Limits...
Quantitative determination of decitabine incorporation into DNA and its effect on mutation rates in human cancer cells.

Öz S, Raddatz G, Rius M, Blagitko-Dorfs N, Lübbert M, Maercker C, Lyko F - Nucleic Acids Res. (2014)

Bottom Line: The mode of action of decitabine strictly depends on the incorporation of the drug into DNA.Finally, we also used next-generation sequencing to comprehensively analyze the effects of decitabine incorporation on the DNA sequence level.These results indicate that standard rates of decitabine incorporation are not genotoxic in myeloid leukemia cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, 69120 Heidelberg, Germany.

Show MeSH

Related in: MedlinePlus

Decitabine incorporation in various human cell lines. (A) Incorporation (upper panel) and total intracellular levels (lower panel) of [3H]-decitabine (100 nM) in DNA of various cancer cells lines after 24 h of treatment. (B) Cell cycle analysis of untreated (upper panel) and decitabine-treated (lower panel, 100 nM for 24 h) cancer cell lines by flow cytometry. (C) Box plot showing incorporation levels of [3H]-decitabine (100 nM, 24 h) for cancer cell lines and non-cancerous cell lines. (D) Box plot showing total intracellular levels of [3H]-decitabine (100 nM, 24 h) for cancer cell lines and non-cancerous cells. P-values were calculated by a Student's t-test. (E) Incorporation levels of [3H]-decitabine (100 nM, 24 h) in DNA of various AML patient samples. (F) Total intracellular levels of [3H]-decitabine in the same AML patient samples.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231731&req=5

Figure 4: Decitabine incorporation in various human cell lines. (A) Incorporation (upper panel) and total intracellular levels (lower panel) of [3H]-decitabine (100 nM) in DNA of various cancer cells lines after 24 h of treatment. (B) Cell cycle analysis of untreated (upper panel) and decitabine-treated (lower panel, 100 nM for 24 h) cancer cell lines by flow cytometry. (C) Box plot showing incorporation levels of [3H]-decitabine (100 nM, 24 h) for cancer cell lines and non-cancerous cell lines. (D) Box plot showing total intracellular levels of [3H]-decitabine (100 nM, 24 h) for cancer cell lines and non-cancerous cells. P-values were calculated by a Student's t-test. (E) Incorporation levels of [3H]-decitabine (100 nM, 24 h) in DNA of various AML patient samples. (F) Total intracellular levels of [3H]-decitabine in the same AML patient samples.

Mentions: In following experiments, we further expanded the scope of our analysis by investigating decitabine incorporation in a panel of six leukemia cell lines (ML-2, K562, KG-1, KG-1a, U937 and HL-60) and a colon cancer cell line (HCT116). When these cell lines were treated with the same dose (100 nM, 24 h) of [3H]-decitabine, we did not observe any detectable drug-induced cytotoxicity (Supplementary Table S3). However, incorporation rates ranged from 0.1 pmol/μg DNA to 0.3 pmol/μg DNA (Figure 4A, upper panel). Total intracellular decitabine levels appeared similar to the DNA-incorporated levels, with HL-60 and U937 cells showing the highest uptake (Figure 4A, lower panel). Drug-dependent differences in the cell cycle profiles were minor and not significant (P > 0.5, two-way ANOVA) for any of the seven cell lines (Figure 4B), which indicates that our results are a consequence of differential drug uptake and not influenced by differential drug toxicity. Interestingly, non-cancerous cell lines showed significantly (P = 0.007, t-test) lower levels of incorporated decitabine (Figure 4C). Similarly, total levels of intracellular decitabine were significantly (P = 0.018, t-test) lower in non-cancerous cells (Figure 4D). These findings are in agreement with the reduced cellular responses to low-dose decitabine in normal bone marrow cells (12).


Quantitative determination of decitabine incorporation into DNA and its effect on mutation rates in human cancer cells.

Öz S, Raddatz G, Rius M, Blagitko-Dorfs N, Lübbert M, Maercker C, Lyko F - Nucleic Acids Res. (2014)

Decitabine incorporation in various human cell lines. (A) Incorporation (upper panel) and total intracellular levels (lower panel) of [3H]-decitabine (100 nM) in DNA of various cancer cells lines after 24 h of treatment. (B) Cell cycle analysis of untreated (upper panel) and decitabine-treated (lower panel, 100 nM for 24 h) cancer cell lines by flow cytometry. (C) Box plot showing incorporation levels of [3H]-decitabine (100 nM, 24 h) for cancer cell lines and non-cancerous cell lines. (D) Box plot showing total intracellular levels of [3H]-decitabine (100 nM, 24 h) for cancer cell lines and non-cancerous cells. P-values were calculated by a Student's t-test. (E) Incorporation levels of [3H]-decitabine (100 nM, 24 h) in DNA of various AML patient samples. (F) Total intracellular levels of [3H]-decitabine in the same AML patient samples.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231731&req=5

Figure 4: Decitabine incorporation in various human cell lines. (A) Incorporation (upper panel) and total intracellular levels (lower panel) of [3H]-decitabine (100 nM) in DNA of various cancer cells lines after 24 h of treatment. (B) Cell cycle analysis of untreated (upper panel) and decitabine-treated (lower panel, 100 nM for 24 h) cancer cell lines by flow cytometry. (C) Box plot showing incorporation levels of [3H]-decitabine (100 nM, 24 h) for cancer cell lines and non-cancerous cell lines. (D) Box plot showing total intracellular levels of [3H]-decitabine (100 nM, 24 h) for cancer cell lines and non-cancerous cells. P-values were calculated by a Student's t-test. (E) Incorporation levels of [3H]-decitabine (100 nM, 24 h) in DNA of various AML patient samples. (F) Total intracellular levels of [3H]-decitabine in the same AML patient samples.
Mentions: In following experiments, we further expanded the scope of our analysis by investigating decitabine incorporation in a panel of six leukemia cell lines (ML-2, K562, KG-1, KG-1a, U937 and HL-60) and a colon cancer cell line (HCT116). When these cell lines were treated with the same dose (100 nM, 24 h) of [3H]-decitabine, we did not observe any detectable drug-induced cytotoxicity (Supplementary Table S3). However, incorporation rates ranged from 0.1 pmol/μg DNA to 0.3 pmol/μg DNA (Figure 4A, upper panel). Total intracellular decitabine levels appeared similar to the DNA-incorporated levels, with HL-60 and U937 cells showing the highest uptake (Figure 4A, lower panel). Drug-dependent differences in the cell cycle profiles were minor and not significant (P > 0.5, two-way ANOVA) for any of the seven cell lines (Figure 4B), which indicates that our results are a consequence of differential drug uptake and not influenced by differential drug toxicity. Interestingly, non-cancerous cell lines showed significantly (P = 0.007, t-test) lower levels of incorporated decitabine (Figure 4C). Similarly, total levels of intracellular decitabine were significantly (P = 0.018, t-test) lower in non-cancerous cells (Figure 4D). These findings are in agreement with the reduced cellular responses to low-dose decitabine in normal bone marrow cells (12).

Bottom Line: The mode of action of decitabine strictly depends on the incorporation of the drug into DNA.Finally, we also used next-generation sequencing to comprehensively analyze the effects of decitabine incorporation on the DNA sequence level.These results indicate that standard rates of decitabine incorporation are not genotoxic in myeloid leukemia cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, 69120 Heidelberg, Germany.

Show MeSH
Related in: MedlinePlus