A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 ,uM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 1OT1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines.
A clonal cell line (56-42) that was stably and exclusively resistant to the toxic effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-CdR) was derived from C3H 10T1/2 C18 cells after multiple treatments with 5-aza-CdR. The 50% lethal dose of 5-aza-CdR for these cells was 1.3 ,uM, which was 15-fold greater than that for the parental cells. Cell line 56-42 was slightly cross-resistant to the ribo-analog 5-azacytidine, but was sensitive to the nucleoside analog 1-4-D-arabinofuranosylcytosine and to colcemid. Both parental and resistant cell lines incorporated equimolar amounts of 5-aza-CdR into DNA. Resistance was therefore not due to decreased activation, increased detoxification, or reduced incorporation of the drug. The overall level of cytosine methylation in the resistant cione was 80% lower than the level in the sensitive cells. Therefore, the potential number of hemimethylated sites created by the incorporation of equivalent amounts of 5-aza-CdR into the DNA of the two cell types was much greater in the sensitive cells. Furthermore, 5-azacytosine-substituted DNA from the sensitive cells bound 100% more nuclear protein in the form of highly stable complexes. The incorporation of 5-azg-CdR opposite methylated cytosine residues in DNA of the sensitive cells thus resulted in increased nuclear protein binding -at hemimethylated sites. This relative increase in tight-binding protein complexes was shown to occur in livlng cells and may well disrupt replication and transcription and instigate cell death. The differential binding of proteins to hypomethylated, azacytosine-containing DNA may thus mediate a novel mechanism of drug resistance.The methylation of specific cytosine residues in DNA is thought to play a role in the transcriptional regulation of certain genes in eucaryotic cells (3,4,15,25). The deoxycytidine analog 5-aza-2'-deoxycytidine (5-aza-CdR) has been widely used as an inducer of suppressed genetic information (13,14,16). The analog is thought to act by incorporating into DNA (5) where it inhibits the postreplicative methylation of cytosine residues (10, 30). The resulting hypomethylation of the genome has been associated with the activation of certain genes (11, 14).5-Aza-CdR is also an effective antileukemic agent in both humans (36) and rmice (23,28,34). The antineoplastic action of the drug is related to its incorporation into DNA (31) and may be mediated through its ability to inhibit DNA methylation (33, 37). In contrast to our present understanding of the mechanisms of 5-aza-CdR-induced gene activation, virtually nothing is known about the mechanisms of drug cytotoxicity. The elucidation of such a mechanism is especially itnportant since the development of resistance to 5-aza-CdR is a major problem in the treatment of leukemias (32).We previously derived a series of cell lines that were resistant to 5-aza-CdR and reported the initial characterization of these cells (12). In this study, we isolated clones from a mass culture of 5-aza-CdR-resistant cells and characterized the mode of ...
As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.
In previous work, three clonal cell lines with extremely low DNA methylation levels were derived by multiple consecutive treatments of C3H 1OT1/2 C18 (1OT1/2) cells with 5-aza-2'-deoxycytidine (5-aza-CdR). In this study we examined the methylation status of genes in these three methyl-deficient clones to assess the specificity of the induced hypomethylation. Complete demethylation of virtually all 5'-CCGG-3' sites was observed in four genes examined, but some sites common to all three clones were persistently methylated even after further exhaustive 5-aza-CdR treatment. Thus, there is a subset of methylation sites within these cells which can never be stably demethylated. The extensive demethylation was not always associated with changes in the level of RNA expression of the genes examined but was strongly correlated with an altered chromatin structure of the unexpressed aq-globin gene and the muscle determination gene MyoDl. These results provide a direct correlation between hypomethylation and the induction of a transcriptionally competent chromatin state.DNA methylation is an epigenetic regulatory mechanism thought to play a role in cell determination and differentiation (21). Consistent with this role is the fact that the extent and pattern of genomic DNA methylation are somatically heritable and species and tissue specific (10,13,32).Alterations in the DNA methylation information system may change epigenetic regulatory signals, which consequently modifies programs of gene expression. Transcriptional activation of certain genes has been correlated with the hypomethylation of specific CpG sites (2, 9, 39). Methylation may therefore lock certain genes in a transcriptionally incompetent state. Recent studies have demonstrated that cytosine methylation may directly influence the binding of regulatory or transcriptional factors to promoter regions of some (1) but not all (15, 16) genes. Alternatively, the inhibitory effect of DNA methylation may be mediated indirectly by protein-DNA interactions which render chromatin inactive (5, 22).However, DNA methylation is only one part of a multifactorial mechanism for the regulation of eucaryotic genes, and a large amount of 5-methylcytosine (5-mCyt) residues are presumably not involved in the direct control of gene expression. The function of the excess methylation may quite possibly be unrelated to gene control. We have therefore used the methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) to derive a series of cell lines stripped of cytosine methylation to determine the minimum level of 5-mCyt which could be attained in mouse cells.The nucleotide analog 5-aza-CdR is a strong inhibitor of DNA methylation and has been widely used as an activator of suppressed genetic information (19,20). Previous studies have examined the effects of a single dose of 5-azacytidine (5-aza-CR) on the methylation status of specific DNA sequences in mouse 10T1/2 cells (17, 18). However, we used repetitive treatments of 10T1/2 cells with 5-aza-CdR to derive a series of cells with genome-w...
The methylation of specific cytosine residues in DNA has been implicated in regulating gene expression and facilitating functional specialization of cellular phenotypes. Generally, the demethylation of certain CpG sites correlates with transcriptional activation of genes. 5-Azacytidine is an inhibitor of DNA methylation and has been widely used as a potent activator of suppressed genetic information. Treatment of cells with 5-azacytidine results in profound phenotypic alterations. The drug-induced hypomethylation of DNA apparently perturbs DNA-protein interactions that may consequently alter transcriptional activity and cell determination. The inhibitory effect of cytosine methylation may be exerted via altered DNA-protein interactions specifically or may be transduced by a change in the conformation of chromatin. Recent studies have demonstrated that cytosine methylation also plays a central role in parental imprinting, which in turn determines the differential expression of maternal and paternal genomes during embryogenesis. In other words, methylation is the mechanism whereby the embryo retains memory of the gametic origin of each component of genetic information. A memory of this type would probably persist during DNA replication and cell division as methylation patterns are stable and heritable.
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