Biochemical mechanisms of drug resistance IV. Development of resistance to 5‐azacytidine and simultaneous depression of pyrimidine metabolism in leukemic mice

Veselý, Jiří; Čihák, A.; Šorm, F.

doi:10.1002/ijc.2910020625

Cited by 31 publications

(9 citation statements)

References 12 publications

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“…Unlike decitabine, the-long term treatment of HCT116 cells with 0.2 mM azacytidine failed to induce L1-cMet transcription (Figure 7b). This may be due to the selection of resistant cells during prolonged drug incubation (Vesely et al, 1967;Qin et al, 2009). Alternatively, the azacytidine …”

Section: L1mentioning

confidence: 99%

Demethylation of a LINE-1 antisense promoter in the cMet locus impairs Met signalling through induction of illegitimate transcription

et al. 2010

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The cytosine analogues 5-azacytidine and 5-aza-2 0 -deoxycytidine are currently the most advanced drugs for epigenetic cancer therapy. Both drugs function as DNA methyltransferase (DNMT) inhibitors and lead to the reactivation of epigenetically silenced tumour suppressor genes. However, not much is known about their target sequence specificity and their possible side effects on normally methylated sequences such as long interspersed nuclear element (LINE)-1 retroelements. It has been shown that demethylation and activation of the LINE-1 antisense promoter can drive the transcription of neighbouring sequences. In this study, we show that demethylation of the colon carcinoma cell line HCT116, either by treatment with DNMT inhibitors or by genetic disruption of the major DNMTs, induces the expression of an illegitimate fusion transcript between an intronic LINE-1 element and the proto-oncogene cMet (L1-cMet). Similar findings were also obtained with myeloid leukaemia cells, an established cellular model for the approved indication of azacytidine and decitabine. Interestingly, upregulation of L1-cMet transcription resulted in reduced cMet expression, which in turn led to decreased cMet receptor signalling. Our results thus provide an important paradigm for demethylation-dependent modulation of gene expression, even if the promoter of the corresponding gene is unmethylated.

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Section: L1mentioning

confidence: 99%

Demethylation of a LINE-1 antisense promoter in the cMet locus impairs Met signalling through induction of illegitimate transcription

et al. 2010

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“…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.…”

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confidence: 99%

Differential nuclear protein binding to 5-azacytosine-containing DNA as a potential mechanism for 5-aza-2'-deoxycytidine resistance.

Michalowsky¹,

Jones²

1987

Mol. Cell. Biol.

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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 ...

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“…They have shown some activity against human acute myelogenous leukemia (19) and have also been effective in markedly increasing the lifespan of mice bearing L1210 leukemia (9). The development of resistance to 5-aza-CR has been associated with decreased levels of uridine-cytidine kinase (16), which is responsible for the primary activation of the analog. On the other hand, resistance to 5-aza-CdR has been attributed to lowered levels of deoxycytidine kinase (16)(17)(18), which is a distinct enzyme.…”

mentioning

confidence: 99%

DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.

Flatau¹,

Gonzales²,

Michalowsky³

et al. 1984

Mol. Cell. Biol.

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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.

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Biochemical mechanisms of drug resistance IV. Development of resistance to 5‐azacytidine and simultaneous depression of pyrimidine metabolism in leukemic mice

Cited by 31 publications

References 12 publications

Demethylation of a LINE-1 antisense promoter in the cMet locus impairs Met signalling through induction of illegitimate transcription

Demethylation of a LINE-1 antisense promoter in the cMet locus impairs Met signalling through induction of illegitimate transcription

Differential nuclear protein binding to 5-azacytosine-containing DNA as a potential mechanism for 5-aza-2'-deoxycytidine resistance.

DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.

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