A novel selectable system for detecting expansion of CAG.CTG repeats in mammalian cells

CAG.CTG repeat expansions cause more than a dozen neurodegenerative diseases in humans. To define the mechanism of repeat instability in mammalian cells we developed a selectable assay to detect expansions of CAG.CTG triplet repeats in Chinese hamster ovary (CHO) cells. We showed previously that long tracts of CAG.CTG repeats, embedded in an intron of the APRT gene, kill expression of the gene, rendering the cells APRT-. By contrast, tracts with fewer than 34 repeats allow sufficient expression to give APRT+ cells. Although it should be possible to use APRT+ cells with short repeats to assay for expansion events by selecting for APRT- cells, we find that APRT+ cells with 31 repeats are not killed by the standard APRT- selection protocol, most likely because they produce too little Aprt to incorporate sufficient 8-azaadenine into their adenine pool. To overcome this problem, we devised a new selection, which increases the proportion of the adenine pool contributed by the salvage pathway by partially inhibiting the de novo pathway. We show that APRT- CHO cells with 61 or 95 CAG.CTG repeats survive this selection, whereas cells with 31 repeats die. Using this selection system, we can select for expansion to as few as 39 repeats. Thus, this assay can monitor expansions across the critical boundary from the longest lengths of normal alleles to the shortest lengths of disease alleles.     

5-aza-2'-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation

In addition to its demethylating function, 5-aza-2'-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 microm) induces inhibition of cell proliferation as well as increased p53/p21(Waf1/Cip1) expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21(Waf1/Cip1) expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21(Waf1/Cip1) expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21(Waf1/Cip1) expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.     

HDAC inhibitors act with 5-aza-2'-deoxycytidine to inhibit cell proliferation by suppressing removal of incorporated abases in lung cancer cells

5-Aza-2'-deoxycytidine (5-aza-CdR) is used extensively as a demethylating agent and acts in concert with histone deacetylase inhibitors (HDACI) to induce apoptosis or inhibition of cell proliferation in human cancer cells. Whether the action of 5-aza-CdR in this synergistic effect results from demethylation by this agent is not yet clear. In this study we found that inhibition of cell proliferation was not observed when cells with knockdown of DNA methyltransferase 1 (DNMT1), or double knock down of DNMT1-DNMT3A or DNMT1-DNMT3B were treated with HDACI, implying that the demethylating function of 5-aza-CdR may be not involved in this synergistic effect. Further study showed that there was a causal relationship between 5-aza-CdR induced DNA damage and the amount of [(3)H]-5-aza-CdR incorporated in DNA. However, incorporated [(3)H]-5-aza-CdR gradually decreased when cells were incubated in [(3)H]-5-aza-CdR free medium, indicating that 5-aza-CdR, which is an abnormal base, may be excluded by the cell repair system. It was of interest that HDACI significantly postponed the removal of the incorporated [(3)H]-5-aza-CdR from DNA. Moreover, HDAC inhibitor showed selective synergy with nucleoside analog-induced DNA damage to inhibit cell proliferation, but showed no such effect with other DNA damage stresses such as gamma-ray and UV, etoposide or cisplatin. This study demonstrates that HDACI synergistically inhibits cell proliferation with nucleoside analogs by suppressing removal of incorporated harmful nucleotide analogs from DNA.     

5-Aza-2'-deoxycytidine reactivates gene expression via degradation of pRb pocket proteins

Not only does 5-aza-2'-deoxycytidine (5-aza-CdR) induce the reexpression of silenced genes through the demethylation of CpG islands, but it increases the expression of unmethylated genes. However, the mechanism by which 5-aza-CdR activates the expression of genes is not completely understood. Here, we report that the pRb pocket proteins pRb, p107, and p130 were degraded in various cancer cell lines in response to 5-aza-CdR treatment, and this effect was dependent on the proteasome pathway. Mouse double minute 2 (MDM2) played a critical role in this 5-aza-CdR-induced degradation of pRb. Furthermore, PP2A phosphatase-induced MDM2 dephosphorylation at S260 was found to be essential for MDM2 binding to pRb in the presence of 5-aza-CdR. pRb degradation resulted in the significant reexpression of several genes, including methylated CDKN2A, RASFF1A, and unmethylated CDKN2D. Finally, knockdown of pRb pocket proteins by either RNAi or 5-aza-CdR treatment induced a significant decrease in the recruitment of SUV39H1 and an increase in the enrichment of KDM3B and KDM4A to histones around the promoter of RASFF1A and thus reduced H3K9 di- and trimethylation, by which RASFF1A expression is activated. Our data reveal a novel mechanism by which 5-aza-CdR induces the expression of both methylated and unmethylated genes by degrading pRb pocket proteins.     

CTCF cis-Regulates Trinucleotide Repeat Instability in an Epigenetic Manner: A Novel Basis for Mutational Hot Spot Determination

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting “instability elements,” and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF—a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.     

Demethylating agent 5-aza-2-deoxycytidine enhances susceptibility of breast cancer cells to anticancer agents

DNA methylation plays an important role in regulation of gene expression and is increasingly being recognized as a determinant of chemosensitivity of human cancers. With the aim of improving the chemotherapeutic efficacy of breast carcinoma, the effect of DNA methyltransferase inhibitor, 5-Aza-2′-deoxycytidine (5-aza-CdR), on the chemosensitivity of anticancer drugs was investigated. The cytotoxicity of paclitaxel (PTX), adriamycin (ADR), and 5-fluorouracil (5-FU) was analyzed against human breast cancer cell lines, MDA MB 231 and MCF 7 cell lines using the MTT assay, and the synergy of 5-aza-CdR and these agents was determined by Drewinko’s fraction method. The effects of each single agent or the combined treatment on cell cycle arrest were analyzed by flow cytometric analysis. We also investigated the effect of each single agent or the combined treatment of anticancer drugs with 5-aza-CdR on the methylation status of the selected genes by methylation specific PCR. In MDA MB 231 cells, a synergistic antiproliferative effect was observed with a combination of 10 μM 5-aza-CdR and these three anticancer drugs, while in MCF 7 cells, a semiadditive effect was observed. Treatment with 5-aza-CdR and anticancer drug resulted in partial demethylation of a panel of genes including RARβ2, Slit2, GSTP1, and MGMT. Based on these findings, we propose that 5-aza-CdR enhances the chemosensitivity of anticancer drugs in breast cancer cells and may be a promising approach for increasing the chemotherapeutic potential of these anticancer agents for more effective management of breast carcinomas.     

Defining genetic factors that modulate intergenerational CAG repeat instability in Drosophila melanogaster

Trinucleotide repeat instability underlies >20 human hereditary disorders. These diseases include many neurological and neurodegenerative situations, such as those caused by pathogenic polyglutamine (polyQ) domains encoded by expanded CAG repeats. Although mechanisms of instability have been intensely studied, our knowledge remains limited in part due to the lack of unbiased genome-wide screens in multicellular eukaryotes. Drosophila melanogaster displays triplet repeat instability with features that recapitulate repeat instability seen in patients with disease. Here we report an enhanced fly model with substantial instability based on a noncoding 270 CAG (UAS-CAG(270)) repeat construct under control of a germline-specific promoter. We find that expression of pathogenic polyQ protein modulates repeat instability of CAG(270) in trans, indicating that pathogenic-length polyQ proteins may globally modulate repeat instability in the genome in vivo. We further performed an unbiased genetic screen for novel modifiers of instability. These studies indicate that different aspects of repeat instability are under independent genetic control, and identify CG15262, a protein with a NOT2/3/5 conserved domain, as a modifier of CAG repeat instability in vivo.     

Radiation-induced genomic instability in tandem repeat sequences is not predictive of unique sequence instability

Tandem repeat sequences, classified as minisatellite sequences or partially duplicated genes, are inherently unstable. Radiation exposure can increase the instability of such repeat sequences, but the biological consequences of this elevated instability are not well characterized. To learn more about the characteristics of the instability at different sequences in the genome, we created mutant HT1080 cells bearing 8.4 kb of partially duplicated allele at the HPRT locus by gene targeting. The cells were then tested to determine whether repeat-sequence instability (assessed by elevated reversion rate caused by loss of one duplicated segment) accompanied increased forward mutation rates at the restored wild-type HPRT allele. After a 4-Gy X irradiation, 32 clones were selected (out of 500 clones, 6%) that showed elevated reversion rates even after many cell generations. These clones also showed general increases in the forward mutation rate, whereas the paired individual mutation rates did not correlate with each other. Furthermore, levels of intracellular reactive oxygen species (ROS) and nuclear gamma-H2AX foci, which are hallmarks for DNA damage responses, were also generally elevated, although the levels did not correlate with the individual reversion rates. It was concluded that repeat sequence instability is not predictive of unique sequence instability, probably because the instability is generated by multiple mechanisms after radiation exposure.     

Determination of spontaneous loss of heterozygosity mutations in Aprt heterozygous mice.

A mouse model was generated to investigate loss of heterozygosity (LOH) events in somatic cells. The adenine phosphoribosyltransferase ( Aprt ) gene was disrupted in embryonic stem cells using a conventional gene targeting approach and subsequently Aprt hetero-zygous and homozygous mice were derived. Aprt homozygous deficient animals were viable though the mendelian inheritance pattern was skewed. On average these mice died at 6 months of age from severe renal failure. In T-lymphocytes of Aprt heterozygous mice the mean spontaneous mutant frequency at the Aprt locus was 8.7 x 10(-6) while the frequency was 0.8 x 10(-6) at the hypoxanthine phosphoribosyltransferase locus. In order to determine whether LOH events contribute to the high spontaneous mutant frequency at the Aprt locus, 140 Aprt mutant T-lymphocyte clones were expanded and analysed by allele-specific PCR. In 97 (69%) of these clones the wild-type allele had been lost. Nine of the mutant clones were characterized in more detail using dual-coloured fluorescence in situ hybridization analysis. Five out of six of the mutant clones which arose from an LOH event, based on the PCR assay, contained a duplication of the targeted allele. Therefore, mitotic recombination or chromosome loss followed by duplication of the remaining homologue appears to be the predominant mechanism for the in vivo generation of Aprt mutant T-lymphocytes.     

Differential somatic CAG repeat instability in variable brain cell lineage in dentatorubral pallidoluysian atrophy (DRPLA): a laser-captured microdissection (LCM)-based analysis

Employing a laser-captured microdissection (LCM), we have investigated the somatic instability of CAG repeats in the variable brain cell lineage in three patients with dentatorubral pallidoluysian atrophy (DRPLA). LCM enables the isolation of single lineage brain cells for subsequent molecular analysis. We have found that CAG repeat size and the range of CAG repeats in the cerebellar granular cells is smaller than those in cerebellar glial cells. Similarly, those in the cerebral neuronal cells are significantly shorter than those in cerebral glial cells. These data directly indicate that the CAG repeat is relatively more stable in neuronal cells than in glial cells. Furthermore, cerebellar granular cells show significantly smaller main CAG repeat size and CAG repeat range than either Purkinje cells or cerebral neuronal cells, suggesting that somatic instability in the CAG repeat is markedly variable even among the different types of neuronal populations. The cell-specific CAG repeat instability may thus be more complex than has previously been considered. LCM is a powerful tool for elucidating the mechanism of the triplet repeat instability of each cell type.     

5-杂氮-2′-脱氧胞苷诱导鼻咽癌细胞株Syk基因启动子去甲基化的研究

利用5-杂氮-2′-脱氧胞苷（5-aza-2′-deoxycytidine,5-aza-CdR）处理体外培养的鼻咽癌细胞株CNE-1、CNE-2及永生化非癌性人鼻咽上皮细胞株NP-69,采用BS-PCR、Q-RT-PCR及Westernblot方法分别检测经5μmol/L的5-aza-CdR处理前后,各细胞株中Syk基因启动子甲基化状况及SykmRNA和蛋白质表达情况。探讨去甲基化药物5-杂氮-2′-脱氧胞苷（5-aza-CdR）对鼻咽癌细胞株中脾酪氨酸激酶（spleen tyrosine kinase,Syk）启动子甲基化水平及其表达的影响。结果显示,Syk基因启动子甲基化水平与鼻咽癌细胞分化程度呈负相关,两种鼻咽癌细胞株的Syk mRNA和蛋白质表达水平显著低于NP-69细胞（P〈0.01）;经5-aza-CdR处理后两种鼻咽癌细胞株的Syk基因启动子甲基化水平降低,Syk mRNA及蛋白质表达升高（P〈0.05）;高分化鼻咽癌细胞株对药物敏感性高于低分化鼻咽癌细胞株（P〈0.01）。由此可见,两种鼻咽癌细胞株中存在不同程度的Syk基因启动子甲基化状态,5-aza-CdR能有效逆转鼻咽癌细胞株Syk基因启动子的甲基化状态,升高Syk mRNA及蛋白质表达,同时鼻咽癌细胞分化程度越高恢复Syk基因表达的比率越高。     

Genetic recombination destabilizes (CTG)n.(CAG)n repeats in E. coli

The expansion of trinucleotide repeats has been implicated in 17 neurological diseases to date. Factors leading to the instability of trinucleotide repeat sequences have thus been an area of intense interest. Certain genes involved in mismatch repair, recombination, nucleotide excision repair, and replication influence the instability of trinucleotide repeats in both Escherichia coli and yeast. Using a genetic assay for repeat deletion in E. coli, the effect of mutations in the recA, recB, and lexA genes on the rate of deletion of (CTG)n.(CAG)n repeats of varying lengths were examined. The results indicate that mutations in recA and recB, which decrease the rate of recombination, had a stabilizing effect on (CAG)n.(CTG)n repeats decreasing the high rates of deletion seen in recombination proficient cells. Thus, recombination proficiency correlates with high rates of genetic instability in triplet repeats. Induction of the SOS system, however, did not appear to play a significant role in repeat instability, nor did the presence of triplet repeats in cells turn on the SOS response. A model is suggested where deletion during exponential growth may result from attempts to restart replication when paused at triplet repeats.     

Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in anaplastic large cell lymphoma

DNA methylation is an epigenetic mechanism establishing long-term gene silencing during development and cell commitment, which is maintained in subsequent cell generations. Aberrant DNA methylation is found at gene promoters in most cancers and can lead to silencing of tumor suppressor genes. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is able to reactivate genes silenced by DNA methylation and has been shown to be a very potent epigenetic drug in several hematological malignancies. In this report, we demonstrate that 5-aza-CdR exhibits high antineoplastic activity against anaplastic large cell lymphoma (ALCL), a rare CD30 positive non-Hodgkin lymphoma of T-cell origin. Low dose treatment of ALCL cell lines and xenografted tumors causes apoptosis and cell cycle arrest in vitro and in vivo. This is also reflected in genome-wide expression analyses, where genes related to apoptosis and cell death are amongst the most affected targets of 5-aza-CdR. Furthermore, we observed demethylation and re-expression of p16^INK4A after drug administration and senescence associated β-galactosidase activity. Thus, our data provide evidence that 5-aza-CdR is highly efficient against ALCL and warrants further clinical evaluation for future therapeutic use.     

Age-related accumulation of autosomal mutations in solid tissues of the mouse is gender and cell type specific

Most cancers in solid tissues increase with age and invariably contain causal mutations eliminating expression of one or more autosomal tumor suppressor genes. However, very little is known about the effect of age on autosomal mutations, often large in size, in cells of solid tissues. In this study, the frequency and spectrum of autosomal mutations were examined as a function of age for kidney epithelial cells and ear mesenchymal cells in B6D2F1 mice heterozygous for the selectable Aprt locus. Aprt mutant frequencies were found to increase with age in the kidneys of both male and female mice, but at all ages the mutant frequencies were approximately twice as high in the females, which in this strain have shorter lifespans than the males. An age-related increase in Aprt mutant frequencies was also observed for ear cells from female mice, but no significant increases in mutant frequencies were observed for the ear cells of male mice. A molecular analysis showed that the kidney and ear mutational spectra were distinct and that the age-related increases in mutant frequencies did not involve significant shifts in the mutational spectra. In total, the results demonstrate both gender and cell-type-specific patterns of autosomal mutational accumulation as a function of age in two solid tissues of the mouse.     

Assays to predict the genotoxicity of the chromosomal mutagen etoposide -- focussing on the best assay

The topoisomerase II inhibitor etoposide is used routinely to treat a variety of cancers in patients of all ages. As a result of its extensive use in the clinic and its association with secondary malignancies it has become a compound of great interest with regard to its genotoxic activity in vivo. This paper describes a series of assays that were employed to determine the in vivo genotoxicity of etoposide in a murine model system. The alkaline comet assay detected DNA damage in the bone marrow mononuclear compartment over the dose range of 10--100mg/kg and was associated with a large and dose dependent rise in the proportion of cells with severely damaged DNA. In contrast, the bone marrow micronucleus assay was found to be sensitive to genotoxic damage between the doses of 0.1--1mg/kg without any corresponding increases in cytotoxicity. An increase in the mutant frequency was undetectable at the Hprt locus at administered doses of 1 and 10mg/kg of etoposide, however, an increase in the mutant frequency was seen at the Aprt locus at these doses. We conclude that the BMMN assay is a good short-term predictor of the clastogenicity of etoposide at doses that do not result in cytotoxic activity, giving an indication of potential mutagenic effects. Moreover, the detection of mutants at the Aprt locus gives an indication of the potential of etoposide to cause chromosomal mutations that may lead to secondary malignancy.     

Micronuclei and sister chromatid exchanges induced by capsaicin in human lymphocytes

Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA. Changes in triplet repeat length during growth following transformation in E. coli have been used as a measure of repeat instability. However, very little is known about the molecular and biological changes that may occur on transformation. Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells. To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E. coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E. coli. Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic. The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2 x 10(5) following transformation. The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent. Thus, the process of transformation was required to observe the instability. Instabilities of (CAG).(CTG) repeats are also dramatically elevated upon transformation. The magnitude of the instability is dependent on the nature and length of the repeat. Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements. Moreover, different E. coli genetic backgrounds show different levels of instability during transformation.     

Radiation-induced genetic instability in vivo depends on p53 status

In response to ionizing radiation and other agents that damage DNA, the p53 tumor suppressor protein activates multiple cellular processes including cell cycle checkpoints and programmed cell death. Although loss of p53 function is associated with radiation-induced genetic instability in cell lines, it is not clear if this relationship exists in vivo. To study the role of p53 in maintenance of genetic stability in normal tissues following irradiation, we have measured mutant frequencies at the adenine phosphoribosyltransferase (Aprt) and hypothanine-guanine phosphoribosyltransferase (Hprt) loci and examined mechanisms of loss of heterozygosity (LOH) in normal T cells of p53-deficient, Aprt heterozygous mice that were subjected to whole-body irradiation with a single dose of 4Gy X-rays. The radiation-induced mutant frequency at both the Aprt and Hprt loci was elevated in cells from mice with different p53 genotypes. The radiation-induced elevation of p53-/- mice was significantly greater than that of p53+/- or p53+/+ mice and was caused by several different kinds of mutational events at the both chromosomal and intragenic levels. Most significantly, interstitial deletion, which occurs rarely in unirradiated mice, became the most common mechanism leading to LOH in irradiated p53 null mice. These observations support the idea that absence or reduction of p53 expression enhances radiation-induced tumorigenesis by increasing genetic instability at various loci, such as those for tumor suppressor genes.