Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

Rogstad, Daniel K.; Darwanto, Agus; Herring, Jason; Rogstad, Katherine Noyes; Burdzy, Artur; Hadley, Scott; Neidigh, Jonathan W.; Sowers, Lawrence C.

doi:10.1021/tx700221x

Cited by 9 publications

(14 citation statements)

References 41 publications

(117 reference statements)

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“…As shown in Figure 1E and F , incorporation of FU was not stimulated by the presence of hmUdR nor vice versa. The incorporation of hmUdR estimated here appears much higher than the incorporation of hmUdR previously measured in U2OS cells [ 11 ]. This is probably because HT-29 cells have extremely weak activity for excision of hmU ( Supplementary Figure 2 ).…”

Section: Resultscontrasting

confidence: 79%

Synergistic enhancement of 5-fluorouracil cytotoxicity by deoxyuridine analogs in cancer cells

et al. 2015

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5-Fluorouracil (FU) is a halogenated nucleobase analog that is widely used in chemotherapy. Here we show that 5-hydroxymethyl-2′-deoxyuridine (hmUdR) synergistically enhances the activity of FU in cell lines derived from solid tumors but not normal tissues. While the cytotoxicity of FU and hmUdR was not directly related to the amount of the modified bases incorporated into cellular DNA, incubation with this combination resulted in dramatic increase in the number of single strand breaks in replicating cancer cells, leading to NAD-depletion as consequence of poly(ADP-ribose) synthesis and S phase arrest. Cell death resulting from the base/nucleoside combination did not occur by apoptosis, autophagy or necroptosis. Instead, the cells die via necrosis as a result of NAD depletion. The FU-related nucleoside analog, 5-fluoro-2′-deoxyuridine, also displayed synergy with hmUdR, whereas hmUdR could not be replaced by 5-hydroxymethyluracil. Among other 5-modified deoxyuridine analogs tested, 5-formyl-2′-deoxyuridine and, to a lesser extent, 5-hydroxy-2′-deoxyuridine, also acted synergistically with FU, whereas 5-hydroxyethyl-2′-deoxyuridine did not. Together, our results have revealed an unexpected synergistic interaction between deoxyuridine analogs and FU in a cancer cell-specific manner, and suggest that these novel base/nucleoside combinations could be developed into improved FU-based chemotherapies.

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Section: Resultscontrasting

confidence: 79%

Synergistic enhancement of 5-fluorouracil cytotoxicity by deoxyuridine analogs in cancer cells

et al. 2015

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“…Unlike hmdC, it is long known that hmdU is relatively toxic to mammalian cells . Upon supplementation of the growth medium with hmdU, the nucleoside is incorporated into the genetic material and subsequently removed by the glycosylase Smug1. Cells lacking this glycosylase show a strongly increased hmdU tolerance, indicating that during repair of hmdU abasic sites are formed at levels that cells are quickly unable to tolerate .…”

Section: Discovery Of Oxidized Bases Beyond Hmdcmentioning

confidence: 99%

Non‐canonical Bases in the Genome: The Regulatory Information Layer in DNA

et al. 2018

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Multicellular organisms developed the concept of specialized cells that perform specific functions. Examples are neurons and fibroblast to name just two out of more than 200. These cellular differences are established based on the same sequence information stored in the cell nucleus of all cells of an organism. The sequence information needs consequently different interpretations by the different cell types. During cellular development this interpretation of the genetic code has to be tightly regulated in space and time. Interpretation of the sequence information involves the controlled activation and silencing of specific genes so that certain proteins are made in one cell type but not in others. This involves an additional regulatory information layer beyond the pure base sequence. One aspect of this regulatory information layer relies on functional groups that are attached to the C(5) position of the canonical base dC. Currently four regulatory, non-canonical bases with a methyl (CH )-, a hydroxymethyl (CH OH)-, a formyl (CHO)- and a carboxyl (COOH)- group are known. While 5-methyl-cytidine is long recognised to be a regulatory base in the genome, the other three bases and the enzymes responsible for generating them, were just recently discovered.

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“…The chemotherapy agent 5-fluorouracil (5FU) can also be incorporated into replicating DNA and facilitate uracil incorporation by interfering with thymidine monophosphate (dTMP) metabolism ( 23–25 ). The methyl group of thymine can be oxidized, forming 5-hydroxymethyluracil (5hmU) either directly in DNA or in the nucleotide pool ( 26 , 27 ). It is known that the modified pyrimidines U, 5FU and 5hmU interfere with sequence-specific DNA–protein interactions when in duplex DNA ( 28–30 ).…”

Section: Introductionmentioning

confidence: 99%

The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence

Theruvathu¹,

Darwanto²,

Hsu³

et al. 2014

Nucleic Acids Res

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Telomeric DNA can form duplex regions or single-stranded loops that bind multiple proteins, preventing it from being processed as a DNA repair intermediate. The bases within these regions are susceptible to damage; however, mechanisms for the repair of telomere damage are as yet poorly understood. We have examined the effect of three thymine (T) analogs including uracil (U), 5-fluorouracil (5FU) and 5-hydroxymethyluracil (5hmU) on DNA–protein interactions and DNA repair within the GGTTAC telomeric sequence. The replacement of T with U or 5FU interferes with Pot1 (Pot1pN protein of Schizosaccharomyces pombe) binding. Surprisingly, 5hmU substitution only modestly diminishes Pot1 binding suggesting that hydrophobicity of the T-methyl group likely plays a minor role in protein binding. In the GGTTAC sequence, all three analogs can be cleaved by DNA glycosylases; however, glycosylase activity is blocked if Pot1 binds. An abasic site at the G or T positions is cleaved by the endonuclease APE1 when in a duplex but not when single-stranded. Abasic site formation thermally destabilizes the duplex that could push a damaged DNA segment into a single-stranded loop. The inability to enzymatically cleave abasic sites in single-stranded telomere regions would block completion of the base excision repair cycle potentially causing telomere attrition.

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Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

Cited by 9 publications

References 41 publications

Synergistic enhancement of 5-fluorouracil cytotoxicity by deoxyuridine analogs in cancer cells

Synergistic enhancement of 5-fluorouracil cytotoxicity by deoxyuridine analogs in cancer cells

Non‐canonical Bases in the Genome: The Regulatory Information Layer in DNA

The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence

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