Growth inhibition of human cancer cells by 5-aza-2′-deoxycytidine does not correlate with its effects on <i>INK4a/ARF</i> expression or initial promoter methylation status

Xiong, Jun; Epstein, Richard J.

doi:10.1158/1535-7163.mct-08-0926

Cited by 8 publications

(5 citation statements)

References 37 publications

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“…Replenishment of 5-aza-CdR daily ensures that sufficient levels of the drug are sustained throughout the treatment period to improve efficacy. While previous studies have performed similar 5-aza-CdR daily treatments in other cancer cell lines, the treatment period (3–4 days only) was relatively short and did not achieve the same efficacy in terms of cell proliferation and cell death when compared to the treatment regimen used in our study [38], [39].…”

Section: Discussionmentioning

confidence: 61%

GSTP1 DNA Methylation and Expression Status Is Indicative of 5-aza-2′-Deoxycytidine Efficacy in Human Prostate Cancer Cells

et al. 2011

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DNA methylation plays an important role in carcinogenesis and the reversibility of this epigenetic modification makes it a potential therapeutic target. To date, DNA methyltransferase inhibitors (DNMTi) have not demonstrated clinical efficacy in prostate cancer, with one of the major obstacles being the inability to monitor drug activity during the trial. Given the high frequency and specificity of GSTP1 DNA methylation in prostate cancer, we investigated whether GSTP1 is a useful marker of DNMTi treatment efficacy. LNCaP prostate cancer cells were treated with 5-aza-2′-deoxycytidine (5-aza-CdR) either with a single high dose (5–20 µM), every alternate day (0.1–10 µM) or daily (0.005–2.5 µM). A daily treatment regimen with 5-aza-CdR was optimal, with significant suppression of cell proliferation achieved with doses of 0.05 µM or greater (p<0.0001) and induction of cell death from 0.5 µM (p<0.0001). In contrast, treatment with a single high dose of 20 µM 5-aza-CdR inhibited cell proliferation but was not able to induce cell death. Demethylation of GSTP1 was observed with doses of 5-aza-CdR that induced significant suppression of cell proliferation (≥0.05 µM). Re-expression of the GSTP1 protein was observed only at doses of 5-aza-CdR (≥0.5 µM) associated with induction of cell death. Treatment of LNCaP cells with a more stable DNMTi, Zebularine required at least a 100-fold higher dose (≥50 µM) to inhibit proliferation and was less potent in inducing cell death, which corresponded to a lack of GSTP1 protein re-expression. We have shown that GSTP1 DNA methylation and protein expression status is correlated with DNMTi treatment response in prostate cancer cells. Since GSTP1 is methylated in nearly all prostate cancers, our results warrant its testing as a marker of epigenetic therapy response in future clinical trials. We conclude that the DNA methylation and protein expression status of GSTP1 are good indicators of DNMTi efficacy.

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Section: Discussionmentioning

confidence: 61%

GSTP1 DNA Methylation and Expression Status Is Indicative of 5-aza-2′-Deoxycytidine Efficacy in Human Prostate Cancer Cells

et al. 2011

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“…The human CDKN2A gene was chosen in this analysis as an example of human endogenous genes potentially relevant for clinical use against cancer cells [37]–[40]. The DLD-1 and the MCF-10A cell lines were used for recipients of the AAV vectors, because the expression of endogenous CDKN2A gene in these cell lines was abrogated by the methylation of promoter region [41] and the homozygous deletion of the gene, respectively, allowing for an accurate measurement of the exogenous CDKN2A gene expression. To alleviate the impact of CDKN2A overexpression on cell cycle progression [42], a H83Y variant form of the CDKN2A gene was utilized for this analysis.…”

Section: Resultsmentioning

confidence: 99%

A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors

et al. 2014

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The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.

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“…These relatively small numbers of genes are possibly due to the fact that Aza not only induces the re-expression of silenced genes through the demethylation of CpG islands, but it increases the expression of unmethylated genes. The agent also induces cell stress by DNA damage (Xiong and Epstein, 2009). It is notable that the TNF-α pathway, which was most prominent in the MCF-7 cells, also ranks among the top networks in other breast cancer cells and other cancer tissues.…”

Section: Discussionmentioning

confidence: 99%

Expression Profiling after Induction of Demethylation in MCF-7 Breast Cancer Cells Identifies Involvement of TNF-α Mediated Cancer Pathways

Kim

Kang

Kim

et al. 2012

Molecules and Cells

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Growth inhibition of human cancer cells by 5-aza-2′-deoxycytidine does not correlate with its effects on INK4a/ARF expression or initial promoter methylation status

Cited by 8 publications

References 37 publications

GSTP1 DNA Methylation and Expression Status Is Indicative of 5-aza-2′-Deoxycytidine Efficacy in Human Prostate Cancer Cells

GSTP1 DNA Methylation and Expression Status Is Indicative of 5-aza-2′-Deoxycytidine Efficacy in Human Prostate Cancer Cells

A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors

Expression Profiling after Induction of Demethylation in MCF-7 Breast Cancer Cells Identifies Involvement of TNF-α Mediated Cancer Pathways

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