, P., Jansen, G., Anvret, M., Riley, B., et al. (1992) Detection of an unstable fragment of DΝA specific to individuals with myotonic dystrophy. Nature, 355, 547-548.

4. Fu, Y.H., Pizzuti, A., Fenwick, R.G., King, J., Rajnarayan, S., Dunne, P.W., Dubel, J., Nasser, G.A., Ashizawa, T., de Jong, P., et al. (1992) An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science, 255, 1256-1258.

5. Brook, J.D., McCurrach, M.E., Harley, H.G., Buckler, A.J., Church, D., Aburatani, H., Hunter, K., Stanton, V.P., Thirion, J.-P., Hudson, T., et al. (1992) Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell, 68, 799-808.

6. Aslanidis, C, Jansen, G., Amemiya, C, Shutler, G., Mahadevan, M., Tsilfidis, C, Chen, C, Alleman, J., Wormskamp, N.G.M., Nooijs, M., et al. (1992) Cloning of the essential myotonic dystrophy region and mapping of the putative defect. Nature, 355, 548- 550.

7. Mahadevan, M., Tsilfidis, C, Sabourin, L., Shutler, G., Amemiya, C, Jansen, G., Neville, C, Narang, M., Barcelo, J., O'Hoy, K., et al. (1992) Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science, 255, 1253-1255.

8. Harley, H.G., Brook, J.D., Rundle, S.A., Crow, S., Reardon, W., Buckler, A.J., Harper, P.S., Housman, D. and Shaw, D.J. (1992) Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy. Nature, 355, 545-546.

9. Harley, H.G., Rundle, S.A., MacMillan, J.C., Myring, J., Brook, J.D., Crow, S., Reardon, W., Fenton, L, Shaw, D.J. and Harper, P.S. (1993) Size of the unstable CTG repeat sequence in relation to phenotype and parental transmission in myotonic dystrophy. Am. J. Hum. Genet, 52, 1164-1174. 10. Lavedan, C, Hofmann-Radvanyi, H., Shelbourne, P., Rabes, J.-P., Duros, C, Savoy, D., Dehaupas, I., Luce, S., Johnson, K. and Junien, C. (1993) Myotonic dystrophy: size- and sex-dependent dynamics of CTG meiotic instability, and somatic mosaicism. Am. J. Hum. Genet., 52, 875-883.

11. Ashizawa, T., Dubel, J.R. and Harati, Y. (1993) Somatic instability of CTG repeat in myotonic dystrophy. Neurology, 43, 2674-2678.

12. Anvret, M., Ahlberg, G., Grandell, U., Hedberg, B., Johnson, K. and Edstrom, L. (1993) Larger expansions of the CTG repeat in muscle compared to lymphocytes from patients with myotonic dystrophy. Hum. Mol. Genet., 2, 1397-1400.

13. Thornton, C.A., Johnson, K.J. and Moxley, R.T. (1994) Myotonic dystrophy patients have larger CTG expansions in skeletal muscle than in leukocytes. Ann. Neurol., 35, 104-107.

14. Ashizawa, T., Dunne, P.W., Ward, P.A., Seltzer, W.K. and Richards, C.S. (1994) Effects of sex of myotonic dystrophy patients on the unstable triplet repeat in their affected offspring. Neurology, 44, 120-122.

15. Jansen, G., Willems, P., Coerwmkel, M., Nillesen, W., Smeets, H., Nits, L., Howeler, C, Brunner, H. and Wieringa, B. (1994) Gonosomal mosaicism in myotonic dystrophy patients: Involvement of mitotic events in (CTG)n variation and selection against extreme expansion in sperm. Am. J. Hum. Genet, 54, 575-585.

16. Monckton, D.G., Wong, L.-J.C, Ashizawa, T. and Caskey, C.T. (1995) Somatic mosaicism, germline expansions, germline reversions and intergenerational reductions in myotonic dystrophy males: small pool PCR analyses. Hum. Mol. Genet., 4, 1-8.

17. Wong, L.-J.C, Ashizawa, T., Monckton, D.G., Caskey, C.T. and Richards, C.S. (1995) Somatic heterogeneity of the CTG repeat in myotonic dystrophy is age and size dependent. Am. J. Hum. Genet, 56, 114-122.

18. Martorell, L., Monckton, D.G., Gamez, J., Johnson, K.J., Gich, I., Lopez de Munain, A. and Baiget, M. (1998) Progression of somatic CTG repeat length heterogeneity in the blood cells of myotonic dystrophy patients. Hum. Mol. Genet, 7, 307-312.

19. Harper, P.S., Harley, H.G., Reardon, W. and Shaw, D.J. (1992) Anticipation in myotonic dystrophy: new light on an old problem. Am. J. Hum. Genet, 51, 10-16.

20. Kennedy, L. and Shelbourne, P.F. (2000) Dramatic mutation instability in HD mouse striatum: does polyglutamine load contribute to cell-specific vulnerability in Huntington's disease? Hum. Mol. Genet, 9, 2539-2544. 21. Richards, R.I. and Sutherland, G.R. (1994) Simple DNA is not replicated simply. Nat. Genet, 6, 114-116.

22. Wells, R.D., Parniewski, P., Pluciennik, A., Bacolla, A., Gellibolian, R. and Jaworski, A. (1998) Small slipped register genetic instabilities in Escherichia coli in triplet repeat sequences associated with hereditary neurological diseases. J. Biol. Chem., 273, 19532-19541.

23. Pearson, C.E. and Sinden, R.R. (1996) Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci. Biochemistry, 35, 5041-5053.

24. Lia, A.S., Seznec, H., Hofmann Radvanyi, H., Radvanyi, F., Duros, C, Saquet, C, Blanche, M., Junien, C. and Gourdon, G. (1998) Somatic instability of the CTG repeat in mice transgenic for the myotonic dystrophy region is age dependent but not correlated to the relative intertissue transcription levels and proliferative capacities. Hum. Mol. Genet., 7, 1285-1291.

25. Fortune, M.T., Vassilopoulos, C, Coolbaugh, M.I., Siciliano, M.J. and Monckton, D.G. (2000) Dramatic, expansion-biased, age-dependent, tissue-specific somatic mosaicism in a transgenic mouse model of triplet repeat instability. Hum. Mol. Genet, 9, 439-445.

26. Seznec, H., Lia-Baldini, A.S., Duros, C, Fouquet, C, Lacroix, C, Hofmann- Radvanyi, H., Junien, C. and Gourdon, G. (2000) Transgenic mice carrying large human genomic sequences with expanded CTG repeat mimic closely the DM CTG repeat intergenerational and somatic instability. Hum. Mol. Genet, 9, 1185-1194.

27. Bacolla, A., Bowater, R.P. and Wells, R.D. Systems for the study of genetic instabilities: Escherichia coli, in Genetic instabilities and hereditary neurological diseases, R.D. Wells and S.T. Warren, Editors. 1998, Academic Press: San Diego, p. 467-484.

28. Jinks-Robertson, S., Greene, C. and Chen, W. Genetic instabilities in yeast, in Genetic instabilities and hereditary neurological diseases, R.D. Wells and S.T. Warren, Editors. 1998, Academic Press: San Diego, p. 485-507.

29. Wohrle, D., Kennerknecht, I., Wolf, M., Enders, H., Schwemmle, S. and Steinbach, P. (1995) Heterogeneity of DM kinase repeat expansion in different fetal tissues and further expansion during cell proliferation in vitro: evidence for a causal involvement of methyl-directed DNA mismatch repair in triplet repeat stability. Hum. Mol. Genet, 4, 1147- 1153. 30. Ashizawa, T., Monckton, D.G., Naishnav, S., Patel, B.J., Noskova, A. and Caskey, C.T. (1996) Instability of the expanded (CTG)n repeats in the myotonin protein kinase gene in cultured lymphoblastoid cell lines from patients with myotonic dystrophy. Genomics, 36, 47-53.

31. Kuhn-Hallek, I., Sage, D.R., Stein, L., Groelle, H. and Fingeroth, J.D. (1995) Expression of recombination activating genes (RAG-1 and RAG-2) in Epstein-Barr virus- bearing B cells. Blood, 85, 1289-1299.

32. Bidichandani, S.I., Purandare, S.M., Taylor, E.E., Gumin, G., Machkhas, H., Harati, Y., Gibbs, R.A., Ashizawa, T. and Patel, P.I. (1999) Somatic sequence variation at the Friedreich ataxia locus includes complete contraction of the expanded GAA triplet repeat, significant length variation in serially passaged lymphoblasts and enhanced mutagenesis in the flanking sequence. Hum. Mol. Genet, 8, 2425-2436.

33. Monckton, D.G., Coolbaugh, M.I., Ashizawa, K., Siciliano, M.J. and Caskey, C.T. (1997) Hypermutable myotonic dystrophy CTG repeats in transgenic mice. Nat. Genet, 15, 193-196.

34. Mangiarini, L., Sathasivam, K., Mahal, A., Mott, R., Seller, M. and Bates, G.P. (1997) Instability of highly expanded CAG repeats in mice transgenic for the Huntington's disease mutation. Nat. Genet, 15, 197-200.

35. La Spada, A.R., Peterson, K.R., Meadows, S.A., McClain, M.E., Jeng, G., Chmelar, R.S., Haugen, H.A., Chen, K., Singer, M.J., Moore, D., et al. (1998) Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability. Hum. Mol. Genet, 7, 959-967.

36. Sato, T., Oyake, M., Νakamura, K., Νakao, K., Fukusima, Y., Onodera, O., Igarashi, S., Takano, H., Kikugawa, K., Ishida, Y., et al. (1999) Transgenic mice harboring a full-length human mutant DRPLA gene exhibit age-dependent intergenerational and somatic instabilities of CAG repeats comparable with those in DRPLA patients. Hum. Mol. Genet, 8, 99-106.

37. Wheeler, N.C., Auerbach, W., White, J.K., Srinidhi, J., Auerbach, A., Ryan, A., Duyao, M.P., Nrbanac, N., Weaver, M., Gusella, J.F., et al. (1999) Length-dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse. Hum. Mol. Genet, 8, 115-122. 38. Shelbourne, P.F., Killeen, N., Hevner, R.F., Johnston, H.M., Tecott, L., Lewandoski, M., Ennis, M., Ramirez, L., Li, Z., Iannicola, C, et al. (1999) A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice. Hum. Mol. Genet, 8, 763-774.

39. Todaro, G.J. and Green, H. (1963) Quantitative studies of the mouse embryo cells in culture and their development into established lines. J. Cell Biol, 17, 299-313.

40. Meek, R.L., Bowman, P.D. and Daniel, C.W. (1977) Establishment of mouse embiyo cells in vitro. Relationship of DNA synthesis, senescence and malignant transformation. Exp. Cell Res., 107, 277-284.

41. Spector, D.L., Goldman, R.D. and Leinwand, L.A. Isolation and purification of intermediate filaments, in Cells: a laboratory manual. 1998, Cold Spring Harbour Laboratory Press: Cold Spring Harbour, p. 55.1.

42. Jeffreys, A.J., Tamaki, K., MacLeod, A., Monckton, D.G., Neil, D.L. and Armour, J.A.L. (1994) Complex gene conversion events in germline mutation at human minisatellites. Nat. Genet, 6, 136-145.

43. Manley, K., Pugh, J. and Messer, A. (1999) Instability of the CAG repeat in immortalized fibroblast cell cultures from Huntington's disease transgenic mice. Brain Res., 835, 74-79.

44. Lanson, N.A., Jr., Egeland, D.B., Royals, B.A. and Claycomb, W.C. (2000) The MRE11-NBS1-RAD50 pathway is perturbed in SV40 large T antigen- immortalized AT-1, AT-2 and HL-1 cardiomyocytes. Nuc. Acids Res., 28, 2882-2892.

45. Kaytor, M.D., Burright, E.N., Duvick, L.A., Zoghbi, H.Y. and Orr, H.T. (1997) Increased trinucleotide repeat instability with advanced maternal age. Hum. Mol. Genet, 6, 2135-2139.

46. Manley, K., Shirley, T.L., Flaherty, L. and Messer, A. (1999) Msh2 deficiency prevents in vivo somatic instability of the CAG repeat in Huntington disease transgenic mice. Nat. Genet, 23, 471-473.

47. Pearson, C.E., Ewel, A., Acharya, S., Fishel, R.A. and Sinden, R.R. (1997) Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases. Hum. Mol. Genet, 6, 1117-1123.

48. Rybak, S.L. and Murphy, R.F. (1998) Primary cell cultures from murine kidney and heart differ in endosomal pH. J. Cell Phys., 176, 216-222. 49. Fortune, J.M. and Osheroff, N. (2000) Topoisomerase II as a target for anticancer drugs : when enzymes stop being nice. Prog Nucleic Acid Res Mol Biol, 64, 221 -253.

50. Jackson, A.L. and Loeb, L.A. (2000) Microsatellite instability induced by hydrogen peroxide in Escherichia coli. Mutation Research, 447, 187-198.

51. Blasina, A., Price, B.D., Turenne, G.A. and McGowan, CH. (1999) Caffeine inhibits the checkpoint kinase ATM. CurrBiol, 9, 1135-8.

52. Momparler, R.L. and Bovenzi, N. (2000) DΝA methylation and cancer. J Cell Physiol, 183, 145-54.

53. Ruschoff, J., Wallinger, S., Dietmaier, W., Bocker, T., Brockhoff, G., Hofstadter, F. and Fishel, R. (1998) Aspirin suppresses the mutator phenotype associated with hereditary nonpolyposis colorectal cancer by genetic selection. Proc. Natl Acad. Sci. USA, 95, 11301- 11306

54 Khajavi, M., Tari, A.M., Patel, Ν.B., Tsuji, K., Siwak, D.R., Meistrich, M.L., Terry, N.H. and Ashizawa, T. (2001) "Mitotic drive" of expanded CTG repeats in myotonic dystrophy type 1 (DM1). Hum. Mol. Genet, 10, 855-863.

55. Martin, B.M., Tissue culture techniques: an introduction. 1994, Boston: Birkhauser.

56. Brock, G.J.R., Anderson, N.H. and Monckton, D.G. (1999) Cts-acting modifiers of expanded CAG/CTG triplet repeat expandability: associations with flanking GC content and proximity to CpG islands. Hum. Mol. Genet, 8, 1061-1067.

57. Gomes-Pereira, M., Fortune, M.T. and Monckton, D.G. (2001). Mouse tissue culture models of unstable triplet repeats: in vitro selection for larger alleles, mutational expansion bias and tissue specificity, but no association with cell division rates. Hum. Mol. Genet. 10, 845 - 854.

58. Miko, M and Chance, B (1975) Ethidium bromide as an uncoupler of oxidative phosphorylation. FEBS Lett. 54, 347 - 352.

59. Chen, K.H. et al. (1998) Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines. Nucleic Acids Res. 26, 2001 - 2007.

60. Thielmann, H.W., Popanda, O., Gersbach, H. & Gilberg, F. (1993) Various inhibitors of DNA topoisomerases diminish repair-specific DNA incision in UV-irradiated human fϊbroblasts. Carcinogenesis 14, 2341 - 2351. 61. Cummins, J.H. (1997) The unique alteration of electrophoretic mobility of fragile-X-expanded fragments in the presence of ethidium bromide. Technical Tips Online SO16895259601054 .

62. McCann, J., Choe, E., Yamasaki, E. & Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonella microsome test: assay of 300 chemicals. Proc. NatlAcad. Sci. USA 72, 5135-5139.

63. Gear, A.R. Rhodamine 6G. (1974) A potent inhibitor of mitochondrial oxidative phosphorylation, J. Biol. Chem. 249, 3628-3737.

Claims

1. An in vitro method for identifying a substance capable of modulating the number of nucleotide repeats in a cell, which method comprises: a) providing a test substance to a cell that displays expanded nucleotide repeat instability; and b) determining if said substance modulates the number of nucleotide repeats of the cell

2. An in vitro method according to claim 1 wherein said cell is a cell line derived from said cell.

3. An in vitro method according to claim 1 wherein said cell is obtained from a subject- derived cell immediately prior to use of the cell in said method.

4. An in vitro method according to claim 1 wherein said cell is obtained from a transgenic animal displaying expanded nucleotide repeat instability immediately prior to use of the cell in said method.

5. An in vitro method according to claim 1 wherein said cell is obtained from an established cell line displaying expanded nucleotide repeat instability.

6. An in vitro method according to claim 3 wherein said transgenic animal is a Dmt-D mouse.

7. An in vitro method according to any preceding claim wherein the cell may be a dividing cell or a non-dividing cell.

8. An in vitro method according to claim 1 wherein the method further comprises an additional step before step b) of allowing the cell to divide for a number of generations.

9. An in vitro method according to claim 1 wherein the method further comprises an additional step before step b) of providing a substance to the cell line that arrests the cell division of the cell.

10. An in vitro method according to claim 9 wherein said substance used to arrest the cell division of the cell is selected from the group consisting of Mitomycin-C and apicidin.

11. An in vitro method according to any preceding claim wherein modulation of the number of repeats in a cell is an increase in the repeat number of nucleotide repeats or rate of nucleotide repeat expansion in the cell ("Positive modulation").

12 An in vitro method according to claim 11 wherein positive modulation is an increase in total array length, relative to the number of nucleotide repeats in the cell before the provision of a test substance to the cell.

13. An in vitro method according to any preceding claim wherein modulation of the number of repeats in a cell is a decrease in the repeat number of nucleotide repeats in the cell ("Negative modulation").

14. An in vitro method according to claim 13 wherein negative modulation is a slowing of the rate of nucleotide repeat expansion, relative to the number of nucleotide repeats in the cell before the provision of a test substance to the cell.

15. An in vitro method according to any preceding claim wherein said modulation is determined using small pool PCR (SP-PCR).

16. An in vitro method according to preceding claim wherein said modulation is determined by comparing cells treated with said substance with untreated cells cultured in parallel.

17. An in vitro method according to any preceding claim wherein said test substance is provided to a cell over a range of doses.

18. An in vitro method according to any preceding claim wherein said test substance is selected from the group consisting of chemicals, nucleic acid analogues, peptides and proteins.

19. An in vitro method according to any preceding claim wherein said test substance is selected from the group consisting of novobiocin, caffeine, hydrogen peroxide, rhodamine 6- G, ethidium bromide, ara C, 5-azacytidine and aspirin.

20. An in vitro method according to any preceding claim wherein the nucleotide repeat is a trinucleotide repeat.

21. A method of obtaining a cell for the study of the cellular metabolism of trinucleotide repeat expansion associated with inherited human disease comprising the steps of: a) harvesting a tissue sample from a Dmt-D transgenic mouse; b) growing cells obtained from the tissue sample; and c) identifying and isolating cells comprising a Dmt-162 transgene and displaying expanded trinucleotide repeat instability.

22. A method of obtaining a cell according to claim 21 wherein said tissues samples are harvested using the explant technique when said tissue sample is harvested from the eye.

23. A method of obtaining a cell according to claim 21 wherein said tissues samples are harvested using the enzymatic dissociation procedure when said tissue sample is harvested from lung or kidney tissue.

24. A cell for the study of the cellular metabolism of trinucleotide repeat expansion associated with inherited human disease, the cell comprising a Dmt-162 transgene and displaying: b) expanded trinucleotide repeat instability.

25. A cell according to claim 24 wherein the cell may be a dividing cell or a non-dividing cell.

26. A cell according to either of claims 24 or 25 wherein the cell is allowed to divide for a number of generations.

27. A cell according to any of claims 24 - 26 wherein a substance is provided to the cell that arrests the cell division of the cell.

28. A cell according to claim 27 wherein said substance used to arrest the cell division of the cell is selected from the group consisting of Mitomycin-C and apicidin.

29. A method of treating a human or animal disease characterised by nucleotide repeat expansion comprising administering to a patient a substance capable of negatively modulating the number of nucleotide repeats in a cell.

30. A method of treating a human or animal disease according to claim 29 wherein said negative modulation is a slowing of the rate of nucleotide repeat expansion, relative to the number of nucleotide repeats in the cell before the provision of a test substance to the cell.

31. A method of treating a human or animal according to either of claims 29-30 wherein said substance is selected from the group consisting of novobiocin, hydrogen peroxide, rhodamine 6-G, ethidium bromide, ara C, 5-azacytidine and aspirin.

32. A method of treating a human or animal according to any one of claims 29-31 wherein said disease characterised by nucleotide repeat expansion is selected from the group consisting of Huntington's disease, myotonic dystrophy type 1, fragile X syndrome, Friedreich ataxia and spinocerebellar ataxias.

33. A method of treating a human or animal according to claim 29-32 wherein said substance is in the form of a pharmaceutical composition comprising an active agent selected from the group consisting of said substance, a synthetic functional derivative of said substance and a carrier.

34. A method of treating a human or animal according to any on of claims 29-33 wherein administration of said substance is selected from the group consisting of intravenous, oral, through an implant and through a patch.

35. Use of a transgenic animal model displaying nucleotide repeat instability for testing substances identified by the method according to claim 1.

36. Use of a transgenic animal model according to claim 35 wherein the animal model is the Dmt-D mouse.

37. Use of a substance determined by the method according to claim 1 for the manufacture of a medicament for the treatment or prophylaxis of human and/or animal disease characterised by nucleotide repeat expansion.

38. Use of a substance according to claim 37 wherein said substance is selected from the group consisting of novobiocin, hydrogen peroxide, rhodamine 6-G, ethidium bromide, ara C, 5-azacytidine and aspirin.